GENE-39870; No. of pages: 7; 4C: Gene xxx (2014) xxx–xxx
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Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes Daizhen Zhang a,b, Ge Ding c, Baoming Ge b, Huabin Zhang b, Boping Tang b, Guang Yang a,⁎ a b c
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, People's Republic of China Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Yancheng 224051, People's Republic of China Chemical and Biological Engineering College, Yancheng Institute of Technology, Yancheng 224003, People's Republic of China
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 19 July 2014 Accepted 4 August 2014 Available online xxxx Keywords: Phylogeography Crustacean Divergence Expansion Environmental change
a b s t r a c t Environmental changes, such as changes in the coastal topography due to Eurasian plate movements, climate oscillation during the Pleistocene, and alteration of ocean currents, have complicated the geographical structure of marine species and deepened their divergence between populations. As two widely distributed species of crustacean (Oratosquilla oratoria and Eriocheir japonica), weak differences were expected due to their high dispersal potential of planktonic larvae with ocean currents. However, results showed a significant genetic divergence between north of China and south of China in the study. In addition, the estimated north–south divergence time (27–30.5 Myr) of mantis shrimp was near the time of the Himalayan movement, and the China–Japan clade divergence time (10.5–11.9 Myr) of mitten crabs was also coincident with the time of the opening of the Sea of Japan. Thus, we hypothesized that environmental changes in the coastal topography contributed to the marine species divergence. Furthermore, based on phylogenetic analysis, network analysis and haplotype distribution, we surmised that mitten crabs originated from a population with the oldest haplotype (H6) and then divided into the north and south populations due to the recent Eurasian plate movements and ocean currents. And lineage of Japan originated from the north population for the opening of the Sea of Japan. While O. oratoria was guessed to originate from two separate populations in the China Sea. The results of “star-like” network, negative values in neutral test, and Tajima's D statistics of two marine species supported a recent rapid population expansion event after the Pleistocene glaciations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vagarious climate fluctuations during the Pleistocene have caused alterations of dramatic temperature, ocean currents and coastal topography (Clark et al., 1999; Hewitt, 1996, 2000). To elude the dramatic changes during the Pleistocene glaciations, marine organisms were forced into a repeated cycle of glacial retreat and interglacial expansion. The ranges of some faunal distributions have exhibited markedly altered contraction or expansion (Avise, 2000; Hewitt, 2000; Xu et al., 2009). Such historical events have led to species extinction and have also acted as the drivers of subspeciation for local adaptation and adaptive radiation (Crowley and North, 1988; Gillespie, 2004; Mayhew et al., 2008). The relative influence of environmental changes on marine organisms could best explain the patterns over a longer evolutionary time frame (Benton, 2009). Moreover, during the Pleistocene glacial cycles, the formation of land bridges due to the lowered sea level between islands and Asian continent might act as the barriers for marine organisms and potentially aid allopatric diversification (Kimura, 2000; Abbreviations: mt, mitochondria(l); nt, nucleotide(s); Myr, million years. ⁎ Corresponding author. E-mail address: [email protected] (G. Yang).
McManus, 1985; Tamaki and Honza, 1991; Voris, 2000; Wang, 1999). Furthermore, the dispersal of the pelagic larvae for ocean currents also played a fundamental role in ecology and evolution (Caley et al., 1996; Strathmann et al., 2002). These physical and environmental attributes as the potential mechanisms have shaped patterns of marine species divergence and population expansion by the occupation of newly formed habitats or adaptation to changing environments (Richardson, 2012). Northwest pacific marginal seas connecting the mainland and deep oceans were regarded as the most suitable areas to understand the past global climate and oceanographic changes. The phylogeographic structure of marine species living in marginal seas was the typical representative of the intertidal invertebrate affected by historical climate, ocean currents and coastal topography. As the representative marine species, mantis shrimps Oratosquilla were widely distributed along the northwest pacific marginal seas, and mitten crabs Eriocheir were also distributed from China and Japan to Korea. Previous studies have indicated that Eriocheir japonica was divided into three geographic subspecies, namely E. j. japonica, E. j. sinensis and E. j. hepuensis (Tang et al., 2003). Thereinto E. j. japonica was distributed in Japan, and the 26°N latitude was considered the boundary between E. j. sinensis and E. j. hepuensis in China (Cohen and Carlton, 1997; Ingle and Andrews, 1976; Tang et al., 2003; Zhao et al., 1988). Subspeciation required the
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Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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termination of gene flow for geographic barrier or population isolation. It was understandable that subspeciation of E. j. japonica might be a result of the isolation between China and Japan. However, it was unacceptable that 26°N latitude was the unambiguous boundary between E. j. sinensis and E. j. hepuensis. So the subspeciation of Eriocheir still remains a controversial topic at present (Tang et al., 2003; Wang et al., 2008; Xu et al., 2009; Zhang et al., 2012), and additional data are needed to reveal the phylogeographic structure and to clarify the relationship between phylogeographic structure and environmental changes. In this study, through a comparative phylogeography study of two marine species (Oratosquilla oratoria and E. japonica) of crustacean, we assessed the phylogeographical pattern of marine species in the northwest pacific marginal seas and revealed the association between divergence and historical environmental changes.
