Integrating Genetic and Cytogenetic Data: A Diversity Study of Astyanax and Psalidodon (Characidae) Species from the Paraná River Basin.

Astyanax is one of the most specious fish groups in the Neotropical region, with many cryptic species, which represents a challenge for correct identification through traditional taxonomic methods. Psalidodon is a recently resurrected genus group of species previously belonging to Astyanax, specifically those with extensive chromosomal variation of the A. scabripinnis and fasciatus complexes. In the present study, the mitochondrial genes cytochrome c oxidase subunit 1 (COI), mitochondrial ATP synthase 6 and 8 (ATPase 6/8), and NADH dehydrogenase subunit 2 (ND2) were used in conjunction with chromosomal data to characterize molecularly and cytogenetically populations of Astyanax and Psalidodon from rivers and streams of the Ivaí River Basin (Paraná Basin). The results demonstrated the effectiveness of the integrative use of molecular and cytogenetic techniques, with the confirmation of at least three species for the sampled sites: A. lacustris, P. paranae, and P. fasciatus, which showed inter- and intrapopulation karyotype variations. In addition, extensive haplotypic variation can be observed for these species within the Ivaí River Basin and throughout the Paraná River Basin. The data demonstrate a hidden diversity among the species analyzed, enrich the ichthyofaunistic knowledge of small rivers and streams, and contribute to future conservation projects in these areas.

Cytogenetic and Molecular Characterization of Eigenmannia aff. desantanai (Gymnotiformes: Sternopygidae): A First Report of System of Sex Chromosomes ZW1W2/ZZ in Gymnotiformes.

Gymnotiformes a monophyletic group of fish endemic to the Neotropics, represent an important component of the freshwater ichthyofauna that presents relevant taxonomic problems. Thus, in view of the morphological complexity involving Eigenmannia (Gymnotiformes) fish species, this study aimed to characterize Eigenmannia aff. desantanai of the upper Paraguay River basin through cytogenetic and molecular analyses, to help in the correct identification and delimitation of species. This study reports a multiple sex system of the type ZW1W2/ZZ, with 2n = 31 for females and 2n = 30 for males. A single pair of chromosomes carrying the nucleolar organizing regions (NORs) was detected. The heterochromatin was colocated in NOR sites and mainly located in the centromeric regions of chromosomes. Besides that, individual sequences COI from the specimens of E. aff. desantanai were obtained, totalizing three haplotypes. The distance p between the haplotypes in E. aff. desantanai, ranged from 0.2% to 7.1%. Species delimitation tests indicated the existence of two possible operational taxonomic units of E. aff. desantanai. Thus, this study reports a new multiple sex system in Gymnotiformes and these specimens previously identified as E. aff. desantanai may belong to two distinct species.

Molecular Markers Reveal a New and Possibly Threatened Species of Cnesterodon (Poeciliidae, Cnesterodontini) from the Upper Paraná River Basin, Brazil.

Cnesterodon comprises 10 valid species occurring in the major river basins of South America. Recent ichthyofaunistic studies in the Ivaí River basin, upper Paraná River system, suggested the existence of a possible new species, which was identified as Cnesterodon sp. based on morphological characters. Currently, the use of molecular tools has proved to be fundamental in aiding phylogenetics and cataloging biodiversity; therefore, in this study, we molecularly characterize a possible new species of Cnesterodon from the Ivaí River basin encoding the mitochondrial genes Cytochrome c Oxidase, subunit I (COI), and NADH dehydrogenase subunit 2 (ND2). The genetic differences found showed that this species really differs from the other Cnesterodon species, indicating that it is a distinct species, which is possibly already in serious danger of extinction since its habitat often suffers from human exploitation and its distribution is restricted to only two sites in the upper Ivaí River basin, but it has disappeared in one of them.

Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil.

Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Cytogenetic and Molecular Characterization of Oligosarcus pintoi (Characidae): A New Record of Supernumerary Chromosome in this Species.

