Skeletochronological assessment of age in the Persian mountain salamander, Paradactylodon gorganensis (Clergue-Gazeau and Thorn, 1979) (Caudata: Hynobiidae) from Golestan Province, Iran

[Research]

Skeletochronological assessment of age in the Persian mountain salamander, Paradactylodon gorganensis (Clergue-Gazeau and Thorn, 1979) (Caudata: Hynobiidae) from Golestan Province, Iran

S. Zivari, H. Gholi Kami*

Department of Biology, Faculty of Sciences, Golestan University, Gorgan, Golestan Province, Iran.

* Corresponding author’s E-mail:hgkami2000@yahoo.com

ABSTRACT

Lines of arrested growth (LAGs) were assessed in the Golestan Province population of the critically - endangered museum specimens of Persian mountain salamander, Paradactylodon gorganensis (Clergue-Gazeau and Thorn, 1979) from Northeastern Iran using skeletochronology. LAGs were clearly discernable in the tibia and femur bone cross-sections and could be interpreted for age determination. The number of LAGs in the sampled Golestan Province population (n=29)was found to be 8-11 (9.75  1.13 years) in males (n=12), 7-13 (9.26  1.98 years) in females (n=15) and 7 and 8 (7.50  0.70 years) in two unknown sex specimens for the first time.The snout-vent length (SVL) was 90.20 - 119.38 (103.85  10.48) mm in males, 77.60 - 135.00 (105.96  15.89) in females and 108.00 and 123.00 mm in unknown sex specimens. It was not found significant correlation between body size and age (P > 0.05). Although limited in sample size, the data suggest that males with ages over eleven years are absent in the fixed specimens studied. Our study showed that skeletochronological method can be successfully applied to Iranian P. gorganensis based on the clear arrest of growth in the hibernation period, as previously observed in other species in subtropical regions.

INTRODUCTION

Age determination of amphibians and reptiles is important in order to obtain information about mortality, longevity, and other ecological factors. In most cases, it provides accurate estimations of individual ages and therefore overcomes a major difficulty in demographic and life history studies (Yilmaz et al. 2005; Uzum & Olgun 2009).Therefore, several methods have been developed and used for estimating age of animals. An alternative tool to get such data for amphibians is skeletochronology which is considered the most reliable method of age estimation by counting the lines of arrested growth (LAGs) recorded in long bones (Castanet et al. 1993; Ashkavandi et al. 2012).However, although skeletochronology is established as a reliable method to assess age in amphibians, it has been applied mainly to temperate species (Halliday & Verrell 1988; Sinsch 2015), because of hibernation or prolonged dormancy in that region. For amphibians in temperate climates, each year of growth during the warm season, and the subsequent slowing of growth in the cool season (hibernation), commonly called Lines of Arrested Growth (LAGs), can be counted much like the growth rings in many trees (McCreary et al. 2008). Bone tissues of tailed amphibians are very simple in structure and LAGs are generally very clear and easily studied (Misawa & Matsui 1999). Because the periosteal cortex is the broadest and the medullar cavity narrowest in the middle part of the diaphysis of limb bones, this section is best-suited for skeletochronological detection of growth marks (Castanet & Smirina 1990; Castanet et al. 1993).

The first successful attempts on amphibians were based on the skull bonessuch as parasphenoid inNecturus maculosus (Senning 1940) and pterygoid inLithobatus [Rana] catesbianus (Schroeder & Baskett 1968).Thereafter, round bones have become the material of choice (Sinsch 2015; Castanet & Smirina 1990; Castanet et al. 1993). Skeleto-chronology has been used to study variety of anurans (Khonsue et al. 2001; Yilmaz et al. 2005; Ashkavandi et al. 2012) and urodeles (Lima et al. 2000; Bovero et al. 2006; Eden et al. 2007; Uzum 2009; Farasat & Sharifi 2016). Age and body size are two demographic traits or standard characteristics that were used by many researchers for understanding the evolutionary life history and ecology of amphibians (Morrison & Hero 2003; Liao & Lu 2011; Gül et al. 2014). Due to the critically - imperiled status of much of salamanders, skeletochronological analysis of amphibian tissues from museum collections may provide some insights into historic demographic constituency of populations, species, and could also assist in planning or management wild populations if reintroduction is attempted in the future (Lindquist et al. 2012).Caetano & Leclair (1996) applied this method to red spotted newts (Notophthalmus viridescens) using the circumference of the LAGs in cross-sections of the humerus (Snover 2002).

There is little information on skeleton-chronological studies of Iranian amphibians. One of these published papers is on the marsh frog, Rana ridibunda (Ashkavandi et al. 2012), and the second on green toad, Bufo viridis (Ashkavandi et al. 2012). Age determination and growth of the endangered Kaiser^’s mountain newt, Neurergus kaiseri in the Southern Zagros range is the only published paper on Iranian salamanders (Farasat & Sharifi 2016). This research is the first study on skeletochronology of the Persian mountain salamander, Paradactylodon gorganensis in Iran.

