Phylogenetic relationships and species delimitation in Haemopis (Annelida: Hirudinea: Haemopidae).

The Holarctic leech genus Haemopis currently includes 11 species, all of which are macrophagous, as opposed to their more infamous bloodfeeding counterparts among hirudiniform leeches. In spite of their ecological importance as fish food and predators of freshwater invertebrates, there is a paucity of data regarding morphology and genetic variation that might guide future identification efforts for members of the genus. The lack of detailed descriptions of distinguishing morphological features, coupled with the absence of a robust phylogenetic hypothesis for the genus, have conspired to prevent meaningful inferences on the natural history of the group. In an attempt to remedy this, we present new genetic (using COI, 12S rDNA, 28S rDNA and 18S rDNA) data for the majority of the known species diversity within the genus in order to both infer a phylogenetic hypothesis and to introduce authoritative DNA barcodes for the newly collected species. The potential of these barcodes is increased through rigorous morphological investigations of the specimens, with comparisons to the original literature. Our resulting phylogenetic hypothesis is agnostic as to the geographic origin of the genus, with equal probability afforded to both a Nearctic and Palearctic origin. Beyond this, we show that there is a strong tendency towards a barcoding gap within the genus, but that a distinct gap is lacking due to the relatively high genetic variation found within H. marmorata. Taken together, our results shed light on species delimitation within, and evolutionary history of, this often-neglected group of leeches.

Placobdella nabeulensis sp. nov. (Hirudinea: Glossiphoniidae), a new glossiphoniiform leech from Palearctic North Africa.

BACKGROUND: Herein, we describe a new species of turtle blood-feeding leech, Placobdella nabeulensis sp. nov. from Palearctic North Africa (Tunisia and Algeria). The new species is described based on detailed morphological analyses using light and scanning electron microscopes. RESULTS: Apart from the detailed morphology of the atrium, morphological features alone do not sufficiently separate the species from congeners due to the absence of distinct diagnostic characters. Therefore, we turned to molecular data to better distinguish this new species from other members of the genus and establish a basis for its genetic separation. Four DNA fragments were successfully amplified, including mitochondrial COI and 12S rDNA, as well as nuclear 28S rDNA and histone H3. We then provided the molecular descriptor of the taxon, based on redundant diagnostic nucleotide combinations in DNA sequence alignment within the Folmer region. Results of the phylogenetic analysis and species delimitation methods (ABGD, ASAP, and bPTP) based on the COI locus support the species rank of the Tunisian-Algerian Placobdella. CONCLUSIONS: The new species is most closely related to the European species Placobdella costata (Fr. Müller, 1846) and the present study indicates that Placobdella nabeulensis sp. nov. has likely been confused with the European counterpart in several previous studies. This article is registered at www.zoobank.org under urn:lsid:zoobank.org:pub:4A4B9C1D-2556-430F-8E4B-0CE99F2012F5.

Genetic variation, pseudocryptic diversity, and phylogeny of Erpobdella (Annelida: Hirudinida: Erpobdelliformes), with emphasis on Canadian species.

Leeches of the family Erpobdellidae are important members of benthic freshwater environments, where they are voracious predators of other invertebrates and an important source of nutrition for several species of vertebrates. Beset by a lack of reliable diagnostic morphological characters and destructive identification processes, molecular approaches have, in recent years, been employed to illuminate the relationships within this family, and DNA barcoding has been employed for identification purposes. However, an understanding of the levels of genetic variation across the geographic distributions of members of the genus is still lacking. Herein, we sequence the mitochondrial COI locus for 249 newly collected North American individuals, representing 5 species, as well as mitochondrial 12S rDNA, nuclear 18S rDNA, and nuclear 28S rDNA for a select subset of these. Our COI dataset was leveraged to detect potential cryptic species, and to calculate genetic distances as a proxy for the degree of gene flow between populations. Augmented by numerous sequences from GenBank, the multilocus dataset was used to reconstruct a phylogenetic hypothesis for worldwide members of the genus. Beyond corroborating previous overarching phylogenetic frameworks, our results show that an undescribed species that is morphologically and genetically similar to Erpobdella punctata exists in sympatry with this species - the new species has likely been overlooked in previous studies due to its morphological similarity with Erpobdella punctata. Erpobdella bucera is reported from Canada for the first time; and Erpobdella microstoma is newly reported from Saskatchewan and placed in a phylogeny for the first time. Finally, we find evidence for genetic structure in both E. cf. punctata and Erpobdella obscura that is correlated with major river drainage basin boundaries in North America.

