迷樣甲殼類Y幼體的系統分類與多樣性(帶甲下綱)

Abstract

神秘的甲殼類「Y幼體」（帶甲亞綱；Facetotecta）一直是一個難以理解的無脊椎動物類別，現時學界對其所知甚少。該類別的成體為何、生殖週期、物種多樣性、以及演化史等多方面，均屬未知的領域。現時本亞綱只包括17個物種，而公開可用的基因序列少於30筆，且假定於未知的宿主體內作相當特化的寄生。由此可見，其生物學和生命週期有待更深入研究。本研究利用形態學及分子生物學證據進行供迄今為止對Y幼體最全面的調查。在簡要的引言（第 1 章）之後，我們回顧了過去一百多年的文獻，並敘述已知的生命週期階段（第 2 章）。進階的顯微鏡映像顯示，最終的幼體階段的形態源自自游泳能力優秀的構造演化而來。該階段的幼體具角質的鉤及刺，假定用於附著，但體節（segments）的分化、功能上的腸道（functional gut）、附肢、眼睛，且更重要的卵母及精子細胞，仍然缺如。與其他寄生藤壺的生命週期進行比較，本研究確定Y幼體為寄生物種。我們揭示Y幼體分布於全球海域，自海面至5600米水深均有採獲，且可能在產地大量出現，且多樣性相當豐富，結果闡釋了該類別於海洋生態系統中扮演重要卻完全被忽略的角色。現時Y幼體的可用分子數據非常缺乏。截至目前為止，僅有兩個物種進行過較保守核基因的定序，而大多數其他的分子生物數據均是採自未存證的（unvouchered）標本的「幽靈」序列。故此，重新建構作業流程，以提高幼體以形態和分子證據的鑑定比率，為當務之急。於第 3 章中，我們開發並提供全新的標準流程，當中先以視頻記錄幼體發育過程直至最後一個浮游階段結束，並在提取基因後保留幼體的蛻皮作憑證，務求確保日後可以新設計的定序引子（primer）對數千個於核醣體（ribosomal）和蛋白質編碼（protein-coding）基因的核苷酸（nucleotides）進行定序作業。

論文的第 4 章邁出了釐清帶甲亞綱演化（建構「生命之樹」）的第一步。從首次採到的單個標本提取的多個基因座（locus）的序列，得出的親緣關係結果，顯示五個主要的演化分枝，當中包括至少48個形態相異的型態，而採自日本及台灣四個港口的標本中，更鑑別出多達一百個「物種」（分子操作分類單位；Operative Taxonomic Unit）。這些尚未釐清的隱存種（cryptic species）在結構和系統發育上相當多樣，於當前的分類系統中同歸一屬，顯示該類群的系統複雜性被嚴重低估。我們的數據表明，在主要演化過程中，攝食（浮游生物）性的無節幼體（nauplius）很可能是祖先型的特徵，而於不同分枝出現的卵黃攝食食性（lecithotrophy），則獨立出現，是趨同演化的結果。由於前者可容讓浮游幼體作遠距離漂流，結果亦初步顯示廣泛的幼體分布在物種的適應輻射及多樣化的過程中起關鍵作用。全球的Y幼體多樣性仍是未知數。於第 5 章中，以 PacBio long-read iso-form、Illumina amplicon sequencing 及 Sanger sequencing 定序方法，從採獲的標本中，本研究的結果闡明帶甲亞綱未知的的多樣性。此外，透過檢視公開的環境基因高通量分子條碼（eDNA metabarcoding）數據，結果顯示帶甲亞綱的多樣性擴展到135至155個潛在「物種」，其中至少有108個於瀨底島（Sesoko Island）珊瑚礁系統周圍採獲，因而推測Y幼體於當地生態系統可能發揮的角色。最後，本研究以全基因組序列數據（genome-wide sequence data）及一系列泛甲殼動物（Pancrustacean）基因組，闡明帶甲亞綱於藤壺分類系統中的位置。以超過59,000個氨基酸位點推估的系統發生樹顯示高節點支持值（nodal support），表明帶甲亞綱是了蔓足類（藤壺）早期演化的姐妹分類群（sister group），且結果亦與現時認知的泛甲殼類演化吻合。由此可見，Y幼體與個別藤壺類別相似的生命週期與寄生的生活方式，是趨同演化的結果。

於第 6 和第 7 章，我們將這些新理解引申至更廣的層面，並評估藤壺入侵宿主的演化機制。第 6 章假定鯨魚藤壺具獨特的依附結構，以應對極端的生活方式。考慮鯨魚藤壺與分布於岩岸的藤壺的幼體，在體節及剛毛等型態上相當一致，因此推翻上述假定，進而支持藤壺的介蟲形幼體（crypid larva）能有效適應多變的生存環境，可被理解為型態上的「高原」（adaptive plateau）。上述觀點有助於解釋藤壺相當廣泛的生態地位。

於第 7 章，我們以珊瑚藤壺考量同樣的問題。對13種珊瑚藤壺入侵宿主以錄像視頻作觀察，結論指出兩個親緣關係相距甚遠的演化分枝至少於兩億年前經已分化，而幼體第一觸角呈獨特的矛形，與貫穿宿主上皮的機制相關。以共生變態發育成成體，本研究以祖先狀態模擬（ancestral state modelling）表明，幼體的表型和行為亦是趨同進化的結果。透過獨立的演化歷程，藤壺能適應堅硬底質，進而入侵並寄生於不同的活體宿主。

