University of Toronto

Assistant Professor

Sebastian worked at University of Toronto as a Assistant Professor

Royal Ontario Museum

Associate Curator

Sebastian worked at Royal Ontario Museum as a Associate Curator

Harvard University

Postdoctoral Fellow

Sebastian worked at Harvard University as a Postdoctoral Fellow

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Hydrobiologia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Hydrobiologia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

Molecular Biology and Evolution

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Hydrobiologia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

Molecular Biology and Evolution

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

Redescription of Micrura dellechiajei (Hubrecht, 1879) (Nemertea, Pilidiophora, Lineidae), a rare Mediterranean species

Journal of the Marine Biological Association of the UK

The heteronemertean species Micrura dellechiajei is thus far only known from its type locality in the Gulf of Naples (Italy) and has not been recorded in 120 years. During two oceanographic surveys conducted in Spanish Mediterranean waters, several nemertean specimens were collected, and thorough morphological examination indicated that some of these pertained to the species M. dellechiajei, suggesting that populations may be more widespread than previously thought. Because of the rarity of this species coupled with the fact that its last morphological narrative was given 120 years ago, we here provide a redescription of the species based on the new specimens, complete with illustrations and new data concerning its morphology, and we also place some of the collected specimens in a molecular phylogenetic framework.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Hydrobiologia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

Molecular Biology and Evolution

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

Redescription of Micrura dellechiajei (Hubrecht, 1879) (Nemertea, Pilidiophora, Lineidae), a rare Mediterranean species

Journal of the Marine Biological Association of the UK

The heteronemertean species Micrura dellechiajei is thus far only known from its type locality in the Gulf of Naples (Italy) and has not been recorded in 120 years. During two oceanographic surveys conducted in Spanish Mediterranean waters, several nemertean specimens were collected, and thorough morphological examination indicated that some of these pertained to the species M. dellechiajei, suggesting that populations may be more widespread than previously thought. Because of the rarity of this species coupled with the fact that its last morphological narrative was given 120 years ago, we here provide a redescription of the species based on the new specimens, complete with illustrations and new data concerning its morphology, and we also place some of the collected specimens in a molecular phylogenetic framework.

Description and phylogeny of Namalycastis jaya sp. n. (Polychaeta, Nereididae, Namanereidinae) from the southwest coast of India

Zookeys

Namalycastis jaya sp. n. (Polychaeta: Nereididae: Namanereidinae)is described from the southern coast of Kerala in southwest India. One important characteristic feature of the species is the lack of notochaetae in all parapodia, a characteristic that it shares with at least two other species, Namalycastis elobeyensis Glasby, 1999and Namalycastis hawaiiensis Johnson, 1903. It differs from Namalycastis elobeyensis by virtue of its smaller antennae, unequal eye size, bilobed acicular neuropodial ligule and multi-incised pygidium rim. Moreover, it differs from Namalycastis hawaiiensis by having fewer teeth on the serrated blades of the sub-neuroacicular falciger in chaetiger 10, and by possessing finely serrated falcigers in posterior segments. Beyond morphological analyses, molecular phylogenetics was used for the first time for Namalycastis to support population monophyly and recognition of the new species.The analysis, using both mitochondrial and nuclear data, corroborated the morphological analysis in suggesting that our specimens represent an as yet undescribed species, Namalycastis jaya sp. n., which forms a monophyletic group among the sampled nereidid taxa. Finally, a taxonomic key for Namalycastis species recorded from the Indian region is provided.