2. Materials and methods 2.1. Sample collection The study region was selected because several marginal seas were once separated during glaciations. A total of 621 samples of the two marine species of crustacean were collected in this study. Samples of 208 individuals of O. oratoria were obtained from 15 localities representing the geographical range of this species across the China Sea, and 413 samples of E. japonica from 19 locations along the northwest Pacific Ocean were also integrated from GenBank to corroborate the results (Fig. 1, Appendix 1). The materials were sampled and preserved in 95% ethanol for later analysis. We chose to focus on the mt cytochrome oxidase c subunit I (mt COI) gene and the cytochrome b (mt Cytb) gene
because these were more informative sites and because of their ability to estimate the mutation rate. The main limitation was that these sites represented only one evolutionary history. However, a lower effective sample size of mtDNA relative to that of nuclear DNA was needed due to their faster rate at the population level. We thus considered the use of mt sequences as the best strategy for accurate estimation. 2.2. DNA extraction, amplification and sequencing The genomic DNA from tissue muscle was extracted using the standard phenol–chloroform method. PCR amplification was executed using the primers described by Folmer et al. (1994). Each 30-μl amplification reaction consisted of thermophilic buffer (50 mM KCl and 10 mM Tris–HCl pH 8.3), 2.0 mM MgCl2, 10 pmol of each primer, 0.2 mM dNTPs, and 1 unit Taq DNA polymerase. Polymerase chain reaction (PCR) was performed on a GeneAmp® PCR System 9700 (ABI) or Mastercycler® gradient (Eppendorf) thermocycler with a temperature profile of 30 cycles at 94 °C for 40 s, 50 °C for 40 s, and 72 °C for 60 s. The PCR products were purified using a PCR purification kit and cycle-sequenced on an ABI Prism 3730 automated sequencer. 2.3. Sequence alignment, genetic diversity and phylogenetic analysis The sequences were aligned with the Clustal W algorithm in Mega 5.0 (Tamura et al., 2011). The nt diversity (π) and haplotype diversity (h) were estimated using Arlequin version 3.1 (Excoffier et al., 2005). The divergences within- and between-populations were determined using Mega 5.0 (Tamura et al. 2011). Haplotype neighbour-joining trees (NJ) were constructed using the Kimura 2-parameter model in
Fig. 1. Map showing the sampling locations for the two marine species in this study. ●: locations for E. japonica; : locations for O. oratoria. Colours in circles represented different haplotypes and their proportions. The four ancestral haplotypes of E. japonica were H5 , H6 , H12 , and H30 . The three ancestral haplotypes of O. oratoria were HP5 , HP14 , and HP63 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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Table 1 Genetic diversity, divergence, neutral test, and gene flow among different lineages. n: number of samples; N: number of haplotypes; h: haplotype diversity; π: nt diversity. Groups
E. japonica ENC EJA ESC EMG O. oratoria ONC OSC a
n
413 229 48 121 15 208 183 25
N
80 30 16 28 6 80 62 18
h
0.899 0.745 0.871 0.756 0.810 0.930 0.911 0.953
π
0.02291 0.00259 0.00265 0.00256 0.00233 0.01712 0.00447 0.00669
Tajima's D
Fu's Fs
Pairwise value of Fst (lower left) and Nm (upper right)
D
P
Fs
ENC
EJA
ESC
EMG
−0.592 −2.286 −1.736 −2.144 −0.144 −1.113 −2.211 −1.432
N0.10 b0.01a N0.05 b0.05a N0.10 N0.10 b0.01a N0.10
−84.819 −43.788 −19.928 −41.210 −4.172 −44.95 −80.434 −11.171
– – 0.83281 0.86501 0.89466 ONC – 0.91256
– 0.05 – 0.89396 0.92190 OSC 0.02 –
– 0.04 0.03 – 0.87752 – – –
– 0.03 0.02 0.03 – – – –
Significant.
Mega 5.0 (Bootstrap = 1000), and a Bayesian evolutionary analysis (BI) was implemented in Beast v1.7 (Drummond et al., 2012). The input file was properly formatted with the BEAUti utility included in the software package using the partition scheme used for the concatenated analysis. Two runs, each of 10 × 106 generations (samplefreq = 1000 and 10% burn in), were conducted. The maximum clade credibility trees were created with TreeAnnotator v1.7.4. Ultimately, a Bayesian evolutionary tree was inferred and viewed using FigTree v1.4.0 (Rambaut, 2008). 2.4. Calculation of the divergence date A likelihood ratio test was performed to determine whether the datasets evolved in a clocklike manner, and the branch age estimation was performed using Mega 5.0. The Sea of Japan is a semi-landlocked sea located between Japan and the Asian continent that developed as a consequence of the rotation of Japan during the period between 10 and 15 Myr (Ichikawa et al., 1990; Kiyotaka, 1991). Using the credible maximum calibration date of the opening of the Sea of Japan, we calculated the likely clade date of Caridea and tested the estimated date with their fossil calibrations (Bravi and Garassino, 1998; Garassino and Bravi, 2003). If the time coincided with the fossil record, we conversely estimated each clade date by the fossil records. 2.5. Historical demography The possible occurrence of historical demographic expansion was examined using mismatch distribution and Tajima's (1989) D and Fu's (1997) Fs neutrality tests. The range of contractions or expansion was
verified by analysing the frequency distribution of pairwise differences among haplotypes (mismatch distribution) (Harpending 1994) using the DnaSP software (Rozas et al., 2003). Tajima's D test was used to test for departures from equilibrium between mutations and drift (Tajima, 1989). The relationship t = τ / 2 μ (Rogers and Harpending, 1992) was used to further estimate the time of expansion (t) using a mutation rate of 2.33% per Myr and a generation time of two years (Knowlton et al., 1993; Schubart et al., 1998). Network v. 4.6.0.0 (Bandelt et al., 1999) was used to construct a statistical parsimony network and to analyse the ancestral haplotypes of the two marine species. 3. Results 3.1. Genetic diversity and phylogenetic tree Overall, the alignment of 208 sequences of O. oratoria representing 15 populations defined 80 unique haplotypes (HP1–HP80) (GenBank Numbers: KM196983–KM197062). In comparison, 413 sequences of E. japonica representing 19 populations also revealed 80 haplotypes (H1–H80) (Appendix 1). The similar haplotype diversity (h) was calculated to be 0.930 for O. oratoria and 0.899 for E. japonica. The details of the haplotype distribution of each geographical population were shown in Fig. 1. Moreover, 89 and 97 sites were defined as polymorphic sites for O. oratoria and E. japonica, in which 65 and 51 sites were parsimony informative sites, respectively. In addition, the whole nt diversity (π) was 0.01712 for O. oratoria and 0.02291 for E. japonica (Table 1). For O. oratoria, haplotype HP5 was shared by 54 samples from the north of China, whereas for E. japonica, haplotypes H6 and H12 were shared by
Fig. 2. Phylogenetic tree and divergent time of E. japonica along the western Pacific Ocean. The numbers above the branches showed the percentages of bootstrap support with 1000 bootstrap replicates. The numbers with arrows were the divergence time of the clade associated with the circle.
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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Fig. 3. Phylogenetic tree and divergence time of O. oratoria along the China Sea. The numbers on the branches were the percentages of bootstrap support with 1000 bootstrap replicates. The numbers with arrows were the divergence time of the clade associated with the circle.
147 samples, most of which were from the north of China. Haplotype H5 was shared by 58 samples from the south of China. Both the NJ and BI phylogenetic trees exhibited similar topologies of the O. oratoria haplotypes, and the E. japonica trees were also similar in terms of topology (Figs. 2 and 3). In the O. oratoria tree, two major clades were apparent: one clade with strong support (bootstrap support of 99%) represented all of the populations from the Bohai Sea, Yellow Sea, and East China Sea in the north of China (ONC), and the other clade with strong support (bootstrap support of 99%) comprised the South China Sea (OSC). The E. japonica exhibited similar topology: one major clade with strong support (bootstrap support of 93%) represented the South China Sea (E. j. hepuensis) (ESC) and E. japonica sp. (EMG), in which the populations of the Nanliujiang River and Zhanjiang River occupied 34% and 26% of the haplotypes in ESC, and the other clade comprised the north of China (E. j. sinensis) (ENC) with 33% of the haplotypes in the Yangtze River and Japan (E. j. japonica) (EJA). Within the two clades, four clades (E. j. sinensis, E. j. japonica, E. j. hepuensis, and E. japonica sp.) were separated with strong support (bootstrap support of 98% or 99%), which corresponded to the three geographical subspecies of E. japonica and a novel clade.
