Phylogeography of Aphyocypris normalis Nichols and Pope, 1927 at Hainan Island and adjacent areas based on mitochondrial DNA data

Abstract

We investigated the genetic structure of the freshwater fish Aphyocypris normalis, in 33 populations around Hainan Island and southern mainland China. Sequencing of the mitochondrial DNA (mtDNA) cytochrome b from 127 specimens yielded 47 haplotypes, from which we inferred a Bayesian tree. This revealed three major divergences: a principal clade of specimens with widespread geographic distribution, plus two clades with limited distribution. We estimated that these diverged between 1.05-0.16 Ma. Additionally, based on molecular data and comparing with the climate patterns of Hainan Island, eight phylogeographic ranges (populations) of A. normalis were constructed: the eastern plain (E), northeastern hills and plain (NE), northwestern hills and lowlands (NW), central mountains (C), southeastern hills and plain (SE), southern mountains and hills (S), southwestern mountains and lowlands (SW), and western lowlands (W). The patterns of geographical divergence in this species do not reflect the isolation caused by the Qiongzhou (Hainan) Strait, which would generally be experienced by terrestrial animals on isolated islands. The present results indicate that the major clades within A. normalis have diverged before the temporary land bridge existed across the strait during the Last Glacial Maximum.

Fig 1. Locations of sampling sites in this study.

Site numbers and river systems are listed in Table 1. a, southern mainland China sampling sites for Aphyocypris normalis (solid red circles); b, Hainan Island sampling sites for A. normalis (solid red circles). Three major river systems of Hainan Island are identified by different colors: white–the Nandu River; blue–the Wanquan River; yellow–the Changhua River; c, Taiwan sampling sites for A. moltrechti as the outgroup (solid blue triangles).

Fig 2. Bayesian tree of 1091-bp-long haplotypes inferred from partial cytb gene.

Branch lengths are proportional to the scale given in nucleotide substitutions per site. Numbers at internal nodes are Bayesian inferences (only value >0.90 are shown) and bootstrap values (>70%) for NJ and ML analyses. Only the values for major clades or sub-clades are shown. Solid circles at internal nodes indicate they were supported by all three analyses; open circles indicate they were supported by Bayesian and NJ analyses; and solid triangles indicate they were supported by Bayesian analysis only. Abbreviations beside clades or sub-clades correspond to the clades listed in Table 1. * indicates the haplotypes from the Zhoushui River, which were assigned to Aphyocypris amnis by Liao et al. [71].

Fig 3. Phylogenetic analysis of Aphyocypris normalis and its closely related species derived by the Bayesian method under the HKY+I+G model.

Numbers at internal nodes are Bayesian posterior probabilities and only value >90% are shown.

Fig 4. Statistical parsimony haplotype network for haplotypes constructed by TCS 1.21.

Numbers inside circles are haplotype codes. Circle size reflects the number of individuals having the corresponding haplotype. Small solid dots signify possible missing haplotypes. A line connecting two haplotypes represents one nucleotide substitution. White circles: haplotypes collected from Hainan Island; grey circles: haplotypes collected from southern mainland China. The red broken line separates the two major clusters in this analysis.

Fig 5

a, Climate ranges of Hainan Island, based on He and Zhang [36]; b, eight phylogeographic ranges inferred from this study’s cytb dataset analyses, modified by climate characteristics; c, haplotype distribution in the continental area. Sampling site locations and haplotypes at each collection site are also marked. The yellow areas in Hainan maps (a, b) indicate mountainous regions more than 750 m ASL. Red triangle: Wuzhishan Mountain. Green triangle: Yinggeling Mountain.