Delimitation and species discovery in the Profundulidae fish family: Using genetic, environmental and morphologic data to address taxonomic uncertainty.

The family Profundulidae includes some one of the most enigmatic freshwater fishes of Mesoamerica: despite many attempts, a robust phylogenetic framework to delimit species is lacking, mainly due to limited morphological variation within the group. The accumulation of molecular data of profundulid fishes has led to advances in the description of new taxa, but relatively less progress has been made estimating evolutionary and phylogenetic relationships for this fish family. Here, we adopt an integrative taxonomy approach including the use of nuclear and mitochondrial DNA sequences, morphometric and ecological data, to test species boundaries in profundulid fishes in the westernmost area of their known distribution range in the states of Guerrero and Oaxaca, Mexico. Using a combination of methods for species discovery and validation based on Bayesian gene tree topologies, our analyses support the delimitation of 15 valid species of profundulid fishes - a combination of previously described species validated by this study, the synonymy of unsupported taxa, and the description of two new species. Using species delimitation methods, examination of phenotypic variation, and ecological niche characterization, we also identify five potentially new lineages which require further evidence to be erected as new species. We demonstrate that the use of an integrative taxonomy approach provides a robust methodology to delimit species in a taxonomically complex group like Profundulidae. Accurate taxonomic and ecological information is crucial for the conservation of these microendemic fishes, as several species are endangered.

Molecular data reveal hidden diversity of the genus Clinostomum (Digenea, Clinostomidae) in Argentina, with the description of a new species from Ardea cocoi (Ardeidae).

The genus Clinostomum has been recently a subject of a large number of molecular phylogenetic studies that have uncovered a larger species diversity than we thought. In Argentina, only two nominal species have been reported, namely C. detruncatum and C. marginatum. Three putative species represented by metacercariae were recently molecularly diagnosed, and there are at least two additional metacercarial morphotypes diagnosed on morphological grounds. Here, we molecularly characterized specimens of Clinostomum sampled from freshwater fishes and fish-eating birds from Argentina through mtDNA cytochrome c oxidase subunit I (COI). Unexpectedly, the phylogenetic analysis uncovered three new additional genetic lineages, two of them corresponding to metacercarial stages and another matching COI sequences of C. heluans Braun, 1899, being considered conspecific, whereas the others still require formal description. Additionally, we add a new host species for a lineage molecularly diagnosed in a previous study. The adult specimens recovered from A. cocoi in Buenos Aires Province represented a new species clearly distinguished from the two species previously reported in Argentina. Clinostomum detruncatum is distinguished from the new species by possessing a characteristic tenoidean uterus, and testes located more posteriorly. Clinostomum marginatum is morphologically similar but differs from the new species in having rounded ovary, posterior testis lobated, and lateral cirrus-sac and displacing the anterior testis, and vitelline follicles not extending beyond the caeca end. Our study raises the number of Clinostomum species in Argentina up to 10. We describe the new species herein.

Exploring the genetic diversity of Tylodelphys (Diesing, 1850) metacercariae in the cranial and body cavities of Mexican freshwater fishes using nuclear and mitochondrial DNA sequences, with the description of a new species.

