Sex ratio estimations of loggerhead sea turtle hatchlings by histological examination and nest temperatures at Fethiye beach, Turkey

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1 Naturwissenschaften (2006) 93: DOI /s SHORT COMMUNICATION Yakup Kaska. Çetin Ilgaz. Adem Özdemir. Eyüp Başkale. Oğuz Türkozan. İbrahim Baran. Michael Stachowitsch Sex ratio estimations of loggerhead sea turtle hatchlings by histological examination and nest temperatures at Fethiye beach, Turkey Received: 12 July 2005 / Accepted: 7 March 2006 / Published online: 11 May 2006 # Springer-Verlag 2006 Abstract Hatchling sex ratios in the loggerhead turtle (Caretta caretta) were estimated by placing electronic temperature recorders in 21 nests at Fethiye beach during Over the seasons, the mean temperature in the middle third of the incubation period ranged from 26.7 to 32.1 C, and incubation periods ranged from 49 to 67 days. Based on the mean temperatures during the middle third of the incubation period, and on histologically sexed dead hatchlings, the sex ratios of hatchlings at Fethiye beach were roughly equal, i.e % of the hatchlings were females. This contrasts with the highly female-skewed sex ratios in loggerhead turtles elsewhere; Fethiye has a Y. Kaska (*). E. Başkale Pamukkale Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü Denizli Turkey caretta@pamukkale.edu.tr Tel.: Fax: İ. Baran Dokuz Eylül Üniversitesi, Buca Eðitim Fakültesi, Biyoloji Bölümü, Buca-Izmir, Turkey O. Türkozan Adnan Menderes Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Aydin, Turkey M. Stachowitsch Faculty of Life Sciences, Department of Marine Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria Ç. Ilgaz Dokuz Eylül University, Fauna and Flora Research and Aplication Center, Buca-Izmir 35150, Turkey A. Özdemir Adnan Menderes Üniversitesi, Egitim Fakültesi, Ilkögretim Bölümü, Aydin 09010, Turkey relatively high proportion of male hatchlings. For endangered sea turtles, the knowledge of hatchling sex ratios at different beaches, coupled with appropriate conservation measures, can make an important contribution to their survival. Introduction Sexual differentiation of sea turtle hatchlings is determined by egg incubation temperature, usually during the middle third of development (Yntema and Mrosovsky 1980; Mrosovsky 1994). The temperature at which an equal sex ratio is produced has been termed the pivotal temperature, and mainly females are produced above this temperature and males below this temperature (Yntema and Mrosovsky 1980; Mrosovsky and Pieau 1991). Nest temperatures can differ due to latitudinal variation, seasonal temperature changes, shading by vegetation, sand colour, episodic events such as rain and depth of the eggs (Hays et al. 2001; Matsuzawa et al. 2002); temperature increases due to metabolic heating of embryos (i.e. Godfrey et al. 1997) have also been reported. Sex ratios skewed as much as 90% toward females have been found or inferred for loggerhead turtle (Caretta caretta) hatchlings in the Mediterranean (Kaska et al. 1998; Godley et al. 2001; Mrosovsky et al. 2002) and elsewhere (Brazil and the USA; Marcovaldi et al. 1997; Hanson et al. 1998). In the Mediterranean, Kaska et al. (1998) found a mean sex ratio of 81.6% females in loggerhead clutches laid over the 1995 and 1996 nesting seasons in Cyprus and Turkey. Godley et al. (2001) reported very short incubation durations for loggerhead turtles in Cyprus, implying warm, feminising conditions (89 99% females). In general, the pivotal temperatures in studies of loggerhead turtles all cluster within one degree of 29 C (Mrosovsky 1994; Marcovaldi et al. 1997). Based on two clutches from Greece incubated in the laboratory, Mrosovsky et al. (2002) reported a pivotal temperature of 29.3 C, a pivotal incubation duration of 52.6 days and a field-analogous duration of 56.6 days, obtained by adding 4 days of hatching in the field to the

2 339 first emergence at 52.6 days in the laboratory. For the eastern Mediterranean, Kaska et al. (1998) used mean temperatures in the middle third of incubation to indicate a pivotal temperature just below 29 C and the calculated pivotal incubation duration to be 59.9 days, close to the values of 59.3 and 61.7 days for Brazil and the USA, respectively. These data indicate similar pivotal temperatures of loggerhead turtle populations in the Mediterranean and elsewhere. Although female-dominated sex ratios (81.6%) on Turkish beaches have been reported, Fethiye beach was highlighted as producing the most males (37%, obtained from two nests) due to cooling effects of the pebbles on the beach (Kaska et al. 1998), much like what was reported in Brazil (Baptistotte et al. 1999). We