The white grunt, Haemulon plumierii (Lacepède, 1801) as paratenic and definitive host of two acanthocephalan species, with the description of a new species of Dollfusentis (Palaeacanthocephala: Leptohynchoididae) from the Yucatán Peninsula, Mexico.

Acanthocephalans are a group of obligate endoparasites that alternate between vertebrates and invertebrates to complete their life cycles. Occasionally, the same individual host acts as a definitive or paratenic host for different acanthocephalan species. In this study, acanthocephalans were sampled in marine fish in three localities of the Yucatán Peninsula; adults and cystacanths were recovered from the intestine and body cavity, respectively, of Haemulon plumierii from off the coast of Sisal, Yucatán. Ribosomal DNA sequences (small and large subunits) were used to test the phylogenetic position of the species of the genus Dollfusentis, whereas the mtDNA gene cox 1 was used for assessing species delimitation. The cox 1 analysis revealed an independent genetic lineage, which is recognized herein as a new species, Dollfusentis mayae n. sp. The new species is morphologically distinguished from the other six congeners by having a cylindrical proboscis armed with 22-25 longitudinal rows bearing 12 hooks each. The cystacanths were morphologically identified as Gorgorhynchus medius by having a cylindrical trunk covered with tiny irregular spines on the anterior region, and a cylindrical proboscis armed with 17-18 longitudinal rows of 21 hooks each; small and large subunit phylogenetic analyses yielded G. medius within the family Isthomosacanthidae, suggesting that Gorgorhynchus should be transferred to this family from Rhadinorhynchidae where it is currently allocated.

Linking metacercariae and adults of Microphallus basodactylophallus (Digenea: Microphallidae), based on larval stages from ctenophores and adult parasites from aquatic birds found in Mexico.

Members of the genus Microphallus Ward, 1901, are endoparasites mainly of birds and mammals distributed worldwide. Unencysted metacercariae of Microphallus sp., were collected from the mesoglea of ctenophores of the genus Pleurobrachia Fleming; adult digeneans were recovered from the intestines of Eudocimus albus Linnaeus (Threskiornithidae) and Buteogallus urubitinga Gmelin (Accipitridae), in four locations from southeastern Mexico. Adult specimens were identified as M. basodactylophallus (Bridgman, 1969) based on the following features: body pyriform entirely covered by minute spines, prepharynx short, oesophagus very long, caeca short and widely divergent, testes slightly symmetrical and excretory vesicle short and V-shaped. Sequences from D1-D3 domain of the large subunit of ribosomal DNA (LSU) were generated, aligned, and compared with those of congeneric species available in GenBank. Phylogenetic analyses indicated that the metacercariae and adults formed a clade together with an isolate identified as M. basodactylophallus from Florida, USA (GenBank: AY220628). The intraspecific genetic divergence among isolates was low ranged from 0.0% to 0.6%, allowing the link between the two stages of the life cycle. We observed phenotypic plasticity in the morphological traits of M. basodactylophallus adults in definitive hosts (mammals and birds) throughout the distribution, which ranged from the USA to southeastern Mexico. Finally, the unencysted metacercariae identified as M. basodactylophallus represent the first report of a microphallid in ctenophores.

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.

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.

A hyper-diverse genus of acanthocephalans revealed by tree-based and non-tree-based species delimitation methods: Ten cryptic species of Neoechinorhynchus in Middle American freshwater fishes.

The genus Neoechinorhynchus represents a hyper-diverse group of acanthocephalans, parasites of fresh and brackish water fish and freshwater turtles, with approximately 116 species described worldwide. Forty-nine species have been recorded in the Americas, nine of them in Middle America. Even though species delimitation methods using DNA sequences have been rarely used for parasitic helminths, the genetic library for species of Neoechinorhynchus has grown in the past few years, enhancing the possibility of using these methods for inferring evolutionary relationships and for establishing more robust species boundaries. In this study, we used non-tree-based and tree-based methods through a coalescent approach to explore the species limits of specimens of Neoechinorhynchus collected in 57 localities across Middle America. We sequenced a large number of individuals to build a comprehensive dataset for three genes: the mitochondrial cytochrome c oxidase subunit I (352 individuals), the internal transcribed spacers (330 individuals), and the D2 + D3 domains of the large subunit (278 individuals). Several species delimitation methods were implemented, i.e., Automatic Barcode Gap Discovery (ABGD), General Mixed Yule-Coalescent Model (GMYC), Bayesian species delimitation (BPP) and species tree (∗BEAST). Additionally, we conducted a detailed morphological study of the diagnostic traits associated with the proboscis of 184 males and 169 females. Overall, our analyses allowed us to validate nine nominal species of Neoechinorhynchus and to identify 10 additional genetic lineages herein regarded as candidate species. This unexpected genetic diversity and the lack of reliable morphological traits show that the genus Neoechinorhynchus includes a group of cryptic species, at least in Middle America.

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.

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.

Checklist of the helminth parasites of the genus Profundulus Hubbs, 1924 (Cyprinodontiformes, Profundulidae), an endemic family of freshwater fishes in Middle-America.