New insights into the phylogeny, systematics and DNA barcoding of Nemertea

Invertebrate Systematics

Although some clades of ribbon worms (phylum Nemertea) are consistently recovered with high support in molecular phylogenies, the placement and inter-relationships of some taxa have proven problematic. Herein, we performed molecular phylogenetic analyses aimed at resolving these recalcitrant splits, using six loci (nuclear 18S rRNA, 28S rRNA, histones H3 and H4, and mitochondrial 16S rRNA and COI) for 133 terminals, with particular emphasis on the problematic families Hubrechtidae and Plectonemertidae. Three different datasets were used for phylogenetic analyses and both maximum likelihood and maximum parsimony methodologies were applied. All but one of the resulting tree topologies agree on the paraphyly of the class Palaeonemertea, whereas Heteronemertea, Hoplonemertea, Polystilifera, Monostilifera and Hubrechtidae are always recovered as reciprocally monophyletic. Hubrechtidae is sister group to Heteronemertea (the Pilidiophora hypothesis) only when length variable regions of 18S rRNA and 28S rRNA are excluded. Moreover, the terrestrial and freshwater family Plectonemertidae is recovered with high support and the implications of this finding are further discussed. Finally, we evaluate the utility of DNA barcoding for specimen identification within Nemertea using an extended dataset containing 394 COI sequences. Results suggest that DNA barcoding may work for Nemertea, insofar as a distinct barcoding gap (the gap between the maximum intraspecific variation and the minimum interspecific divergence) may exist, but its recognition is regularly hampered by low accuracy in species level identifications.

Genome-wide search for leech antiplatelet proteins in the non-blood-feeding leech Helobdella robusta (Rhyncobdellida: Glossiphoniidae) reveals evidence of secreted anticoagulants

Invertebrate Biology

The genome of the non-blood-feeding glossiphoniid leech Helobdella robusta was screened for leech antiplatelet protein (LAPP), an anticoagulant that specifically inhibits collagen-stimulated platelet aggregation. Previously identified LAPP sequences from Haementeria officinalis were used as queries against the predicted genes in the genome, employing a variety of BLAST protocols. Matches were reciprocally BLASTed against GenBank databases as a cross-validation of the predicted annotations of the genes. A total of eight loci, positioned as a tandem array, were recovered with significantly low e-values; these showed high sequence similarity (32.49% average sequence similarity of shared amino acid positions) to the known anticoagulants. Moreover, six of these possessed a predicted signal-peptide toward the N-terminus, indicating their secretion by the leech. All eight loci, together with known LAPP sequences from Ha. officinalis, as well as several sequences from publicly available expressed sequence tag libraries of Ha. depressa and He. robusta, were aligned and subjected to phylogenetic analysis. The resulting tree showed a monophyletic clade consisting of the He. robusta loci, which was sister to a clade comprised of Haementeriaderived sequences. To corroborate the evolution of the anticoagulants with the evolution of leeches more generally, the topology of the LAPP-tree was compared to that of a previously published leech phylogeny; these showed compatible topologies with respect to the included genera. These results corroborate recent phylogenetic work, which suggests that this non-blood-feeding leech has a hematophagous ancestry.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens

GigaScience

Although molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

Invertebrate Biology

Freshwater, marine, and terrestrial leeches that depend on a diet of fresh blood have evolved salivary peptide components that inhibit normal thrombus formation by prey. Although bloodfeeding in leeches has long been of interest to biologists and medical practitioners alike, only a few studies have comprehensively examined the anticoagulant repertoires of hematophagous leeches, and these have largely been confined to representatives of Glossiphoniidae, Hirudinidae, and Macrobdellidae. Here, we present 454 pyrosequencing data from the salivary transcriptome of the hematophagous terrestrial leech Haemadipsa interrupta (Haemadipsidae). Assembled transcripts were annotated using both similarity scores (BLAST) and gene ontology (Blast2GO). Subsequently, transcripts were examined within alignments containing well-characterized anticoagulants and other select bioactive (i.e., affecting the living cells of the prey) salivary proteins and phylogenies were reconstructed for each protein data set to verify orthology predictions. In total, transcripts significantly matching 20 salivary proteins of interest were found in the transcriptome, representing several different antagonistic pathways. After reviewing gene ontologies, alignments, and phylogenetic trees, sequences for 15 out of the 20 hits were deemed correctly annotated. Additionally, we recovered matches against several proteins that have previously been linked to anticoagulation (e.g., cathepsin and disintegrins), but the specific function of which in leeches needs further investigation. Finally, in light of these data as well as those previously published, we discuss our current understanding of the distribution and evolution of anticoagulants in leeches.

