Contributions to Zoology, 83 (4) – 2014S.D. Biju; Sonali Garg; Stephen Mahony; Nayana Wijayathilaka; Gayani Senevirathne; Madhava Meegaskumbura: DNA barcoding, phylogeny and systematics of Golden-backed frogs (Hylarana, Ranidae) of the Western Ghats-Sri Lanka biodiversity hotspot, with the description of seven new species

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DNA barcoding

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Neighbor Joining (NJ) trees for 16S (Fig. 2), COI and Cytb (not shown) were created for species delineation and to help elucidate species geographic ranges. The three molecular markers produced identical taxa groupings and similar tree topologies.

Here, we discuss our results with respect to only the 16S NJ tree (Fig. 2) since the topology was in agreement with our deeper phylogenetic analyses, and because analysis based on this gene showed a more conservative approach to species delineation. Within the in-group taxa, there were 15 major clades corresponding to four valid Hylarana species: H. aurantiaca (clade 1), H. temporalis (clade 12), H. gracilis (clade 13) and H. malabarica (clade 14); three named species currently considered as synonyms: H. intermedius (clade 2), H. flavescens (clade 9) and H. montanus (clade 10); and between seven (clades 3-8 and 11) to eight (clades 3-8, 11 and 14a) unnamed species. We herein describe these as — Hylarana doni sp. nov., Hylarana urbis sp. nov., Hylarana sreeni sp. nov., Hylarana indica sp. nov., Hylarana caesari sp. nov., Hylarana magna sp. nov. and Hylarana serendipi sp. nov., respectively (Appendix) and clade 14a is referred to as Hylarana malabarica haplogroup 1. For further discussion see ‘Morphological recognition of species’.

Genetic distance comparisons (Tables S2, S3) showed maximum intraspecific uncorrected pairwise distance of 1.5% (for 16S) and 3.6% (for COI) for Hylarana sreeni (clade 5). For Cytb, maximum intraspecific distance of 6.2% was observed for H. indica (clade 6) and 6.1% for H. sreeni. With respect to closely related species, the minimum interspecific uncorrected pairwise divergence was 16S: 2.0% between H. indica and H. sreeni, COI: 4.3% between H. sreeni and H. indica, Cytb: 8.0% between H. intermedius and H. doni. An analysis of the barcode gap showed no overlap between the maximum intraspecific pairwise distance within species and the minimum interspecific divergence between species (Fig. 3). The observed mean threshold values of sequence divergence for species delineation were: 2.6% for 16S, 5.2% for COI and 8.7% for Cytb. A high level of genetic variation was observed between H. malabarica typical and H. malabarica haplogroup 1 (clades 14a-b) (Fig. 2). Though there was considerable sequence divergence with an average of 3.7% for 16S, the COI and Cytb sequence divergences (3.2% and 6.2% respectively) were lower than the threshold values observed for other species pairs. Maximum divergence in our dataset was observed within the Hylarana temporalis group of Sri Lanka, with mean values of 10.5% (16S), 18.1% (COI) and 23.8% (Cytb) between the species H. serendipi and H. temporalis.

Overall, not only were our results concordant for all the three molecular markers but also in agreement with earlier studies that have proposed 3% and 4-8% as threshold values for 16S and COI, respectively (Fouquet et al., 2007a; Smith et al., 2008). Only two of the recognised species pairs showed slightly lower mean sequence divergence values for 16S (2.7% between Hylarana intermedius-H. doni, and 2.5% between H. indica-H. sreeni). However, morphologically these represented well-supported distinct lineages. For Cytb, the overall threshold value was slightly lower than the values shown by certain other studies (Kotaki et al., 2010; Hasan et al., 2012). However, there are not many comparable studies available for amphibian barcoding based on Cytb, and since species delineation based on Cytb sequences was in agreement with the other two molecular markers in the present study, an 8.7% threshold value for Cytb seems realistic. For detailed genetic comparisons, see the ‘Genetic divergence’ section of each species in the Appendix.

