Microsatellite matrix of Poa annua

Comparative studies of invasive species in human-inhabited versus truly uninhabited habitats, particularly on their genetic structure, remain scarce. Sub-Antarctic islands provide an ideal system to study invasions in such contrasting environments as they represent semi-pristine conditions in highly remote areas that are accessible only through a small number of introduction routes. Here we studied the invasion genetics of annual bluegrass Poa annua on the Prince Edward Islands (PEI) that include the inhabited Marion Island and the uninhabited Prince Edward Island. We analysed variation at nuclear microsatellite loci and performed flow cytometry analyses to compare the genetic diversity and structure of this widespread invasive grass. We also used ecological niche models to estimate currently suitable habitat in these islands. We found high levels of genetic diversity and evidence for extensive admixture between genetically distinct groups of P. annua on Marion Island. In contrast, Prince Edward Island populations showed low levels of genetic diversity and no apparent admixture. Higher genetic diversity was apparent at the human entry points and around human settlements on both islands, suggesting that these areas received multiple introductions and acted as both initial introduction sites and secondary sources for invasive populations within the archipelago. Over 70 years of continuous human activity on Marion Island have led to the invasive spread of this species around human settlements and along footpaths, facilitating ongoing gene flow among geographically separated populations. In contrast, this was not the case for Prince Edward Island. The high levels of genetic variation, admixture, and habitat suitability in invasive P. annua facilitated by human actions, may increase the adaptive potential of the species, which could further enhance the species’ invasiveness.

Methods
Microsatellite-containing sequences were isolated by Ecogenics GmbH (Balgach, Switzerland). Size selected fragments from P. annua genomic DNA were enriched for nuclear microsatellite repeats using magnetic streptavidin beads and biotin-labelled tri- and tetra-mer repeat oligonucleotides. The microsatellite-enriched library was sequenced on a Roche 454 platform using the Roche GS FLX Titanium technology (Roche Diagnostics Corporation). This resulted in 861 reads containing microsatellite sequences with at least six tri- or tetra-nucleotide repeat units. Primers were designed for 24 of these loci, that showed both amplification and polymorphism. Of these, 12 loci were discarded due to amplification failure in most samples or poor electrophoretic profiles. Primers for the  remaining 12 microsatellites are shown in Table S1.

Genomic DNA was extracted from all samples using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1987) with the addition of 0.2 M sodium sulphite to the extraction and wash buffers. DNA quality and quantity was measured using a Nanodrop spectrophotometer (Infinite 200 PRO NanoQuant, Tecan Group Ltd, Männedorf, Switzerland), and all DNA samples were diluted to 10 ng/μL−1 prior to PCR amplification and stored at −80°C until further use. Amplification of the 12 retained nuclear microsatellites was performed in two multiplex PCR reactions (Table S1). All PCR reactions were carried out in 15 μL reaction volumes containing 1.5 μL template DNA (20 ng/μL, 7.5 μL KAPA2G Fast Multiplex Mix (Kapa Biosystems, Cape Town, South Africa), 1.5 μL primer mix (2μM), and 4.5 μL distilled H2O. Samples were amplified using the following PCR conditions: 3 min of denaturation at 95°C, 30 cycles of 15 sec of denaturation at 95°C, 30 sec multiplex-specific annealing (Table S1), 25 sec of elongation at 72°C, and a final extension for 10 min at 72°C. Each 96‐well PCR plate contained 93 samples plus two randomly selected technical replicates and one negative control (H2O). Gel capillary electrophoretic separation of amplified fragments was carried out at the Central Analytical Facility, Stellenbosch University (Stellenbosch, South Africa). All microsatellites were scored using GeneMarker software (version 2.6.4; SoftGenetics LLC, State College, Pennsylvania, USA) with the LIZ 500 size standard. We applied semi‐automatic genotype scoring for each allele, with visual inspection of each sample, following Dewoody, Nason, and Hipkkins (2006), to reduce scoring errors. After this, three more loci were eliminated (Poa5, Poa6 and Poa12) because of low levels of variation or band stuttering.

We evaluated data quality by testing for the rate of meiotic error, the presence of null alleles and homoplasy between isoloci. For this, we analysed genotypes under the assumption of random segregation and assigned alleles to isoloci in POLYSAT (Clark & Schreier 2010). The algorithm processDatasetAllo indicated significant positive correlations between alleles at locus Poa1. However, we ruled out the possibility of scoring errors at this locus because the positively correlated alleles did not have similar amplicon sizes (tetranucleotide motif). After scoring of genotypes, some loci (Poa 1, 3, 8, 9 and 11 in Table S1) were split into two isoloci, resulting in a final dataset of 14 loci.

