A total of 320 individuals from eight locations were collected during 2016–2018 from the coastal waters of southeast China and north Vietnam. Two locations were along the coast of Vietnam (Hai Phong (HP), Nam Dịnh (ND)) and six locations were along the coast of China (Wenzhou (WZ), Fu’ an (FA), Fuzhou (FZ), Yangjiang (YJ), Zhongshan (ZS), Qinzhou (QZ)). Detailed geographic locations and sampling information are shown in Figure 1 and Table 1. The samples were stored in 95% ethyl alcohol at –20 °C until DNA extraction. Muscle tissues (~10 mg for each individual) were dissected and genomic DNA was extracted using a Genomic DNA Extraction Kit (Tiangen Biotech, DP304, Beijing, China) according to the manufacturer’ s protocols. The extracted DNA was stored at −80 °C before use.
Location code Locality Country Sample size (n) Longitude (E) Latitude (N) WZ Wenzhou, Zhejiang China 40 121°18’ 28°38’ FA Fu’an, Fujian China 40 119°40’ 26°59’ FZ Fuzhou, Fujian China 40 119°18’ 25°58’ YJ Yangjiang, Guangdong China 40 112°02’ 21°51’ ZS Zhongshan, Guangdong China 40 113°21’ 22°15’ QZ Qinzhou, Guangxi China 40 108°29’ 21°56’ HP Hai Phong Vietnam 40 106°35’ 20°43’ ND Nam Dịnh Vietnam 40 106°22’ 20°20’
Table 1. Sampling information on T. heterochaetus in this study
Partial sequences of mtDNA COI were amplified and sequenced using universal DNA primers COI-LCO1490 and HCO2198 (Folmer et al., 1994). Polymerase chain reaction (PCR) amplification was conducted in a 50 μL volume containing 0.5 µmol/L of each primer, 0.2 mmol/L of each dNTP, 30 ng of template DNA, 1.5 mmol/L MgCl2, 1×PCR buffer, and 1 unit of Taq DNA polymerase (Fermentas, Thermo Scientific, USA). PCR was conducted with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 3 min. The amplified products were then purified and sequenced on an ABI Prism 3730 DNA sequencer (Applied Biosystems, USA) using both forward and reverse primers individually.
Using the COI gene from the complete mitochondrial genome sequence of T. heterochaetus as a reference sequence (Chen et al., 2016), sequence data were aligned using Cluster X 2.0 (Thompson et al., 1997). DNA sequence polymorphisms, including number of polymorphic sites (S), number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and average number of nucleotide differences (K), were estimated using DnaSP 5.10 (Librado & Rozas, 2009). To examine the genealogical relationships among mtDNA haplotypes, a haplotype network was constructed based on the median-joining algorithm in Network 5.0 (Bandelt et al., 1999). A neighbor-joining phylogenetic tree of COI haplotypes was constructed in MEGA 7.0 (Kumar et al., 2016) with the Kimura-2-parameter model (Saitou & Nei, 1987) and 1 000 bootstrap replicates. Perinereis aibuhitensis, a sea worm from the family Nereididae, was used as the outgroup (GenBank accession No.: NC023943.1). Pairwise genetic distances among haplotypes or different locations of T. heterochaetus were also calculated using MEGA 7.0 based on the Kimura-2-parameter model.
Pairwise ΦST, analysis of molecular variance (AMOVA), as well as correlation between genetic and geographic distance (coastline distance between sampling sites measured by Google Earth) estimated with the Mantel test, were all calculated in Arlequin v3.5 with 10 000 permutations (Excoffier et al., 2005). For examining demographic history of T. heterochaetus, Tajima’ s D (Tajima, 1989) and Fu’ s Fs (Fu, 1997) tests were used to examine the neutrality of coding sequences with Arlequin v3.5. Additionally, mismatch distribution analysis was also performed to investigate the hypothesis of population expansion. The fitness between the observed and simulated distributions was tested using the sum of squared deviations (SSD) as well as Harpending’ s raggedness index (HRI) (Harpending, 1994). Time of population expansion was calculated with the formulas Tau (τ)=2ut and u=2µk, where t is the time since expansion, u is the mutation rate for the COI gene (Rogers & Harpending, 1992), k is the number of nucleotides, and µ is the mutation rate. In this study, a mutation rate of 2% per million years was used as suggested by Olson et al. (2009) for another polychaete Hobsonia florida.
