Rhacophorus schlegelii (Günther, 1858) is a common treefrog distributed on Japan’s main islands Honshu, Shikoku, and Kyushu, as well as adjacent small islands (Matsui and Maeda, 2018). These frogs are well known to produce foam nests underground or in shallow mud burrows near standing water in the springtime, from which the hatched tadpoles wiggle into the water (Matsui and Maeda, 2018). In contrast to that, R. arboreus (Okada and Kawano, 1924), which has a widely overlapping range with R. schlegelii, produces larger foam nests, which are usually attached to tree branches, leaves, or other vegetation over standing water, but rarely on the ground. These two species occur sympatrically on Honshu and their egg-laying habits are differentiated by their nesting tactics. We here report for the first time an observation of foam nest construction by R. schlegelii on a tree branch overhanging water.

We observed three egg masses at a small tributary of the Minato River, Kagawa City, Kagawa Prefecture, Shikoku Island, Japan (34.2061°N, 134.2687°E, elevation 208 m) on 29 May 2018. They had been deposited on a branch of a dead, broad-leaved deciduous tree (Fig. 1A), dead fern frond (Gleichenia japonica) (Fig. 1B), and a branch of a living shrub (Fig. 1C), all overhanging at a height of 1–2 m the water surface of a small artificial lake that serves to control soil erosion. No other foam nests were found in the vicinity. The bank of the reservoir was partly covered by concrete but consisted mostly of natural sandy soil. We searched the

banks for underground egg masses but none were found, although an adult male R. schlegelii was encountered at the site (Fig. 2). The eggs were collected and fixed in 10% formalin, except for some eggs fixed in 99% ethanol for genetic analysis. All samples were deposited in the Graduate School of Human and Environmental Studies, Kyoto University (voucher specimen number: KUHE 60515).

In order to ascertain species identity, we examined mitochondrial DNA sequences of the eggs. Total DNA was extracted from five ethanol-preserved eggs using DNeasy Blood & Tissue Kit (Qiagen). A fragment containing the partial mitochondrial Cytochrome C Oxidase subunit 1 gene (CO1) was amplified by polymerase chain reaction (PCR). The amplified PCR products were purified by polyethylene glycol precipitation, and then cycle-sequencing reactions were done using an ABI PRISM Big Dye Terminator

Herpetology Notes, volume 13: 341-342 (2020) (published online on 23 April 2020)

First report of arboreal breeding in Rhacophorus schlegelii (Günther, 1858) (Amphibia, Rhacophoridae)

Kazumi Fukutani1,*, Hirokazu Onuma2, and Kanto Nishikawa1

1 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida, Nihonmatsu-cho, Sakyo-ku, Kyoto 606–8501, Japan.

2 The Nature Conservation Society of Hyogo Prefecture, 2–1–18 Goko-dori, Chuo-ku, Kobe 651–0087, Japan.

* Corresponding author. E-mail: fukutani.kazumi.55a@st.kyoto-u.ac.jp

Figure 2. An adult male Rhacophorus schlegelii found near the site where the egg masses were deposited. Photo by Hirokazu Onuma.

Kazumi Fukutani et al.342

v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing was performed on ABI 3130 automatic sequencers. A total of 1185 bp of CO1 was sequenced. Obtained sequences have been deposited in the DNA Data Bank of Japan under accession number LC440576. The primers and reaction conditions will be distributed upon request made to the corresponding author.

We compared our own new sequence data with GenBank data of R. schlegelii from Hiroshima (Sano et

al., 2005: NC_007178). The genetic distance (uncorrected p-distance) between the two samples was very small (1.37%). The small genetic distance indicates that our sample is from R. schlegelii. This is the first record of foam nest attachment on the branches and leaves above the water in this species, which usually deposits eggs underground or in mud burrows, demonstrating a higher degree of plasticity in reproduction site choice than previously assumed for the species. We nevertheless consider the present observation a rare case for R. schlegelii. We hope that further observations will clarify if arboreal nest sites are used by R. schlegelii only in the absence of R. arboreus on Shikoku Island.

References

Matsui, M., Maeda, N. (2018): Encyclopedia of Japanese Frogs. Tokyo, JPN, Bun-Ichi Shuppan Co., Ltd.

Sano, N., Kurabayashi, A., Fujii, T., Yonekawa, H., Sumida, M. (2005): Complete nucleotide sequence of the mitochondrial genome of Schlegel’s tree frog Rhacophorus schlegelii (family Rhacophoridae): duplicated control regions and gene rearrangements. Genes & Genetic Systems 80(3): 213–224.

Figure 1. Foam nests of Rhacophorus schlegelii deposited on a branch of a dead broad-leaved deciduous tree (A), on a dead fern frond (B), and a branch of a shrub (C: position indicated by an arrow). Photos by Hirokazu Onuma.

Accepted by Maximilian Dehling