Functional traits: Adaption of ferns in forest

Ferns evolved from 400 million years ago show various functional traits and ecological strategies in extant species, and over 80% of them belong to the youngest order Polypodiales. How the functional traits and strategies of ferns have changed during their evolutionary history remains unexplored. Here, we measured functional traits that sensitive to environmental light and water availability of 345 fern species across the fern phylogeny, and reconstructed their evolutionary histories. We found that ferns, mainly Polypodiales, have developed diversified functional traits in response to forest environments. Terrestrial species, especially Thelypteridaceae and Athyriaceae in eupolypods II, showed decreased leaf mass per area (LMA) and area-based leaf nitrogen (Narea) but increased mass-based leaf nitrogen (Nmass) than early-derived polypods since the late Jurassic. Epiphytic species, mainly those in Polypodiaceae, showed reductions in Nmass and individual leaf area (Area) since the late Cretaceous. The adaptation of functional traits of Polypodiales to forest environment may have played a crucial role in fern radiation since the late Jurassic. Integrative analysis of functional traits especially the numerical ones may shed new light on plant evolution.

Methods
 In the field, we sampled 950 sporophytes of 345 fern species identified according to the Flora of China (Lin et al., 2013), which covers 87 genera, 28 families and 9 out of 11 orders in the PPG I system (PPG I, 2016) (Table S1). For each species except a few rare ones, at least three individuals were collected as three samples; and for each individual three or more fully expanded and healthy leaves were collected. For each sampled individuals, we measured four numeric leaf functional traits, including LMA, Narea, Nmass and Area (Pérez-Harguindeguy et al., 2013), and recorded three categorical traits, including growth form, leaf venation type and leaf type. Each sample with single leaf petioles or compound leaf rachis removed was scanned using an image scanner (LiDE110, Cannon, Japan). Projected leaf area (cm2) of each sample was measured using the WinFOLIA system (Regent, Canada). Leaves were oven dried for at least 48 h at 60 °C to a constant weight, and the leaf dry mass was weighed to the nearest milligram. For each sample, LMA (g m-2) was calculated as the leaf dry mass divided by the projected area; Nmass (mg g-1) was measured using an elemental analyser (Elementar, Germany); Narea (mg m-2) was calculated as Nmass × LMA. The growth form (terrestrial, lithophyte or epiphytic), leaf type (single or compound) and leaf venation type (open, semi-reticulate or reticulate) were also recorded (Table S1). The traits data at species level were averaged from all the sampled individuals within the species accordingly (Table S2).

Table S1. Functional traits of 950 individuals of 345 fern species.

Table S2. Functional traits of 345 fern species in Fig. 2.

Table S3. Functional traits of ferns and seed plants in Fig. 3.

Data S1. Time-calibrated phylogeny of 345 fern species in Fig. 2.

Figure S1. Variation of four functional traits of ferns explained by three climatic factors and growth form.

起源于4亿年前的蕨类植物，现存类群展现出多样的功能性状与生态策略，其中超过80%的物种隶属于最年轻的水龙骨目（Polypodiales）。蕨类植物的功能性状与策略在演化历史中如何发生变化，这一问题至今尚未得到探索。本研究测定了覆盖蕨类系统发育全谱系的345个蕨类物种的、对光照与水分可利用性敏感的功能性状，并重建了它们的演化历史。研究发现，以水龙骨目为主的蕨类植物已演化出多样的功能性状以适应森林生境。自侏罗纪晚期以来，陆生类群（尤其是真水龙骨类II（eupolypods II）中的金星蕨科（Thelypteridaceae）和蹄盖蕨科（Athyriaceae））相比早期分化的水龙骨类群，呈现出更低的比叶面积（LMA, leaf mass per area）和单位叶面积氮含量（Narea, area-based leaf nitrogen），但单位叶质量氮含量（Nmass, mass-based leaf nitrogen）更高。附生类群（主要为水龙骨科（Polypodiaceae）物种）自白垩纪晚期以来，其Nmass和单叶面积（Area）均出现下降。水龙骨目功能性状对森林生境的适应，可能在侏罗纪晚期以来的蕨类辐射演化中发挥了关键作用。对功能性状尤其是量化性状的整合分析，可为植物演化研究提供新视角。

## 方法
野外采样阶段，我们依据《中国植物志》（Lin等，2013）鉴定了345个蕨类物种，共采集950份孢子体（sporophyte），这些物种涵盖PPG I系统（PPG I, 2016）中11个目里的9个目、28个科以及87个属（附表S1）。除少数稀有物种外，每个物种至少采集3个个体作为3份重复样本；每个个体采集3片及以上完全展开且健康的叶片。针对每个采样个体，我们测定了4项量化叶功能性状，包括LMA、Narea、Nmass和Area（Pérez-Harguindeguy等，2013），同时记录了3项分类性状：生长型、叶脉类型和叶型。将每份样本去除单叶叶柄（petiole）或复叶叶轴（rachis）后，使用图像扫描仪（LiDE110，Cannon，日本）进行扫描。采用WinFOLIA系统（Regent，加拿大）测定每份样本的投影叶面积（单位：cm²）。将叶片置于60℃烘箱中烘干至少48小时至恒重，随后称量叶片干重（精度至毫克）。针对每份样本，LMA（单位：g m⁻²）以叶片干重除以投影叶面积计算得到；Nmass（单位：mg g⁻¹）通过元素分析仪（Elementar，德国）测定；Narea（单位：mg m⁻²）由Nmass乘以LMA计算得到。此外，我们同时记录了生长型（陆生、石生或附生）、叶型（单叶或复叶）以及叶脉类型（开放脉、半网状脉或网状脉）（附表S1）。物种水平的性状数据由该物种所有采样个体的性状值平均得到（附表S2）。

附表S1. 345个蕨类物种的950份个体的功能性状数据。
附表S2. 图2中345个蕨类物种的功能性状数据。
附表S3. 图3中蕨类与种子植物的功能性状数据。
数据S1. 图2中345个蕨类物种的时间校准系统发育树。
图S1. 3个气候因子与生长型解释的蕨类4项功能性状变异。