Phylogeography And Conservation Genetics Of Sharp-snouted Fu (Deinagkistrodon Acutus; Reptilia, Viperidae)

The sharp-snouted fu, Deinagkistrodon acutus (Gunther, 1888), belonging to the monotypic genus Deinagkistrodon, occurs in ten provinces of southern and western China, as well as in Taiwan, and southwestwards to Laos and northern Vietnam. More than 90% of the populations of this species occur in China. It is one of the main snake species in China with commercial and medicinal importance. In 2004, D. acutus was listed in China Species Red List as a vulnerable endangered species (VU A2a) because there has been not less than 30% population reduction during the last decade, and the reduction is still continuingUsing phylogenetic and population genetic approaches, the present study reports the phylogeographic structure of the sharp-snouted fu. A dense and relevant geographical sampling was designed to include individuals from most of the distribution range of the sharp-snouted fu, in order to comprehensively represent the inter-and intra-population variation of the species The entire mitochondrial NADH dehydrogenase subunit 2 (ND2) gene sequences of 86 individuals of D. acutus from 14 localities across its range in mainland China were determined. For ease of description and discussion, sampling localities were artificially grouped into five geographic units according to the geographic distances and geographic similarityBased on the results of phylogenetic analyses, distribution of diagnostic sites, haplotype network, and AMOVA hierarchical analysis, an east-west division with strongly geographic configuration of the whole D. acutus population could be observed. Geographically, a line formed by a lake, river, and mountain chain (the Poyang Lake, Gan River, Wuyi Mountains), results in vicariance and approximately longitudinally splits the range into two and the whole population into two main lineages (western and eastern). The bifurcating tree suggested not only a previously west to east dispersal trend but also several subsequent short dispersal which reversed to the previously dispersal direction.Within site 714 to 963 in ND2 gene of ingroups, seven variable sites were found to be diagnostic i.e. they could definitely distinguish sequences belonging to western or eastern lineages. We also found five diagnostic sites which can locate a sequence in a subclade or a localized area. The subclade W1 could be distinguished from the others by site 600. The site 29, 193, and 453 could be used to distinguish the subclade El from the others. The "A" at site 567 only occurred in the haplotypes from Wuyi Mts.A total of 32 haplotypes (from haplotype 1 to 32 corresponding to GenBank Accession Nos. from DQ836185 to DQ836216) defined from all ingroup individuals. Only 3 haplotypes (9.4%) were shared by individuals from two adjacent units, the others were all restricted either to the same sampling locality (75%) or to the same unit (15.6%). A Mantel test of association of geographical distances and genetic differentiation showed that D. acutus fits the isolation by distance (IBD) model well (r=0.57, P < 0.01). Both results indicate that local populations of D.acutus have evolved relatively independently.In the haplotype network (statistical parsimony), there are many unobserved intermediate haplotypes missing along the long branches between two old singletons and central haplotypes, whereas almost all possible haplotypes are represented within each star-like cluster. Both phenomena could result from a bottleneck event causing massive extinction of populations before subsequent recent expansions (the earliest recent expansion is roughly estimated to have occurred at about 252,000 yr. B.P.). Star-like clusters in the haplotype network, significantly negative values of Fs statistics, and unimodal mismatch distributions all suggest recent demographic expansions in four areas after the bottleneck. The earliest expansion occurred over the area of Poyang Lake before the development of the ancient Gan River.The results suggest that isolation, dispersal, bottleneck, and expansion jointly constitute the history of D. acutus. Each of them all played their role as a population dynamics.In a haplotype network, the excessive predominance of central haplotypes, few medium-frequency haplotypes, predominance (73.1%) of singletons among the surrounding haplotypes, most of which are connected to the central haplotype by only one mutational step, unsymmetrical campanulate unimodal curve of mismatch distributions and leftwards shift of the peaks, all suggest that the whole D. acutus population is a young population with low genetic diversity, likely still in the process of population development and expansion.Our data could not identify ESUs within D. acutus, but appear to suggest at least two MUs (one in westmost unit and the other in Wuyishan unit), each of which deserves conservation attention. Based on our phylogenetic tree there also might be some minor candidate MUs in local populations within the Huangshan unit, central unit and Zhejiang unit respectively. Based on our genetic data, the highest priority for conservation seems to be given to the Huangshan unit, although no ESU and explicit MUs could be suggested within this unit. This conclusion is supported by two facts. First, among the five geographical units, only the Huangshan population is derived from both main lineages, therefore is diphyletic, and houses both genetic types. Second, the most cogent evidence is that the highest nucleotide diversity (1.37%) among all units was found in the Huangshan unit, more than other units by a factor of 3.5-29.5, and thus it makes a significant contribution to the overall nucleotide diversity of whole population (1.41%). Protection of the Huangshan population will protect the majority of overall nucleotide diversity, and will produce an optimal input-output ratio in conservation economics. Thus our recommendations for action are: 1, the creation of a nature reserve and establishment of a special fund for protection of wild D. acutus in the Huangshan unit; 2, the establishment of a well-designed and large-scale captive-breeding program in the Huangshan unit, to maintain a healthy and genetically diverse captive population for preserving the genetic diversity of this area, and to reproduce large numbers of captive-bred individuals for the market to alleviate the pressure on the wild population of D. acutus.