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.

Assessing the genetic diversity of the metacercariae of Posthodiplostomum minimum (Trematoda: Diplostomidae) in Middle American freshwater fishes: one species or more?

Trematode taxonomy is mainly based on the morphological traits of adults. The identification of metacercariae is challenging because such traits are not developed in larval forms, and they even may show some level of morphological variability. Studies testing the potential correspondence between morphological differences and genetic variation of parasites are still lacking. The metacercariae of Posthodiplostomum minimum are probably the diplostomids more widely distributed in North and Middle American freshwater fish, and their intraspecific morphological variability has been attributed to the effect exerted by the host. Here, we tested the hypothesis whether they represent a single species, or a species complex by assessing the genetic divergence and phylogenetic relationships of metacercariae sampled from several host species in a wide geographical range across Middle America. The internal transcribed spacers (ITS1-5.8S-ITS2), and the mitochondrial COI gene were sequenced for 124 and 55 metacercariae, respectively. Phylogenetic analysis inferred from ITS sequences uncovered six well-supported monophyletic lineages. The six lineages show no correspondence to any Posthodiplostomum species for which sequences are available thus far in GenBank. Lineages exhibit some degree of host specificity; Lineages I, II, IV and V are primarily parasites of cyprinodontiforms of the families Poeciliidae, Goodeidae, Profundulidae and Fundulidae. In poeciliids there are at least four candidate species of Posthodiplostomum, some of them occurring in sympatry; instead, Lineages II and VI are exclusively parasites of cichlids. This study contributes to our understanding of the diversity of larval forms of diplostomids and provides an opportunity to further study their life cycles.

Phylogenetic relationships and ecological niche conservatism in killifish (Profundulidae) in Mesoamerica.

The family Profundulidae is a group of small-sized fish species distributed between southern Mexico and Honduras, where they are frequently the only fish representatives at higher elevations in the basins where they occur. We characterized their ecological niche using different methods and metrics drawn from niche modelling and by re-examining phylogenetic relationships of a recently published molecular phylogeny of this family to gain a better understanding of its biogeographic and evolutionary history. We assessed both lines of evidence from the perspective of niche conservatism to set a foundation for discussing hypotheses about the processes underlying the distribution and evolution of the group. In fish clades where the species composition is not clear, we examined whether niche classification could be informative to discriminate groups geographically and ecologically consistent with any of the different hypotheses of valid species. The characterization of the ecological niche was carried out using the Maxent algorithm under different parameterizations and the projection of the presence on the main components of the most relevant environmental coverage, and the niche comparison was calculated with two indices (D and I), both in environmental space and in that projected geographically. With the molecular data, a species tree was generated using the *BEAST method. The comparison of these data was calculated with an age-overlap correlation test. Based on the molecular phylogeny and on niche overlap analyses, we uncovered strong evidence to support the idea that ecologically similar species are not necessarily sister species. The correlation analysis for genetic distance and niche overlap was not significant (P > 0.05). In clades with taxonomic conflicts, we only identified Profundulus oaxacae as a geographically and ecologically distinct group from P. punctatus. All the evidence considered leads us to propose that Profundulidae do not show evidence of niche conservatism and that there are reasons to consider P. oaxacae as a valid species. Our study suggests that niche divergence is a driving evolutionary force that caused the diversification and speciation processes of the Profundulidae, along with the geological and climatic events that promoted the expansion or contraction of suitable environments.

Morphological and molecular characterization of Maritrema kostadinovae n. sp. (Digenea: Microphallidae) from the yellow-crowned night heron Nyctanassa violacea (Aves: Ardeidae) in Mexico.

A new species of microphallid trematode was collected from the intestine of the yellow-crowned night heron Nyctanassa violacea (L.) (Pelecaniformes: Ardeidae) from Veracruz, Mexico. Maritrema kostadinovae n. sp. differs distinctly from other members of Maritrema Nicoll, 1907 from the Americas by its smaller body size (262-435 × 242-363 µm), the extension of caeca (reaching to anterior level of ventral sucker), the size and shape of the cirrus (short, tubular and unarmed) and metraterm (simple and thin-walled), the position of the genital pore (sinistrolateral to ventral sucker) and the arrangement of the vitellaria (horseshoe-shaped with posteriorly directed opening). Maximum likelihood and Bayesian inference analyses, based on partial 28S rDNA sequences, depicted M. kostadinovae n. sp. within the genus Maritrema with strong support. The new species is in a sister position to other available members of Maritrema, except for M. subdolum Jägerskiöld, 1909 that branches as the early divergent species in the Maritrema clade. The new species is the third species of Maritrema described from birds in Mexico. Comparative morphometric data for Maritrema taxa from birds and mammals from the Americas is provided.

Three new species of Clinostomum Leidy, 1856 (Trematoda) from Middle American fish-eating birds.

