This Article Appeared in a Journal Published by Elsevier. the Attached

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy

Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147

Contents lists available at SciVerse ScienceDirect

Journal of Experimental Marine Biology and Ecology

journal homepage: www.elsevier.com/locate/jembe

Estimating the sex ratio of green sea turtles (Chelonia mydas) in Taiwan by the nest temperature and histological methods

Rowena King a,⁎, Wan-Hwa Cheng b, Cheng-Tsung Tseng c, Hochang Chen c, I-Jiunn Cheng c,⁎ a St. Vincent Girls' High School, P.O. Box 1057, Kingstown, Saint Vincent and The Grenadines b Department of Biological Science, Central Florida University, Orlando, FL 32816, USA c Institute of Marine Biology, National Taiwan Ocean University, Keelung 202-24, Taiwan, ROC article info abstract

Article history: Sex ratio is an important population characteristic. It is especially important for endangered species with Received 23 July 2012 temperature-dependent sex determination, such as sea turtles. As turtle sex ratio at hatching is primarily Received in revised form 28 March 2013 inﬂuenced by the environmental temperature, the ability to estimate sex ratio is crucial to the success of conser- Accepted 29 March 2013 vation measures. Green turtles (Chelonia mydas) are the only sea turtles that nest in Taiwan. Despite much region- Available online xxxx al research on the ecology of this species, the sex ratio has not yet been determined in Taiwan. We combined measures of nest temperature during the thermally sensitive period with a histological method to estimate the Keywords: Green sea turtle hatchling sex ratio from three nesting islands in Taiwan from 2010 to 2011. We determined that the sex ratios Hatchling sex ratio for green turtle populations in Taiwan were female biased during the study period. In line with that overall Histological method trend, Wan-an Island with drier and hotter weather during the nesting season, produced more female hatchlings Nest temperature than the other main nesting island—Lanyu Island. Results of this study stress that differences in sex ratio are im- Nesting islands portant to consider when developing conservation strategies, even among closely located sites such as Wan-an and Lanyu Islands. This is the ﬁrst study of the hatchling sex ratio of green sea turtles in the East Asian region. © 2013 Elsevier B.V. All rights reserved.

1. Introduction For immature turtles, the lack of morphological sex characteristics has made distinguishing the sexes difficult. (Larios, 1999; Wibbels, It is important to assess the sex ratios of endangered species with 2003). The sex of juvenile animals can be determined either by lapa- temperature-dependent sex determination, such as sea turtles, so roscopic observation of gonads (Limpus and Reed, 1985; Wibbels, that the conservation implications of skewed sex ratios can be consid- 1999, 2003) or indirectly by radioimmunoassay (RIA) of testosterone ered. Recent reviews of global climate change (e.g., IPCC, 2007) have in the blood serum (Owens et al., 1978; Wibbels, 1999). For hatch- demonstrated the impact of temperature on organisms worldwide. lings, two methods are currently employed: direct and indirect Because temperature is rising globally, it is important to determine (Wibbels, 2003). Direct methods involve sacrificing hatchlings or the effects of temperature and other factors on the sex ratios of sea using dead hatchlings found in the nest. Their sex can be determined turtle populations, and to learn whether their environmental sex de- by careful gross morphological examination of the gonad exterior and termination operates similarly among species and populations global- accessory duct characters (Wyneken et al., 2007) or by histological ly (e.g. Hawkes et al., 2009; Hays et al., 2010; Houghton et al., 2007; examination of the gonad (e.g. Ceriani and Wyneken, 2008; Newson et al., 2007; Poloczanska et al., 2009). Sea turtles are endan- Wibbels, 2003). Of these direct two methods, the latter is considered gered species with sex ratios, at least at egg hatching, determined by more accurate (Mrosovsky and Benabib, 1990; Mrosovsky and nest temperature. Information on the sex ratio at every life stage is Godfrey, 1995). It incurs fewer sex-identification errors than the crucial to conservation measures intended to protect these species. gross morphology method (Mrosovsky and Benabib, 1990). Indirect Several methods have been used to determine the sex of sea turtles. methods use pivotal temperature, sand temperature or incubation For adult turtles, sex identification is not difficult, because male duration to predict the hatchling sex ratios from the nesting beach turtles have a large and muscular prehensile tail extending well be- (e.g. Broderick et al., 2000; Godfrey et al., 1999; Godley et al., 2001; yond the carapace. The tails of females are short and, at most, project Marcovaldi et al., 1997; Mrosovsky et al., 1999, 2009). That reduces only slightly beyond the edge of the marginal scutes (Wibbels, 1999). the tedious work of sexing individual hatchlings. However, it also re- quires sacrifice of large numbers of hatchlings to obtain the pivotal temperature (PT) and Transitional Range of Temperature (TRT) ⁎ Corresponding author. Tel.: +886 2 24622192x5303; fax: +886 2 24628974. from a specific beach in the preliminary work (Wibbels, 2003). De- E-mail addresses: [email protected] (R. King), [email protected] (W.-H. Cheng), [email protected] (C.-T. Tseng), [email protected] (H. Chen), spite morphological differences it is difficult to estimate the sex [email protected] (I.-J. Cheng). ratio of adults and juveniles in any given sea turtle population. For

