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


Weizhao YANG", Chang LIU’, Jianping JIANG’, Cheng LI' and Feng XIE" 8 ping

'Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China “The 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


Keywords skeletochronology, age structure, sexual maturity, fecundity, clutch size

1. Introduction

In recent years, the decline of amphibians on a worldwide scale is an indisputable fact (Wake, 1991; Houlahan et al., 2000). Therefore, finding the critical factors responsible for the population decline is a major challenge for amphibian conservation. Habitat destruction, biological invasions, pollution, overexploitation, and climate change all have been identified as the general causes for population loss (Wake, 1991; Stuart et al., 2004). Understanding the profound causes continues to be a driving force for amphibian conservation (Davies and Halliday, 1977; Steams and Koella, 1986; Hemelaar, 1988; Pounds et al., 1999; Lu et al., 2006).

Reliable knowledge of the life history of a species is crucial for their conservation and management. Theoretical and empirical analyses indicate that certain life-history traits enhance the risk of extinction for the species (Jennings et al., 1999; Sadovy, 2001). Sadovy (2001) pointed out that species with low rates of

* Corresponding author: Prof. Feng XIE, from Chengdu Institute of Biology, Chinese Academy of Sciences, with his research focusing on conservation biology and evolutionary biology of amphibians


Received: 11 Feburary 2011 Accepted: 17 May 2011

population increase and limited geographic ranges were more likely to be overexploited, even if all else being equal. Age is an important life-history trait in association with population dynamics. The correlation of age with sexual maturity, longevity, body size and fecundity may help understand the life-history strategy of endangered species (Hemelaar, 1988; Zug, 1993). A case study using age structure to detect the threatened Steller Sea Lions revealed that their decline in the early 1980s was associated with the low survival rate of their juveniles, whereas the decline in the 1990s was associated with their low fecundity (Holmes and York, 2003). Age is also used for amphibian species (Liao, 2009). For example, Liao (2009) determined the age structures of Bufo andrewsi and Rhacophorus omeimontis in the subtropic montane forest in southwestern China and analyzed the correlation among age structure, snout-vent length and clutch size, which indicated the probable causes of endangerment of the two species.

Echinotriton chinhaiensis (Urodela: Salamandridae) (Figure 1) is one of the most endangered salamanders in the world, which has a distribution limited to the Beilun District of Ningbo, Zhejiang, China, and is assessed as Critically Endangered in the IUCN Red List of Threatened Species in 2004 (Xie, 1999; Liu et al.,

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

2010). A nature reserve was established in Ningbo, and many measures have been taken to protect the species from extinction, including habitat restoration. However, the population of E. chinhaiensis continues to drop over the recent years (Liu et al., 2010). Except for habitat loss, many other underlying causes for this decline need further study.

In order to understand the life-history strategy of E. chinhaiensis and detect the profound causes of its population decline, we investigated female age structure and fecundity for a population of E. chinhaiensis in Ruiyansi.

2. Materials and Methods

2.1 Materials The study area is located in the Ruiyansi Forest Park (29°48'24"N, 121°51'12”E), northeast of Ningbo, Zhejiang, China. During their breeding season from late March to late April, females arrive at the breeding sites and spawn along pool edges (Figure 2), which made them easy to collect. In 2008 and 2009, we collected 44 gravid individuals (23 were sampled in 2008, and 21 in 2009), which were transported live to the laboratory to spawn. After spawning was completed, we marked each individual using the toe clipping method (Ferner, 1979), with the toes preserved in 10% (w/w) formalin for skeletochronological analysis, and counted the clutch size and measured the snout-vent length (SVL) of the females as body size. Finally, we released all the individuals and eggs back to their breeding sites. Our

collection in the area was permitted by the Provincial Fishery Management Department of Zhejiang, China.

2.2 Age determination We adopted skeletochronology to determine the age of the collected E. chinhaiensis individuals (Castanet and Smirina, 1990). The experimental procedure was complied with that of Castanet and Smirina (1990) and modified slightly. The age of each individual was determined by observing the glass slides under a microscope. The number of lines of arrested growth (LAGs) was interpreted as age. We inspected each slide twice by different expertise for reducing errors.

2.3 Statistical analysis In order to test whether the age structure fluctuated sharply, we used Student’s t-test to compare the two age structures of 2008 and 2009. Furthermore, Pearson’s correlation coefficient was used for comparing age, body size and clutch size (Liao, 2009).