3.2. Genetic divergence The pairwise Fst was concordant, showing that the average value between the four lineages in mitten crabs was ENC/EJA: 0.83281, ENC/ESC: 0.86501, ENC/EMG: 0.89466, EJA/ESC: 0.89396, EJA/EMG: 0.92190, and ESC/EMG: 0.87752. Assuming populations at equilibrium between gene flow and genetic drift, the gene flow was estimated by Fst-based methods to be ENC/EJA: 0.05, ENC/ESC: 0.04, ENC/EMG: 0.03, EJA/ESC: 0.03, EJA/EMG: 0.02, and ESC/EMG: 0.03. In the case of mantis shrimp, the Fst between the two clades was 0.91256, and the corresponding gene flow was 0.02 (details in Table 1). The net average genetic distance between the four lineages (±SE) in mitten crabs were ENC/EJA: 0.0249 (±0.0058), ENC/ESC: 0.0367 (±0.0075), ENC/EMG: 0.0405 (±0.0078), EJA/ESC: 0.0408 (± 0.0081), EJA/EMG: 0.0456 (± 0.0081), and ESC/ EMG: 0.0321 (±0.0069). In addition, the value between OSC and ONC for mantis shrimp was 0.0622 (±0.0102), showing deep divergence between the south of China and the north of China. The molecular clock test was performed by comparing the ML values for the given topology. The null hypothesis of an equal evolutionary rate throughout the tree was not rejected at a 5% significance level (p b 1) both for mitten crabs and mantis shrimp. Using the calibration date of 10–15 Myr defined by the opening of the Sea of Japan, we calculated
Fig. 4. Median-joining network of haplotypes of O. oratoria. The circle size was proportional to the number of individuals exhibiting the corresponding haplotype. The ancestral haplotypes (HP5 , HP14 , and HP63 ) were coloured according to Fig. 1. The length of the lines was proportional to the number of mutational steps separating the haplotypes. The smallest circles indicated missing intermediate haplotypes. The map in the lower right corner showed the torso of the network of O. oratoria, including all of the ancestral haplotypes. A: Group from the Bohai Sea, Yellow Sea, and East China Sea. B: Group from the South China Sea. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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that the absolute fossil time of Caridea was approximately 94–141 Myr, which was similar to that obtained from the fossils records, i.e., 99– 112 Myr. We concluded that the divergence of clade E. j. sinensis and clade E. j. japonica was related to the opening of the Sea of Japan and the consequent rotation of Japan. Conversely, we estimated the date of each clade using the date of the fossil calibrations. The results show that divergence date of clade E. j. sinensis and clade E. j. japonica was approximately 10.5–11.9 Myr, whereas the divergence data of clade E. j. hepuensis and clade E. japonica sp. was 12.9–14.6 Myr and that of clade E. j. sinensis and E. j. hepuensis was approximately 15.5–17.5 Myr (Fig. 2). The application of the same calculations to the phylogenetic tree of O. oratoria revealed that the divergence date between the south clade and the north clade was 27–30.5 Myr, and the splitting time between the south clade and the north clade in China was older than that of mitten crabs (Fig. 3).
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A similar “star-like” median-joining network was shown for the E. japonica population, and the whole network separated all of the haplotypes into four distinct networks, corresponding to the four geographical lineages. Moreover, the “star-like” network with the central haplotypes H5, H6, H12, and H30 indicated the rapid expansion of the population historical demography of E. japonica, and the central haplotypes were likely the ancestral types (Fig. 5). The mismatch distributions supported the similar conclusions of the historical population expansion demographics for smooth unimodal distributions. Estimates of population size changes and selection generally indicated nonneutrality. The Tajima's D statistics showed negative values (Tajima's DENC = −2.286, p b 0.01; Tajima's DESC = − 2.144, p b 0.05; Tajima's DEJA = − 1.736, p N 0.05; Tajima's DEMG = − 0.144, p N 0.10), and Fu's F test of neutrality also gave negative value (FS-ENC = − 43.788; FS-ESC = − 41.210; FS-EJA = − 19.928; FS-EMG = − 4.172) for the expansion of the four clades (Table 1). And the time of expansion estimated using the formula t = τ / 2 μ was approximately 36–57 Ka.
3.3. Population demographic analysis 4. Discussion The median-joining analysis of O. oratoria revealed a “star-like” network with the central haplotypes HP5, HP14, and HP63 as the ancestral haplotypes. However, two distinct networks (A, B) were observed: A comprised all of the haplotypes from the Bohai Sea, Yellow Sea, and East China Sea, and B consisted of all of the haplotypes from the South China Sea (Fig. 4). The mismatch distributions, which revealed smooth unimodal distributions for each group, supported a rapid population expansion event. The negative value of Tajima's D statistics for the estimation of population size changes (Tajima's DONC = − 2.211, p b 0.01; Tajima's D OSC = − 1.432, p N 0.10) and Fu's F test of neutrality (FS-ONC = −80.434; FS-OSC = −11.171) supported the conclusion of a rapid expansion. The estimated time for the rapid population expansion was approximately 48–50 Ka.
As widely distributed marine species, weak differences among geographical populations were expected due to their high dispersal potential (Knowlton et al., 1993; Avise, 1994; Uthicke and Benzie, 2003). However, our results revealed the significant divergence, as determined by genetic distance, phylogenetic tree, and network methods, between the north and south populations of two dispersed marine species. So we inferred environmental changes, such as Eurasian plate movements, ocean currents, and climatic oscillations during the Pleistocene, complicated the geographical structure of marine species and deepened the divergence among populations. For Eurasian plate movements, the previous study showed that Taiwan Island was the natural boundary for many marine organisms (Liu et al., 2007), so we thought that the
Fig. 5. Median-joining network of haplotypes of E. japonica. The circle size was proportional to the number of individuals exhibiting the corresponding haplotype. The ancestral haplotypes (H5 , H6 , H12 , and H30 ) were coloured according to Fig. 1. The length of the lines was proportional to the number of mutational steps separating the haplotypes. The smallest circles indicated missing intermediate haplotypes. The map in the upper right corner showed the torso of the network of E. japonica, including all of the ancestral haplotypes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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Fig. 6. Map summarizing the proposed origin, expansion, and subspeciation of the two marine species. The arrows with the dotted line indicated the route of colonization and divergence of E. japonica. The centred arrows represented the rapid expansion of O. oratoria.