R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147 141 the adults, only the females emerge to nest, and they do not nest 2. Material and methods every year. Despite this logistical problem, it is possible to estimate sex ratios of breeding adults from fishery by-catches and direct under- Two methods were used to determine hatchling sex ratios in this water observations. Juveniles are difficult to locate, and if found, it is study: the histology of dead hatchlings and the nest temperature uncertain what proportion of the population they represent (Wibbels, during the sex-determination period. The endangered status of green 2003). Sex identification of hatchlings is, therefore, the most reliable turtles in Taiwan and the low number of turtles nesting in Taiwan method to estimate the early-life sex ratio of a population. prevented us from sacrificing whole clutches as has been done in Several factors have been found to influence the sex ratio of hatch- some other studies (e.g. Godfrey et al., 1996; Mrosovsky, 1988). In addi- lings. They include latitude, season, shading such as by nearshore forest, tion, logistic limitations (e.g. insufficient manpower in the field, poor sand color, precipitation, nest depth and anthropogenic events like beach weather conditions, etc.) prevented us from extensively sampling the nourishment (adding sand) and adjacent housing projects (Broderick et clutches. To amend this shortfall, we recorded the clutch temperatures al., 2000; Godfrey et al., 1996; Hawkes et al., 2009; Hays et al., 1999; with loggers and estimated the clutch temperature from the sand tem- Houghton et al., 2007; Kamel and Mrosovsky, 2006; Kaska et al., 2006; perature near clutches for which loggers were not available. The sex Morreale et al., 1982; Mrosovsky, 1988; Mrosovsky et al., 1984; ratios of the green turtle hatchlings from clutches not examined directly Pavenport, 1997; Poloczanska et al., 2009; Wibbels, 2003). These factors can be approximated from the incubation temperatures. are all related to in situ sand temperature. Temperature influences the sex of the hatchling through the sex determination cascade, by activating 2.1. Field data collections genes encoding enzymes for steroid synthesis and for enzymes such as aramatase (Godfrey et al., 2003; Hulin et al., 2009; Merchant-Larios et Data were collected from Lanyu and Wan-an during the nesting al., 1997; Pavenport, 1997; Wibbels, 2003). This will result in the varia- seasons (June to October) of 2010 and 2011, with the addition of tion of sex ratios on both the spatial (different nesting beaches) and LiuChiu Island in 2011. Beaches were patrolled for nesting females temporal (yearly) scales. Therefore, it is important to determine the each night and early morning. Once a female had finished camouflaging sex ratios of turtles from different nesting beaches in order to conduct her nest, eggs were excavated within 4 h of oviposition and the clutch proper conservation measures. size (total number of eggs laid) was determined. Morning patrols Five species of sea turtle live near Taiwan, namely green turtle were conducted to make sure that no nest had been missed the previ- (Chelonia mydas), loggerhead turtle (Caretta caretta), hawksbill turtle ous night, and patrols were also conducted on the earliest expected (Eretmochelys imbricata), olive Ridley turtle (Lepidochelys olivacea) hatchling emergence date, so that the emerging hatchlings could be and leatherback turtle (Dermochelys coriacea). Among them, only recorded. Nests were excavated 3 to 5 days after the first emergence. green sea turtles nest on beaches in Taiwan (Chen and Cheng, 1995; On Wan-an Island, hatchlings were dissected on the day they were col- Cheng, 2011). Most research on the nesting beaches to date has lected, and the gonads were preserved in 10% buffered formalin. On the concentrated on the nesting ecology, nesting environment and satel- other two islands where no laboratory was available, whole hatchlings lite tracking of individuals (e.g. Chen and Cheng, 1995; Chen et al., were preserved in 10% buffered formalin. They were then transported 2010; Cheng, 2000; Cheng et al., 2008). No previous study has esti- back to the laboratory for histologic studies. mated the sex ratios of the green turtle populations nesting in Taiwan. The sex ratio provides vital information about the contribu- 2.2. Sand and nest temperature recordings tion of the two sexes to the genetic diversity of the population, its mating probabilities and reproductive potential and, thus, about the Sand and nest temperatures were determined with temperature fitness in the long run of a stock or of a whole species (Sibly and loggers (HOBO® U22-001 Water Temp Pro v2). These loggers have Calow, 1986; Townsend and Calow, 1981). This parameter is especially a memory size of 65 kB of computer memory with an accuracy of important for long-lived species with the temperature sex determina- 0.2 °C over a range of −20 °C to 70 °C and a resolution of 0.02 °C at tion (TSD) characteristic of sea turtles, because it is one aspect of how 25 °C. They weigh 42 g, close to the weight of one green turtle egg. these animals maintain viable population sizes. It is an important fea- Loggers were buried in the centers of the nests, at a depth of ca. ture of the life history strategy of green turtle populations in Taiwan. 70 cm, to record the nest temperature. They were left in place for Many field studies have found that hatchling sex ratios are female- the whole incubation period and retrieved during the excavation of biased (e.g. Booth and Freeman, 2006; Broderick et al., 2000, 2001; the nest. One control logger was buried in a randomly selected Godfrey et al., 1996). The TSD process, however, may provide differ- beach on each island at the same depth as the nest loggers. The con- ent sex ratios at different sites. Thus the first hypothesis of this trol loggers were left in the beach until the last nest was excavated. study was that the hatchling sex ratio of green turtles in Taiwan is Loggers were set to measure the temperature every 30 min. They also female-biased. However, a 10-year ecological study comparing were calibrated in °C against a traceable mercury-in-glass thermom- the two main nesting islands in Taiwan has shown that their nesting eter in the laboratory prior to deployment. environments are significantly different (Cheng et al., 2008). Thus, the second hypothesis was that the hatchling sex ratios of these two 2.3. Sex determination of the hatchlings in the laboratory islands are different. The sexes of hatchling were determined by the histologic method of Yntema and Mrosovsky (1980), which provides accurate results. In 1.1. Study sites the laboratory, hatchlings were dissected, and the organs around the kidneys were excised. The gonads attach to the ventral side of the kid- The fieldwork took place on three different nesting islands (Fig. 1). neys. Histological procedures were briefly as follows. Samples were Lanyu Island (22°00′–08′N, 121°50′–60′E) is approximately 65 km off soaked in 70% alcohol for 24 h, then dehydrated in a Tissue-Tek® the south-east coast of Taiwan; LiuChiu Island (22° 20′ N, 120° 22′ E) VIP®Jr. machine (Sakura Finet Co., Ltd.) to prepare them for infiltration is located to the south-southwest, approximately 14 km off Taiwan; with paraffin of 60 °C melting point. Tissues were embedded in paraffin and Wan-an Island (23°22′N, 119°30′E) in the Penghu archipelago in stainless steel molds using a Shandon Histocentre Embedding ma- is approximately 60 km off the southwest coast of Taiwan. Wan-an chine (Shandon, Inc.). After resting on a cooling plate for approximately and Lanyu islands are the main nesting sites of green turtles in Tai- 15 min, the tissue samples were ready for sectioning with a Shandon wan (Cheng et al., 2008). LiuChiu Island was identified as a third M1 microtome (Shandon, Inc.) (Ceriani and Wyneken, 2008). Three nesting site in 2011. ribbons of 10 μm sections, 10 sections in length were taken from each Author's personal copy

