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.     

Sequence and RFLP analysis of the ITS2 ribosomal DNA in two Neotropical social bees, <Emphasis>Melipona beecheii</Emphasis> and <Emphasis>Melipona yucatanica</Emphasis> (Apidae,Meliponini)

Two stingless bees species of the genus Melipona, M. beecheii and M. yucatanica, are the only ones reported for the Yucatan Peninsula. The natural distribution of M. beecheii ranges from southern Mexico to Costa Rica, that of M. yucatanica from south Mexico to Guatemala. Colonies of both species occur in a variety of habitats and show adaptations to local conditions denoting the occurrence of ecotypes. The ITS2 of ribosomal DNA has been characterized in both species and its utility to discriminate among colonies has been investigated through RFLP experiments. The ITS2 region is unusually long, 1788 bp in M. beecheii and 1845 bp in M. yucatanica (including the 3′ end of the 5.8S gene and partial 5′ of the 28S gene). Mean nucleotide divergence between both ITS2 sequences is 16% (excluding sites with insertions/deletions) and 20% when the insertions/deletions are taken into account. The G+C content in both sequences is close to 53%. The PCR-RFLP assay was performed with 12 restriction enzymes on colonies of M. beecheii from Mexico (Yucatan, Campeche and Chiapas) Costa Rica, El Salvador and Guatemala, and of M. yucatanica from Mexico (Yucatan) and Guatemala. The restriction patterns obtained allow to discriminating colonies of both species with different origins. Both kinds of data are thus useful for assessing intra and interspecific genetic variability and for developing appropriate conservation strategies for these species. Received 20 June 2007; revised 31 August 2007; accepted 12 September 2007.     

An assessment of potential diatom "barcode" genes (cox1, rbcL, 18S and ITS rDNA) and their effectiveness in determining relationships in Sellaphora (Bacillariophyta)

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model 'morphospecies', Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5bp (=0.8%) and interspecific levels were at least 18bp (=c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.     

Molecular evidence that Langeronia macrocirra and Langeronia cf. parva (Trematoda: Pleurogenidae) parasites of anurans from Mexico are conspecific

The genus Langeronia parasitizing the intestine of several species of anurans is distributed from North to Central America. We identified Langeronia macrocirra and Langeronia cf. parva from the same host and localities, and present here new data not applicable about their tegumental surface by scanning electron microscopy. We compared sequences of the rDNA ITS2 region and mtDNA cox1 gene for the two morphotypes. ITS2 exhibited a high degree of conservation. Phylogenetic reconstruction using cox1 revealed three clades (I, II, and III), which did not correspond to a previous identification or host. Little divergence was found within clades: sequences were identical in clade I, whereas clade II had 0.27% and clade III had 1.08%. Inter-clade divergence reached 8.69% (I vs. III). This pattern of genetic divergence indicated that both taxa probably belong to the same species, so we posit that the morphological changes could be correlated with development. Increasing sample size and geographical coverage will contribute to the taxonomy of the genus based on morphological and molecular evidence, and will open tracks toward the use of DNA barcodes to the genus in Mexico.     

Digeneans (Plathelminthes: Trematoda) of seven sympatric species of anurans from Los Tuxtlas,Veracruz, Mexico

Digeneans of sympatric Bufo marinus, B. valliceps, Hyla picta, H. ebraccata, Smilisca baudini, Rana berlandieri, and R. vaillanti from Los Tuxtlas, Veracruz, Mexico, are reported. A total of 7 adult trematode species and 3 unidentified metacercariae was found in the different host species examined: Gorgoderina attenuata, G. parvicava, Cephalogonimus americanus, Haematoloechus medioplexus, Glypthelmins californiensis, Mesocoelium monas and Langeronia macrocirrus. Prevalence, mean intensity and abundance of the trematodes in each host species are given.     

