Martín O. Pereyra

Big, Bad, and Beautiful: Phylogenetic Relationships of the Horned Frogs (Anura: Ceratophryidae)

Abstract. The horned frog family, Ceratophryidae, currently comprises three genera and 12 extant species, distributed from the Caribbean lowlands to the Pampean grasslands. Horned frogs are fossorial species that are remarkable in terms of their adult and larval morphology, karyotype, behavior, and other aspects of their biology. In this paper we present a molecular phylogenetic analysis with the goals of: (1) exploring the relationships among the species of Ceratophryidae; (2) studying the evolution of polyploidy; (3) studying the evolution of cocoon formation and larval development duration associated with surviving in semiarid environments; and (4) reviewing the ceratophryid fossil record that could be relevant as calibration points in molecular divergence estimations. The analysis included 11 of the 12 extant species and, when possible, multiple exemplars per species, as well as multiple outgroups. Sequence data were obtained on seven mitochondrial and six nuclear genes for up to 8200 bp per specimen. Our results indicate that the individual monophyly of Ceratophrys and Lepidobatrachus is well corroborated. The monotypic Chacophrys is recovered as the sister taxon of Lepidobatrachus, but with Jackknife frequency < 50%. Lepidobatrachus asper is the sister taxon of L. laevis + L. llanensis. Relationships within Ceratophrys are congruent with an earlier proposal, with a clade composed of the species possessing a dorsal bony shield (Ce. aurita, Ce. cranwelli, Ce. joazeirensis, and Ce. ornata), and another clade composed of Ce. stolzmanni, Ce. calcarata, and Ce. cornuta. Unlike earlier proposals, the octoploid species (Ce. aurita, Ce. joazeirensis, and Ce. ornata) are not monophyletic, as the diploid Ce. cranwelli, and Ce. ornata are sister taxa. This result implies an ambiguous optimization of ploidy levels, with either a single origin of octoploidy with a subsequent reversal to diploidy, or two independent origins of octoploidy being equally parsimonious; both alternatives are quite unusual from the perspective of chromosome evolution. Our results suggest that ceratophryids diversified in semiarid environments and three independent events resulted in three species subsequently occupying temperate or tropical humid areas. This early diversification in semiarid areas explains the retention of characteristics associated with these environments (like the production of a cocoon of dead skin during estivation, and possibly an accelerated larval period and development) in species present in humid areas. A revision of the fossil record of this family of frogs indicates that there are only two fossil remains that could serve as calibration points for molecular clock estimation, but a number of issues associated with them preclude their use.

Phylogenetic relationships of toads of the Rhinella granulosa group (Anura: Bufonidae): a molecular perspective with comments on hybridization and introgression

The Rhinella granulosa group consists of 13 species of toads distributed throughout open areas of South America and Panama. In this paper we perform a phylogenetic analysis considering all but one species of the group, employing five nuclear and four mitochondrial genes, for up to 7910 bp per specimen. Separate phylogenetic analyses under direct optimization (DO) of nuclear and mitochondrial sequences recovered the R. granulosa group as monophyletic and revealed topological incongruence that can be explained mainly by multiple events of hybridization and introgression, both mitochondrial and nuclear. The DO combined analysis, after the exclusion of putatively introgressed or heterozygous genomes, resulted in a phylogenetic hypothesis for the R. granulosa group in which most of the species are recovered as monophyletic, but with interspecific relationships poorly supported. The optimization of morphological (adult and larval), chromosomal, and behavioural characters resulted in 12 putative phenotypic synapomorphies for this species group and some other synapomorphies for internal clades. Our results indicate the need for additional population genetic studies on R. dorbignyi and R. fernandezae to corroborate the taxonomic status of both taxa. Finally, we discuss biological and genetic characteristics of Bufonidae, as possible explanations for the common occurrence of hybridization and introgression observed in some lineages of this family.

Chromosome Evolution in Dendropsophini (Amphibia, Anura, Hylinae)

Dendropsophini is the most species-rich tribe within Hylidae with 234 described species. Although cytogenetic information is sparse, chromosome numbers and morphology have been considered as an important character system for systematic inferences in this group. Using a diversity of standard and molecular techniques, we describe the previously unknown karyotypes of the genera Xenohyla, Scarthyla and Sphaenorhynchus and provide new information on Dendropsophus and Lysapsus. Our results reveal significant karyotype diversity among Dendropsophini, with diploid chromosome numbers ranging from 2n = 22 in S. goinorum, 2n = 24 in Lysapsus, Scinax, Xenohyla, and almost all species of Sphaenorhynchus and Pseudis, 2n = 26 in S.carneus, 2n = 28 in P.cardosoi, to 2n = 30 in all known Dendropsophus species. Although nucleolar organizer regions (NORs) and C-banding patterns show a high degree of variability, NOR positions in 2n = 22, 24 and 28 karyotypes and C-banding patterns in Lysapsus and Pseudis are informative cytological markers. Interstitial telomeric sequences reveal a diploid number reduction from 24 to 22 in Scarthyla by a chromosome fusion event. The diploid number of X.truncata corroborates the character state of 2n = 30 as a synapomorphy of Dendropsophus.

