Integrative taxonomy reveals cryptic Amazonian species of Pristimantis (Anura: Strabomantidae)

Hypotheses on the taxonomic status of two Bolivian Pristimantis with taxonomic problems are assessed by an integrative taxonomic approach that integrates three independent lines of evidence: external morphology, prezygotic reproductive barriers (advertisement calls) and reciprocal monophyly (phylogenetic analyses of partial 16S mtDNA sequences). Central Andean Bolivian populations previously assigned to either P. peruvianus or P. dundeei, and lowland Amazonian populations from southern Peru and northern Bolivia previously considered P. peruvianus do not correspond to these species. Indeed, multivariate analyses of qualitative and quantitative morphological and bioacoustic characters, and phylogenetic analyses support the hypothesis that they represent different, previously unknown, cryptic lineages. They are herein described as new species. The former is a sibling species of P. fenestratus that inhabits the Amazonian and semideciduous forests of the Andean foothills in central Bolivia. The latter is sibling to the Andean species P. danae and is parapatric to it in the Amazonian lowland forests and adjacent foothills of northern Bolivia, southern Peru and adjacent Brazil. Most species of Neotropical frogs, and especially Pristimantis, have been described by using external qualitative morphological characters only. An extended integrative taxonomic approach, as exemplified herein, may lead to the discovery of many other cryptic and sibling lineages that would increase the species numbers of tropical areas. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 97–122.     

Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species complex (Anura,Hylidae) with the description of four new species

We review the systematics of the Hypsiboas calcaratus species complex, a group of widely distributed Amazonian hylid frogs. A comprehensive analysis of genetic, morphological, and bioacoustic datasets uncovered the existence of eleven candidate species, six of which are confirmed. Two of them correspond to Hypsiboas fasciatus and Hypsiboas calcaratus and the remaining four are new species that we describe here. Hypsiboas fasciatus sensu stricto has a geographic range restricted to the eastern Andean foothills of southern Ecuador while Hypsiboas calcaratus sensu stricto has a wide distribution in the Amazon basin. Hypsiboas almendarizae sp. n. occurs at elevations between 500 and 1950 m in central and northern Ecuador; the other new species (H. maculateralis sp. n., H. alfaroi sp. n., and H. tetete sp. n.) occur at elevations below 500 m in Amazonian Ecuador and Peru. The new species differ from H. calcaratus and H. fasciatus in morphology, advertisement calls, and mitochondrial and nuclear DNA sequences. Five candidate species from the Guianan region, Peru, and Bolivia are left as unconfirmed. Examination of the type material of Hyla steinbachi, from Bolivia, shows that it is not conspecific with H. fasciatus and thus is removed from its synonymy.     

Assessing the taxonomic status of tropical frogs through bioacoustics: geographical variation in the advertisement calls in the Eleutherodactylus discoidalis species group (Anura)

The taxonomic status of a very poorly known group of Andean frogs (the Eleutherodactylus discoidalis group) is assessed through acoustic and statistical analyses of differences in temporal parameters of advertisement calls, such as the number of pulses and the call duration, and also in a spectral parameter, dominant frequency. As these species are usually misidentified or ignored because of their taxonomic complexity in both ecological and biodiversity studies, we provide a bioacoustical diagnosis for each species in order to facilitate identification in the field. Differences in acoustic parameters support the specific status of Eleutherodactylus cruralis , E. discoidalis , Eleutherodactylus ibischi , and Eleutherodactylus madidi . The name E. cruralis is probably applied to three different species: the nominal form from Amazonian forests of the Andean slopes and adjacent lowlands, and two cryptic species restricted to inter-Andean dry valleys and cloud forests from central Bolivia. Moreover, the distribution of energy through the call and the aggregation of pulses seem to be useful meristic characters for detecting interspecific differences. Populations from each macrohabitat can be recognized by distinctive advertisement calls, usually corresponding to a recognized species. For the whole group, pulse rate is significantly correlated to latitude, which could indicate a speciation process along the Andes in relation to habitat changes and isolation. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society , 2008, 152 , 353–365.     