Our barcoding results, in the light of taxonomic interpretations, showed remarkable patterns of species level distribution. All of the Western Ghats and Sri Lankan taxa formed distinct clades. The Western Ghats groups showed north-south distribution trends among different species, while the Sri Lankan species showed a dry zone-wet zone (again essentially a north-south) distribution trend (Fig. 1). Hylarana malabarica (India) and H. gracilis (Sri Lanka), both with wider distributions, formed the basal clades to all of the remaining Western Ghats-Sri Lankan clades (Fig. 2).


Fig. 2. Neighbour Joining tree of Kimura-2-parameter (K2P) distances based on mtDNA 16S for 102 Hylarana samples from the Western Ghats-Sri Lanka biodiversity hotspot. Collection numbers are presented in Table S1.


Fig. 3. Comparison of mean, minimum and maximum uncorrected pairwise intraspecific values for 13 species, and interspecific values for 23 species pairs. Mean threshold values for species delineation are represented for three mitochondrial markers: 16S showing 2.6%, COI showing 5.2% and Cytb showing 8.7%. For intra and interspecific comparisons, see Tables S2 and S3.

Morphological recognition of species

A detailed comparison of morphological data along with molecular evidence provides greater confidence in the identification of 14 taxonomic haplotypes (Figs 2, 4), and facilitates a comprehensive taxonomic revision of Hylarana from the Western Ghats-Sri Lanka biodiversity hotspot. Morphological examination of available types, museum specimens and our recent collections, indicate seven new species in the genus Hylarana, all of which are endemic to the Western Ghats-Sri Lanka biodiversity hotspot. Furthermore, the study of new collections of three ‘lost’ species: Rana (Hylarana) gracilis montanus Rao, 1922, Rana flavescens Jerdon, 1853 and Rana (Hylorana) intermedius Rao, 1937 from their type localities ‘Bhagamandla’, ‘Malabar’ and ‘Sakleshpur’, respectively, conclude that these are valid species. Rana (Hylorana) bhagmandlensis Rao, 1922 is considered as junior subjective synonym of Hylarana montanus, based on detailed comparison of type specimens and original descriptions. Detailed taxonomic accounts and the descriptions of the new species are provided in the Appendix, and illustrated in Figs 5-25.

Our data is insufficient to provide a conclusive taxonomic resolution to the status of Hylarana malabarica haplogroup 1 from the northern Western Ghats, which on the basis of the molecular data appeared to represent a lineage divergent from the typical form, from the southern Western Ghats. The available specimen collection of both the typical and haplogroup forms was, however, inadequate from the entire range of this species to enable detailed morphological comparisons. Our preliminary results indicate that H. malabarica typical and H. malabarica haplogroup 1 are very similar morphologically and therefore we recognise that further work is required to assess the taxonomic status of these populations.

Phylogenetic analysis

The results of our phylogenetic analysis allow us to investigate evolutionary relationships among all nominal species within the Western Ghats-Sri Lanka biodiversity hotspot, and with the Southeast Asian taxa by extending the geographical range of our dataset.

The Maximum Likelihood tree (Fig. 4) depicts four major clades, which are highly resolved, with various associated distribution patterns within the Western Ghats-Sri Lanka biodiversity hotspot: the Hylarana aurantiaca and Hylarana flavescens groups are restricted to the Western Ghats; the Hylarana temporalis group is found only in Sri Lanka; the Hylarana malabarica group has a wide distribution in India and Sri Lanka, along with Hylarana cf. leptoglossa from Northeast India (Fig. 4).


Fig. 4. Maximum Likelihood tree for 46 taxa, including 30 selected haplotypes from the Western Ghats-Sri Lanka biodiversity hotspot, five Hylarana species from Northeast and Central India, nine Hylarana species from Southeast Asia, and two outgroup taxa. Numbers above and below the nodes indicate Bayesian Posterior Probabilities and Maximum Parsimony Boostrap values >50, respectively. Squares indicate the species distribution — black squares represent species found south of Palghat Gap, grey squares represent species found north of Palghat Gap.