142 R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147

Fig. 1. Map of Taiwan with sampling sites marked in circles: Wan-an Island, Lanyu Island and LiuChiu Island.

sample to guarantee that the gonad would be present on the stained 2.4. Statistical analyses slides. Tissues were stained with Hematoxylin and Eosin (H&E) in 2010. Hematoxylin primarily stains nuclei blueish purple, while Eosin Linear correlation was used to determine the relationship between stains cytoplasm and extracellular substances pink to orange (Neill, sand and nest temperatures and to predict temperatures of nests 2008). In 2011, a Periodic acid-Schiff (PAS) staining method was used, without loggers during the sex determination period. A linear regres- because some H&E stained samples from 2010 were not very clear. sion analysis was used to determine the relationship between the sex PAS is very useful for outlining tissue structures such as basement mem- ratio and nest temperature during the sex determination period. The branes (Neill, 2008). When PAS was applied to the samples from 2010, percent female value was divided by 100 and arcsine square root the sex was easily determined. The failures with H&E might have been transformation was applied before the comparison was conducted. A due to the fact that the gonads of some hatchlings were partially Student's t-test was used to compare the sex ratio and sand characters decomposed. The PAS staining method, however, did not stain all the between Wan-an and Lanyu Islands. After the values for the three 2011 samples successfully. This might have been due to the much better islands were tested for normality and homogeneity (Sokal and Rholf, preserved status of some samples. H&E was then used to stain those 1982), one-way ANOVA was applied to determine whether the sex ra- samples. Sections of the kidney-gonad tissue samples were stained tios were significantly different among the three nesting islands. before mounting on slides. They were then examined under a Zeiss Where results were significant, a post-hoc Tukey test was used to de- Axioskop light microscope (Model: FH1032) at 10× and 40× magnifi- termine which island pairs were significantly different. cation to determine the sex of the hatchlings. The sex of each hatchling was determined based on the descriptive criteria distinguishing the 3. Results sexes on the basis of the gonad medulla and cortex histology, as given by Yntema and Mrosovsky (1980) and Ceriani and Wyneken (2008). 3.1. Number of nests and dead hatchlings collected from each island Those authors found a thick cortex and a disorganized medulla in female during the study period hatchlings versus a thin cortex and an organized medulla in males (Fig. 2). The sex ratio was expressed as percent female calculated as (Number of The numbers of nests and dead hatchlings sampled from the three female hatchlings / total number of hatchlings examined) × 100. islands during the two-year period are listed in Table 1. In 2010, a Author's personal copy

R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147 143

Fig. 2. Gonads of green turtle hatchlings. (a) Male hatchlings: cortex and seminiferous cords from two, somewhat decomposed specimens. (b) Female hatchlings: ovary showing the well-developed cortex and the randomly arrayed medullar tissue from two different individuals. c = cortex, m = medulla, sc = seminiferous cords. total of 16 clutches was examined with 142 hatchlings collected. In 3.2. Sex ratio estimated from histology of hatchlings 2011, a total of 8 clutches was examined with 108 hatchlings collected. For the whole study period, 13 clutches from Wan-an Island, 9 clutches Most hatchlings emerged in groups over a period of 2–3 days. To from Lanyu and 2 from LiuChiu Island were examined. Data for LiuChiu avoid interrupting the natural process of emergence, clutches were Island were only collected in 2011. excavated 3 to 5 days after the ﬁrst emergence. Some live hatchlings

Table 1 Number nesting females (nesters), total nests, number of nests examined and number of hatchlings sampled from each island in 2010 and 2011.

Nesting island Year Nesters Total nests Number of nest examined Number of hatchling collected

Lanyu 2010 21 36 8 108 2011 4 11 1 3 Wan-an 2010 5 16 8 34 2011 3 18 5 41 Xiao Liuqiu 2011 2 15 2 65 Total 24 251 Author's personal copy