We recently engaged in a two-part study of Clinostomum Leidy, 1856 across a geographic range comprising central Mexico southwards to Costa Rica, in Central America. In the first study, we investigated the species boundaries by using DNA sequences of mitochondrial and nuclear molecular markers, implementing several analytical tools and species delimitation methods. The result of that approach revealed five highly divergent genetic lineages that were interpreted as independent evolutionary units, or species. Here, we present the second part of the study, where we describe three of the five species for which we have sexually mature adult specimens obtained from the mouth cavity of fish-eating birds. Additionally, we characterise morphologically the metacercariae of the other two species, collected from freshwater fishes; these species cannot be formally described since no adults were found in their definitive hosts. We further discuss the characters that are more reliable for species identification within Clinostomum, such as the cirrus sac shape and relative position with respect to testes and ovary, the shape of the reproductive organs, and the diverticulated condition of the caeca.

Mayarhynchus karlae n. g., n. sp. (Acanthocephala: Neoechinorhynchidae), a parasite of cichlids (Perciformes: Cichlidae) in southeastern Mexico, with comments on the paraphyly of Neoechinorhynchus Stiles & Hassall, 1905.

Mayarhynchus n. g. (Acanthocephala: Neoechinorhynchidae) is erected for Mayarhynchus karlae n. g, n. sp. described from the intestine of four species of cichlid fishes distributed from southeastern Mexico. The new genus placed in the family Neoechinorhynchidae (Ward, 1917) Van Cleave, 1928, is readily distinguished from the other 17 genera in the family by having a small proboscis armed with 45-46 relatively weak rooted hooks arranged in nine longitudinal rows of five hooks each. In addition, Mayarhynchus n. g., n. sp. is diagnosed by the presence of a short trunk, body wall with five dorsal and one ventral giant hypodermal nuclei, proboscis receptacle nearly cylindrical with single layered wall, lemnisci broad and flat with large nuclei, testes in tandem, cement gland with eight large nuclei, and eggs elongate to oval. Partial sequences of the cytochrome c oxidase subunit 1 (cox1), internal transcribed spacers (ITS1 + 5.8S + ITS2), and the D2-D3 domains of the large subunit rRNA gene (28S) were obtained for five specimens of the new species and other species belonging to the Neoechinorhynchidae. Phylogenetic analyses confirmed that the new genus belongs to the Neoechynorhynchidae and indicated that the genus Neoechynorhynchus Stiles & Hassall, 1905 is not monophyletic. Comparison between three populations of the new species yielded nine variable sites for cox1, 11 for ITS and four for 28S.

Novel morphological and molecular data for Corynosoma hannae Zdzitowiecki, 1984 (Acanthocephala: Polymorphidae) from teleosts, fish-eating birds and pinnipeds from New Zealand.

The polymorphid acanthocephalan, Corynosoma hannae Zdzitowiecki, 1984 is characterised on the basis of newly collected material from a New Zealand sea lion, Phocarctos hookeri (Gray), and long-nosed fur seal, Arctophoca forsteri (Lesson) (definitive hosts), and from Stewart Island shags, Leucocarbo chalconotus (Gray), spotted shags, Phalacrocorax punctatus (Sparrman) and yellow-eyed penguins, Megadyptes antipodes (Hombron & Jacquinot) (non-definitive hosts) from New Zealand. Specimens are described in detail and scanning electron micrographs for C. hannae are provided. Additionally, cystacanths of C. hannae are reported and described for the first time from the body cavity and mesenteries of New Zealand brill, Colistium guntheri (Hutton) and from New Zealand sole, Peltorhamphus novaezeelandiae Günther from Kaka Point, Otago in New Zealand. Partial sequence data for the mitochondrial cytochrome c oxidase 1 gene (cox1) for adults, immature specimens and cystacanths of C. hannae were obtained. Phylogenetic analyses of the newly-generated sequences and for available cox1 sequences of Corynosoma spp. revealed a close relationship between C. hannae and C. australe Johnston, 1937, both species infecting pinnipeds in the Southern Hemisphere. However, a morphological comparison of the species suggests that C. hannae mostly closely resembles C. evae Zdzitowiecki, 1984 and C. semerme (Forssell, 1904), the latter of which occurs in pinnipeds in the Northern Hemisphere.

A morphological and molecular study of Pseudocorynosoma Aznar, Pérez Ponce de León and Raga 2006 (Acanthocephala: Polymorphidae) from Mexico with the description of a new species and the presence of cox 1 pseudogenes.