formation of Taiwan Island must have contributed to the divergence of the north–south division of marine species. The Taiwan Island was traced back to the latest orogeny of the Himalayan movement in the Eocene epoch of the Tertiary period of the early Cenozoic 40 Myr (Jia et al., 2004). In the study, based on fossil record and phylogenetic tree analyses, the predicted north–south divergence time of O. oratoria was estimated to be approximately 27–30.5 Myr, which was near to the time of the Himalayan movement and the later plate activity of Taiwan Island. To some extent, the formation of Taiwan Island and the early Himalayan movement must deepen the divergence between the north and south populations of O. oratoria. However, the divergence time of E. japonica (15.5–17.5 Myr) was inferred to be more recent than that of O. oratoria by the same methods. In lifecycle of E. japonica, it lived in freshwater in most time of its lifecycle and migrated to a salty estuary for oviposition in its reproductive seasons. So the influence of Taiwan Island might be less on mitten crabs than that of mantis shrimps. Moreover, fluctuation of Taiwan Strait in the late glacial period further influenced the phylogeographic structure of marine species. Taiwan Strait was a submarine valley lying between the Chinese Mainland and Taiwan. In glacial period of quaternary, Taiwan Strait once emerged in response to decreases of the sea level (Tougard, 2001), and Taiwan Island was linked with Fujian Province of China (Shan et al., 2013). So acting as a land bridge, Taiwan Strait enabled terrestrial organisms to extend their ranges but limited the gene flow between the north and south populations of marine species and potentially resulted in allopatric diversification (Imron et al., 2007; McManus, 1985). The Sea of Japan was a semi-landlocked sea between Japan and the Asian
continent that was developed as a consequence of the rotation of Japan during the period between 10 and 15 Myr. In the study, the divergence time (15.5–17.5 Myr) of E. j. sinensis in China and E. j. japonica in Japan by calibration of fossil records was abnormally coincident with the opening time of the Sea of Japan (10–15 Myr). The novel coincident time demonstrated the possibility that opening of the Sea of Japan contributed to increasing the divergence of marine species. Ocean currents in the China Sea derived from the Kuroshio tributary or originated from the monsoons. Currents of the Bohai Sea, Yellow Sea and East China Sea, which exhibited the characteristics of counterclockwise cyclonic circulation, were mainly the result of the Kuroshio current and coastal streams. However, the surface circulation of the South China Sea exhibited clockwise circulation due to the monsoon drift. So the planktonic larvae of the two species in northern seas would disperse northward due to the counterclockwise cyclonic circulation currents, whereas the larvae in the South China Sea dispersed southward (Shane et al., 2008). Such opposite dispersion further deepened the divergence between the north and south geographical populations. In late Pleistocene glaciations, vagarious climate fluctuations caused dramatic temperature changes, and marine organisms were forced into glacial retreat and later rapid interglacial expansion. In this study, a similar rapid population expansion event was revealed in two crustacean species by mismatch distribution, neutral test and median-joining network. Moreover, the expansion time (36–57 Ka) estimated by the formula t = τ / 2 μ was in the range of the climate fluctuations of the late Pleistocene. The result explained the reason for lack of phylogeographical
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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structure within the north population or south population. In Pleistocene, marine species were forced into a repeated cycle of glacial retreat and interglacial expansion as a result of the climate fluctuations, so ranges of some faunal distributions exhibited markedly altered contraction or expansion (Avise, 2000; Hewitt, 2000; Xu et al., 2009). About the original centres, ancestral haplotypes of Eriocheir (H5, H6, H12 and H30) were in the centre of the genealogical network. And haplotype H5 were shared by all samples from the south population (100%), and H12 were shared by most samples from the north population (95.6%). That revealed that H5 and H12 were the ancestral haplotypes of the south and north populations, respectively. However, as the oldest haplotype, H6 was shared by the north and south populations in China. We hypothesized that Eriocheir in the northwest Pacific Ocean originated from an oldest population with the ancestral haplotype H6 in the China Sea, but gene flow between the north and south seas was then weakened due to the formation of Taiwan Island, explosion of Taiwan Strait, opposite direction of ocean currents, and climatic oscillations during the Pleistocene, that resulted in the division of the north population with H5 and south population with H12. Later, genetic divergence between Japan and north of China was deepened for the opening of the Sea of Japan in 10–15 Myr (Fig. 6). However, results of O. oratoria showed that HP5, HP14, and HP63 were the ancestral haplotypes in the centre of network. Unlike Eriocheir, all samples sharing HP5 and HP14 were obtained from north of China. Similarly, all samples sharing HP63 were from south of China, but no ancestral haplotype was shared by both the north and south populations. Thus, we hypothesized that O. oratoria in China were from two original regions: north of the China Sea and south of the China Sea (Fig. 6), and that their divergence was increased by the later environmental changes. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.006. Acknowledgements The work was funded by National Natural Science Foundation of China (41301050), Natural Science Foundation of Jiangsu Province (BK2011421, 12KJA180009), Foundation of Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection (JLCBE12002) and sponsored by “Qing Lan Project” and “333 Project” of Jiangsu Province ( (2013) Ⅲ-1129) to Daizhen Zhang. We also thank all anonyms for their help in sample collection. References Avise, J.C., 1994. Molecular markers. Natural History and EvolutionChapman and Hall, New York. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, MA. Bandelt, H.J.,Forster, P.,Röhl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Benton, M.J., 2009. The Red Queen and the Court Jester: species diversity and the role of biotic and abiotic factors through time. Science 323, 728–732. Bravi, S., Garassino, A., 1998. ‘Plattenkalk’ of the Lower Cretaceous (Albian) of Petina, in the Alburni Mounts (Campania, S. Italy), and its decapod crustacean assemblage. Atti Societa italiana Scienze naturali Museo civico Storia naturale Milano. 138, pp. 89–118. Caley, M.J.,Carr, M.H.,Hixon, M.A., 1996. Recruitment and the local dynamics of open marine populations. Annu. Rev. Ecol. Syst. 27, 477–500. Clark, P.U.,Alley, R.B.,Pollard, D., 1999. Northern hemisphere ice sheet influences on global climate change. Science 286, 1104–1111. Cohen, A.N., Carlton, J.T., 1997. Transoceanic transport mechanisms: introduction of the Chinese mitten crab, Eriocheir sinensis, to California. Pac. Sci. 51, 1–11. Crowley, T.J.,North, G.R., 1988. Abrupt climate change and extinction events in earth history. Science 240, 996–1002. Drummond, A.J., Marc, A.S., Dong, X., Andrew, R., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin version 3.0: an integrated software package for population genetics data analysis. Evol. Bioinformatics Online 1, 47–50. Folmer, O.,Black, M.,Hoeh, W.,Lutz, R.,Vrijenhoek, R., 1994. DNA primers for amplification of mt cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.
