Integrative species delimitation uncovers hidden diversity within the Pithecopus hypochondrialis species complex (Hylidae, Phyllomedusinae) and its phylogeography reveals Plio-Pleistocene connectivity among Neotropical savannas.

Despite their limited vagility and pronounced habitat heterogeneity in the tropics, many anuran species have unexpectedly extensive geographic ranges. One prominent example of this phenomenon is Pithecopus hypochondrialis, which is found in the Cerrado, Guianan savanna, and Llanos domains, as well as isolated tracts of savanna and open habitat within the Amazon Forest. The present study employs an integrative species delimitation approach to test the hypothesis that P. hypochondrialis is in fact a species complex. We also reconstruct the relationships among the lineages delimited here and other Pithecopus species. In this study, we employ Ecological Niche Modelling (ENM) and spatiotemporal phylogeographic reconstruction approaches to evaluate a multitude of scenarios of connectivity across the Neotropical savannas. We identified three divergent lineages, two of which have been described previously. The lineages were allocated to a lowland Pithecopus clade, although the relationships among these lineages are weakly supported. Both the ENM and the phylogeographic reconstruction highlight the occurrence of periods of connectivity among the Neotropical savannas over the course of the Pliocene and Pleistocene epochs. These processes extended from eastern Amazonia to the northern coast of Brazil. The findings of the present study highlight the presence of hidden diversity within P. hypochondrialis, and reinforce the need for a comprehensive taxonomic review. These findings also indicate intricate and highly dynamic patterns of connectivity across the Neotropical savannas that date back to the Pliocene.

Non-random distribution of microsatellite motifs and (TTAGGG)n repeats in the monkey frog Pithecopus rusticus (Anura, Phyllomedusidae) karyotype.

The monkey frog, Pithecopus rusticus (Anura, Phyllomedusidae) is endemic to the grasslands of the Araucarias Plateau, southern Brazil. This species is known only from a small population found at the type locality. Here, we analyzed for the first time the chromosomal organization of the repetitive sequences, including seven microsatellite repeats and telomeric sequences (TTAGGG)n in the karyotype of the species by Fluorescence in situ Hybridization. The dinucleotide motifs had a pattern of distribution clearly distinct from those of the tri- and tetranucleotides. The dinucleotide motifs are abundant and widely distributed in the chromosomes, located primarily in the subterminal regions. The tri- and tetranucleotides, by contrast, tend to be clustered, with signals being observed together in the secondary constriction of the homologs of pair 9, which are associated with the nucleolus organizer region. As expected, the (TTAGGG)n probe was hybridized in all the telomeres, with hybridization signals being detected in the interstitial regions of some chromosome pairs. We demonstrated the variation in the abundance and distribution of the different microsatellite motifs and revealed their non-random distribution in the karyotype of P. rusticus. These data contribute to understand the role of repetitive sequences in the karyotype diversification and evolution of this taxon.

Hidden Diversity in the Populations of the Armored Catfish Ancistrus Kner, 1854 (Loricariidae, Hypostominae) from the Paraná River Basin Revealed by Molecular and Cytogenetic Data.

Only one species of armored catfish, Ancistrus cirrhosus Valenciennes 1836, has been historically described in the basin of the Paraná River, from Misiones (Argentina). However, the ample variation found in the morphology and coloration of the populations sampled in the tributaries of the Brazilian state of Paraná makes it difficult to establish the real taxonomic status and evolutionary history of the Ancistrus specimens, suggesting that A. cirrhosus is not the only species found in this basin. By combining data on mitochondrial DNA (COI gene) and chromosomal markers from different Ancistrus populations, totaling 144 specimens, in the tributaries of the Paraná, and specimens from Misiones (type-locality of A. cirrhosus), we detected five distinct evolutionary lineages. All the specimens were 2n = 50, but had four distinct karyotype formulae. The results of the Generalized Mixed Yule Coalescent (GYMC) and the genetic distances (uncorrected P-values) between lineages ranged from 3 to 5%. Clusters of 18S rDNA were observed in a single chromosome pair in seven populations of Ancistrus, but at different positions, in some cases, in synteny with the 5S rDNA sites. Multiple 5S sites were observed in all populations. Overall, the cytogenetic data reinforce the genetic evidence of the diversification of lineages, and indicate the existence of candidate species in the study region. The evidence indicates that at least four candidate species of the Ancistrus may coexist in the Paraná basin besides A. cirrhosus. Overall, our results provide a comprehensive scenario for the genetic variation among Ancistrus populations and reinforce the conclusion that the true diversity of the freshwater fish of the Neotropical regions has been underestimated.

Molecular and morphological evidence reveals a new species in the Phyllomedusa hypochondrialis group (Hylidae, Phyllomedusinae) from the Atlantic Forest of the highlands of southern Brazil.

The taxonomic status of a disjunctive population of Phyllomedusa from southern Brazil was diagnosed using molecular, chromosomal, and morphological approaches, which resulted in the recognition of a new species of the P. hypochondrialis group. Here, we describe P. rustica sp. n. from the Atlantic Forest biome, found in natural highland grassland formations on a plateau in the south of Brazil. Phylogenetic inferences placed P. rustica sp. n. in a subclade that includes P. rhodei + all the highland species of the clade. Chromosomal morphology is conservative, supporting the inference of homologies among the karyotypes of the species of this genus. Phyllomedusa rustica is apparently restricted to its type-locality, and we discuss the potential impact on the strategies applied to the conservation of the natural grassland formations found within the Brazilian Atlantic Forest biome in southern Brazil. We suggest that conservation strategies should be modified to guarantee the preservation of this species.