本研究為鮮為人知的帶甲亞綱提供了新的見解，而提供的數據不僅為日後發現Y幼體奠下基礎，亦為無脊椎類幼體的演化生物學提供更深入的理解。

Enigmatic crustacean y-larvae (Facetotecta) rank as some of the most elusive and poorly understood invertebrates. Their adult/reproductive stages, true biodiversity, and evolu-tionary history remains entirely unknown. With only 17 species described, less than 30 DNA sequences being publicly available, and a putatively specialized endoparasitic life-style in unknown hosts, this calls for a penetrating study on their biology and lifecycle.The present thesis leverages diverse morphological and molecular tools to provide the most comprehensive scientific inquiry on y-larvae to date. After a brief introduction (Chapter 1), I review 100+ years of literature and provide a fully-fledged biological in-vestigation of the known life cycle stages (Chapter 2). Advanced microscopy on the ultrastructure of these shows that the terminal larval instar develops from a fast-swimming larva equipped with cuticular hooks and spines, putatively assumed to be used for attachment, yet nevertheless lacks segments, a functional gut, appendages, eyes and, importantly, oocytes and sperm cells. Comparing this lifecycle to that of the parasit-ic barnacles build a compelling case for y-larvae being parasites. I reveal that y-larvae occur globally and from 0-5600m but may be locally highly abundant and extremely diverse. This emphasizes the potentially important yet completely overlooked roles the taxon plays in marine ecosystems.There is a significant dearth of available molecular data on y-larvae. Only two spe-cies are sequenced with conservative nuclear genes thus far, with most molecular rec-ords representing unvouchered “ghost” sequences. Thus, there is an urgent need to de-velop new protocols that maximizes morphological and molecular resolution on single larval specimens. In Chapter 3, I therefore develop a novel protocol in which individual larvae are video-recorded throughout development to the last planktonic stage, voucher exuviae of these are retained after DNA extraction, and thousands of nucleotides across ribosomal and protein-coding genes may be sequenced using newly designed primers.Chapter 4 then takes the first step to unearthing the Facetotecta Tree of Life. By estimating the first single-specimen, multi-locus phylogeny of the group, I show that this consists of five major clades that comprises at least 48 distinct morphotypes but as many as 100 “species” (or molecular operational taxonomic units) in four harbors in Japan and Taiwan.These putatively cryptic species are so structurally and phylogenetically diverse that the current classificatory system, in which all species are lumped into the same genus, is a severe and critical underestimation of the systematic complexity of the group. Our data suggest that feeding (planktotrophic) dispersive larvae (y-nauplii) were likely ancestrally retained throughout the evolution of major clades, and from which morphologically de-rived yolk-feeding (lecithotrophic) forms evolved independently and convergently. Be-cause the former can perform long-distance dispersal, this tentatively suggests that oce-anic larval distribution plays a key role in the radiation and diversification of the group.The global diversity of y-larvae is unknown. In Chapter 5 I take a holistic ap-proach to illuminating the dark, global diversity of Facetotecta using PacBio long-read isoform sequencing, Illumina amplicon sequencing and Sanger sequencing of vouch-ered and imaged specimens. By also mining publicly available eDNA/metabarcoding datasets, I show that the diversity extends to between 135 and 155 putative “species”. At least 108 of these occur at a single site around the Sesoko Island reef system, stirring speculation on the potential ecosystem roles y-larvae could play. Finally, I use genome-wide sequence data and a selection of Pancrustacean genomes to cast light on the recalci-trant systematic position of Facetotecta in the barnacle Tree of Life. With highest nodal support across more than 59000 aligned amino acid sites, I show that Facetotecta consti-tute an early-branching sister taxon to the cirripede barnacles, with the tree also reflect-ing the accepted taxonomy for Pancrustacea. The striking life cycle similarities of y-larvae and parasitic barnacles thus evolved convergently and likely adaptively to facili-tate a parasitic mode of life.The evolutionary dynamics of host invasion mechanisms in barnacles is poorly understood despite its wide potential for understanding the drivers of barnacle evolution and why they became one of the most ecologically successful invertebrate taxa. Chapter 6 constructs a platform from where to study comparative cyprid ultrastructure using standardized antennular descriptions. It hypothesizes that whale barnacles possess uniquely modified attachment structures to cope with their extreme lifestyles. Rejecting this hypothesis, I conclude that whale barnacle larvae surprisingly contain the same segmental and setal elements as rocky shore barnacles. This lends support for the alter-native hypothesis that barnacle cypris larvae are remarkably versatile and already at a morphologically adaptive plateau. This notion helps explain why barnacles have such wide and ecologically broad niches.In Chapter 7 I ask the same question but using coral barnacles as a model system. I use detailed video recordings to catalog the host invasion process of 13 barnacle spe-cies including species of two clades of distantly related coral barnacles that diverged 200+ mya. I show that the larval antennules of coral barnacles are uniquely spear-shaped and associated with mechanical penetration of the host epithelium. Facilitating an endosymbiotic metamorphosis to adult stages, ancestral state modelling shows that these larval phenotypes and behaviors evolved convergently. This is best explained by inde-pendent and adaptive evolution of similar host invasion mechanisms driven by switches from invading hard bottom substrates to entering live, complex hosts.This thesis provides new insights into a poorly known, enigmatic group of crusta-ceans. Our data pave the road for finally discovering the missing y-adult but also inves-tigating the evolutionary ecology of these wonderfully diverse larval forms in even greater depth.