Santiago Castroviejo-Fisher

Deciphering the products of evolution at the species level: the need for an integrative taxonomy

Progress in molecular techniques together with the incorporation of phylogenetic analyses of DNA into taxonomy have caused an increase in the number of species’ discoveries in groups with morphological characters that are difficult to study or in those containing polytypic species. But some emerged criticisms plead for a taxonomic conservatism grounded either on the requirement of providing evidences of morphological distinctiveness or reproductive barriers to erect new species names. In a case study of taxonomic research on Neotropical frogs, we combine several lines of evidence (morphological characters, prezygotic reproductive isolation and phylogenetic analyses of mitochondrial DNA) to test the status of 15 nominal species and to assess the degree of agreement of the different lines of evidence. Our study reveals that morphology alone is not sufficient to uncover all species, as there is no other single line of evidence independently. Full congruence between lines of evidence is restricted to only four out of the 15 species. Five species show congruence of two lines of evidence, whereas the remaining six are supported by only one. The use of divergence in morphological characters seems to be the most conservative approach to delineate species boundaries because it does not allow the identification of some sibling reciprocally monophyletic species differing in their advertisement calls. The separate analysis of differences in advertisement calls (evidence of reproductive isolation) or of phylogenetic data alone also shows limitations, because they do not support some morphological species. Our study shows that only an integrative approach combining all sources of evidence provides the necessary feedback to evaluate the taxonomic status of existing species and to detect putative new ones. Furthermore, the application of integrative taxonomy enables the identification of hypotheses about the existence of species that will probably be rejected or changed, and those that can be expected to persist.

Phylogenetic relationships of glassfrogs (Centrolenidae) based on mitochondrial and nuclear genes

Glassfrogs (family Centrolenidae) represent an exceptionally diverse group among Neotropical anurans, but their evolutionary relationships never have been assessed from a molecular perspective. Mitochondrial and nuclear markers were used to develop a novel hypothesis of centrolenid phylogeny. Ingroup sampling included 100 terminals, with 78 (53%) of the named species in the family, representing most of the phenotypic diversity described for the group. Thirty-five species representing taxa traditionally associated with glassfrogs were used as outgroups. Gene sampling consisted of complete or partial sequences of three mitochondrial (12S, 16S, ND1) and three nuclear markers (c-myc exon 2, RAG1, POMC) for a total of approximately 4362bp. Phylogenies were estimated using maximum parsimony, maximum likelihood, and Bayesian analyses for individual genes and combined datasets. The separate analysis of mitochondrial and nuclear datasets allowed us to clarify the relationships within glassfrogs; also, we corroborate the sister-group relationship between Allophryne ruthveni and glassfrogs. The new phylogeny differs significantly from all previous morphology-based hypotheses of relationships, and shows that hypotheses based on few traits are likely to misrepresent evolutionary history. Traits previously hypothesized as unambiguous synapomorphies are shown to be homoplastic, and all genera in the current taxonomy (Centrolene, Cochranella, Hyalinobatrachium, Nymphargus) are found to be poly- or paraphyletic. The new topology implies a South American origin of glassfrogs and reveals allopatric speciation as the most important speciation mechanism. The phylogeny profoundly affects the traditional interpretations of glassfrog taxonomy, character evolution, and biogeography-topics that now require more extensive evaluation in future studies.

From Amazonia to the Atlantic forest: molecular phylogeny of Phyzelaphryninae frogs reveals unexpected diversity and a striking biogeographic pattern emphasizing conservation challenges.

Documenting the Neotropical amphibian diversity has become a major challenge facing the threat of global climate change and the pace of environmental alteration. Recent molecular phylogenetic studies have revealed that the actual number of species in South American tropical forests is largely underestimated, but also that many lineages are millions of years old. The genera Phyzelaphryne (1 sp.) and Adelophryne (6 spp.), which compose the subfamily Phyzelaphryninae, include poorly documented, secretive, and minute frogs with an unusual distribution pattern that encompasses the biotic disjunction between Amazonia and the Atlantic forest. We generated >5.8 kb sequence data from six markers for all seven nominal species of the subfamily as well as for newly discovered populations in order to (1) test the monophyly of Phyzelaphryninae, Adelophryne and Phyzelaphryne, (2) estimate species diversity within the subfamily, and (3) investigate their historical biogeography and diversification. Phylogenetic reconstruction confirmed the monophyly of each group and revealed deep subdivisions within Adelophryne and Phyzelaphryne, with three major clades in Adelophryne located in northern Amazonia, northern Atlantic forest and southern Atlantic forest. Our results suggest that the actual number of species in Phyzelaphryninae is, at least, twice the currently recognized species diversity, with almost every geographically isolated population representing an anciently divergent candidate species. Such results highlight the challenges for conservation, especially in the northern Atlantic forest where it is still degraded at a fast pace. Molecular dating revealed that Phyzelaphryninae originated in Amazonia and dispersed during early Miocene to the Atlantic forest. The two Atlantic forest clades of Adelophryne started to diversify some 7 Ma minimum, while the northern Amazonian Adelophryne diversified much earlier, some 13 Ma minimum. This striking biogeographic pattern coincides with major events that have shaped the face of the South American continent, as we know it today.

