Population Genetics Of Three Schizothorax Species In The Yarlung Tsango River

Schizothorax oconnori(Lloyd 1908),Schizothorax waltoni(Regan 1905)and Schizothorax macropogon(Regan 1905)belong to Schizothorax,Schizothoracinae,Cyprinidae,Cypriniformes.They are endemic rare fish species with economic importance of the fisheries in Tibet,and the three unique Schizothorax species distributed in the middle reaches of the Yarlung Tsangpo River(YLTR).Amongst the three fish species,S.oconnori,creature card of the YLTR,is the only Schizothorax species that is thought to be distributed in both the upstream and downstream drainages of the Yarlung Tsangpo Grand Canyon(YTGC).Significant theoretical approaches and practical implications are found in the three species due to their specific distribution ranges.All the three species are now threatened by sharp declines in natural population caused by ecological hazards such as overexploitation and illegal fishing,invasive fishes and habitat destruction.It is the critical time to design several effective conservation and management strategies for the three species.Special guidelines and protective measures have been introduced to protect the endemic fish species of Tibet,however,it is still not clear on the characterization of genetic variation in the natural populations of the three species.In this study,we developed polymorphic microsatellite(SSR)markers from the three Schizothorax species for the first time.Afterwards,the levels of mitochondrial and nuclear DNA genetic variability,population genetic structure and demographic history of the three species from the middle reaches of the YLTR were investigated using mitochondrial DNA(mt DNA)cytochrome b(Cyt b)and control region(CR)sequences and polymorphic SSR markers.The primary aim of this study was to evaluate genetic diversity and differentiation within and amongst geographic populations,and to explore conservation management strategies for the three species.The main results are as follows:1.Population genetics of S.oconnoriGenetic variation in the 168 individuals of S.oconori from seven geographic populations were analysed using mt DNA Cyt b(1141 bp)and CR(714 bp)alone,and concatenated mt DNA Cyt b+CR(1855 bp),respectively.A total of 59 Cyt b haplotypes,80CR haplotypes,and 107 Cyt b+CR haplotypes were identified.Genetic diversity analyses revealed that obvious lower haplotype/nucleotide diversity were found in S.oconori from the downsteam region of the YTGC(Bomi BM)in the Parlung Tsangpo River(PLTR)compared to the genetic diversity indexes observed in the other six populations(Shigatse SG,Zhaxue ZX,Quxu QX,Shannan SN,Mainling ML,Paizhen PZ)in the upstream region of the YTGC.High mt DNA genetic diversity was observed in S.oconnori at the species level.Intraspecific phylogeography and population genetic structure revealed that reciprocal monophyly was found based on the haplotypes from the upstream region of the YTGC and the PLTR region(Bomi BM).Significant mt DNA genetic differentiation was observed between the upstream region of the YTGC and the PLTR region(Bomi BM),and two major genetic lineages according to the same regions were identified within S.oconnori.Low mt DNA genetic variation and no obvious genetic lineages were found amongst the six populations in the upstream region of the YTGC.Historical population dynamics analyses indicated that estimates of the timing of this YTGC upstream-downstream split was?1.0Ma,and population size of S.oconnori in the PLTR(Bomi BM)region remained relatively stable over time,while,pronounced population expansion(?0.1 Ma)was found in the six populations in the upstream region of the YTGC.Quaternary Pleistocene climatic oscillations and Qinghai-Tibet Plateau(QTP)uplift have played important roles in shaping the modern mt DNA population genetic pattern of S.oconnori.A total of 42 polymorphic microsatellite markers were isolated from S.oconnori through constructing the(AC)_n and(AGAT)_n microsatellite enriched libraries using the fast isolation by AFLP of sequences containing repeats(FIASCO)protocol.The cross-species amplification and applicability of these loci were tested against the related Schizothoracinae species and high applicability was observed.All the 42 microsatellite markers will be valuable for further population genetics and molecular identification of related Schizothoracinae species.Genetic variation in the 322 individuals of S.oconori from seven geographic populations were analysed using 12 microsatellite markers.A total of 359microsatellite bands were identified,of which 11 microsatellite bands were predicted to be under selection.Genetic diversity and population structure analyses using total(n=359)and neutral(n=348)microsatellite bands revealed that obvious lower Nei's gene diversity index and Shannon's information index were found in S.oconnori from the PLTR region(Bomi BM)compared to the genetic diversity indexes observed in the six populations(Shigatse SG,Zhaxue ZX,Quxu QX,Shannan SN,Mainling ML,Paizhen PZ)in the upstream region of the YTGC.High microsatellite genetic diversity was observed in S.oconnori at the species level.Population genetic structure analyses revealed that a certain level of microsatellite genetic differentiation was observed between the upstream region of the YTGC and the PLTR region(Bomi BM),and two major genetic clades according to the same regions were identified within S.oconnori.Low microsatellite genetic variation was found amongst the six populations at the upstream region of the YTGC,however,three obviously different genetic clades were observed:SG+PZ clustered in the first clade,and QX+SN+ML clustered in the second clade,while ZX was the mixture of the first two genetic clades and was the third clade.2.Population genetics of S.waltoniGenetic variation in the 114 individuals of S.waltoni from five geographic populations were analysed using mt DNA Cyt b(1141 bp)and CR(712 bp)alone,and concatenated mt DNA Cyt b+CR(1853 bp),respectively.A total of 17 Cyt b haplotypes,28 CR haplotypes,and 