Intraspecific variation of Haematoloechus floedae Harwood, 1932 (Digenea: Plagiorchiidae), from Rana spp. in North and Central America

Haematoloechus floedae was originally described from the lungs of Rana catesbeiana in Texas, and later reported in Florida, in the United States. It was proposed to be synonymous with H. breviplexus, H. parviplexus, and H. varioplexus. We obtained specimens of H. floedae from Rana brownorum and R. vaillanti in Yucatán, Mexico; R. cf. forreri and R. taylori in Guanacaste, Costa Rica; and R. catesbeiana in Georgia and California. Some specimens were processed for morphological study; sequences of the 28S of the rDNA and the mitochondrial COI were obtained from several specimens of each population. Phylogenetic analysis of molecular data indicates studied populations constitute a single taxon, different from H. varioplexus, H. breviplexus, and H. parviplexus. Some morphological characters remain constant among populations of H. floedae, and are useful for differentiating this species. These include the oral sucker-pharynx ratio, oral sucker-ventral sucker ratio, ovary and testes shape, extension of uterine longitudinal loops, and extension of vitelline follicles (in fully developed worms). Sequence homogeneity among populations of H. floedae suggests a recent spread, perhaps due to the introduction of R. catesbeiana for culture to the western United States, southeast Mexico, and Costa Rica.     

Haematoloechus danbrooksi n. sp. (Digenea: Plagiorchioidea) from Rana vaillanti from Los Tuxtlas,Veracruz, Mexico

Haematoloechus danbrooksi n. sp. from the lungs of Rana vaillanti in Veracruz state, Mexico, was found. The new species is most similar morphologically to H. medioplexus, H. parviplexus, and H. meridionalis in having a ventral sucker less than half the diameter of the oral sucker. It differs from these species by the extension of the vitellaria, which are shorter in the new species, and in the shape of the tegumental spines, which are blunt in the new species. It differs from all other known species of Haematoloechus in the distribution of the uterine loops that are arranged diagonally and that present several short, extracecal. longitudinal loops in the postacetabular region. The new species shows 1.2% sequence divergence in partial 28S sequence with respect to H. medioplexus, 1.1% with H. parviplexus. and 2.5% with H. meridionalis, sequence divergences complementing the morphological differences.     

Neohaematotrephus arayae n. sp. (Digenea: Echinostomiformes: Cyclocoelidae) in Jacana spinosa (Aves: Charadriiformes: Jacanidae) from the Area de Conservación Guanacaste, Costa Rica

Specimens of a species of cyclocoelid digenean inhabiting Jacana spinosa from the Area de Conservación Guanacaste, Costa Rica, most closely resemble Haematotrephus facioi (Brenes and Arroyo, 1962) Yamaguti, 1971, in the same host from Aranjuez, Puntarenas Province, Costa Rica, in having confluent vitelline follicles posteriorly, diagnostic of Neohaematotrophus, and in pharynx length, ovary width, and cirrus sac on the sinistral side. The new species is also highly similar in appearance to H. gendrei Dubois, 1959, also inhabiting a jacanid (from West Africa), which has vitelline follicles confluent posteriorly, and extending anteriorly to the intestinal bifurcation and genital pore opening immediately posterior to the anterior margin of the pharynx. Like H. facioi, H. gendrei has a relatively much shorter and broader cirrus sac than does the new species. Examination of the holotype and paratype of H. facioi confirmed that the specimens from Guanacaste differ in having a longer body, a larger ovary and eggs, and smaller testes. They also have the ovary on the sinistral rather than the dextral side of the body, genital pore anterior to the pharynx rather than at or posterior to the level of the posterior margin of the pharynx, longer and thinner cirrus sac, and eggs without eyespotted miracidia. Half the eggs in both specimens of H. facioi have well-developed eyespotted miracidia, whereas the typical condition for cyclocoelids is for virtually all eggs to exhibit eyespotted miracidia. Both H. facioi and H. gendrei are transferred to Neohaematotrophus, along with the new species.     