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Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes Daizhen Zhang a,b, Ge Ding c, Baoming Ge b, Huabin Zhang b, Boping Tang b, Guang Yang a,⁎ a b c
Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, People's Republic of China Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers University, Yancheng 224051, People's Republic of China Chemical and Biological Engineering College, Yancheng Institute of Technology, Yancheng 224003, People's Republic of China
a r t i c l e
i n f o
Article history: Received 27 March 2014 Received in revised form 19 July 2014 Accepted 4 August 2014 Available online xxxx Keywords: Phylogeography Crustacean Divergence Expansion Environmental change
a b s t r a c t Environmental changes, such as changes in the coastal topography due to Eurasian plate movements, climate oscillation during the Pleistocene, and alteration of ocean currents, have complicated the geographical structure of marine species and deepened their divergence between populations. As two widely distributed species of crustacean (Oratosquilla oratoria and Eriocheir japonica), weak differences were expected due to their high dispersal potential of planktonic larvae with ocean currents. However, results showed a significant genetic divergence between north of China and south of China in the study. In addition, the estimated north–south divergence time (27–30.5 Myr) of mantis shrimp was near the time of the Himalayan movement, and the China–Japan clade divergence time (10.5–11.9 Myr) of mitten crabs was also coincident with the time of the opening of the Sea of Japan. Thus, we hypothesized that environmental changes in the coastal topography contributed to the marine species divergence. Furthermore, based on phylogenetic analysis, network analysis and haplotype distribution, we surmised that mitten crabs originated from a population with the oldest haplotype (H6) and then divided into the north and south populations due to the recent Eurasian plate movements and ocean currents. And lineage of Japan originated from the north population for the opening of the Sea of Japan. While O. oratoria was guessed to originate from two separate populations in the China Sea. The results of “star-like” network, negative values in neutral test, and Tajima's D statistics of two marine species supported a recent rapid population expansion event after the Pleistocene glaciations. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Vagarious climate fluctuations during the Pleistocene have caused alterations of dramatic temperature, ocean currents and coastal topography (Clark et al., 1999; Hewitt, 1996, 2000). To elude the dramatic changes during the Pleistocene glaciations, marine organisms were forced into a repeated cycle of glacial retreat and interglacial expansion. The ranges of some faunal distributions have exhibited markedly altered contraction or expansion (Avise, 2000; Hewitt, 2000; Xu et al., 2009). Such historical events have led to species extinction and have also acted as the drivers of subspeciation for local adaptation and adaptive radiation (Crowley and North, 1988; Gillespie, 2004; Mayhew et al., 2008). The relative influence of environmental changes on marine organisms could best explain the patterns over a longer evolutionary time frame (Benton, 2009). Moreover, during the Pleistocene glacial cycles, the formation of land bridges due to the lowered sea level between islands and Asian continent might act as the barriers for marine organisms and potentially aid allopatric diversification (Kimura, 2000; Abbreviations: mt, mitochondria(l); nt, nucleotide(s); Myr, million years. ⁎ Corresponding author. E-mail address: [email protected] (G. Yang).
McManus, 1985; Tamaki and Honza, 1991; Voris, 2000; Wang, 1999). Furthermore, the dispersal of the pelagic larvae for ocean currents also played a fundamental role in ecology and evolution (Caley et al., 1996; Strathmann et al., 2002). These physical and environmental attributes as the potential mechanisms have shaped patterns of marine species divergence and population expansion by the occupation of newly formed habitats or adaptation to changing environments (Richardson, 2012). Northwest pacific marginal seas connecting the mainland and deep oceans were regarded as the most suitable areas to understand the past global climate and oceanographic changes. The phylogeographic structure of marine species living in marginal seas was the typical representative of the intertidal invertebrate affected by historical climate, ocean currents and coastal topography. As the representative marine species, mantis shrimps Oratosquilla were widely distributed along the northwest pacific marginal seas, and mitten crabs Eriocheir were also distributed from China and Japan to Korea. Previous studies have indicated that Eriocheir japonica was divided into three geographic subspecies, namely E. j. japonica, E. j. sinensis and E. j. hepuensis (Tang et al., 2003). Thereinto E. j. japonica was distributed in Japan, and the 26°N latitude was considered the boundary between E. j. sinensis and E. j. hepuensis in China (Cohen and Carlton, 1997; Ingle and Andrews, 1976; Tang et al., 2003; Zhao et al., 1988). Subspeciation required the
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Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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D. Zhang et al. / Gene xxx (2014) xxx–xxx
termination of gene flow for geographic barrier or population isolation. It was understandable that subspeciation of E. j. japonica might be a result of the isolation between China and Japan. However, it was unacceptable that 26°N latitude was the unambiguous boundary between E. j. sinensis and E. j. hepuensis. So the subspeciation of Eriocheir still remains a controversial topic at present (Tang et al., 2003; Wang et al., 2008; Xu et al., 2009; Zhang et al., 2012), and additional data are needed to reveal the phylogeographic structure and to clarify the relationship between phylogeographic structure and environmental changes. In this study, through a comparative phylogeography study of two marine species (Oratosquilla oratoria and E. japonica) of crustacean, we assessed the phylogeographical pattern of marine species in the northwest pacific marginal seas and revealed the association between divergence and historical environmental changes.
