An integrative approach to infer systematic relationships and define species groups in the shrub frog genus Raorchestes, with description of five new species from the Western Ghats, India

The genus Raorchestes is a large radiation of Old World tree frogs for which the Western Ghats in Peninsular India is the major center for origin and diversification. Extensive studies on this group during the past two decades have resolved long-standing taxonomic confusions and uncovered several new species, resulting in a four-fold increase in the number of known Raorchestes frogs from this region. Our ongoing research has revealed another five new species in the genus, formally described as Raorchestes drutaahu sp. nov., Raorchestes kakkayamensis sp. nov., Raorchestes keirasabinae sp. nov., Raorchestes sanjappai sp. nov., and Raorchestes vellikkannan sp. nov., all from the State of Kerala in southern Western Ghats. Based on new collections, we also provide insights on the taxonomic identity of three previously known taxa. Furthermore, since attempts for an up-to-date comprehensive study of this taxonomically challenging genus using multiple integrative taxonomic approaches have been lacking, here we review the systematic affinities of all known Raorchestes species and define 16 species groups based on evidence from multi-gene (2,327 bp) phylogenetic analyses, several morphological characters (including eye colouration and pattern), and acoustic parameters (temporal and spectral properties, as well as calling height). The results of our study present novel insights to facilitate a better working taxonomy for this rather speciose and morphologically conserved radiation of shrub frogs. This will further enable proper field identification, provide momentum for multi-disciplinary studies, as well as assist conservation of one of the most colourful and acoustically diverse frog groups of the Western Ghats biodiversity hotspot

Seven new species of Night Frogs (Anura, Nyctibatrachidae) from the Western Ghats Biodiversity Hotspot of India, with remarkably high diversity of diminutive forms

The Night Frog genus Nyctibatrachus (Family Nyctibatrachidae) represents an endemic anuran lineage of the Western Ghats Biodiversity Hotspot, India. Until now, it included 28 recognised species, of which more than half were described recently over the last five years. Our amphibian explorations have further revealed the presence of undescribed species of Nights Frogs in the southern Western Ghats. Based on integrated molecular, morphological and bioacoustic evidence, seven new species are formally described here as Nyctibatrachus athirappillyensis sp. nov., Nyctibatrachus manalari sp. nov., Nyctibatrachus pulivijayani sp. nov., Nyctibatrachus radcliffei sp. nov., Nyctibatrachus robinmoorei sp. nov., Nyctibatrachus sabarimalai sp. nov. and Nyctibatrachus webilla sp. nov., thereby bringing the total number of valid Nyctibatrachus species to 35 and increasing the former diversity estimates by a quarter. Detailed morphological descriptions, comparisons with other members of the genus, natural history notes, and genetic relationships inferred from phylogenetic analyses of a mitochondrial dataset are presented for all the new species. Additionally, characteristics of male advertisement calls are described for four new and three previously known species. Among the new species, six are currently known to be geographically restricted to low and mid elevation regions south of Palghat gap in the states of Kerala and Tamil Nadu, and one is probably endemic to high-elevation mountain streams slightly northward of the gap in Tamil Nadu. Interestingly, four new species are also among the smallest known Indian frogs. Hence, our discovery of several new species, particularly of easily overlooked miniaturized forms, reiterates that the known amphibian diversity of the Western Ghats of India still remains underestimated

The vocal repertoire of <i>Pseudophilautus kani</i>, a shrub frog (Anura: Rhacophoridae) from the Western Ghats of India

<div><p>Taxonomic descriptions of new anuran species are published with high frequency in the recent systematics literature. In contrast, there are fewer detailed quantitative descriptions of the vocalizations of these new species. Here, we describe the vocalizations of a recently described shrub frog, <i>Pseudophilautus kani</i> (Anura: Rhacophoridae; Biju and Bossuyt 2009) endemic to the Western Ghats region of India. We recorded two distinct, pulsatile call types that could be distinguished by their temporal and spectral properties as well as their mode of production. Type 1 calls (<i>creek</i>) were short ( ≈ 85 ms), consisted of about six or seven pulses (92 pulses/s) and had a spectrum with a single frequency peak at about 3.5 kHz. By comparison, Type 2 calls (<i>ta-ta-ta-ta</i>) were typically longer ( ≈ 320 ms), contained fewer pulses (4–5) produced at much lower rates (12 pulses/s) and had broadband spectra with multiple frequency peaks, two of which were consistently present at about 2.3 and 4.6 kHz. Video analyses indicated that males produced pulses in Type 1 calls using a single, continuous contraction of the trunk musculature, whereas pulses of Type 2 calls were produced with separate muscle contractions. In both call types, spectral properties were significantly negatively related with body length and body mass. The pulse rate and pulse period of Type 2 calls were also related to body size. No call properties were correlated with body condition. Patterns of individual variation in call properties were generally consistent with those described for other anurans.</p> </div

