Connectivity of the coral Pocillopora damicornis from the Great Barrier Reef

Pocillopora damicornis was sampled from 28 sites in the northern, central and southern regions of the Great Barrier Reef. In the northern region samples were collected from Martin Reef (1 site), Eyrie Reef (1 site), Lizard Island (9 sites), MacGillivray Reef (1 site) and Yonge Reef (1 site). In the central region samples were collected from Fantome Island (1 site), Orpheus Island (4 sites), Pelorus Island (3 sites), Trunk Reef (2 sites), Dip Reef (1 site) and Myrmidon Reef (1 site). Samples were collected in the southern region from Chinaman Reef (1 site), East Cay Reef (1 site) and Frigate Reef (1 site). Fragments of Pocillopora damicornis, approximately two cm long, were collected from each of 1040 colonies. Each colony was photographed and the location recorded by towing a handheld Garmin Etrex GPS unit in a waterproof container on the surface. Between 6 and 99 individuals were sampled per site. Collections were made haphazardly on the upper reef slope along a zigzag transect approximately 200 m long, between 2 m and 10 m of depth. Unattached or asymmetrical colonies within one metre of a colony already sampled were not sampled due to the possibility that these were clones by fragmentation. Coral branches were fixed in absolute ethanol.DNA was extracted using a modified protocol of the salt precipitation method. Samples were genotyped using nine microsatellite markers ( xxxxxx) according to the multiplex groups, primers and protocols described in Torda et al. (2013). Samples from four sites, Dip Reef, Chinaman Reef, East Cay Reef and Frigate Reef were not genotyped for marker Pd4.To determine the lineage identity of each sample, a rapid genetic assay was used. The vast majority of samples (72%; i.e.745 samples) were118 identified as Type alpha. The remaining samples were Type beta (22%; i.e. 228 samples), 'other Pocillopora' (5%; i.e. 56 samples) or did not give reliable results (1%; 11 samples) (Table 1).All subsequent analyses were carried out on Type alpha and Type beta samples separately, omitting 'other Pocillopora' and unidentified samples, which potentially include the poorly resolved genetic lineage Type gamma and P. verrucosa (Schmidt-Roach et al. 2013).Data analysesTo assess the discriminative power of sets of loci, Genotype Probability (GP) was calculated for each sample and each locus in GENALEX 6.4 (Peakall & Smouse 2006). Repeated multilocus genotypes (MLG) were considered to be clone mates if the product of GP for all 128 loci was To estimate genetic differentiation among populations, we used Dest (Jost 2008), calculated in SMOGD 1.2.5 (Crawford 2010), because this statistic is not sensitive to genetic diversity and because it accounts for both migration and mutation rates, being based on the finite island model. Significance levels of Dest values were determined by a permutation test, randomizing alleles over all compared populations, using R code from Alberto et al. (2011). For easier comparison with results of other studies, we explored other statistics as well, including (i) uncorrected pairwise Fst values by the ¿weighted" analysis of variance method (Weir & Cockerham 1984), as implemented in Genepop; (ii) the standardised pairwise F¿st, estimated using an AMOVA (Meirmans 2006) in GenoDive; (iii) pairwise Fst values corrected for null alleles (ENA correction), computed in FreeNA (Chapuis & Estoup 2007). To account for unbalanced sample sizes, the significance of uncorrected Fst values was assessed by a Fisher exact test (Goudet 1995) in Genepop with the default Markov chain parameters. To facilitate direct comparison of our results with those of previous allozyme studies, we also carried out a hierarchical analysis of standardised genetic variance as Weir & Cockerham¿s (1984) ¿ using the program TFPGA, following Ayre & Hughes (2000). To approximate the sampling design of Ayre & Hughes (2000), only samples collected from around Lizard Island and the Palm Islands were used for this analysis and samples from Type ¿ and s were pooled, as information on these distinct genetic lineages was not available in the earlier studies. To detect putative first generation migrants, the probability that each individual belongs to each reference population was computed in GeneClass2 (Piry et al. 2004) using the criteria and probability computation algorithm of Rannala and Mountain (1997), with 10,000 simulated genotypes. Individuals were excluded from a reference population if the probability of exclusion was greater than 0.99 (¿Genetic structuring of samples without prior definition of populations was analysed using the Bayesian clustering method implemented in InStruct (Gao et al. 2007). As opposed to the more commonly used method implemented in Structure (Pritchard et al. 2000), InStruct accounts for potential selfing. Five independent chains were run for each K from K = 1 to K = 20, with a burn-in of 100,000 and another 100,000 MCMC replications after the burn-in, using the ¿infer population structure and population selfing rates¿ function with the default samplers. To assist in the interpretation of the results of the genetic analyses, the potential dispersal capacity of brooded larvae was simulated using virtual Lagrangian particle transport modelling in the 0.025° x 0.025°-cell circulation model of the GBR in Connie 2.0 (CSIRO Connectivity Interface, http://www.csiro.au/connie2/). Collection sites were selected as both sources and sinks for dispersal of passive particles at a depth of 5 m over a dispersal period of 1, 15, 50 and 100 days. In previous population genetic studies, populations of Pocillopora damicornis on the Great Barrier Reef showed strong genetic subdivision on small spatial scales& Hughes 2000, 2004) ¿ a pattern difficult to interpret. Recent evidence that P. damicornis is78 a species complex with several sympatric, but genetically isolated lineages (Flot et al. 2008;79 Souter 2010; Schmidt-Roach et al. 2013), suggests that earlier findings of genetic80 subdivisions within reefs may have been confounded by the unknown inclusion of several81 putative cryptic species within studies.the subsequent development of a83 rapid genetic assay capable of distinguishing the two most common genetic lineages (Torda et84 al. 2013), which now make it possible to focus research efforts on genetically more85 meaningful units, it is timely to revisit the connectivity and population genetic puzzle of this86 model scleractinian coral species.

