| Hynobius yiwuensis belongs to Animalia,Chordata,Amphibia,Caudata,Hynobiidae,Hynobius.This species is endemic to China,and the threatened class is"vulnerable"(VU).At present,there are few studies on this species,mainly focusing on histology,systematic classification,karyotype analysis,genetic differentiation,etc.,but there is a lack of relevant data on its habitat survey,population statistics,and reproductive biology.In order to better protect wild population resources and maintain biodiversity,this study investigated the distribution and habitat status of the population of H.yiwuensis in Pujiang county and Yiwu city from the perspectives of resource investigation,embryonic development and genetics.The parents were collected and the egg bags were obtained by indoor artificial breeding.The embryonic development process and the optimal incubation conditions were observed and studied.In addition,the mitochondrial D-loop molecular marker technique was used to analyze the genetic diversity of 107 individuals from four sampling sites in Pujiang county and Yiwu city,so as to provide scientific basis for their future management.The main results of this study are as follows:(1)resource survey1)since 2017,a total of 15(Pujiang 7,8 from yiwu)wild habitats of H.yiwuensis including Zhangshan,Lihuang,Limei,City forest farm et al have been found through the investigation of the whole area of Pujiang county and Yiwu city,determine the scope of the physical distribution of H.yiwuensis 14.50 km~2(Yiwu 3.75 km~2,Pujiang10.75 km~2),a total of investigation to the adults 11 tails,larva 4151 tails,egg bags338 pairs(Pujiang adult 9 tails,larva 2509,egg bags 189 pairs;Yiwu adult 2 tails,larva 1642 tails,egg bags 149 pairs).In addition,no larva,subadult,adult and egg bag were found in other investigation areas.Seven of the habitats(4 in Pujiang river and 3in Yiwu)were assessed to be affected by human disturbance to varying degrees.2)By means of blanket absolute number statistics and assuming that the sex ratio of male and female H.yiwuensis involved in reproduction is 1:1,the number of adult population was estimated according to the number of egg bags.The population number was defined by larval number and adult number,and the high-density distribution area in Pujiang county was determined to include Zhangshan habitat(population number 1123 tails,density:1018 tails/km~2)and Songxi habitat(population number 540 tails,densit:2076 tails/km~2).Low-density areas include Lihuang habitat(population number 1176 tails,density 529 tails/km~2),Dongling habitat(population number 93 tails,density 26 tails/km~2),Tangwu habitat(population number 49 tails,density 21 tails/km~2)and Tangling head habitat(population number 11 tails,density14 tails/km~2).The high-density distribution areas in Yiwu city include Limei habitat(population number 335 tails,density 1080 tails/km~2)and city forest farm habitat(population number 373 tails,density:1094 tails/km~2).Low density area including the Huangshan mountain habitat(population 393 tails,density 427 tails/km~2),Wangdao habitat(population number 664 tails,density of 845 tails/km2),Duiwu habitat(population number 30 tails,population density 63 tails/km~2),Wayao habitat(population number 66tails,density 182 tails/km~2)and Dongzhu habitat(population number 71 tails,density 125 tails/km~2).3)To determine survey across Pujiang county and Yiwu city,H.yiwuensis habitat at 150?500 m above sea level in the hilly mountain,preference,dark wet environment,mainly live in full of litter in the bamboo forest,broad-leaved forest and mixed needle,habitat in natural vegetation types can be divided into to include coniferous forest,mixed forest,needle broad-leaved forest,economic forest,shrubs,bushes and plants,all layers of the 7 vegetation type groups,10 vegetation type,22 formation group,22formation.4)In the H.yiwuensis distribution area of the two places(Pujiang county and Yiwu city),based on the soil type,crown density,water clarity of 16 kinds of habitat factors,through the 78 samples(24 selected samples and 54 so on),research on breeding habitat selection in H.yiwuensis,results show that the selected samples and so on conditions on the shore,the water covers,water clarity,water temperature,water area,vegetation coverage,six kinds of ecological factors on significant difference.The stepwise discrimination shows that the correct discrimination rate of the quadrat and the quadrat can be determined by vegetation coverage,water temperature and water area.Stepwise regression analysis