Barcoding in the dark?: A critical view of the sufficiency of zoological DNA barcoding databases and a plea for broader integration of taxonomic knowledge

Molecular Phylogenetics and Evolution

The functionality of standard zoological DNA barcoding practice (the identification of unknown specimens by comparison of COI sequences) is contingent on working barcode databases with sufficient taxonomic coverage. It has already been established that the main barcoding repositories, NCBI and BOLD, are devoid of data for many animal groups but the specific taxonomic coverage of the repositories across animal biodiversity remains unexplored. Here, I shed light on this mystery by contrasting the number of unique taxon labels in the two databases with the number of currently recognized species for each animal phylum. The numbers reveal an overall paucity of COI sequence data in the repositories (15.13% total coverage across the recognized biodiversity on Earth, and 20.76% average taxonomic coverage for each phylum) and, more importantly, bear witness to the idleness towards numerous phyla, rendering current barcoding efforts either ineffective or inaccurate. The importance of further integrating taxonomic expertise into barcoding practice is briefly discussed and some guidelines, previously mentioned in the barcoding literature, are suggested anew. Finally, the asserted values concerning the taxonomic coverage in barcoding databases for Animalia are contrasted with those of Plantae and Fungi.

Comparative Mitogenomics of Leeches (Annelida: Clitellata): Genome Conservation and Placobdella-Specific trnD Gene Duplication

PLoS ONE

Mitochondrial DNA sequences, often in combination with nuclear markers and morphological data, are frequently used to unravel the phylogenetic relationships, population dynamics and biogeographic histories of a plethora of organisms. The information provided by examining complete mitochondrial genomes also enables investigation of other evolutionary events such as gene rearrangements, gene duplication and gene loss. Despite efforts to generate information to represent most of the currently recognized groups, some taxa are underrepresented in mitochondrial genomic databases. One such group is leeches (Annelida: Hirudinea: Clitellata). Herein, we expand our knowledge concerning leech mitochondrial makeup including gene arrangement, gene duplication and the evolution of mitochondrial genomes by adding newly sequenced mitochondrial genomes for three bloodfeeding species: Haementeria officinalis, Placobdella lamothei and Placobdella parasitica. With the inclusion of three new mitochondrial genomes of leeches, a better understanding of evolution for this organelle within the group is emerging. We found that gene order and genomic arrangement in the three new mitochondrial genomes is identical to previously sequenced members of Clitellata. Interestingly, within Placobdella, we recovered a genus-specific duplication of the trnD gene located between cox2 and atp8.We performed phylogenetic analyses using 12 protein-coding genes and expanded our taxon sampling by including GenBank sequences for 39 taxa; the analyses confirm the monophyletic status of Clitellata, yet disagree in several respects with other phylogenetic hypotheses based on morphology and analyses of non-mitochondrial data.

Sine Systemate Chaos? A Versatile Tool for Earthworm Taxonomy: Non-Destructive Imaging of Freshly Fixed and Museum Specimens Using Micro-Computed Tomography

PLoS ONE

In spite of the high relevance of lumbricid earthworms (‘Oligochaeta’: Lumbricidae) for soil structure and functioning, the taxonomy of this group of terrestrial invertebrates remains in a quasi-chaotic state. Earthworm taxonomy traditionally relies on the interpretation of external and internal morphological characters, but the acquisition of these data is often hampered by tedious dissections or restricted access to valuable and rare museum specimens. The present state of affairs, in conjunction with the difficulty of establishing primary homologies for multiple morphological features, has led to an almost unrivaled instability in the taxonomy and systematics of certain earthworm groups, including Lumbricidae. As a potential remedy, we apply for the first time a non-destructive imaging technique to lumbricids and explore the future application of this approach to earthworm taxonomy. High-resolution micro-computed tomography (mCT) scanning of freshly fixed and museum specimens was carried out using two cosmopolitan species, Aporrectodea caliginosa and A. trapezoides. By combining two-dimensional and three-dimensional dataset visualization techniques, we demonstrate that the morphological features commonly used in earthworm taxonomy can now be analyzed without the need for dissection, whether freshly fixed or museum specimens collected more than 60 years ago are studied. Our analyses show that mCT in combination with soft tissue staining can be successfully applied to lumbricid earthworms. An extension of the approach to other families is poised to strengthen earthworm taxonomy by providing a versatile tool to resolve the taxonomic chaos currently present in this ecologically important, but taxonomically neglected group of terrestrial invertebrates.