The genus Oligosarcus currently comprises 24 valid species distributed in the major river basins of South America. In this group, nine species were cytogenetically investigated, and found to share a diploid number of 50 chromosomes. Despite the conservation of the diploid number, variations in the karyotypic formula, number and position of the nucleolar organizer regions, and longitudinal bands have been described between both species and populations. In this study, we present cytogenetic and molecular data from Oligosarcus pintoi specimens from the Keller River, a tributary of the Ivaí River (Upper Paraná basin), using DNA barcoding and cytogenetic markers (C-band, silver-stained nucleolar organizer regions, and fluorescence in situ hybridization of 18S and 5S rDNA). The genetic inferences reached after analyzing the cytochrome c oxidade subunit 1 gene allowed us to confirm the identity of the individuals with 2n = 50 chromosomes. However, one specimen contained a medium subtelocentric supernumerary chromosome (2n = 51). This is the second record of additional chromosomes in O. pintoi, thereby confirming the existence of a supernumerary chromosome in allopatric populations of this species, a fact that demonstrates an evolutionary path that is divergent from other populations and/or species of Oligosarcus analyzed so far, contributing to the karyotypic diversification of the group.

Morphological and molecular characterization of Austrodiplostomum compactum metacercariae in the eyes and brains of fishes from the Ivaí River, Brazil / Caracterização morfológica e molecular de metacercárias de Austrodiplostomum compactum em olhos e cérebros de peixes do Rio Ivaí, Brasil

Abstract Austrodiplostomum spp. (Platyhelminthes: Digenea) are endoparasites with a broad geographic distribution in South America. During the larval stage, they parasitize the eyes, brains, muscles, gill, kidneys and swim bladder of a wide variety of fishes. The metacercariae of Austrodiplostomum spp. have several morphological characteristics during development, but are very similar among species, which makes it necessary to use molecular tools to contribute to the elucidation during the larval stage. The objective of this study was to perform morphological and molecular analyses of Austrodiplostomum sp. found in specimens of Hypostomus sourced from the Ivaí River in the state of Paraná, Brazil. Of the 93 analyzed specimens (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], and Hypostomus sp. 2 [n = 10]), 60 were parasitized. A total of 577 Austrodiplostomum sp. metacercariae was collected from the infected hosts; DNA from seven of these samples was extracted, amplified, and sequenced. The morphological data associated with the genetic distance values and the relationships observed in the COI gene tree, indicate that all metacercariae were A. compactum. This is the first record of A. compactum parasitizing H. hermanni, H. albopunctatus, Hypostomus sp. 1, and Hypostomus sp. 2 in the Ivaí River.

Resumo Austrodiplostomum spp. (Platyhelminthes: Digenea) são endoparasitos com uma ampla distribuição geográfica na América do Sul. Durante a fase larval, parasitam os olhos, cérebros, músculos, brânquias, rins e bexiga natatória de uma grande variedade de peixes. As metacercárias de Austrodiplostomum spp. apresentam várias características morfológicas durante o desenvolvimento, as quais são muito semelhantes entre as espécies, o que torna necessário o uso de ferramentas moleculares para contribuir para a elucidação durante a fase larval. O objetivo deste estudo foi realizar análises morfológicas e moleculares de Austrodiplostomum sp. encontradas em espécimes de Hypostomus provenientes do rio Ivaí, no Paraná, Brasil. Dos 93 espécimes analisados (H. hermanni [n = 50], H. albopunctatus [n = 9], Hypostomus sp. 1 [n = 24], e Hypostomus sp. 2 [n = 10]), 60 foram parasitados. Um total de 577 metacercárias de Austrodiplostomum foram coletadas dos hospedeiros infectados; o DNA de sete dessas amostras foi extraído, amplificado e sequenciado. Os dados morfológicos, associados aos valores de distância genética e as relações observadas na árvore gênica do COI, indicam que todas as metacercárias são A. compactum. Este é o primeiro registo de A. compactum parasitando H. hermanni, H. albopunctatus, Hypostomus sp. 1, e Hypostomus sp. 2 no rio Ivaí.

