G. Pérez-Ponce de León

Using DNA barcoding to link cystacanths and adults of the acanthocephalan Polymorphus brevis in central Mexico

In parasitic organisms, particularly helminths, the usage of the mitochondrial cytochrome c oxidase subunit I gene as the standard DNA barcoding region for species identification and discovery has been very limited. Here, we present an integrated study, based on both DNA barcoding and morphological analyses, for acanthocephalans belonging to the genus Polymorphus, whose larvae (cystacanths) are commonly found in the mesentery of freshwater fishes, while adults are found in the intestine of fish‐eating birds. The alpha taxonomy of parasitic helminths is based on adult morphological traits, and because of that larval forms cannot be identified to species level based on morphology alone. DNA barcoding offers an alternative tool for linking larval stages of parasitic organisms to known adults. We sequenced cystacanths collected from freshwater fishes in localities across central Mexico and adults obtained from fish‐eating birds, to determine whether they were conspecific. To corroborate the molecular results, we conducted a morphometric analysis with ‘Proboscis profiler’, which is a software tool developed to detect heterogeneity in morphologically similar acanthocephalans based on the multivariate statistical analysis of proboscis hook dimensions. Both sources of information indicate that cystacanths infecting freshwater fishes in central Mexico belong to a single species, Polymorphus brevis.

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.

The systematic position of Wallinia spp. and Margotrema spp. (Digenea), parasites of Middle-American and Neotropical freshwater fishes, based on the 28S ribosomal RNA gene

The systematic relationships of aspinose allocreadiid-like digeneans from Middle-American and Neotropical freshwater fishes, such as Wallinia Pearse, 1920, Margotrema Lamothe-Argumedo, 1970, Magnivitellinum Kloss, 1966 and other related genera, remain enigmatic. Wallinia (2 spp.) and Margotrema (2 spp.) have been placed in the subfamily Walliniinae, within the Macroderoididae. Recent descriptions of species in these genera have followed this classification but have raised questions about their affinities with macroderoidids. Partial sequences of the 28S rRNA gene of Walliniachavarriae Choudhury, Hartvigsen & Brooks, 2002 and Margotremabravoae Lamothe Argumedo, 1970, as representatives of their genera, were used in a phylogenetic analysis along with the available sequences of macroderoidids and allocreadiids. Results from Maximum Parsimony, Maximum Likelihood and Bayesian Analyses were identical in placing both genera into one clade along with allocreadiids rather than the macroderoidids. Margotrema was the sister group of Crepidostomum Braun, 1900, and Wallinia was the sister group of both Margotrema and Crepidostomum combined. Bootstrap and Bremer values, as well as posterior probabilities derived from Bayesian analysis strongly supported these relationships in three analyses.

A new species of Rhabdochona Railliet, 1916 (Nematoda: Rhabdochonidae) from endemic goodeids (Cyprinodontiformes) from two Mexican lakes

A new species of Rhabdochona from 2 species of freshwater fishes, Alloophorus robustus and Goodea atripinnis, is described from 2 lakes of the Mesa Central of Mexico. Rhabdochona lichtenfelsi n. sp. belongs to a group of species possessing inconspicuous bifurcated deirids and a prostom armed with 10 large teeth. It is distinguished from them because the left spicule is shorter, the tip is bifurcate, and both spicules lack a reflected dorsal barb. The new species most closely resembles Rhabdochona catostomi and Rhabdochona milleri but differs from them because the former possesses a left spicule tip with ventral barb, right spicule with reflected distal barb, and 5 pairs of postanal papillae, whereas R. milleri has 14 prostomal teeth, eggs rounded, and left spicule with slightly outlined bifurcation and right with a dorsal barb. Previous records of R. milleri in Mexico must be referred to R. lichtenfelsi.

Phylogeny and biogeography of Rhabdochona Railliet, 1916 (Nematoda: Rhabdochonidae) species from the Americas

A phylogenetic analysis of 40 species of Rhabdochona Railliet, 1916, including all 21 valid species in the Americas, resulted in 1733 equally most parsimonious trees and indicates that Rhabdochona is arguably monophyletic. Species from the Americas do not form a monophyletic group, since each of the six clades of Rhabdochona includes species from the Americas and species from other continents. The synapomorphies defining each clade stem from the morphology of the left spicule. Teeth number was consistent in one clade only, suggesting that this␣character, while useful for taxonomic purposes, is not indicative of phylogeny. Species of Rhabdochona associated with certain host groups, such as salmonids, catostomids and goodeids, do not always form monophyletic assemblages, nor do species associated with smaller discrete areas, such as the Mesa Central of Mexico. This indicates widespread host-switching rather than co-speciation as the main phenomenon in the evolution of this group, at least in the species from the Americas. Phylogenetic patterns reveal an ancient origin for the group that probably pre-dates current continental configurations.

RHABDOCHONA AHUEHUELLENSIS N. SP. (NEMATODA: RHABDOCHONIDAE) FROM THE BALSAS GOODEID, ILYODON WHITEI (OSTEICHTHYES: GOOEDEIDAE), IN MEXICO

Rhabdochona ahuehuellensis n. sp. is described from the Balsas goodeid, Ilyodon whitei. The distinctive characters are cuticular elevations in the middle of the prostom, tricuspid ending of the distal part of the left spicule combined with a scoop shape, and an egg surface with limited, short subpolar filaments. The characters place the present species farther apart from species described from North America, except R. lichtenfelsi. This description brings the total number of species of Rhabdochona in the Mesa Central of Mexico to 4, suggesting that this genus has undergone speciation in response to basin fragmentation within the Mesa Central of Mexico.

