The Genetic Diversity And Population Structure Of A New Genus And Species Of Labeonine Fishes

Biodiversity is the basic field in life sciences, because it is the basis for maintaining the stable and sustainable production of the ecosystem. The research of biodiversity includes four levels: genetic diversity, species diversity, ecosystem diversity and landscape diversity. In the Yangtze River basin, there are 338 freshwater fish species in records, however, more than 25% species among them were listed as threatened or endangered, and some fishes go extinct before they are discovered. A species is a precious treasure for biological resources and sustainable development. First of all, we should obtain systematically and deeply information in its taxonomic status, morphology, distribution, quantity, ecology and genetic diversity.In 2013, we discovered a new species of the Labeoninae(Teleostei: Cypriniformes) from the Wu-Jiang and Chishui River in the upper Yangtze River basin. It can not be classified into any existing genus of Labeoninae. Here, we named the new species as Paraqianlabeo lineatus and carried out a series of studies on its taxonomy, genetic diversity and population structure. The main results are shown below.1. Establishment and identification of the new genus Paraqianlabeo. This new genus is distinguished from all other labeonine genera by a combination of morphological characters as following; i.e., an arched, wide, rudimentary sucker formed by raised front and lateral margins of lower lip, postlabial groove and mental groove between middle and lateral lobes of lower lip chevron-shaped; anteroventral margin of rostral cap arched, thin and papillose; rostral cap not overlying upper lip, upper lip connected with lower lip around corners of mouth; well developed upper lip free from upper jaw; lower lip divided into two lateral fleshy lobes and one central plate, these two lateral fleshy lobes small and short, median lobe of lower lip large; anterior and anterolateral edges of upper and lower lip finely papillose; shallow, straight groove between lower lip and lower jaw; lower jaw bears thin, cornified cutting edge. The results from DNA barcoding show that three populations(XS, DJ, JM) formed a monophyletic group with strong support. The genetic distance(K2P) between Paraqianlabeo and other labeonine genera ranges from 6.9% to 19.0%; the lowest pairwise genetic distance is 6.9% between Pseudogyrinocheilus and Paraqianlabeo. Traditional classification methods and DNA barcoding technique convince us that a new genus is warranted.2. Genetic diversity and population structure of Paraqianlabeo lineatus based on mitochondrial cytb gene. Genetic diversity and population structure will provided information of genetic background for the management and protection of P. lineatus. Thus, we used mtDNA cyt b gene marker to assess the genetic diversity and population structure of P. lineatus.A total of 22 haplotypes were identified based on nucleotide variation of cyt b sequences in 152 individuals. There was no shared haplotype between any two populations. The total haplotype diversity(h) and nucleotide diversity(π) were 0.753 and 0.00712, respectively. Haplotype and nucleotide diversities varied across the three populations, with the lowest values(h = 0.113) was found in the Jingmen River population(JM), the lowest nucleotide diversity(π = 0.0001) was found in the Xiangshuwan population(XS), the highest haplotype diversity and nucleotide diversities both found in the Daijia population(DJ)(h = 0.496, π = 0.0012). Based on the Kimura’s 2-parameter model(K2P), the overall average genetic distance among individuals was 0.007, the overall mean intra-population genetic distance of the DJ population(0.001) was the largest in the three populations and that of JM and XS(0.000) were the smallest. The neighbor-joining tree(NJ) and the median-joining network which were constructed based on all haplotypes showed that the haplotypes form a monophyletic group. DJ population formsa sister group relationship with JM population, and the XS population has a relatively distant relationship with the JM and DJ populations. The AMOVA revealed that most molecular variance was observed to occur among populations(95.73%), whereas variance within populations(4.27%) was relatively small. Pairwise FST demonstrated significant divergence between any two populations(0.869-0.988). Significant divergence across all samples was also observed, indicating a high level of geographical population structure. Gene flow(Nm) estimates between populations were significant less than 1(0.006-0.076), showing very limited genetic exchanges between any two populations. It appears that the level of divergence among populations was very high, and isolation has occurred among them. The mismatch distributions, Tajima’s D and Fu’s Fs statistics showed that as a whole, P. lineatus had not passed through a bottleneck, however, the nucleotide and haplotype diversity(0.000, 0.113, 0.000, 0.122) of JM and XS populations were very poor. Gene homogeneity and BSP Plot analysis showed that JM and XS Populations were not demonstrate expansion, but may be experiencing an effect of