Herpetology Notes, volume 13: 831-833 (2020) (published online on 08 October 2020)

First record of bicephaly in Paramesotriton hongkongensis (Myers and Leviton, 1962) (Caudata: Salamandridae)

Ming Fung Franco Au1, Jie Shen2, Paolo Martelli3, and Anthony Lau1,4,*

A congenital anomaly is a structural malformation or pools with aquatic and riparian vegetation during or functional birth defect that occurs in many animal the dry seasons (September to March) but migrates species during early developmental stages (Westworth to adjacent forests during the wet seasons (April to et al., 2010). Congenital anomalies are mainly caused August) (Fu et al., 2013; Lau et al., 2017). Three P. by genetic or environmental factors (Lanteri et al., hongkongensis egg masses (Fig. 1) were collected from 2017). Bicephaly (also known as dicephalism) is a a stream pool at Ma On Shan Country Park, Hong Kong type of congenital anomaly, which refers to conjoined on 26 September 2019 for a study on larval cannibalism. twins with two independent heads from a single body. The collection site is a relatively deep (~60 cm) and slow- The existence of bicephalic individuals is frequently flowing freshwater habitat surrounded by subtropical reported in mammals and reptiles, especially for broad-leaved forest with low human disturbance. In Serpentes (Sayyed, 2015; Devkota et al., 2020) and the laboratory, the collected egg masses were incubated Testudines (Lanteri et al., 2017). However, bicephaly in a plastic container (38.5 cm in length × 24.5 cm in seems to be rare in amphibians. Bicephalic individuals width × 24 cm in height) at room temperature (~24 have been reported in five anurans (Lebedinsky, 1921; °C) with overnight tap water under 12L12D (light/ Dragoiu and Busnitza, 1927; Schwind, 1942; Lynn, dark) condition. The incubation container was checked 1944; McFadden et al., 2011), four urodelans (Pereira every other day. Each hatchling was separated from and Rocha, 2004; Velo-Antón et al., 2007; Fernandez- the incubation container and reared in a transparent Alvarez, 2011) and one gymnophionans (Bei et al., plastic tray (13.4 cm in length × 6.3 cm in width × 3.7 2011). Here we report the first case of bicephaly in a cm in height) immediately. On 14 October 2019, a P. conjoined Paramesotriton hongkongensis larva. The hongkongensis larva with two heads and anterior bodies external morphology and osteology of the bicephalic was found (Fig. 2). No other individuals with anomalies individual were described below. Paramesotriton hongkongensis (Hong Kong newt) (Myers & Leviton, 1962) is the only native urodelan found in Hong Kong. The geographic distribution of this species is limited to Hong Kong and Southern Guangdong Province of Mainland China (Fei et al., 2006). This stream-dwelling salamander breeds in unpolluted stony montane streams

1 Department of Biology, Hong Kong Baptist University, Hong Kong SAR, China. 2 Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong SAR, China. 3 Veterinary Department, Ocean Park Corporation, Hong Kong SAR, China. 4 Science Unit, Lingnan University, Hong Kong SAR, China. * Corresponding author. E-mail: [email protected] Figure 1. An egg mass of Paramesotriton hongkongensis. 832 Ming Fung Franco Au et al.

Figure 2. Dorsal (A) and ventral (B) view of a bicephalic larva Figure 3. Micro CT radiographs (dorsal-ventral projection) of of Paramesotriton hongkongensis. a bicephalic larva of Paramesotriton hongkongensis.

were noticed in the same clutch of egg mass. Upon the heads may represent inner ear structures (Duellman closer examination conducted on a stereo dissecting and Trueb, 1994; Gao et al. 1998). The endolymphatic microscope (Leica Stereozoom S9i), a duplication of sacs in amphibians contain lime (Ca2+) for cartilaginous the anterior parts was observed in the malformed larva. skeleton ossification during metamorphosis (Stiffler, Both heads were completely separated from each other 1993). Radiographic examination also detected an with equal size. Two well-developed eyes could be unknown structure near the 8th vertebrae of the left body. clearly seen on each head of the conjoined twins and all As the two axial skeletons of the animal joined together eyes appeared to be functional. The animal also showed at the posterior one-third of the body, the animal had a pair of external gills at the back of each head. Both four entirely developed forelimbs, but only two fully- pairs of gills were normally located and functional. formed hindlimbs. The coordination between two heads However, due to the limited space for gill development, appears to be difficult for the bicephalic individual. The there was a size reduction of the medial gills (i.e. left larva was not able to move easily despite functional gills of the right head and right gills of the left head). extremities. Unfortunately, the micro-CT images did Similar to other larvae, both heads included a fully not allow the differentiation of soft tissues (i.e. muscle formed mouth (Fig. 2). A full-body micro-CT (Bruker and organs) due to low X-ray adsorption of the soft SkyScan 1076) scan was performed on the bicephalic tissues and the small size of the animal. The bicephalic larva to investigate its internal morphology (Fig. 3). individual died after 5 days on 19 October 2019 of a From the resulting radiographs, we can see that the axial suspected fungal infection. The specimen was finally skeletons fused together at the level of sacrum and tail preserved in 70% ethanol for further study. This P. region, which means that each anterior body probably hongkongensis larva represents the first bicephaly has its own cervical vertebra and trunk vertebrae while record for this species. Even though it is not possible to sharing the same caudal vertebrae. Referring to the determine the exact cause for the development of this dorsal-ventral projection, there were approximately 17 congenital anomaly without other evidence, it will help vertebrae in the right body and 16 vertebrae in the left to determine the possible influencing factors by adding body before fusion. The four white spots presence in to the worldwide list of bicephalic amphibians. First record of bicephaly in Paramesotriton hongkongensis 833

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Accepted by Kanto Nishikawa