37 Cyt b+CR haplotypes were identified.Genetic diversity analyses revealed that a slightly lower haplotype/nucleotide diversity were found in S.waltoni from the population Mainling(ML)compared to the genetic diversity indexes observed in the other four upstream populations(Shigatse SG,Maldrogongkar MG,Quxu QX,Shannan SN).High mt DNA genetic diversity was observed in S.waltoni at the species level.Intraspecific phylogeography and population genetic structure revealed that paraphyly or polyphyly was found based on the haplotypes from the five populations.A certain level of mt DNA genetic differentiation was observed between the population Mainling(ML)and the other four upstream populations,but low mt DNA genetic variation and no obvious genetic lineages were found amongst the five populations.Mismatch distribution and neutrality test analyses indicated that the population size of all the five populations remained relatively stable.However,BSPs(Bayesian skyline plots)analyses showed that S.waltoni might experience a pronounced expansion during 0.05 Ma to 0.10 Ma.A total of 20 polymorphic microsatellite markers were isolated from S.waltoni through constructing the(AGAT)_n microsatellite enriched library using the FIASCO protocol.The cross-species amplification and applicability of these loci were tested against the related Schizothoracinae species and high applicability was observed.One of the 20 microsatellite markers(Schw13)will be valuable for the molecular identification of the three Schizothorax species.Genetic variation in the 221 individuals of S.waltoni from five geographic populations were analysed using 11 nuclear microsatellite markers.A total of 266microsatellite bands were identified,of which nine microsatellite bands were predicted to be under selection.Genetic diversity and population structure analyses using total(n=266)and neutral(n=257)microsatellite bands revealed that obvious lower Nei's gene diversity index and Shannon's information index were found in S.waltoni from the population Mainling(ML)compared to the genetic diversity indexes observed in the other four upstream populations(Shigatse SG,Maldrogongkar MG,Quxu QX,Shannan SN).High microsatellite genetic diversity was observed in S.waltoni at the species level.Population genetic structure analyses revealed that a certain level of microsatellite genetic differentiation was observed between the population Mainling(ML)and the other four upstream populations and two major genetic clades according to the same regions were identified within S.waltoni.Low microsatellite genetic variation was found amongst the four upstream populations except the population ML,however,two obviously different genetic clades were observed:SG was in one clade,and MG+QX+SN clustered in another clade.3.Population genetics of S.macropogonGenetic variation in the 96 individuals of S.macropogon from four geographic populations were analysed using mt DNA Cyt b(1141 bp)and CR(715 bp)alone,and concatenated mt DNA Cyt b+CR(1856 bp),respectively.A total of 32 Cyt b haplotypes,58CR haplotypes,and 64 Cyt b+CR haplotypes were identified.Genetic diversity analyses revealed that little difference was found amongst the haplotype/nucleotide diversity observed in the four populations(Mainling ML,Paizhen PZ,Shigatse SG,Maldrogongkar MG).High mt DNA genetic diversity was observed in S.macropogon at the species level.Intraspecific phylogeography and population genetic structure revealed that paraphyly or polyphyly was found based on the haplotypes from the four populations.Low mt DNA genetic differentiation and no obvious genetic lineages were found amongst the four populations.Mismatch distribution and neutrality test analyses indicated that the population size of all the four populations remained relatively stable.However,BSPs(Bayesian skyline plots)analyses showed that S.macropogon might experience a pronounced expansion during0.05 Ma to 0.15 Ma.A total of 23 polymorphic microsatellite markers were isolated from S.oconnori through constructing the(AGAT)_n microsatellite enriched library using the FIASCO protocol.The cross-species amplification and applicability of these loci were tested against the related Schizothoracinae species and high applicability was observed.Two of the 23 microsatellite markers(Schm03,Schm04)will be valuable for the molecular identification of the three Schizothorax species.Genetic variation in the 170 individuals of S.macropogon from four geographic populations were analysed using 12 microsatellite markers.A total of 224microsatellite bands were identified,of which four microsatellite bands were predicted to be under selection.Genetic diversity and population structure analyses using total(n=224)and neutral(n=220)microsatellite bands revealed that obvious lower Nei's gene diversity index and Shannon's information index were found in S.macropogon from the populations Mainling(ML)and Paizhen(PZ)compared to the genetic diversity indexes observed in the populations Shigatse(SG)and Maldrogongkar(MG).High microsatellite genetic diversity was observed in S.macropogon at the species level.Population genetic structure analyses revealed that low microsatellite genetic variation and no obvious genetic clades were found amongst the four populations.4.Conservation implications of the three Schizothorax speciesBased on the genetic diversity and population structure analyses of the three Schizothorax species,we suggest that the populations of the three species can be divided into five management units(MG,ZX+MG,QX+SN,ML+PZ,and BM)in order to design their conservation and management strategies.It is suggested that the PLTR should be identified as priority conservation area for S.oconnori.The fish resources of the three Schizothorax species should also be given priority for conservation in two river sections of the YLTR(the SG section and the section between ML and PZ)and in the river section between ZX and MG of the Lhasa River,and followed by the fish resources in the river section between SG and SN of the YLTR.