bottleneck. Phylogenetic reconstruction based on cyt b sequences using BEAST revealed 3 major lineages. The divergence time of the Wu River(XS) and the Chishui River(DJ and JM) population was at around 3.61 million year ago(Mya), DJ and JM populations divergence time at around 3.25 Mya. Since XS population have the maximum genetic distance, biggest fist, minimal gene flow from the other populations, and have changes on the lips structure, the difference reached the level of subspecies or even species.3. Genetic diversity and population structure of Paraqianlabeo lineatus based on microsatellite marker. We isolated and characterized 21 novel polymorphic microsatellite markers in the genome of P. lineatus using "MiSeq" sequencing. A total of ten polymorphic microsatellite markers were employed to assess the genetic diversity of P. lineatus in three populations from the Wujiang River and the Chishui River of the Yangtze River. The results show that average number of alleles of three populations varied from 5.8-10.8. The genetic diversity index including average allele number, average allele frequency and average polymorphism information content of DJ was the largest, and that of XS population was the lowest. The genetic structure was evaluated by the analysis of gene flow(Nm), genetic differentiation index(FST) and analysis of molecular variance(AMOVA). The results revealed that the FST has bigger variation among populations(66.63%) than within populations(33.37%). The genetic divergence mainly comes from populations of P. lineatus. In the three populations, the gene flow of 10 microsatellite loci was less than 1, and the average gene flow level was only 0.6071. The genetic distance of the three populations according to Nei’s unbiased, the XS and DJ population have largest genetic distance(2.8180), while the XS and JM populations have the smallest(1.2769).The STRUCTURE analyses and ΔK tests based on the microsatellite data showed that P. lineatus were comprised of three genetic populations. Therefore, population structure based on microsatellite data and mtDNA cyt b sequence data suggested that P. lineatus samples in the study could be divided into three genetic management units. These genetic management units should be protected and managed separately. According to the level of genetic diversity, FST, gene flow and habitat condition, threatened status of the three populations, the priority order for preservation is XS>JM>DJ population.4. Study on the changes of lip structure in the XS population. We found different lip structure from XS population during the examination of the morphological characters: 1) Rostral cap long, completely covering upper lip and upper jaw(vs. short, not overlying upper lip and upper jaw); 2) Anteroverntral margin of rostral cap like fimbriate(vs. Anteroverntral margin of rostral cap arched, not fimbriate); 3) The upper lip reduced or disappeared(vs. developed); 4) The front and sides of the lower lip is uplift(vs. slightly raised); 5) Anterior and anterolateral edges of lower lip developed papillose(vs. finely papillose). The cause of lip structure changes due to deformity, age, sex or different chromosome ploidy or have distinct differentiation in the geographical populations. We carried out the following studies on abovementioned issue and the results are as follows: 1) there is no direct association between the fish with different lip structure in terms of age and gender; 2) Karyotypic and DNA content analyses showed that there was no chromosome number variation of the fish with different lip structure; 3) The genetic distance based on DNA barcoding reached 1.5% between XS and the other populations, which is closed to the critical value(2%) in the interspecies. And then consider the following two points: a) the results from cyt b and microsatellite data showed that the level of genetic differentiation is very high. Meanwhile, the XS population had greater genetic distance and FST were detected than the other two populations. There is no gene exchange between populations, with a strong population structure; b) Because the lip structure have stability as taxonomic character, there is no relations with sex, age, deformity, the changes with lip structure have the important meaning to the taxonomy. Taken together, the variations of the lip structure among different individuals from XS population might be an important morphological character which has the potential to form a new species. We speculate that the fish from the XS population might be in the early stages of speciation.Briefly, the main results of the study are as follows: 1) the new genus is validated based on traditional morphological characteristics and DNA barcoding; 2) The genetic diversity is very low in the XS and JM populations, strong population structure of P. lineatus revealed by mtDNA and microsatellite markers, the XS population diverged early from other populations; 3) The results based on mtDNA sequences and microsatellite loci showed that XS population has lager differentiation than the other populations. There have some difference in the classification traits, fish from the XS population might be in the early stages of speciation.