Disentangling the evolutionary and biogeographical history of the freshwater fish trematode genus Creptotrema (Digenea: Allocreadiidae) using an integrative taxonomy approach: the case of Creptotrema agonostomi in Middle American mountain mullets.

Species of the allocreadiid genus Creptotrema are parasites of freshwater fishes in the Americas. Species in the genus possess one pair of muscular oral lobes on the oral sucker. Currently, the genus contains eight species, six distributed in South America, one in Middle America and one in North America. Genetic data are only available for the North American species, Creptotrema funduli, a parasite of fundulids originally described from Oneida Lake, New York State. In this study, we obtained 28S ribosomal DNA sequences of trematodes morphologically similar to Creptotrema agonostomi from the mountain mullet, Dajaus monticola, across a wide geographical range in Middle America. Our molecular phylogenetic analyses showed that (1) the genus Creptotrema, as currently conceived, is not monophyletic; (2) the allocreadiids in mountain mullets should be re-allocated in the genus Pseudoparacreptotrema; and (3) the allocreadiid trematodes from D. monticola across Middle America represent four morphologically similar species, three of which can be distinguished genetically. These three new species are described herein using an integrative taxonomy approach. We contend that accurate estimates of species diversity and phylogenetic relationships among allocreadiids, and most likely other species of trematodes, necessarily require an integrative taxonomy approach that should consider at least DNA sequences and scanning electron microscopy.

A morphological and molecular study of adults and metacercariae of Hysteromorpha triloba (Rudolpi, 1819), Lutz 1931 (Diplostomidae) from the Neotropical region.

Adults of Hysteromorpha triloba (Rudolpi, 1819), Lutz, 1931 inhabit primarily the intestine of cormorants across the globe, whereas metacercariae have been found in the body cavity of freshwater fishes of the families Cyprinidae, Ictaluridae, Ariidae, Pimelodidae and Catostomidae. In this study, adults and metacercariae identified as H. triloba were collected from the Neotropical cormorant (Nannopterum brasilianus) and from the Mexican tetra fish (Astyanax mexicanus) from the Gulf of Mexico and Pacific Ocean slopes in the Neotropical region. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox 1) and the internal transcribed spacers (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA were generated for both developmental stages, and were compared with available sequences of H. triloba from the Nearctic region. The genetic divergence between metacercariae and adults of H. triloba from the Neotropical and Nearctic region (Canada) associated with the double-crested cormorant (Nannopterum auritus), ranged from 0 to 5.5% for cox 1 and from 0 to 0.2% for ITS. Phylogenetic analyses inferred with both molecular markers using maximum likelihood and Bayesian inference placed the adults and metacercariae in a single clade, confirming that both stages are conspecific. Our data confirmed that H. triloba is a widely distributed species across the Americas, parasitizing both the Neotropical and Nearctic cormorants in Argentina, Brazil, Venezuela, Mexico, USA and Canada.

A closer look at the morphological and molecular diversity of Neoechinorhynchus (Acanthocephala) in Middle American cichlids (Osteichthyes: Cichlidae), with the description of a new species from Costa Rica.

Neoechinorhynchus is one of the most speciose genera of acanthocephalans, with approximately 116 described species. A recent study, aimed at establishing the genetic diversity of Neoechinorhynchus in Middle American freshwater fishes, validated nine species molecularly and morphologically and revealed the existence of 10 putative candidate species. Neoechinorhynchus golvani, a parasite commonly found in cichlids throughout Middle America with an allegedly large intraspecific morphological variability, was found to represent a species complex; species delimitation methods uncovered three additional genetic lineages. Here, we re-analyse the morphological and molecular data for N. golvani species complex infecting cichlids in that geographical area. A multivariate analysis of variance (MANOVA) was conducted particularly for the length of apical, middle and posterior hooks of the species/lineages of Neoechinorhynchus in cichlids, revealing morphological variation in the length of apical hooks for Lineage 8, although no morphological distinction was observed for Lineages 9 and 10. A new concatenated phylogenetic analysis of one mitochondrial and two ribosomal DNA genes was used to further corroborate the species delimitation among lineages; Neoechinorhynchus Lineage 8 was found to be morphologically and genetically distinct from its sister taxa, N. golvani and other two undescribed genetic lineages, and is formally described as a new species. Neoechinorhynchus costarricense n. sp. is described from the intestines of eight species of cichlids in Costa Rica. The new species is distinguished from the other species/lineages of Neoechinorhynchus in cichlids mainly by the size of the apical hooks of the proboscis.

