Asian Herpetological Research 2011, 2(2): 91-96 DOI: 10.3724/SP.J.1245.2011.00091

Age Structure of Females in a Breeding Population of Echinotriton chinhaiensis (Caudata: Salamandridae) and Its Conservation Implication

Weizhao YANG1, 2, Chang LIU1, Jianping JIANG1, Cheng LI1 and Feng XIE1*

1Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China 2The Graduate University of Chinese Academy of Sciences, Beijing 100049, China

Abstract Knowledge of life history is important for understanding possible connections to population declines. Here, we investigated the female age structure and fecundity of Echinotriton chinhaiensis, one of the most endangered salamanders in the world, using skeletochronology based on specimens collected in 2008 and 2009 from a population in Ruiyansi, northeast of Ningbo, Zhejiang, China. The results showed that most female salamanders were between 5 and 6 years of age, with the minimal reproductive age, predicted to be 3 years, and the clutch size correlated to the body size. We argue that both delayed attainment of sexual maturity and low fecundity make this species more vulnerable to extinction. Keywords skeletochronology, age structure, sexual maturity, fecundity, clutch size

1. Introduction population increase and limited geographic ranges were more likely to be overexploited, even if all else being In recent years, the decline of amphibians on a worldwide equal. Age is an important life-history trait in association scale is an indisputable fact (Wake, 1991; Houlahan et al., with population dynamics. The correlation of age with 2000). Therefore, finding the critical factors responsible sexual maturity, longevity, body size and fecundity may for the population decline is a major challenge for help understand the life-history strategy of endangered amphibian conservation. Habitat destruction, biological species (Hemelaar, 1988; Zug, 1993). A case study invasions, pollution, overexploitation, and climate using age structure to detect the threatened Steller Sea change all have been identified as the general causes Lions revealed that their decline in the early 1980s was for population loss (Wake, 1991; Stuart et al., 2004). associated with the low survival rate of their juveniles, Understanding the profound causes continues to be a whereas the decline in the 1990s was associated with their driving force for amphibian conservation (Davies and low fecundity (Holmes and York, 2003). Age is also used Halliday, 1977; Steams and Koella, 1986; Hemelaar, for amphibian species (Liao, 2009). For example, Liao 1988; Pounds et al., 1999; Lu et al., 2006). (2009) determined the age structures of Bufo andrewsi Reliable knowledge of the life history of a species and Rhacophorus omeimontis in the subtropic montane is crucial for their conservation and management. Theoretical and empirical analyses indicate that certain forest in southwestern China and analyzed the correlation life-history traits enhance the risk of extinction for the among age structure, snout-vent length and clutch size, species (Jennings et al., 1999; Sadovy, 2001). Sadovy which indicated the probable causes of endangerment of (2001) pointed out that species with low rates of the two species. Echinotriton chinhaiensis (Urodela: Salamandridae) (Figure 1) is one of the most endangered salamanders * Corresponding author: Prof. Feng XIE, from Chengdu Institute of in the world, which has a distribution limited to the Biology, Chinese Academy of Sciences, with his research focusing on Beilun District of Ningbo, Zhejiang, China, and is conservation biology and evolutionary biology of amphibians E-mail: [email protected] assessed as Critically Endangered in the IUCN Red List Received: 11 Feburary 2011 Accepted: 17 May 2011 of Threatened Species in 2004 (Xie, 1999; Liu et al., 92 Asian Herpetological Research Vol. 2

