Unoculubranchiobdella sp. (Hirudinea: Ozobranchidae) as a vector for Haemogregarina spp. in freshwater turtles from Brazil.

Species of Haemogregarina are blood parasites known to parasitise vertebrate hosts, including fishes (Haemogregarina sensu lato) and freshwater turtles (Haemogregarina sensu stricto). Their vectors, include gnathiid isopods and leeches, respectively. In turtles, Haemogregarina balli has the best-characterized life cycle in the genus. However, no studies in Brazil have suggested a possible vector for any species of Haemogregarina from freshwater turtles. Therefore, in the present study, we provide insights into a leech vector based on specimens found feeding on two species of freshwater turtles, Podocnemis unifilis and Podocnemis expansa, using morphological and molecular data. In 2017 and 2019, freshwater turtles were collected in Goiás State, Brazil. Hosts were inspected for ectoparasites and leeches were collected from two specimens of P. expansa and nine specimens of P. unifilis. Leeches were subsequently identified as members of the genus Unoculubranchiobdella. Leech histological slides revealed haemogregarine-like structures, similar to post-sporogonic merogony, found near the gills and within the posterior sucker. Molecular analysis of the haemeogregarines resulted in the identification of three species of Haemogregarina: Haemogregarina embaubali, Haemogregarina goianensis, and Haemogregarina brasiliana. Therefore, our findings, based on morphology and DNA data suggest leeches of the genus Unoculubranchiondella as vectors for at least three species of Haemogregarina from Brazilian turtles.

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.

Phylogenetic relationships of the Geoplaninae land planarians (Platyhelminthes, Tricladida) assessed with a total evidence approach, with the description of a new species of Gigantea.

Several studies have focused on the phylogenetic relationships within the Geoplaninae land planarians (Tricladida). In those studies, ancient phylogenetic relationships remained obscure. In this work, the phylogeny of Geoplaninae is assessed through three different datasets, namely morphological, molecular, and both datasets combined, i.e, a total evidence approach (TE). The data matrix consisted of six DNA regions, including a newly developed marker (DOM5), and a morphological matrix with 37 characters. The study produced the best-resolved hypothesis so far for the phylogeny of Geoplaninae, although ancient clades still remain elusive. The effect of the morphological data on the TE tree topology and clade support is seemingly negligible. The phylogenetic tree also suggests that most of the diagnostic morphological characters of the genera are homoplastic, while unambiguous unique synapomorphies can characterize some supra-generic informal groupings.

New species of Foleyellides (Nematoda: Onchocercidae: Waltonellinae), parasite of Lithobates brownorum (Amphibia: Ranidae) from South-eastern Mexico and genetic barcodes of the Mexican species of the genus.

Specimens of Foleyellides were collected from the body cavity of frogs in different regions of Mexico; Lithobates brownorum from Yucatán, Quintana Roo and Campeche; L. megapoda from Jalisco and Rhinella marina, from Guerrero. Foleyellides calakmulesis n. sp. is described based on specimens found parasitizing L. brownorum. The new species is distinguished from the other members of the genus by the combination of the following male characters: four pairs of caudal papillae different in size and the presence of a preanal plaque. Partial DNA sequences of the mitochondrial Cytochrome Oxidase C, subunit I of the four known Mexican species of Foleyellides and two potentially new species collected in this study were generated and compared, validating the erection of the new species.

Finding a needle in a haystack: larval stages of Didymozoidae (Trematoda: Digenea) parasitizing marine zooplankton.

Larval didymozoids (Trematoda: Digenea) were discovered parasitizing the hemocoel of the heteropod Firoloida desmarestia (redia mean intensity = 13) and the chaetognaths Flaccisagitta enflata and Flaccisagitta hexaptera (metacercaria mean intensity = 1) during a 2014-2016 systematic study of parasites of zooplankton collected in the central and southern regions of the Gulf of California, Mexico. Didymozoid infection route during the early life cycle was inferred combining morphological (light microscopy) and molecular (mitochondrial cytochrome c oxidase subunit I gene, cox1) evidence. Didymozoid rediae parasitizing F. desmarestia were observed, just after field collection of the host, containing hundredths of completely developed cystophorous cercariae, releasing them though the birth pore at approximately one cercaria every 12 s. Cercariae lost their tails developing into a 'young metacercaria' in 1 d at 22 °C without need of an intermediate host. Molecular analysis of cox1 showed that rediae found in F. desmarestia belong to two distinct didymozoid species (Didymozoidae sp. 1 and sp. 2). Metacercariae parasitizing chaetognaths were morphologically identified as Didymozoidae type Monilicaecum and cox1 sequences showed that metacercariae of chaetognaths matched with these two Didymozoidae sp. 1, and sp. 2 species found parasitizing F. desmarestia, plus a third distinct Didymozoidae sp. 3. These are the first DNA sequences of cox1 gene from didymozoid larvae for any zooplankton taxonomic group in the world. We concluded that F. desmarestia is the first intermediate host of rediae and cercariae, and the chaetognaths are the second intermediate hosts where non-encysted metacercariae were found. The definitive host is still unknown because cox1 sequences of present study did not genetically match with any available cox1 sequence of adult didymozoid. Our results demonstrate a potential overlap in the distribution of two carnivorous zooplankton taxonomic groups that are intermediate hosts of didymozoids in the pelagic habitat. The didymozoid specimens were not identified to species level because any of the cox1 sequences generated here matched with the sequences of adult didymozoids currently available in GenBank and Bold System databases. This study provides baseline information for the future morphological and molecular understanding of the Didymozoidae larvae that has been previously based on the recognition of the 12 known morphotypes.

