Conservation Biology Research Of The Asian Giant Softshell Turtle,Pelochelys Cantorii

The Testudines are appeared in the late Triassic period with the same generation of dinosaurs and have a history of more than 250 million years.Testudines has an important reference value in the long geological evolution and species evolution.The Asian giant softshell turtle Pelochelys cantorii(Trionychidae),is one of the largest aquatic turtles in China.The P.cantorii was not only widespread but also abundant in ancient times,however,the range of its distribution has been gradually reduced and the population number has been reduced due to the increase of human activities in modern times,the continuous deterioration of habitat and the overfishing of human beings,it has become critically endangered,in some areas it was in a state of extinction and the species has been designated as a First Grade Protected Animal in China in 1989 and listed as endangered species by the IUCN Red List in 2000 and put on par with the panda and the white-flag dolphin.But the species have not received much attention just with few reports on related research,and there have been almost no reports of individuals in the wild in the past 10 years.Given this,this study summarized the status quo of P.cantorii conservation in China by investigating nature reserves and artificial domestication base.The laying egg time,behavior and,other characteristics of the laying egg for the first time and many years'P.cantorii were studied through six artificially domesticated parent turtles.The germplasm characteristics of morphology,DNA content,cell karyotype,and the relationship between the length of the carapace and body weight were studied.Based on the second-generation genome sequencing,the genome was assembled and annotated,and the correlation analysis of species evolution was carried out.Multiplex PCR was developed for the microsatellite paternity test of P.cantorii.Finally,according to the biological characteristics of the P.cantorii and the current situation of resource distribution,some reasonable suggestions for the management and protection of the resources of the P.cantorii are put forward.The main research results as follows:1. The semi-structured interview method was used for the conservation of P.cantorii in the nature reserves.The results showed that the only Yuanyuan Provincial Nature Reserve in China(Qingtian County Nature Reserve)and Guangning nature have done a good job in the protection and management of the reserve,with index scores of3.75 and 3.0(four points in total),but the habitat environment is still not optimistic.Three nature reserve with the indexes of 1.25,the management of the nature reserves were a sham.In other counties or cities,conservation and management were weak,and the habitats on which the P.cantorii live were basically destroyed.Currently,there are 5artificial P.cantorii domestication sites in China,two of which have successfully conducted artificial P.cantorii breeding.The results of the questionnaire survey on the villagers in the river section of the artificial breeding reserve for the first time in China show that that 63.6 percent of the villagers along the coast did not know the turtle,and only 15.2 percent of the fishermen had seen the turtle,and most of it happened 10 to 20years ago.77.3 percent of the fishermen believed that the construction of dams in the river resulted in a decrease in the species and biomass of fish,72.7%of the fishermen believed that the water quality of the Suijiang river section was polluted by the factories built along the bank.In general,although P.cantorii is widely distributed in history,the species is becoming more and more fragmented in space distribution due to the interference of human activities in recent years.The population of P.cantorii is endangered and the situation of conservation P.cantorii is not optimistic.2. From 2014 to 2019,breeding biology was carried out on two P.cantorii(one?and one?)in Guangning city and four P.cantorii(two?and two?)in Gaoming city.The laying egg period of the artificial domesticated P.cantorii was mainly from May to August,laying eggs behavior that happened at night,without protective egg behavior.Through the analysis of video,the oviposition behavior was divided into IV stages.There were significant differences in oviposition characteristics between the Guangning P.cantorii and Gaoming P.cantorii.The average weight of the eggs,the weight of the eggs and the weight of the newly hatched juvenile of the Guangning P.cantorii and Gaoming P.cantorii were 13.27±0.97 g and 17.31±1.99 g,2.90±0.12 cm and 3.13±0.25cm,10.00±0.56 g and 13.31±1.03 g respectively.The Guangning female P.cantorii laid three nests of eggs,with an average of 22 per nest.While each female of Gaoming laid5-6 nests with the clutch size is 43.6±8.2 individual from 2016-2019,the laying egg time(T,min)of each nest was positively correlated with the number of eggs per nest(N),N=4.3863 T-0.9059(r=0.872,P<0.01),with an average of 1 egg laid at 13.67 s.There was a significant correlation between egg weight(We,g)and egg diameter(De,cm),We=8.3362 De-8.8494(r=0.861,P<0.01).The average egg-laying interval was16.49±4.32 d,with a median of 15 d.The average incubation period was 64.94±3.47days at the temperature was 28.8±1.5 and relative air humidity was 59.9±4.9%for Gaoming P.cantorii and the mean egg mass and mean egg size were positively related and there was no significant difference in average clutch size between the two females,but the heavier females produced significantly heavier eggs than the lighter females.The incubated