2. Materials and methods 2.1. Sample collection The study region was selected because several marginal seas were once separated during glaciations. A total of 621 samples of the two marine species of crustacean were collected in this study. Samples of 208 individuals of O. oratoria were obtained from 15 localities representing the geographical range of this species across the China Sea, and 413 samples of E. japonica from 19 locations along the northwest Pacific Ocean were also integrated from GenBank to corroborate the results (Fig. 1, Appendix 1). The materials were sampled and preserved in 95% ethanol for later analysis. We chose to focus on the mt cytochrome oxidase c subunit I (mt COI) gene and the cytochrome b (mt Cytb) gene
because these were more informative sites and because of their ability to estimate the mutation rate. The main limitation was that these sites represented only one evolutionary history. However, a lower effective sample size of mtDNA relative to that of nuclear DNA was needed due to their faster rate at the population level. We thus considered the use of mt sequences as the best strategy for accurate estimation. 2.2. DNA extraction, amplification and sequencing The genomic DNA from tissue muscle was extracted using the standard phenol–chloroform method. PCR amplification was executed using the primers described by Folmer et al. (1994). Each 30-μl amplification reaction consisted of thermophilic buffer (50 mM KCl and 10 mM Tris–HCl pH 8.3), 2.0 mM MgCl2, 10 pmol of each primer, 0.2 mM dNTPs, and 1 unit Taq DNA polymerase. Polymerase chain reaction (PCR) was performed on a GeneAmp® PCR System 9700 (ABI) or Mastercycler® gradient (Eppendorf) thermocycler with a temperature profile of 30 cycles at 94 °C for 40 s, 50 °C for 40 s, and 72 °C for 60 s. The PCR products were purified using a PCR purification kit and cycle-sequenced on an ABI Prism 3730 automated sequencer. 2.3. Sequence alignment, genetic diversity and phylogenetic analysis The sequences were aligned with the Clustal W algorithm in Mega 5.0 (Tamura et al., 2011). The nt diversity (π) and haplotype diversity (h) were estimated using Arlequin version 3.1 (Excoffier et al., 2005). The divergences within- and between-populations were determined using Mega 5.0 (Tamura et al. 2011). Haplotype neighbour-joining trees (NJ) were constructed using the Kimura 2-parameter model in
Fig. 1. Map showing the sampling locations for the two marine species in this study. ●: locations for E. japonica; : locations for O. oratoria. Colours in circles represented different haplotypes and their proportions. The four ancestral haplotypes of E. japonica were H5 , H6 , H12 , and H30 . The three ancestral haplotypes of O. oratoria were HP5 , HP14 , and HP63 . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
D. Zhang et al. / Gene xxx (2014) xxx–xxx
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Table 1 Genetic diversity, divergence, neutral test, and gene flow among different lineages. n: number of samples; N: number of haplotypes; h: haplotype diversity; π: nt diversity. Groups
E. japonica ENC EJA ESC EMG O. oratoria ONC OSC a
n
413 229 48 121 15 208 183 25
N
80 30 16 28 6 80 62 18
h
0.899 0.745 0.871 0.756 0.810 0.930 0.911 0.953
π
0.02291 0.00259 0.00265 0.00256 0.00233 0.01712 0.00447 0.00669
Tajima's D
Fu's Fs
Pairwise value of Fst (lower left) and Nm (upper right)
D
P
Fs
ENC
EJA
ESC
EMG
−0.592 −2.286 −1.736 −2.144 −0.144 −1.113 −2.211 −1.432
N0.10 b0.01a N0.05 b0.05a N0.10 N0.10 b0.01a N0.10
−84.819 −43.788 −19.928 −41.210 −4.172 −44.95 −80.434 −11.171
– – 0.83281 0.86501 0.89466 ONC – 0.91256
– 0.05 – 0.89396 0.92190 OSC 0.02 –
– 0.04 0.03 – 0.87752 – – –
– 0.03 0.02 0.03 – – – –
Significant.
Mega 5.0 (Bootstrap = 1000), and a Bayesian evolutionary analysis (BI) was implemented in Beast v1.7 (Drummond et al., 2012). The input file was properly formatted with the BEAUti utility included in the software package using the partition scheme used for the concatenated analysis. Two runs, each of 10 × 106 generations (samplefreq = 1000 and 10% burn in), were conducted. The maximum clade credibility trees were created with TreeAnnotator v1.7.4. Ultimately, a Bayesian evolutionary tree was inferred and viewed using FigTree v1.4.0 (Rambaut, 2008). 2.4. Calculation of the divergence date A likelihood ratio test was performed to determine whether the datasets evolved in a clocklike manner, and the branch age estimation was performed using Mega 5.0. The Sea of Japan is a semi-landlocked sea located between Japan and the Asian continent that developed as a consequence of the rotation of Japan during the period between 10 and 15 Myr (Ichikawa et al., 1990; Kiyotaka, 1991). Using the credible maximum calibration date of the opening of the Sea of Japan, we calculated the likely clade date of Caridea and tested the estimated date with their fossil calibrations (Bravi and Garassino, 1998; Garassino and Bravi, 2003). If the time coincided with the fossil record, we conversely estimated each clade date by the fossil records. 2.5. Historical demography The possible occurrence of historical demographic expansion was examined using mismatch distribution and Tajima's (1989) D and Fu's (1997) Fs neutrality tests. The range of contractions or expansion was
verified by analysing the frequency distribution of pairwise differences among haplotypes (mismatch distribution) (Harpending 1994) using the DnaSP software (Rozas et al., 2003). Tajima's D test was used to test for departures from equilibrium between mutations and drift (Tajima, 1989). The relationship t = τ / 2 μ (Rogers and Harpending, 1992) was used to further estimate the time of expansion (t) using a mutation rate of 2.33% per Myr and a generation time of two years (Knowlton et al., 1993; Schubart et al., 1998). Network v. 4.6.0.0 (Bandelt et al., 1999) was used to construct a statistical parsimony network and to analyse the ancestral haplotypes of the two marine species. 3. Results 3.1. Genetic diversity and phylogenetic tree Overall, the alignment of 208 sequences of O. oratoria representing 15 populations defined 80 unique haplotypes (HP1–HP80) (GenBank Numbers: KM196983–KM197062). In comparison, 413 sequences of E. japonica representing 19 populations also revealed 80 haplotypes (H1–H80) (Appendix 1). The similar haplotype diversity (h) was calculated to be 0.930 for O. oratoria and 0.899 for E. japonica. The details of the haplotype distribution of each geographical population were shown in Fig. 1. Moreover, 89 and 97 sites were defined as polymorphic sites for O. oratoria and E. japonica, in which 65 and 51 sites were parsimony informative sites, respectively. In addition, the whole nt diversity (π) was 0.01712 for O. oratoria and 0.02291 for E. japonica (Table 1). For O. oratoria, haplotype HP5 was shared by 54 samples from the north of China, whereas for E. japonica, haplotypes H6 and H12 were shared by
Fig. 2. Phylogenetic tree and divergent time of E. japonica along the western Pacific Ocean. The numbers above the branches showed the percentages of bootstrap support with 1000 bootstrap replicates. The numbers with arrows were the divergence time of the clade associated with the circle.
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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Fig. 3. Phylogenetic tree and divergence time of O. oratoria along the China Sea. The numbers on the branches were the percentages of bootstrap support with 1000 bootstrap replicates. The numbers with arrows were the divergence time of the clade associated with the circle.
147 samples, most of which were from the north of China. Haplotype H5 was shared by 58 samples from the south of China. Both the NJ and BI phylogenetic trees exhibited similar topologies of the O. oratoria haplotypes, and the E. japonica trees were also similar in terms of topology (Figs. 2 and 3). In the O. oratoria tree, two major clades were apparent: one clade with strong support (bootstrap support of 99%) represented all of the populations from the Bohai Sea, Yellow Sea, and East China Sea in the north of China (ONC), and the other clade with strong support (bootstrap support of 99%) comprised the South China Sea (OSC). The E. japonica exhibited similar topology: one major clade with strong support (bootstrap support of 93%) represented the South China Sea (E. j. hepuensis) (ESC) and E. japonica sp. (EMG), in which the populations of the Nanliujiang River and Zhanjiang River occupied 34% and 26% of the haplotypes in ESC, and the other clade comprised the north of China (E. j. sinensis) (ENC) with 33% of the haplotypes in the Yangtze River and Japan (E. j. japonica) (EJA). Within the two clades, four clades (E. j. sinensis, E. j. japonica, E. j. hepuensis, and E. japonica sp.) were separated with strong support (bootstrap support of 98% or 99%), which corresponded to the three geographical subspecies of E. japonica and a novel clade.