本研究从大堡礁（Great Barrier Reef）北部、中部和南部区域的28个采样点采集了杯形珊瑚（Pocillopora damicornis）样本。其中北部区域的采样点包括：马丁礁（Martin Reef，1个位点）、艾里礁（Eyrie Reef，1个位点）、蜥蜴岛（Lizard Island，9个位点）、麦吉尔里夫礁（MacGillivray Reef，1个位点）以及永格礁（Yonge Reef，1个位点）。中部区域的采样点包括：幻岛（Fantome Island，1个位点）、奥菲斯岛（Orpheus Island，4个位点）、佩洛鲁斯岛（Pelorus Island，3个位点）、特伦克礁（Trunk Reef，2个位点）、迪普礁（Dip Reef，1个位点）以及默米登礁（Myrmidon Reef，1个位点）。南部区域的采样点包括：中国人礁（Chinaman Reef，1个位点）、东凯礁（East Cay Reef，1个位点）以及护卫舰礁（Frigate Reef，1个位点）。

从1040个珊瑚群落中各采集一段长约2cm的杯形珊瑚断枝。对每个珊瑚群落进行拍照，并通过将置于防水容器中的佳明Etrex（Garmin Etrex）手持式GPS设备在水面拖拽来记录采样位置。每个采样位点采集6至99个个体。采样采用随机方式，在深度2至10米的礁坡上部沿长约200米的之字形样带进行。对于已采样群落1米范围内的游离或不对称群落，由于其可能为通过碎裂产生的克隆体，故未进行采样。珊瑚枝用无水乙醇固定。

采用改良的盐沉淀法（salt precipitation method）提取基因组DNA。根据Torda等人（2013）描述的多重组、引物及实验流程，使用9个微卫星标记（microsatellite markers）对样本进行基因分型。其中，迪普礁、中国人礁、东凯礁以及护卫舰礁共4个位点的样本未进行Pd4标记的基因分型。

为确定每个样本的谱系归属，采用快速遗传检测方法。绝大多数样本（72%，即745个样本）被鉴定为α型（Type alpha），剩余样本分别为β型（Type beta，22%，即228个样本）、“其他杯形珊瑚类群”（5%，即56个样本），或未获得可靠分型结果（1%，即11个样本）（详见表1）。后续分析仅针对α型和β型样本分别开展，剔除了“其他杯形珊瑚类群”及未分型样本——此类样本可能包含分辨率较低的γ型遗传谱系以及P. verrucosa（Schmidt-Roach等，2013）。

