| Echinotriton chinhaiensis is a kind of amphibians endemic to the city of Ningbo,Zhejiang Province of China.It is a secondary national rotected animal species in China,but its wild population is less than 400,and its population is extremely rare.It's listed as critically endangered on Red list of China's Vertebrates and IUCN Red List of Threatened Species,indicating that it is likely to become extinct in the wild.Only a few scholars had reseached about the breeding habitat,artificial captivity experiment to its wild population.In this paper,molecular identification,breeding habitat selection and early development were studied.Meanwhile,we succeeded in breeding the larva.This study can provide basic guidance for in-situ conservation and artificial captivity of E.chinhaiensis,and provide reference for the protection of other endangered species,such as E.maxiquadratus and E.andersoni.(1)Study on molecular evolution: We selected CO I gene,12 s r RNA gene,16 s r RNA gene and D-loop region sequence of mitochondrial DNA common target,and amplified the sequence of PCR.Compared with the relative species as E.andersoni,Yaotriton wenxianensis and Tylototrition kweichowensis.The clustering and comparative analysis were carried out.The results showed that the DNA sequence was identical to that of CO I gene,12 s r RNA gene and 16 s r RNA gene in Gen Bank database,but the sequence of D-loop region failed to enter.The sequence similarity and cluster relationship showed that the CO I sequence measured in this experiment was identical with that of Gen Bank.The sequence most accurately reflected the geographical differentiation and phylogenetic relationship between E.chinhaiensis and other related species.Therefore,CO I gene is the preferred sequence for species identification and taxonomic research comparison with other genes.(2)Selection of breeding habitat: It was found that the selection of the breeding grounds of E.chinhaiensis preferred areas with high vegetation density through the comparison of ecological factors such as water body and vegetation,shallow water level,smaller water area and lower dissolved oxygen.It was found that the dominant vegetation in the breeding habitat at the same time.The study has important guiding significance for habitat vegetation transformation and artificial habitat production.(3)Early development of wild population: Field observation on the egg production of E.chinhaiensis from 2015 to 2018 showed that in different years and ponds the spawning quantities were different.The spawning period lasted from the end of March to the beginning of May in 2018.The average number of eggs was 72.40 ±33.57 eggs / nest(n=88).The hatching rate was93.87 ±3.92% and the incubation period was 35 days.The rate of larvae successfully migrating to water sources was 27.45 ±16.13%.The total length(X)of aquatic larva was related to body weight(Y),Y=38.12X2-52.99X+23.11(R2=0.7562).The larvae metamorphosed and left the water after 62-82 d,and the rate of freshly metamorphosed salamanders leaving the water was16.44±11.80%.The mortality rate was highest in the stage of freshly metamorphosed salamanders leaving the water.(4)Analysis of embryo development,larvae growth in artificial culture and feeding habits: The early development procedure was studied and the feeding habits of the aquatic larvae were analyzed.In this study,The larvae were fed with 5 different feeds.The results show that:(1)Among the 5 kinds diets Tubifex was significantly different from other(P < 0.05),and the feeding effect was the best.(2)E.chinhaiensis takes a preference for live feed,cooked egg Yolk is not suitable for larval feed;(3)The larvae fed with artificial feed healthier than the individual fed in the field;(4)Mixed feed had better feeding effect than single feed.This study is helpful to guide the artificial breeding,to reach the goal of reintroduction and expansion of the population. |
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