Comparison of the Genetics and Nesting Ecology of Two Green Turtle Rookeries I.-J

Journal of Zoology

Journal of Zoology. Print ISSN 0952-8369 Comparison of the genetics and nesting ecology of two green turtle rookeries I.-J. Cheng1, P. H. Dutton2, C.-L. Chen1, H.-C. Chen1, Y-H. Chen1 & J.-W. Shea1

1 Institute of Marine Biology, National Taiwan Ocean University, Keelung, Taiwan, ROC 2 NOAA-Fisheries, Southwest Fisheries Science Center, La Jolla Shores Drive, La Jolla, CA, USA

Keywords Abstract Chelonia mydas; Taiwan; nesting ecology; Wan-an Island; Lanyu Island; genetic We characterize the behavioral, phenotypic and genotypic characteristics of the structure. two main green turtle Chelonia mydas nesting populations remaining in Taiwan and examine how the differing ecology of the nesting environments on each island Correspondence may have influenced these life-history traits. Wan-an Island in the south-west I-Jiunn Cheng, Institute of Marine Biology, sector of the Taiwan Straits was found to be hotter and drier than Lanyu Island National Taiwan Ocean University, Keelung, located south-east of Taiwan in the Pacific Ocean. The frequency of nesting Taiwan 202-24, ROC. attempts and the internesting intervals were both significantly greater (mean Email: [email protected] nesting attempts: 15.4 vs. 15.2, mean internesting interval: 13.7 vs. 10.6 days) for the Wan-an nesters. Nests were deeper (69.6 vs. 69.0 cm) while the incubation Editor: Prof. Tim Halliday duration was shorter on this island (52.1 vs. 54.7 days). Green turtles were larger on Lanyu, but deposited smaller eggs. Nests on Lanyu had higher hatching Received 20 January 2008; revised 21 July success, hatchling emergence success and clutch survival rate (hatching success; 2008; accepted 22 July 2008 72.2 vs. 80.76%, hatchling emergence success; 47 vs. 64.1%, clutch survival rate; 67.7 vs. 70.3%). However, hatchlings on Wan-an were larger (HC; 47.6 vs. 46.6 doi:10.1111/j.1469-7998.2008.00501.x cm. HW; 23.9 vs. 21.7 cm). Analysis of mitochondrial DNA sequences obtained from a total of 53 nesters indicated that both rookeries are genetically distinct (FST =0.673, Po0.00001), with a single haplotype characterizing the Lanyu rookery (n=13). This lack of gene flow between the two rookeries is evidence for localized natal homing and is consistent with the morphological and behavioral differences that we detected between the two rookeries. These findings are surprising given the close geographic proximity of the two rookeries.

Introduction tence of distinguishable stocks for management purposes (Norman, Moritz & Limpus, 1994; Encalada et al., 1996; Wan-an Island of Penghu Archipelago and Lanyu Island of Dethmers et al., 2006; Dutton et al., 2008). These studies Taitung County are the two main nesting sites of green have shown population structuring consistent with theories turtles Chelonia mydas, Linnaeus, 1758 remaining in Taiwan of broad natal homing to regional rookeries. A general (Fig. 1) (Cheng, 1998). Wan-an is located south-west from limitation to these studies is that they rarely compare Taiwan, on the continental shelf in the Taiwan Straits. rookeries that are closely spaced (o400 km), so the extent Lanyu, on the other hand, is south-east of Taiwan in the of connectivity on a local scale remains unknown. High Pacific Ocean. Satellite telemetry studies have shown that fidelity to particular nesting areas has recently been demon- the foraging sites for the green turtles nesting on Wan-an are strated elsewhere in green turtles (Lee, 2007) and this species distributed mainly on the continental shelves east of the certainly seems to maintain higher nesting area fidelity than Mainland China (Cheng, 2000), while foraging sites for the some other sea turtle species. The degree of demographic green turtles nesting on Lanyu may distributed mainly in connectivity between the two nesting populations in Taiwan Penghu waters south of Wan-an (I.-J. Cheng, unpubl. data). and with other populations in the Pacific is unknown. However, the sizes of these nesting populations are unknown, and it is unclear if the two rookeries represent distinct breeding populations. Purpose of the study Studies of maternally inherited mitochondrial DNA Sea turtles are known to have phenotypic and behavioral (mtDNA) in green turtles have been useful in understanding plasticity in response to varying environmental conditions the population structure and reproductive behavior of these (Hays et al., 2002; Glen et al., 2003; James et al., 2006) found highly migratory marine animals (Bowen et al., 1992; on nesting beaches. Local adaptations of nesting behavior FitzSimmons et al., 1997) and in demonstrating the exis- are associated with temperature, precipitation, sand

Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London 375 Comparison of life-history traits between two green turtle rookeries I.-J. Cheng et al.