A 709 bp fragment of the COI gene was obtained after alignment. The average nucleotide frequency was: T=29.7%, C=24.4%, A=29.1%, and G=16.9%. Of the 320 sequences from the eight locations, a total of 94 variable sites were observed, including 32 singleton variable sites and 62 parsimony informative sites. Genetic diversity indices are presented in Table 2. A total of 85 haplotypes (GenBank accession No.: MK614603–MK614686) were identified, most of which (68 out of 85) were represented by a single individual, and pairwise genetic distances among haplotypes varied from 0.001 to 0.067. The number of haplotypes at a location ranged from 8 to 23. Overall, most locations showed moderate to high haplotype diversity (0.237–0.949) due to the large number of rare haplotypes. However, nucleotide diversity was relatively low, ranging from 0.000 4 to 0.009 12, across all studied locations. The WZ location exhibited the highest haplotype diversity (Hd=0.949) and nucleotide diversity (π=0.009).
Location (lineage) n S H Hd π k WZ 40 32 23 0.949 0.009 12 6.465 FA 40 13 10 0.776 0.004 74 3.362 FZ 40 16 13 0.783 0.004 70 3.329 ZS 40 18 6 0.237 0.001 27 0.900 YJ 40 12 13 0.654 0.001 40 0.994 QZ 40 11 10 0.441 0.001 04 0.735 HP 40 17 16 0.645 0.001 59 1.124 ND 40 7 8 0.618 0.001 05 0.744 Lineage A 120 42 39 0.861 0.007 11 5.043 Lineage B 80 29 18 0.660 0.001 75 1.239 Lineage C 120 29 29 0.767 0.002 74 1.942 Overall 320 94 85 0.926 0.032 15 22.794 n: Sample size; H: Number of haplotypes; S: Number of polymorphic sites; Hd: Haplotype diversity; π: Nucleotide diversity; k: Mean number of pairwise differences. WZ: Wenzhou; FA: Fu’ an; FZ: Fuzhou; ZS: Zhongshan; YJ: Yangjiang; QZ: Qinzhou; HP: Hai Phong; ND: Nam Dịnh. Lineage A=WZ+FA+FZ, Lineage B=ZS+YJ, Lineage C=QZ+HP+ND.
Table 2. Genetic diversity of eight T. heterochaetus locations based on COI gene sequences
Pairwise ΦST values ranged from –0.013 to 0.951 for all locations and were highly significant (P<0.01), except for two close location pairs (FA-FZ and HP-ND), whereas pairwise genetic distance varied from 0.001 to 0.063 (Table 4). Thus, all eight locations sampled could be treated as six genetically different populations (WZ, FA+FZ, ZS, YJ, QZ, HP+ND, Table 4). The topologies produced from both the haplotype phylogenetic tree (Figure 2) and haplotype median-joining network (Figure 3) showed a consistent structure. All eight locations could be characterized into three geographically distinguishable lineages (i.e., lineage A: WZ, FA+FZ; lineage B: ZS, YJ; lineage C: QZ, HP+ND, Figures 1–3). The AMOVA results based on the three lineages revealed that genetic variation mainly occurred among lineages (89.86%). Only 6.67% and 3.47% of variation occurred within populations and among populations within lineages, respectively (Table 3), suggesting strong genetic divergence among the different regions. Additionally, a significant pattern of isolation-by-distance was detected across all studied locations using the Mantel test (r=0.873 1, P=0.001).
Source of variation d. f. Sum of squares Variance component Percentage of variation (%) Fixation indices Among lineages 2 3171.121 14.871 Va 89.86 FSC =0.342* Among populations within lineages 5 120.279 0.573 Vb 3.47 FST =0.933* Within populations 312 344.225 1.103 Vc 6.67 FCT =0.818* Total 319 3635.625 16.548 *: P<0.05; d. f.: Degree of freedom; Va: Variance component due to differences among lineages, Vb: Variance component due to differences among populations within lineages, Vc: Variance omponent due to differences among individuals within populations; FSC=Vb/(Vb+Vc), FST=(Va+Vb)/(Va+Vb+Vc), FCT =Va/(Va+Vb+Vc).
Table 3. AMOVA results of T. heterochaetus based on mtDNA
Location code WZ FA FZ ZS YJ QZ HP ND WZ − 0.009 0.009 0.058 0.057 0.061 0.061 0.060 FA 0.226 − 0.005 0.059 0.058 0.062 0.063 0.063 FZ 0.238 −0.013 − 0.059 0.058 0.062 0.063 0.063 ZS 0.906 0.946 0.946 − 0.002 0.020 0.019 0.019 YJ 0.903 0.944 0.944 0.378 − 0.022 0.021 0.021 QZ 0.912 0.950 0.951 0.941 0.942 − 0.005 0.005 HP 0.907 0.944 0.947 0.922 0.926 0.711 − 0.001 ND 0.911 0.951 0.951 0.936 0.938 0.771 0.017 − ФST values in bold type indicate statistical significance (P<0.01). WZ: Wenzhou; FA: Fu’ an; FZ: Fuzhou; ZS: Zhongshan; YJ: Yangjiang; QZ: Qinzhou; HP: Hai Phong; ND: Nam Dịnh.