Members of the genus Tylodelphys Diesing, 1850 are endoparasites of fish-eating birds, particularly ciconiids, anhingids, and podicipedids across the globe. Metacercariae of Tylodelphys spp. were collected from the cranial and body cavities of freshwater fishes in central and northern Mexico; adults were recovered from the intestine of two species of freshwater diving birds of the family Podicipedidae, commonly known as grebes, in two locations of central Mexico. Specimens were sequenced for two molecular markers, the internal transcribed spacers (ITS1 and ITS2) plus 5.8S gene of the nuclear ribosomal DNA and of the cytochrome c oxidase subunit 1 from mitochondrial DNA. The genetic divergence among the 25 samples (16 metacercariae and 9 adults) and between the newly sequenced specimens and those deposited in the GenBank were estimated. Maximum likelihood and Bayesian inference analyses inferred with each data set revealed the existence of five genetic lineages. Eight metacercariae analyzed in this study were nested in two divergent lineages previously recognized as Tylodelphys sp. 5 and Tylodelphys sp. 6 (sensu Locke et al., Int J Parasitol, 45:841-855, 2015). Five adult specimens recovered from the intestine of the least grebe (Tachybaptus dominicus Linnaeus, 1766) in Tecocomulco Lake, Hidalgo State, nested in a single clade with other sequences identified previously as Tylodelphys aztecae, expanding its distribution range in other areas of central Mexico. The isolates of the metacercariae found in the cranial cavity of the shortfin silverside, Chirostoma humboldtianum Valenciennes, 1835 from Zacapu Lake in central Mexico formed a monophyletic lineage and were recognized as an undescribed species of Tylodelphys. The lack of adult specimens of this lineage in our samples prevented a formal description. However, the metacercariae collected in the cranial cavity of the silverside, Chirostoma jordani Woolman, 1894 and the adult specimens recovered from the intestine of the western grebe, Aechmophorus occidentalis (Lawrence, 1858) from Cuitzeo Lake formed a monophyletic clade, allowing us to link both stages of the life cycle and to describe this as a new species, Tylodelphys kuerepus n. sp. The new species represents the eighth species of the genus described in the Americas and the fourth in the Nearctic region. We briefly discuss the ecological associations between the metacercariae and their second intermediate hosts in relation to the genetic diversity patterns uncovered in our study.

Molecular and morphological characterization of the metacercariae of two species of diplostomid trematodes (Platyhelminthes, Digenea) in freshwater fishes of the Batalha River, Brazil.

The Diplostomidae include a large group of flatworms with complex life cycles and are frequently found parasitizing the eyes and central nervous system of freshwater fishes. The morphological identification of the metacercariae at species level is not always possible. Thus, molecular tools have become essential to assist in the parasite species determination. This study was aimed at describing two diplostomid metacercariae found in freshwater fish in São Paulo, Brazil, based on morphological characters and in the genetic characterization of COI sequences. Our results showed that the two recognized taxa (Tylodelphys sp. and Diplostomidae gen. sp.) appear to be different from the species already described in South America. Tylodelphys sp. differs morphologically from Tylodelphys xenopi, T. mashonense, T. jenynsiae, and T. scheuringi. The metacercariae of T. clavata and T. conifera are smaller than Tylodelphys sp., while T. podicipina is larger than the metacercariae described here. The phylogenetic analysis of COI sequences yielded Tylodelphys sp. as the sister species of Tylodelphys sp. 4, a species reported from the brain of the eleotrid Gobiomorus maculatus in Oaxaca, Mexico. The metacercariae identified as Diplostomidae gen. sp. are morphologically different from the known diplostomid metacercariae and did not match with other diplostomid sequences available. Diplostomidae gen. sp. is recovered as the sister species of Diplostomum ardeae. Although the morphological evidence and the COI sequences differentiate the metacercariae found, the absence of adult specimens of both species precludes the specific designation. This is one of the first papers that use an integrative taxonomy approach to describe the species diversity of diplostomid trematodes in Brazil.

Trematode diversity in freshwater fishes of the Globe II: 'New World'.

We provide a summary overview of the diversity of trematode parasites in freshwater fishes of the 'New World', i.e. the Americas, with emphasis on adult forms. The trematode fauna of three regions, South America, Middle America, and USA and Canada (North America north of Mexico), are considered separately. In total, 462 trematode species have been reported as adults from the Americas. The proportion of host species examined for parasites varies widely across the Americas, from a high of 45% in the Mexican region of Middle America to less than 5% in South America. North and South America share no adult species, and one exclusively freshwater genus, Creptotrema Travassos, Artigas & Pereira, 1928 in the Allocreadiidae Looss, 1902 is the most widely distributed. Metacercariae of strigeiforms maturing in fish-eating birds (e.g. species of the Diplostomidae Poirier, 1886) are common and widely distributed. The review also highlights the paucity of known life-cycles. The foreseeable future of diversity studies belongs to integrative approaches and the application of molecular ecological methods. While opportunistic sampling will remain important in describing and cataloguing the trematode fauna, a better understanding of trematode diversity and biology will also depend on strategic sampling throughout the Americas.

