Genetic Diversity And Conservation Of Tanichthys Albonubes

As one of the most representative fish in Guangdong Province,White Cloud Mountain minnow(Tanichthys albonubes)has been listed in the second class state protected animals in China,and its status is extinct in nature in the China red data book of endangered animals.In 2003,the natural population of T.albonubes were rediscovered in CongHua city,In order to understanding its natural distribution,habitat,the genetic diversity and ecological characteristics, and performing the conservation of its natural resource,a series of investigations in wild and experiment research were performed.Firstly,we investigated the natural population and habitat of T.albonubes,emphasizing on the CongHua city.Secondly,we synthesized its characteristics of genetic diversity,gave the hypothesis that the distribution pattern has taken shape throughout the important geological affairs.Thirdly,we researched its reproductive behaviors and embryonic development,then finished the functional division and technique measures of the CongHua Natural Reserve of T.albonubes.The main results are as follows.1 Distibution and habitat of its natural populationInvestigations indicated that the distributions of its natural population were CongHua city, ZengCheng city,QingYuan city,GaoMing city and LuHe city,Belonging to sub-basin of Liuxi river,Dongjiang,Beijiang,Xijiang and the sub-basin of Lianhua mountain repectively.In this paper,the distributions of ZengCheng city,GaoMing city,LuHe city and seven location of CongHua city were firstly reported.On respect of the body color of T.albonubes.The wild fish displayed two color types,the fish color of LuHe city was different from other distributions,with the red basal margin of basal fin and black spots on its body.There were three type of habitat of T.albonubes in natural distributions,including marsh, mud-sand-bottom stream and sand-stone-bottom stream.The characteristics of habitat are as follows:1.It mainly lived in stream and marsh.2.There are much aquatic plant in its habitat.3. Other fish are seldom in its habitat.No other fish appear in marsh,only several fish appear in stream,such as Puntius semifasciolatus.With rich aquatic plants,phytoplankton and zooplankton,CongHua Natural Reserve of T. albonubes was the typical natural distibution including three types of habitat,Building up reserve in this place is scientific and very important to the conservation of T.albonubes.2 Morphological variations between the natural population and hatchery stock of Tanichtys albonubesThe natural population of T.albonubes in Conghua city were investigated between 2006 and 2007.In this paper,traditional morphological data and truss network data were combined to conduct principal component analysis(PCA) and stepwise discriminant analysis to study the morphological variations between the natural populations and hatchery stock of T.albonubes. The results are showed as follows:1.In traditional morphological data,the morphological variations were significantly different(P＜0.01).2.In truss network data,through PCA with selected 12 morphological parameters,the identification accuracy were 95-100%(PC1) between the five natural population and one hatchery stock,synthetic identification accuracy were 100%.3 Assessing genetic diversity of T.albonubes using RAPD markers.Randomly Amplified Polymorphic DNA(RAPD) was used to detect the genetic diversity of five natural populations and one hatchery stock.six individuals from each stock were used.Of the 100 RAPD primers screened,8 primers generated clear and reproducible bands.A total of 90 reproducible loci ranging from 200 to 2000bp were am-plified from all the 36 individuals. Comparing the percentage of polymorphic loci and the average heterozygosity between six stocks,the CongHua population was the highest with 22.22%and 0.091 respectively.The Shannon's diversity indices between CongHua population,ZengCheng population and QingYuan population were about 0.9251-0.9551 with high similarity,.The Shannon's diversity indices between LuHe population and other five stocks is about 0.6309-0.7459,so the LuHe population was low relative of other stocks.Analysis of molecular variance(AMOVA) revealed that the percentage of variation within stock(26%) was lower than among stocks(74%).The six stocks were clustered into four major clades using UPGMA analysis.4 Assessing genetic diversity of T.albonubes using fAFLP markers.Genetic diversity of five natural populations and a hatchery stock of T.albonubes was investigated using fluorescence-based AFLP(amplified fragment length polymorphism).6 individuals from each stock were used.A total of 334 reproducible bands amplified with four fAFLP primer combinations were obtained from 36 fish.The percentage of polymorphic loci ranged widely from 25%to 52.4%.The percentage of polymorphic loci and the average heterozygosity of six stocks were as follows:CongHua population(48.5%,0.168),ZengCheng population(52.4%,0.190),QingYuan population(42.51%,0.142),Hatchery stock(25.45%,0.100),LuHe population(30.84%,0.111),GaoMing (30.54%,0.113)respectively.The genetic diversity of hatchery stock was the lowest in six stocks. Comparing other fish with AFLP,such as Gobio gobio,Barbatula barbatula,Procypris rabaudi et al.,the genetic diversity of T.albonubes was higher.The average Fst value of overall stocks was 0.395,which indicated a high degree of genetic differentiation among the stocks.Also,the analysis of molecular variance(AMOVA) revealed that the percentage of variation within stock(47%) was lower than among stocks(53%).The estimated Nm value was 0.383,indicated that six stocks were geographic,and no gene flow happened.The six stocks were clustered into four major clades with UPGMA.Through MANTLE analysis, result indicated five natural populations were relative to geologic distance,combining the geological changes,we gave the hypothesis that the natural distribution pattern of T.albonubes has been taken shape throughout the important geological affairs.5 The reproductive behaviors,embryonic development and conservation of Tanichthys albonubesReproductive behaviors of T.albonubes were observed and summaried in this paper,including range behavior,stretching fins,confronting,collision,attacking,courtship and mating.Also,We studied its embryonic development through microscope,divided and illustrated it into 29 developmental stages.Under the water temperature of 27℃(±0.5℃),it took 34 hours and 8 minutes to complete embryonic development.Time for incubation showed a negative correlation to the water temperature.The optimum incubation temperature for T.albonubes embryos was between 24℃and 27℃.In addition,our research showed that the decrease of habitat was negative to the natural resource of T.albonubes,protecting the habitat of T.albonubes was the most effective method.In the process of the establishment of CongHua Natural Reserve of T.albonubes,we considered the habitat,reproductive,genetic diversity,gene flow and restoration,so our work was conforming to the princilple of endangered conservation and the physiology,ecology of T.albonubes.