3. Results

3.1 Age determination In this study, all the toe samples of E. chinhaiensis were observed by using skeletochronology, and their ages were determined. A typical result shows that the number of LAGs is interpreted as age (Figure 3). The ages of the two recaptured individuals (captured both in 2008 and 2009) were determined twice, and the result showed that their LAGs added one normally, which verified the effectiveness of the skeletochronological method in this animal.

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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


£ \

Figure 3 Phalangeal bone cross-section of specimen No. 249 of E. 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


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. chinhaiensis female breeding population in Ruiyansi, Ningbo in 2008 and 2009 are shown in Figure 4.

3.3 Age The minimal reproductive age was assumed

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: 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 age and clutch size of the females captured in 2009. The

= Co

39.13% 42.86%

13.04% 13.04%

Number of Individuals

o N A QA œ


Figure 4 Age structures of E. chinhaiensis female breeding population in Ruiyansi, Ningbo in 2008 and 2009.

The black bars represent the female population of 2008, and the white bars represent that of 2009.

The percentages on the bars mean that the individuals of each age stage account for the total individuals of the year.

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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 size* 3 3 79.6742.08 - - z 4 3 85.00+2.64 4 69.80+2.93 49.25+23.04 5 9 82.67+7.89 9 75.7744.10 66.38+18.24 6 5 76.40+4.16 6 76.1849.48 80.67438.01 7 2 77.00+1.41 1 70.3 - 8 1 76 1 73 42 Total 23 80.43+6.13 21 74.36+6.12 66.00£28.02

Because few females laid eggs in the laboratory in 2008, we just analyzed their clutch sizes of 2009.

> This female laid no eggs in laboratory.

result showed that no correlation was found between age and clutch size as well (Pearson’s correlation coefficient: r=0.166, df=18, P=0.149).

Alternatively, the analysis showed that SVL positively correlated with the clutch size (Pearson’s correlation coefficient: r=0.569, df=18, P=0.011). The result is shown in Figure 5.



Clutch size fon 5

SVL (mm)

Figure 5 Regression between SVL and clutch size of E. chinhaiensis female breeding population in 2009

4. Discussion

4.1 Applicability of phalangeal skeletochronology of age determination in amphibians Castanet and Smirina (1990) indicated that skeletochronology was an effective method to determine the age of animals, and could also be used to assess the growth, age at sexual maturity and longevity. Phalanges of amphibians could be used not only for skeletochronology, but also for mark-and- recapture in population studies. Toe-clipping has little adverse effect on individuals (Phillott et al., 2008). In addition, it is a proper method to validate the efficiency

of skeletochronology for the individuals whose ages have been known or which are kept in mark-and-recapture experiment.

We found that the body size of E. chinhaiensis adult females correlated with clutch size, but the correlation was absent between body size and age, which was also 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 maturity in E. chinhaiensis females probably allows for increased opportunities for life threatening encounters. For instance, the ages at sexual maturity of female Ambystoma opacum and Hemidactylium scutatum are only 1.3 and 2.3, which have similar living environment to that of E. chinhaiensis (Blanchard and Blanchard, 1931; Duellman and Trueb, 1986). These results imply that the delayed attainment of sexual maturity for female E. chinhaiensis may enhance the opportunity of death and perhaps is one of the life-history elements contributing to their numerical decline (Liu et al., 2010; Xie, 1999).

Compared with T. verrucosus, another member of the family Salamandridae, the age at sexual maturity and potential longevity are almost the same (Khonsue

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et al., 2010). However, the average clutch sizes of the two species are greatly different. The average clutch size of T. verrucosus (114.78; Roy and Mushahidunnabi, 2001) is nearly twice that of E. chinhaiensis (66.00). Consequently, the average fecundity of the former is nearly twice that of the latter. The low fecundity of E. chinhaiensis may be considered another life-history cause of population decline.

In conclusion, through the study on the age structure of a female breeding population of E. chinhaiensis, we considered that the delayed attainment of sexual maturity and lower fecundity may be two probable life- history traits endangering E. chinhaiensis. As mentioned previously, since the nature reserve has been established and protective measurements have been taken for this species, such as habitat restoration, the population of E. chinhaiensis continues to decline (Liu et al., 2010). Accordingly, other causes for the endangerment of £E. chinhaiensis should be investigated and understood, and then particular methods or measures should be studied and applied to solve the problems related with the continuous declining of this species. This study provides a good example for detecting the underlying causes of any endangered species.

Acknowledgements We would like to thank Junru CHEN and Hanggang DONG at the Ruiyansi Forest Park, and Xintai LIU, Yi LIU and Miaofei WU from the Cai- qiao Middle School for their assistance in collection of samples. This work was supported by the National Natu- ral Science Foundation of China (30770316).


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