118° 40′ 119° 20′ 120° 00′ 120° 40′ 121° 20′ 122° 00′ 122° 40′ Materials and methods

Description of the study sites 25° 20′ Wan-an Island 24° 40′ Wan-an (231220N, 1191300E) is c. 7.4 km2 in size and located in the southern Penghu Archipelago, about 18 miles from the main Penghu Island. The average annual air tempera- 24° 00′ ture is 22 1C, and the average total annual precipitation is 1117 mm, occurring mostly in the monsoon and typhoon Taiwan seasons. ° ′ 23 20 There are 11 relatively ﬂat sand beaches, all composed of Wan-an Island quartz propyrite, coral and shell debris. They range from 67 to 800 m in length, 20 to 100 m in width and are separated by 22° 40′ rocky outcrops. Turtles are known to nest on nine of the Lanyu Island beaches, all of which are located on the south and west sides of the island. The total length of the nesting beach is about 22° 00′ 4 km (Chen & Cheng, 1995; Cheng, 2006).

km 0 71 21° 20′ Lanyu Island Lanyu (221000–080N, 1211500–600E) is in the Paciﬁc Ocean Figure 1 Map of Taiwan with the location of two main sea turtle 2 nesting sites: Lanyu and Wan-an Islands showed on either side south-west of Taitung, Taiwan, measuring about 46 km . 1 of Taiwan. The annual average air temperature is 22.4 C and annual total precipitation exceeds 3077 mm (Natural Conservation Society ROC, 1988). The sand on the beaches is composed of quartz propyrite, inter-layered with muddy sediment. characteristics, vegetation coverage, beach width and slope Although there are six sandy beaches on Lanyu, prelimin- (Mortimer, 1990; Rivalan et al., 2005; Chen et al., 2007). ary surveys show nesting activity on only three beaches: The characteristics of the nesting environment in turn Ba-dai, big Ba-dai and Don-chin beaches. The areas of these inﬂuences the incubation success, sex ratio, hatchling mor- three beaches were about 5522, 17 000 and 15 766 m2 (Kuo, phology, and ultimately recruitment to the population 1999). (Hays et al., 2001; Glen et al., 2003). Glen et al. (2003) found phenotypic differences between green turtle hatchl- Environmental data ings from Cyprus and Ascension Island attributed in part to environmental factors such as temperature. Wan-an Sand grain characteristics and Lanyu are relatively close (c. 300 km); however, their respective climates are different, with over twice as much Sand samples were taken near nesting sites and stored dry in precipitation on average on Lanyu (Natural Conservation double-sealed plastic bags. The graphic mean (Mz; in mm) Society ROC, 1988). We characterize and compare the and inclusive graphic standard deviation (s1) of the beach nesting environments on both islands, and investigate sand were determined according to Folk (1974). whether there are differences in nesting behavior, morphology and hatching success. Secondly, we use mtDNA MzðfÞ¼ðf16 þ f50 þ f84Þ=3 sequencing to determine whether the two nesting populations are demographically distinct. Anthropogentic im- z1 ¼ðf95 ðf5Þ=6:6 þðf84 ðf16Þ=4 pacts associated with urban development and tourism have where f16, f50, f84, f95 and f5 denote the proportion by been a concern for some time on Wan-an, but have also weight of total sand at 16, 50, 84, 95 and 16% of total phi(f) increased dramatically in recent years on Lanyu. The nest- value. Sand samples were collected from 1997 to 1998 and ing beaches on Wan-an were declared protected areas in from 2005 to 2006. 1995 (Chen & Cheng, 1995; Cheng, 2006). Study of green turtle nesting ecology on Lanyu was initiated in 1997 Climate data (Cheng, 1998). Understanding of the extent of demographic connectivity between the green turtle nesting populations on The meteorological data collected for the study include daily both islands, and the extent of adaptation to any local air temperature and precipitation on both Wan-an and differences in the nesting environment will be vital to Lanyu from 1997 until 2006. The data were purchased from developing management strategies appropriate for each the Central Weather Bureau of the ROC (Central Weather island. Bureau, 1997–2006).