therefore more closely examined this site based on the preliminary findings reported by Kaska et al. (1998). The aim was to generate an overall estimate of the sex ratio of hatchlings produced within three seasons. The approach was to collect data on the mean temperatures during the middle third of the incubation period to predict the sex ratios within the nests, and then to compare this result with the sexes of dead hatchlings from the nests in which temperatures were recorded. Materials and methods Fethiye beach, one of the main loggerhead turtle nesting grounds in the Mediterranean, is located in southwest Turkey and is about 8 km long (for a map and further details, see Türkozan and Baran 1996). Temperatures in loggerhead turtle nests were examined using electronic Tinytalk temperature recorders (Orion Components, UK) during the nesting seasons. The temperature recorders were calibrated against a thermometer of known accuracy [National Accreditation of Measurement and Sampling (NAMAS)] between 15 and 40 C. The average of recorded temperatures was identical to the NAMAS thermometer (standard deviation ±0.1 C). The temperature recorders were placed into the centre of the nests (ca. 44 cm depth) either during oviposition (N=16), after oviposition but on the same night or in the following morning in the case of relocated nests (N=5). To determine the effect of metabolic heating, the temperatures in one nest were recorded at three levels (top, centre and bottom, i.e. ca. 33, 44 and 55 cm) and, in two additional nests, at two adjacent positions (centre of nest and at same depth in the sand outside the nest). The middle third of the incubation period was calculated based on an incubation time defined as the number of days from the date of egg deposition to the date of the first emergence. Nest contents were excavated after the last hatchlings emerged; nest depths were measured and data loggers were retrieved. The total number of eggs and the hatching success were calculated by counting unhatched eggs and hatched shell fragments. The sex ratios of hatched loggerhead turtles were estimated using two methods: The first used the mean temperature during the middle third of the incubation period and the sex ratio employed by Kaska et al. (1998) for Mediterranean loggerheads and by others (i.e. Houghton and Hays 2001, Godley et al. 2001). Here, the plots of mean temperatures during the middle third incubation period and corresponding sex ratio (% female), together with the pivotal incubation durations (duration that yields 50% of each sex) of the mean incubation temperatures, were obtained using the data from Kaska et al. (1998). The second method used to estimate the sex ratios is based on sexing all the dead hatchlings and late-stage dead embryos (>stage 25) (Kaska and Downie 1999) found in suitable condition in the 21 nests by histological analysis of the gonads (Yntema and Mrosovsky 1980). A total of 85 dead hatchlings were evaluated (36, 29 and 20 for 2000, 2001 and 2002, respectively). Nesting data were recorded during standard night and early morning beach patrols (Table 1). The 21 nests were selected as being representative of the beach and the main nesting seasons. Results The nesting season started in late May and continued until mid-august in all 3 years ( ) studied. All 21 nests studied here were recorded during June and July, reflecting the peak nesting season (3-year average nesting dates: 8% in May, 63% in June, 28% in July and 1% in August). The spatial distributions of nests were similar in different years based on the distribution of suitable sandy zones of the beach. The information on the temperaturerecorded nests is presented in Table 1. The mean temperature of the whole incubation period for the 21 nests ranged from 26.8 to 31.8 C. The maximum temperature increase during the incubation period was 7 C (for nest 11, minimum of 26.3 C, maximum of 33.3 C). On average, these increases were higher in the first third of the incubation period (maximum increase 5.3 C) and lower in the middle third (maximum increase 3.8 C); in the last third, values initially increased and then dropped (Fig. 1). Figure 1 shows two nests that were laid on the same day, the first (nest 2), 15 m from the sea, the second (nest 3), 8 m from the sea. This comparison reveals that distance from the water table (or sea) along with clutch size can considerably affect nest temperature, the sex of the hatchlings and the incubation period. The difference between the mean temperatures of these two nests over the entire incubation period was 2.7 C, and the difference between the incubation periods was 13 days. Thus, a 1 C change effected a 4.8-day change in incubation period. The mean temperature during the middle third of the incubation ranged from 26.7 to 32.1 C. The sex ratio of hatchlings for all nests was estimated according to the nest temperatures (Table 1). There were no statistically significant differences between the in situ and relocated nests in either the mean temperature (t=0.02, df=9, P>0.05) (both during the middle third and entire incubation periods) or in the sex ratio (t=0.03, df=9, P>0.05). The data were therefore pooled. The mean sex ratio for all 21 nests was 60.8% females [minimum of 31% (mean of nest 1), maximum of 96%]. Because the tops of nests will be