Checklist of the species of Neoechinorhynchus (Acanthocephala: Neoechinorhynchidae) in fishes and turtles in Middle-America, and their delimitation based on sequences of the 28S rDNA.

Among the acanthocephalans, Neoechinorhynchus is one of the most speciose genera, with 116 described species distributed worldwide. The adults of Neoechinorhynchus are found in the intestine of freshwater and brackish water fish, as well as in freshwater turtles. In this study, a checklist of the congeneric species of Neoechinorhynchus occurring in Middle-American fish and turtles is presented. The checklist contains the records established in all published accounts, as well as novel data from survey work conducted in the region comprising Neotropical areas of Mexico, as well as some localities in Central America. The species delimitation criteria used to discriminate among species is based on molecular data. In the last years, a large database derived from sequences of the D2 + D3 domains of the large subunit of rDNA (28S) was generated for 262 specimens corresponding to nine species of Neoechinorhynchus. This molecular marker has shown to be useful in establishing species limits within Neoechinorhynchus and in resolving phylogenetic relationships at species level. Based on our results, the domains D2 + D3 of the 28S rDNA could be considered as potential DNA barcodes to complement mitochondrial DNA to discriminate among acanthocephalan species.

Parasites (Monogenea) of tilapias Oreochromis niloticus and Coptodon rendalli (Cichlidae) in a river spring in Brazil.

In the present study, we examined 30 individuals of introduced African cichlids, Oreochromis niloticus and Coptodon rendalli, collected in a river spring of the Pardo River, Paranapanema River basin, southeastern Brazil. Based on morphological and molecular analyses of the partial LSU rDNA gene, we identified four species of monogeneans, Cichlidogyrus tilapiae, C. thurstonae, C. mbirizei, and Scutogyrus longicornis on the gills of O. niloticus, whereas individuals of C. rendalli were infested only with C. papernastrema. This is the first record of C. mbirizei and C. papernastrema in tilapias from Brazil. The ecological consequences of the introduction of exotic species of tilapia such as O. niloticus and C. rendalli along with their monogenean parasites in a wild environment represented by a river spring are discussed. Our new molecular data on Cichlidogyrus and Scutogyrus contribute to the investigation of the phylogenetic interrelationships of these widely distributed genera of monogeneans since their species composition is still unsettled.

Title: Parasites (Monogenea) des tilapias Oreochromis niloticus et Coptodon rendalli (Cichlidae) dans une source au Brésil. Abstract: Dans la présente étude, nous avons examiné 30 individus de cichlidés africains introduits, Oreochromis niloticus et Coptodon rendalli, collectés dans une source fluviale du fleuve Pardo, bassin du fleuve Paranapanema, dans le sud-est du Brésil. Sur la base d'analyses morphologiques et moléculaires du gène partiel de l'ADNr LSU, nous avons identifié quatre espèces de monogènes, Cichlidogyrus tilapiae, C. thurstonae, C. mbirizei et Scutogyrus longicornis sur les branchies d'O. niloticus, alors que les individus de C. rendalli étaient infestés uniquement par C. papernastrema. Il s'agit du premier signalement de C. mbirizei et C. papernastrema chez des tilapias du Brésil. Les conséquences écologiques de l'introduction d'espèces exotiques de tilapia telles que O. niloticus et C. rendalli ainsi que leurs monogènes parasites dans un environnement sauvage représenté par une source fluviale sont discutées. Nos nouvelles données moléculaires sur Cichlidogyrus et Scutogyrus contribuent à l'étude des interrelations phylogénétiques de ces genres de monogènes largement distribués puisque leur composition spécifique est encore incertaine.

Using mitochondrial and ribosomal DNA sequences to test the taxonomic validity of Clinostomum complanatum Rudolphi, 1814 in fish-eating birds and freshwater fishes in Mexico, with the description of a new species.