The salivary transcriptome of Limnobdella mexicana (Annelida: Clitellata: Praobdellidae) and orthology determination of major leech anticoagulants.

Bloodfeeding requires several adaptations that allow the parasite to feed efficiently. Leeches and other hematophagous animals have developed different mechanisms to inhibit hemostasis, one of the main barriers imposed by their hosts. Limnobdella mexicana is a member of the leech family Praobdellidae, a family of host generalists known for their preference to attach on mucosal membranes of mammals, such as those in nasopharyngeal cavities, bladders and ocular orbits. Previous studies have hypothesized a positive relationship between diversity of anticoagulants and diversity of hosts in bloodfeeding leeches. However, orthology determination of putative anticoagulants and the lack of standardization of sequencing effort and method hinder comparisons between publicly available transcriptomes generated in different laboratories. In the present study, we examine the first transcriptome of a praobdellid leech and identify 15 putative anticoagulants using a phylogeny-based inference approach, amino-acid conservation, Pfam domains and BLAST searches. Our phylogenetic analyses suggest that the ancestral leech was able to inhibit factor Xa and that some hirudins that have been reported in previous studies on leech anticoagulants may not be orthologous with the archetypal hirudin.

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

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.

Phylogenetic analysis of Placobdella (Hirudinea: Rhynchobdellida: Glossiphoniidae) with consideration of COI variation.

Placobdella is a genus of blood-feeding leeches in the family Glossiphoniidae. Historically, species of Placobdella have posed difficulty for systematists owing to a lack of informative morphological characters and the preponderance of inadequate or incomplete species descriptions. Here, we conduct a phylogenetic analysis of 55 individuals representing 20 of the 24 currently recognized nominal taxa using COI, ND1, 12S rDNA and ITS sequences under parsimony, maximum likelihood and Bayesian inference. We also examine the isolated COI phylogeny for the genus using an expanded dataset encompassing three additional species not included in the concatenated dataset. Finally, we assess genetic variation at the COI locus to validate initial specimen identifications and estimate how COI variation may reflect species boundaries. We conclude that Placobdella is a monophyletic group that places as the sister group to a clade formed by the genera Haementeria and Helobdella. We discuss the evolutionary implications of several internal relationships that are robustly resolved by all three optimality criteria, paying particular attention to the apparent fluidity of morphological characters exhibited by members of Placobdella. We also find preliminary evidence for the presence of cryptic and undescribed diversity within the genus.

Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Similarly to other strict blood feeders, leeches from the Haementeria genus (Hirudinida: Glossiphoniidae) have established a symbiotic association with bacteria harbored intracellularly in esophageal bacteriomes. Previous genome sequence analyses of these endosymbionts revealed co-divergence with their hosts, a strong genome reduction, and a simplified metabolism largely dedicated to the production of B vitamins, which are nutrients lacking from a blood diet. 'Candidatus Providencia siddallii' has been identified as the obligate nutritional endosymbiont of a monophyletic clade of Mexican and South American Haementeria spp. However, the Haementeria genus includes a sister clade of congeners from Central and South America, where the presence or absence of the aforementioned symbiont taxon remains unknown. In this work, we report on a novel bacterial endosymbiont found in a representative from this Haementeria clade. We found that this symbiont lineage has evolved from within the Pluralibacter genus, known mainly from clinical but also environmental strains. Similarly to Ca. Providencia siddallii, the Haementeria-associated Pluralibacter symbiont displays clear signs of genome reduction, accompanied by an A+T-biased sequence composition. Genomic analysis of its metabolic potential revealed a retention of pathways related to B vitamin biosynthesis, supporting its role as a nutritional endosymbiont. Finally, comparative genomics of both Haementeria symbiont lineages suggests that an ancient Providencia symbiont was likely replaced by the novel Pluralibacter one, thus constituting the first reported case of nutritional symbiont replacement in a leech without morphological changes in the bacteriome. IMPORTANCE: Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

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

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.

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

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 specific clades.

Evolution of an Alternative Genetic Code in the Providencia Symbiont of the Hematophagous Leech Haementeria acuecueyetzin.