Systematics of spiny-backed treefrogs (Hylidae: Osteocephalus): An Amazonian puzzle

Spiny‐backed tree frogs of the genus Osteocephalus are conspicuous components of the tropical wet forests of the Amazon and the Guiana Shield. Here, we revise the phylogenetic relationships of Osteocephalus and its sister group Tepuihyla, using up to 6134 bp of DNA sequences of nine mitochondrial and one nuclear gene for 338 specimens from eight countries and 218 localities, representing 89% of the 28 currently recognized nominal species. Our phylogenetic analyses reveal (i) the paraphyly of Osteocephalus with respect to Tepuihyla, (ii) the placement of ‘Hyla’ warreni as sister to Tepuihyla, (iii) the non‐monophyly of several currently recognized species within Osteocephalus and (iv) the presence of low (<1%) and overlapping genetic distances among phenotypically well‐characterized nominal species (e.g. O. taurinus and O. oophagus) for the 16S gene fragment used in amphibian DNA barcoding. We propose a new taxonomy, securing the monophyly of Osteocephalus and Tepuihyla by rearranging and redefining the content of both genera and also erect a new genus for the sister group of Osteocephalus. The colouration of newly metamorphosed individuals is proposed as a morphological synapomorphy for Osteocephalus. We recognize and define five monophyletic species groups within Osteocephalus, synonymize three species of Osteocephalus (O. germani, O. phasmatus and O. vilmae) and three species of Tepuihyla (T. celsae, T. galani and T. talbergae) and reallocate three species (Hyla helenae to Osteocephalus, O. exophthalmus to Tepuihyla and O. pearsoni to Dryaderces gen. n.). Furthermore, we flag nine putative new species (an increase to 138% of the current diversity). We conclude that species numbers are largely underestimated, with most hidden diversity centred on widespread and polymorphic nominal species. The evolutionary origin of breeding strategies within Osteocephalus is discussed in the light of this new phylogenetic hypothesis, and a novel type of amplexus (gular amplexus) is described.

Correlates of species richness in the largest Neotropical amphibian radiation

Although tropical environments are often considered biodiversity hotspots, it is precisely in such environments where least is known about the factors that drive species richness. Here, we use phylogenetic comparative analyses to study correlates of species richness for the largest Neotropical amphibian radiation: New World direct‐developing frogs. Clade‐age and species richness were nonsignficantly, negatively correlated, suggesting that clade age alone does not explain among‐clade variation in species richness. A combination of ecological and morphological traits explained 65% of the variance in species richness. A more vascularized ventral skin, the ability to colonize high‐altitude ranges, encompassing a large variety of vegetation types, correlated significantly with species richness, whereas larger body size was marginally correlated with species richness. Hence, whereas high‐altitude ranges play a role in shaping clade diversity in the Neotropics, intrinsic factors, such as skin structures and possibly body size, might ultimately determine which clades are more speciose than others.

Vanishing native American dog lineages

BackgroundDogs were an important element in many native American cultures at the time Europeans arrived. Although previous ancient DNA studies revealed the existence of unique native American mitochondrial sequences, these have not been found in modern dogs, mainly purebred, studied so far.ResultsWe identified many previously undescribed mitochondrial control region sequences in 400 dogs from rural and isolated areas as well as street dogs from across the Americas. However, sequences of native American origin proved to be exceedingly rare, and we estimate that the native population contributed only a minor fraction of the gene pool that constitutes the modern population.ConclusionsThe high number of previously unidentified haplotypes in our sample suggests that a lot of unsampled genetic variation exists in non-breed dogs. Our results also suggest that the arrival of European colonists to the Americas may have led to an extensive replacement of the native American dog population by the dogs of the invaders.