The taxonomic history and species composition of the genus Clinostomum has been unstable. Two species, Clinostomum complanatum Rudolphi, 1814 and Clinostomum marginatum Rudolphi, 1819, have been particularly problematic and its validity has been disputed for nearly 200 years. In this paper, we have made use of an integrative taxonomy approach, and we used, in first instance, DNA sequences of two genes (cox1 and ITS) to test the validity of C. complanatum, a species apparently widely distributed in Mexico and to link the metacercariae and adult forms of the recognized species of Clinostomum. Combining molecular data with morphology, host association, and geographical distribution, we searched for the potential existence of undescribed species. A new species of Clinostomum is described based on adults found in the mouthy cavity of three species of fish-eating birds as well as in metacercariae found in freshwater and estuarine fishes. A few morphological characteristics distinguish the new species from other congeners even though reciprocal monophyly in a phylogenetic tree based on maximum-likelihood and Bayesian analysis, genetic divergence, and a multivariate analysis of variance and a principal component analysis of 18 morphometric traits for adults and metacercariae demonstrates the validity of the new species. Based on our results, it seems that C. complanatum is not currently distributed in Mexico, although this requires further verification with a more thoroughful sampling in other areas of the country, but it is plausible to support the hypothesis that C. marginatum is the American form, as previously suggested by other authors.

Host-parasite interactions in perpetual darkness: Macroparasite diversity in the cavefish Astyanax mexicanus.

Astyanax mexicanus has repeatedly colonized cave environments, displaying evolutionary parallelisms in many troglobitic traits. Despite being a model system for the study of adaptation to life in perpetual darkness, the parasites that infect cavefish are practically unknown. In this study, we investigated the macroparasite communities in 18 cavefish populations from independent lineages and compared them with the parasite diversity found in their sister surface fish populations, with the aim of better understanding the role that parasites play in the colonization of new environments. Within the cavefish populations, we identified 13 parasite taxa, including a subset of 10 of the 27 parasite taxa known for the surface populations. Parasites infecting the cavefish belong to five taxonomic groups, including trematodes, monogeneans, nematodes, copepods, and acari. Monogeneans are the most dominant group, found in 14 caves. The macroparasites include species with direct life cycles and trophic transmission, including invasive species. Surprisingly, paired comparisons indicate higher parasite richness in the cavefish than in the surface fish. Spatial variation in parasite composition across the caves suggests historical and geographical contingencies in the host-parasite colonization process and potential evolution of local adaptations. This base-line data on parasite diversity in cavefish populations of A. mexicanus provides a foundation to explore the role of divergent parasite infections under contrasting ecological pressures (cave vs. surface environments) in the evolution of cave adaptive traits.

Testing the evolutionary and biogeographical history of Glypthelmins (Digenea: Plagiorchiida), a parasite of anurans, through a simultaneous analysis of molecular and morphological data.

The genus Glypthelmins includes some of the most common digeneans inhabiting the intestine of anurans in the Americas. Phylogenetic analyses of eight species of Glypthelmins and five outgroups, using 26 morphological characters and sequences of cox1, 18S, 5.8S, 28S genes and ITS2 were performed. Additionally, 2 species for which no molecular data have been obtained were included in the analyses. Following a simultaneous analysis approach and using different methods of phylogenetic inference we obtained a phylogenetic tree where the eight studied species conform a monophyletic clade which is well supported by Bremer support, bootstrap, and posterior probabilities. The mapping of morphological characters showed that traits such as serrate scale-like spines, bipartite seminal vesicle, metraterm running dorsal to the cirrus pouch, and ovary sinistral are unequivocal synapomorphies that support the monophyly of Glypthelmins. Phylogenetic hypothesis based on combined data sets was used to re-evaluate the evolutionary and biogeographical history of this group of digeneans. New information provided in this study, in the context of a more robust analytical method allowed us to corroborate that members of the "Rana pipiens" group were the plesiomorphic group of hosts for Glypthelmins, with two host switching events occurring from the "Rana pipiens" group to the "Rana palmipes" group and to Hylidae during the evolutionary history of this group of parasites, and the origin of the group is proposed in Nearctic frogs, with a colonization of Neotropical hosts represented by a monophyletic clade constituted by G. brownorumae, G. facioi, and G. tuxtlasensis.

Integrating molecular and morphological approaches for characterizing parasite cryptic species: implications for parasitology.