Strict blood-feeding animals are confronted with a strong B-vitamin deficiency. Blood-feeding leeches from the Glossiphoniidae family, similarly to hematophagous insects, have evolved specialized organs called bacteriomes to harbor symbiotic bacteria. Leeches of the Haementeria genus have two pairs of globular bacteriomes attached to the esophagus which house intracellular "Candidatus Providencia siddallii" bacteria. Previous work analyzing a draft genome of the Providencia symbiont of the Mexican leech Haementeria officinalis showed that, in this species, the bacteria hold a reduced genome capable of synthesizing B vitamins. In this work, we aimed to expand our knowledge on the diversity and evolution of Providencia symbionts of Haementeria. For this purpose, we sequenced the symbiont genomes of three selected leech species. We found that all genomes are highly syntenic and have kept a stable genetic repertoire, mirroring ancient insect endosymbionts. Additionally, we found B-vitamin pathways to be conserved among these symbionts, pointing to a conserved symbiotic role. Lastly and most notably, we found that the symbiont of H. acuecueyetzin has evolved an alternative genetic code, affecting a portion of its proteome and showing evidence of a lineage-specific and likely intermediate stage of genetic code reassignment.

Placobdella akahkway n. sp. (Clitellata: Hirudinea: Glossiphoniidae), a new leech species from central Canada.

The leech genus Placobdella harbours 24 currently-recognized species, and only a handful of new species has been described in the past decade. Placobdella akahkway n. sp., a new species of ectoparasitic glossiphoniid leech is herein described from sites in the provinces of Alberta, Saskatchewan, and Manitoba, Canada. Beyond morphological description of the species, a total of 21 specimens (including the five specimens from the type series) were also employed in a phylogenetic analysis using the COI locus. The new species can be distinguished from its congeners by its possession of both compact and diffuse salivary glands. The included specimens of Placobdella akahkway n. sp. form a monophyletic group with high support, and place as the sister taxon to Placobdella kwetlumye.

The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation.

Leeches play important roles in food webs due to their abundance, diversity and feeding habits. Studies using invertebrate-derived DNA (iDNA) extracted from leech gut contents to target vertebrate DNA have focused on the Indo-Pacific region and mainly leveraged the leech family Haemadipsidae, composed of bloodfeeding terrestrial leeches, while predatory, fluid/tissue-feeding and aquatic bloodfeeding species have been largely disregarded. While there is some general knowledge regarding the taxonomic groups that leeches prefer to feed on, detailed taxonomic resolution is missing and, therefore, their potential use for monitoring animals is unknown. In this study, 116 leeches from 12 species (six families) and spanning the three feeding habits were collected in Mexico and Canada. We used DNA metabarcoding to investigate their diet and assess their potential use for biodiversity monitoring. We detected vertebrates from five orders including fish, turtles and birds in the diet of aquatic bloodfeeding leeches; eight invertebrate orders of annelids, arthropods and molluscs in leeches that feed on body fluids and tissues; and 10 orders of invertebrates belonging to Arthropoda and Annelida, as well as one vertebrate and one parasitic nematode, in predatory leeches. These results show the potential use of iDNA from aquatic bloodfeeding leeches for retrieving vertebrate taxa, and from predatory and fluid-feeding leeches for invertebrates. Our study provides information about the dietary range of freshwater leeches and one terrestrial leech and contributes proof-of-concept for the use of these leeches for animal monitoring, expanding our knowledge of the use of iDNA from leech gut contents to North America.

Function of the waterfowl nests as reproduction and living areas for leeches (Annelida: Hirudinea).

Some species of leeches migrate into waterfowl nests and use these both as general habitats and to deposit cocoons, but ecological associations between leeches and birds are not well understood. In the present study, characteristics of waterfowl nests both as living area for leeches (Hirudinea) and as reproduction areas for select hirudinid leeches (Hirudo verbana and Haemopis sanguisuga) was investigated in both natural and controlled laboratory conditions. A total of 48 leeches were detected in 23 of the 51 nests surveyed in natural habitats. The leeches were detected more frequently but there were less dense populations in the nests of the great crested grebe (Podiceps cristatus) (frequency: 57 %, population density: 0.71 ± 0.76 leeches/nest), and leeches were detected less frequently but there were more dense populations in the nests of coots (Fulica atra) (frequency: 36 %, population density: 0.91 ± 1.70 leeches/nest). Although the hirudinid leeches naturally reproduce between June and September, cocoons were only detected in August and September, when the nests were not actively used by the waterfowl and when water depths were less. In laboratory conditions, gravid medicinal leeches prefer moist peat rather than waterfowl nests for cocoon deposition. Results from the present study indicate leeches more frequently use the nests of coots, which have both dry and wet layers, compared to the nests of the great crested grebe, which have only a wet layer. Leeches were also found to be more prevalent in bird nests during spring months, during the reproductive periods of the waterfowl.

The Origin and Evolution of Antistasin-like Proteins in Leeches (Hirudinida, Clitellata).

