Phylogeography of the critically endangered neotropical annual fish, <Emphasis Type="Italic">Austrolebias wolterstorffi</Emphasis> (Cyprinodontiformes: Aplocheilidae): genetic and morphometric evidence of a new species complex

Austrolebias wolterstorffi is a critically endangered annual fish, occurring in temporary ponds in a restricted area of Southern Brazil and Uruguay. Here, we evaluate the levels of genetic diversity and morphometric differentiation presented by A. wolterstorffi, attempting to reconstruct the spatiotemporal scenario by which this species reached their current distribution. Part of the mitochondrial cytochrome b and nuclear rhodopsin genes were characterized and analysed for a set of 122 and 110 specimens, respectively, collected along the entire distribution range of the species. Additionally, shape variations were evaluated for 92 individuals (43 males and 49 females) through geometric morphometric methods. Our analyses demonstrated several cases of significantly high levels of genetic differentiation among individual populations, in an isolation-by-distance pattern of divergence, with at least six different population groups along the Patos-Mirim lagoon. These groups differed by a minimum of 0.9% and a maximum of 2.6% of corrected cyt b nucleotide distances and did not share any mitochondrial haplotype. Such a pattern, added to the slight morphometric differentiation detected for most of the groups, suggests the occurrence of incipient speciation as consequence of allopatric fragmentation. The chronophylogenetic tree performed with the concatenated dataset supported independent oriental and occidental colonization routes, with the population located in the northwest part of the Rio Grande do Sul coastal plain presenting the most ancient divergence. In general, the recovered biogeographic patterns are highly consistent with the records of Quaternary climatic changes and depositional events that have occurred along the area inhabited by the studied species. This allowed us to establish a molecular clock calibration system for Neotropical annual fish. Thus, although the taxonomic status of each of the detected population units needs further study, it is clear that independent conservation strategies must be taken in each of the major areas covered by this study, most of which are located in Brazil.