Ribosomal variation in six species of shape Fusarium

Eighteen isolates representing six Fusarium species from diverse hosts and geographical origins were evaluated to determine ribosomal DNA variation using polymerase chain reaction and restriction fragment length polymorphisms. No length variation was observed for amplified 18S and 28S regions. However, amplification of the ITS region showed one isolate, a F. oxysporum, to be about 120 bp larger than the remaining 17. Restriction digestions in the 18S region revealed polymorphisms within species of F. oxysporum and F. solani. An amplified variable stretch of the 28S gene showed restriction site differences between F. avenecum, F. sambucinum and F. sporotrichioides. A large degree of polymorphism was observed both between and within species in the ITS region. Therefore, entire sequences of the ITS and the 5.8S subunit were obtained for 17 of the 18 isolates. These sequences, along with those from eight additional isolates, were analysed using PAUP to assess the occurrence of DNA sequence divergence within the ITS region. The lack of correlation between molecular-based relationships and species affinities inferred from morphology for some isolates indicates that species designation can be unreliable using morphological data alone. Possible reasons for the discordance of the sequence and morphological data are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Two Distinct Triatoma dimidiata (Latreille, 1811) Taxa Are Found in Sympatry in Guatemala and Mexico

Approximately 10 million people are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which remains the most serious parasitic disease in the Americas. Most people are infected via triatomine vectors. Transmission has been largely halted in South America in areas with predominantly domestic vectors. However, one of the main Chagas vectors in Mesoamerica, Triatoma dimidiata, poses special challenges to control due to its diversity across its large geographic range (from Mexico into northern South America), and peridomestic and sylvatic populations that repopulate houses following pesticide treatment. Recent evidence suggests T. dimidiata may be a complex of species, perhaps including cryptic species; taxonomic ambiguity which confounds control. The nuclear sequence of the internal transcribed spacer 2 (ITS2) of the ribosomal DNA and the mitochondrial cytochrome b (mt cyt b) gene were used to analyze the taxonomy of T. dimidiata from southern Mexico throughout Central America. ITS2 sequence divides T. dimidiata into four taxa. The first three are found mostly localized to specific geographic regions with some overlap: (1) southern Mexico and Guatemala (Group 2); (2) Guatemala, Honduras, El Salvador, Nicaragua, and Costa Rica (Group 1A); (3) and Panama (Group 1B). We extend ITS2 Group 1A south into Costa Rica, Group 2 into southern Guatemala and show the first information on isolates in Belize, identifying Groups 2 and 3 in that country. The fourth group (Group 3), a potential cryptic species, is dispersed across parts of Mexico, Guatemala, and Belize. We show it exists in sympatry with other groups in Peten, Guatemala, and Yucatan, Mexico. Mitochondrial cyt b data supports this putative cryptic species in sympatry with others. However, unlike the clear distinction of the remaining groups by ITS2, the remaining groups are not separated by mt cyt b. This work contributes to an understanding of the taxonomy and population subdivision of T. dimidiata, essential for designing effective control strategies.     

Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes

The phylogenetic relationships of 51 isolates representing 27 species of Phytophthora were assessed by sequence alignment of 568 bp of the mitochondrially encoded cytochrome oxidase II gene. A total of 1299 bp of the cytochrome oxidase I gene also were examined for a subset of 13 species. The cox II gene trees constructed by a heuristic search, based on maximum parsimony for a bootstrap 50% majority-rule consensus tree, revealed 18 species grouping into seven clades and nine species unaffiliated with a specific clade. The phylogenetic relationships among species observed on cox II gene trees did not exhibit consistent similarities in groupings for morphology, pathogenicity, host range or temperature optima. The topology of cox I gene trees, constructed by a heuristic search based on maximum parsimony for a bootstrap 50% majority-rule consensus tree for 13 species of Phytophthora, revealed 10 species grouping into three clades and three species unaffiliated with a specific clade. The groupings in general agreed with what was observed in the cox II tree. Species relationships observed for the cox II gene tree were in agreement with those based on ITS regions, with several notable exceptions. Some of these differences were noted in species in which the same isolates were used for both ITS and cox II analysis, suggesting either a differential rate of evolutionary divergence for these two regions or incorrect assumptions about alignment of ITS sequences. Analysis of combined data sets of ITS and cox II sequences generated a tree that did not differ substantially from analysis of ITS data alone, however, the results of a partition homogeneity test suggest that combining data sets may not be valid.     