数据分析
为评估位点集的区分能力，使用GENALEX 6.4软件（Peakall & Smouse，2006）计算每个样本及每个位点的基因型概率（GP）。若所有128个位点的GP乘积满足条件，则将重复出现的多位点基因型（MLG）视为克隆同伴（原文此处内容存在缺失）。
为评估种群间的遗传分化，我们采用了Dest统计量（Jost，2008），该统计量基于有限岛屿模型，不受遗传多样性影响，同时兼顾了迁移与突变率，通过SMOGD 1.2.5软件（Crawford，2010）计算得到。Dest值的显著性通过置换检验确定：将所有对比种群的等位基因进行随机化，使用Alberto等人（2011）提供的R代码实现。为便于与其他研究结果对比，我们同时探索了其他统计量，包括：(i) 采用Weir & Cockerham（1984）提出的加权方差分析法计算的未校正成对Fst值，通过Genepop软件实现；(ii) 采用GenoDive软件中的分子方差分析（AMOVA）估算的标准化成对Fst值（Meirmans，2006）；(iii) 通过FreeNA软件计算的针对无效等位基因（null alleles）校正的成对Fst值（Chapuis & Estoup，2007）。为平衡样本量不均衡的问题，在Genepop中采用费希尔精确检验（Fisher exact test，Goudet，1995）对未校正Fst值的显著性进行评估，使用默认的马尔可夫链参数。
为便于与此前的同工酶研究结果直接对比，我们还遵循Ayre & Hughes（2000）的方法，使用TFPGA软件开展了基于Weir & Cockerham（1984）提出的标准化遗传方差的层级分析。为匹配Ayre & Hughes（2000）的采样设计，本次分析仅使用了采集自蜥蜴岛及棕榈群岛周边的样本，并将α型与β型样本合并——因早期研究未提供这两类不同遗传谱系的相关信息。
为检测推定的初代迁移个体，使用GeneClass2软件（Piry等，2004）计算每个个体属于每个参考种群的概率，采用Rannala和Mountain（1997）提出的标准及概率计算算法，生成10000个模拟基因型。若个体被排除出参考种群的概率大于0.99，则将其从该参考种群中剔除。
未预先定义种群的样本遗传结构分析采用InStruct软件实现的贝叶斯聚类法（Gao等，2007）。相较于更常用的Structure软件（Pritchard等，2000）所实现的聚类方法，InStruct可考虑潜在的自交现象。对于K值从1到20的每个设定，均运行5次独立链，预烧期（burn-in）为100000代，预烧期后再运行100000代马尔可夫链蒙特卡洛（MCMC）重复采样，使用“推断种群结构及种群自交率”功能，并采用默认采样器。
为辅助解读遗传分析结果，我们采用康妮2.0（Connie 2.0，澳大利亚联邦科学与工业研究组织连通性接口，http://www.csiro.au/connie2/）中的大堡礁0.025°×0.025°格点环流模型，通过虚拟拉格朗日粒子输运模型模拟了育幼幼虫的潜在扩散能力。以采样位点作为被动粒子扩散的源与汇，设置扩散深度为5米，扩散时长分别为1天、15天、50天及100天。
此前的种群遗传研究显示，大堡礁的杯形珊瑚种群在小空间尺度上存在强烈的遗传分化（Ayre & Hughes，2000，2004），这一模式难以解释。近期研究表明，杯形珊瑚实为一个物种复合群，包含多个同域分布但遗传隔离的谱系（Flot等，2008；Souter，2010；Schmidt-Roach等，2013），这提示早期研究中观测到的礁内遗传分化现象，可能因研究中未知混入了多个推定隐存物种而被混淆。随着能够区分两类最常见遗传谱系的快速遗传检测方法的开发（Torda等，2013），如今可将研究聚焦于更具生物学意义的遗传单元，此时重新审视该模式石珊瑚（scleractinian coral）物种的连通性与种群遗传谜题恰逢其时。