Multilocus phylogenetic analysis and morphological data reveal a new species composition of the genus Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae), parasites of fish-eating birds in the Americas.

Members of the genus Drepanocephalus are endoparasites of fish-eating birds of the families Phalacrocoracidae and Sulidae distributed across the Americas. Currently, Drepanocephalus contains three species, i.e. D. spathans (type species), D. olivaceus and D. auritus. Two additional species, D. parvicephalus and D. mexicanus were transferred to the genus Petasiger. In the current study, available DNA sequences of D. spathans, D. auritus and Drepanocephalus sp., were aligned with newly generated sequences of D. spathans and Petasiger mexicanus. Phylogenetic analyses inferred with three nuclear (LSU, SSU and ITS1, 5.8S, ITS2) and two mitochondrial (cox1, nad1) molecular markers showed that the sequences of D. spathans and D. auritus are nested together in a single clade with very low genetic divergence, with Petasiger mexicanus as its sister species. Additionally, P. mexicanus was not a close relative of other members of the genus Petasiger, showing that P. mexicanus actually belongs to the genus Drepanocephalus, suggesting the need to re-allocate Petasiger mexicanus back into the genus Drepanocephalus, as D. mexicanus. Morphological observations of the newly sampled individuals of D. spathans showed that the position of the testes is variable and testes might be contiguous or widely separated, which is one of the main diagnostic traits for D. auritus. Our results suggest that D. auritus might be considered a synonym of D. spathans and, as a result, the latter represents a species with a wide geographic range across the Americas, parasitizing both the Neotropical and the double-crested cormorant in Argentina, Brazil, Paraguay, Venezuela, Colombia, Mexico, USA and Canada.

Morphological and molecular analyses of a new species of Saccocoelioides Szidat, 1954 (Haploporidae Nicoll, 1914) in the fat sleeper Dormitator maculatus (Bloch) (Perciformes: Eleotridae) from the Gulf of Mexico.

Saccocoelioides olmecae n. sp. is described from specimens recovered from the intestine of the fat sleeper Dormitator maculatus (Bloch) (Perciformes: Eleotridae) collected in six localities along the coast of the Gulf of Mexico. The new species is mainly distinguished from the other three described species of Saccocoelioides Szidat, 1954 from North and Middle America (i.e. S. sogandaresi Lumsden, 1963, S. chauhani Lamothe-Argumedo, 1974 and S. lamothei Aguirre-Macedo & Violante-González, 2008) by having an elongated body, a sac-like caecum, a uterus that extends to the first third of body and by having vitelline follicles longitudinally elongated reaching the posterior end of the body. Sequences of the large subunit (LSU) of the ribosomal DNA, including the domain D1-D3, and the internal transcribed spacer 2 (ITS2) were used independently and concatenated to corroborate the morphological distinction among S. olmecae n. sp., S. chauhani and S. lamothei from freshwater and brackish-water fish from Middle America. The genetic divergence estimated among the three species of Saccocoelioides was very low: 1% for LSU and from 1 to 4% for ITS2. Maximum likelihood and Bayesian inference analyses for each dataset and both datasets combined revealed that S. olmecae n. sp. represents an independent clade with moderate bootstrap support and posterior probabilities. This is the third species of Saccocoelioides described in Mexico, and the 17th species from the Americas.

An integrative taxonomic study reveals a new species of Tylodelphys Diesing, 1950 (Digenea: Diplostomidae) in central and northern Mexico.

