| The genus Acrossocheilus are a group of Cyprinidae,that are native to freshwater in South China,Laos,Vietnam.The previous studies were almost based on the typological and phylogenetic species concepts.The inter-and intra-species diversities are still uncertain.In this study,we studied the geographical relationship inter-and intra-species.And based on mitochondrial gene molecular markers and nuclear gene molecular markers,we analyzed population genetic structure and the population dynamies of historical evolution.Through the genetic structure,to rebuild the phylogenetic relationships of the genus,and the cause of phylogeographic pattern was also discussed.Then inferred the dispersal route of the genus.The main results were shown as follows:1.Study on phylogeography of genus Acrossocheilus by mitochondrial DNA makers1.1 The complete mitochondrial genome and phylogenetic analysis of Acrossocheilus longipinnisAccording to the published complete mitochondrial genome sequences of Acrossocheilus and related Cyprinidae,18 pairs of primers were designed.Then the complete mitochondrial genome of Acrossocheilus longipinnis was sequenced by direct sequencing of PCR amplification products and its structure was analyzed finally,the phylogenetic of this genus was analyzed.The results showed that the whole length of the mtDNA genome of the A.longipinnis is 16593 bp.Here the whole mitochondrial genome sequences of 16 species(including one newly determined,A.longipinnis),and two cyprinids as outgroups are applied to infer the phylogenetic relationships of the genus.Our results supported the monophyly of the Acrossocheilus and identified two monophyletic clades within the genus.The clade I also can be divided into two branches,the A branch contained eleven narrow barred species,the B branch contained three wide barred species.Moreover,most of the species distributed in Yangtze,southeast China river systems and Taiwan belonged to the A branch,and all the four species inhabited in Pearl River and Hainan included in B branch.The Clade II,only with two species separately distributed in Yuan-Jiang,upper and middle Yangtze River.Estimates of the time of differentiation in the 16 species show that the earliest ancestor species from the16 species were A.monticola and A.yunnanensis(5.19 Mya),and the latest were A.iridescens and A.longipinnis(0.39 Mya).Based on the above results,we supposed that(1)the ancestor of the genus Acrossocheilus derived from the upper Yangtze river and Yuan-Jiang river,(2)species in the genus maybe dispersed and evolved from two different routes,from the Yangtze river southward to southeast China,then to Taiwan and from upper Yangtze river to Yuanjiang,Pearl river,Hainan island.1.2 Population genetic variation of the species of Acrossocheilus based on Dloop and Cyt b DNAIn this section,the combined gene sequences of D-loop and Cyt b in 270 specimens of 13 species from 23 sites in south China were analyzed.1)Population diversity of 13 species: we analyzed 1980 base pairs of the mt DNA(D-loop and Cyt b).The variation rate of the 13 species varied from 0.1% to 13.5%,among which the variation rate of the smallest species was A.yunnanensis and A.jishouensis,and the variation rate of the largest species was A.monticola.The contents were basically the same,270 specimens defined 152 haplotypes and only one shared haplotype.The nucleotide diversity(h)range of 13 species was 0.536~1.000 and the range of nucleotide diversity(?)was 0.00072~0.03351.The h values of A.jishouensis,A.yunnanensis and A.beijiangensis are all 1,and ?< 0.005.The h values of A.kreyenbergii are 0.536 and ?=0.00232.The values of other species are all h>0.5 and ?> 0.005.The results showed that there was a high genetic diversity among the species.2)Phylogegraphy of 13 species: NJ tree and haplotypes network,based on haplotypes of 13 species,the results have shown 13 species can be divided into two clades.The clade II also can be divided into two branches,The Clade I,only with two species separately distributed in Yuan-Jiang,upper and middle Yangtze River.The distribution of most species was influenced by geographical isolation.3)Genetic structure of 13 species: The results of neutral test and mismatch distribution showed that most of the 13 species probably did not experience the bottleneck effect and rapid expansion in the evolutionary process?There was extreme differentiation between species,but there was gene exchange between populations distributed in different river.4)Demographic history of 13 species:the range of Fst was 0.24935~0.98423.Among them,the Fst between A.longipinnis and A.iridescens was the smallest,which showed that the relationship between these two species was the closest.The Fst between A.beijiangensis and A.yunnanensis was the largest,indicating the highest degree of differentiation between the two species.However,the degree of differentiation between species was affected by geographical isolation.2.Study on phylogeography of genus Acrossocheilus by nu DNA makersIn this chapter,S7 and IRBP sequences of 213 specimens of 11 species from 19 sites in south China were analyzed.1)Population diversity of 11 species: we analyzed 1332 base pairs of the nu DNA(S7 and IRBP),and there were 209 insertion/deletion.The variation loci of the 11 species was significantly different with a fluctuation range of 0bp~328bp,among which the variation loci of A.beijiangensis were the least.A.spinifer has the most mutation sites and the highest rate.But the range of singleton loci was 0bp~116bp.The contents were basically the same.A total of 130 haplotype is classified for these 213 specimens,and 10 shared haplotypes.Haplotype diversity of all the 11 species was greater than 0.5,and the range was 0.6000~1.000.Nucleotide diversity of 9 species is greater than 0.005,the range of 0.00570 ~ 0.05988,A.yunnanensis ? = 0.00163,A.beijiangensis ?=0.It can be seen that the species of Acrossocheilus has a very high genetic diversity of the population.2)Phylogegraphy of 11 species: The phylogenetic tree of 11 species of pholichthyes reconstructed with BI method based on haplotype.The haplotype network was consistent with the result of BI tree.The results showed that 11 species were divided into two lineages.The Clade I,only with A.longipinnis and A.iridescens,but the clustering of single species didn't show obvious geographical division.3)Genetic structure of 11 species: The results of neutral test and mismatch distribution showed some differences among A.fasciatus?A.kreyenbergii?A.monticola,this may be the three species were once geographically isolated,but then came into contact again,and no other species experienced population expansion,populations size remained stable.4)Demographic history of 11 species: the range of Fst was-0.24293~0.88384,with great differentiation among each species.The Fst<0,between A.beijiangensis and A.spinifer,this showed that the species differentiation between the two species was not significant.The Fst=0.08083 between A.wenchowensis and A.fasciatus,that there was moderate differentiation between these two species.And Fst=0.22685 between A.longipinnis and A.iridescens,suggesting that there was high differentiation between these two species.Fst>0.25 was highly differentiated among the other groups.The results indicated that there was apparent geographical differentiation between the species based on nu DNA,namely the genetic differentiation was not affected by the geographical isolation.In this study,we found that compared with the nuclear genes,mitochondrial genes have more polymorphic information.The variations of mitochondrial gene distributed among the past geological districts,but the variations ofnuclear gene distributed among the current geological districts.The study found that the clustering of 7 narrow barred species,based on mt DNA analysis showed obvious geographical division,and nu DNA analysis showed that it was not affected by geographical isolation.The evolution rates of mt DNA and nu DNA were significantly different among the species distributed in the Yangtze River and the wide barred species distributed in the Pearl River and Hainan Island.Combined with the estimated time of species differentiation,mt DNA is likely to be perfect for phylogenetic analysis. |
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