Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs

Euglenids are a well-known group of single-celled eukaryotes, with
phototrophic, osmotrophic and phagotrophic members. Phagotrophs represent
most of the phylogenetic diversity of euglenids, and gave rise to the
phototrophs and osmotrophs, but their evolutionary relationships are
poorly understood. Symbiontids, in contrast, are anaerobes that are
alternatively inferred to be derived euglenids, or a separate euglenozoan
group. Most phylogenetic studies of euglenids have examined the SSU rDNA
gene only, which is often highly divergent. Also, many phagotrophic
euglenids (and symbiontids) are uncultured, restricting collection of
other molecular data. We generated transcriptome data for 28 taxa, mostly
using a single-cell approach, and conducted the first multigene
phylogenetic analyses of euglenids to include phagotrophs and symbiontids.
Euglenids are recovered as monophyletic, with symbiontids forming an
independent branch within Euglenozoa. Spirocuta, the clade of flexible
euglenids that contains both the phototrophs (Euglenophyceae) and
osmotrophs (Aphagea), is robustly resolved, with the ploeotid Olkasia as
its sister group, forming the new taxon Olkaspira. Ploeotids are
paraphyletic, although Ploeotiidae (represented by Ploeotia spp.),
Lentomonas, and Keelungia form a robust clade (new taxon Alistosa).
Petalomonadida branches robustly as sister to other euglenids in
outgroup-rooted analyses. Within Spirocuta, Euglenophyceae is a robust
clade that includes Rapaza, and Anisonemia is a well-supported
monophyletic group containing Anisonemidae (Anisonema and Dinema spp.),
‘Heteronema II’ (represented by H. vittatum), and a clade of Neometanema
plus Aphagea. Among ‘peranemid’ phagotrophs, Chasmostoma branches with
included Urceolus, and Peranema with the undescribed ‘Jenningsia II’,
while other relationships are weakly supported and consequently the
closest sister group to Euglenophyceae remains unresolved. Our results are
inconsistent with recent inferences that Entosiphon is the evolutionarily
pivotal sister either to other euglenids, or to Spirocuta. At least three
transitions between posterior and anterior flagellar gliding occurred in
euglenids, with the phylogenetic positions and directions of those
transitions remaining ambiguous.

眼虫类（Euglenids）是一类广为人知的单细胞真核生物，包含光合营养型、渗透营养型与吞噬营养型类群。吞噬营养型类群占据了眼虫类绝大多数的系统发育多样性，且演化出了光合营养型与渗透营养型类群，但其演化关系至今仍不甚明晰。与之相对的共生体虫类（Symbiontids）均为厌氧菌，过往研究要么将其视为衍生的眼虫类群，要么将其归为独立的眼虫类群（Euglenozoa）分支。以往多数眼虫类系统发育研究仅针对小亚基核糖体DNA基因（SSU rDNA）开展，而该基因往往具有极高的序列分化度。此外，诸多吞噬营养型眼虫（及共生体虫类）尚未实现纯培养，这限制了其他分子数据的获取。本研究采用单细胞实验策略，为28个分类单元构建了转录组数据，并首次开展了涵盖吞噬营养型类群与共生体虫类的眼虫类多基因系统发育分析。研究结果显示，眼虫类为单系群，共生体虫类在眼虫类群（Euglenozoa）内形成独立分支。螺旋动群（Spirocuta）是一类具有柔韧性的眼虫类演化支，包含光合营养型的裸藻纲（Euglenophyceae）与渗透营养型的无吞噬亚纲（Aphagea），该演化支得到强力支持，且以波罗虫类（Ploeotida）的Olkasia属作为其姊妹群，二者共同构成新类群Olkaspira。波罗虫类为并系群，尽管波罗虫科（Ploeotiidae，以Ploeotia spp.为代表）、Lentomonas属与Keelungia属构成了一个得到强力支持的演化支（新类群Alistosa）。Petalomonadida类群在外类群根化的系统发育分析中，作为其余眼虫类的姊妹群得到强力支持。在螺旋动群内部，裸藻纲（Euglenophyceae）为单系群，包含Rapaza属；异鞭虫类（Anisonemia）是一个得到充分支持的单系群，包含异鞭虫科（Anisonemidae，Anisonema与Dinema spp.）、“异鞭虫II类群”（以H. vittatum为代表）以及由Neometanema属与无吞噬亚纲（Aphagea）构成的演化支。在“佩兰虫类（peranemid）”的吞噬营养型眼虫中，Chasmostoma属与Urceolus属聚为一支，Peranema属与未命名的“Jenningsia II类群”聚为一支，其余类群间的亲缘关系支持度较弱，因此裸藻纲（Euglenophyceae）的最近姊妹群仍未明确。本研究结果与近期的两项推论相悖：其一认为内管虫属（Entosiphon）是其余眼虫类的演化关键姊妹群，其二则认为其是螺旋动群的关键姊妹群。眼虫类中至少发生过三次前后鞭毛滑行方式的转换，且这些转换的系统发育位置与转换方向仍有待明确。