3.2. Genetic divergence The pairwise Fst was concordant, showing that the average value between the four lineages in mitten crabs was ENC/EJA: 0.83281, ENC/ESC: 0.86501, ENC/EMG: 0.89466, EJA/ESC: 0.89396, EJA/EMG: 0.92190, and ESC/EMG: 0.87752. Assuming populations at equilibrium between gene flow and genetic drift, the gene flow was estimated by Fst-based methods to be ENC/EJA: 0.05, ENC/ESC: 0.04, ENC/EMG: 0.03, EJA/ESC: 0.03, EJA/EMG: 0.02, and ESC/EMG: 0.03. In the case of mantis shrimp, the Fst between the two clades was 0.91256, and the corresponding gene flow was 0.02 (details in Table 1). The net average genetic distance between the four lineages (±SE) in mitten crabs were ENC/EJA: 0.0249 (±0.0058), ENC/ESC: 0.0367 (±0.0075), ENC/EMG: 0.0405 (±0.0078), EJA/ESC: 0.0408 (± 0.0081), EJA/EMG: 0.0456 (± 0.0081), and ESC/ EMG: 0.0321 (±0.0069). In addition, the value between OSC and ONC for mantis shrimp was 0.0622 (±0.0102), showing deep divergence between the south of China and the north of China. The molecular clock test was performed by comparing the ML values for the given topology. The null hypothesis of an equal evolutionary rate throughout the tree was not rejected at a 5% significance level (p b 1) both for mitten crabs and mantis shrimp. Using the calibration date of 10–15 Myr defined by the opening of the Sea of Japan, we calculated
Fig. 4. Median-joining network of haplotypes of O. oratoria. The circle size was proportional to the number of individuals exhibiting the corresponding haplotype. The ancestral haplotypes (HP5 , HP14 , and HP63 ) were coloured according to Fig. 1. The length of the lines was proportional to the number of mutational steps separating the haplotypes. The smallest circles indicated missing intermediate haplotypes. The map in the lower right corner showed the torso of the network of O. oratoria, including all of the ancestral haplotypes. A: Group from the Bohai Sea, Yellow Sea, and East China Sea. B: Group from the South China Sea. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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that the absolute fossil time of Caridea was approximately 94–141 Myr, which was similar to that obtained from the fossils records, i.e., 99– 112 Myr. We concluded that the divergence of clade E. j. sinensis and clade E. j. japonica was related to the opening of the Sea of Japan and the consequent rotation of Japan. Conversely, we estimated the date of each clade using the date of the fossil calibrations. The results show that divergence date of clade E. j. sinensis and clade E. j. japonica was approximately 10.5–11.9 Myr, whereas the divergence data of clade E. j. hepuensis and clade E. japonica sp. was 12.9–14.6 Myr and that of clade E. j. sinensis and E. j. hepuensis was approximately 15.5–17.5 Myr (Fig. 2). The application of the same calculations to the phylogenetic tree of O. oratoria revealed that the divergence date between the south clade and the north clade was 27–30.5 Myr, and the splitting time between the south clade and the north clade in China was older than that of mitten crabs (Fig. 3).
5
A similar “star-like” median-joining network was shown for the E. japonica population, and the whole network separated all of the haplotypes into four distinct networks, corresponding to the four geographical lineages. Moreover, the “star-like” network with the central haplotypes H5, H6, H12, and H30 indicated the rapid expansion of the population historical demography of E. japonica, and the central haplotypes were likely the ancestral types (Fig. 5). The mismatch distributions supported the similar conclusions of the historical population expansion demographics for smooth unimodal distributions. Estimates of population size changes and selection generally indicated nonneutrality. The Tajima's D statistics showed negative values (Tajima's DENC = −2.286, p b 0.01; Tajima's DESC = − 2.144, p b 0.05; Tajima's DEJA = − 1.736, p N 0.05; Tajima's DEMG = − 0.144, p N 0.10), and Fu's F test of neutrality also gave negative value (FS-ENC = − 43.788; FS-ESC = − 41.210; FS-EJA = − 19.928; FS-EMG = − 4.172) for the expansion of the four clades (Table 1). And the time of expansion estimated using the formula t = τ / 2 μ was approximately 36–57 Ka.
3.3. Population demographic analysis 4. Discussion The median-joining analysis of O. oratoria revealed a “star-like” network with the central haplotypes HP5, HP14, and HP63 as the ancestral haplotypes. However, two distinct networks (A, B) were observed: A comprised all of the haplotypes from the Bohai Sea, Yellow Sea, and East China Sea, and B consisted of all of the haplotypes from the South China Sea (Fig. 4). The mismatch distributions, which revealed smooth unimodal distributions for each group, supported a rapid population expansion event. The negative value of Tajima's D statistics for the estimation of population size changes (Tajima's DONC = − 2.211, p b 0.01; Tajima's D OSC = − 1.432, p N 0.10) and Fu's F test of neutrality (FS-ONC = −80.434; FS-OSC = −11.171) supported the conclusion of a rapid expansion. The estimated time for the rapid population expansion was approximately 48–50 Ka.
As widely distributed marine species, weak differences among geographical populations were expected due to their high dispersal potential (Knowlton et al., 1993; Avise, 1994; Uthicke and Benzie, 2003). However, our results revealed the significant divergence, as determined by genetic distance, phylogenetic tree, and network methods, between the north and south populations of two dispersed marine species. So we inferred environmental changes, such as Eurasian plate movements, ocean currents, and climatic oscillations during the Pleistocene, complicated the geographical structure of marine species and deepened the divergence among populations. For Eurasian plate movements, the previous study showed that Taiwan Island was the natural boundary for many marine organisms (Liu et al., 2007), so we thought that the
Fig. 5. Median-joining network of haplotypes of E. japonica. The circle size was proportional to the number of individuals exhibiting the corresponding haplotype. The ancestral haplotypes (H5 , H6 , H12 , and H30 ) were coloured according to Fig. 1. The length of the lines was proportional to the number of mutational steps separating the haplotypes. The smallest circles indicated missing intermediate haplotypes. The map in the upper right corner showed the torso of the network of E. japonica, including all of the ancestral haplotypes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
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D. Zhang et al. / Gene xxx (2014) xxx–xxx
Fig. 6. Map summarizing the proposed origin, expansion, and subspeciation of the two marine species. The arrows with the dotted line indicated the route of colonization and divergence of E. japonica. The centred arrows represented the rapid expansion of O. oratoria.
