Origin, diversity, and biogeography of Antarctic scale worms (Polychaeta: Polynoidae): a wide-scale barcoding approach

Aim: The Antarctic marine environment hosts diversified and highly endemic benthos owing to its unique geologic and climatic history. Current warming trends have increased the urgency of understanding Antarctic species history to predict how environmental changes will impact ecosystem functioning. Antarctic benthic lineages have traditionally been examined under three hypotheses: 1) high endemism and local radiation, 2) emergence of deep-sea taxa through thermohaline circulation, 3) species migrations across the Polar Front. In this study, we investigated which hypotheses best describe benthic invertebrate origins by examining Antarctic scale worms.

Location: Southern Ocean, Kerguelen archipelago, South American peninsula, Indian Ocean, New Zealand.

Taxon: Scale worm polychaetes (Polynoidae).

Methods: We amassed 670 polynoids from the Southern Ocean and neighbouring areas and performed phylogenetic reconstructions to identify lineages across geographic regions, aided by mitochondrial markers Cytochrome c oxidase subunit I (Cox1) and 16S ribosomal RNA (16S). Additionally, we produced haplotype networks at the species scale to examine genetic diversity, biogeographic separations, and past demography.

Results: The Cox1 dataset provided the most illuminating insights into the evolution of polynoids. Eunoe sp. was present at South America and Kerguelen, in favour of the latter acting as a migration crossroads. Harmothoe fuligineum, widespread around the Antarctic continent, was also present but isolated at Kerguelen, possibly resulting from historical freeze-thaw cycles. The genus Polyeunoa appears to have diversified prior to colonizing the continent, leading to the cooccurrence of at least three cryptic species around the Southern and Indian Oceans. Analyses identified that nearly all populations are presently expanding following a bottleneck event, possibly caused by habitat reduction from the last glacial episodes.

Main Conclusions: This study details the largest phylogenetic dataset assembled to date for Antarctic polynoids. These findings provide insight into past demographic events experienced by Antarctic marine benthos and identify multiple origin scenarios for contemporary polynoids. 

Methods
Polynoids were retrieved across several years aboard multiple research vessels, aided by an assortment of sampling equipment. Specimens processed within the context of this study were preserved either in 96° ethanol or at ‑80 °C. Each collected individual was identified to the lowest possible taxonomic level based on morphological characters. DNA was extracted from each specimen either by following a modified CTAB protocol (Doyle & Doyle 1987) or  shipped to the Canadian Center for DNA Barcoding (CCDB) at the University of Guelph to be processed following the CCDB automated standard protocols. PCR assays were performed using extracted DNA to amplify fragments of two mitochondrial genes, Cytochrome c oxidase subunit I (Cox1) and 16S ribosomal RNA (16S). Resulting PCR products were either submitted to Eurofins Scientific for purification and Sanger sequencing in both directions, using ABI BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems) or underwent amplification and sequencing at CCDB.

Sources: Doyle, J, Doyle, J (1987). Genomic plant DNA preparation from fresh tissue-CTAB method. Phytochem Bulletin, 19(11), 11-15.
Canadian Center for DNA Barcoding (CCDB – University of Guelph, Canada): http://ccdb.ca/resources/

**研究目的**：南极海洋环境因其独特的地质与气候历史，孕育了多样且高度特有性（endemism）的底栖生物（benthos）。当前的气候变暖趋势使得解析南极物种历史的紧迫性显著提升，以预测环境变化将如何影响生态系统功能。传统上，南极底栖类群的演化研究基于三大假说：1）高特有性与局部分化辐射；2）通过温盐环流（thermohaline circulation）出现深海类群；3）物种跨极锋（Polar Front）迁移。本研究以南极鳞沙蚕为研究对象，旨在探究哪一假说最能解释底栖无脊椎动物的起源。

**研究区域**：南大洋（Southern Ocean）、凯尔盖朗群岛（Kerguelen archipelago）、南美半岛、印度洋、新西兰。

**研究类群**：多毛纲鳞沙蚕科（Polynoidae）物种（即鳞沙蚕）。

**研究方法**：我们收集了来自南大洋及邻近海域的670份鳞沙蚕科标本，借助线粒体标记（mitochondrial markers）——细胞色素c氧化酶亚基I（Cytochrome c oxidase subunit I, Cox1）与16S核糖体RNA（16S ribosomal RNA, 16S），开展系统发育重建（phylogenetic reconstructions）以识别不同地理区域的类群谱系。此外，我们构建了物种级别的单倍型网络（haplotype networks），以分析其遗传多样性、生物地理分化及历史种群动态。

**研究结果**：Cox1数据集为鳞沙蚕科的演化提供了最具启发性的解析。真海鳞虫属未定种（Eunoe sp.）在南美洲与凯尔盖朗群岛均有分布，表明凯尔盖朗群岛可能是物种迁移的交汇点。广泛分布于南极大陆周边的褐毛鳞虫（Harmothoe fuligineum）也在凯尔盖朗群岛被发现，但该种群呈现孤立状态，这可能与历史上的冻融循环事件有关。多柔鳞虫属（Polyeunoa）似乎在殖民南极大陆前就已发生分化辐射，进而在南大洋与印度洋周边区域共存了至少3个隐存种（cryptic species）。分析结果显示，几乎所有种群在经历一次种群瓶颈事件（bottleneck event）后均呈现扩张趋势，该瓶颈事件可能由末次冰期导致的栖息地缩减所引发。

**主要结论**：本研究构建了目前为止针对南极鳞沙蚕科物种的最大规模系统发育数据集。本研究结果揭示了南极海洋底栖生物所经历的历史种群动态事件，并明确了现生鳞沙蚕科物种的多种起源场景。

**样本采集与实验流程**：本研究历时多年，依托多艘科研考察船及多种采样设备收集鳞沙蚕科标本。所处理的标本分别保存于96%乙醇或-80℃环境中。每一份采集到的标本均基于形态特征鉴定至最低可分类阶元。DNA提取可采用改良CTAB法（CTAB protocol）（Doyle & Doyle 1987），或送至加拿大圭尔夫大学加拿大DNA条码中心（Canadian Center for DNA Barcoding, CCDB——加拿大圭尔夫大学），按照CCDB的自动化标准流程进行处理。采用提取的DNA进行聚合酶链式反应（PCR assays）扩增，以获取两个线粒体基因的片段：Cox1与16S。扩增得到的PCR产物可送至Eurofins Scientific公司进行纯化，并采用ABI BigDye®终止剂v3.1循环测序试剂盒（Applied Biosystems）进行双向桑格测序（Sanger sequencing），或在CCDB完成扩增与测序步骤。

**参考文献与资源**：
1. Doyle J, Doyle J (1987). 新鲜组织基因组DNA提取的CTAB法. 《植物化学通报》, 19(11): 11-15.
2. 加拿大DNA条码中心（CCDB——加拿大圭尔夫大学）：http://ccdb.ca/resources/