Figure 1 The female of the Chinhai salamander, E. chinhaiensis

2010). A nature reserve was established in Ningbo, and collection in the area was permitted by the Provincial many measures have been taken to protect the species Fishery Management Department of Zhejiang, China. from extinction, including habitat restoration. However, 2.2 Age determination We adopted skeletochronology the population of E. chinhaiensis continues to drop over to determine the age of the collected E. chinhaiensis the recent years (Liu et al., 2010). Except for habitat loss, individuals (Castanet and Smirina, 1990). The many other underlying causes for this decline need further experimental procedure was complied with that of study. Castanet and Smirina (1990) and modified slightly. The In order to understand the life-history strategy of age of each individual was determined by observing the E. chinhaiensis and detect the profound causes of its glass slides under a microscope. The number of lines population decline, we investigated female age structure of arrested growth (LAGs) was interpreted as age. We and fecundity for a population of E. chinhaiensis in inspected each slide twice by different expertise for Ruiyansi. reducing errors. 2. Materials and Methods 2.3 Statistical analysis In order to test whether the age structure fluctuated sharply, we used Student’s t-test 2.1 Materials The study area is located in the Ruiyansi to compare the two age structures of 2008 and 2009. Forest Park (29°48′24″N, 121°51′12″E), northeast of Furthermore, Pearson’s correlation coefficient was used Ningbo, Zhejiang, China. During their breeding season for comparing age, body size and clutch size (Liao, 2009). from late March to late April, females arrive at the breeding sites and spawn along pool edges (Figure 2), 3. Results which made them easy to collect. In 2008 and 2009, we collected 44 gravid individuals (23 were sampled in 3.1 Age determination In this study, all the toe 2008, and 21 in 2009), which were transported live to the samples of E. chinhaiensis were observed by using laboratory to spawn. After spawning was completed, we skeletochronology, and their ages were determined. marked each individual using the toe clipping method A typical result shows that the number of LAGs is (Ferner, 1979), with the toes preserved in 10% (w/w) interpreted as age (Figure 3). The ages of the two formalin for skeletochronological analysis, and counted recaptured individuals (captured both in 2008 and 2009) the clutch size and measured the snout-vent length (SVL) were determined twice, and the result showed that their of the females as body size. Finally, we released all the LAGs added one normally, which verified the effectiveness individuals and eggs back to their breeding sites. Our of the skeletochronological method in this animal. No. 2 Weizhao YANG et al. Age Structure of Females in a Breeding Population of E. chinhaiensis 93

Figure 2 Breeding site of the Chinhai salamander, E. chinhaiensis, showing the female and its eggs

to be 3 years according to emergence of females in the breeding sites. The average age was 5.13±1.29 (n=23), and 5.33±1.02 (n=21) for 2008 and 2009 samples, respectively, with no statistical difference between the two years (Student’s t-test: t=0.576, P=0.568). The maximum reproductive age was 8 years. 3.4 Correlation among age, body size and clutch size The mean of SVL of each age and clutch size was listed in Table 1. Statistical analysis of E. chinhaiensis female ages and their SVLs show no correlation between age and SVL for 2008 and 2009 (2008: Pearson’s correlation coefficient: r=0.324, df=22, P=0.660; and 2009: r=0.137, df=20, P=0.277). In spite of the lack of females which laid eggs in the laboratory in 2008, we analyzed the correlation between Figure 3 Phalangeal bone cross-section of specimen No. 249 of E. age and clutch size of the females captured in 2009. The chinhaiensis. The black arrows point to the lines of arrested growth (LAGs); MC is abbreviation for the marrow cavity; ML for the metamorphism line, which is a dark line next to MC and caused by metamorphic development.

3.2 Age structure of female breeding population In 2008, the eldest individual was 8 years old and the youngest was 3 years old, with majority of 5 and 6 years old, accounting for 60.87%. In 2009, the eldest was 8 and the youngest was 4, with majority of 5 and 6 years, accounting for 71.43%. The age structures of E. Figure 4 Age structures of E. chinhaiensis female breeding chinhaiensis female breeding population in Ruiyansi, population in Ruiyansi, Ningbo in 2008 and 2009. The black bars represent the female population of 2008, and the white Ningbo in 2008 and 2009 are shown in Figure 4. bars represent that of 2009. The percentages on the bars mean that the individuals of each age stage 3.3 Age The minimal reproductive age was assumed account for the total individuals of the year. 94 Asian Herpetological Research Vol. 2