376 Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London I.-J. Cheng et al. Comparison of life-history traits between two green turtle rookeries

Field surveys deepest position in the nest. On Lanyu, the clutch size was determined by counting the eggs in each nest. While on On both islands, field surveys were conducted between late Wan-an, the clutch size was determined by excavating the April and early October. Although Wan-an has been mon- nest after hatchling emergence, and counting the total itored since 1992, only data collected from 1997 to 2006 were numbers of hatched egg shells and unhatched eggs. Clutch used for comparative purposes, since the study on Lanyu frequency was determined as the number of nests deposited only began in 1997. per individual (Rivalan et al., 2006). Two types of surveys were conducted: (1) intensive The incubation duration was determined from the date nocturnal surveys where nesters and nesting behavior were when the eggs were deposited until the first hatchling was directly observed, and (2) daytime track counts obtained by observed emerging on the sand surface. When the hatchlings patrolling the beach each morning. Nighttime surveys were emerged after 12:00 h midnight, the next day was considered designed to ensure that most nesters were encountered. On the emergence date. Wan-an, the beaches were surveyed at least twice each night Mean egg size and weight for each nest were determined for by foot from 19:00 until 04:00 h, while on Lanyu, beaches a random sub-sample of 30 eggs for clutches. We measured were surveyed by foot once every 2 h from 19:00 until the egg diameter with a Vernier slide caliper ( 0.5 mm) and 03:00 h, to count nesting events and to observe nesting weighed eggs with an electronic balance (Kang-Jen Model turtles. Surveys were carried out by at least two patrollers KH, Kang-Jen Co. Ltd., Taipei, ROC; 0.01 g). for each beach and stayed for about 4 h on each survey on Wan-an. On Lanyu, most turtles come out of the water to nest between 19:00 and 03:00 h, and are on the beach for Hatch data more than 2 h. During typhoons, the beaches were surveyed Hatching success was calculated as the percentage of live only during the daytime to record new nests or body pits left hatchlings from the total clutch. For Wan-an, because the from the previous night. The morning track counts were nest was left in situ, the number of live hatchlings was conducted after daybreak. The nest location on the beach determined by counting the number of hatched eggshells was recorded. Nester ‘emergence’ was recorded when a and subtracting the dead hatchlings that were excavated turtle first emerged from the water. The number of nesting from the nest. The total clutch size was the sum of the attempts was determined by counting number of digging number of hatched shells and unhatched eggs. For Lanyu, attempts, as indicated by body pits, or abandoned egg the clutch size was determined directly from egg counts cavities. Nesting success is defined as the proportion of recorded when clutches were reburied shortly after they were emergences that resulted in egg deposition. laid. The post-hatch mortality was calculated as the propor- Turtles that were encountered nesting were measured for tion of dead hatchlings among the total hatchlings. Emer- curved and straight carapace length (CCL and SCL, Bolten, gence success was calculated as the proportion of hatchlings 1999) and tagged on the trailing edge of the front and rear leaving the nest. Clutch survival was calculated as the flippers using numbered Inconel tags. Beginning in 2002, a product of hatching success and emergence success (Ma- passive integrated transponder tag was also applied on zaris, Fiksen & Matsinos, 2005). Hatchling straight cara- either or both hind flippers. The inter-annual remigration pace length (HC) was measured using a Vernier slide interval was determined from the tagged (Inconel tag) calipers and hatchling curved carapace lengthwas measured turtles that were observed nesting in more than one season. with a cloth tape ( 0.1 mm). Hatchlings were weighed Turtles without previous tags, or evidence of tags (e.g. tag (HW) using an electronic balance (Kang-Jen Model KH). scars) were classified as ‘new’ turtles as opposed to remi- grants, that were previously tagged turtles, or ones bearing tag scars (Miller, 1997). The internesting interval was Genetic analysis defined as the time (in days) between nestings within the Small skin biopsies were taken from a total of 40 nesters on same season (Alavarado & Murphy, 1999). Wan-an, and 14 nesters on Lanyu and preserved in a 20% dimethyl sulphoxide solution saturated with sodium chlor- ide (Dutton, 1996). DNA was isolated from these samples Nest data using either standard phenol/chloroform extraction techni- On Lanyu, clutches were dug up within 1 h after oviposition ques (Sambrook et al., 1989) or the Fast Prep DNA isola- and reburied with a plastic mesh screen to protect the eggs tion kit (Bio101s, MP Biomedicals, Irvine, CA, USA). from predation by red back pine root snakes Oligodon Amplification of mtDNA was performed by the polymerase formosanus. On Wan-an, the nests were left undisturbed, chain reaction (PCR) (Innis, 1990) using the primers because there is not much threat from predation. On both HDCM2 and LTCM2, designed to target 488 bp at the 50 islands, any nest susceptible to flooding was relocated. On end of the control region of the mitochondrial genome Lanyu, the nest depth and clutch size were determined (Lahanas et al., 1994). Direct cycle sequencing reactions of before screen installation or nest relocation, while on Wan- the light strand were performed on 2 mL of purified PCR an, the nest depth was determined after the hatchlings had product combined with 2 mL of ABI Prisms dRhodamine emerged from the nest. Nest depth was determined with a Terminator Cycle Sequencing Kit, 3 mL of primer LTCM2 fiberglass tape ( 0.1 cm) from the beach surface to the and 5 mL of purified water. The labeled extension products

Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London 377 Comparison of life-history traits between two green turtle rookeries I.-J. Cheng et al. were purified via ethanol precipitation and analyzed with an Female green turtles emerged more frequently from the Applied Biosystems model 377 (Applied Biosystems, Foster sea (Student t-test, Po0.01) and made more nesting at- City, CA, USA) automated DNA Sequencer. The sequences tempts (Student t-test, P=0.005) on Wan-an (Table 3). The were analyzed for uncalled and miscalled bases using either internesting interval was longer on Wan-an (Student t-test, Gene Codes Sequencher 3.1.1 or ABI SeqED v. 1.0.2. Po0.001; Table 3). Sequences were aligned against reference data from the 384 bp segment of the mtDNA control region corresponding to the region reported by Norman et al. (1994). Haplotype nomenclature follows that reported in (http://swfsc.noaa.- (a) gov/prd-turtles.aspx) and were based on the same 384 bp 20 fragment used by Norman et al. (1994), and Dethmers et al. (2006) to assign haplotypes. 18 Population genetic parameters were estimated using AR- 16 LEQUIN Version 3.01 software (Excoffier, Laval & Schnei- 14 der, 2005). These included estimates of haplotype (h) and 12 nucleotide (p) diversity, and frequency-based F-statistic tests for genetic differentiation between the two populations 10 (Weir & Cockerham, 1984). A minimum spanning network 8 was constructed to illustrate the relationships between No. of females 6 haplotypes based on Kimura 2-parameter mutation model 4 as implemented in ARLEQUIN (Excoffier & Smouse, 1994). 2 0 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 Results (b) 14 Environmental data 12 The mean sand grain size was similar on both islands, but with a wider range of particle size on Wan-an (Table 1). 10 Conditions were warmer and drier on Wan-an than on Lanyu, as indicated by higher annual and seasonal air 8 temperature (Student t-test, Po0.001) and lower precipita- 6 tion (Student t-test, Po0.001) on Wan-an (Table 1).

No. of females 4 Field surveys 2 The nesting population sizes were similar between the two islands (Fig. 2). Nesting females were larger on Lanyu in 0 terms of SCL (P=0.017; Table 1). Green turtles returned 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 for subsequent nesting seasons (=remigration interval) at Figure 2 Yearly trend of nesting green turtles from (a) 1992 till 2006 on similar intervals (Table 2). Wan-an Island, and (b) from 1997 till 2006 on Lanyu Island.

Table 1 Environmental parameter comparisons [mean 1 standard deviation (SD)] between Wan-an and Lanyu Islands during the nesting seasons from 1997 to 2006, with the statistical analyses results showed in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value Whole year Air temperature ( 1C) 23.94 4.17 3646 22.81 3.13 3646 o0.001 Precipitation (mm) 8.62 52.95 3646 33.82 127.47 3646 o0.001 Nesting season Air temperature ( 1C) 27.3 1.6 2024 25.3 1.3 2024 o0.001 Precipitation (mm) 10.8 89.3 1530 50.9 171.6 1530 o0.001 Particle characteristics

Mz (mm) 1.535 2.183 131 1.026 0.372 43 0.688

s1 0.816 0.548 131 0.778 0.32 43 0.032

The environmental parameters include air temperature and precipitation for the whole year and during the nesting season, and the sediment characteristics [mean grain size (Mz) and inclusive graphic standard deviation (s1)].

