Female Reproduction,nest-site Use,Developmental Plasiticity And Genetic Diversity Of Green Sea Turtles On Ganquan Island,South China Sea

Green sea turtle(Chelonia mydas)is a large marine reptile,which is distributed globally and highly migratory.It is called the“living fossil”of the ocean and has importantly ecological,cultural and scientific value.Factors such as global warming,habitat destruction and illegal trade have led to severe declines in wild populations of the species and the species has been assessed as endangered species by the International Union for Conservation of Nature(IUCN)Red Catalogue and also listed as a National First-class Protected Animal in China.The coastal areas of South China and the South China Sea are important breeding areas for C.mydas,and the Xisha Islands in the South China Sea is the largest spawning ground in China.However,little were known about the resource status and population genetic information of field green sea turtle in the region as well as the anthropogenic and ecological threats they is facing.The lack of basic research data in these aspects have severely restricted the development of conservation and management strategies for sea turtles in China.In this study,we conducted long-term monitoring for the nesting of green turtle in the rookery of Ganquan Island,Xisha Islands where the nesting number of green turtle is relatively abundant.We used research methods of macroscopic,physiological and molecular ecology to collect comprehensively the studied data of Ganquan C.madys,which were related to population dynamics,including of female reproduction,field hatching of eggs,pivotal temperature of sex determination,offspring sex ratio,genetic diversity and genetic structure,as well as developmental temperature plasticity.And we discussed the ecological threats that breeding population on Ganquan Island is facing under the background of global warming.In addition,the study also analyzed the population sources of confiscated green turtle population based on mixed-stock analysis and assessed the high-risk populations that were most vulnerable to illegal trade.Finally,based on the results of this study,some suggestions on the conservation and management of green sea turtle resources in China were put forward.The main results were shown as below:1.An average of 55 nests(36-73 nests)in a single breeding season were nested by C.mydas on Ganquan Island and the breeding season lasted from May to December with the peak nesting season being from July to September.Western sand beach(about1 km in length)of Ganquan Island has the highest nesting frequency,which is the core area of nesting.During the whole breeding season,the mean nest temperature of in-situ nests was 31.5±0.2 ^oC;the field mean incubation length,hatching success and emergence success of the in-situ nests were 50.8±0.6 d,76.6%±2.5%and 70.7%±2.7%,respectively.The nesting season of Ganquan C.mydas had a significant effect on egg incubation in their in-situ nests;the mean nest temperature,incubation period,hatching success and emergence success decreased,prolonged,increased and increased,respectively as nesting season delayed.2.A total of 16 pairs of microsatellite primers with high polymorphism were developed.The maternal right relationship of offspring from 56 C.mydas natural nests on Ganquan Island showed that 56 natural nests belonged to 19 breeding females,of which 25 nests from 2018 belonged 8 breeding mothers and 31 nests from 2019belonged 11 breeding mothers.The same breeding female was not detected between2018 and 2019.Based on the maternal rights among natural nests,it was estimated that there were about 18.8 green turtle females nesting on Ganquan Island each year,and that each mother laid an average of 2.9 clutches in a single breeding season and the average nesting interval of same female was 14.8 days.3.The functional equation between the proportion of female offspring(PF)in the in-situ nest and the mean nest temperature of in-situ nest during the thermal sensitive period(TSP)is PF=101.4032/{1+e^{-[(TSP-29.4379)/1.1726]}}.Based on the equation,it was estimated that the pivotal temperature produced the equilibrium ratio was 29.4 ^oC for Ganquan C.mydas population.During the whole breeding season,the proportion of female offspring in Ganquan C.mydas population was 74.6%on average.With the postponement of nesting season of green turtles,the proportion of female offspring hatched from their in-situ nests decreased.Specifically,nests produced from May to September hatched mainly female-biased offspring,while those produced from October to December hatched mainly male-biased offspring.The results suggest that the offspring sex ratio of Ganquan C.mydas population have a serious female skew in the present stage and the nesting season of C.mydas can regulate the sex bias direction of offspring within their in-situ nests.4.The results of egg incubation within in-situ nests showed that there was a significantly negative correlation between field incubation period and the mean nest temperature;with the increase of the mean nest