The principal component analysis and the discriminant function analysis of call characters for the four <i>Microhyla</i> species.

<p>(A) Plot of principle components 1 and 2 (PC1 vs. PC2) of the four call characters (call duration, dominant frequency, number of pulses per call and pulse rate). (B) Canonical variables plot of the discriminant function analysis of eight call character variables with 95% confidence ellipses and the centroids indicated.</p

Descriptive statistics for calls of the four <i>Microhyla</i> species based on values determined from a sample of 100 calls from 5 males of each species (except <i>M</i>. <i>zeylanica</i>, 53 calls from 3 males).

<p>Descriptive statistics for calls of the four <i>Microhyla</i> species based on values determined from a sample of 100 calls from 5 males of each species (except <i>M</i>. <i>zeylanica</i>, 53 calls from 3 males).</p

An Acoustic Analysis of the Genus <i>Microhyla</i> (Anura: Microhylidae) of Sri Lanka

<div><p>Vocalizing behavior of frogs and toads, once quantified, is useful for systematics, rapid species identification, behavioral experimentation and conservation monitoring. But yet, for many lineages vocalizations remain unknown or poorly quantified, especially in diversity rich tropical regions. Here we provide a quantitative acoustical analysis for all four Sri Lankan congeners of the genus <i>Microhyla</i>. Three of these species are endemic to the island, but <i>Microhyla ornata</i> is regionally widespread. Two of these endemics, <i>M</i>. <i>karunaratnei</i> (Critically Endangered) and <i>M</i>. <i>zeylanica</i> (Endangered), are highly threatened montane isolates; the other, <i>M</i>. <i>mihintalei</i>, is relatively common across the dry lowlands. We recorded and analyzed 100 advertisement calls from five calling males for each species, except for <i>M</i>. <i>zeylanica</i>, which only had 53 calls from three males suitable for analyses. All four species call in choruses and their vocal repertoires are simple compared to most frogs. Their calls contain multiple pulses and no frequency modulation. We quantified eight call characters. Call duration and number of pulses were higher for the two montane isolates (inhabiting cooler habitats at higher altitudes) compared to their lowland congeners. <i>Microhyla zeylanica</i> has the longest call duration (of 1.8 ± 0.12 s) and the highest number of pulses (of 61–92 pulses). The smallest of the species, <i>Microhyla karunaratnei</i> (16.2–18.3 mm), has the highest mean dominant frequency (3.3 ± 0.14 kHz) and pulse rate (77 ± 5.8 pulses per second). The calls separate well in the Principal Component space: PC1 axis is mostly explained by the number of pulses per call and call duration; PC2 is mostly explained by the pulse rate. A canonical means plot of a Discriminant Function analysis shows non-overlapping 95% confidence ellipses. This suggests that some call parameters can be used to distinguish these species effectively. We provide detailed descriptions for eight call properties and compare these with congeners for which data is available. This work provides a foundation for comparative bioacoustic analyses and species monitoring while facilitating the systematics of <i>Microhyla</i> across its range.</p></div

Descriptions of acoustic properties measured.

<p>Descriptions of acoustic properties measured.</p

Advertisement calls of the four <i>Microhyla</i> species.

<p>(A) Ten second segment of a call by a single male. (B) Two second segment showing a single call underlined in A. (C) Spectrogram of the call shown in B. (D) Power spectrum of the call averaged over the duration of each call depicted in B (256 FFT size, Hanning window).</p

Four <i>Microhyla</i> species in life and their distribution.

<p>(A) A map showing the distribution of <i>Microhyla karunaratnei</i> (blue), <i>M</i>. <i>zeylanica</i> (green), <i>M</i>. <i>mihintalei</i> (red) and <i>M</i>. <i>ornata</i> (yellow). (B) Dorsolateral view of the four species (in life).</p

Immature stages and the larval food plant of Nacaduba pactolus ceylonica Fruhstorfer, 1916 (Lepidoptera: Lycaenidae) in Sri Lanka

The biology of  Nacaduba pactolus ceylonica (Large Four-lineblue ), a rare butterfly endemic to Sri Lanka is described with a report of Entada rheedii (Fabaceae) as the first known larval host plant. Information pertaining to the distribution of this butterfly is also provided.</p

Immature stages and the larval food plant of Nacaduba pactolus ceylonica Fruhstorfer, 1916 (Lepidoptera: Lycaenidae) in Sri Lanka