378 Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London I.-J. Cheng et al. Comparison of life-history traits between two green turtle rookeries

Table 2 The nesting female size and remigration intervals of the green turtles at both the Wan-an Island and Lanyu Island from 1997 to 2006, with the statistical analysis results shown in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value Nesting female SCL (cm) 97.02 5.64 115 99.08 5.66 54 0.017 CCL (cm) 101.4 10.3 115 103.5 4.9 54 0.082 Re-migration interval (year) 4.59 1.82 22 4.3 1.81 20 0.529

Individual female size is measured as both straight carapace length (SCL) and curved carapace length (CCL).

Table 3 The emergences, nesting attempts, nest position, clutch frequency, nesting success and internesting interval of the green turtles on both the Wan-an and Lanyu Islands from 1997 to 2006, with the statistical analyses results showed in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value Emergences per turtle 9.96 7.79 86 6.71 4.77 78 0.009 Nesting attempts (attempts per turtle) 15.36 12.86 86 11.23 15.15 78 0.005 Nest position (% total nests) Beach (B) 36 20 10 4 5 10 0.003 Interface (IZ) 34 14 10 64 36 10 0.069 Grass (G) 30 30 10 32 37 10 0.89 Clutch frequency (nests per turtle) 3.2 2.0 118 2.8 2.1 78 0.162 Nesting success (%) 51 27 86 48 28 78 0.849 Internesting interval (d) 13.7 1.63 227 10.6 1.21 163 o0.001

Nest data Discussion Green turtles dug deeper nests (Student t-test, P=0.02) but had shorter incubation duration (Po0.001) on Wan-an Nesting environment (Table 4). Females nesting on Wan-an also deposited Our results show that the nesting environments are different larger (Po0.001) and heavier eggs (Po0.001) on Wan-an on the two islands, with Wan-an being consistently drier and (Table 4). hotter than Lanyu. In addition, the size distribution of sand was wider on Wan-an. Even though the sand grain sizes on Hatch data both islands were well within the range of nesting beaches for green turtles (i.e. fine sand to coral pebbles; Mortimer, The embryos incubated on Lanyu had lower hatching and 1995), the sand on Wan-an is a mixture of quartz propyrite, post-hatching mortalities (P 0.001; Table 5), and thus o coral and shell debris. It is lighter in color than the sand on higher hatching and hatchling emergence successes Lanyu that is composed mainly of quartz propyrite, occa- (P 0.001). Clutch survival rate was subsequently higher o sionally inter-layered with muddy sediment, which is darker on Lanyu (P=0.005; Table 5). Hatchlings on Wan-an were in color. Hays et al. (2001) suggested that the sand tempera- larger in terms of HC and HW (P 0.001; Table 6). o ture was cooler on lighter beaches, and this may be one of the factors, along with higher precipitation, that would Genetics suggest cooler conditions to prevail in the nests. A total of four haplotypes were identified among both nesting populations. Of the total of 40 samples analyzed Life-history traits of nesting green turtles from Wan-an, 65% were CmP18, 2.5% were CmP19 and 32.5% were CmP20. All 14 samples from Lanyu had the Physiological characters same haplotype, CmP49 (Table 7). The lack of any shared haplotypes between two nesting populations resulted in The considerable inter-annual variability in nesting numbers significant differentiation (FST =0.673; Po0.00001). Hap- we found in our study is a pattern that has been seen before lotype (h) and nucleotide (p) diversities were zero for the in green turtles and other species (Broderick, Godley & Lanyu population, and 0.483 0.056 SE for Wan-An. Three Hays, 2001b; Chaloupka et al., 2008) and has been explained of the haplotypes were closely related; however, CmP20 by climate-controlled changes in foraging conditions influ- diverged greatly, separated by 21–24 base substitutions from encing the trajectory of body condition increase by foraging the other three (Fig. 3). adults (Hays, 2000). Hence, in good foraging years many

Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London 379 Comparison of life-history traits between two green turtle rookeries I.-J. Cheng et al.