Male advertisement call characters as phylogeographical indicators in European water frogs

Anuran vocalizations are valuable in the determination of species identity and have the potential to discriminate intraspecific variation. We developed novel bioacoustic sampling techniques, based on Fast Fourier Transforms, to increase the precision and sensitivity of male advertisement call analysis and applied the method to European water frogs. This approach robustly separated the three types of north European water frogs (Rana ridibunda, R. lessonae and their viable, fertile hybrid R. esculenta) by their call subunit characteristics. The hybrid frog exhibited a high frequency call component absent from both the parental species. Furthermore, call analysis demonstrated significant intraspecific differences among populations of all three frog types. Call characters of R. ridibunda changed systematically as a function of longitude. This trend may reflect either clinal variation in selection pressures across Europe, or the consequences of drift following postglacial colonization from eastern refugia. High resolution vocalization analysis therefore provides a potentially useful method for investigating intraspecific differentiation and the phylogeographical origins of anuran distributions. © 2002 The Linnean Society of London. Biological Journal of the Linnean Society, 2002, 77 , 355?365.     

An integrative approach to reveal speciation and species richness in the genus Diasporus (Amphibia: Anura: Eleutherodactylidae) in eastern Panama

We have applied an integrative taxonomic approach, including bioacoustics, ecology, morphology, and molecular genetics (barcoding and phylogeography), to explore species richness in the genus Diasporus in eastern Panama, from where only Diasporus quidditus (Lynch, 2001) was previously known. During fieldwork in eastern Panama in 2011 and 2012 we found six additional species, four of which we are describing here as new to science, plus two species that are new for this region. We have evaluated the presence of Diasporus diastema (Cope, 1875) in eastern Panama by comparing morphological, genetic, and bioacoustic characters of specimens from near the type locality in central Panama with specimens from eastern Panama. We further describe and compare male advertisement calls of most Diasporus species. The phylogeographic analysis suggests the allopatric speciation of Diasporus species in eastern Panama following the completion of the Panamanian isthmus in the middle Miocene. Subsequent geological events concur with the vicariant evolution of different lineages in situ, suggesting eastern Panama to be a centre of endemism for this group of frogs. We present an integrative analysis of the species from eastern Panama and include an identification key for all species of the genus.     

Patterns of diversification in the high Andean Ponderacris grasshoppers (Orthoptera: Acrididae: Melanoplinae)

The Andes, the world's longest mountain chain, harbours great taxonomic and ecological diversity. Despite their young age, the tropical Andes are highly diverse due to recent geological uplift. Speciation either followed the orogeny closely or occurred after the Andean uplift, as a result of subsequent climatic changes. Different scenarios have been proposed to explain the diversification of high Andean taxa. The Melanoplinae grasshopper Ponderacris Ronderos & Cigliano is endemic to the eastern slopes of the Andes of Peru and Bolivia, mostly distributed between 1000 and 4000 m above sea level. Diversification in several montane habitats of Bolivia and Peru allows tests via cladistic analysis of distinct possible geographic modes of speciation. Eight species are recognized, with three described here as new with revised diagnostic morphological characters provided: Ponderacris carlcarbonelli sp.n., P. chulumaniensis sp.n. and P. amboroensis sp.n. Cladistic analyses of 15 species (8 ingroup and 7 outgroup) and 38 morphological characters, under equal and implied weighting, confirm the monophyly of Ponderacris. Characters from the external morphology and colour pattern provided less phylogenetic information than did the male abdominal terminalia and phallic complex. Species distributed in the Peruvian Andes constituted a monophyletic group, whereas those from the Bolivian Andes formed a basal paraphyletic grade. Dispersal–vicariance analysis resulted in one ancestral distribution reconstruction indicating that the most recent common ancestor was distributed in the Lower Montane Yungas of Bolivia. Eleven dispersal and one vicariant events are postulated, with a South‐to‐North speciation pattern coincident with progressive Andean uplift. Vicariance could relate to fragmentation of montane forest during the dry intervals of the late Cenozoic. From the Bolivian area, ancestral Peruvian Ponderacris may have dispersed northward, coinciding with the rise of the Andes. Ten of 11 dispersal events occurred at terminal taxa and are likely to be recent. However, diversification of Ponderacris cannot be explained solely by the South‐to‐North speciation hypothesis, but may also include both vicariance and dispersal across barriers influenced by Pleistocene climatic cycles.     