COMPARATIVE TRANSCRIPTOMIC ANALYSES OF THREE SPECIES OF PLACOBDELLA (RHYNCHOBDELLIDA: GLOSSIPHONIIDAE) CONFIRMS A SINGLE ORIGIN OF BLOOD FEEDING IN LEECHES

Journal of Parasitology

One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar —this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

DNA Barcoding of Parasitic Nematodes: Is it Kosher?

Journal of Parasitology

Nematode parasites were encountered in kosher-certified fish meat and roe, and the question was raised as to whether or not these food products were kosher as concerns food preparation standards—a matter that pertains to the identity and, by extension, the life cycle of the parasites. To ascertain the identities of parasitic nematodes, given the distorted or damaged nature of the specimens, molecular techniques were applied in the form of DNA barcoding. To our knowledge, this is the first application of this technique to an obviously cultural concern as opposed to one of health or economic significance. Results, based both on cytochrome c oxidase subunits I and II, suggested that the parasite species found in the fish products are anisakine species that do not inhabit the intestinal lumen of the fish hosts examined. Thus, there was no evidence of failure to adhere to food preparation practices consistent with the proscriptions of Orthodox Judaism. Notwithstanding the success of DNA barcoding in determining at least the higher taxonomic identities of the parasites, some shortcomings of the DNA barcoding pipeline as it pertains to nematode parasites were encountered; specifically, the paucity of data available for the DNA barcoding locus, even for very common nematode taxa.

Evaluating the Utility of Single-Locus DNA Barcoding for the Identification of Ribbon Worms (Phylum Nemertea)

PLoS ONE

Whereas many nemerteans (ribbon worms; phylum Nemertea) can be identified from external characters if observed alive, many are still problematic. When it comes to preserved specimens (as in e.g. marine inventories), there is a particular need for specimen identifier alternatives. Here, we evaluate the utility of COI (cytochrome c oxidase subunit I) as a single- locus barcoding gene. We sequenced, data mined, and compared gene fragments of COI for 915 individuals representing 161 unique taxonomic labels for 71 genera, and subjected different constellations of these to both distance-based and character-based DNA barcoding approaches, as well as species delimitation analyses. We searched for the presence or absence of a barcoding gap at different taxonomic levels (phylum, subclass, family and genus) in an attempt to understand at what level a putative barcoding gap presents itself. This was performed both using the taxonomic labels as species predictors and using objectively inferred species boundaries recovered from our species delimitation analyses. Our data suggest that COI works as a species identifier for most groups within the phylum, but also that COI data are obscured by misidentifications in sequence databases. Further, our results suggest that the number of predicted species within the dataset is (in some cases substantially) higher than the number of unique taxonomic labels—this highlights the presence of several cryptic lineages within well-established taxa and underscores the urgency of an updated taxonomic backbone for the phylum.