144 R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147 could still be found in the nests after excavation that were later released Table 3 at night. Delay for a few days also meant that some dead hatchlings had The mean sex ratio (=percentage of females in all samples) from each nesting island during the study period. The abbreviation labels are the same as in Table 1. s.d.: stan- already decomposed when collected. Several embryos were dead in the dard deviation, n: sample size. early stages of development. The gonad characters of these embryos were decayed beyond recognition. The cortices of male hatchlings LN WN XLN Overall were much thinner than those of females; males had only one layer of Mean 68 93 100 84 cells, while females had multiple layers (Fig. 2). It was noted that, the s.d. 33 14 0 26 cortices of female hatchlings occasionally had a columnar appearance. n. 9 13 2 24 Due to the decayed status of many hatchlings, the cortices of females were detached from the medulla in some gonad sections. The organiza- tion of the medulla was a bit more difﬁcult to identify. However, careful ranging from 30.5 °C (XLN1) to 33.8 °C (XLN2). The mean middle-third observations showed seminiferous cords in the medulla of the male incubation temperature of 5 clutches on Wan-an Island in 2011 hatchlings (Fig. 2a). The appearance of the seminiferous cords matched was 32.1 ± 0.8 °C, ranging from 31.1 °C (WN5) to 33.6 °C (WN8) well with the description by Yntema and Mrosovsky (1980). Percent- (Table 4). For all clutches with embedded loggers, the lowest tempera- ages of females among dead hatchlings from each clutch are listed in ture throughout this period was 28.7 °C and the highest was 34.5 °C. Table 2. The percentage of females ranged from 11 to 100% in 2010 and 85 to 100% in 2011. The median for Lanyu clutches in 2010 was 3.4. Nest temperatures estimated from sand temperature on Wan-an 75%, and the interquartile ranged from 37 to 89% (n = 8). The median Island in 2010 for Wan-an clutches in 2010 was 100%, and the interquartile ranged from 96 to 100% (n = 8). Lanyu (n = 3) and LiuChiu Island (n = 2) Even though the nest temperatures on Wan-an Island were not both produced 100% females in 2011. In 2011, the median for Wan-an monitored in 2010, sand temperatures were monitored. Both the clutches was100%, and the interquartile ranged from 87 to 100% sand and nest temperatures were monitored in 2011. The relationship (n = 5). The percentage of hatchlings examined during the study peri- between these two parameters in 2011 was determined as: (nest od ranged from 1 to 51% of clutch size, averaging 9 ± 12% (n = 24). temperature) = 0.6013 (sand temperature) + 11.866 (n = 3744, r=0.871,p b 0.001). Because there was no difference in the sex ratio 3.3. Variation in nest temperature during the sex determination period between 2010 and 2011 (p = 0.724), we then assumed that the relationship between nest and sand temperature was similar between the Nest temperatures recorded during the middle-third of the incuba- two years. The mean nest temperatures during the middle third of incu- tion interval are shown in Table 3. Nest temperatures for Wan-an Island bation in 2010 could then be estimated from this equation by substitut- were not monitored in 2010. On Lanyu Island, the mean nest tempera- ing the measured sand temperature in that year. The estimated mean ture during the middle third of incubation was 29.7 ± 0.7 °C (n = 8), nest temperatures ranged from 30.4 °C to 32.3 °C (Table 5). ranging from 29.4 °C (LN3, LN8) to 30.8 °C (LN22). Only 1 clutch was monitored on Lanyu Island in 2011. Its mean middle-third incubation 3.5. Nest temperature and sex ratio data for nesting beaches in Taiwan temperature was 32.3 ± 1.0 °C. On LiuChiu in 2011, the mean middle-third incubation temperature was 32.2 ± 1.7 °C (n = 2), Due to the small number of clutches examined in this study, the sex ratios from all nests were pooled. The nest temperatures during the sex determination period (middle third of incubation) for all three islands Table 2 were plotted against the sex ratio determined by the histological Sex ratio of the hatchlings examined from each nest in 2010 and 2011. The label for each nest indicates the nesting island; LN = Lanyu Island, WN = Wan-an Island, methods and are shown in Fig. 3. A positive relationship between sex XL = Xiao Liuqiu Island. ratio and nest temperature (Arcsin (sex ratio) = −3.012 + 0.140 (nest temp.), n = 24, r = 0.479, p = 0.018) was determined. Nest Clutch size Number of Percentage Number Percentage hatchlings of hatchling of females of females examined examined (%) Table 4 2010 The IP (incubation period), clutch size and nests temperatures during the middle third LN3 103 10 10 4 40 of the incubation period of the monitored nests from three islands during 2010 and LN5 95 12 13 4 33 2011 nesting seasons. The abbreviation labels are the same as in Table 1. LN6 134 1 1 1 100 LN7 94 28 30 3 11 Nest no. Deposited date Clutch size Temperature during middle LN8 127 34 27 25 74 third of incubation (°C) LN14 126 13 10 10 77 (eggs) Mean s.d. Max Min LN21 126 6 5 6 100 LN22 116 4 3 3 75 2010 WN1a 103 5 5 5 100 LN3 4th July 103 29.5 1.5 27 31.6 WN2a 134 8 6 8 100 LN5 6th July 95 28.8 1.2 26.8 30.3 WN3a 124 1 1 1 100 LN6 7th July 134 29.7 1.4 27.4 31.5 WN4a 92 11 12 10 91 LN7 7th July 94 28.8 1.2 26.7 30.3 WN5a 131 2 2 1 50 LN8 8th July 127 29.5 0.2 29 29.9 WN6a 129 4 3 4 100 LN14 14th July 87 30.2 0.8 28.6 31.9 WN7a 45 2 4 2 100 LN21 20th July 126 30.7 0.9 29.4 32.4 WN8a 111 1 1 1 100 LN22 21st July 116 30.8 0.8 29.4 32.3

2011 2011 LN2b 127 3 2 3 100 LN2 9th July 127 32.3 1 29.9 33.4 XLN1b 135 2 1 2 100 XLN1 30th June 100 30.5 1.7 28.2 33.7 XLN2b 123 63 51 63 100 XLN2 11th July 122 33.8 1.2 32 36.3 WN5b 94 2 2 2 100 WN5 3rd July 94 31.1 1.4 28.7 33.4 WN6b 119 20 17 17 85 WN6 10th July 119 32.5 0.9 31 34.3 WN8b 126 8 6 7 88 WN8 27th July 126 33.6 0.6 32.7 34.5 WN9b 108 1 1 1 100 WN9 31st July 108 31.8 0.5 30.5 32.5 WN10b 100 3 3 3 100 WN10 9th August 100 31.8 1.1 29.2 33.6 Author's personal copy

