Morphological and molecular phylogeny of Austrolebias.

Species of the genus Austrolebias were originally included in Cynolebias, that was described just five years earlier by the same author from a single species, C. porosus Steindachner from Pernambuco, northeastern Brazil (Steindachner, 1876). In the first phylogenetic analysis of Cyprinodontiformes, Parenti (1981) provided one synapomorphy to diagnose Cynolebias (caudal fin without scales) and synonymized this genus with several annual genera that had been described during the 20 th century (Cynopoecilus Regan, 1912, Leptolebias Myers, 1952, Simpsonichthys, Carvalho, 1959, Campellolebias Vaz-Ferreira & Sierra, 1974, and Terranatos Taphorn & Thomerson, 1978). Costa’s (1990) phylogenetic analysis of Rivulidae elevated Cynolebias sensu Parenti (1981) to a subfamily level, revalidated those genera previously synonymized, except Simpsonichthys, and rediagnosed Cynolebias based on five synapomorphies.

Costa (1998), in a new phylogenetic analysis of Rivulidae, described Austrolebias and Megalebias Costa (both from La Plata and Patos Merin basins), including some species previously located in Cynolebias. Austrolebias was diagnosed by three synapomorphies and included species previously diagnosed as the “ C. bellottii Steindachner ” species complex (Costa, 1995); Megalebias was also diagnosed based on three synapomorphies and included species previously diagnosed as the “ C. elongatus Steindachner ” species complex (Costa, 1995). Cynolebias sensu stricto (diagnosed by three synapomorphies) included species previously diagnosed as the “ C. porosus ” species complex (Costa, 1995), distributed in northeastern Brazil. These three genera were included in the Subtribe Cynolebiatina with Austrolebias as the sister clade of Megalebias + Cynolebias (Costa, 1998). Costa (2002a), changed this relationship and proposed that Cynolebias was the sister genus of Austrolebias + Megalebias. Costa (2006b), considered Megalebias as a synonym of Austrolebias based on a morphological phylogenetic analysis in which Megalebias was nested within Austrolebias. Cynolebiatina was diagnosed by nine synapomorphies (Costa, 1998). However, as was mentioned before, Costa et al. (2017a), proposed Austrolebias as the sister genus of a clade with the following relationships: (( Cynolebias, Simpsonichthys), (Ophthalmolebias, Hypsolebias )).

Monophyly of Austrolebias is supported by three exclusive synapomorphies (Costa, 2006b): absence of scales between the corner of the mouth and anterior portion of the preopercular region and ventral portion of the opercular region, a deep urohyal (Fig. 2e), a dark gray to black infraorbital bar and supraorbital spot; and three synapomorphies independently arose in other cynolebiatines: males with dorsal and anal fins rounded, males with long urogenital papilla, and reduced ventral process of the anguloarticular. However, according to relationships proposed by Costa et al. (2017a), these non-exclusive synapomorphies may represent plesiomorphic states of Cynolebiasini.

Costa (2008b), divided the genus in several subgenera according to the phylogenetic relationships obtained in Costa (2006b): Acantholebias Costa ((type species A. luteoflammulatus (Vaz-Ferreira, Sierra & Scaglia)), Acrolebias ((type species A. carvalhoi (Myers)), Argolebias ((type species A. nigripinnis (Regan); also known as the “ A. alexandri ” species group), Austrolebias (type species A. bellottii; includes “ A. bellotti ” + “ A. adloffi ” species groups), Cypholebias ((type species A. robustus (Günther)), Gymnolebias ((type species A. gymnoventris (Amato)), Megalebias ((type species A. wolterstorffi (Ahl)). According to a morphological phylogeny (Costa, 2010) the subgenus Argolebias could be paraphyletic.

Relationships within Austrolebias are still unresolved; conflicting phylogenetic hypotheses have been proposed for this genus using morphological (Costa, 2006b, 2010; Loureiro, 2004) and molecular (mitochondrial) analyses (Garcia et al., 2014). Most subgeneric clades remain stable in species composition; however, relationships among clades and position of some species are still unresolved.

In this study, we performed a phylogenetic analysis of Austrolebias sensu Costa (2006b), using the most complete species sampling to date and a total evidence approach, including morphological, and molecular (mitochondrial and nuclear) data.