Molecular phylogeny and systematics of Neotropical toucanets in the genus Aulacorhynchus (Aves,Ramphastidae)

Bonaccorso, E., Guayasamin, J. M., Peterson, A. T. & Navarro‐Sigüenza, A. G. (2011). Molecular phylogeny and systematics of Neotropical toucanets in the genus Aulacorhynchus (Aves, Ramphastidae). —Zoologica Scripta, 40, 336–349. We studied the phylogenetic relationships in the genus Aulacorhynchus, an assemblage of Neotropical toucanets distributed from Mexico south to Bolivia. Based on mitochondrial and nuclear DNA characters, we obtained a robust hypothesis of relationships for all recognized species, including good representation of distinct geographic populations. Our results support the monophyly of the genus Aulacorhynchus, but contradict previous taxonomic arrangements. The genus is made up of three major clades: the Aulacorhynchus prasinus complex, Aulacorhynchus huallagae + Aulacorhynchus coeruleicinctis, and Aulacorhynchus haematopygus + Aulacorhynchus sulcatus + Aulacorhynchus derbianus. Andean populations of A. derbianus are more closely related to A. sulcatus than to Pantepuian populations of A. derbianus, rendering A. derbianus paraphyletic. Based on the molecular phylogeny, and information on geographic distributions and morphological and behavioural characters, we review the specific status of these taxa and propose a new taxonomic arrangement within Aulacorhynchus.     

Morphological and molecular analysis of centropagids from the high Andean plateau (Copepoda: Calanoidea)

In small metazoan invertebrates classical taxonomic analyses can be ambiguous due to the limited number of morphological characters available. This difficulty can yield incorrect estimates of species richness or taxa distribution. The Boeckella genus has been described as the dominant taxon of zooplankton assemblages in the Andean biogeographical region. In this genus, taxonomic classification and delimitation of boundaries between species has long been problematic and controversial. Among South American centropagids Boeckella gracilipes has been regarded as one of the most broadly distributed species, its presence having been reported from Ecuador to Tierra del Fuego. However, in the high Andean plateau some centropagid populations identified as Boeckella gracilipes have also been considered as B. gracilipes titicacae or even identified as a different species, namely Boeckella titicacae. In an attempt to resolve the taxonomic status of the Centropagidae family from the high Andean plateau, we combined traditional and multivariate morphological analyses (integral approach) with the molecular phylogenetic approach. The results obtained allow us to conclude that centropagids collected from the high Andean plateau actually represent a different species, B. titicacae, not B. gracilipes. The phylogenetic reconstruction of the South American Centropagidae family indicated that B. gracilipes represents a sister taxon to B. titicacae. The present study stresses the usefulness of integrating alfa-taxonomy with morphometric and molecular approaches in order to resolve species boundaries, to determine geographical distributions and to investigate evolutionary processes.     

Taxonomic survey of water frog populations of <Emphasis Type="Italic">Pelophylax bedriagae</Emphasis> (Anura: Ranidae) in western Iran: a morphometric and bioacoustic approach

We explored the population systems of Pelophylax bedriagae (Camerano, 1882) in northwestern, western and southwestern Iran. Statistical significance for a hypothesis of sexual dimorphism of the frogs was tested using t-test and multivariate analysis of variances (MANOVA). Due to lack of sexual differences between the male and female frogs, the data of both sexes were pooled for further analysis. Taxonomic relationships among local populations were investigated using discriminate analysis (by stepwise method) with iterative sample assignment. The multivariate morphometric analyses revealed that the range of P. bedriagae extends to western Iran. The next step was to analyze the advertisement call (five standard parameters: call duration, number of pulse groups per call, duration of pulse groups, interval between pulse groups, and intercall interval) of the water frogs inhabiting three sites in western Iran. Rigorous application of the bioacoustic method removes all doubt that these three sites are inhabited by the same species, which on the basis of priority of nomenclature should be called P. bedriagae.     