A Revision of Species Diversity in the Neotropical Genus Oreobates (Anura: Strabomantidae), with the Description of Three New Species from the Amazonian Slopes of the Andes

ABSTRACT We revisit species diversity within Oreobates (Anura: Strabomantidae) by combining molecular phylogenetic analyses of the 16S rRNA amphibian barcode fragment with the study of the external morphology of living and preserved specimens. Molecular and morphological evidence support the existence of 23 species within Oreobates, and three additional candidate species (Oreobates sp. [Ca JF809995], Oreobates sp. [Ca EU368903], Oreobates cruralis [Ca EU192295]). We describe and name three new species from the Andean humid montane forests of Departamento Cusco, southern Peru: O. amarakaeri New Species from Río Nusinuscato and Río Mabe, at elevations ranging from 670 to 1000 m in the Andean foothills; O. machiguenga, new species, from Río Kimbiri (1350 m), a small tributary of the Apurimac River, in the western versant of Cordillera Vilcabamba; and O. gemcare, new species, from the Kosñipata Valley at elevations ranging from 2400 to 2800 m. The three new species are readily distinguished from all other Oreobates by at least one qualitative morphological character. Three species are transferred to Oreobates from three genera of Strabomantidae: Hypodactylus lundbergi Pristimantis crepitans, and Phrynopus ayacucho (for which the advertisement call, coloration in life, and male characteristics are described for first time). Oreobates simmonsi is transferred to the genus Lynchius. Hylodes verrucosus is considered a junior synonym of Hylodes philippi. In addition, H. philippi is removed from the synonymy of O. quixensis and considered a nomem dubium within Hypodactylus. The inclusion of Phrynopus ayacucho in Oreobates extends the ecological range of the genus to the cold Andean puna. Oreobates is thus distributed from the Amazonian lowlands in southern Colombia to northern Argentina, reaching the Brazilian Atlantic dry forests in eastern Brazil, across an altitudinal range from ca. 100 to 3850 m.

SYSTEMATICS OF THE ELEUTHERODACTYLUS FRAUDATOR SPECIES GROUP (ANURA: BRACHYCEPHALIDAE)

ABSTRACT Members of the Eleutherodactylus fraudator species group occur in Bolivia and Peru. This group has not been reviewed since its proposal and description. Its putative monophyly has not been tested and its phylogenetic relationships are still unresolved. It was included in the Central American genus Craugastor based on a putative morphological synapomorphy, but this hypothesis of relationship is doubtful. Its alpha-diversity is unknown and some putative members have still not been studied in detail. To assess the taxonomic status and phylogenetic relationships of this species group, we applied a taxonomic integrative approach based on comparisons of morphological characters (external and internal), bioacoustics (based on advertisement calls) and phylogenetic analyses of partial 16S mitochondrial gene (560 bp). We redescribe Eleutherodactylus bisignatus (Werner, 1899) and include it together with E. mercedesae in the group. Molecular, bioacoustical and morphological characters support the taxonomic status of all the species. Maximum parsimony, Bayesian and neighbor-joining phylogenetic analyses of the partial mitochondrial gene as well as the presence of a new condition of the mandibular ramus of the trigeminal nerve and two bioacoustical characters, support the monophyly of this group. Moreover, our data suggest the nonmonophyly of the subgenus Eleutherodactylus and the monophyly of the genus Craugastor when the C. bufoniformis species group is excluded. We redescribe the crown clade formed by members of the E. fraudator group and name it as the subgenus Yunganastes, which contains Eleutherodactylus ashkapara Köhler, 2000, E. bisignatus (Werner, 1899), E. fraudator Lynch and McDiarmid, 1987, E. mercedesae Lynch and McDiarmid, 1987 and E. pluvicanorus De la Riva and Lynch, 1997. We describe the advertisement call of E. bisignatus for the first time and compare it with available calls of E. ashkapara and E. pluvicanorus. We suggest the common structure of the advertisement calls and the aggressive calls of the species belonging to Yunganastes (single melodic whistles with frequency modulation and no pulses) as synapomorphic for the new subgenus. We exclude members of Yunganastes from the Middle American genus Craugastor, to which it was previously tied by the presence of the putative synapomorphic “E” condition of the trigeminal nerve of the mandibular ramus of the abductor muscle. We describe a new condition of the trigeminal nerve and consider it synapomorphic for Yunganastes. Members of this subgenus are endemic to the cloud forests and humid montane forests of the Cordillera Oriental of the Andes in Bolivia and southern Peru.

Molecular phylogenetics, species diversity, and biogeography of the Andean lizards of the genus Proctoporus (Squamata: Gymnophthalmidae).