Table 4 The nest depth, clutch size, incubation duration and egg characteristics of the green turtles on both the Wan-an and Lanyu Islands from 1997 to 2006, with the statistical analyses results shown in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value Nest depth (mm) 69.58 8.69 216 68.99 17.81 197 0.02 Clutch size (eggs) 104.6 23.9 258 105.5 28.3 199 0.368 Incubation duration (d) 52.13 3.65 231 54.65 3.65 57 o0.001 Egg characters Diameter (mm) 44.3 7.85 5586 42.21 2.34 7031 o0.001 Weight (g) 47.3 4.78 5558 43.3 7.61 7121 o0.001

Table 5 Hatching success, post-hatching mortality, hatchling emergence and clutch survival rate of the green turtles on Wan-an and Lanyu Islands from 1997 to 2006, with the statistical analysis results shown in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value Hatching success (%) 72.2 30.2 242 80.7 27.8 166 o0.001 Post-hatching mortality (%) 7.4 13.8 242 2.04 6 166 o0.001 Hatchling emergence success (%) 47 39.1 242 64.1 39.7 166 o0.001 Clutch survival (%) 67.7 30.98 242 70.7 33.1 166 0.005

Table 6 The morphological characteristics of green turtle hatchlings on both Wan-an Island and Lanyu Island from 1997 to 2006, with the statistical analyses results showed in the last column Wan-an Lanyu

Parameter Mean SD n Mean SD n P-value HC (mm) 47.59 2.34 3716 46.55 2.06 1270 o0.001 HCC (mm) 50.33 12.02 2479 50.13 3.28 1177 0.057 HW (g) 23.92 2.47 4528 21.74 4.09 1360 o0.001

HC, hatchling straight carapace length; HCC, hatchling curved carapace length.

Table 7 Haplotype frequencies in green turtle nesting populations difference might be related to the differences in the near- sampled on Wan-an (n=40) and Lanyu (n=14) Islands in Taiwan, shore marine environments. Most water depths around based on mitochondrial DNA (mtDNA) control region sequences Wan-an are o50 m and there are extensive coral reefs, while Haplotype Wan-an Lanyu most depths around Lanyu exceed 250 m. Studies of diving behavior of green turtles during internesting periods using CmP18 26 – temperature–depth recorders on Wan-an nesters showed CmP19 1 – that they mainly performed U-type dives with some erratic CmP20 13 – CmP49 – 14 bottom proﬁles; that is type 1(b) in Houghton et al. (2002) (T.-L. Dallas, unpubl. data). This suggests that the turtles Haplotype nomenclature follows that on (http://swfsc.noaa.gov/prd- are actively foraging during the internesting period. Even turtles.aspx) and were based on the same 384 bp fragment used by though no study of internesting dives was carried out on the Norman et al (1994), and Dethmers et al. (2006) to assign haplotypes. Lanyu population, the waters around this island are too deep for foraging activity. This result is similar to that of Hays et al. (2002), and suggests that the Wan-an females turtles may attain sufﬁcient body condition to breed, while have adapted their dive behavior for maximum energy gain, the reverse is true in poor foraging years. In spite the high while the females on Lanyu might adapted for minimum tag loss rate, the total number of different females that have energy loss (by resting) during the internesting period. been tagged for the study period would give an idea of how Novel data loggers attached to turtles have the ability to many different females in each population. directly measure the extent of feeding and resting by Green turtles on Lanyu were larger, but deposited smaller individuals (Houghton et al., 2008) and could be used to eggs with similar clutch size. The size of egg depends on the test these inferred differences in the internesting behavior of body conditions of gravid female (Hays, 2001). The egg size individuals between the two rookeries in Taiwan.

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sand dries out deeper than on Lanyu. This deeper layer of looser, drier sand decreases the substratum compactness and increases the chance of cave-ins during nest excavation (Chen et al., 2007). Thus, turtles had to make more nesting attempts and dig deeper nests on Wan-an in order to nest successfully.

