Mitochondrial heteroplasmy as a systematic bias in molecular species delimitation and barcoding

Molecular species delimitation is commonly performed by extrapolating differentiation patterns from small regions to the entire genome. Here we compare different data sources (mitochondrial COI sequences and nuclear and mitochondrial SNPs) for the marine bivalve species complex Aequiyoldia eightsii in South America and Antarctica. Whilst all data suggest that populations on either side of the Drake Passage belong to different species, the picture is less clear within Antarctic populations which harbor three distinct mitochondrial lineages (p-dist ˜ 6 %) that coexist in populations as well as in a subset of individuals. Haplotype-specific amplification and mitochondrial SNPs data demonstrate that two haplotypes occur in heteroplasmy in some individuals (h1h3 and h2h3). Standard barcoding procedures using universal primers lead to amplification bias favoring either haplotype unpredictably and thus overestimate the species richness with high confidence. In nuclear SNPs, no differentiation akin to the trans-Drake comparison can be observed inside the Southern Ocean, suggesting that 1) the Antarctic populations represent a single species and 2) their mitochondrial haplotypes h1, h2, h3 evolved during periods of temporary allopatry and survived as non-recombining mitochondrial haplotypes whereas recombination eroded similar differentiation patterns in the nuclear genome after secondary contact. Mitochondrial heteroplasmy in combination with amplification bias are possibly more common problems in molecular barcoding but remain undetected because evidence for it is eliminated during quality checking sequencing results. As a countermeasure, an active search in electropherograms for secondary peaks and reads with alternative bases in Illumina data as well as haplotype-specific primers for amplification are recommended.

分子物种界定（molecular species delimitation）通常通过将小范围区域的分化模式外推至全基因组来完成。本研究以南美与南极海域的海洋双壳类物种复合群埃氏奇锦蛤（Aequiyoldia eightsii）为研究对象，比较了不同数据来源的分析结果，包括线粒体COI序列以及核基因组与线粒体基因组单核苷酸多态性（SNPs）。
尽管所有数据均显示，德雷克海峡两侧的种群分属不同物种，但南极海域种群的情况则相对模糊：该种群存在3个不同的线粒体谱系，谱系间的平均遗传距离（p-dist）约为6%，这些谱系不仅共存于整个种群中，也存在于部分个体内。单倍型特异性扩增与线粒体SNPs数据表明，部分个体的线粒体中存在两种单倍型的异质性现象（h1与h3、h2与h3）。
采用通用引物的标准DNA条形码鉴定流程会引发不可预测的扩增偏倚，随机偏向扩增某一类单倍型，进而以较高置信度高估物种丰富度。针对核基因组SNPs的分析则显示，南大洋内部未出现类似跨德雷克海峡种群间的分化模式，这表明：1）南极海域种群属于单一物种；2）其线粒体单倍型h1、h2、h3是在短暂的地理隔离时期演化形成，并以不发生重组的线粒体单倍型形式保留下来，而次级接触后的重组作用已清除了核基因组中类似的分化信号。
线粒体异质性与扩增偏倚的组合效应，可能是DNA条形码鉴定中更为普遍的问题，但这类现象往往未被检出——因为测序结果的质量校验流程会清除相关证据。对此，我们建议采取两项应对措施：一是在电泳图谱中主动搜寻次要峰，以及在Illumina测序数据中查找携带变异碱基的读段；二是使用单倍型特异性引物进行扩增。