From December 2012 to November 2014, 267 fish belonging to the family Profundulidae (representing nine of the 11 species of the genus Profundulus) were collected in 26 localities of Middle-America, across southern Mexico, Guatemala, and Honduras, comprising the distribution range of the genus, and analyzed for helminth parasites. Additionally, a database with all ten available published accounts of the helminth parasite fauna of this genus (the only genus within the family) was assembled. Based on both sources of information, a checklist containing all the records was compiled as a tool to address future questions in the areas of evolutionary biology, biogeography, ecology and phylogeography of this host-parasite association. The helminth parasite fauna of this fish group consists of 20 nominal species, classified in 17 genera and 14 families. It includes six species of adult digeneans, five metacercariae, two monogeneans, one adult cestode, three adult nematodes and three larval nematodes. The profundulid fishes are parasitized by a specialized group of helminth species (e.g. Paracreptotrema blancoi sensu Salgado-Maldonado et al. (2011b), Paracreptotrema profundulusi Salgado-Maldonado, Caspeta-Mandujano & Martínez Ramírez, 2011, Phyllodistomum spinopapillatum Pérez-Ponce de León, Pinacho-Pinacho, Mendoza-Garfias & García-Varela, 2015, Spinitectus humbertoi Mandujano-Caspeta & Moravec, 2000, Spinitectus mariaisabelae Caspeta-Mandujano Cabañas-Carranza & Salgado-Maldonado, 2007 and Rhabdochona salgadoi Mandujano-Caspeta & Moravec, 2000), representing the core helminth fauna that are not shared with other Middle-American fish species.

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.

Morphological and molecular data reveal a new species of Neoechinorhynchus (Acanthocephala: Neoechinorhynchidae) from Dormitator maculatus in the Gulf of Mexico.

Neoechinorhynchus (Neoechinorhynchus) mexicoensis sp. n. is described from the intestine of Dormitator maculatus (Bloch 1792) collected in 5 coastal localities from the Gulf of Mexico. The new species is mainly distinguished from the other 33 described species of Neoechinorhynchus from the Americas associated with freshwater, marine and brackish fishes by having smaller middle and posterior hooks and possessing a small proboscis with three rows of six hooks each, apical hooks longer than other hooks and extending to the same level as the posterior hooks, 1 giant nucleus in the ventral body wall and females with eggs longer than other congeneric species. Sequences of the internal transcribed spacer (ITS) and the large subunit (LSU) of ribosomal DNA including the domain D2+D3 were used independently to corroborate the morphological distinction among the new species and other congeneric species associated with freshwater and brackish water fish from Mexico. The genetic divergence estimated among congeneric species ranged from 7.34 to 44% for ITS and from 1.65 to 32.9% for LSU. Maximum likelihood and Bayesian inference analyses with each dataset showed that the 25 specimens analyzed from 5 localities of the coast of the Gulf of Mexico parasitizing D. maculatus represent an independent clade with strong bootstrap support and posterior probabilities. The morphological evidence, plus the monophyly in the phylogenetic analyses, indicates that the acanthocephalans collected from intestine of D. maculatus from the Gulf of Mexico represent a new species, herein named N. (N.) mexicoensis sp. n.

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.

Reproduction and survival under different water temperatures of Gyrodactylus mexicanus (Platyhelminthes: Monogenea), a parasite of Girardinichthys multiradiatus in Central Mexico.

Gyrodactylid population growth may depend on abiotic variables such as temperature. We tested the survival and reproductive rate of Gyrodactylus mexicanus, a parasite infecting fins of Girardinichthys multiradiatus, at 3 different water temperatures, 10-13, 19-22, and 24 C. The temporal sequence of birth and age at death of each parasite isolated from the hosts was recorded through at least 8 generations. Our results showed that the average number of offspring per parasite was 2.0 when averaged across all temperatures. However, the generation time was negatively correlated with temperature. The innate capacity for increase (r(m)) was positively correlated with water temperature: from 0.29 parasite/day at 13 C to 0.48 parasite/day at 24 C. These data confirm that water temperature has a direct influence on parasite population dynamics. The current study represents the first contribution to understanding the population ecology of the monogenean G. mexicanus in central Mexico.

Two new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) parasitizing Girardinichthys multiradiatus (Cyprinodontiformes: Goodeidae), an endemic freshwater fish from central Mexico.

Gyrodactylus mexicanus n. sp. and Gyrodactylus lamothei n. sp. are described from the fins and skin of Girardinichthys multiradiatus, an endemic freshwater fish from central Mexico. Gyrodactylus mexicanus is compared to other Gyrodactylus species that parasitize Fundulus spp., the phylogenetically closest group to the Goodeidae from North America. Gyrodactylus mexicanus is distinguished by having large anchors with well-developed superficial roots, enlarged hooks with a proximally disrupted shank (ligament), and a ventral bar with 2 poorly developed anterolateral projections and a small medial process. Gyrodactylus lamothei is distinguished from G. mexicanus and from other species of Gyrodactylus on the North American continent by having anchors with a sclerite on the superficial root and robust hooks with a straight shaft and a recurved point.