馬達加斯加的地理廣布蝶種具高度的粒線體基因多樣性：DNA條碼方法的測試

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

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Molecular Phylogenetics and Evolution 50 (2009) 485–495

Marjorie C. Linares ^a, Iván D. Soto-Calderón ^a,b, David C. Lees ^c, Nicola M. Anthony ^a,*
a Department of Biological Sciences, Computer Resources Center Building, Room 200, University of New Orleans, New Orleans, LA 70148, USA
^b Biology Institute, University of Antioquia, Medellín AA1226, Colombia
^c Department of Entomology, Natural History Museum, Cromwell Rd., London SW7 5BD, UK

關於DNA條碼在種級鑑別上的適用性有許多爭議，這些討論近期愈來愈多，先前介紹過的文章主張「擴大樣本取樣之地理範圍並不會顯著降低以DNA生物條碼來鑑識物種的成功率」，然而本次介紹文章的作者則發現DNA生物條碼的原則在區分兩個截然不同的物種時也有失效的時候。

使用粒線體COI基因序列作為DNA條碼進行鑑定物種與探索的方法，已經成為一種標準流程且具有很高的產出效率。物種的界定基於下列原則：(1)在親緣關係上呈現單系群，且頻率較低 (2)物間之間的分化至少比物種之內高出10倍。然而，如果姊妹種沒有被囊括在分析當中、或者所選分類群中的任何地理範圍產生的變異沒有被全面性地取樣，分化程度的評估就會過於誇大。本文對Heteropsis屬中的一些形態上有區別的特有種蝴蝶作大範圍變異的取樣，提出DNA分化評估中潛在的偏見；本文同時比較Heteropsis族群與另一個不相關的森林相類群Saribia tepahi之間的遺傳變異，以探究生物條碼領域中、粒線體DNA可被用來評價雨林破碎化造成之影響的限度。出乎意料地，廣義的引子使得細胞內共生物Wolbachia的DNA被不被注意地增幅，暗中減損了通用引子的使用、且迫使設計具屬級專一性的COI引子成為必須之務。雖然作者觀察到高度的種內遺傳變異，大部分物種仍滿足DNA條碼原則；但是兩種形態上可區別的候選物種卻無法滿足DNA條碼的10倍遺傳距離原則。COI資料的親緣地理分析暗示森林破碎化可能在蝴蝶近期演化的多樣性上扮演重要的腳色。未來研究中同時使用粒線體DNA與核DNA的資料研究馬拉加西的類群，將對歷史上棲地破碎化對物種多樣性的影響提供更好的洞見，且可能有助於指出保育上較優先的地區。

Abstract
The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10 greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forestassociated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10x genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.