Pseudocorynosoma tepehuanesi n. sp., is described from the intestine of the ruddy duck Oxyura jamaicensis Gmelin, 1789 from single locality from northern Mexico. The new species is mainly distinguished morphologically from the other five described species of Pseudocorynosoma from the Americas (P. constrictum, type species, P. peposacae, P. anatarium, P. enrietti and P. iheringi) associated with waterfowl species by possessing a proboscis with 15 longitudinal rows with 7-8 hooks each, a trunk expanded anteriorly and by having smaller lemniscus. Partial sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox 1) and the large subunit (LSU) of ribosomal DNA including the domains D2+D3 were used independently to corroborate the morphological distinction between the new species and other two congeneric species (P. constrictum and P. anatarium) from North America. The genetic divergence estimated among the new species and the other two species ranged from 15 to 18% for cox 1 and from 3.2 to 4% for LSU. The cox 1 alignment shows 24 sequences from P. anatarium with abnormalities, which were defined as pseudogenes due the presence of insertions, deletions and premature stop codons. Maximum likelihood and Bayesian inference analyses with each data set showed that the acanthocephalans from ruddy duck represent an independent clade with strong bootstrap support and posterior probabilities. The phylogenetic tree inferred with cox 1 gene placed all the pseudogenes from P. anatarium in single clade suggesting that those genes arose after speciation process within genus Pseudocorynosoma. The morphological evidence, plus the monophyly in both phylogenetic analyses indicate that the acanthocephalans collected from intestine of the ruddy duck from northern Mexico represent a new species.

Species delimitation in trematodes using DNA sequences: Middle-American Clinostomum as a case study.

The recent development of genetic methods allows the delineation of species boundaries, especially in organisms where morphological characters are not reliable to differentiate species. However, few empirical studies have used these tools to delineate species among parasitic metazoans. Here we investigate the species boundaries of Clinostomum, a cosmopolitan trematode genus with complex life cycle. We sequenced a mitochondrial [cytochrome c oxidase subunit I (COI)] gene for multiple individuals (adults and metacercariae) from Middle-America. Bayesian phylogenetic analysis of the COI uncovered five reciprocally monophyletic clades. COI sequences were then explored using the Automatic Barcode Gap Discovery to identify putative species; this species delimitation method recognized six species. A subsample was sequenced for a nuclear gene (ITS1, 5·8S, ITS2), and a concatenated phylogenetic analysis was performed through Bayesian inference. The species delimitation of Middle-American Clinostomum was finally validated using a multispecies coalescent analysis (species tree). In total, five putative species are recognized among our samples. Mapping the second intermediate hosts (fish) onto the species tree suggests that metacercariae of these five species exhibit some level of host specificity towards their fish intermediate host (at the family level), irrespective of geographical distribution.

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.

M aritrema corai n. sp. (Digenea: Microphallidae) from the white ibis Eudocimus albus (Linnaeus) (Aves: Threskiornithidae) in Mexico.

M aritrema corai n. sp. is described based on material from the intestine of the white ibis Eudocimus albus (L.) (Threskiornithidae) in Mexico. The new species can be distinguished morphologically from all congeners by the unique combination of the following morphological features: a very long cirrus sac attenuated distally [cirrus sac to body length ratio 1:0.90-1.29 (mean 1:1.07)]; a large, elongate-oval seminal receptacle, located dorsally between the cirrus sac and ovary; and long, filiform, unarmed, evaginable cirrus. Phylogenetic analyses of 28S ribosomal DNA (rDNA) sequences for the new species and for Maritrema spp. and Microphallus spp. depicted strong support for the two genera (excluding Microphallus fusiformis) and revealed close relationships between Ma. corai n. sp. and the clade formed by Maritrema novaezealandense Martorelli, Fredensborg, Mouritsen & Poulin, 2004, Maritrema heardi (Kinsella & Deblock, 1994) and Maritrema cf. eroliae.

Phyllodistomum spinopapillatum sp. nov. (Digenea: Gorgoderidae), from the Oaxaca killifish Profundulus balsanus (Osteichthyes: Profundulidae) in Mexico, with new host and locality records of P. inecoli: Morphology, ultrastructure and molecular evidence.

Phyllodistomum spinopapillatum sp. nov. is described from the urinary bladder of the Oaxaca killifish, Profundulus balsanus Ahl (Profundulidae) in Rio Pueblo Viejo and Rio Santa Cruz, Oaxaca, southwestern Mexico. The new species is described based on evidence gathered from morphology, scanning electron microscopy (SEM), and partial sequences of the 28S rRNA gene. Diagnostic characters of the new species of Phyllodistomum Braun 1899 are the presence of spines on the entire body surface and having a ventral sucker almost half the size of oral sucker. The new species possess a large number of dome-like papillae on the body surface with scattered distribution along the hindbody, and these papillae are characteristically spinulated. Phyllodistomum spinopapillatum sp. nov. most closely resembles P. inecoli Razo-Mendivil, Perez-Ponce de Leon and Rubio- Godoy, 2013, a parasite of the twospot livebearer Pseudoxiphophorus bimaculatus (Heckel) from Veracruz, in the Atlantic slope of Mexico. In addition to the new species, specimens of P. inecoli were also found parasitising the urinary bladder of cyprinodontiforms such as the Mexican molly Poecilia sphenops Valencienes in a pond at Santa Maria Coyotepec, and in Profundulus sp. in Rio Templo, both in Oaxaca, and in the Porthole livebearer Poeciliopsis gracilis (Heckel) in Rio San Juan, as well as in Profundulus punctatus (Gunter) from Rio Nueva Francia, both in Chiapas. The distribution and host range of P. inecoli is extended to freshwaters of the Pacific slope of Mexico, and to other cyprynodontiforms.

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.