Herein we review theoretical and methodological considerations important for finding and delimiting cryptic species of parasites (species that are difficult to recognize using traditional systematic methods). Applications of molecular data in empirical investigations of cryptic species are discussed from an historical perspective, and we evaluate advantages and disadvantages of approaches that have been used to date. Developments concerning the theory and practice of species delimitation are emphasized because theory is critical to interpretation of data. The advantages and disadvantages of different molecular methodologies, including the number and kind of loci, are discussed relative to tree-based approaches for detecting and delimiting cryptic species. We conclude by discussing some implications that cryptic species have for research programmes in parasitology, emphasizing that careful attention to the theory and operational practices involved in finding, delimiting, and describing new species (including cryptic species) is essential, not only for fully characterizing parasite biodiversity and broader aspects of comparative biology such as systematics, evolution, ecology and biogeography, but to applied research efforts that strive to improve development and understanding of epidemiology, diagnostics, control and potential eradication of parasitic diseases.

Molecular and Morphological Elucidation of the Life Cycle of the Frog Trematode Langeronia macrocirra (Digenea: Pleurogenidae) in Los Tuxtlas, Veracruz, Mexico.

The genus LangeroniaCaballero and Bravo-Hollis, 1949, currently contains 6 species of amphibian trematodes distributed in North and Middle America. The type species of the genus, Langeronia macrocirraCaballero and Bravo-Hollis, 1949, occurs in Mexico and is relatively commonly found as a parasite of leopard frogs. However, information regarding its life cycle is lacking. In this paper, we study the life cycle of L. macrocirra in Laguna Escondida, Los Tuxtlas, Veracruz. Definitive hosts (Rana spp.) as well as potential intermediate hosts (gastropods, bivalves, crustaceans, tadpoles, hemipterans, and odonate naiads) were sampled in the locality and studied to search for the presence of adults and larval stages of the trematode. Specimens were morphologically characterized, and some individuals were sequenced for 1 ribosomal gene (28S rRNA) and 1 mitochondrial gene (COI). DNA sequences of the adults obtained from leopard frogs were matched with those of the larval forms in their intermediate hosts (metacercariae, cercariae, and sporocysts) to demonstrate conspecificity. Further, we conducted a detailed study of the tegument of the body surface with scanning electron microscopy to characterize each of the developmental stages of the life cycle of L. macrocirra.

Molecular data reveal a new species of Rhopalias Stiles & Hassall, 1898 (Digenea, Echinostomatidae) in the Common opossum, Didelphismarsupialis L. (Mammalia, Didelphidae) in the Yucatán Peninsula, Mexico.

A new species of Rhopalias Stiles & Hassall, 1898 is described from the small intestine of the Common opossum, Didelphismarsupialis Linnaeus from the Yucatán Peninsula, Mexico. Rhopaliasoochi sp. nov. is morphologically very similar to the type species of the genus, Rhopaliascoronatus (Rudolphi, 1819) Stiles & Hassall 1898, a species widely distributed in opossums across Mexico. A molecular phylogenetic analysis using a mitochondrial gene (cox1), and the nuclear ribosomal internal transcribed spacer region (ITS1-5.8S-ITS2), of specimens of R.coronatus collected in several localities of Mexico revealed that those from the Yucatán Peninsula, originally recorded on morphological grounds as R.coronatus actually represented an independent genetic lineage. Maximum Likelihood and Bayesian Inference analyses were performed for each data set independently, and for the concatenated data set (ITS1-5.8S-ITS2 + cox1). All phylogenetic analyses showed that the specimens from Yucatán represented a monophyletic lineage, with high bootstrap support and Bayesian posterior probabilities. In addition, the genetic divergence estimated between R.oochi sp. nov. and two species of Rhopalias, R.coronatus, and R.macracanthus Chandler, 1932 that also occur in Mexican marsupials ranged between 7-8% and 16-17%, for cox1, and between 0.1-0.2% and 7% for the ITS region, respectively. The molecular evidence gathered in this study (reciprocal monophyly in both phylogenetic analyses, and estimated genetic divergence) suggested that the specimens found in the intestine of D.marsupialis originally reported as R.coronatus from Yucatán, actually represent a new species. Morphological evidence was found through light and scanning electron microscopy to support the species distinction based on molecular data.