Two families, four genera and seven species of salamanders have been reported from Iran (Baloutch & Kami 1995; Rastegar-Pouyani et al. 2008, 2015; Safaei- Mahroo et al. 2015).The Persian brook salamander, Batrachuperus persicus (Eiselt & Steiner 1970) is the first Hynobiid salamander described based on five salamander larvae collected around Asalem in the Talesh Mountains,  Guilan Province,  Iran (Eiselt & Steiner 1970), and subsequently reported from Weyser in southeast of Chalus City,  Mazandaran Province, Iran (Schmidtler & Schmidtler 1971; Ahmadzadeh & Kami 2009). New distribution records (Kami &Vakilpoure 1996) and adult specimens of this species were described for the first time (with photos of B. gorganensis) in Guilan and Ardabil provinces, Iran (Kami 1999).A second hynobiid salamander, Persian mountain salamander, Batrachuperus gorganensis (Clergue-Gazeau & Thorn 1979) was described from Shirabad cave in Golestan Province (Clergue-Gazeau & Thorn 1979) and was identified as P. persicus some years ago (Kami 2004). This species was classified in a new genus, Paradactylodon (Risch 1984) and Iranodon (Dubios & Raffaelli 2012). Total length of this salamander reaches 282 mm (Kamiunpublished data) (Fig. 1). This species status is categorized as Critically Endangered (CR) in the IUCN Red list (Safaei- Mahroo et al. 2015). The important aims of this study were to compare the age structure and body size, to determine the longevity of males and females, to gain the correlation between age and SVL as well as to provide data for species conservation in a population of Paradactylodon gorganensisin Golestan Province, Iran.

MATERIALS AND METHODS

A total of 12 adult males and 15 adult females and two unknown sex adult specimens were studied. All specimens were deposited in Zoological Museum of Golestan University (ZMGU). These materials were collected from Shirabad Cave (36°57' N, 55° 03 ' E) between years 1994-2009. This cave is situated in about 70 km east of Gorgan City, southeast of Khanbebain and Shirabad Village at about 420 m above sea level. The snout - vent length (SVL) was measured to the nearest 0.01 mm, using a digital caliper. Sexes of salamanders were determined after dissection byobserving their gonads. Two different long bones were used in this study including the femur and tibia of the left hind limb. Bones were fixed in 10% formalin, the bones were decalcified in 10% (v/v) chloric acid during the period of decalcification, for 48 h. After decalcification, the bones were washed thoroughly for 24 hand dehydrated with 70, 80, 90, 95, 100% Methanol (eachfor 1.5 h). Cleaningthe surrounding tissues with Xylol (for 1.5 h), processed in paraffin block preparation (for 1.5 h), tissues were embedded in small paraffin block. Serial sections (5, 7, 10  and for some specimens20 ) were cut using a rotary microtome, stained with Hematoxylin and Eosin, and observed under an Olympus BX51 microscope equipped with digital camera DP12. The camera mounted and linked to a monitor and computer with image software. The number of lines of arrested growth (LAGs) was counted in the periosteal bone.

 Age classes and SVL were normally distributed (Kolmogorov-Smirnov D test, P- value > 0.05), thus allowing comparisons using parametric tests (t - test).

We used Pearson correlation to examine the relationship between age and SVL. Data analysis was performed using SPSS 22 and Excel 2013 (Microsoft Office), and interpreted at   = 0.05.

Fig. 1.Adult P. gorganensis from Shirabad cave, total length 282 mm (Photo by H.G.K).

RESULTS

The averaged SVL were 103.85  10.48 mm in males, 105.96  15.89 mm in females and also 108.00 and 123.00 mm in the two unknown sex specimens (Table. 1).

In all examined sections, the hema-toxylinophilic lines, interpreted as LAG, were clearlyobserved in the cross-section (Fig. 2).

Both SVL and age showed normal distribution (Kolmogorov-Smirnov test, P > 0.05).The range of age was 7-13 years in females, 8-11 years in males and also 7 and 8 years in the two unknown sex specimens (Fig. 3). The Pearson correlation showed no significant correlation between SVL and age (P > 0.05, Fig. 4).

Fig.2. Cross-section of tibia and femur of P. gorganensis.  Arrows: line of arrested growth (LAGs); EB = Endosteal Bone, m.c. = marrow cavity, PB = Periosteal Bone, P = Periphery. (A) Femural bone cross - section at diaphyseal level, male (SVL= 96.97 mm), 10 LAG, 7 . (B) Tibia bone cross section at diaphyseal level, Female (SVL= 120.06 mm), 10 LAGs were observed in the periosteal bone, 5  (C) Femural bone cross section at diaphyseal level, male (SVL= 108.58 mm), 8 LAG, 10 . (D) Femural bone cross section at diaphyseal level, Female (SVL= 104.84 mm), 8 LAG, 10 H&E.

Fig. 3. Age frequency of the P. gorganensis (adult specimens).

Fig.4. Absence of correlation between age classes and SVL in females, males and unknown sex specimens of P. gorganensis from Golestan Province, Iran.

Table 1. Age and SVL^† of the P. gorganensis in a population from Golestan Province. Values are mean standard deviation and sample size.

                                                    The snout-vent length^†

                                                    Millimeter‡

                                                    Number of individuals^§