Bloodfeeding is employed by many parasitic animals and requires specific innovations for efficient feeding. Some of these innovations are molecular features that are related to the inhibition of hemostasis. For example, bloodfeeding insects, bats, and leeches release proteins with anticoagulatory activity through their salivary secretions. The antistasin-like protein family, composed of serine protease inhibitors with one or more antistasin-like domains, is tightly linked to inhibition of hemostasis in leeches. However, this protein family has been recorded also in non-bloodfeeding invertebrates, such as cnidarians, mollusks, polychaetes, and oligochaetes. The present study aims to 1) root the antistasin-like gene tree and delimit the major orthologous groups, 2) identify potential independent origins of salivary proteins secreted by leeches, and 3) identify major changes in domain and/or motif structure within each orthologous group. Five clades containing leech antistasin-like proteins are distinguishable through rigorous phylogenetic analyses based on nine new transcriptomes and a diverse set of comparative data: the trypsin + leukocyte elastase inhibitors clade, the antistasin clade, the therostasin clade, and two additional, unnamed clades. The antistasin-like gene tree supports multiple origins of leech antistasin-like proteins due to the presence of both leech and non-leech sequences in one of the unnamed clades, but a single origin of factor Xa and trypsin + leukocyte elastase inhibitors. This is further supported by three sequence motifs that are exclusive to antistasins, the trypsin + leukocyte elastase inhibitor clade, and the therostasin clade, respectively. We discuss the implications of our findings for the evolution of this diverse family of leech anticoagulants.

Genetic variation and phylogeny of the cosmopolitan marine genus Tubificoides (Annelida: Clitellata: Naididae: Tubificinae).

Prior attempts to resolve the phylogenetic relationships of the cosmopolitan, marine clitellate genus Tubificoides, using only morphology, resulted in unresolved trees. In this study, three mitochondrial and three nuclear loci (5912 aligned sites) were analyzed, representing 14 morphologically separate species. Genetic distances within and between these forms on the basis of the mitochondrial genes (COI, 16S and 12S) revealed that 18 distinct mitochondrial lineages were represented in the data set. After analyzing also nuclear data (28S, 18S and ITS) we conclude that 17 separately evolving lineages (i.e., phylogenetic species) were represented, including three new, cryptic species closely related to T. pseudogaster, T. amplivasatus and T. insularis, respectively. Special emphasis was put on the DNA barcoding gene (COI), which was subject to haplotype diversity analysis and, for four species, diagnostic position (as determined by the Characteristic Attribute Organization System [CAOS]) screening. Typically, the intralineage variation was 1-2 orders of magnitude smaller than the interlineage divergence, making COI useful for identification of species within Tubificoides. The genetic data corroborate that many of the morphospecies are coherent but widely distributed metapopulations. Monophyly of the genus is supported and the evolutionary history of parts of the genus is revealed by phylogenetic analysis of the combined data set. A northern hemisphere origin of the genus is suggested, and most of the widely distributed species are members of one particular clade. Two morphological characters previously emphasized in Tubificoides taxonomy (hair chaetae and cuticular papillation) were optimized on the phylogenetic tree, revealing considerable homoplasy, belying the utility of these features as phylogenetic markers.

Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants.

The European medicinal leech has been used for medicinal purposes for millennia, and continues to be used today in modern hospital settings. Its utility is granted by the extremely potent anticoagulation factors that the leech secretes into the incision wound during feeding and, although a handful of studies have targeted certain anticoagulants, the full range of anticoagulation factors expressed by this species remains unknown. Here, we present the first draft genome of the European medicinal leech, Hirudo medicinalis, and estimate that we have sequenced between 79-94% of the full genome. Leveraging these data, we searched for anticoagulation factors across the genome of H. medicinalis. Following orthology determination through a series of BLAST searches, as well as phylogenetic analyses, we estimate that fully 15 different known anticoagulation factors are utilized by the species, and that 17 other proteins that have been linked to antihemostasis are also present in the genome. We underscore the utility of the draft genome for comparative studies of leeches and discuss our results in an evolutionary context.

Prey, populations, and the pleistocene: evidence for low COI variation in a widespread North American leech.