Table 1 Mean SVLs and clutch sizes of E. chinhaiensis females collected in 2008 and 2009

2008 2009 Age n SVL (mm) n SVL (mm) Clutch sizea 3 3 79.67±2.08 - - - 4 3 85.00±2.64 4 69.80±2.93 49.25±23.04 5 9 82.67±7.89 9 75.77±4.10 66.38±18.24 6 5 76.40±4.16 6 76.18±9.48 80.67±38.01 7 2 77.00±1.41 1 70.3 -b 8 1 76 1 73 42 Total 23 80.43±6.13 21 74.36±6.12 66.00±28.02 a Because few females laid eggs in the laboratory in 2008, we just analyzed their clutch sizes of 2009. b This female laid no eggs in laboratory. result showed that no correlation was found between age of skeletochronology for the individuals whose ages have and clutch size as well (Pearson’s correlation coefficient: been known or which are kept in mark-and-recapture r=0.166, df=18, P=0.149). experiment. Alternatively, the analysis showed that SVL positively We found that the body size of E. chinhaiensis adult correlated with the clutch size (Pearson’s correlation females correlated with clutch size, but the correlation coefficient: r=0.569, df=18, P=0.011). The result is shown was absent between body size and age, which was also in Figure 5. found in the studies of Rana sakuraii (Kusano et al., 1995), Triturus vittatus ophryticus (Kutrup et al., 2005) and Tylototriton verrucosus (Khonsue et al., 2010). In this study, we conclude that body size alone is an unreliable character to use for the determination of age in female E. chinhaiensis. 4.2 Life-history strategy and its implication in species declines Duellman and Trueb (1986) concluded that most salamanders became sexually mature after 2 years of age. In our study, we found that the earliest age at sexual maturity of female E. chinhaiensis was 3. In addition, from the age structure, we also found that most breeding females were at the ages of 5 (40.9%) and 6 (25.0%) based on our results from a skeletochronological analysis, which implies that females’ main reproductive periods are at the ages of 5 and 6 years, and thus older females contribute more to the gene pool. This lengthy period prior to sexual Figure 5 Regression between SVL and clutch size of E. chinhaiensis female breeding population in 2009 maturity in E. chinhaiensis females probably allows for increased opportunities for life threatening encounters. For 4. Discussion instance, the ages at sexual maturity of female Ambystoma opacum and Hemidactylium scutatum are only 1.3 and 4.1 Applicability of phalangeal skeletochronology of 2.3, which have similar living environment to that of E. age determination in amphibians Castanet and Smirina chinhaiensis (Blanchard and Blanchard, 1931; Duellman (1990) indicated that skeletochronology was an effective and Trueb, 1986). These results imply that the delayed method to determine the age of animals, and could also attainment of sexual maturity for female E. chinhaiensis be used to assess the growth, age at sexual maturity may enhance the opportunity of death and perhaps is one and longevity. Phalanges of amphibians could be used of the life-history elements contributing to their numerical not only for skeletochronology, but also for mark-and- decline (Liu et al., 2010; Xie, 1999). recapture in population studies. Toe-clipping has little Compared with T. verrucosus, another member of adverse effect on individuals (Phillott et al., 2008). In the family Salamandridae, the age at sexual maturity addition, it is a proper method to validate the efficiency and potential longevity are almost the same (Khonsue No. 2 Weizhao YANG et al. Age Structure of Females in a Breeding Population of E. chinhaiensis 95 et al., 2010). However, the average clutch sizes of the Evans K., Hindell M. A. 2004. The age structure and growth of two species are greatly different. The average clutch female sperm whales (Physeter macrocephalus) in southern size of T. verrucosus (114.78; Roy and Mushahidunnabi, Australian waters. 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