Redescription of Namalycastis glasbyi Fernando & Rajasekaran, 2007 (Annelida, Nereididae, Namanereidinae) from India

Proceedings of the Biological Society of Washington

Namalycastis glasbyi Fernando & Rajasekaran, 2007 was originally described from the creek-lined coast north of Mumbai in western India. However, the original description includes some oddities that make species recognition problematic and may confuse future taxonomic evaluations of the genus. Moreover, the type material of the species has been lost and, therefore, this species is herein redescribed, a neotype is assigned, and the distinguishing morphological features are clearly illustrated. The species shares some features with two other Indian species: Namalycastis indica Southern, 1921 and the N. abiuma M¨uller in Grube, 1871 species group. However, N. glasbyi differs from N. indica in having comparatively smaller antennae, shorter posterior dorsal cirri, a higher count of notochaetae (maximum of three) in the mid and anterior body, and finely serrated blades in sub- and supra-neuroacicular falcigers in the anterior and mid-body. Further, sub-neuroacicular spinigers have finely serrated blades in all parapodia in N. glasbyi, as opposed to the coarsely serrated spinigers from antero-mid body parapodia of N. indica. Moreover, N. glasbyi differs from the N. abiuma species group in lacking coarsely serrated sub-neuroacicular spinigers in the parapodia of mid and posterior parts of the body and in possessing a tripartite pygidium. In addition to these morphological differences, the mitochondrial COI sequence (DNA barcode) of the neotype of Namalycastis glasbyi clearly distinguishes it from other Indian species.

Diversity and selective pressures of anticoagulants in three medicinal leeches (Hirudinida: Hirudinidae, Macrobdellidae)

Ecology and Evolution

Although medicinal leeches have long been used as treatment for various ailments because of their potent anticoagulation factors, neither the full diversity of salivary components that inhibit coagulation, nor the evolutionary selection acting on them has been thoroughly investigated. Here, we constructed expressed sequence tag libraries from salivary glands of two species of medicinal hirudinoid leeches, Hirudo verbana and Aliolimnatis fenestrata, and identified anticoagulant-orthologs through BLASTx searches. The data set then was augmented by the addition of a previously constructed EST library from the macrobdelloid leech Macrobdella decora. The identified orthologs then were compared and contrasted with well-characterized anticoagulants from a variety of leeches with different feeding habits, including non-sanguivorous species. Moreover, four different statistical methods for predicting signatures of positive and negative evolutionary pressures were used for 10 rounds each to assess the level and type of selection acting on the molecules as a whole and on specific sites. In total, sequences showing putative BLASTx-orthology with five and three anticoagulant-families were recovered in the A. fenestrata and H. verbana EST libraries respectively. Selection pressure analyses predicted high levels of purifying selection across the anticoagulant diversity, although a few isolated sites showed signatures of positive selection. This study represents a first attempt at mapping the anticoagulant repertoires in a comparative fashion across several leech families.

Incipient speciation within the Namalycastis abiuma (Annelida: Nereididae) species group from southern India revealed by combined morphological and molecular data

Memoirs of Museum Victoria

Namalycastis abiuma (Grube, 1872), originally described from Brazil, comprises a species complex of morphologically similar forms occurring circumtropically, including India. Apart from the Namalycastis abiuma species group, four other Namalycastis species are presently known from India: N. indica Southern, 1921, N. fauveli Nageswara Rao, 1981, N. glasbyi Fernando & Rajasekaran, 2007, and N. jaya Magesh, Kvist & Glasby, 2012. Recent sampling along the southern Indian coast has uncovered new specimens of the N. abiuma species group. The present study uses combined morphological and molecular data (DNA barcoding) to explore species boundaries within the complex in southwest India and thereby resolve existing taxonomic confusion. In order to evaluate morphological variability within the N. abiuma species group, a total of 50 specimens were sampled from different geographical regions in southern India, and assessed using traditional methods. For 18 of the specimens, a 509 bp fragment of COI, the proposed DNA barcoding gene, was sequenced and subjected to tree reconstruction using both distance methods and maximum parsimony. Based on similarity alone, six different haplotypes were recognized within the dataset and these were also subsequently recovered as six distinct clades in the parsimony analysis. There is significant concordance between the morphotypes and the genetic haplotypes, suggesting that significant structural forces are acting on the specimens at a population level, and that these specimens may even be in an early stage of speciation.