The other Southeast Asian taxa, including Hylarana nigrovittata — type species of the genus Sylvirana (Dubois, 1992), were found to be basal to the above-mentioned Western Ghats and Sri Lankan groups. However these associations were not strongly supported, probably due to insufficient taxon sampling to resolve these relationships. The clade containing Hylarana erythraea (type species of the genus Hylarana) formed the most basal split with strong support. Though some relationships among the Southeast Asian taxa remained unresolved due to poor data sampling from outside the study area, most branches in our trees were well supported and the overall topology was in agreement with earlier studies (Che et al., 2007; Pyron and Wiens, 2011).

Seen in the context of Che et al. (2007) and Pyron and Wiens (2011) our results suggest that most of the Western Ghats-Sri Lankan congeners form an endemic radiation (Fig. 4). This includes the Hylarana aurantiaca and H. flavescens groups from India, and the H. temporalis group from Sri Lanka. The H. malabarica group which has one species each from India and Sri Lanka, forms a well-supported clade with South East Asian H. cf. leptoglossa. Given that previously recognized generic level taxa (Sylvirana and Hydrophylax) included in our study form sister clades at the base of the phylogeny, further taxonomic focus is needed to ascertain if the endemic Western Ghats-Sri Lankan radiation warrants a higher level taxonomic status.

Three (out of four) Northeast Indian taxa nested with Southeast Asian species rather than the species from the Western Ghats, except Hylarana cf. leptoglossa, which was closely related to the Hylarana malabarica group (Fig. 4). This group also contained the single sample from Central India (SDBDU 2011.596), which nested with the Hylarana malabarica haplogroup 1. The identity of this sub-adult specimen could not be confirmed morphologically. Our results suggest that among the Western Ghats-Sri Lankan groups, the Hylarana malabarica group is more closely related to the Southeast Asian taxa. This is also evident from the distribution of species in the subgenus Hydrophylax (Dubois, 1992), whose taxonomic status remains unresolved (Che et al., 2007). Though our sampling is insufficient to comment on the dispersal and distribution of Hylarana throughout its range, the results of our study provide the first molecular evidence for the diversity and distribution of Hylarana species across the Western Ghats and Sri Lankan biogeographical region, and may assist future studies to resolve the existing uncertainty about taxonomic positions of taxa contained in the genus Hylarana.

Generic level taxonomy

Our study provides an opportunity to discuss the taxonomic history of two named, but currently synonymised group level taxa within the genus Hylarana (sensu lato).

Hydrophylax Fitzinger, 1843: We report sequences from the type species of Hydrophylax (H. malabarica from ‘Malabar’ India). Our molecular phylogenetic analysis suggests monophyly of the proposed Hylarana malabarica group (containing H. malabarica and H. gracilis) (Fig. 4), along with morphological support (Figs 5, 6). Hylarana cf. leptoglossa, a species closely related to the H. malabarica group, is shown to have distinct finger and toe tips demonstrating that digit tip morphology may not be taxonomically significant for delineating putative generic level taxa like Hydrophylax, as previously suggested (Dubois, 1992). Further morphological and molecular evidence incorporating more samples from the H. malabarica group, as well as closely related members like ‘Hylarana leptoglossa’ and Amnirana would be required to completely resolve the taxonomic status of Hydrophylax. For detailed taxonomic history see generic level taxonomy in the appendix.

Sylvirana Dubois, 1992: Our study does not provide conclusive evidence to address the generic status of Sylvirana, but suggests an interesting biogeographical pattern to understand the dispersal of members of the genus Hylarana. The well-supported sister relationship between the Hylarana malabarica group and H. cf. leptoglossa, a species which was previously regarded as Sylvirana leptoglossa (Dubois, 1992; Frost et al. 2006), indicates that if Sylvirana is considered as a genus, it would include members from throughout much of India and Sri Lanka, as well as Southeast Asia, thus partially supporting the assertions of some studies (e.g. Dubois, 1992, 2005; Frost et al., 2006; Fei et al., 2010). However, this mixed clade is a sequential sister group to the well-supported and phylogenetically distinct Western Ghats-Sri Lankan endemic radiation (H. aurantiaca, H. flavescens and H. temporalis groups). The phylogenetic position of this clade relative to its sister congeners, suggests that the Western Ghats-Sri Lankan endemic radiation came into being through an India-Sri Lanka dispersal event from Southeast Asia through Northeast India.

