Néstor G. Basso

A phylogenetic analysis of Pleurodema (Anura: Leptodactylidae: Leiuperinae) based on mitochondrial and nuclear gene sequences, with comments on the evolution of anuran foam nests

Species of the genus Pleurodema are relatively small, plump frogs that mostly occur in strong‐seasonal and dry environments. The genus currently comprises 14 species distributed from Panama to southern Patagonia. Here we present a phylogenetic analysis of Pleurodema, including all described species and several outgroups. Our goals include testing its monophyly and the monophyly of the species groups that were historically proposed, and studying the evolution of some character systems, particularly macroglands and egg‐clutch structure; this last point also provided the chance for a discussion of foam nest evolution in anurans. Our dataset includes portions of the mitochondrial genes cytochromeb, 12S, 16S, and the intervening tRNAVal; the nuclear gene sequences include portions of rhodopsin exon 1 and seven in absentia homolog I. Our results support a clade composed of Pleurodema and including the monotypic Somuncuria Lynch, 1978 nested within it. The latter genus is therefore considered a junior synonym of Pleurodema and its sole species is added to this genus. Furthermore, our results indicate the non‐monophyly of several species groups proposed previously. We recognize four clades in Pleurodema: the P. bibroni clade (P. bibroni, P. cordobae and P. kriegi), the P. thaul clade (P. bufoninum, P. marmoratum, P. somuncurensis and P. thaul), the P. brachyops clade (P. alium, P. borellii, P. brachyops, P. cinereum, P. diplolister and P. tucumanum) and the P. nebulosum clade (P. guayapae and P. nebulosum). Our results further indicate the need for a taxonomic reassessment of P. borellii and P. cinereum (as did previous studies), P. guayapae and P. nebulosum, and the three species in the P. bibroni clade. Pleurodema shows a striking pattern of variation in presence/absence of lumbar glands. Our results indicate multiple losses or independent gains of this character associated with defensive displays. The reproductive modes of Pleurodema include four different egg‐clutch structures. The optimization of these indicates that there are at least two independent transformations from the plesiomorphic mode of foam nests to egg‐clutch structures involving gelatinous masses of different sorts (ovoid plates, masses, or strings). We hypothesize that these independent transformations could involve changes at the behavioural (the loss of foam beating behaviour by the parent) and/or structural level (transformations involving the pars convoluta dilata, the section of the oviduct where the foam‐making substance is secreted). Finally, our study of foam nest evolution in Pleurodema is extended to the other groups of anurans where foam‐nesting occurs, on the basis of available data and recent phylogenetic hypotheses. In the different hyloid groups where it occurs, foam‐nesting evolved from clutches laid in water. However, in all ranoids in which foam‐nesting occurs, it evolved from terrestrial clutches, with eggs laid hanging in vegetation, or, if the clutches are laid on a restricted volume of water, involving endotrophic development.

Phylogenetic relationships of a Patagonian frog radiation, the Alsodes + Eupsophus clade (Anura: Alsodidae), with comments on the supposed paraphyly of Eupsophus

The frog clade composed of the alsodid genera Alsodes + Eupsophus is the most species‐rich of the Patagonian endemic frog clades, including nearly 31 of the slightly more than 50 species of that region. The biology of this group of frogs is poorly known, its taxonomy quite complex (particularly Alsodes), and its diversity in chromosome number striking when compared with other frogs (collectively, there are species having 2n = 22, 2n = 26, 2n = 28, 2n = 30 or 2n = 34). We present a phylogenetic analysis of this Patagonian frog clade based on mitochondrial and nuclear gene sequences. We sequenced five mitochondrial genes (cytochrome b, cytochrome oxidase I, 12S, 16S, NADH dehydrogenase subunit 1) with three intervening tRNAs, and fragments of three nuclear genes (seven in absentia homolog 1, rhodopsin exon 1, RAG‐1), for a maximum of 6510 bp for multiple specimens from 26 of the 31 species. We recovered Eupsophus as polyphyletic, with E. antartandicus, E. sylvaticus, and E. taeniatus in Batrachylidae, in accordance with most previous hypotheses. Based on this result, we transfer E. antartandicus and E. taeniatus back to Batrachyla, and E. sylvaticus to Hylorina (resurrected from the synonymy of Eupsophus), remediating the paraphyly of Eupsophus. Our results strongly corroborate the monophyly of Alsodes + Eupsophus (sensu stricto), the individual monophyly of these genera, and the monophyly of the species groups of Eupsophus. They also show the non‐monophyly of all non‐monotypic species groups of Alsodes proposed in the past. Our results expose several taxonomic problems particularly in Alsodes, and to a lesser extent in Eupsophus. This phylogenetic context suggests a rich evolutionary history of karyotypic diversification in the clade, in part corroborating previous hypotheses. In Alsodes, we predict three independent transformations of chromosome number from the plesiomorphic 2n = 26. All these, strikingly, involve increments or reductions of pairs of haploid chromosomes. Finally, the phylogenetic pattern recovered for Alsodes and Eupsophus suggests a trans‐Andean origin and diversification of the group, with multiple, independent ingressions over cis‐Andean regions.