3 340 Table 1 The 21 temperature-recorded nests and descriptive statistics of the temperature data for the entire and middle third of the incubation periods together with the estimated sex ratio (% females) Nest no Lay date Clutch size Depths (cm) T M B Emergence success No of Distance IP sexed hatchlings N F M to sea (m) (days)+ days of hatching Total IP Middle third IP Sex Mean ±SD Min Max Mean ±SD Min Max ratio (% ) 1 T 28 Jun M 28 Jun B 28 Jun Jun Jun Jul (R) 03 Jul Jul M 06 Jul S 06 Jul (R) 07 Jul S 24 Jul M 24 Jul Jun Jun Jun Jul (R) 09 Jul Jul (R) 17 Jul Jun Jun Jul (R) 14 Jul Jul Mean ±SD 84.4 T:32.6± ±16.3 M:44.3±1.3 ±12.8 ± B:55.4±1.7 ±5.5 ±1.4 ±1.2 ±1.8 ±1.4 ±1.2 ±1.7 ±20.06 Values in bold are either discussed in the text or are the minima and maxima T top, M middle, B bottom, S outside the nest, R relocated nest, N sample size, F female, M male, IP incubation periods Fig. 1 Temperatures in two nests (top, nest 2, and bottom, nest 3) laid on 29 June 2000 at Fethiye beach. The arrows indicate the start of hatchling emergence, which was on 15 and 28 August 2000 for nests 2 and 3, respectively

4 341 warmer than the bottom during the middle third of incubation (see nest 1-T/-M/-B in Table 1), we can expect the percentage of females to be higher among the uppermost eggs if the temperatures are already well above pivotal. Positioning temperature recorders in both the sand and the nests allowed us to compare the temperatures at the same depth in both settings. The daily mean sand temperatures were C lower than in loggerhead nests at the same time and depth. In nests 7 and 9 (Table 1), for example, the mean values in the centre of the nests were higher than in the adjoining sand. The temperature increased by a similar amount in both nests during the middle third of the incubation period, when sex is thought to be determined. The differences between the mean temperatures during the entire incubation period and on the sides of nests during this period were only C. These differences were greater during the middle third of the incubation period: 2.4 C ( C) for nest 7 and 1.4 C ( C) for nest 9 vs 3 C ( C) next to nest 7 and 1.5 C ( C) next to nest 9. The relationship between the mean temperature during the middle third of the incubation period and the sex ratio (percentage of females) was significant (F 1,23 =22,260, r 2 =0.97, P<0.001) (Fig. 2a). The relationship was also significant (F 1,23 =1,097, r 2 =0.98, P<0.001) between the mean nest temperature during the entire incubation period and the incubation period (Fig. 2b), even when only the temperature during the middle third of the incubation period was considered (F 1,23 =310, r 2 =0.93, P<0.001) (Fig. 2c). The mean incubation temperatures can be used to estimate the incubation period. In general, a 1 C Fig. 2 Temperatures, sex ratios and incubation periods of 21 nests on Fethiye beach. a Comparison of mean temperatures during the middle third of the incubation periods and arc-sin-transformed sex ratio. b Correlation of mean incubation temperatures to the total incubation periods. The pivotal incubation duration of 59 days is indicated. c Correlation of mean incubation temperatures during the middle third of the incubation period to the total incubation periods

5 342 decrease in the former increases the latter by 4 5 days (Table 1 and Fig. 2b). The mean incubation period for the 21 nests studied here was 58±5.5 days, suggesting that Fethiye beach produced a sex ratio of nearly 60% females. The durations of hatchings (Table 1) were generally more than 2 days, usually around 6 days. The 85 histologically sexed dead hatchlings from the temperature-recorded nests yielded 64.7% (N=55) females for the three seasons. The annual values [63.9% (N=23 females) in 2000, 65.5% (19 females) in 2001 and 65% (13 females) in 2002] are very similar, and further support the results obtained by the temperature-based method. Discussion Direct temperature measurements in nests and the sexing of dead hatchlings are the two practicable approaches for estimating the sex ratios in the nests of endangered