The future of nemertean taxonomy (phylum Nemertea) — a proposal

Zoologica Scripta

Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data mining and examining the effects of missing data

Cladistics

We present phylogenomic analyses of the most comprehensive molecular character set compiled for Annelida and its constituent taxa, including over 347 000 aligned nucleotide sites for 39 taxa. The nucleotide data set was recovered using a pre-existing amino acid data set of almost 48 000 aligned sites as a backbone for tBLASTn searches against NCBI. In addition, orthology determinations of the loci in the original amino acid data set were scrutinized using an All vs All Reciprocal Best Hit approach, employing BLASTp, and examining for statistical interdependency among the loci. This approach revealed considerable sequence redundancy among the loci in the original data set and a new data set was compiled, with the redundancy removed. The newly compiled nucleotide data set, the original amino acid data set, and the new reduced amino acid data set were subjected to parsimony analyses and two forms of bootstrap resampling. The last-named data set also was analysed using a maximum- likelihood approach. There were two main objectives to these analyses: (i) to examine the general topology, including support, resulting from the analyses of the new data sets and (ii) to assess the consistency of the branching patterns across optimality criteria by comparison with previous probabilistic approaches. The phylogenetic hypotheses resulting from analyses of the three data sets are largely unsupported, reflecting the continued difficulty of finding numerous, reliable, and suitable loci for a group as ancient as Annelida. Resulting parsimonious hypotheses disagree, in some respects, with the previous probabilistic approaches; Sedentaria and, in most cases, Errantia are not supported as monophyletic groups but Pleistoannelida is recovered as a (unsupported) monophyletic group in one of the three parsimony analyses as well as the likelihood analysis. In addition, we performed missing data titration studies to estimate the impact of missing data on overall support and support for

Does a global DNA barcoding gap exist in Annelida?

Mitochondrial DNA

Accurate identification of unknown specimens by means of DNA barcoding is contingent on the presence of a DNA barcoding gap, among other factors, as its absence may result in dubious specimen identifications – false negatives or positives. Whereas the utility of DNA barcoding would be greatly reduced in the absence of a distinct and sufficiently sized barcoding gap, the limits of intraspecific and interspecific distances are seldom thoroughly inspected across comprehensive sampling. The present study aims to illuminate this aspect of barcoding in a comprehensive manner for the animal phylum Annelida. All cytochrome c oxidase subunit I sequences (cox1 gene; the chosen region for zoological DNA barcoding) present in GenBank for Annelida, as well as for ‘‘Polychaeta’’, ‘‘Oligochaeta’’, and Hirudinea separately, were downloaded and curated for length, coverage and potential contaminations. The final datasets consisted of 9782 (Annelida), 5545 (‘‘Polychaeta’’), 3639 (‘‘Oligochaeta’’), and 598 (Hirudinea) cox1 sequences and these were either (i) used as is in an automated global barcoding gap detection analysis or (ii) further analyzed for genetic distances, separated into bins containing intraspecific and interspecific comparisons and plotted in a graph to visualize any potential global barcoding gap. Over 70 million pairwise genetic comparisons were made and results suggest that although there is a tendency towards separation, no distinct or sufficiently sized global barcoding gap exists in either of the datasets rendering future barcoding efforts at risk of erroneous specimen identifications (but local barcoding gaps may still exist allowing for the identification of specimens at lower taxonomic ranks). This seems to be especially true for earthworm taxa, which account for fully 35% of the total number of interspecific comparisons that show 0% divergence.

Phylogeny of Nemertea with special interest in the placement of diversity from Far East Russia and northeast Asia

Hydrobiologia

Recent investigations, based mostly on molecular data, have unraveled the evolutionary history of several common ribbon worm (phylum Nemertea) species and solidified the taxonomic status of many higher taxa within the group. However, a large proportion of enigmatic species have yet to be placed in a phylogenetic framework. We investigated the phylogenetic positions of 26 novel and/or perplexing nemertean species from the Sea of Okhotsk, the Sea of Japan, the Kuril–Kamchatka Trench, and Vietnam (including the first record of a reptant nemertean from the Far East seas of Russia). We conducted both maximum likelihood and parsimony analyses, utilizing four molecular loci—mitochondrial COI and 16S rRNA, as well as nuclear 18S rRNA and 28S rRNA—finding that the current classification for most of these species is corroborated by their phylogenetic placement. We then discuss the evolution of some unique morphological traits possessed by some of these species, using the molecular phylogeny as a backbone for our general conclusions