R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147 145

Table 5 may not be 100% accurate, it can combine with other techniques to esti- Sand temperatures and the derived nest temperatures on Wan-an (WN) Island in 2010. mate the sex ratio of the population (Broderick et al., 2000). The unit is °C. The columnar cortices observed in this study matched with those Nest Sand temperature Derived nest temperature Standard deviation n described by Miller and Limpus (1981). Based on their descriptions, WN1a 30.4 30.1 0.5 672 the cortex of female hatchlings has columnar cells, whereas the WN2a 31.1 30.6 0.3 672 male cortex has flattened, squamous cells (Miller and Limpus, WN3a 30.8 30.4 0.9 672 2003). The cortices were very easy to identify in sections of gonad ru- WN4a 30.5 30.2 0.9 672 diment, and the primary characters of male and female organs were WN5a 31.9 31 0.5 672 fi WN6a 32.1 31.2 0.4 672 easy to differentiate. The cortex can be identi ed under a stereo mi- WN7a 32.3 31.3 0.2 672 croscope with X10 magnification. However, a higher magnification WN8a 31.6 30.8 0.6 672 is needed to identify the seminiferous cords (Fig. 2). In addition, tissue samples from whole, dead hatchlings preserved in 10% buffered formalin, as taken from nests on Lanyu and LiuChiu Island, are not Hatchlings from 13 of 23 nests were all (100%) females. All but two of ideal for internal preservation, because it took longer for the formalin those nests had middle-third intervals above 30 °C. The only nests with to infiltrate the gonads. a majority of males were two examined from Lanyu Island (LN5a, LN7a) in 2010, and their middle-third interval was just below 29 °C. 4.2. Sex ratio of green turtle hatchling populations in different islands The result from those two nests and the form of the fitted regression in Taiwan suggest that the pivotal temperature for green turtle sex determination in Taiwan is also close to 29 °C. Despite the female-biased sex ratios found for all nesting islands in Taiwan, sex ratio differed among islands. One-way ANOVA found 4. Discussion modest significance for the differences in sex ratio among the three islands (post-hoc Tukey test; p = 0.034). However, values from We determined that the sex ratios for green turtle populations in LiuChiu Island should not be considered, because only two clutches Taiwan were female biased during our study period. Similar results from one year were monitored. After excluding that island, statistical have been found in other green turtle populations (e.g. Booth and analysis showed that the Wan-an Island sex ratios were more female Freeman, 2006; Broderick et al., 2001; Casale et al., 2000; Godley et biased than those on Lanyu Island (Student's t-test, p = 0.031). It is al., 2001, 2002; Horikoshi, 1992; Kaska et al., 2006; Mrosovsky, clear that the green turtles nesting on Wan-an Island produced rela- 1994; Spotila et al., 1987). This is the first study ever done on the tively more female hatchlings than did those nesting on Lanyu Island. hatchling sex ratio of green sea turtles in the East Asian region. As Comparisons of the long-term nesting ecology between those two explained below, the resulting knowledge is important to the conser- islands (Wan-an Island 1992 to 2011; and Lanyu Island 1997 to vation measures for this endangered species, even on the regional 2011) have shown that Wan-an Island is drier and hotter during the scale. nesting season than Lanyu Island (Cheng, unpublished data). Other comparisons from 1997 to 2011 have shown that nests are deeper 4.1. Histological determinations sex of hatchlings on Wan-an Island (p = 0.027). Sand on Lanyu beach is dark, poorly sorted gravel, while on Wan-an Island beaches are composed of The logistic limitations encountered in this study only allowed us to white, moderately well sorted, coarse sand. Statistical analyses during determine the sex ratio from dead hatchlings left in the clutches. This al- the same period showed that the sand particles are coarser and ternative method was suggested by Wibbels et al. (1991).Eventhoughit less homogeneous on Lanyu Island than Wan-an Island. (p b 0.001;

Fig. 3. Percentages of females in clutches versus mean temperature during the middle third of the incubation interval from 24 nests examined in 2010 and 2011. Vertical dashed line represents the pivotal sex-determining temperature of 29 °C for sea turtles. Author's personal copy