temperature,the field incubation period decreased non-linearly.Moreover,the relationship between the field hatching success and the mean nest temperature was a very significantly parabolic function,with field hatching success increasing firstly and then decreasing with the increase of mean nest temperature;when the mean nest temperature is in the temperature range from 27 ^oC to32 ^oC,the field hatching success will be optimal(>80%).Incubation results of C.mydas eggs incubated at a constant temperature(26-34 ^oC)in the laboratory showed that incubation temperature significantly affected incubation length and hatching success of eggs,phenotype and terrestrial locomotor performance of offspring as well as early growth of offspring.C.mydas eggs incubated at 34 ^oC had lowest hatching success(26.9%)and none of hatchlings hatched at this temperature was normal and survived over one week.Body weight and shape of C.mydas hatchlings(carapace size index,head size and fore-and-hind flippers)tended to decrease with the increase of incubation temperature.At a constant temperature range from 26 ^oC to 32 ^oC,the fitness of hatchlings hatched at 30 ^oC were the highest,followed by 28 and 32 ^oC,and the lowest at 26 ^oC.The results of constant temperature incubation indicate that both 26 ^oC and 34 ^oC are unsuitable for incubation of C.mydas eggs and temperatures within the range from 28 ^oC to 32 ^oC are generally optimal for embryonic development of C.mydas.5.Seven haplotypes were detected based on the 770 bp d-loop sequence of 309samples from the Ganquan C.mydas population,of which two haplotypes(Cmp19.1and Cmp49.1)have been described in the Pacific region,and the other five haplotypes(GQ1,GQ2,GQ3,GQ4 and GQ5)are new and unique haplotypes.Cmp19.1 was the dominant haplotype,accounting for 67.6%of all samples.The nucleotide diversity and the haplotype diversity for Ganquan C.mydas population was 0.00093±0.00007 and0.515±0.031 respectively.In addition,a total of 110 haplotypes were identified based d-loop sequences(770 bp)data in 33 green turtle management units(including Ganquan C.mydas population)in the Pacific,and the average nucleotide diversity and haplotype diversity of green turtles in the Pacific was 0.00635 and 0.528 respectively.The results indicate that Ganquan C.mydas population have high genetic diversity.6.There was significant genetic differentiation between Ganquan C.mydas population and 32 green turtle management units reported in the Pacific Ocean,indicating that Ganquan C.mydas population is a genetically independent evolutionary population;therefore,it can be defined as an evolutionary significant unit.In addition,Bayesian phylogenetic analysis showed that 110 d-loop haplotypes(770 bp)of C.mydas in the Pacific belonged to six evolutionary clades(lineages)with high posteriori probability,and the geographical distribution of each genetic lineage in the Pacific showed obvious regionalism,suggesting that there may be barriers to gene diffusion between regions of the six lineages.7.The confiscated green turtle samples were mainly from two nesting populations,namely Sulu Sea population and Lanyu population,in Taiwan.In two analysis models,the cumulative contribution ratios of the two populations to the confiscated samples were 95.7%(uniform prior)and 97.8%(weighted prior),respectively.Furthermore,in two major source populations,we found that Lanyu population contributed the largest proportion to the confiscation samples,with an average contribution of 60.2%(uniform prior)and 61.7%(weighted prior).Thus,the results demonstrate that the Sulu Sea population and Lanyu population are currently under threat from the illegal trade;especially,the Lanyu population is the most affected by the illegal trade,while the Ganquan C.mydas population is not under threat from illegal trade so far.8.Based on the above research results,the following protection suggestions are put forward.(1)Delimit functional zones on Ganquan Island,and the western beach of Ganquan Island should be set as the protection core area.(2)According to the seasonal characteristics of natural nest temperatures of green turtle in Ganquan Island,formulate corresponding protection measures for natural nests produced in different seasons.(3)Protect the genetic diversity of Ganquan C.mydas population.(4)Establish trans-national or trans-regional joint protection strategies for sea turtles.In conclusion,this thesis is the first to reveal systematically the resources status,reproduction characteristics,pivotal temperature of sex determination,the current offspring sex ratio,developmental temperature plasticity,genetic information and the current ecological threats for C.mydas population nesting on Ganquan Island,Xisha Islands.In addition,the thesis also reveals the high-risk populations most affected by illegal trade in the Pacific.These results deepen our understanding of the current population ecology and threatened factors of C.mydas in South China Sea,and provide important data support for the scientific development of management and conservation strategies of C.mydas in China.