formation of Taiwan Island must have contributed to the divergence of the north–south division of marine species. The Taiwan Island was traced back to the latest orogeny of the Himalayan movement in the Eocene epoch of the Tertiary period of the early Cenozoic 40 Myr (Jia et al., 2004). In the study, based on fossil record and phylogenetic tree analyses, the predicted north–south divergence time of O. oratoria was estimated to be approximately 27–30.5 Myr, which was near to the time of the Himalayan movement and the later plate activity of Taiwan Island. To some extent, the formation of Taiwan Island and the early Himalayan movement must deepen the divergence between the north and south populations of O. oratoria. However, the divergence time of E. japonica (15.5–17.5 Myr) was inferred to be more recent than that of O. oratoria by the same methods. In lifecycle of E. japonica, it lived in freshwater in most time of its lifecycle and migrated to a salty estuary for oviposition in its reproductive seasons. So the influence of Taiwan Island might be less on mitten crabs than that of mantis shrimps. Moreover, fluctuation of Taiwan Strait in the late glacial period further influenced the phylogeographic structure of marine species. Taiwan Strait was a submarine valley lying between the Chinese Mainland and Taiwan. In glacial period of quaternary, Taiwan Strait once emerged in response to decreases of the sea level (Tougard, 2001), and Taiwan Island was linked with Fujian Province of China (Shan et al., 2013). So acting as a land bridge, Taiwan Strait enabled terrestrial organisms to extend their ranges but limited the gene flow between the north and south populations of marine species and potentially resulted in allopatric diversification (Imron et al., 2007; McManus, 1985). The Sea of Japan was a semi-landlocked sea between Japan and the Asian
continent that was developed as a consequence of the rotation of Japan during the period between 10 and 15 Myr. In the study, the divergence time (15.5–17.5 Myr) of E. j. sinensis in China and E. j. japonica in Japan by calibration of fossil records was abnormally coincident with the opening time of the Sea of Japan (10–15 Myr). The novel coincident time demonstrated the possibility that opening of the Sea of Japan contributed to increasing the divergence of marine species. Ocean currents in the China Sea derived from the Kuroshio tributary or originated from the monsoons. Currents of the Bohai Sea, Yellow Sea and East China Sea, which exhibited the characteristics of counterclockwise cyclonic circulation, were mainly the result of the Kuroshio current and coastal streams. However, the surface circulation of the South China Sea exhibited clockwise circulation due to the monsoon drift. So the planktonic larvae of the two species in northern seas would disperse northward due to the counterclockwise cyclonic circulation currents, whereas the larvae in the South China Sea dispersed southward (Shane et al., 2008). Such opposite dispersion further deepened the divergence between the north and south geographical populations. In late Pleistocene glaciations, vagarious climate fluctuations caused dramatic temperature changes, and marine organisms were forced into glacial retreat and later rapid interglacial expansion. In this study, a similar rapid population expansion event was revealed in two crustacean species by mismatch distribution, neutral test and median-joining network. Moreover, the expansion time (36–57 Ka) estimated by the formula t = τ / 2 μ was in the range of the climate fluctuations of the late Pleistocene. The result explained the reason for lack of phylogeographical
Please cite this article as: Zhang, D., et al., Comparative phylogeography of two marine species of crustacean: Recent divergence and expansion due to environmental changes, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.08.006
D. Zhang et al. / Gene xxx (2014) xxx–xxx
structure within the north population or south population. In Pleistocene, marine species were forced into a repeated cycle of glacial retreat and interglacial expansion as a result of the climate fluctuations, so ranges of some faunal distributions exhibited markedly altered contraction or expansion (Avise, 2000; Hewitt, 2000; Xu et al., 2009). About the original centres, ancestral haplotypes of Eriocheir (H5, H6, H12 and H30) were in the centre of the genealogical network. And haplotype H5 were shared by all samples from the south population (100%), and H12 were shared by most samples from the north population (95.6%). That revealed that H5 and H12 were the ancestral haplotypes of the south and north populations, respectively. However, as the oldest haplotype, H6 was shared by the north and south populations in China. We hypothesized that Eriocheir in the northwest Pacific Ocean originated from an oldest population with the ancestral haplotype H6 in the China Sea, but gene flow between the north and south seas was then weakened due to the formation of Taiwan Island, explosion of Taiwan Strait, opposite direction of ocean currents, and climatic oscillations during the Pleistocene, that resulted in the division of the north population with H5 and south population with H12. Later, genetic divergence between Japan and north of China was deepened for the opening of the Sea of Japan in 10–15 Myr (Fig. 6). However, results of O. oratoria showed that HP5, HP14, and HP63 were the ancestral haplotypes in the centre of network. Unlike Eriocheir, all samples sharing HP5 and HP14 were obtained from north of China. Similarly, all samples sharing HP63 were from south of China, but no ancestral haplotype was shared by both the north and south populations. Thus, we hypothesized that O. oratoria in China were from two original regions: north of the China Sea and south of the China Sea (Fig. 6), and that their divergence was increased by the later environmental changes. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.08.006. Acknowledgements The work was funded by National Natural Science Foundation of China (41301050), Natural Science Foundation of Jiangsu Province (BK2011421, 12KJA180009), Foundation of Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection (JLCBE12002) and sponsored by “Qing Lan Project” and “333 Project” of Jiangsu Province ( (2013) Ⅲ-1129) to Daizhen Zhang. We also thank all anonyms for their help in sample collection. References Avise, J.C., 1994. Molecular markers. Natural History and EvolutionChapman and Hall, New York. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, MA. Bandelt, H.J.,Forster, P.,Röhl, A., 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16, 37–48. Benton, M.J., 2009. The Red Queen and the Court Jester: species diversity and the role of biotic and abiotic factors through time. Science 323, 728–732. Bravi, S., Garassino, A., 1998. ‘Plattenkalk’ of the Lower Cretaceous (Albian) of Petina, in the Alburni Mounts (Campania, S. Italy), and its decapod crustacean assemblage. Atti Societa italiana Scienze naturali Museo civico Storia naturale Milano. 138, pp. 89–118. Caley, M.J.,Carr, M.H.,Hixon, M.A., 1996. Recruitment and the local dynamics of open marine populations. Annu. Rev. Ecol. Syst. 27, 477–500. Clark, P.U.,Alley, R.B.,Pollard, D., 1999. Northern hemisphere ice sheet influences on global climate change. Science 286, 1104–1111. Cohen, A.N., Carlton, J.T., 1997. Transoceanic transport mechanisms: introduction of the Chinese mitten crab, Eriocheir sinensis, to California. Pac. Sci. 51, 1–11. Crowley, T.J.,North, G.R., 1988. Abrupt climate change and extinction events in earth history. Science 240, 996–1002. Drummond, A.J., Marc, A.S., Dong, X., Andrew, R., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin version 3.0: an integrated software package for population genetics data analysis. Evol. Bioinformatics Online 1, 47–50. Folmer, O.,Black, M.,Hoeh, W.,Lutz, R.,Vrijenhoek, R., 1994. DNA primers for amplification of mt cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299.
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