Assessing the Taxonomic Validity of Austrodiplostomum SPP. (Digenea: Diplostomidae) Through Nuclear and Mitochondrial Data.

Adults of the genus Austrodiplostomum are parasites in cormorants of the New World, whereas metacercariae are parasites from eye globe and brain of freshwater and brackish water fishes. In this study, specimens of Austrodiplostomum mordax from South America (type-species) were analyzed together with other specimens of Austrodiplostomum spp. collected from several locations across Middle America and North America. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I ( COI), the internal transcribed spacers ( ITS1, ITS2, and 5.8S gene), and the D2-D3, domains of the large subunit ( LSU) of nuclear ribosomal DNA, were generated for both developmental stages and compared with available sequences of Austrodiplostomum spp. Phylogenetic analyses inferred with each molecular marker using maximum likelihood and Bayesian inference revealed the existence of 4 lineages representing 2 described species, A. mordax and Austrodiplostomum compactum (syn. Austrodiplostomum ostrowskiae) and 2 undescribed species of Austrodiplostomum recognized in previous studies. The COI haplotype network inferred with 172 sequences detected 28 haplotypes divided into 4 clusters, separating each other by 33 and 40 substitutions and with a genetic divergence ranging from 9 to 12%. The largest group included specimens identified as A. compactum plus those identified as A. ostrowskiae, supporting the synonymy of both species. As a result, we conclude that A. compactum is widely distributed across the Americas, in locations of the United States, Mexico, El Salvador, Honduras, Costa Rica, Venezuela, Peru, and Brazil. The other 2 undescribed species of the genus Austrodiplostomum were previously recorded in the United States and now are reported in Mexico. These 2 species cannot be described because adult forms have not been found in their definitive hosts. Finally, the species A. mordax has been found only in some lakes from Argentina, and it was validated in this study through molecular analyses.

Integrative taxonomy reveals a new species of Dollfusentis (Acanthocephala: Illiosentidae), in Orthopristis ruber (Osteichthyes: Haemulidae) from Rio de Janeiro, Brazil.

The taxonomy of species of Dollfusentis is rather confused due to the overlap of morphological traits. The aim of this study was to follow an integrative taxonomy approach over the acanthocephalans collected from Orthopristis ruber in Brazil. Dollfusentis lenti n. sp. is described and is characterised by having an elongate trunk with spines sparsely distributed (largest 60-85 µm long) extending from the neck to almost reach the end of proboscis receptacle; additionally, the new species possesses a long proboscis with 12-14 longitudinal rows of 16-17 hooks each; 3-4 posterior hooks reduced in size, well-spaced from the eight ventrolateral crescent hooks, and lemnisci longer than proboscis receptacle. New sequences of 18S rDNA, ITS1, 5.8S and ITS2, 28S rDNA and COI mtDNA are provided. Dollfusentis bravoae is morphologically similar because it possesses the same number of proboscis hooks, although it differs by the size of testes and uterus and by having a higher number of trunk spines; additionally, new scanning electron micrographs and genetic data for both species support its distinction. Phylogenetic analysis obtained either with two nuclear genes or mitochondrial COI gene showed that Dollfusentis spp. belong to Illiosentidae, and the new species is yielded as the sister species of D. bravoae, with D. chandleri as the sister species of the latter two.

Evolutionary history of the endangered fish Zoogoneticus quitzeoensis (Bean, 1898) (Cyprinodontiformes: Goodeidae) using a sequential approach to phylogeography based on mitochondrial and nuclear DNA data.