Species level taxonomy and endemism

The detailed morphological and molecular evidences from our study successfully resolved many taxonomic ambiguities and distribution limits of several nominal taxa in the genus Hylarana. These include species that have long been misidentified, or are currently considered as synonyms. Confirmed endemicity of H. aurantiaca and H. temporalis based on our results is interesting given that these two species have been known for more than 100 years (H. aurantiaca), up to nearly 150 years (H. temporalis). While H. aurantiaca is endemic to the vicinity of its type locality in the Western Ghats, H. temporalis is endemic to Sri Lanka. The small-sized Hylarana in Sri Lanka belong to a previously unnamed species H. serendipi and ‘Hylarana temporalis’ populations from India comprise of a complex of two named (H. flavescens and H. montanus) and four previously unnamed species (H. caesari, H. indica, H. magna and H. sreeni). Hylarana flavescens, H. intermedius and H. montanus are resurrected from the synonymy of H. temporalis and considered as valid species. Rana (Hylorana) bhagmandlensis is removed from the synonymy of H. aurantiaca, and regarded as a junior subjective synonym of H. montanus. In the interest of nomenclatural stability, Rana flavescens, Rana malabarica and Hylorana temporalis are lectotypified, and a neotype is designated for Rana (Hylorana ) intermedius. Hylarana malabarica, which was originally described from the southern Western Ghats is suggested to possibly represent at least two genetically distinct morphologically cryptic species. For taxonomic remarks on all Hylarana species of the Western Ghats and Sri Lanka, see individual species accounts in the Appendix.

Our data suggests that many species have a narrow latitudinal range, especially H. temporalis and H. aurantiaca, which were previously considered as the most widely distributed taxa. The consensus of all our analyses is a trichotomy of four major lineages within the study area: 1 — the Hylarana aurantiaca group, is one that gave rise to a clade of species mostly restricted to south of the Palghat Gap, except for the distribution of H. intermedius which is only found north of the Palghat Gap (Fig. 10); 2 — the Hylarana flavescens group, is widely distributed along the entire Western Ghats, but has restricted distribution ranges at species level. Hylarana sreeni is the only species found both north and south of the Palghat gap. Hylarana magna is restricted to the Agasthyamala hill range south of the Palghat gap, while H. flavescens, H. indica and H. montanus are only found north of the gap. Furthermore, H. caesari is restricted to the northern Western Ghats of Maharashtra (Fig. 14). 3 — the Hylarana temporalis group is a radiation endemic to Sri Lanka. Hylarana temporalis is widely distributed on the island and occurs both at mid elevation (up to about 900 meters) and in the wet zones of Sri Lanka. This group also contains a previously unnamed species, H. serendipi, which is endemic to, and found sympatrically with H. temporalis in the wet zone of Sinharaja World Heritage Site, Sri Lanka (Fig. 20). 4 — the Hylarana malabarica group forms a distinct, well supported clade containing one peninsular Indian (H. malabarica) and one Sri Lankan (H. gracilis) species (Fig. 24). Though H. malabarica is considered to be widely distributed, our study finds genetic segregation between the populations found in northern (Hylarana malabarica haplogroup 1) and southern parts of the Western Ghats. Based on molecular evidence, we confirm the extended occurrence of Hylarana malabarica haplogroup 1 populations from the northern Western Ghats, east to Madhya Pradesh state. Hylarana gracilis is the most widely occurring among Sri Lankan Hylarana species, which is endemic to Sri Lanka and found from sea level to 1250 m elevation.