sea turtles. Kaska et al. (1998) compared the sex ratios at different nest levels and showed that the results at central levels (82.7%) are very close to the general mean (81.6%) and to the mean of the top and bottom levels (80.9%). Recording only central parts of the nests therefore yields the best estimation of sex ratios (Hanson et al. 1998; Kaska et al. 1998). The reported sex ratio for loggerhead sea turtles is generally female dominated (Mrosovsky 1994; Kaska et al. 1998; Öz et al. 2004). In Brazil, Marcovaldi et al. (1997) used pivotal incubation durations to estimate that 82.5% of the loggerhead hatchlings were female. Based on incubation durations, Godley et al. (2001) also suggested a female-biased ratio in Cyprus and elsewhere. Finally, laboratory work designed to determine the pivotal incubation period for Mediterranean loggerheads points to a female-biased sex ratio (Mrosovsky et al. 2002). Sex ratio estimations and their biological and ecological implications are clearly a complex issue. While most sites in Brazil are largely female-producing, some key sites have conditions that are biased toward producing male hatchlings (Baptistotte et al. 1999). The present study estimates that Fethiye may also be one such beach that produces a lower percentage (60 65%) of female hatchlings. In our study, the daily mean sand temperatures were C lower than the corresponding daily means in loggerhead nests at the same depths. These means are lower than the 2.2 C difference that Matsuzawa et al. (2002) measured in a loggerhead clutch vs sand. The general pattern of metabolic heating and the comparison of nest centre and adjoining sand temperatures were similar to previous studies: metabolic heating was recorded during the second half of incubation (i.e. Godfrey et al. 1997), with a peak followed by a gradual decline in nest temperatures toward the end of incubation (Fig. 1). Emergence asynchrony of our nests (hatching took 2 6 days) may reflect temperature- and development-related differences: patterns of short vs long hatching durations with longer hatching durations potentially indicating cool temperatures, lengthier development and more male hatchlings should be investigated in future studies of sex ratios. Moreover, the longer the incubation period, the greater the chances of that nest being predated. The smell and activities of the first group(s) of hatchlings may provide clues for predators about the location of a nest, and those emerging toward the end of the hatching duration are threatened most (Kaska 2000). At a time when the threats to nesting beaches are increasing and funding levels for conservation programmes may not be keeping pace, it becomes ever more important to make informed decisions about appropriate strategies. Monitoring of hatchling sex ratios should be a part of this effort, especially if nests are to be translocated to hatcheries or to safer locations on the beach (Wibbels 2003). Information on sex ratios and the complex of factors involved in determining them can provide input for the debate on sex ratio manipulation as a conservation strategy, support predictions on potential climate-change-related shifts in sex ratios and help to better focus conservation efforts for endangered sea turtles. These considerations are especially important on beaches such as Fethiye, which produce relatively high proportions of males. Acknowledgements We thank all the volunteers from Pamukkale, Dokuz Eylul, Adnan Menderes and Vienna Universities for their help during the fieldwork. Y. Kaska thanks the Pamukkale University Research Fund for partly financing the project (2000-FEF01- BAD001). M. Stachowitsch wishes to thank his assistant, C. Fellhofer and acknowledges support from the Society of the Friends of Schönbrunn 200 and Gulet Touropa Touristik. We are grateful to Tony Holmes for English corrections on an earlier manuscript version. The manuscript has improved considerably as a result of comments received from anonymous referees. References Baptistotte C, Scalfoni JT, Mrosovsky N (1999) Male-producing thermal ecology of a southern loggerhead nesting beach in Brazil: implications for conservation. Anim Conserv 2:9 13 Godfrey MH, Barreto R, Mrosovsky N (1997) Metabolicallygenerated heat of developing eggs and its potential effect on sex ratio of sea turtle hatchlings. J Herpetol 31: Godley BJ, Broderick AC, Mrosovsky N (2001) Estimating hatchling sex ratios of loggerhead turtles in Cyprus from incubation durations. Mar Ecol Prog Ser 210: Hanson J, Wibbels T, Martin RE (1998) Predicted female bias in hatchling sex ratios of loggerhead sea turtles from a Florida nesting beach. 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