Phylogeny and biogeography of the Cavernicola (Platyhelminthes: Tricladida): Relicts of an epigean group sheltering in caves?

The planarian suborder Cavernicola Sluys, 1990 was originally created to house five species of triclad flatworms with special morphological features and a surprisingly discontinuous and broad geographic distribution. These five species could not be accommodated with any degree of certainty in any of the three taxonomic groups existing at that moment, viz., Paludicola Hallez, 1892, Terricola Hallez, 1892, and Maricola Hallez, 1892. The scarce representation of the group and the peculiarities of the morphological features of the species, including several described more recently, have complicated new tests of the monophyly of the Cavernicola, the assessment of its taxonomic status, as well as the resolution of its internal relationships. Here we present the first molecular study including all genera currently known for the group, excepting one. We analysed newly generated 18S and 28S rDNA data for these species, together with a broad representation of other triclad flatworms. The resulting phylogenetic trees supported the monophyly of the Cavernicola, as well as its sister-group relationship to the Maricola. The sister-group relationship to the Maricola and affinities within the Cavernicola falsify the morphology-based phylogeny of the latter that was proposed previously. The relatively high diversity of some cavernicolan genera suggests that the presumed rarity of the group actually may in part be due to a collecting artefact. Ancestral state reconstruction analyses suggest that the ancestral habitat of the group concerned epigean freshwater conditions. Our results point to an evolutionary scenario in which the Cavernicola (a) originated in a freshwater habitat, (b) as the sister clade of the marine triclads, and (c) subsequently radiated and colonized both epigean and hypogean environments. Competition with other planarians, notably members of the Continenticola, or changes in epigean habitat conditions are two possible explanations -still to be tested- for the loss of most epigean diversity of the Cavernicola, which is currently reflected in their highly disjunct distributions.

Morphological and molecular data reveal a new species of Lueheia (Acanthocephala: Plagiorhynchidae) from Turdus migratorius (Turdidae) in central Mexico and its phylogenetic implications within the family.

Members of the genus Lueheia Travassos, 1919, are endoparasites of birds, particularly passerines, throughout the Americas. Adults of Lueheia sp., (Plagiorhynchidae Golvan, 1960; Porrorchinae Golvan, 1956) were recovered from the intestine of the American robin (Turdus migratorius phillipsi Bangs) in Mexico City, and two other species of acanthocephalans identified as Porrorchis nickoli, (Plagiorhynchidae: Porrorchinae) Salgado-Maldonado and Cruz-Reyes, 2002 and Centrorhynchus microcephalus (Bravo-Hollis, 1947) Golvan, 1956 (Centrorhynchidae Van Cleave, 1916), were recovered from the Virginia opossum (Didelphis virginiana Allen) and groove-billed ani (Crotophaga sulcirostris Swainson), respectively in southeastern Mexico. Specimens of three species were sequenced at two molecular markers, the small subunit (SSU) and large subunit (LSU) of the nuclear rDNA and compared with other sequences available in GenBank. Maximum likelihood and Bayesian inference analyses of the combined (LSU + SSU) dataset and each individual dataset revealed that the specimens of Lueheia sp. formed an independent lineage, which is recognized herein as a new species, Lueheia aztecae n. sp., representing the fifth species of the genus in the Americas, and the second in the Nearctic region. The new species can be morphologically distinguished from the other five species in the genus by having a cylindrical proboscis, armed with 24-26 longitudinal rows with 9-10 hooks each. Phylogenetic inference performed with the combined dataset consisting of two genes (LSU + SSU) revealed that Lueheia aztecae n. sp. and P. nickoli belonging to subfamily Porrorchinae, formed two independent lineages, indicating that the subfamily is paraphyletic. Porrorchis nickoli and C. microcephalus formed a clade with other species of the genus Centrorhynchus, suggesting that P. nickoli should be transferred to genus Centrorhynchus, to form C. nickoli n. comb. In addition, we briefly discuss the ecological associations between the members of the families Plagiorhynchidae and Centrorhynchidae.