results show that the H.yiwuensis breeding habitat selection and pH is significantly related to the water and the water temperature has significant negative correlation,above all,H.yiwuensis breeding preference during the period of microenvironment for low canopy density,smaller area of low water,water temperature,multiple,complex water covering the shore conditions,weak alkaline water hydrostatic model pond or pool.5)According to the prediction results of MAXENT niche model output,the seasonal variation of rainfall(BIO15)contributed the most to the model,accounting for 42.3%of the total contribution rate of all environmental variables,followed by32.4%of the precipitation in the wettest season(BIO16),and the contribution rate of other environmental variables was small or 0.The high-fitness area of Pujiang river is mainly distributed in Huaqiao township,Qianwu township and Punan street,etc.The area is 108.34 km~2,accounting for 11.8%of the total area.The sub-suitable area is mainly distributed on the edge of the high-suitable area,which is located in Hangping town and Xianhua street,with an area of 191.90 km~2,accounting for 20.9%of the total area.The low-fitness area is mainly distributed in the areas of Yuzhai,Yantou and Zhengzhai,with an area of 205.67 km~2,accounting for 22.4%of the total area.The unsuitable areas are mainly distributed in areas such as Dafan,Tanxi and Zhengjiawu,with an area of 413.17 km~2,accounting for 45.0%of the total area.The high-suitability area of Yiwu city is mainly distributed in the north of Shangxi town,the north of Chengxi street,and the south of Chian town,with an area of 49.75 km~2,accounting for 4.5%of the total area.The sub-suitable area is mainly distributed on the edge of the high-suitable area,which is located in the middle of Shangxi town,Chengxi street,Dachen town and Suxi town,with an area of 127.13 km2,accounting for 11.5%of the total area.The low-fitness areas are mainly distributed in the south of Shangxi town and Yiting town,north of Houzhai street and east of Fotang town,with an area of 262.00 km~2,accounting for 23.7%of the total area.The inappropriate areas are distributed in Futian street,Choucheng street,Choujiang street,Niansanli street and other areas,with an area of 666.59 km~2,accounting for 60.3%of the total area.(2)embryonic development observationthe early embryonic development of H.yiwuensis is divided into 26 stages.Under the condition of water temperature of 10±1?,the embryo development is slow and takes about 1088 h to hatch.(3)Selection of optimum incubation conditionsunder the conditions of water depth of 5 cm,water temperature of 15±1?and density of 4000/m~2,embryo of H.yiwuensis have the best hatching rate(99.57±0.75%)and incubation time(115.4±56.0 h),control the light intensity of 1500 lx,the hatching rate was the largest(96.7±3.3%),and the incubation time was the shortest(145.8±5.7h).(4)genetic diversity analysis1)the results of the detection of the genetic diversity of H.yiwuensis based on the mitochondrial D-loop region showed that the genetic diversity of H.yiwuensis was at an extremely low level.In 107 sequences,6 haplotypes were obtained by screening 8mutation sites.Haplotype polymorphism was 0.3232 and nucleotide polymorphism was 0.00587 in all the samples.2)use of mitochondrial D-loop molecular markers to detect the genetic differentiation of the population.The range of mitochondrial data analysis differentiation coefficient(FST)was between-0.00584 and 0.05128,with a very low differentiation coefficient.In the detection results,the FST was negative,which may exist that the differences within the samples were greater than the differences between the samples.At the same time,the analysis showed that the gene exchanges between the samples were significant.The results of mitochondrial sequence analysis of AMOVA showed that the genetic variation of AMOVA mainly existed in the variation between individuals,with little or no differentiation.3)the mitochondrial D-loop control region was used to investigate the historical dynamics of the population of H.yiwuensis.Both the Tajima's D value and Fu's Fs value of the population of H.yiwuensis were less than 0,and neither reached the significant level P>0.10),indicating that the population of Pujiang and Yiwu was still in a state of neutral selection,and the neutral evolutionary diversity of the population was not significantly deviated.Fu Fs=-3.230,indicating the existence of recent genetic variation. |
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