Usage Notes
Missing data: -9

针对人类定居生境与纯粹原生无人生境中入侵物种的比较研究，尤其是其遗传结构相关研究，仍然较为匮乏。亚南极岛屿为这类对比环境下的入侵生物学研究提供了理想体系：它们地处极端偏远区域，保留了近乎原始的生态状态，且仅通过少量引入路径实现人类可达。本研究以爱德华王子群岛（PEI）为研究区域，针对其中的人类定居岛马里恩岛与无人生态岛爱德华王子岛，开展一年生早熟禾（Poa annua）的入侵遗传学研究。本研究通过分析核微卫星（microsatellite）位点的变异情况，并结合流式细胞术（flow cytometry），对比了这种广泛分布的入侵禾草的遗传多样性与遗传结构；同时利用生态位模型（ecological niche model）评估了两岛当前的适宜生境范围。

研究结果显示，马里恩岛的一年生早熟禾种群具有较高的遗传多样性，且存在多个遗传分化类群间的广泛基因交流（即遗传混合）；与之形成鲜明对比的是，爱德华王子岛的种群遗传多样性较低，未观察到明显的遗传混合现象。在两岛的人类入境点及定居点周边区域，均观测到更高水平的遗传多样性，这表明这些区域是多次物种引入的发生地，同时也是群岛内部入侵种群的初始引入源与次级扩散源。马里恩岛70余年持续不断的人类活动，促使该物种在定居点周边及步道沿线实现入侵扩散，推动了地理隔离种群间的持续基因交流；而爱德华王子岛并未出现此类现象。人类活动驱动下的一年生早熟禾种群所具备的高遗传变异、遗传混合特性与适宜生境条件，或可提升该物种的适应潜力，进而进一步增强其入侵能力。

研究方法
微卫星序列的分离工作由瑞士巴尔加赫的Ecogenics GmbH公司完成。本研究利用链霉亲和素磁珠与生物素标记的三核苷酸、四核苷酸重复寡核苷酸探针，从一年生早熟禾基因组DNA中筛选富集核微卫星重复序列的片段。构建的微卫星富集文库采用罗氏454平台（Roche GS FLX Titanium技术，罗氏诊断公司）进行测序，最终获得861条包含至少6个三核苷酸或四核苷酸重复单元的微卫星序列。针对其中24个兼具扩增成功与多态性的位点设计引物，最终因多数样本扩增失败或电泳图谱质量不佳，剔除12个位点，剩余12个微卫星位点的引物信息详见附表S1。

所有样本的基因组DNA均采用改良的十六烷基三甲基溴化铵（CTAB）法提取（Doyle & Doyle, 1987），并在提取液与洗涤缓冲液中添加0.2 M亚硫酸钠。DNA的质量与浓度通过Nanodrop分光光度计（Infinite 200 PRO NanoQuant，瑞士特卡恩集团有限公司，曼讷多夫）进行测定，所有DNA样本在聚合酶链式反应（PCR）扩增前均稀释至10 ng/μL，并于-80℃保存备用。针对剩余12个核微卫星位点的扩增采用两轮多重PCR反应完成（附表S1）。PCR反应体系总体积为15 μL，包含1.5 μL模板DNA（20 ng/μL）、7.5 μL KAPA2G快速多重PCR预混液（Kapa Biosystems，南非开普敦）、1.5 μL引物混合液（2μM）以及4.5 μL无菌去离子水。PCR扩增程序设置如下：95℃预变性3 min；95℃变性15 sec，对应各多重体系的特异性退火温度30 sec（附表S1），72℃延伸25 sec，共30个循环；最后72℃终延伸10 min。每块96孔PCR板包含93个样本、2个随机设置的技术重复以及1个空白对照（无菌去离子水）。扩增片段的凝胶毛细管电泳分离在南非斯泰伦博斯大学中央分析实验室完成。所有微卫星位点的基因型分型采用GeneMarker软件（版本2.6.4；SoftGenetics LLC，美国宾夕法尼亚州州学院）完成，以LIZ 500作为分子量内标。参考Dewoody、Nason与Hipkkins（2006）的方法，我们对每个等位基因采用半自动基因型评分流程，并对每个样本进行人工校验，以降低分型误差。此后，又因变异水平较低或条带拖尾现象，剔除了Poa5、Poa6与Poa12三个位点。

本研究通过检测减数分裂错误率、无效等位基因（null alleles）与同塑性（homoplasy），对数据质量进行评估。具体而言，我们在随机分离假设下分析基因型，并利用POLYSAT软件（Clark & Schreier 2010）将等位基因分配至同基因座（isoloci）。processDatasetAllo算法显示，Poa1位点的等位基因间存在显著正相关，但由于这些正相关的等位基因扩增片段长度差异明显（四核苷酸基序），因此排除了该位点存在分型误差的可能。基因型评分完成后，部分位点（附表S1中的Poa1、3、8、9与11）被拆分为两个同基因座，最终得到包含14个位点的数据集。

使用说明
缺失数据标记：-9