Genome skimming for improved phylogenetics of Taiwanese phasmids (Insecta: Phasmatodea)

Abstract

Taiwan, a relatively young continental island, harbors a high proportion of endemic phasmids, reflecting its unique evolutionary history. However, a comprehensive phylogenetic framework to clarify these phasmids is still lacking. In this study, we sequenced ten of eleven valid genera and two undescribed species of Taiwanese phasmids (total 16 species) using the genome-skimming approach. We also integrated these sequences with public databases to create two aligned datasets: one comprising 92 taxa (mitogenomes) and the other 606 taxa (seven nuclear and mitochondrial genes), enabling us to examine their phylogenetic relationships using longer sequences and more samples. Our analyses show that Taiwanese phasmids should be categorized into six families, with a revised number of genera to 13. Furthermore, four species require taxonomic treatments: namely Micadina honei (Günther, 1940) comb. nov., Micadina truncatum (Shiraki, 1935) comb. nov., Otraleus okunii (Shiraki, 1935) comb. nov., and Ramulus granulatus (Shiraki, 1935) syn. nov. now recognized as Ramulus artemis (Westwood, 1859). While some Taiwanese genera exhibit polyphyletic relationships, our findings highlight the importance of taxon sampling, particularly for type species in resolving these systematic issues. The genomeskimming approach has proven to be an excellent method for producing comparable sequence datasets, facilitating the investigation of highly diverse insects, even when samples are old, small, or have highly fragmented DNAs.

Methodology and Implementation Steps

本研究共採集 16 種台灣竹節蟲，包括已知 10 屬14 種與 2 個未確認物種，DNA 萃取後碎裂成 200–500 bp 的片段後，使用 Illumina NovaSeq 6000 進行次世代定序 (NGS)。NGS 序列使用CLC genomic workbench 22 組裝出完整的粒腺體基因體與 18S、28S、H3 核基因。結合 NCBI 已公開之序列資料庫，產生包含606樣本，7 個基因的序列矩陣。使用 IQ-tree 2.1.3 與 Mrbayes 3.2.7a 建構親緣關係樹，並依據親緣關係樹的結果與形態特徵歸納並提出合適的分類修訂。

Innovation and Cross-Disciplinary Collaboration

本研究是首個針對台灣竹節蟲的完整親緣關係分析，並與國立自然科學博物館合作獲得較為稀有或保育類竹節蟲之樣本。自台灣的竹節蟲研究由素木得一於二十世紀初期的餘百年之後，首次提出了基於分子生物學的證據，相比起僅基於傳統形態分類研究更全面的系統分類與親緣演化框架。此外本研究亦揭示了台灣整體的竹節蟲向具有複雜的生物地理學背景，為東亞地區島嶼生物地理與後冰期物種擴散研究提供可比較的材料與方法參考。

Expected Results and Contributions

1. 本研究完成台灣竹節蟲絕大多數屬與物種的 DNA 定序，此資料集成為目前台灣與鄰近區域竹節蟲最完整的分子親緣資料庫，為全球竹節蟲目研究提供關鍵資料。
2. 明確釐清台灣竹節蟲的分類地位與演化關係，證實台灣竹節蟲應分屬 6 個科、13 個屬，並針對多系群進行分類修訂。
3. 本研究示範淺層基因體定序法在分類與保育上的應用潛力，本研究成功從博物館舊標本與降解樣本中恢復完整基因序列。對其他稀有昆蟲、保育物種、無法野外再採樣的樣本具有高度參考價值。