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）特征建立的琉璃草亚族（Cynoglossinae）传统分类系统提出了挑战。为探究该复杂类群中与扩散模式相关的果实性状演化，我们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共28个类群开展了研究。

琉璃草亚族的小坚果可分为四种主要类型：具棱状边缘型（marginate）、微凹缘型（emarginate）、平翅型（flat wing）与内弯翅型（incurved wing）。我们的研究结果揭示了钩毛（glochids）的排列方式与结构（包括顶端钩的数量），以及小坚果表面纹饰在这四种主要类型间及类型内均存在高度变异。

我们通过最大似然法（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，我们借助祖先性状重建（ancestral state reconstruction）分析，对与不同扩散模式相关的四种小坚果性状（即小坚果边缘、小坚果钩毛、钩毛纹饰以及钩毛顶端钩）进行了祖先状态映射。

研究推断，琉璃草亚族的祖先类群具有具棱状边缘且被钩毛覆盖的小坚果，其钩毛外部带有复杂的刺状瘤突，顶端至少生有4个钩，且采用动物体外传播（epizoochory）的扩散模式。这些祖先性状在演化支II（东亚-澳洲-非洲琉璃草属（Cynoglossum）- Lindelofia演化支）与演化支III（地中海-亚洲琉璃草属（Cynoglossum）- Solenanthus - Trachelanthus演化支）中得以保留，并有多个类群独立演化出微凹缘果实。

从具棱状边缘型小坚果向宽翅型小坚果的转变至少独立发生了4次，这一转变促进了演化支I（Microparacaryum演化支）、演化支IV（Paracaryum - Mattiastrum - Rindera演化支）、Rindera tetraspis以及Mattiastrum crista-galli（二者均隶属于演化支III）的风媒扩散；在上述类群中，同时发生了从瘤突状表面向乳突状表面的转变。