Hatching success and hatchling morphology Precipitation is the main source of water in the upper reaches of a beach, and is important for the embryogenesis (Ratterman & Ackermann, 1989). The drier and especially warmer sands tend to increase the interstitial air content and decrease the absorption of water vapor by embryos. This influences the embryogenesis negatively. Furthermore, the metabolic heat produced during the incubation results in outfluxes of water from the nest (Ackermann, 1997). This will add to the detrimental effects of coarser sand characteristics on embryogenesis. The hotter and drier nesting season on Wan-an thus might resulted in its higher hatching mortality and a lower hatching success. Post-hatching mortality was also higher on Wan-an. This might relate to the fact that in a drier nesting environment, Figure 3 Minimum spanning network showing the relationships embryonic development was less complete, producing weak- between the four mtDNA haplotypes among the green turtles nesting er hatchlings (Packard, 1999) and decreasing hatchling in Taiwan. Hatch mark represents the number of mutational steps emergence successes. This is consistent with the lower clutch between pairs of haplotypes. Squares indicate the haplotypes corre- survival we found on Wan-an. sponding to the presumed ancestral haplotypes (CmP3=A3; Despite the fact that nests were deeper on Wan-an, the CmP49=C3) in two of the distinct clades identified in Dethmers incubation duration was shorter. Several environmental et al. (2006) in the Indo-Pacific. factors, such as nest temperature, humidity and movement of respiratory gases, can influence the rate of embryonic development, and thus the incubation duration (Packard, Nesting characteristics Packard & Boardman, 1982; Hewavisenthi & Parmenter, The internesting interval was longer for Wan-an rookery 2002). The hotter and drier nesting environment on Wan-an than Lanyu rookery, likely due mainly to the difference in might decrease the incubation duration by speeding up water temperature of the internesting habitats during the embryo development (Glen et al., 2003). Despite the short oviducal phase of embryogenesis and egg shell formation duration, Wan-an females produced larger and heavier (Miller, 1985; Limpus et al., 2003). The higher Q10 for water hatchlings. This suggests that in the present study the temperature suggests sensitivity to the ambient water tem- maternal effect (larger eggs produce larger hatchlings; He- perature. Lanyu is located in the main stream of the warm wavisenthi & Parmenter, 2002) is more important than the Kuroshio, while Wan-an is in the middle of the continental environmental factors (shorter incubation produces smaller shelf, dominated by tidal currents. The surface water hatchlings; Broderick, Godley & Hays, 2001a). temperature around Lanyu thus was warmer than that of The differences in the nesting environment and the nest Wan-an (Central Weather Bureau, 2006). characteristics that we found between the two islands may The females emerged fewer times and made fewer nesting have important demographic consequences. The shorter attempts on Lanyu. This might relate to the difference in the incubation periods associated with warmer temperatures size of the nesting beaches. There are less options for nesting suggest a female sex ratio bias in hatchling production on sites on Lanyu, where there are fewer beaches than on Wan- Wan-an. The impact of this sex ratio bias on demographic an, and where most of the turtles were nesting on the traits such as the genetic diversity and reproductive output, smallest beach (I.-J. Cheng, unpubl. data). Turtles made would be more measurable given the very small size of this more nesting attempts and dug deeper nests on Wan-an. The population, and might in part explain why similar differ- difference might relate to the different nesting environ- ences have not been reported elsewhere between larger ments. Sand grain size was more variable on Wan-an. In rookeries in close proximity. The decrease precipitation addition, the sand layer was thinner on Lanyu. One can since 1997 (I.-J. Cheng, unpubl. data) on both islands might easily dig a pit more than 1 m with a bare hand on Wan-an result in higher sand temperature during the incubation. nesting beach, while on Lanyu, shovel is needed to remove This will further enhance the bias of the hatchling sex ratio. the top sand layer and then wear a glove to dig a similar Certainly, heavy rainfall has been shown to be a major depth. Sand particles on Wan-an are less cohesive and the driver of incubation temperatures at other turtle rookeries

Journal of Zoology 276 (2008) 375–384 c 2008 The Authors. Journal compilation c 2008 The Zoological Society of London 381 Comparison of life-history traits between two green turtle rookeries I.-J. Cheng et al.

