The genus Rineloricaria is a Neotropical freshwater fish group with a long and problematic taxonomic history, attributed to the large number of species and the pronounced similarity among them. In the present work, taxonomic information and different molecular approaches were used to identify species boundaries and characterize independent evolutionary units. We analyzed 228 samples assembled in 53 distinct morphospecies. A general mixed yule-coalescent (GMYC) analysis indicated the existence of 70 entities, while BOLD system analyses showed the existence of 56 distinct BINs. When we used a new proposed integrative taxonomy approach, mixing the results obtained by each analysis, we identified 73 OTUs. We suggest that Rineloricaria probably has some complexity in the known species and several species not formally described yet. Our data suggested that other hyperdiverse fish groups with wide distributions can be further split into many new evolutionary taxonomic units.

Lungworms from the genus Rhabdias are common parasites of amphibians and reptiles distributed worldwide. To assess the diversity of Rhabdias spp., we performed molecular analyses of 35 specimens sampled in different regions of Brazil. Molecular analyses were based on the internal transcribed spacer (ITS), large subunit (28S) ribosomal and the cytochrome oxidase I (COI) mitochondrial genes. DNA sequence divergence was compared among ribosomal and mitochondrial genes, analyses using the general mixed Yule-coalescent (GMYC) method based on the COI gene were used to identify possible cryptic diversity, and phylogenetic analyses using concatenated ITS and 28S ribosomal genes were used to test the monophyly of Rhabdiasidae.We revealed five morphospecies: R. cf. stenocephala, R. breviensis, R. pseudosphaerocephala and two new species, Rhabdias sp.4 and Rhabdias sp.5. DNA sequence levels of divergence among genes ITS, 28S and COI were compared, and the efficiency of the molecular markers to identify species (ITS and COI) and lineages (COI) was tested. GMYC was assigned to 17 well-supported clades (i.e., 17 species), and cryptic diversity was detected in the Neotropical region as evidenced by the multiple lineages in R. breviensis and R. pseudosphaerocephala. In addition, our results suggest evidence for host-parasite cophylogeny in the R. pseudosphaerocephala complex and dispersal events among their populations. Phylogenetic analyses supported the monophyly of Rhabdiasidae and improved the resolution of main clades. Rhabdias breviensis is closely related to Rhabdias cf. africanus, Rhabdias cf. stenocephala, R. pseudosphaerocephala, Rhabdias sp.4 and Rhabdias sp.5 grouping together in a main clade with Neotropical-related species. The large geographical distribution appeared to be a phylogenetic pattern among the species of Rhabdias from the neotropics. 596 | MÜLLER Et aL.

Brachyplatystoma platynemum is a catfish species widely distributed in the Amazon basin. Despite being considered of little commercial interest, the decline in other fish populations has contributed to the increase in the catches of this species. The structure, population genetic variability, and evolutionary process that have driven the diversification of this species are presently unknown. Considering that, in order to better understand the genetic structure of this species, we analyzed individuals from seven locations of the Amazon basin using eight molecular markers: control region and cytochrome b mtDNA sequences, and a set of six nuclear microsatellite loci. The results show high levels of haplotype diversity and point to the occurrence of two structured populations (Amazon River and the Madeira River) with high values for FST. Divergence time estimates based on mtDNA indicated that these populations diverged about 1.0 Mya (0.2–2.5 Mya 95% HPD) using cytochrome b and 1.4 Mya (0.2–2.7 Mya 95% HPD) using control region. During that time, the influence of climate changes and hydrological events such as sea level oscillations and drainage isolation as a result of geological processes in the Pleistocene may have contributed to the current structure of B. platynemum populations, as well as of differences in water chemistry in Madeira River. The strong genetic structure and the time of genetic divergence estimated for the groups may indicate the existence of strong structure populations of B. platynemum in the Amazon basin.

The cytogenetic characteristics of Eigenmannia aff. trilineata were analyzed by basic and molecular cytogenetics, applying fluorescent in situ hybridization, with 18S and 5S rDNA and U2 snRNA probes. The species revealed a kind of polymorphism associated to ZZ/Z0 type sex chromosomes, with 2n = 32 (8m+2sm+22a, NF = 42) in all males under analysis, whereas females evidenced 2n = 31 (8m+1sm+22a, NF = 40). C-banding showed constitutive heterochromatin restricted to the pericentromeric region of all chromosomes and single-nucleolus organized regions on pair 11. A site for rDNA 5S was synthetic with a cluster of rDNA 18S near the centromere on the long arm of only one homologue of pair 11. Other clusters for 5S rDNA were sited on pairs 7, 10, 12, 13, and 16. Further, 5S rDNA was co-located with U2 cluster in the pericentromeric region of pair 12. Joint analysis of DNA barcoding from cytochrome c oxidase subunit I (COI) sequences, generated from the karyotyped samples of E. aff. trilineata, and sequences of other Gymnotiforms recognized E. aff. trilineata as an Operational Taxonomic Unit. Results foreground the hypothesis that cytotypes are independent evolution units as cryptic species with a low morphological differentiation level, although with high genetic/karyotype differentiation rates.

Characidium comprises several species of small freshwater fish that display conserved diploid chromosome numbers and karyotypic formulae. In this study, a comparative cytogenetic analysis using telomeric DNA probes was carried out in nine species of Characidium; a molecular phylogenetic analysis with mitochondrial DNA was also performed in order to investigate the direction of the evolutionary chromosome changes observed here. Our results showed the existence of species with several and variable interstitial telomeric sites (ITSs), with other species showing only terminal signals in their chromosomes. Molecular phylogenetic data suggested that these ITSs emerged once in the evolutionary history of Characidium and were later differentially spread in distinct species/populations of this clade. Additionally, the origin of an exclusive acrocentric pair found in C. pterostictum, C. serrano and C. timbuiense was also investigated, revealing that this pair possibly had a common origin to these species. These results evidence the occurrence of intense and continuous genomic changes among species of Characidium.