Placobdella rugosa has long presented challenges to leech biologists. Its extreme morphological variability and similarity to some congeneric species has confounded classification for over a century. Recent molecular analyses revealed a surprising lack of genetic variation among morphologically disparate, geographically widespread specimens of P. rugosa. Given the lack of any obvious mechanism by which this species could disperse between distant habitats, it was expected that widespread populations would be genetically isolated from each other. In the present study, we investigate the relationship between geographic distance and genetic diversity in P. rugosa using COI sequences from specimens collected across Canada and the United States. Although we find preliminary evidence for a barrier to gene flow between eastern and western collecting localities, our vastly expanded dataset largely corroborates prior studies, showing minimal phylogeographic signal among the sequences and negligible levels of genetic isolation by distance. A recent range expansion following the last ice age and/or host-mediated dispersal are discussed as potential explanations for this unexpected phylogeographic pattern.

Phylogenetic Position and Description of a New Species of Medicinal Leech from the Eastern United States.

A new species of medicinal leech, Macrobdella mimicus n. sp., is described from specimens collected in Maryland; this is the first description of a North American macrobdellid since 1975. Superficially, the new species resembles the well-known Macrobdella decora, as both species possess 4 accessory pores arranged symmetrically on the ventral surface, yet the new species is distinguished from M. decora in possessing 4-4½ annuli (rather than 3½) between the gonopores and 4 annuli (rather than 5 annuli) between the female gonopore and the first pair of accessory pores. Phylogenetic analyses, based on 2 mitochondrial and 2 nuclear loci for a set of closely related taxa, confirms the placement of the new species within the family Macrobdellidae and places it as the sister taxon to M. decora and M. diplotertia.

Transcriptomics and differential gene expression in Whitmania pigra (Annelida: Clitellata: Hirudinida: Hirudinidae): Contrasting feeding and fasting modes.

The medicinal utility of leeches has been demonstrated through decades of use in modern hospital settings, mainly as relievers of venous congestion following flap or digit replantation surgery. In the present study, we sequence and annotate (through BLAST- and Gene Ontology-based approaches) the salivary transcriptome of the nonblood feeding hirudinid Whitmania pigra and assess the differential gene expression of anticoagulation factors (through both quantitative real-time PCR [qRT-PCR] and in silico-based methods) during feeding and fasting conditions. This was done in order to evince the diversity of putative anticoagulation factors, as well as estimate the levels of upregulation of genes immediately after feeding. In total, we found sequences with demonstrated orthology (via both phylogenetic analyses and BLAST-based approaches) to seven different proteins that have previously been linked to anticoagulatory capabilities-eglin C, bdellin, granulin, guamerin, hyaluronidase, destabilase I, and lipocalin. All of these were recovered from leeches both in the fasting and in the feeding conditions, but all show signs of upregulation in the feeding leeches. Interestingly, our RNA-seq effort, coupled with a hypergeometric test, indicated that the differentially expressed genes were disproportionately involved in three main immunological pathways (endocytosis, peroxisome regulation, and lysosome regulation). The results and implications of the finding of anticoagulants in this nonblood feeding leech and the putative upregulation of anticoagulation factors after feeding are briefly discussed in an evolutionary context.

Broad geographic sampling and DNA barcoding do not support the presence of Helobdella stagnalis (Linnaeus, 1758) (Clitellata: Glossiphoniidae) in North America.

The description of Helobdella stagnalis (Linnaeus, 1758) has emphasized the presence of a nuchal, chitinous scute located on the dorsal surface in the first third of the body as the diagnostic character for the species. Historically, identifications of species of Helobdella have relied heavily on this character and, as a result, Helobdella stagnalis has been reported from an inordinately broad geographic range, including Europe, Asia, Africa, North America, and South America. In addition to a few earlier investigations, a recent analysis showed that great genetic distances (orders of magnitude greater than previous estimations of intraspecific divergence in leeches) are present between scute-bearing specimens identified as H. stagnalis from Europe and North America, implying that H. stagnalis does not occur in North America. The present study expands the geographic boundaries of taxon sampling for both European and North American taxa, and re-examines the phylogenetic relationships and cytochrome c oxidase subunit I (COI) variation within scute-bearing species of the genus Helobdella. Our analyses include specimens putatively identified as "Helobdella stagnalis" from Sweden, Norway, Iceland, England, France, Italy, Slovenia, Turkey, Russia, and Iran, as well as numerous localities covering Canada and the USA. Our results corroborate previous studies in that European and west Asian specimens form a clade, including the neotype, which is separate from North American taxa. To alleviate future taxonomic confusion, we redescribe H. stagnalis and designate a neotype from the inferred type locality. The designation of a neotype stabilizes the taxonomy of scute-bearing leeches of the genus Helobdella and enables us to definitively correct erroneous identifications reported in previous studies. We also note that at least four lineages of scute-bearing, North American species of Helobdella lack formal descriptions.