146 R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147

Cheng, unpublished data from 1997 till 2012). The dark sand on to clutch size and incubation were negligible on both Wan-an Island Lanyu Island might be expected to produce more female skewed sex (Chen et al., 2010) and Lanyu Island (Cheng, unpublished data). Thus, ratios due to the greater absorption of incident solar radiation (Hays the hatchling sex ratio of green turtles in Taiwan is influenced mainly et al., 2001). However, the precipitation was greater on Lanyu Island by the meteorological events during the nesting season. during the nesting seasons (p b 0.001; Cheng, unpublished data The TSD character of sea turtles and the results of this study sug- from 1997 till 2012). The hilly terrain and steep mountain slope of gest that with the rise of global temperature, as predicted by the Lanyu Island result in copious underground water flows through ICPP, relatively more female hatchlings will be produced from Taiwan and over its nesting beaches (Kuo, 2008), and its water table is in the future. That would increase the probability of population extir- expected to be higher than that on Wan-an Island. That tends to pation if or when ambient temperatures induce the production of lower the sand temperature on the nesting beach. That may explain all female offspring. However, Hays et al. (2010) recently suggested why more male hatchlings were produced on Lanyu Island. Indeed, that adult males copulate substantially more often than females, all of our recent studies show that sand temperatures at nest depth and thus female biased sex ratios may translate into sufficiently are consistently lower on Lanyu Island than on Wan-an Island balanced “operational” sex ratios. Thus, important work for the future (p b 0.001; Cheng, unpublished data from 2006 till 2012). The should be to assess operational sex ratios, those among adults in the comparison of the two islands suggests that air temperature and indi- wild producing fertilized eggs. Adult sex ratios should be estimated rectly precipitation are two dominant factors determining the sex in sea turtles in may mating and nesting areas. ratio of green turtle hatchlings in Taiwan. In this study, we only determined the hatchling sex ratio of the green turtles for two years. However, sex ratio is important for demo- 4.3. Temperature in the nest during the sex determination period graphic studies of sea turtle populations. Thus, it will be necessary to determine the long-term spatio-temporal variations in hatchling sex Mean temperatures in the nest during the middle thirds of incuba- ratio for all the nesting sites in Taiwan. However, the tedious work tion in 2010 and 2011 on Lanyu Island were 29.7 and 32.3 °C, respec- of histologic analysis of sex organs prevents us from conducting tively, 32.1 °C on Wan-an Island in 2011, and 32.2 °C on LiuChiu such long-term studies on all nesting sites. The relationship between Island. Many studies have suggested that the pivotal temperatures the sand temperature and hatchling sex ratio, however, does allow us across marine turtle species and populations are relatively conserved to estimate the hatchling sex ratio. We have determined the sand and cluster around 29 °C (e.g. Broderick et al., 2001; Godfrey and temperature with the temperature loggers buried at the mean nest Mrosovsky, 2006; Hawkes et al., 2009; Merchant-Larios et al., 1997; depths on Lanyu Island and Wan-an Island since 2006 and on LiuChiu Mrosovsky, 1994; Pavenport, 1997; Poloczanska et al., 2009). The Island since 2011. Thus, we are able to assess the likely levels of pivotal temperature for green turtles had not been determined previ- spatio-temporal variability in hatchling sex ratio by this method as ously in Taiwan for the reasons stated above. Therefore, we adopted suggested by Hays et al. (2003). The impact of global climate changes Mrosovsky's (1994) argument and assumed that the pivotal temper- to the population of this endangered species can then be evaluated. ature for green turtles in Taiwan is 29 °C. We found that (Table 4, Fig. 3), except for two clutches from Lanyu Island (LN5 and LN7), all 5. Conclusions and conservation implications the mean temperatures in the nest were above 29 °C. Both of those cooler exceptions produced more than 50% male hatchlings according We have found that the sex ratio of hatchling green turtles is to our histological examinations (Table 2). Temperature in the nests female-biased in Taiwan, in agreement with the first hypothesis we were not measured for 8 clutches on Wan-an in 2010, but tempera- proposed. The differences in the nesting environments (mainly the tures were estimated based on the relationship between clutch and air temperature and the precipitation) between two islands resulted in sand temperatures in 2011 and the sand temperature in 2010. It more male hatchlings produced from Lanyu Island. This finding supports should be noted that factors such as metabolic heating, shading by the second hypothesis proposed for this study. The hatchling sex ratio is vegetation and nest depth may create variations in clutch tempera- important to the maintenance of a viable population size, and sea turtles, tures (Broderick et al., 2000; Kamel and Mrosovsky, 2006; Wibbels, especially the green turtles, have strong nest site fidelity. Thus, the signif- 2003), and thus deviations from our estimates. Despite the largest icant difference in the hatchling sex ratio found within such a short likely deviations, all of the estimated mean temperatures during the distance (b150 km) stresses the importance of designing specificlocal middle third of incubations were above 29 °C, and support the general conservation strategies for everywhere in the world. These strategies conclusion that female bias in sea turtle sex ratios occurs above that should consider the sex ratio of sea turtle hatchlings in order to maintain clutch temperature on Wan-an Island in 2010. healthy populations. Poloczanska et al. (2009) suggested that global climate change is 4.4. Influence of clutch temperature on the hatchling sex ratio and one of the five major hazards to marine turtles globally. Warming, a future efforts central aspect of climate change, strongly affects the sex ratio and incubation success during the incubation period (Pavenport, 1997). As the Although we found a difference in the sex ratios between Lanyu Intergovernmental Panel on Climate Change (ICPP) (2007) has predict- and Wan-an Islands, small sample size prevents us from estimating ed, there has been an increasing trend in air temperature, and there is the sex ratio of each island properly. However, this study also found reason to expect that warming to continue. A rise in sea level and other that, the percentage of female hatchlings increased with increasing environmental changes, such as extreme weather patterns, are also clutch temperature during the sex determination period (Fig. 3). Several predicted for the near future (Hawkes et al., 2009; Hoegh-Guldberg intrinsic factors are found to influence the clutch temperature during et al., 2007; IPCC, 2007; Meehl et al., 2005). Sand temperature is the incubation. Among them the clutch size and metabolism are two influenced by air temperature. Thus, the changes in weather pattern main factors (Broderick et al., 2001; Degregorio and Willard, 2011; do influence the sex ratio of hatchlings through the TSD character of Zbinden et al., 2006). The clutch temperature increases with the clutch sea turtles. In many sites, more female hatchlings are to be expected size due to the greater total metabolic activity as the incubation pro- as the global warming trend progresses. However, the rise in sea ceeds. This may change the hatchling sex ratio toward female-bias level and increased recurrence of extreme weather patterns could when the temperature during the sex determination period exceeds also reverse the hatchling sex ratio (Hays et al., 2003). Thus, a the pivotal temperature (Degregorio and Willard, 2011). Most clutches long-term study will be needed to determine the influence of contin- from Taiwan exceeded the pivotal temperature during the sex determi- ued climate change on sex ratio of the sea turtle populations in Taiwan nation period (Fig. 3). However, the increases in clutch temperature due and elsewhere. Possibly proper conservation measures, such as shading Author's personal copy