A new species of Gnathostoma (Nematoda: Gnathostomatidae) in Procyon lotor hernandezii from Mexico

Gnathostoma lamothei n. sp., inhabiting the stomach of Procyon lotor hernandezii Wagler, 1831, in Tlacotalpan, Veracruz State, and Rio Sapo, Oaxaca, Mexico, is described. This new species differs from all other congeners by having the posterior half of the body surface covered by rows of tiny round bosses instead of spines, or lacking ornamentations. Sequences of the ITS2 of the ribosomal DNA of G. lamothei n. sp. are compared with sequences of other species of the genus recorded in Mexico; they show a wide divergence (<50%) with Gnathostoma binucleatum Almeyda-Artigas, 1991, and Gnathostoma turgidum Stossich, 1902, and high similarity with Gnathostoma sp. I sequence (99.2%). On the basis of morphometric traits and sequences, previous records of Gnathostoma sp. I (=Gnathostoma procyonis of Almeyda-Artigas et al., 1994, not Chandler, 1942, and Gnathostoma neoprocyonis nomen nudem) in Mexico are referred to as the new species.     

The mosquitoes (Diptera: Culicidae) of Tabasco,Mexico

To document and update the mosquito species of Tabasco, Mexico, field collection trips were conducted in the two physiographic regions of Tabasco: the coastal plain of the southern gulf and the mountains of Chiapas and Guatemala. Mosquitoes were collected as immature and adult stages during the dry and rainy seasons from 2014 through 2015. Additionally, the Reference Collection of Arthropods of Medical Importance (CAIM‐InDRE) containing mosquitoes of Tabasco was re‐examined. In total, 4,913 specimens were collected and examined, which are divided into seven tribes, 18 genera, 27 subgenera, and 104 species. Of these, one genus (Shannoniana Lane and Cerqueira), two subgenera (Georgecraigius Reinert, Harbach and Kitching, and Carrollia Lutz), and 21 species are new records for the mosquito fauna of Tabasco. Culex metempsytus Dyar is a new record for Mexico and Wyeomyia jocosa (Dyar and Knab) is removed from the Mexican mosquito fauna. Seventeen species historically reported were not found in the field collections conducted here. Taxonomic notes, new distribution limits, and comments about the medical importance of species of mosquitoes of Tabasco are discussed. Tabasco is the second state in Mexico with the largest mosquito richness (104 species), followed by Veracruz with 139 species.     

Diplogonoporiasis in Japan: genetic analyses of five clinical isolates

Infection of the whale tapeworm Diplogonoporus balaenopterae (Diphyllobothriidae) is occasionally found in humans, especially among Japanese. In the present study, we analysed the nucleotide sequences of the 18S rDNA, ITS1 and cox1 genes of the immature and mature proglottids of Diplogonoporus species recovered from five Japanese patients. The nucleotide sequences of 18S rDNA, ITS1 and cox1 showed little, if any, intraspecific divergence. Phylogenetic analyses of several diphyllobothriid species revealed a close relationship of Diplogonoporus isolates with the cetacean tapeworm Diphyllobothrium stemmacephalum. The results suggest that the genus Diphyllobothrium is paraphyletic and raise a question regarding the validity of the genus Diplogonoporus.     

