Ancestral state reconstruction reveals extensive homoplasy in nutlet characters of Cynoglossinae (Boraginaceae, subfam. Cynoglossoideae, tribe Cynoglosseae)

Recent phylogenetic studies have challenged the traditional classification of the subtribe Cynoglossinae, which was based on nutlet characteristics. To investigate the evolution of fruit traits related to dispersal modes in this complex group, we conducted a study using scanning electron microscopy to examine 28 taxa representing all previously recognized lineages of the subtribe. Cynoglossinae displays four main types of nutlets: marginate, emarginate, flat wing, and incurved wing. Our findings reveal the arrangement of glochids and their structure, including the number of apical hooks, and their surface ornamentation is highly variable both across and within these four main types. We reconstructed the phylogenetic tree with four main clades (i.e., Clades I–IV) using Maximum likelihood analysis. In addition, we mapped four nutlet traits (i.e., nutlet margin, nutlet glochid, glochid sculpture, and glochid hooks) associated with different dispersal modes using ancestral state reconstruction analysis. The ancestor of subtribe Cynoglossinae was inferred to have marginate and glochidiate nutlets, with glochids externally provided with complex spiny tubercles and bearing at least four hooks at the tip as well as epizoochory dispersal mode. These ancestral states have been retained in Clade II (East Asian-Australian-African <i>Cynoglossum</i>-<i>Lindelofia</i> clade) and Clade III (Mediterranean-Asian <i>Cynoglossum</i>-<i>Solenanthus</i>-<i>Trachelanthus</i> clade), with several independent shifts to emarginate fruits. Transitions from marginate to wide-winged nutlets have occurred at least four independent times, facilitating wind-assisted dispersal in Clade I (<i>Microparacaryum</i> clade), Clade IV (<i>Paracaryum</i>-<i>Mattiastrum</i>-<i>Rindera</i> clade), <i>Rindera tetraspis</i>, and <i>Mattiastrum crista-galli</i> (both belonging to Clade III), where a transition from tuberculate to papillate surface has also occurred.

近期的系统发育研究对基于小坚果（nutlet）特征建立的倒提壶亚族（Cynoglossinae）传统分类系统提出了挑战。为探究该复杂类群中与传播方式相关的果实性状演化，本研究采用扫描电子显微镜（scanning electron microscopy），对该亚族此前已确认的所有演化支对应的28个分类群进行了观测。倒提壶亚族的小坚果共分为四种主要类型：具缘型、微凹型、平翅型和内弯翅型。本研究揭示了钩毛（glochids）的排列方式与结构特征，包括顶生钩的数量，且四种主要类型间及类型内部的小坚果表面纹饰均存在高度变异。本研究采用最大似然法（Maximum Likelihood）构建了包含四个主要演化支（即演化支I~IV）的系统发育树。此外，本研究通过祖先性状重建分析（ancestral state reconstruction analysis），对与不同传播方式相关的四种小坚果性状（即小坚果边缘、小坚果钩毛、钩毛纹饰及钩毛钩数）进行了性状映射。研究推断，倒提壶亚族的祖先类群具有具缘且被钩毛覆盖的小坚果，其钩毛外部具复杂的刺状瘤突，顶端至少生有4个钩，且传播方式为表皮动物传播（epizoochory）。这些祖先性状在演化支II（东亚-澳洲-非洲倒提壶属（Cynoglossum）- Lindelofia属演化支）与演化支III（地中海-亚洲倒提壶属（Cynoglossum）- Solenanthus属- Trachelanthus属演化支）中得以保留，且多个分支独立演化出微凹型果实。从具缘型到宽翅型小坚果的演化转变至少独立发生了四次，这一转变促进了演化支I（Microparacaryum属演化支）、演化支IV（Paracaryum属- Mattiastrum属- Rindera属演化支）、以及隶属于演化支III的Rindera tetraspis与Mattiastrum crista-galli类群的风媒传播；同时这些类群的小坚果表面也从瘤突状转变为乳突状。