Tylodelphys aztecae n. sp. (Digenea: Diplostomidae) is described from adult specimens obtained from the intestine of the pied-billed grebe (Podilymbus podiceps) and the metacercariae found in the body cavity of freshwater fishes of the families Goodeidae and Cyprinidae in eight localities across central and northern Mexico. The new species is mainly distinguished from the other four described species of Tylodelphys from the Americas (T. adulta, T. americana, T. elongata and T. brevis) by having a forebody slightly concave, a larger ventral sucker, two larger pseudosuckers and by having between 2 and 7 eggs in the uterus. Partial DNA sequences of the mitochondrial gene cytochrome c oxidase subunit I (cox1), and the internal transcribed spacers (ITS1+5.8S+ ITS2) of the ribosomal DNA, were generated for both developmental stages and compared with available sequences in GenBank of other congeners. The genetic divergence estimated among Tylodelphys aztecae n. sp. and other congeneric species varied from 12 to 15% for cox1, and from 3 to 11% for ITS. In contrast, the genetic divergence among metacercariae and adults of the new species was very low, ranging between 0 and 1% for cox1 and between 0 and 0.3% for ITS. Phylogenetic analyses inferred with both molecular markers using maximum likelihood and Bayesian inference placed the adults and their metacercariae in a single clade, confirming that both stages are conspecific. The morphological evidence and the genetic divergence, in combination with the reciprocal monophyly in both phylogenetic trees, support the hypothesis that the diplostomids found in the intestines of the pied-billed grebe bird and the body cavity from goodeid and cyprinid fishes in central and northern Mexico represent a new species.

Molecular and morphological characterization of Austrodiplostomum ostrowskiae Dronen, 2009 (Digenea: Diplostomatidae), a parasite of cormorants in the Americas.

The diplostomid genus Austrodiplostomum currently contains two species, i.e. A. mordax and A. ostrowskiae. Adults of these species inhabit the intestine of cormorants of the genus Nannopterum, whereas larval forms (metacercariae) are found in the eyes (vitreous humor) of freshwater fishes. Records of both species have been established across a wide geographic range in the Americas. Diplostomid adults and metacercariae were collected from a wide geographical range that spans from south-eastern Mexico, southwards to Central and South America. Even though the diplostomid has been largely reported in Mexico as Diplostomum (Austrodiplostomum) compactum, our specimens were identified morphologically as A. ostrowskiae. Sequences of the mitochondrial gene cytochrome c oxidase (cox 1) were obtained for 86 individuals, including 15 adults recovered from the intestine of Nannopterum brasilianus, in five localities, and 71 metacercariae from the eyes of ten fish species, in 13 localities. Sequences were used to evaluate the genetic diversity, and to test conspecificity of these specimens with the available sequence of A. ostrowskiae. Sequences were aligned with another 12 taxa representing five genera of Diplostomatidae, forming a dataset of 104 taxa with 478 nucleotides. The genetic divergence estimated among the 86 sequenced individuals, and that of A. ostrowskiae from the double-crested cormorant, Nannopterum auritus, in the USA, was very low, ranging from 0 to 0.8%. The maximum likelihood (ML) and Bayesian consensus trees showed that all sequences nested within a monophyletic lineage, with strong bootstrap and Bayesian posterior probability support values (100/1.0). In conclusion, a link between the metacercariae in fish and the adults in cormorants was established, indicating also that a single species is found in the distribution range comprising southern USA, southwards to Venezuela. Previous records of this species, particularly from Mexico, need to be corrected.

Morphological and molecular analyses of larval and adult stages of Echinoparyphium recurvatum von Linstow 1873 (Digenea: Echinostomatidae) from central Mexico.

In central Mexico, populations of the freshwater snail Physella cubensis were infected with metacercariae from a species of Echinoparyphium (Digenea: Echinostomatidae). In the current study, we describe both larval and adult stages of this species obtained from experimental and natural infections. A total 180 snails were collected from Patzcuaro Lake, Michoacan state in central Mexico in July 2012. In the laboratory snails were placed in individual vials and exposed to light with the aim of observing emergence of cercariae. To obtain metacercariae, uninfected snails (P. cubensis) were exposed to cercariae. Chicks were infected with metacercariae to obtain adults. Nine days post-infection, eggs were recovered and incubated in tap water at room temperature to observe miracidia. Adults obtained from natural and experimentally infected hosts possess a head collar with 45 spines in two alternating rows, confirming the identification as Echinoparyphium recurvatum von Linstow 1873. To test the conspecificity of all stages, sequences of nuclear internal transcribed spacer 1 (ITS1), 5.8S and ITS2 rDNA were obtained from two adult worms recovered from chicks and also a natural avian host, the shoveler duck Anas clypeata, together with five cercarial and four metacercarial isolates from nine snails. The genetic divergence estimated among the 13 isolates was very low, ranging from 0 to 0.6%. Phylogenetic analyses inferred by maximum likelihood and Bayesian methods showed that all isolates of E. recurvatum form a single clade with strong support. The presence of E. recurvatum in P. cubensis and A. clypeata from central Mexico represents new host reports, and extends the distribution range in the Americas.