R. King et al. / Journal of Experimental Marine Biology and Ecology 445 (2013) 140–147 147 or sprinkling nests during the incubation (Fuentes et al., 2009; Kamel, S.J., Mrosovsky, N., 2006. Deforestation: risk of sex ratio distoration in hawksbill sea turtles. Ecol. Implicat. 16 (3), 923–931. Poloczanska et al., 2009) can be applied to maintain the proper opera- Kaska, Y., Ilgaz, Ç., Özdemir, A., Baskale, E., Türkozan, O., Baran, I., Stachoqitsch, M., 2006. tional sex ratio of sea turtle populations. Properly adjusting these strat- Sex ratio estimations of loggerhead sea turtle hatchlings by histological examination egies will require thorough assessment of optimal operational sex ratios and nest temperatures at Fethiye beach, Turkey. Naturwissenschaften 93, 338–343. fl Kuo, J.-W., 2008. Changes of the oxygen content in the clutch during the incubation of among adults and the in uence on those adult ratios of hatchling ratios green sea turtles at Lanyu Island, Taitung County, Taiwan. MS Thesis National Taiwan and nest temperatures. Ocean Univ (48 pages). Larios, H.M., 1999. Determining hatchling sex. In: Eckert, K.L., Bjorndal, K.A., Abreu- References Grobois, F.A., Donnelly, M. (Eds.), Research and Management Techniques for the Conservation of Sea Turtles. Washington, DC: IUCN/SSC Marine Turtle Specialist Group Publication, 4, pp. 130–135. Booth, D.T., Freeman, C., 2006. Sand and nest temperatures and an estimate of hatchling Limpus, C.J., Reed, P.C., 1985. The green turtle, Chelonia mydas in Queensland: a preliminary sex ratio from the Heron Island green turtle (Chelonia mydas)rookery,Southern description of the population structure in a coral reef feeding ground. Biology of. Great Barrier Reef. Coral Reefs 2, 629–633. Australiasian Frogs and Reptiles, pp. 47–52. Broderick, A.C., Godley, B.J., Reece, S., Downie, J.R., 2000. Incubation periods and sex ratios of Marcovaldi, M.Â., Godfrey, M.H., Mrosovsky, N., 1997. Estimating sex ratios of loggerhead green turtles: highly female biased hatchling production in the eastern Mediterranean. turtles in Brazil from pivotal incubation durations. Can. J. Zool. 75, 755–770. Mar. Ecol. Prog. Ser. 202, 273–281. Meehl, G.A., Washington, W.M., Collins, W.D., Arblaster, J.M., Hu, A., Buja, L.E., Strand, Broderick, A.C., Godley, B.J., Hays, G.C., 2001. Metabolic heating and the prediction of W.G., Teng, H., 2005. How much more global warming and sea level rise? Science sex ratios for green turtles (Chelonia mydas). Physiol. Biochem. Zool. 74, 161–170. 307, 1769–1772. Casale, P., Gerosa, G., Yerli, S.V., 2000. Female-biased primary sex ratio of the green turtle, Merchant-Larios, H., Ruiz-Ramirez, S., Moreno-Mendoza, N., Marmolejo-Valencia, A., Chelonia mydas, estimated through sand temperature at Akyatan, Turkey. Zool. 1997. Correlation among thermosensitive period, estordiol response and gonad Middle East 20, 33–42. differentiation in the sea turtle Lepidochelys olivacea. Gen. Comp. Endocrinol. 107, Ceriani, S.A., Wyneken, J., 2008. Comparative morphology and sex identification of the 373–385. reproductive system in formalin-preserved sea turtle specimens. Zoology 111, 179–187. Miller, J.D., Limpus, C.J., 1981. Incubation period and sexual differentiation in the green tur- Chen, T.-H., Cheng, I.-J., 1995. Breeding biology of the green turtle, Chelonia mydas, tle, Chelonia mydas L. In: Banks, C.B., Martin, A.A. (Eds.), Proceeding of the Melbourne (Reptilia: Cheloniidae) on Wan-an Island, Peng-Hu Archipelago, Taiwan. I. Nesting Herpetological Symposium. Zoological Board of Victoria, Parkville, pp. 66–73. ecology. Mar. Biol. 124, 9–15. Miller, J.D., Limpus, C.J., 2003. Chap. 7. Ontogeny of marine turtle gonads. In: Lutz, P.L., Chen, C.-L., Wang, C.-C., Cheng, I.-J., 2010. Effects of biotic and abiotic factors on the Musick, J.A., Wyneken, J. (Eds.), The Biology of Sea Turtles, vol II. CRC Press, Boca oxygen content of green sea turtle nests during embryogenesis. J. Comp. Physiol. Raton, FL, pp. 199–224. B 180, 1045–1055. Morreale, S.J., Ruiz, G.J., Spotila, J.R., Standora, E.A., 1982. Temperature dependent sex Cheng, I.-J., 2000. Post-nesting migrations of green turtles at Wan-An Island, Penghu determination: current practices threaten conservation of sea turtles. Science Archipelago, Taiwan. Mar. Biol. 137, 747–754. 216, 1245–1247. Cheng, I.-J., 2011. Chap. 17. Sea turtle research. In: Krejcar, O. (Ed.), Modern Telemetry. Mrosovsky, N., 1988. Pivotal temperatures for loggerhead turtles (Caretta caretta) from In Tech Inc., Croatia, pp. 353–370. northern and southern nesting beaches. Can. J. Zool. 66, 661–669. Cheng, I.-J., Dutton, P.H., Chen, C.-L., Chen, H.-C., Chen, Y.-H., Shea, J.-W., 2008. Comparison Mrosovsky, N., 1994. Sex ratios of sea turtles. J. Exp. Zool. 270, 16–27. of the genetics and nesting ecology of two green turtle rookeries in Taiwan. J. Zool. 276 Mrosovsky, N., Benabib, M., 1990. An assessment of two methods of sexing hatchling (4), 375–384. sea turtles. Copeia 1990, 589–591. Degregorio, B.A., Willard, A.S., 2011. Incubation temperatures and metabolic heating of Mrosovsky, N., Godfrey, M.H., 1995. Manipulating sex ratios: turtle speed ahead! Chelonian relocated and in situ loggerhead sea turtle (Caretta caretta) nests at a northern Conserv.Biol.1,238–240. rookery. Chelonian Conserv. Biol. 10 (1), 54–61. Mrosovsky, N., Dutton, P.H., Whitmore, C.P., 1984. Sex ratios of two species of sea turtle Fuentes, M.M.P.B., Maynard, J.A., Guinea, M., Bell, I.P., Werdell, P.J., Hamann, M., 2009. nesting in Suriname. Can. J. Zool. 62, 2227–2239. Proxy indicators of sand temperature help project impacts of global warming on Mrosovsky, N., Baptistotte, C., Godfrey, M.H., 1999. Validation of incubation duration as sea turtles. Endanger. Species Res. J. 9, 33–40. an index of the sex ratio of hatchling sea turtles. Can J. Zool. 77, 831–835. Godfrey, M.H., Mrosovsky, N., 2006. Pivotal temperature and predicted sex ratios for Mrosovsky, N., Kamel, S.J., Diez, C.E., van Dam, R.P., 2009. Methods of estimating natu- green turtles, Chelonia mydas, nesting in Suriname. J. Herpetol. 