Distinctness, phylogenetic relations and biogeography of intertidal mussels ( Brachidontes , Mytilidae) from the south-western Atlantic

Rocky shore intertidal communities along the cold- and warm-temperate coasts of the south-western Atlantic are dominated by small mussels of the genus Brachidontes s.l . (Mytilidae), yet the status of species occurring in the region remains unresolved. Taxonomic studies have been based on shell morphology, but high phenotypic variability has led to much confusion. Based on mitochondrial and nuclear genes (COI, 28S rDNA and ITS1) from nine localities in Uruguay and Argentina we confirmed the occurrence of three species in the south-western Atlantic: Brachidontes darwinianus and B. rodriguezii in the warm-temperate and B. purpuratus in the cold-temperate sector. The latter two species coexist in the same beds along the transition zone (41–43°S). The phylogeny based on mitochondrial and nuclear genes, indicate an early divergence of B. purpuratus . At the intra-specific level, low genetic differentiation and absence of fossil record for B. purpuratus from the earlier Quaternary marine terraces of Patagonia likely result from a relatively recent (post-LGM) colonization originated from populations in the south-eastern Pacific. In the case of B. rodriguezii , by contrast, genetic intraspecific differentiation, a fossil record of phenotypically-related forms going back to the Late Miocene, and phylogenetic position in the COI-based phylogeny, prompts the hypothesis that this species is derived from a local stock with a long history of occurrence in the warm-temperate region of the south-western Atlantic. While intertidal mussel beds from the south-western Atlantic are ecologically similar in appearance, their assembly involves components clearly differentiated in terms of historical biogeography and phylogeny.

A REVISION OF ODONTOPHRYNUS BARRIOI (ANURA: NEOBATRACHIA): MORPHOLOGY, OSTEOLOGY, VOCALIZATIONS, AND GEOGRAPHIC DISTRIBUTION

Abstract The present paper provides diagnostic characters from external morphology and osteology that reinforce the specific recognition of the frog Odontophrynus barrioi, describes its advertisement and encounter calls, and extends its distribution range. Odontophrynus barrioi is distinguished from all other species of the genus principally by the large size of adults, great number of irregularly arranged postorbital and temporal glands, lack of both a light vertebral line and keratinous spines, alary process of the premaxilla short and wide, pterygoid process of the maxilla well developed, and high preorbital process of the maxilla. The advertisement call consists of a trill of a single repeated and pulsed note with a low dominant frequency. The encounter call consists of only one note of similar frequency as the advertisement call, but longer in time duration. We report new records of O. barrioi from Catamarca, La Rioja, and San Juan Provinces (Argentina), that increase considerably the range of this previously poorly known species.

Scorched mussels (Brachidontes spp., Bivalvia: Mytilidae) from the tropical and warm-temperate southwestern Atlantic: the role of the Amazon River in their speciation.

Abstract Antitropicality is a distribution pattern where closely related taxa are separated by an intertropical latitudinal gap. Two potential examples include Brachidontes darwinianus (south eastern Brazil to Uruguay), considered by some authors as a synonym of B. exustus (Gulf of Mexico and the Caribbean), and B. solisianus, distributed along the Brazilian coast with dubious records north of the intertropical zone. Using two nuclear (18S and 28S rDNA) and one mitochondrial gene (mtDNA COI), we aimed to elucidate the phylogeographic and phylogenetic relationships among the scorched mussels present in the warm‐temperate region of the southwest Atlantic. We evaluated a divergence process mediated by the tropical zone over alternative phylogeographic hypotheses. Brachidontes solisianus was closely related to B. exustus I, a species with which it exhibits an antitropical distribution. Their divergence time was approximately 2.6 Ma, consistent with the intensification of Amazon River flow. Brachidontes darwinianus, an estuarine species is shown here not to be related to this B. exustus complex. We suspect ancestral forms may have dispersed from the Caribbean to the Atlantic coast via the Trans‐Amazonian seaway (Miocene). The third species, B rodriguezii is presumed to have a long history in the region with related fossil forms going back to the Miocene. Although scorched mussels are very similar in appearance, their evolutionary histories are very different, involving major historical contingencies as the formation of the Amazon River, the Panama Isthmus, and the last marine transgression.