Vocal Behavior of the Elusive Purple Frog of India (<i>Nasikabatrachus sahyadrensis</i>), a Fossorial Species Endemic to the Western Ghats

<div><p>Quantitative descriptions of animal vocalizations can inform an understanding of their evolutionary functions, the mechanisms for their production and perception, and their potential utility in taxonomy, population monitoring, and conservation. The goal of this study was to provide the first acoustical and statistical analysis of the advertisement calls of <i>Nasikabatrachus sahyadrensis</i>. Commonly known as the Indian purple frog, <i>N. sahyadrensis</i> is an endangered species endemic to the Western Ghats of India. As the only known species in its family (Nasikabatrachidae), it has ancient evolutionary ties to frogs restricted to the Seychelles archipelago (Sooglossidae). The role of vocalizations in the behavior of this unique species poses interesting questions, as the animal is fossorial and potentially earless and it breeds explosively above the soil for only about two weeks a year. In this study, we quantified 19 acoustic properties of 208 calls recorded from 10 males. Vocalizations were organized into distinct call groups typically composed of two to six short (59 ms), pulsatile calls, each consisting of about five to seven pulses produced at a rate of about 106 pulses/s. The frequency content of the call consisted of a single dominant peak between 1200–1300 Hz and there was no frequency modulation. The patterns of variation within and among individuals were typical of those seen in other frogs. Few of the properties we measured were related to temperature, body size, or condition, though there was little variation in temperature. Field observations and recordings of captive individuals indicated that males engaged in both antiphonal calling and call overlap with nearby calling neighbors. We discuss our findings in relation to previous work on vocal behavior in other fossorial frogs and in sooglossid frogs.</p></div

A unique mating strategy without physical contact during fertilization in Bombay Night Frogs (Nyctibatrachus humayuni) with the description of a new form of amplexus and female call

Anurans show the highest diversity in reproductive modes of all vertebrate taxa, with a variety of associated breeding behaviours. One striking feature of anuran reproduction is amplexus. During this process, in which the male clasps the female, both individuals’ cloacae are juxtaposed to ensure successful external fertilization. Several types of amplexus have evolved with the diversification of anurans, and secondary loss of amplexus has been reported in a few distantly related taxa. Within Nyctibatrachus, a genus endemic to the Western Ghats of India, normal axillary amplexus, a complete loss of amplexus, and intermediate forms of amplexus have all been suggested to occur, but many species remain unstudied. Here, we describe the reproductive behaviour of N. humayuni, including a new type of amplexus. The dorsal straddle, here defined as a loose form of contact in which the male sits on the dorsum of the female prior to oviposition but without clasping her, is previously unreported for anurans. When compared to known amplexus types, it most closely resembles the form of amplexus observed in Mantellinae. Furthermore, we prove that, opposed to the situation in most anurans, male semen release happens before egg deposition. We hypothesize that the male ejaculates on the female’s dorsum and that sperm subsequently runs from her back and hind legs before fertilizing the eggs. A second feature characterizing anuran breeding is the advertisement call, mostly produced solely by males. Despite recent descriptions of several new Nyctibatrachus species, few studies have explored their vocal repertoire. We describe both the male advertisement call and a female call for N. humayuni. The presence of a female call has not been reported within Nyctibatrachidae, and has been reported in less than 0.5% of anuran species. Altogether, our results highlight a striking diversity and several unique aspects of Nyctibatrachus breeding behaviour

Descriptive statistics for call groups, calls, and pulses, including means (<i>X¯</i>), standard deviations (SDs), ranges, and coefficients of variation.

†<p>For calls per call group and pulses per call, the values reported in the columns headed <i>X¯</i> and SD, respectively, are the median and interquartile range. Note, however, that coefficients of variation for these two properties are computed from the <i>X¯</i> and SD.</p

Results of Spearman rank correlations between acoustic properties and temperature, body size, and condition (<i>N</i> = 10).

†<p>Scatterplots depicting correlations with <i>P</i>-values below the conventional (uncorrected) α level of 0.05 are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084809#pone.0084809.s004" target="_blank">Figure S1</a>.</p

Advertisement calls of a male <i>Nasikabatrachus sahyadrensis</i>.

<p>(a) 30-s segment of continuous, spontaneous calling by a single male. (b) 5-s segment showing the three consecutive call groups underlined in (a). (c) 0.5-s segment showing the three calls of the call group underlined in (b). (d) 0.1 s segment showing the call underlined in (c). (e) Spectrogram of the call illustrated in (d); <i>Inset</i>: power spectrum averaged over the duration of the call depicted in (d). The entire 30-s segment depicted in this figure is included as an audio file in the Supporting Information for this article (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084809#pone.0084809.s001" target="_blank">Audio S1</a>).</p