16, 55–61. ral sex ratios of sea turtles from incubation temperatures and laboratory data. En- Godfrey, M.H., Barreto, R., Mrosovsky, N., 1996. Estimating past and present sex ratios danger. Species Res. 8, 147–155. of sea turtles in Suriname. Can. J. Zool. 74, 267–277. Neill, A.L., 2008. The A to Z of Anatomical Medical Terms, 2nd. 11–12 Aspen Pharmacare Godfrey, M.H., D'Amato, A.F., Marcovaldi, M.A., Mrosovsky, N., 1999. Pivotal temperature Australia, Sydney NSW, Australia. and predicted sex ratios for hatchling hawksbill turtles from Brazil. Can. J. Zool. 77, Newson, S.E., Mendes, S., Crick, H.Q.P., Dulvy, N.K., Houghton, J.D.R., Hays, G.C., Hutson, 1465–1473. A.M., Macleod, C.D., Pierce, G.J., Robinson, R.A., 2007. Indicators of the impact Godfrey, M.H., Delmars, V., Girondot, M., 2003. Assessment of patterns of temperature- of climate change on migratory species. Endanger. Species Res. http://dx.doi.org/ dependent sex determination using maximum likehood model selection. Ecoscience 10.3354/esr00162. 10 (3), 265–272. Owens, D.W., Hendrickson, J.R., Lance, V., Callard, I.P., 1978. A technique for determining Godley, B.J., Broderick, A.C., Mrosovsky, N., 2001. Estimating hatchling sex ratios of logger- sex of immature Chelonia mydas using radioimmunoassay. Herpetology 34, 270–273. head turtles in Cyprus from incubation durations. Mar. Ecol. Prog. Ser. 210, 195–201. Pavenport, J., 1997. Temperature and the life-history strategies of sea turtles. J. Therm. Godley, B.J., Richardson, S., Broderick, A.C., Coyne, M.S., Glen, F., Hays, G.C., 2002. Long Biol. 22 (6), 479–488. term satellite telemetry of the movements and habitat utilization by green turtles Poloczanska, E.S., Limpus, C.L., Hays, G.C., 2009. Chap. 2. Vulnerability on Marine Turtles in the Mediterranean. Ecography 25, 352–362. to climate change. Adv. Mar. Biol. 56, 1–61. Hawkes, L.A., Broderick, A.C., Godfrey, M.H., Godley, B.J., 2009. Climate change and marine Sibly, R.M., Calow, P., 1986. Physiological Ecology of Animals. Blackwell Scientific Publ., turtle. Endang. Species Res. 7, 137–154. Oxford, London, UK. Hays, G.C., Godley, B.J., Broderick, A.C., 1999. Long-term thermal conditions on the nesting Sokal, R.R., Rohlf, F.J., 1982. Biometry: The Principles and Practice of Statistics in Biological beaches of green turtles on Ascension Island. Mar. Ecol. Prog. Ser. 185, 297–299. Research. W.H. Freeman and Company, San Francisco. Hays, G.C., Ashworth, J.S., Barnsley, M.J., Broderick, A.C., Emery, D.R., Godley, B.J., Henwood, Spotila, J.R., Standora, E.A., Morreale, S.J., Ruiz, G.J., 1987. Temperature dependent A., Jones, E.L., 2001. The importance of sand albedo for the thermal conditions on sea sex determination in green turtles (Chelonia mydas): effect on the sex ratio on a turtle nesting beaches. Oikos 93, 87–94. natural nesting beach. Herpetology 43, 74–81. Hays, G.C., Broderick, A.C., Godley, B.J., Luschi, P., Nichols, W.J., 2003. Satellite telemetry Townsend, C.R., Calow, P., 1981. Physiological Ecology: An Evolutionary Approach to suggests high levels of fishing induced mortality for marine turtles. Mar. Ecol. Prog. Resource Use. Blackwell Scientific Publ., Oxford, London, UK. Ser. 262, 305–309. Wibbels, T., 1999. Diagnosing the sex of sea turtles in foraging habitats. In: Eckert, K., Hays, G.C., Fossette, S., Katselidis, K.A., Schofield, G., Gravenor, M.B., 2010. Breeding Bjorndal, K.A., Abreu, A., Donnelly, M. (Eds.), Research and Management Techniques periodicity for male sea turtles, operational sex ratios, and implications in the for the Conservation of Sea Turtles: IUCN/SSC Marine Turtle Specialist Group Publication face of climate change. Conserv. Biol. 24, 1636–1643. No.4,pp.139–143. Hoegh-Guldberg, O., Mumby, P.J., Hooten, A.J., Steneck, R.S., Greenfield, P., Gomez, E., Wibbels, T., 2003. Critical approaches to sex determination in sea turtles. In: Lutz, P.L., Harvell, C.D., Sale, P.F., Edwards, A.J., Caldeira, K., Knowlton, N., Eakin, C.M., Iglesias- Musick, J.A., Wyneken, J. (Eds.), The Biology of Sea Turtles, vol II. CRC Press, Boca Prieto, R., Muthiga, N., Bradbury, R.H., Dubi, A., Hatziolos, M.E., 2007. Coral reefs Raton, FL, pp. 103–134. under rapid climate change and ocean acidification. Science 318, 1737–1742. Wibbels, T., Martin, R.E., Owens, D.W., Amoss Jr., M.S.J., 1991. Female-biased sex ratio of Horikoshi, K., 1992. Egg survivorship and primary sex ratio of green turtles. Chelonia immature loggerhead sea turtles inhabiting the Atlantic coastal waters of Florida. mydas, at tortuguero, Costa Rica. Ph.D. thesis University of Florida, Gainsville. Can. J. Zool. 69, 2973–2977. Houghton, J.D.R., Myers, A.E., Lloyd, C., King, R.S., Isaacs, C., Hays, G.C., 2007. Protracted Wyneken, J., Epperly, S.P., Crowder, L.B., Vaughan, J., Esper, K.B., 2007. Determining sex rainfall decreases temperature within leatherback turtle (Dermochelys coriace) in posthatchling loggerhead sea turtles using multiple gonadal and accessory duct clutches in Grenada, West Indies: ecological implications for a a species displaying characteristics. Herpetology 63, 19–30. temperature dependent sex determination. J. Exp. Mar. Biol. Ecol. 345, 71–77. Yntema, C.L., Mrosovsky, N., 1980. Sexual differentiation in hatchling loggerheads Hulin, V., Delmas, V., Girondot, M., Godfrey, M.H., Guillon, J.-M., 2009. Temperature- (Caretta caretta) incubated at different controlled temperatures. Herpetology 36, dependent sex determination and global change: are some species at great risk? 33–36. Oecologia 160, 493–506. Zbinden, J.A., Margaritoulis, D., Arlettaz, R., 2006. Metabolic heating in Mediterranean IPCC (Intergovernmental Panel on Climate Change), 2007. Summary for Policy Makers. loggerhead sea turtle clutches. J. Exp. Mar. Biol. Ecol. 334, 151–157 [SS]. Cambridge University Press, Cambridge.