Neglected native or undesirable alien? Resolution of a conservation dilemma concerning the pool frog Rana lessonae

Introduced species often pose serious threats to biodiversity, but occasionally confusion arises as to whether a species really is introduced or is in fact an overlooked native. A recent UK conservation dilemma has centred on the status of the pool frog Rana lessonae. This species has been the subject of documented introductions from central and southern Europe since the early 1800s, the accepted position being that all UK R. lessonae populations are descended from these introductions. However, a closer examination of early UK literature sources, and recent discoveries of isolated, native R. lessonae populations in Sweden and Norway, led some herpetologists to question whether the species was in fact present as a native at some locations prior to the introductions. Research was initiated along four major lines of enquiry: genetic, bioacoustic, archaeozoological and archival. A high degree of convergence among the genetic and bioacoustic investigations demonstrated that the potentially native UK pool frogs were closely related to Scandinavian frogs, thus ruling out introductions from further south as a potential origin. Subfossil evidence of pool frogs was found from ca. 1000 years before present, demonstrating that the species occurred in the UK prior to known introductions. Archival sources produced no historical support for introductions from northern Europe. The postglacial history inferred for these northern populations is consistent with the known climatic and geographical conditions. Taken together, the evidence for the native status of the pool frog is compelling, and furthermore the UK population appears to be part of a distinct northern clade.     

Bioacoustic and genetic divergence in a frog with a wide geographical distribution

The study of intraspecific variation of acoustic signals and its relationship with genetic divergence is important for understanding the origin of divergence in communication systems. We studied geographical variation in the acoustic structure of advertisement calls from five populations of the four‐eyed frog, Pleurodema thaul, and its relationship with the genetic divergence among these populations. By analyzing temporal and spectral parameters of the advertisement calls, we report that the signals of northern, central, and southern populations have remarkable differences between them. A phylogeographical analysis from a mitochondrial DNA fragment demonstrated three phylogenetic groups coincident with those found with the bioacoustics analysis. Furthermore, bioacoustic and genetic distances show significant correlations after controlling for geographical distance. These results suggest that behavioural divergence among populations of P. thaul has a phylogenetic basis, supporting three evolutionary units within this species, as well as prompting the exploration of divergence processes in the sound communication system of this species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 142–155.     

The water frogs of Greece: Bioacoustic evidence for a new species

The structure of the mating call of lake frogs (referred to as R. ridibunda) from 16 populations in Greece was analyzed for local variation using multivariate statistics. The populations of Thrace and of the island of Samothraki form a group giving the same type of mating call, whereas the mating call of the other populations differs in the degree of temperature dependence of four parameters, and specifically in the number of pulses/pulse group and pulse groups/call. Discriminant functions distinguish even single call series with a probability of 97%, intermediate mating calls are absent, and there is a significant, but slight differentiation of external morphological characters. These results have strong taxonomic implications. We conclude that the lake frogs of Greece comprise two species. The mating call of the lake frogs from Thrace resembles in all parameters that of the Rana ridibunda in the terra typica restricta (Guryev, CIS). Accordingly, the lake frogs of eastern Greece belong to R. ridibunda. The mating call of these lake frogs consists of 20 pulses/pulse group and of 7 pulse groups/call on the average. Most of Greece is inhabited by the second taxon, Rana balcanica sp. n. Its mating call is characterized by 27 pulses/pulse group and 4 pulse groups/call on the average. The two species in Greece do not differ with respect to coloration and size, but several standardized indices vary significantly: body length/digitus primus length; body length/callus internus length; body length/snout-eye distance; body length/tympanum diameter; tibia length/callus internus length; maximal head width/snout-eye distance.     

Population status of Andean Condors in central and southern Bolivia

Andean Condors (Vultur gryphus) are a Near Threatened species that was formerly distributed along the entire length of the Andes from western Venezuela to Tierra del Fuego. Populations have been severely reduced north of Peru, but several thousand Andean Condors still exist in the southern portion of their range in Argentina and Chile. Little is known, however, about the size of the Andean Condor population in the central part of their range in Peru and Bolivia. From June to September 2012, we used feeding stations to attract Andean Condors and estimate the size and structure of the population in the eastern Andes of central and southern Bolivia. We estimated a minimum population of 253 condors, an adult male‐to‐female ratio of 1:0.6, an immature male‐to‐female ratio of 1:0.9, and an adult‐to‐immature ratio of 1:1.1. At our five survey areas, estimated abundance ranged from 15 to 100 condors per area. Males outnumbered females in three areas and the opposite was true in two areas. Our estimated adult‐to‐immature ratio, overall and in each area, suggests that the populations could be reproducing at a high rate. As previously observed in other Andean Condor populations, skewed sex ratios could be associated with differences between sexes and age classes in habitat selection. Although our results suggest that Bolivian populations of Andean Condors are still reasonably large, population monitoring is urgently needed, including use of feeding stations throughout the entire Bolivian range of the species and intensive searches for roosting and nesting sites.     