The tropical <Emphasis Type="Italic">Polyporus tricholoma</Emphasis> (Polyporaceae) — Taxonomy,phylogeny, and the development of methods to detect cryptic species

The stipitate pore-bearing fungus Polyporus tricholoma (Polyporus infrageneric group Polyporellus) is commonly reported from Mesoamerica and more rarely from the palaeotropics. Collections from Puerto Rico, Mexico, and Costa Rica did not differ morphologically, but their monokaryotic cultures revealed the existence of three interINcompatibility groups. These putative cryptic species exist sympatrically in Costa Rica. Sequencing, restriction enzyme digestion, and specific primers targeting the ITS region of nuclear ribosomal DNA (ITS 1-5.8S-ITS2) could only partly be used for detecting intercompatibility groups. Heterogeneity within the rDNA repeat was observed. Molecular evidence supports the existence of a close relative in Australia.     

Ribosomal DNA Comparisons of Globodera from Two Continents

Ribosomal DNA (rDNA) sequence data were compared for five species of Globodera, including G. rostochiensis, G. pallida, G. virginiae, and two undescribed Globodera isolates from Mexico collected from weed species and maintained on Solanum dulcamara. The rDNA comparisons included both internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene, and small portions of the 3'' end of the 18S gene and the 5'' end of the 28S gene. Phylogenetic analysis of the rDNA sequence data indicated that the two potato cyst nematodes, G. pallida and especially G. rostochiensis, are closely related to the Mexican isolates, whereas G. virginiae is relatively dissimilar to the others and more distantly related. The data are consistent with the thesis that Mexico is the center of origin for the potato cyst nematodes.     

Description of a new species of Neoechinorhynchus (Acanthocephala: Neoechinorhynchidae) a parasite of Dormitator latifrons from Southwestern Mexico based on morphological and molecular characters

Neoechinorhynchus mamesi n. sp. is described from the estuarine fish Dormitator latifrons collected in 3 localities along the coast of Chiapas State in Southwestern Mexico. The new species is characterized by possessing a small trunk, a very small proboscis with relatively very long apical proboscis hooks and small middle and posterior hooks, 2 giant nuclei in the ventral body wall, and males with testes smaller than the cement gland. A multivariate analysis of variance (MANOVA) and a Principal Component Analysis (PCA) of 46 morphometric traits for 21 mature females and 18 males of N. mamesi n. sp., N. brentnickoli and N. golvani, revealed morphological variation among species. DNA sequences of 2 genes, cytochrome oxidase subunit 1 (cox 1) of the mitochondrial DNA and the domains D2 and D3 of the large subunit of the nuclear ribosomal RNA (LSU) were used to corroborate the morphological distinction. The genetic divergence estimated among populations of N. brentnickoli and N. mamesi n. sp. ranged from 10.14 to 10.55% for LSU and from 20.53 to 22.06% for cox 1, whereas the genetic divergence between N. golvani and N. mamesi n. sp. ranged from 20.31 to 21.03% for LSU and from 22.24 to 24.95% for cox 1. Maximum likelihood, maximum parsimony and Bayesian inference analyses were performed for the combined data sets (LSU+cox 1) and each data set alone. All the phylogenetic analyses showed that the specimens from 3 coastal lagoons of Chiapas State in Southwestern Mexico represented a monophyletic clade with strong bootstrap support and Bayesian posterior probabilities. The haplotype network based on the analysis of the cox 1 sequences indicated that N. mamesi n. sp. is separated by 84 substitutions from N. brentnickoli, and with 69 substitutions from N. golvani. The morphological evidence, the multivariate analyses, in combination with the genetic divergence estimated with two genes, the reciprocal monophyly in all the phylogenetic analyses, and the haplotype network, suggested that the acanthocephalans found in the intestine of D. latifrons in Southwestern Mexico represent a new species, named N. mamesi n. sp., and it constitutes the second species of the genus Neoechinorhynchus associated with the Pacific fat sleeper along the Pacific Coast of Mexico.