Integration of Ecology, Larval Phenotypes, and Mate-Recognition Signals with Molecular and Morphological Data Indicate Taxonomic Inflation in Nyctibatrachus (Anura: Nyctibatrachidae)
Taxonomic studies over the past decade of the endemic Night Frog genus Nyctibatrachus (originally described in 1882) from Peninsular India have more than tripled, from 11 at the turn of this century to 36 by 2017. Despite these revisionary contributions, it is still challenging for field biologists to identify night frog species reliably, due to a near-complete absence of diagnostic, discrete character states or trait values. Worse, many questionably diagnosed night frog species' status has ostensibly been “supported” by phylogenies derived from sparsely sampled gene-trees that are based on a single locus or a handful of markers—with topology and arbitrary genetic distance thresholds of 3–6% used to support new species descriptions. We sought to re-evaluate and validate the species boundaries of six currently nominated species of Nyctibatrachus of the aliciae group (N. aliciae, N. periyar, N. deveni, N. pillaii), N. vasanthi, and N. poocha clade using a comprehensive integrative taxonomic approach that integrates classical taxonomy, molecular species delimitation analysis, statistical analysis of morphological characters of adults and larvae, analyses of bioacoustics, and natural history information. Our results indicate that recent descriptions of Nyctibatrachus deveni, N. periyar, and N. pillaii represent cases of taxonomic inflation (over-splitting), because the evidence cited in support of their recognition is irreproducible, subjective, and devoid of strong statistical support. We demonstrate the need for multidimensional species delimitation approaches in the celebrated Western Ghats biodiversity hotspot paleo-endemic genus Nyctibatrachus and suspect that this concerning trend of over-splitting amphibian species based on limited data and untenable support may be applicable to other amphibian groups.
(A) Map of the southern Western Ghats of India showing estimated distributional range and collection and observation localities of species of Nyctibatrachus belonging to: (left) the aliciae group (circles) and (right) N. poocha (squares) and N. vasanthi (star). Red = type locality for which genetic samples are available on GenBank; blue = collection locality of specimens and tissues used in this study. Collection localities from current and previous studies: a. Mahendragiri, b. Shendurney, c. Uppukunnu, d. and e. Kadalar, 1. Ponmudi, 2. Sengaltheri, 3. Vallakadavu, 4. Nelliyampathy, 5. Valparai, 6. Kakachi. Samples downloaded from GenBank have respective accession numbers provided. (B) Maximum likelihood phylogeny of the aliciae group, N. poocha, and N. vasanthi derived from 2172 bp comprising two mitochondrial (16S and ND1) and one nuclear gene (TYR); newly sequenced individuals from intermediate populations used in this study marked in bold. (C) Species delimitation using mPTP analysis, based on a 16S rRNA ML phylogeny, for which support values at nodes indicate the fraction of sampled delimitations in which a node was part of the speciation process. The analysis strongly supported the discovery step delimitation of Sp. 1 (= N. poocha), Sp. 2 (= N. vasanthi), and Sp. 3 (= N. aliciae + N. periyar + N. deveni + N. pillaii) as distinct species.
Live adult males of (A) Nyctibatrachus poocha, (B) N. vasanthi, and (C) N. aliciae from Ponmudi (type locality), (D) N. pillaii from Kakachi (type locality), (E) N. cf. aliciae from Konni, (F) N. periyar from Gavi (photograph inverted for comparison), (G) N. deveni from Kadalar, and (H) N. deveni from Nelliyampathy (type locality). Dorsolateral skin texture respectively of (I) N. aliciae, (J) N. poocha, and (K) N. vasanthi.
Variability in foot webbing in the aliciae group: (A) webbing attachment on the right foot (left) reaches up to the third subarticular tubercle on either side of toe IV of the right foot, while webbing attachments touch the intercalary tubercle on either side of toe IV of the left foot; (B) complete webbing, where webbing attachments touch the intercalary tubercle on either side of toe IV of the left foot; and (C) medium webbing, where webbing attachment from toe V reaches up to the intercalary tubercle below the toe disc of toe IV and the webbing attachment from toe IV to toe III begins at the third subarticular tubercle of toe IV of the left foot. (A) Male N. periyar from Uppukunnu; (B, C) two individual N. aliciae males from the same population in Ponmudi.
Tadpoles in Stage 27 of (A) Nyctibatrachus aliciae (from type locality; TNHM T1), (B) N. vasanthi (from Pandipath, Kerala; TNHM T4), and (C) N. poocha (from Valparai, Tamil Nadu; TNHM T3). Scale bar = 10 mm. Oral disc structure of (D) N. aliciae, (E) N. vasanthi, and (F) N. poocha, for which there is little variation among the three species.
Advanced metamorphic stages of Nyctibatrachus aliciae. (A) Stage 42, (B) Stage 43, (C) Stage 46.
PCA plots of morphological variables for adults of the aliciae group alone (left) and aliciae group, N. poocha, and N. vasanthi clade (center) and tadpoles (right) in 2D (top) and 3D (bottom). The principal components are visualized as hypervolumes constructed using kernel density estimation. Geometry of hypervolumes corresponds to a minimum convex hull (polytopes) that minimally encloses the data. Axes show the first three principal components and their proportion of variance. Note: The hypervolumes for N. deveni and N. periyar align completely, suggesting minimal morphological divergence.
Comparative uncorrected p-distances at the 16S rRNA gene for different taxonomic ranks in the family Nyctibatrachidae. For each particular rank comparison, the peak in each histogram represents the number of samples included, and the breadth represents range of genetic distances.
Breeding behavior of Nyctibatrachus aliciae. (A) Male calling with female beside him; (B) male mounting female in loose amplexus; (C) male withdraws backwards off the female while she lays eggs simultaneously; (D) male sits on freshly laid clutch and rotates over it; (E) freshly laid egg clutch with exposed jelly layer, attached upside down to boulder; (F) egg clutch with embryos at Stage 10; and (G) clutch of Stage 19 embryos. Egg clutch protection in Nyctibatrachus poocha: (H) male guarding clutch on a mossy rock overlooking a forest stream (arrow = egg clutch covered with rotting vegetative debris); (I) vocalizing male Nyctibatrachus vasanthi.
Comparative spectrograms and corresponding oscillograms of advertisement calls of male Nyctibatrachus of the aliciae group, N. poocha, and N. vasanthi clade. Top: (A) call Type 1b of N. aliciae from Ponmudi; call Type 1a of (B) N. aliciae from Ponmudi; (C) N. periyar from Vallakadavu; (D) N. cf. deveni of Kadalar; and (E) N. deveni from Nelliyampathy. Bottom: advertisement call of (F) N. poocha and (G) N. vasanthi.
Contributor Notes
Associate Editor: B. L. Stuart.