The complex evolutionary history of the tympanic middle ear in frogs and toads (Anura)

Most anurans possess a tympanic middle ear (TME) that transmits sound waves to the inner ear; however, numerous species lack some or all TME components. To understand the evolution of these structures, we undertook a comprehensive assessment of their occurrence across anurans and performed ancestral character state reconstructions. Our analysis indicates that the TME was completely lost at least 38 independent times in Anura. The inferred evolutionary history of the TME is exceptionally complex in true toads (Bufonidae), where it was lost in the most recent common ancestor, preceding a radiation of >150 earless species. Following that initial loss, independent regains of some or all TME structures were inferred within two minor clades and in a radiation of >400 species. The reappearance of the TME in the latter clade was followed by at least 10 losses of the entire TME. The many losses and gains of the TME in anurans is unparalleled among tetrapods. Our results show that anurans, and especially bufonid toads, are an excellent model to study the behavioural correlates of earlessness, extratympanic sound pathways, and the genetic and developmental mechanisms that underlie the morphogenesis of TME structures.

Description and Phylogenetic Position of a New Species of Oreobates (Anura: Craugastoridae) from Northwestern Argentina

Abstract:  We describe a new species of Oreobates from Jujuy, Argentina. The new species is clearly diagnosable from other species of Oreobates by a combination of morphological characters and support by molecular evidence (genetic distance and phylogenetic analysis). We also provide taxonomic comments about O. discoidalis and O. barituensis, emphasizing the need for an exhaustive revision of these species.

A Cytotaxonomic Survey of the Genus Melanophryniscus Gallardo, 1961 (Anura: Bufonidae)

Abstract We present a cytogenetic survey of the basal bufonid genus Melanophryniscus that covered 14 of the 25 species currently recognized, representing the three phenetic species groups: M. moreirae, M. stelzneri, and M. tumifrons. All species presented a diploid chromosome complement constituted by 11 bi-armed chromosome pairs (2n  =  2x  =  22; FN  =  44). Some remarkable differences were observed between species groups: chromosome pair 4 was metacentric in species of the M. tumifrons group (also with a distinctive C-positive block) but submetacentric in the M. stelzneri group and M. sanmartini (M. moreirae group); pair 5 was submetacentric in M. sanmartini and metacentric in the rest. Chromosome secondary constrictions and silver-stained nucleolar organizer regions were located either in pair 5, 7, or 8 in the M. tumifrons group, M. sanmartini and M. krauczuki (M. stelzneri group), and M. stelzneri group, respectively; and pair 7 was relatively larger in M. sanmartini and M. krauczuki. Studied cytogenetic characters support the M. tumifrons group and suggest a close relationship between M. krauczuki and M. sanmartini. These results call for a reassessment of species relations within Melanophryniscus under an inclusive phylogenetic study.

The Tadpole of Rhinella azarai (Gallardo, 1965) with Comments on Larval Morphology in the Rhinella granulosa Species Group (Anura: Bufonidae)

Abstract We describe the larval morphology of Rhinella azarai, a medium-sized species of the Rhinella granulosa group. None of the morphological characters allow the larvae of R. azarai to be distinguished unequivocally from those of other species in the group. However, the tadpoles show a distinctive set of character states shared with some species of the group, which may represent putative synapomorphies of the R. granulosa group or internal clades.

Concentrated evolutionary novelties in the foot musculature of Odontophrynidae (Anura: Neobatrachia), with comments on adaptations for burrowing

We studied the foot musculature of the fossorial family Odontophrynidae (composed of the genera Macrogenioglottus, Odontophrynus, and Proceratophrys) plus several outgroup taxa from Hyloidea and other burrowing taxa from across Anura. We found novel character-states supporting the monophyly of Odontophrynus, Proceratophrys, and Odontophrynus + Macrogenioglottus. The character-states observed in O. cultripes support conflicting phylogenetic positions within Odontophrynus. A comparison of some novel character-states with a diverse sample of burrowing taxa suggests that some modifications of the foot musculature might be involved in digging.

Structural and Heterochronic Variations During the Early Ontogeny in Toads (Anura: Bufonidae)

Abstract:  In recent decades, a renewed interest in comparative studies of embryonic ontogeny in anurans is taking place. Toad embryos are often employed as model organisms, and scarce attention has been put on interspecific variations. In this work we analyze the development of transient embryonic and larval structures in 21 species in five genera of Bufonidae. These species vary in their ovipositional mode and the type of environments where the embryos and tadpoles develop, including ponds, streams, and axils of leaves of terrestrial or epiphytic plants. Comparative anatomical studies and sequence heterochrony analyses show that primary morphological variations occur in the morphology at the tail-bud stage, the arrangement and development of the external gills, adhesive gland type and division timing, growth of the dorsal hatching gland on the head, configuration of the oral disc, emergence and development of the hind limbs, and presence of the abdominal sucker. Some of these transformations are best explained by phylogeny (e.g., early divergent taxa of bufonids have embryos with kyphotic body curvature, Type C adhesive glands, and a very small third pair of gills). Other traits might be correlated with reproductive modes (e.g., phytotelmata embryos hatch comparatively late and show an accelerated development of hind limbs). Because these actual variations are not well studied (e.g., less than the 10% of the known diversity of bufonids has been studied from this perspective), comprehensive analyses are required to interpret character evolution and the relationship with reproductive modes within the family.

Natural interspecific hybridization in Odontophrynus (Anura: Cycloramphidae)

The frog genus Odontophrynus is a composite of diploid and tetraploid populations and species that are widely distributed in South America. Some of the several genetic studies on this group report the production of artificial hybrids but only a single case of natural hybridization has been documented, in southern Brazil. In this study we report the finding of an interspecific natural hybrid specimen in central Argentina. We present morphological and cytogenetical evidence that the diploid taxa Odontophrynus cordobae and O. occidentalis are the parental species. The hybrid genome exhibited problematic pairing and segregation of homeologue chromosomes during meiosis, and the production of non-reduced gametes.