juveniles were fed by live fish seedlings;they could be reared to510.30±82.77 g in the greenhouse for one year and the established growth formula was Y(body weight,g)=1.4993 t(days,d)+58.521(R~2=0.991).In 2019,the hatching rate was 78.39%,the survival rate of the seedlings was 93.33%,and more than 700offspring were successfully bred in the past 5 years of Gaoming P.cantorii.These studies have achieved artificial conservation of P.cantorii species,enriched the basic data of breeding biology,and provided material and theoretical basis for its conservation.3. To better understand and protect the species of P.cantorii,the study on the characteristics of its germplasm was carried out.There were significant differences between male and female individuals in the tail length and the color of dispersal pores,and there were also significant differences in the body surface markings between juvenile and adult individuals.In terms of morphological and measurable traits,there were significant differences in carapace length/carapace width,proboscis width/carapace length,soft proboscis length/carapace length,the distance between eyes/carapace length,body highness/carapace length between P.cantorii and Chinese soft-shell turtle Pelodiscus sinensis.The body length(SCL)and body weight(TW)relationship of hatchling to 3-age P.cantorii was log TW=log 0.148+2.858×log SCL(n=164),which was the same as the non-inverse growth pattern of Chinese turtles.The DNA content of the P.cantorii cells was 3.1033±0.722 pg/2c,which was significantly lower than P.sinensis which was 4.0967±0.1430 pg/2c.The analysis of the metaphase in 1 individual showed that 2n=62 chromosomes of the P.cantorii.The study of these germplasm characteristics provides theoretical guidance for species identification of P.cantorii.4. Genome sequencing,assembly,and analysis were performed on the genome of a turtle based on the whole-genome shotgun sequencing strategy(WGS)and Illuminate Hiseq 2500 sequencing system.A total of 293.4 Gb of genome data was obtained,and the length of the assembled genome Contig N50 was 32.2 Kb and the length of Scaffold N50 was 1.47 Mb.The genome was estimated to be approximately 2.27 Gb in size.The average GC content of the genome was 44.9%,which was slightly higher than that of similar species such as P.sinensis,green sea turtle Chelonia mydas,and western painted turtle Chrysemys picta bellii.The repeat sequence obtained by genome annotation was40.34%,LINE repeat sequence was the highest,reaching 28.73%,and SINE was only0.99%.There were 19767 genes in the annotated gene set of P.cantorii,with an average transcript length of 40629 bp,an average CDs length of l637 bp,an average exon number of genes of 8.75,an average exon length of 187 bp,and an average intron length of 5029 bp.Using 100 single-copy homologous gene families from 14 species,the evolutionary tree shows that the divergence between P.cantorii and P.sinensis was about 55 000 000 years ago.CAFE software based on the most recent common ancestor analysis results shows that the P.cantorii expanded 684 gene families and shrank 2485gene families compared with 12 other species.Using PSMC to analyze the population dynamics of turtle species,the results showed that the population in P.cantorii and P.sinensis,decayed in 10~6?4×10~6 years,but the effective population doubled for P.cantorii in 2.5×10~5?5×10~5 years,the effective population size of Chelonia mydas remains relatively low.5. Genome-wide scan data of can,182277 microsatellite sequences were found,62loci were obtained through the filter as candidate markers. The candidate markers were tested for polymorphism using genomic DNA samples from 35 individual samples of P.cantorii,the typing results were reviewed,and the microsatellite loci with polymorphism were screened.Two groups of microsatellite marker multiplex PCR systems were constructed,each of which contained 12 markers.The genotyping success rate of all multiple PCR sites in the two groups was 97.1%and 99.2%,respectively.The GERUD2 software analysis showed that 99.8%of the offspring could be identified with10 loci with known parental information,97.8%of the offspring could be identified with a known parent,and 87.9%of the offspring could be identified with unknown parents.Multiple paternity was found in the offspring of two clutches,indicating that there may be multiple mating in the P.cantorii.These conclusions will effectively guide the offspring breeding management of P.cantorii.6.In combination with the previous investigation of the P.cantorii resources and environment and the biological research,we analyzed that the reasons for the endangered P.cantorii are as follows.The first is the internal reason,the mature period of P.cantorii is long and many individuals die before reaching adulthood.With the habit of swallowing food and diving into sand,it is easy to be captured by human beings.Second,objective reasons,mainly human hunting and killing,water conservancy project construction,river sand mining,water environmental pollution,threat of invasive species and other reasons.For protection situation and the existing problems and puts forward:the wild resource investigation and the habitat environment monitoring should be strengthened,strengthen the construction of nature reserve and management,actively carry out habitat restoration and effective protection and in-depth study on artificial breeding technology,establishing artificial breeding groups to conservation,and ex situ conservation and release,strengthen the public propaganda and popular science education and so on,to save and protect the endangered species.