Genome-Wide Survey of Gut Fungi (Harpellales) Reveals the First Horizontally Transferred Ubiquitin Gene from a Mosquito Host

Molecular Biology and Evolution

Harpellales, an early-diverging fungal lineage, is associated with the digestive tracts of aquatic arthropod hosts. Concurrent with the production and annotation of the first four Harpellales genomes, we discovered that Zancudomyces culisetae, one of the most widely distributed Harpellales species, encodes an insect-like polyubiquitin chain. Ubiquitin and ubiquitin-like proteins are universally involved in protein degradation and regulation of immune response in eukaryotic organisms. Phylogenetic analyses inferred that this polyubiquitin variant has a mosquito origin. In addition, its amino acid composition, animal-like secondary structure, as well as the fungal nature of flanking genes all further support this as a horizontal gene transfer event. The single-copy polyubiquitin gene from Z. culisetae has lower GC ratio compared with homologs of insect taxa, which implies homogenization of the gene since its putatively ancient transfer. The acquired polyubiquitin gene may have served to improve important functions within Z. culisetae, by perhaps exploiting the insect hosts’ ubiquitin-proteasome systems in the gut environment. Preliminary comparisons among the four Harpellales genomes highlight the reduced genome size of Z. culisetae, which corroborates its distinguishable symbiotic lifestyle. This is the first record of a horizontally transferred ubiquitin gene from disease-bearing insects to the gut-dwelling fungal endobiont and should invite further exploration in an evolutionary context.

Redescription of Micrura dellechiajei (Hubrecht, 1879) (Nemertea, Pilidiophora, Lineidae), a rare Mediterranean species

Journal of the Marine Biological Association of the UK

The heteronemertean species Micrura dellechiajei is thus far only known from its type locality in the Gulf of Naples (Italy) and has not been recorded in 120 years. During two oceanographic surveys conducted in Spanish Mediterranean waters, several nemertean specimens were collected, and thorough morphological examination indicated that some of these pertained to the species M. dellechiajei, suggesting that populations may be more widespread than previously thought. Because of the rarity of this species coupled with the fact that its last morphological narrative was given 120 years ago, we here provide a redescription of the species based on the new specimens, complete with illustrations and new data concerning its morphology, and we also place some of the collected specimens in a molecular phylogenetic framework.

Description and phylogeny of Namalycastis jaya sp. n. (Polychaeta, Nereididae, Namanereidinae) from the southwest coast of India

Zookeys

Namalycastis jaya sp. n. (Polychaeta: Nereididae: Namanereidinae)is described from the southern coast of Kerala in southwest India. One important characteristic feature of the species is the lack of notochaetae in all parapodia, a characteristic that it shares with at least two other species, Namalycastis elobeyensis Glasby, 1999and Namalycastis hawaiiensis Johnson, 1903. It differs from Namalycastis elobeyensis by virtue of its smaller antennae, unequal eye size, bilobed acicular neuropodial ligule and multi-incised pygidium rim. Moreover, it differs from Namalycastis hawaiiensis by having fewer teeth on the serrated blades of the sub-neuroacicular falciger in chaetiger 10, and by possessing finely serrated falcigers in posterior segments. Beyond morphological analyses, molecular phylogenetics was used for the first time for Namalycastis to support population monophyly and recognition of the new species.The analysis, using both mitochondrial and nuclear data, corroborated the morphological analysis in suggesting that our specimens represent an as yet undescribed species, Namalycastis jaya sp. n., which forms a monophyletic group among the sampled nereidid taxa. Finally, a taxonomic key for Namalycastis species recorded from the Indian region is provided.