Integrative taxonomy reveals the historically poorly defined armoured catfish Hypostomus variipictus (Ihering 1911), from the upper rio Paraná basin, Brazil (Siluriformes, Loricariidae).

In a recent expedition to the rio Grande basin, a tributary of the rio Paraná in southern Brazil, individuals of the armoured catfish genus Hypostomus with a peculiar and beautiful colour pattern composed of pale vermiculations on the head and four to five horizontal stripes on the flanks were collected. Initially, the specimens were identified as a colour morph of the pale-spotted H. margaritifer. However, when we compared their partial cytochrome C oxidase subunit I (COI) mitochondrial gene to sequences of some typically pale-spotted H. margaritifer, the striped specimens were genetically distinct. Further analysis of the striped individuals revealed that they are the poorly known but valid species Hypostomus variipictus, which was described by Ihering in 1911 from the rio Pardo, a tributary of the rio Grande, upper rio Paraná basin, in São Paulo State, Brazil. Since its descriptions, no robust taxonomic work has been published concerning this species. In this study, the newly sampled population was compared to the original description and to the holotype of H. variipictus, providing the foundation for a complete redescription, proper diagnosis, and first live colour illustration and description of the previously hidden H. variipictus.

Spar genetic analysis of two invasive species of Cichla (Tucunaré) (Perciformes: Cichlidae) in the Paraná river basin / Análise genética via spar, de duas espécies de Cichla (Tucunaré) (Perciformes: Cichlidae) introduzidas na Bacia do rio Paraná

The introduction of exotic species in lake ecosystems has been greatly highlighted in the literature worldwide. Since introduction may threaten diversity of native fish, the issue turns up to be of paramount importance. Ecological effects may be predation, competition, parasitism or genetic, that is, changes in the genetic pool of populations owing to the occurrence of hybrids. Although the Tucunaré fish (Cichla) is native to the Amazon region, it can be found in other hydrographic basins in which it has been introduced. RAPD molecular marker research showed that there are two species (Cichla kelberi and C. piquiti) belonging to the genus Cichla in the rivers of the Paraná basin. Different morphotypes in the region may also be due to hybridization. Current research used SPAR molecular markers to confirm the presence of hybrids and the rupture of isolation mechanisms. Seventy-two specimens collected in several sites of the river Paraná and Amazon basins were analyzed. Since exclusive SPAR molecular markers were obtained for Cichla kelberi and C. piquiti populations, the introduction of the two species in the region has been confirmed. Identification of the markers in specimens of the Paraná river basin confirmed hybridization between these exotic species.

A introdução de espécies exóticas nos ecossistemas lacustres tem sido muito destacada na literatura mundial, pois ameaça a diversidade de peixes nativos, tornando-se uma questão de extrema importância. Os efeitos observados podem ser ecológicos, como predação, competição e parasitismo ou genéticos, como a ocorrência de híbridos. Apesar do peixe tucunaré (Cichla) ser nativo da região amazônica, ele pode ser encontrado em outras bacias hidrográficas nas quais foram introduzidos. Estudos utilizando marcadores moleculares RAPD revelaram que existem duas espécies (Cichla kelberi e C. piquiti) do gênero Cichla na bacia do alto rio Paraná e morfotipos diferentes que podem ser devido à hibridização. O presente trabalho utilizou marcadores moleculares SPAR para confirmar a presença de híbridos e os mecanismos de ruptura de isolamento entre essas espécies. Setenta e dois espécimes foram coletados em diversos pontos da bacia do rio Paraná e da bacia Amazônica e foram analisados. Marcadores moleculares SPAR exclusivos foram obtidos para populações de Cichla kelberi e C. piquiti, confirmando a introdução das duas espécies na região. A identificação dos marcadores diagnósticos em espécimes da bacia do rio Paraná confirmou também a hibridização entre estas espécies exóticas.