BACKGROUND: Tectonic, volcanic and climatic events that produce changes in hydrographic systems are the main causes of diversification and speciation of freshwater fishes. Elucidate the evolutionary history of freshwater fishes permits to infer theories on the biotic and geological evolution of a region, which can further be applied to understand processes of population divergence, speciation and for conservation purposes. The freshwater ecosystems in Central Mexico are characterized by their genesis dynamism, destruction, and compartmentalization induced by intense geologic activity and climatic changes since the early Miocene. The endangered goodeid Zoogoneticus quitzeoensis is widely distributed across Central México, thus making it a good model for phylogeographic analyses in this area. RESULTS: We addressed the phylogeography, evolutionary history and genetic structure of populations of Z. quitzeoensis through a sequential approach, based on both microsatellite and mitochondrial cytochrome b sequences. Most haplotypes were private to particular locations. All the populations analysed showed a remarkable number of haplotypes. The level of gene diversity within populations was Hd = 0.987 (0.714 - 1.00). However, in general the nucleotide diversity was low, pi = 0.0173 (0.0015 - 0.0049). Significant genetic structure was found among populations at the mitochondrial and nuclear level (PhiST = 0.836 and FST = 0.262, respectively). We distinguished two well-defined mitochondrial lineages that were separated ca. 3.3 million years ago (Mya). The time since expansion was ca. 1.5 x 10(6) years ago for Lineage I and ca. 860,000 years ago for Lineage II. Also, genetic patterns of differentiation, between and within lineages, are described at different historical timescales. CONCLUSION: Our mtDNA data indicates that the evolution of the different genetic groups is more related to ancient geological and climatic events (Middle Pliocene, ca. 3.3 Mya) than to the current hydrographic configuration of the basins. In general, mitochondrial and nuclear data supported the same relationships between populations, with the exception of some reduced populations in highly polluted basins (Lower Lerma River), where the effects of genetic drift are suggested by the different analyses at the nuclear and mitochondrial level. Further, our findings are of special interest for the conservation of this endangered species.

Phylogenetic Position of Pseudosellacotyla lutzi (Freitas, 1941) (Digenea: Cryptogonimidae), A Parasite of Hoplias malabaricus (Bloch) in South America, through 28S rDNA Sequences, and New Observations of the Ultrastructure of Their Tegument.

The freshwater fish digenean Pseudosellacotyla lutzi ( Freitas, 1941 ) Yamaguti, 1954 has had an unsettled taxonomic history, and has at various times been classified as a member of Nanophyetidae, Heterophyidae, Microphallidae, Faustulidae, and Cryptogonimidae. Nine individual specimens of the trahira, Hoplias malabaricus (Bloch, 1794), were sampled in the Paraná River basin, Paraná State, Brazil; 22 specimens of P. lutzi were collected. One specimen of P. lutzi was used to obtain a sequence of the domains D1-D3 of the 28S rRNA gene, and to perform a phylogenetic analysis to assess their position and classification within Plagiorchiida. The resulting tree unequivocally shows that the species, along with acanthostomines, belong to the Cryptogonimidae, corroborating recent findings based on the morphology of the cercariae, and in the characteristics of the life cycle. In addition, the study of the ultrastructure of the tegumental spines through scanning electron microscopy allowed us to characterize them as pectinate spines possessing 3 to 8 digitiform projections at their distal end and extending from the anterior to the posterior extremity of the body. This study also provides the first molecular data for a cryptogonimid from South America.

Helminth Fauna Associated with Three Neotropical Bat Species (Chiroptera: Mormoopidae) in Veracruz, México.

Bats are recognized as potential hosts of pathogens exploiting the food chain to reach them as definitive hosts. However, very little is known about their endoparasites, especially for Neotropical bats. In this study, we assessed the helminth fauna associated with 3 insectivorous bat species roosting in the same single hot cave in central Veracruz, México: Mormoops megalophylla, Pteronotus davyi, and Pteronotus personatus. During a period of 1 yr (April 2007-2008), 135 mormoopid bats in total were collected and examined for helminths. Six parasite species representing 3 types of intestinal helminths were found: 1 cestode Vampirolepis elongatus; 2 trematodes Maxbraunium tubiporum and Ochoterenatrema labda; and 3 nematodes Linustrongylus pteronoti, Molineidae gen. sp., and Capillaria sp. Overall, trematodes were the most abundant parasite group (72.4%), followed by nematodes (20.7%) and cestodes (6.9%). Species-accumulation curves suggest that the worms collected (n = 1,331) from these 6 parasite species comprise the helminth fauna associated with the 3 bat populations studied. The only species shared by the 3 bat species was Capillaria sp. Most (5/6) of the helminth species recorded use Lepidoptera and Diptera as intermediate hosts; therefore, diet is likely the main source of infection. Although insectivorous bats are considered dietary generalist species, the differences found in helminth diversity in these sympatric populations of closely related bat species, suggest that diet partitioning occurs in mormoopid bat communities. Helminths tend to exploit the food chain to reach their final hosts; therefore, studying these parasites can provide useful information to further understand the biology of bats.