The biology of  Nacaduba pactolus ceylonica (Large Four-lineblue ), a rare butterfly endemic to Sri Lanka is described with a report of Entada rheedii (Fabaceae) as the first known larval host plant. Information pertaining to the distribution of this butterfly is also provided.</p

Integrating bioacoustics, DNA barcoding and niche modeling for frog conservation – The threatened balloon frogs of Sri Lanka

Discovering and monitoring anuran populations that are in decline, and ascertaining boundaries for cryptic and rare species, is a challenge for their conservation management. Here, we integrate three techniques, bioacoustics (call), niche modeling and DNA barcoding as a test case to investigate how the combination of these methods can enhance search efficiency for previously unknown populations, especially for those species that are threatened. As a focal group, we considered a clade in the genus Uperodon earlier referred to as Ramanella, represented by four endemic species in Sri Lanka (U. nagaoi – Endangered; U. palmatus – Critically Endangered; U. obscurus – Vulnerable and U. rohani – possibly Least Concern); we focus on the two highly threatened species (U. nagaoi and U. palmatus). We used mitochondrial DNA barcodes (16S rRNA) to link species accurately to their call and subsequently predicted species distributions using MaxEnt-based niche modeling of known species locations and forest cover data to increase the efficiency of searching for new populations. Lastly, we analyzed call data for accurate and rapid identification of new and viable populations. Following enhanced predicted distribution models, we visited 14 potential sites and sampled for calls of the two highly threatened species. Within a period of two weeks of fieldwork, we discovered two new populations of U. nagaoi and one population of U. palmatus by identifying their calls in areas predicted by niche modeling; we also confirm species identities at several previously unconfirmed locations. Finally, we included the new locations to enhance the distributional predictions for the threatened species. We discuss our results in the context of integrating methods to facilitate conservation of rare and threatened frog species. Keywords: Anura, Distribution modeling, Exploration, Population monitoring, Species boundaries, Vocalizatio

Species boundaries, biogeography and evolutionarily significant units in dwarf toads: <em>Duttaphrynus scaber</em> and <em>D. atukoralei</em> (Bufonidae: Adenominae)

Species boundaries and patterns of gene flow in Dwarf toads, Duttaphrynus scaber and D. atukoralei, were assessed using mitochondrial DNA markers. Samples from four populations in Sri Lanka (Mihintale, Ampara, Yala, Galle) were analyzed for three mitochondrial gene fragments (16S rRNA, COI and Cyt b) along with four Genbank sequences of 16S rRNA from Indian samples (Thiruvananthapuram, Maharashtra, Mudigere). Phylogenetic trees and haplotype networks were generated, and morphology was assessed. Analyses suggest a single species (Duttaphrynus scaber) with three major clades: a widespread clade shared between India and Northern Sri Lanka, an Eastern and Southeastern Sri Lankan clade (previously referred to as D. atukoralei, the validity of which, however, our analysis disputes), and a distinct Southern wet-zone clade from Galle (referred previously to as D. atukoralei). Duttaphrynus atukoralei (topotypes from Yala, Sri Lanka) is genetically too close to D. scaber (Indian and northern Sri Lankan clade) to be distinguished as a species; these two clades have a genetic distance of 0.95 – 1.55% for the 16S rRNA fragment. The haplotype networks for the 16S rRNA gene suggest incomplete lineage sorting between the Ampara and Yala populations; COI and Cyt b show complete sorting for all populations analyzed, suggesting strong population structure. All analyses suggest substantially restricted gene flow to the southern wet-zone population (Galle). This population also assumes a basal phylogenetic position, suggesting that D. scaber first evolved in southern Sri Lanka’s wet zone and dispersed across the lowland areas of the island and to India. Here, we provisionally recognize this population (Galle) as an evolutionarily significant unit of D. scaber; future analyses using multiple criteria may indicate this to be a new Dwarf toad species. External morphology is largely uninformative as the Yala, Ampara and Galle populations cannot be distinguished from each other; the morphological distinction between Yala, Ampara, Galle versus Mihintale is restricted to only the shape of the parotid glands – slightly oval versus rounded – a minor difference. Both genetic and morphological evidence so far suggest that there is only a single Dwarf toad species in Sri Lanka, which is also shared with India, namely Duttaphrynus scaber; however, with strong population structure, including an evolutionarily significant unit (Southern wet-zone population)