The family Gymnophthalmidae comprises ca. 220 described species of Neotropical lizards distributed from southern Mexico to Argentina. It includes 36 genera, among them Proctoporus, which contains six currently recognized species occurring across the yungas forests and wet montane grasslands of the Amazonian versant of the Andes from central Peru to central Bolivia. Here, we investigate the phylogenetic relationships and species limits of Proctoporus and closely related taxa by analyzing 2121 base pairs of mitochondrial (12S, 16S, and ND4) and nuclear (c-mos) genes. Our taxon sampling of 92 terminals includes all currently recognized species of Proctoporus and 15 additional species representing the most closely related groups to the genus. Maximum parsimony, maximum likelihood and Bayesian phylogenetic analyses recovered a congruent, fully resolved, and strongly supported hypothesis of relationships that challenges previous phylogenetic hypotheses and classifications, and biogeographic scenarios. Our main results are: (i) discovery of a strongly supported clade that includes all species of Proctoporus and within which are nested the monotypic Opipeuter xestus (a genus that we consider a junior synonym of Proctoporus), and two species of Euspondylus, that are therefore transferred to Proctoporus; (ii) the paraphyly of Proctoporus bolivianus with respect to P. subsolanus, which is proposed as a junior synonym of P. bolivianus; (iii) the detection of seven divergent and reciprocally monophyletic lineages (five of them previously assigned to P. bolivianus) that are considered confirmed candidate species, which implies that more candidate species are awaiting formal description and naming than currently recognized species in the genus; (iv) rejection of the hypothesis that Proctoporus diversified following a south to north pattern parallel to the elevation of the Andes; (v) species diversity in Proctoporus is the result of in situ diversification through vicariance in the grasslands of the high Andes, with at least five dispersals contributing to montane forest species.

The phylogenetic relationships of Yunganastes revisited (Anura: Terrarana)

1. IntroductionIn this study, we reanalyze the phylogenetic relationships of thesubgenusYunganastes,agroupofCentralAndeandirect-developingfrogs with five described species (Padial et al., 2007). The phyloge-netic relationships of Yunganastes remain contentious. Members ofthis taxon were for a long time considered Eleutherodactylus (Lynchand McDiarmid, 1987; De la Riva and Lynch, 1997; Kohler, 2000).Subsequently, they were included in the Middle American subge-nus Craugastor, despite their disjunct distributions, because ashared condition of the trigeminal nerve (De la Riva and Lynch,1997). Kohler (2000) formally grouped four of the five species ofYunganastes in the Eleutherodactylus fraudator species group, stillwithin the subgenus Craugastor. Crawford and Smith (2005) usedmoleculardatatoanalyzethephylogeneticrelationshipsandbioge-ography of the subgenus Craugastor and raised it to the genus level,but these authors did not include members of the E. fraudator spe-cies group in their data set. Frost et al. (2006) compared the condi-tionofthetrigeminalnerveinamemberoftheE.fraudatorgroup(E.pluvicanorus) and found differences with Craugastor. They removedtheE.fraudatorgroupfromCraugastorandplaceditwithinthenon-monophyleticsubgenusEleutherodactylus.Padialetal.(2007)madea comprehensive taxonomic revision of the group, included onemore species (E. mercedesae), and found that the condition de-scribed by Frost et al. (2006) was shared by all members of thegroup but not by Craugastor or Eleutherodactylus. Additionally, Pa-dial et al. (2007) presented phylogenetic analyses of a short frag-ment (560 bp) of the 16S mtDNA that further supported themonophyly of the group, placing it outside the Central AmericanCraugastor and more closely related to other ‘‘Eleutherodactylus”from South America. They formally described the group as the sub-genus Yunganastes within the non-monophyletic ‘‘Eleutherodacty-lus”. More recently, Hedges et al. (2008) presented a taxonomicreviewofTerrarana(anamethatreferstoacladethatencompassescirca 900 species of New World direct-developing frogs, includingYunganastes) based on three molecular trees with different taxon(80, 216 and 362 species) and character sampling (1207, 2578and 3709 bp). They placed most South American ‘‘Eleutherodacty-lus”, including members of Yunganastes, in the genus Pristimantis(427 spp.). They also analyzed a short ( 500 bp) sequence of a rep-resentative species of Yunganastes (Pristimantis pluvicanorus) andfoundthatitwasbasaltoPristimantis,althoughsupportlevelswerelow and the resulting topologywas not shown. Hedges et al. (2008)tentatively placed Yunganastes within the large genus Pristimantis,but recommended to analyze additional sequence data to assessthe relationships of this taxon.Here, we study the evolutionary relationships of Yunganasteswithin Terrarana based on Bayesian and maximum likelihood phy-logenetic analyses (BA and ML, respectively). We analyze a matrixof 1300 base pairs of the 16S and 12S mtDNA genes for a total of375 terminals. Ingroup sampling includes sequences from all fivespecies of Yunganastes, plus eight species of Oreobates, seven of