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.

A new species of Branchellion Savigny, 1822 (Hirudinida: Piscicolidae), a marine leech parasitic on the giant electric ray Narcine entemedor Jordan & Starks (Batoidea: Narcinidae) off Oaxaca, Mexico.

Branchellion spindolaorum n. sp. (Hirudinida: Piscicolidae) is described based on specimens found parasitising the giant electric ray Narcine entemedor Jordan & Starks off the coast of Oaxaca, Mexico. The new species can be clearly distinguished from the other species of Branchellion Savigny, 1822 by the presence of 30 pairs of lateral branchiae and 10 pairs of pulsatile vesicles. The definition of the genus Branchellion is expanded to include species with either 30, 31 or 33 pairs of foliaceous (plate-like) lateral branchiae in the urosome. In addition, we provide for the first time for the genus, scanning electron micrographs of the secondary suckers located on the ventral surface of the posterior sucker. Additionally, partial DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) were generated and compared with homologous sequences of other species of the genus. Branchellion spindolaorum n. sp. represents the fourth species of the genus known in the Eastern Pacific and the first record of a leech parasitising N. entemedor.

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.

Caligus fajerae n. sp. (Copepoda: Caligidae) parasitic on the Pacific sierra Scomberomurus sierra Jordan & Starks (Actinopterygii: Scombridae) in the Pacific Ocean off Mexico.

A new species of parasitic copepod, Caligus fajerae n. sp. (Caligidae), is described from Scomberomorus sierra Jordan & Starks (Scombridae) caught off the northwestern coast of Mexico. The new species morphologically resembles Caligus cybii Bassett-Smith, 1898, Caligus kanagurta Pillai, 1961, Caligus pelamydis Krøyer, 1863 and Caligus robustus Bassett-Smith, 1898, all of which have been reported from scombrid hosts. Caligus fajerae n. sp. differs from these species by having spinules on the abdomen and caudal ramus, two processes on the proximal antennulary segment, fine striations on the claw of the antenna and maxilliped, a stouter and more recurved maxillulary dentiform process, shorter tines on the sternal furca, two additional patches of spinules on the distal endopodal segment of leg 2, a sclerotised lobe on the anteromedian surface of the leg 3 protopod and serrations on both margins of the first exopodal spine of leg 3. Analysis of the DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 gene for Caligus fajerae n. sp. and 28 congeners, including C. pelamydis and C. robustus, showed that the new species grouped with Caligus belones Krøyer, 1863 (with 20% divergence), a species known to occur predominantly on needlefishes. Caligus fajerae n. sp. is the fifth species of Caligus reported from S. sierra. An updated host-parasite list for Caligus spp. on scombrids is provided.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

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.

Potential zoonotic role of the tick Amblyomma cf. oblongoguttatum (Ixodida: Ixodidae) in the bacterial transmission of Ehrlichia chaffeensis (Rickettsiales: Anaplasmataceae) in a deciduous tropical forest in Mexico.

Ehrlichia chaffeensis is a bacterium belonging to the Anaplasmataceae family. In Mexico, only 2 species have been recorded in association with tick species and humans. The objective of the present study was to detect the presence of bacteria of the genus Ehrlichia in ticks collected from the Chamela-Cuixmala Biosphere Reserve, Jalisco, Mexico. The collected ticks were identified and analyzed individually by polymerase chain reaction to amplify a fragment of the Anaplasmataceae 16S rRNA gene and the Ehrlichia-specific dsb gene. A total of 204 ticks, corresponding to 5 species of Ixodidae and 1 of Argasidae, were collected from 147 mammals of 6 species and 4 orders; 57 ticks collected from vegetation were also included. Among the total ticks collected, 1.47% (3/204) was positive for Ehrlichia sp. DNA was obtained using the primers EHR 16SD and EHR 16SR for 16S rRNA and DSB-330 and DSB-728 for dsb. The positive samples corresponded to a larva (Amblyomma sp.) associated with Didelphis virginiana and 2 nymphs (Amblyomma cf. oblongoguttatum) infesting Nasua narica. None of the ticks collected from the vegetation tested positive for Ehrlichia sp. DNA on the basis of the 16S rRNA and dsb genes. The sequences from the larvae of Amblyomma sp. and the nymphs of A. cf. oblongoguttatum were similar to those of E. chaffeensis. The phylogenetic analysis inferred with maximum likelihood corroborated the identity as E. chaffeensis. Although the role of these tick species as vectors of E. chaffeensis is still undetermined, the presence of infected ticks in the area indicates a potential zoonotic risk.

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.

Temporal variation in helminth infracommunities of the Gafftopsail pompano, Trachinotus rhodopus (Pisces: Carangidae) off the Pacific coast of Mexico.