ADVERTISEMENT CALLS OF THE TREE FROGS,HYLA ARBOREA AND HYLA SAVIGNYI (ANURA: HYLIDAE) IN TURKEY

ABSTRACT

The systematic classification of tree frogs (Hyla) inhabiting different regions of the country of Turkey is unclear. Recordings of natural advertisement calls of individual male tree frogs in different locations in Turkey were analyzed to determine variation in acoustic features that may be related to taxonomic status. Multivariate analysis of covariance showed that call duration, intercali interval and number of pulses per call varied significantly between frogs in different locales. Call duration, intercall interval, and number of pulses per call were related to air temperature. Dominant frequency differed significantly between different groups of frogs, and was temperature-independent. These data are consistent with the hypothesis that tree frogs in Turkey represent two distinct species, Hyla arborea and Hyla savignyi.     

The interplay of colour and bioacoustic traits in the differentiation of a Southeast Asian songbird complex

Morphological traits have served generations of biologists as a taxonomic indicator, and have been the main basis for defining and classifying species diversity for centuries. A quantitative integration of behavioural characters, such as vocalizations, in studies on biotic differentiation has arisen more recently, and the relative importance of these different traits in the diversification process remains poorly understood. To provide a framework within which to interpret the evolutionary interplay between morphological and behavioural traits, we generated a draft genome of a cryptic Southeast Asian songbird, the limestone wren‐babbler Napothera crispifrons. We resequenced whole genomes of multiple individuals of all three traditional subspecies and of a distinct leucistic population. We demonstrate strong genomic and mitochondrial divergence among all three taxa, pointing to the existence of three species‐level lineages. Despite its great phenotypic distinctness, the leucistic population was characterized by shallow genomic differentiation from its neighbour, with only a few localized regions emerging as highly diverged. Quantitative bioacoustic analysis across multiple traits revealed deep differences especially between the two taxa characterized by limited plumage differentiation. Our study demonstrates that differentiation in these furtive songbirds has resulted in a complex mosaic of colour‐based and bioacoustic differences among populations. Extreme colour differences can be anchored in few genomic loci and may therefore arise and subside rapidly.     

Allozyme variation among Rana balcanica,R. levantina,and R. ridibunda (Amphibia: Anura)1

The allozyme variation among water frogs of the species R. balcanica, R. levantina and R. ridibunda, all formerly considered as one species (R. ridibunda Pallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frog R. esculenta collected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River where R. ridibunda and R. balcanica occur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data: R. ridibunda and R. balcanica (Nei's genetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereas R. levantina (distance to the European species D = 0.1780 - 0.1955) together with R. perezi represent an independent afroasian lineage.     

Taxonomy as a hypothesis: testing the status of the Bermuda buckeye butterfly Junonia coenia bergi (Lepidoptera: Nymphalidae)

Species determination and definition in eukaryotes have traditionally been based on morphology, with little focus on genetic differentiation. Molecular methods allow for the independent assessment of morphology‐based taxonomic hypotheses. Three criteria used to define a full species for taxonomic purposes are morphological distinction, formation of a monophyletic lineage, and reproductive isolation. Junonia butterflies (Nymphalidae) are becoming an important experimental model system, but the taxonomy of many New World Junonia species is unclear. One of these species is J. coenia, which contains the subspecies J. coenia coenia, J. coenia grisea and J. coenia bergi. Previous studies suggest that J. coenia grisea may meet the criteria for full species status. Therefore, we evaluated the geographically isolated and rarely studied Bermuda buckeye butterfly J. coenia bergi to determine if it was similarly distinct. Physical examination of specimens and phylogenetic and population genetic analyses of mitochondrial cytochrome c oxidase subunit I, nuclear wingless, and complete mitochondrial genome sequences suggest that while J. coenia bergi is smaller in body size than many Junonia and has distinctive ventral hindwing colouration, it does not form a monophyletic lineage and shows indications of continued gene flow with North American mainland J. coenia coenia populations. Thus, J. coenia bergi does not meet the criteria for full species designation, but geographic isolation, morphological distinctiveness, and cultural importance suggest that it remain recognized as a subspecies of J. coenia. Similar analyses will be useful for addressing further taxonomic questions in Junonia and other taxa, especially where morphology‐based taxonomic determinations are ambiguous.