(Houghton et al., 2008). Further studies to test for popula- small population size, loss of Lanyu rookery would lead to tion differences in pivotal temperatures for sex determina- loss of genetic diversity for this species, and therefore special tion would help elucidate whether these characteristics conservation consideration is warranted. are the result of recent environmental changes, or whether the populations have adapted to longer standing differences Conclusion in the nesting environment that differentiate these two Our study found significant morphological differences and beaches. genetic isolation between these two green turtle populations, despite their relatively close geographic proximity. We have Genetic structure discussed the environmental parameters that may explain these trait differences, and the conservation implications for The mtDNA sequence analysis showed that both nesting these depleted populations. This study further stresses the populations are genetically distinct. The differences are importance of long-term monitoring the important life- quite pronounced, suggesting no gene flow between two history traits, such as hatching success as well as genetic nesting populations. This is consistent with the morphological and behavioral differences detected between two rook- data, and environmental parameters in order to provide the meaningful insight into the distinctiveness of two rookeries. eries. Our tagging results have also not indicated any interchange of nesters between the two islands, which further corroborates the conclusion that the two rookeries are demographically independent. These findings are sur- Acknowledgments prising given the close geographic proximity of these two The authors thank J-W. Kuo, M-D. Chang and volunteers rookeries, and are strong evidence for localized natal hom- for fieldwork, and Robin LeRoux, Erin LaCasella and Amy ing. In general, this level of population differentiation has Frey for their assistance with laboratory analysis. Thanks to been found between rookeries 4500 km apart (Dethmers George Balazs for facilitating collection of genetic samples. et al., 2006). It is notable that Wan-an is on the continental Genetic analyses were carried out at the NOAA Southwest shelf, while Lanyu is located in an oceanic environment and Fisheries Science Center, La Jolla Laboratory. All work was the hatchlings leaving the beach at Lanyu would be exposed conducted in accordance with international (CITES, 2000) to oceanic currents that would likely transport them to and Taiwanese legislation (COA, 2000–2006) under the different developmental habitats than Wan-an hatchlings. scientific collection permits AF0961700204, CITES Permits The Lanyu population is comprised of a single haplotype Taiwan export permit 01US844694/9-06US844694/9. We (CmP49) that has also been found in several Australasian wish to acknowledge the use of Maptool program, available and Indo-Pacific rookeries (Dethmers et al., 2006). Two of from http://www.seaturtle.org. This research was funded by the other haplotypes (CmP18 and CmP19), found in the the Council of Agriculture, I-Mei Environmental Protection Wan-an population, have not been detected elsewhere (P. H. Foundation and NOAA-Fisheries. We thank Graeme Hays Dutton et al., unpubl. data), and are closely related to the and an anonymous reviewer for helpful input that improved Lanyu haplotype (Fig. 3). This suggests that these two the paper. rookeries evolved from a shared common ancestor, CmP49, which Dethmers et al. (2006) propose as an ancestral haplotype (‘C3’, see Fig. 3) for one of the clades identified in their survey of Indo-Pacific rookeries. The References presence of the widely divergent haplotype (CmP20; Fig. 3) Ackermann, R.A. (1997). Chap. 4. The nest environment and in the Wan-An population suggests deeper evolutionary the embryonic development of sea turtles. In The biology of connectivity with other rookeries in the western and Indo- sea turtles: 86–106. Lutz, P.L. & Musick, J.A. (Eds). New Pacific region, and possibly indicates multiple colonization York, USA: CRC Press. events (see Dethmers et al., 2006). The Wan-an population has higher genetic diversity than Lanyu, with h (0.48) within Alavarado, J. & Murphy, T.M. (1999). Nesting periodicity the range of 0.15–0.82 reported by Dethmers et al. (2006) for and internesting behavior. In Research and management green turtle rookeries within the Pacific region. However, techniques for the conservation of sea turtles: 115–118. the high nucleotide diversity we found for Wan-an is Eckert, K.L., Bjorndal, K.A., Abreu-Grobois, F.A. & consistent with a scenario of multiple colonization events. Donnelly, M. (Eds). Washington, DC: IUCN/SSC Marine Further work is underway to determine the phylogeographic Turtle Specialist Group Publication No. 4. relationships among the Taiwanese and other Pacific rook- Bolten, A.B. (1999). Techniques for measuring sea turtles. In eries (P.H. Dutton et al., unpubl. data). Research and management techniques for the conservation of Our findings, taken together, show that the Wan-an and sea turtles: 110–115. Eckert, K.L., Bjorndal, K.A., Abreu- Lanyu rookeries are two distinct genetic stocks and should Grobois, F.A. & Donnelly, M. (Eds). Washington, DC: be managed accordingly. The genetic distinctiveness of these IUCN/SSC Marine Turtle Specialist Group Publication two rookeries suggests that there exists a substantial and No. 4. persistent nesting site fidelity that limits gene flow between Bowen, W.B., Meylan, A.B., Ross, J.P., Limpus, C.J., Balazs, the rookeries. Our results show that despite its relatively G.H. & Avise, C.J. (1992). 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