Temporal variation of the helminth infracommunity structure in the Gafftopsail pompano Trachinotus rhodopus was studied during bi-monthly revisions of samples collected offshore from Puerto Ángel, Oaxaca (Mexican Pacific) in 2018. In total, 110 specimens of T. rhodopus were subjected to a parasitic review. Helminths found were identified to the lowest possible taxonomic level (six species and three genera) by means of morphological and molecular data. Attributes of the helminth infracommunities are described through statistical analyses, showing stability in terms of their richness throughout the year. However, variations were found in helminth abundance related to the seasonality of samplings, which may be associated with the life cycles of the parasites, the host species' gregarious behavior, the availability of intermediate hosts, and/or the diet of T. rhodopus.

Diversity of myxozoans (Cnidaria) infecting Neotropical fishes in southern Mexico.

Myxozoans are a unique group of microscopic parasites that infect mainly fishes. These extremely reduced cnidarians are highly diverse and globally distributed in freshwater and marine habitats. Myxozoan diversity dimension is unknown in Mexico, a territory of an extraordinary biological diversity. This study aimed to explore, for the first time, myxozoan parasite diversity from fishes of the Neotropical region of Mexico. We performed a large morphological and molecular screening using host tissues of 22 ornamental and food fish species captured from different localities of Veracruz, Oaxaca and Chiapas. Myxozoan infections were detected in 90% of the fish species, 65% of them had 1 or 2 and 35% had 3 and up to 8 myxozoan species. Forty-one putative new species were identified using SSU rDNA phylogenetic analyses, belonging to two main lineages: polychaete-infecting (5 species) and oligochaete-infecting (36 species) myxozoans; from those we describe 4 new species: Myxidium zapotecus sp. n., Zschokkella guelaguetza sp. n., Ellipsomyxa papantla sp. n. and Myxobolus zoqueus sp. n. Myxozoan detection increased up to 6 × using molecular screening, which represents 3.7 × more species detected than by microscopy. This study demonstrated that Neotropical fishes from Mexico are hosts of a multitude of myxozoans, representing a source of emerging diseases with large implications for economic and conservation reasons.

Molecular phylogeny of the New World bloodfeeding leeches of the genus Haementeria and reconsideration of the biannulate genus Oligobdella.

The phylogenetic relationships of species of the New World bloodfeeding genus Haementeria were investigated for the first time. The analysis included five molecular markers. The mitochondrial COI, 12S and ND1 as well as the nuclear 28S and ITS. The evolutionary history of the group was investigated through Maximum Parsimony and Bayesian Inference. Both phylogenetic methods resulted in highly congruent hypotheses. The correlation between the phylogeny and morphological traits such as eyespot number, annulation, Lang's organs, salivary glands, bacteriomes and reproductive organs is discussed. Restricted to Haementeria are Lang's organs, spherical bacteriomes and ovaries forming an anterior ring around the ventral nerve cord. In addition, Oligobdella brasilensis was formally transferred to Haementeria, providing additional arguments for the disposal of the genus Oligobdella. Haementeria gracilis is shown to be just a junior synonym of Haementeria depressa as suggested by previous authors. Finally, the geographical distribution of species of Haementeria was compared with that of other non-leech and leech taxa. Multiple events of South-North American interchange were proposed to explain the current geographical distribution of the species of Haementeria.

Phylogenetic position of Acanthobothrium cleofanus (Cestoda: Onchoproteocephalidea) using molecular evidence.

Despite the large number of species described to date for the onchoprotepcephalid genus Acanthobothrium (207), only 16 named species have a genetic sequence. With this background, specimens of adult cestodes of the stingray Hypanus longus were collected off San Blas, Nayarit, and onchoproteocephalid larvae in the carangid fish Trachinotus rhodopus from Puerto Ángel, Oaxaca, both located on the Pacific coast of Mexico. The objective of this work is to investigate the phylogenetic position of these adults and larvae using nuclear ribosomal markers (18S rDNA and 28S rDNA). Morphologically, adult specimens were identified as Acanthobothrium cleofanus; larvae were identified only to family level. The phylogenetic position of both taxa was investigated based on the information of two nuclear molecular markers analyzed under Parsimony (PA) and Bayesian Inference (BI) methods. The newly generated sequences of A. cleofanus from Nayarit are identical to the sequences of several samples of Acanthobothrium sp. collected in the Mexican Pacific, which sequence are available in GenBank; DNA sequences obtained from onchoproteocephalid larva clearly place this taxon within Acanthobothrium but representing an independent lineage. In the resulting phylogenetic trees, Uncibilocularis okei was found nested within Acanthobothrium with an unstable position depending on the optimality criteria, indicating the need for more molecular analyzes with a greater number of species of both genera prior to define its phylogenetic relationships.

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.