Thaulin-1: The first antimicrobial peptide isolated from the skin of a Patagonian frog Pleurodema thaul (Anura: Leptodactylidae: Leiuperinae) with activity against Escherichia coli

Patagonias biodiversity has been explored from many points of view, however, skin secretions of native amphibians have not been evaluated for antimicrobial peptide research until now. In this sense, Pleurodema thaul is the first amphibian specie to be studied from this large region of South America. Analysis of cDNA-encoding peptide in skin samples allowed identification of four new antimicrobial peptides. The predicted mature peptides were synthesized and all of them showed weak or null antimicrobial activity against Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli with the exception of thaulin-1, a cationic 26-residue linear, amphipathic, Gly- and Leu-rich peptide with moderate antimicrobial activity against E. coli (MIC of 24.7μM). AFM and SPR studies suggested a preferential interaction between these peptides and bacterial membranes. Cytotoxicity assays showed that thaulin peptides had minimal effects at MIC concentrations towards human and animal cells. These are the first peptides described for amphibians of the Pleurodema genus. These findings highlight the potential of the Patagonian regions unexplored biodiversity as a source for new molecule discovery.

Old and new hypotheses about the homology of the compound bones from the cheek and otico-occipital regions of the anuran skull

We studied the larval development of compound bones from the otico-occipital and cheek regions in species of the neobatrachian genera Batrachyla, Hylorina, Leptodactylus, Odontophrynus and Pleurodema. Comparisons were made using a set of Ambystoma spp. (Caudata) and Ceratophrys ornata (Anura; Ceratophryidae) larvae. As suggested by previous studies, we verified the compound nature of the exoccipital (two centers, anurans only), frontoparietal (one center, most anurans and Ambystoma; three centers, some anurans), and squamosal (two centers, all anurans and Ambystoma) bones. We discuss old and new homology hypotheses for each of the compound bone centers in the context of the most widely accepted scenario of lissamphibian origins and relationships, i.e., monophyletic Lissamphibia that includes the clade Batrachia (Caudata+Anura) and the most divergent Gymnophiona. Our findings have a direct impact on our understanding of the composition of the skull in Lissamphibia. We recognized the presence of the following bones: (i) opisthotic (fused to the exoccipital) and tabular (fused to the squamosal) in Batrachia (Anura+Caudata) and (ii) supratemporal (fused to the parietal portion of the frontoparietal) in Anura. Separate centers of the parietal were found only in Pleurodema.

Population genetic analyses of the Powerplex(®) Fusion kit in a cosmopolitan sample of Chubut Province (Patagonia Argentina).

Allele frequencies and forensic parameters for 22 autosomal STR loci and DYS391 locus included in the PowerPlex(®) Fusion System kit were estimated in a sample of 770 unrelated individuals from Chubut Province, southern Patagonia. No significant deviations from Hardy-Weinberg equilibrium were observed after Bonferronis correction. The combined power of discrimination and the combined probability of exclusion were >0.999999 and 0.999984, respectively. Comparisons with other worldwide populations were performed. The MDS obtained show a close biological relation between Chubut and Chile. The estimated interethnic admixture supports a high Native American contribution (46%) in the population sample of Chubut. These results enlarge the Argentine databases of autosomal STR and would provide a valuable contribution for identification tests and population genetic studies.

Using Morphometry and Molecular Markers for Sexing South American, Cayenne and Royal Terns Breeding in Patagonia, Argentina

Abstract. The first information on the morphometry and sexual size dimorphism of the sympatric South American (Sterna hirundinacea), Cayenne (Thalasseus sandvicensis eurygnathus) and Royal (T. maximus maximus) terns from the Patagonia region in Argentina is provided, and a discriminant analysis to sex the three species is used. Morphological characters were obtained from South American Terns (n = 83), Cayenne Terns (n = 63) and Royal Terns (n = 20). All species were sexed using polymerase chain reaction-based molecular techniques. Sexes in the three tern species were only slightly dimorphic in size. Male Cayenne and South American terns were significantly larger than females in bill length, bill depth and head length. Royal Terns, in contrast, showed a high overlap in most morphological measurements, with head length being the only measurement that differed significantly between sexes. Head length correctly sexed 89% of South American Terns and 75% of Royal Terns, while a function including bill depth and head length correctly sexed 78% of Cayenne Terns. Our results provide a valuable tool for rapid sexing in the field of these three Patagonian terns, although reliable sexing in Cayenne and Royal terns should be preferentially achieved using a combination of morphometric and molecular sexing.

Population genetic analysis of 23 Y-STR loci in Central Argentine Patagonia

We analyzed 23 Y-STR haplotypes of 139 unrelated males from Central Argentine Patagonia. A total of 133 different haplotypes (127 singletons) were observed. Haplotype diversity was similar to that previously observed in other Argentine populations and matching probability showed a strong dependence on the sample size. AMOVA carried out with full haplotypes showed significant differences between different regions of the country. The multi-dimensional scaling plot showed Chubut sample in an intermediate position among Europe and other Patagonian populations. These results will contribute to increase the Y-chromosome haplotype reference database and constitute a useful tool for anthropological and forensic researches.