A new species of Auriculostoma (Trematoda: Allocreadiidae) from the intestine of Brycon guatemalensis (Characiformes: Bryconidae) from the Usumacinta River Basin, Mexico, based on morphology and 28S rDNA sequences, with a key to species of the genus.

We describe a new species of Auriculostoma Scholz, Aguirre-Macedo & Choudhury, 2004 based on several sources of information including morphology (light and scanning electron microscopy [SEM]), sequences of two nuclear genes, host association, and geographical distribution. Morphologically, the new species most closely resembles Auriculostoma astyanace Scholz, Aguirre-Macedo & Choudhury, 2004, but differs by having deeply lobated testes and cirrus-sac extending posteriorly to seminal receptacle level. Auriculostoma lobata n. sp. can be readily distinguished from all the other congeners by the combination of the following characters: testes located in tandem, testes deeply lobated, and larger body size. A phylogenetic analysis using 28S rDNA sequences along with those available for other allocreadiid trematodes, revealed that the new species is a sister taxon of A. astyanace, a species described from the banded astyanax, Astyanax fasciatus (Cuvier) in Nicaragua. Auriculostoma totonacapanensis Razo-Mendivil, Mendoza-Garfias, Pérez-Ponce de León & Rubio-Godoy, 2014 from the Mexican tetra, Astyanax mexicanus (De Filippi) in Mexico is the sister taxon of A. astyanace plus the new species. Genetic divergence levels for the 28S rDNA and ITS2 were estimated among the Middle-American species of Auriculostoma infecting characiforms. The validity of the new species is then established by reliable morphological differences, its host association to bryconids (Brycon guatemalensis Regan), restricted geographical distribution (Usumacinta and Lacantun River basins), and genetic divergence levels, albeit relatively low. A morphometric comparison between the new species and the other seven congeneric species was undertaken and, in addition, a taxonomic key to identify the species contained in the genus Auriculostoma, widely distributed across the Americas, is provided.

Phylogenetic Analysis Using the 28S rRNA Gene Reveals That the Genus Paracreptotrema (Digenea: Allocreadiidae) Is Not Monophyletic; Description of Two New Genera and One New Species.

This study investigates the systematics of Paracreptotrema Choudhury, Pérez-Ponce de León, Brooks and Daverdin, 2006 using morphological data (stained whole mounts and scanning electron microscopy) and partial sequences of the 28S ribosomal rRNA gene, obtained from freshly collected material. In total, 484 specimens representing 4 species, i.e., Paracreptotrema blancoi (157), Paracreptotrema profundulusi (12), Paracreptotrema rosenthali (8), and Paracreptotrema blancoi sensu Salgado-Maldonado et al. (2011) (307) were collected. Existing museum depositions were also studied. The 28S rRNA gene sequences of these Paracreptotrema spp. were aligned, along with sequences from 22 other allocreadiids and 4 other non-allocreadiid xiphidiatan species. Bayesian inference and maximum likelihood analyses indicated a paraphyletic Paracreptotrema split into 3 clades: 1 comprising P. blancoi and P. rosenthali that was sister to a clade formed by 3 other species of allocreadiids (species of Wallinia, Creptotrematina, and Auriculostoma) typically found in characid fishes, a second clade formed solely by Paracreptotrema heterandriae as the sister taxon of the aforementioned species, and a third by P. profundulusi and specimens erroneously identified as P. blancoi. Two new taxa were erected to reflect these results: Paracreptotrematoides for Paracreptotrema heterandriae, and Pseudoparacreptotrema for Paracreptotrema profundulusi and P. macroacetabulata (the species erroneously identified as P. blancoi from profundulids across Middle America). Closer consideration of the morphology corroborates these findings. The revised systematics also indicated that Paracreptotrema spp. are found in poeciliids, whereas Pseudoparacreptotrema spp. parasitize profundulids. The study demonstrates the value of an integrative taxonomy approach to address the apparently complicated systematics of the allocreadiids.