M_fissipes_phyml

Phylogeny of mitochondrial COI lineages from 11 populations of Microhyla fissipes in Taiwan. This tree was constructed by using PhyML 3.0 with 1000 bootstrap replicates

Acoustic differentiation and behavioral response reveals cryptic species within <i>Buergeria</i> treefrogs (Anura, Rhacophoridae) from Taiwan

<div><p><i>Buergeria japonica</i> is a widely distributed treefrog occurring from Ryukyu Archipelago to Taiwan. Across this wide distributional range, we combined molecular, acoustic, morphological, and behavioral characters to clarify the taxonomic status among these insular populations. Genetic differentiation in mitochondrial sequences indicated an over 16% divergence among two deeply divergent clades: Japanese clade distributes in Ryukyu Archipelago and northwestern drainages of Taiwan, while Taiwanese clade distributes in the remaining drainages on Taiwan. The Taiwanese clade can be distinguished from the nominative species not only by molecular and morphological differences, but also distinguishable by considerable acoustic differentiation, which is extraordinarily noticeable for an additional type of long call that never recorded from Japanese clade. The two clades form a parapatric distribution pattern with narrow contact zones both in western and eastern Taiwan. Playback experiments indicated that male frogs show significantly stronger defensiveness against conspecific calls rather than heterospecific calls, indicating that these signals play a crucial role in species recognition. Here we describe the Taiwanese clade as a new species; the behavioral response and the magnitude of gene flow across their contact zones are especially worth for detailed studies.</p></div

Comparison of long call sonograms between the Japanese and Taiwanese clades.

<p>The Japanese clade (<i>Buergeria japonica</i>) could express only one type of long call (Type 1A), yet the Taiwanese clade (<i>Buergeria otai</i> sp. nov.) is capable to express an additional type (Type 1B and Type 2). The units (u or ku) displayed on the vertical axis of a waveform view are the actual sample values in the signal, which are proportional to the sound pressure at the microphone when the sound was recorded (Raven Pro v1.4, Cornell Lab of Ornithology, Ithaca, NY, USA). Sonograms in this figure: Type 1A: No. 150518_03#2 from Wu Stream, northwestern Taiwan; Type 1B: NMNS 19824 (paratype of <i>B</i>. <i>otai</i> sp. nov.) from Donggang Stream, southern Taiwan; Type 2: NMNS 19816 (paratype of <i>B</i>. <i>otai</i> sp. nov.) from Donggang Stream, southern Taiwan.</p

Within-clade (diagonal) and between-clade (upper-right) divergences among the four major lineages in the Japanese clade (<i>Buergeria japonica</i>) and two major lineages in the Taiwanese clade (<i>Buergeria otai</i> sp. nov.).

<p>Within-clade (diagonal) and between-clade (upper-right) divergences among the four major lineages in the Japanese clade (<i>Buergeria japonica</i>) and two major lineages in the Taiwanese clade (<i>Buergeria otai</i> sp. nov.).</p

Principal component analysis and morphological differentiation between the Japanese and the Taiwanese clades.

<p>The Japanese clade (<i>Buergeria japonica</i>) is abbreviated as JP and represented in blue; and the Taiwanese clade (<i>Buergeria otai</i> sp. nov.) is abbreviated as OT and represented in orange. These two clades differed significantly in PC2, which was consisted majorly by the size and shape of the head (F1,14 = 15.85, p = 0.001).</p

Sampling regimes of <i>Buergeria japonica sensu lato</i> in this study.

<p>(A) Sample localities, sample sizes, and genetic assignments from molecular evidence; (B) sample localities, sample sizes, and call types from acoustic evidence; and (C) the fine-scale sampling regime in Taiwan. Populations from Ryukyu and northwestern Taiwan (Japanese clade) could express only one type of long calls (Type 1A in light blue), but those from eastern and southern Taiwan (Taiwanese clade) are capable to express two: one very similar to the former (Type 1B in dark blue), and the other exclusively occurring in this region (Type 2 in orange). Arabic numerals in the pie charts indicate samples sizes for molecular analysis; while percentages in column charts indicate the population ratio in each drainage which was recorded to express either types of the calls.</p

Region, locality, abbreviation, sample sizes (acoustic, morphological, molecular, and number of mitochondrial haplotypes), and GenBank accession numbers for each <i>Buergeria japonica</i> population used in this study.

<p>Region, locality, abbreviation, sample sizes (acoustic, morphological, molecular, and number of mitochondrial haplotypes), and GenBank accession numbers for each <i>Buergeria japonica</i> population used in this study.</p

Maximum likelihood phylogeny of <i>Buergeria japonica sensu lato</i>.

<p>Phylogenetic tree was constructed by using PhyML 3.0 based on complete cytochrome <i>b</i> sequences, with 1000 bootstrap replicates and Bayesian posterior probability as statistic supports. Japanese clade (blue) and Taiwanese clade (orange) represent 16.19% between-group sequence divergence (<i>p</i>-distance).</p

Holotype (A and C, male, NMNS 19819) and one of the paratypes (B and D, female, NMNS 19871) of <i>Buergeria otai</i> sp. nov.

<p>Holotype (A and C, male, NMNS 19819) and one of the paratypes (B and D, female, NMNS 19871) of <i>Buergeria otai</i> sp. nov.</p

<i>Buergeria japonica</i> (A, C) and <i>Buergeria otai</i> sp. nov. (B, D, E, F) in live.

<p>The irregular patches of <i>B</i>. <i>japonica</i> on the thighs could be compared to the regular tiny spots of <i>Buergeria otai</i> sp. nov., regardless the intraspecific variation from very few (E) to many (F). Photographed by YJ Liang (A, C), CM Tsao (B), and HN Nguyen (D, E, F).</p