In the present study, we compare the results of alpha taxonomy (based on morphology), DNA Barcoding method with a 2% genetic divergence threshold, and the GMYC (General Model Yule Coalescent) technique to identify species clusters in Neoplecostomus. We used partial sequences of mitochondrial COI (Cytochrome oxidase subunit I) for 59 specimens representing 13 valid species (Neoplecostomus bandeirante, N. jaguari, N. langeanii, N. paranensis, N. yapo, N. botucatu, N. selenae, N. doceensis, N. corumba, N. ribeirensis, N. microps, N. espiritosantensis and N. franciscoensis) of Neoplecostomus collected in all its distribution area. For the analysis we used Bayesian inference of phylogeny with relaxed clock methods on an arbitrary timescale, using BEAST. The ultrametrics genes trees obtained for each tree prior evaluated (Yule, Birth-death and coalescent Population Size) were used in the GMYC analysis to identify a time in the tree when the branching rate shifts (in forward time) from Yule and Birth-Death (species) to a coalescent (population) process. We found that the GMYC model using the Yule prior identified 11 groups, closer to the current taxonomy (13 species). GMYC analyses using other ultrametric gene trees estimated under alternative prior such as Birth-Death and Coalescent Population Size, identified 9 groups, results observed in the traditional 2% genetic distance threshold, resulting in a low number of species recognized compared to the number of species identified with current taxonomy. Based on these results we conclude that the molecular approaches are helpful to distinguish species of Neoplecostomus, nevertheless it is important to combine molecular methodologies with current taxonomy in order to identify correctly species that recently originated.

The current distribution of freshwater fishes across multiple basins along Eastern Brazil can be associated to two main events: river captures or temporary paleoconnections. Apparently, river captures had a more significant role on distribution and structuring of species from upland areas, such as Glandulocauda melanopleura. Populations of this species are found in contiguous drainages in presently isolated upper parts of Rio Tietê and the coastal basins of Guaratuba, Itatinga, Itanháem, and Ribeira de Iguape, in the Atlantic Forest domain. The allopatric and disjoint distribution of G. melanopleura associated with variation of morphological characters detected among geographically isolated populations stimulated this study. Thus, an integrative approach was undertaken, including morphological and molecular data, to better understand the evolutionary history of the species and the area where it occurs. Molecular analyses based on two mitochondrial markers revealed a strong genetic structure within G. melanopleura, that allowed recognition of two lineages, one distributed in both the upper Tietê and Itanhaém and the other in the Guaratuba. Overall, morphological data revealed some intraspecific overlapping variation, indicating that all samples are conspecific. Phylogenetic and phylogeographic analyses allied to divergence times and geomorphological information indicate that the current distribution of G. melanopleura is a result of relatively recent river captures involving the Tietê and some other coastal drainages. Although of recent origin, they occurred long enough to completely isolate these populations, since there are no haplotypes sharing between them. The conservation status of this species is also discussed, and our results corroborate the need to understand population structure for conservation planning.

The fish species Synbranchus marmoratus has been reported to exist as a species complex due to high intraspecific karyotypic variability in spite of the difficulty or impossibility to distinguish them using morphological traits alone. The goal of this work was to use cytogenetic and molecular methods to determine the species delimitations and understand the karyoevolution of S. marmoratus using samples collected from distinct Brazilian localities. Among the analyzed specimens, a large degree of cytogenetic variation related to diploid numbers and karyotype structure was observed, with karyotypes showing 2n=42, 44 and 46 chromosomes. In addition, using sequences of three mitochondrial genes, the phylogenetic relationships between every sample with a known karyotype were determined, which revealed significant nucleotide divergence among the karyomorphs. Also, the analyses indicate that chromosomal rearrangements occurred independently within the distinct lineages of S. marmoratus complex, which resulted in the appearance of distinct karyotypic variants in a non-linear fashion related to diploid numbers and in the appearance of similar nonhomologous chromosomes. Finally, the integration of both molecular cytogenetic and phylogenetic approaches allowed the determination of specific chromosomes possibly involved in rearrangements and a better understanding about the evolutionary processes involved in the differentiation of Synbranchus genus.A espécie de peixe Synbranchus marmoratus tem sido reportada como um complexo de espécies devido à elevada variabilidade cariotípica intraespecífica a despeito da dificuldade ou impossibilidade de distingui-las usando apenas caracteres morfológicos. O objetivo deste trabalho foi utilizar métodos citogenéticos e moleculares para determinar a delimitação das espécies e compreender a carioevolução de S. marmoratus utilizando amostras coletadas em distintas localidades brasileiras. Dentre os espécimes analisados, um alto grau de variação citogenética relativo aos números diploides e estrutura cariotípica foi observado, com cariótipos mostrando 2n=42, 44 e 46 cromossomos. Adicionalmente, utilizando sequências de três genes mitocondriais, as relações filogenéticas entre cada amostra com cariótipo conhecido foram determinadas, revelando uma divergência nucleotídica significativa entre os cariomorfos. Além disso, as análises indicam que rearranjos cromossômicos ocorreram independentemente nas distintas linhagens do complexo S. marmoratus, o que resultou no aparecimento de distintas variantes cariotípicas de forma não linear em relação aos números diploides e no surgimento de cromossomos similares e não homólogos. Finalmente, a integração de uma abordagem citogenética molecular e filogenética permitiu a determinação de cromossomos específicos que, possivelmente, estão envolvidos em rearranjos e um melhor entendimento sobre os processos evolutivos envolvidos na diferenciação do gênero Synbranchus.