Two new species of Oochoristica Lühe, 1898 (Eucestoda: Cyclophyllidea: Anoplocephalidae: Linstowiinae) parasitic in Ctenosaura spp. (Iguanidae) from Costa Rica and México.

Five species of Oochoristica, Oochoristica osheroffi, Oochoristica gracewileyae, Oochoristica whitentoni, and 2 new species described herein have strobilae longer than 200 mm, many secondary ovarian lobes, and testes extending anteriorly to midovarian level. A combination of 3 characters distinguishes the 5 species from each other. Oochoristica osheroffi has an average of 68 testes per proglottid, ovarian lobes wider than long, and osmoregulatory canals not forming an anastomosing plexus; O. gracewileyae has an average of 113 testes per proglottid, ovarian lobes longer than wide, and osmoregulatory canals not forming an anastomosing plexus; O. whitentoni has an average of 125 testes per proglottid, ovarian lobes wider than long, and osmoregulatory canals forming an anastomosing plexus; a new species in Ctenosaura similis from Costa Rica has an average of 62 testes per proglottid, ovarian lobes longer than wide, and osmoregulatory canals forming an anastomosing plexus; and a new species in Ctenosaura pectinata from Mexico has an average of 122 testes per progottid, ovarian lobes longer than wide, and osmoregulatory canals forming an anastomosing plexus. Oochoristica gracewileyae differs from the other 4 species by having genital pores 10-15% rather than 25-35% of proglottid length from the anterior end and by having convoluted rather than straight or sinuous transverse osmoregulatory canals. The new species from Mexico differs from the other 4 species and apparently from all described species of Oochoristica thus far by possessing darkly staining granules throughout the parenchyma of the scolex.

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.

The use of mitochondrial and nuclear sequences in prospecting for cryptic species in Tabascotrema verai (Digenea: Cryptogonimidae), a parasite of Petenia splendida (Cichlidae) in Middle America.

In this study, we used sequences of mitochondrial and nuclear markers to test the hypothesis that Tabascotrema verai Lamothe-Argumedo and Pineda-López, 1990, a parasite of the cichlid Petenia splendida Günther, 1862 in Middle America, represents a single species, following a molecular prospecting approach. One-hundred and five individuals of T. verai were collected from the intestine of 43 specimens of P. splendida in eight localities of Mexico, Belize and Guatemala. A portion (n=53) of the collected specimens was used for molecular studies, whereas the remaining individuals were processed for morphological studies. Fifty-three partial sequences of the cytochrome c oxidase subunit I (cox1) gene were obtained. In addition, 36 sequences of the ITS (internal transcribed spacers 1, 5.8S rRNA gene, and ITS2) and of the 28S rRNA gene were generated from most of the specimens that showed nucleotide variation with the cox1 gene. A haplotype network obtained from cox1 sequences revealed three independent groups (haplogroups 1, 2, and 3). Independent phylogenetic analyses performed with maximum likelihood and Bayesian inference of cox1, ITS and the 28S rRNA gene recovered three genetically-distinct and reciprocally-monophyletic groups, corresponding with the 3 haplogroups obtained in the haplotype network. Values of genetic divergence between clades for cox1 sequences ranged between 8.3% and 11.9%, while for ITS and the 28S rRNA gene, these ranged from 0.08% to 1.2% and from 0.3% to 0.4%, respectively. Morphological observations, and measurements of 23 characters of 44 mounted individuals, showed that no morphological differences exist between individuals from the eight collecting sites, and that the ranges of most morphological traits overlap. Our results suggest that the digenean T. verai represents a complex of cryptic species; the haplotype network, phylogenetic analyses, and genetic differences, along with the morphological stasis recorded here support this notion. Finally, the three recovered lineages showed neither geographical association nor correlation with drainage basins.

The influence of host ecology and biogeography on the helminth species richness of freshwater fishes in Mexico

Freshwater fish helminths, the most well known Mexican vertebrate parasites, include approximately 260 species (platyhelminthes, acanthocephalans, nematodes, and hirudineans). The distribution patterns of adult helminth diversity (throughout parasite and host groups and hydrological regions) are described and the effects of host traits and environmental and geographical factors on diversity are evaluated. Adult helminths include 160 species, parasitizing 149 fish species of 23 families distributed in 21 regions. Nematoda was the most species-rich (>50 species). Cichlidae harboured rich helminth assemblages, with widespread parasites. By contrast, Atherinopsidae and Goodeidae showed relatively poor helminth assemblages, including specific parasites with narrow distribution. Helminth richness in southeastern Mexico was higher than northern or central regions. Non-parametric richness estimators were used to avoid confusion in comparisons with unequal sampling efforts. Bootstrap values, the method with the best performance, indicated that estimated richness shows the same distribution pattern that observed richness. Non-phylogenetic and phylogenetic analyses were used to determine the role of different factors in the parasite diversification. The distribution range was the most important richness predictor (widespread fishes harbour richer parasite assemblages), although interactions between this variable and others such as trophic level, latitude, habitat temperature and precipitation are also important. Likewise, biogeographical factors can also affect parasite diversity.