Table 4. Pairwise ФST (below diagonal) and genetic distance (above diagonal) among different T. heterochaetus locations
As genetic differences were not significant in the two geographically close location pairs FA-FZ and HP-ND, we treated them as a whole when analyzing demographic history. Both Tajima’ s D and Fu’ s F tests showed negative values for all genetically differentiated populations (Table 5), indicating departure from mutation-drift equilibrium and possible population demographic expansion. Furthermore, mismatch distribution did not differ significantly from the model of sudden expansion when using either SSD or HRI for goodness-of-fit (Table 5), further supporting the hypothesis of population expansion in T. heterochaetus. The τ value across populations varied from 0.969 to 13.936. Using the mutation rate of 2% per million years, it was estimated that the T. heterochaetus population expansion occurred about 17 000–246 000 years ago in the middle to late Pleistocene.
Population Neutrality test Mismatch distribution analysis Expansion time Tajima’s D Fu’s Fs SSD HRI Tau (τ) t (Ma) WZ –0.486 –7.786** 0.023 0.024 6.425 0.113 FA+FZ –0.395 –5.076* 0.052 0.123 4.834 0.085 ZS –2.571** –1.607 0.002 0.408 13.936 0.246 YJ –1.995** –11.090** 0.001 0.06 1.062 0.019 QZ –2.172** –7.960** 0.000 1 0.124 1.103 0.019 HP+ND –2.333** –24.297** 0.003 0.081 0.969 0.017 *: P<0.05, **: P<0.01. WZ: Wenzhou; FA: Fu’ an; FZ: Fuzhou; ZS: Zhongshan; YJ: Yangjiang; QZ: Qinzhou; HP: Hai Phong; ND: Nam Dịnh.
Table 5. Parameters of neutrality test and mismatch distribution analysis for six T. heterochaetus populations
First genetic assessment of brackish water polychaete Tylorrhynchus heterochaetus: mitochondrial COI sequences reveal strong genetic differentiation and population expansion in samples collected from southeast China and north Vietnam
- Received Date: 2019-07-03
- Publish Date: 2020-01-01
- Tylorrhynchus heterochaetus /
- Mitochondrial DNA /
- Genetic diversity /
- Population structure /
- Demographic history
Tylorrhynchus heterochaetus is a widespread benthic polychaete worm found in coastal brackish waters of the west Pacific. It has high ecological and economic value as a biomarker of water quality and as a high-quality feed in aquaculture and fisheries and is considered a delicacy in some areas of Asia. However, it has experienced a marked reduction in recent years due to overexploitation as well as changes in the environment and climate. Here, to comprehensively understand its genetic background and thus provide insights for better conservation and utilization of this species, we assessed the genetic variability and demographic history of T. heterochaetus individuals sampled from eight locations along the coasts of southeast China and north Vietnam based on mitochondrial cytochrome c oxidase I (COI) sequences. We observed high haplotype diversity (Hd), with an average of 0.926, but relatively low nucleotide diversity (π), with a mean of 0.032 across all samples. A total of 94 polymorphic sites and 85 haplotypes were identified among 320 individuals. The pairwise genetic distances among haplotypes ranged from 0.001 to 0.067, with the high intraspecific divergence possibly reflecting geographic isolation and gene pool fragmentation. Significant genetic structures were revealed among the studied locations; specifically, the eight locations could be treated as six genetically different populations based on pairwise ΦST results (0.026–0.951, P<0.01). A significant pattern of isolation-by-distance was detected between the genetic and geographic distances (r=0.873, P=0.001). Three geographic lineages were defined based on phylogenetic tree and network analyses of COI haplotypes. AMOVA results indicated that genetic variations mainly occurred among the three lineages (89.96%). Tests of neutrality and mismatch distribution suggested that T. heterochaetus underwent recent population expansion. These results provide the first report on the genetic status of T. heterochaetus and will be valuable for the management of genetic resources and better understanding of the ecology and evolution in this species.#Authors contributed equally to this work
|Citation:||Xing-Han Chen, Sen Yang, Wei Yang, Yuan-Yuan Si, Rui-Wen Xu, Bin Fan, Le Wang, Zi-Ning Meng. First genetic assessment of brackish water polychaete Tylorrhynchus heterochaetus: mitochondrial COI sequences reveal strong genetic differentiation and population expansion in samples collected from southeast China and north Vietnam. Zoological Research, 2020, 41(1): 61-69. doi: 10.24272/j.issn.2095-8137.2020.006|