Nuanced qualitative trait approaches reveal environmental filtering and phylogenetic constraints on lichen communities

We propose that a qualitative trait approach based on more detailed nuanced traits may reveal previously overlooked patterns, especially when combined with phylogenetic perspectives. By sampling epiphytic lichens and using a functional approach based on nuanced qualitative traits, such as a much greater resolution over photobiont identity, type of cortex and chemical compounds, we evaluated the effects of environmental filtering and phylogenetic constraints on community assembly along natural succession of Atlantic rainforest. We found changes in taxonomic, functional and phylogenetic composition, structure and diversity. Functional traits such as photobiont genera, type of cortex, reproductive structures, propagule size and protection strategies showed strong responses to succession. Mature forests with a closed canopy impose strong environment filtering that is reflected in lichen species turnover, limiting diversity, but also holding different functional and phylogenetic composition. The use of a nuanced qualitative trait approach may overcome some of the limitations of using this type of traits and show the importance of often overlooked key lichen functional traits, including presence of carbon concentrating mechanisms in photobionts and cortex properties. Furthermore, this is the first study showing how patterns of phylogenetic assembly along forest succession structure lichen communities.

Methods
This study was carried out in forest fragments in the river basin Maquiné, between 51o21’-50o05’W and 29o20’-29o50’S, northeastern Rio Grande do Sul state, southern Brazil, where the climate is classified as subtropical humid (Moreno 1961). The annual mean temperature varies from 13ºC to 22ºC and the annual precipitation is around 1400 and 1800 mm (Hasenack and Ferraro 1989). The original vegetation is composed of Atlantic rainforests and corresponds to the southern distribution limit of one of the most important and rich tropical forests in the world (Myers et al. 2000). The demographic density in the region is only 11 inhabitants/km2 (IBGE 2010), and the main economic activity is small scale agriculture (subsistence farming), which means that we do not expect any important anthropogenic influence on lichen communities. Lichen communities were sampled in four different sites, each of them located in different valleys and each containing stands of three different stages of forest succession: initial - between 6 and 10 years of natural regeneration, intermediate - between 12 and 20 years, and late - between 40 and 60 years (Koch et al. 2013). The criteria used for selecting the areas were the existence of reliable information about the forest age and the possibility of finding all stages in a similar range of altitude and climatic conditions. We estimated stand ages by interviewing local inhabitants, only those living in the areas for at least 40 years. In each successional stage from each site two sampling units (SUs) were set (for a total of twenty-four SUs), where three randomly selected adult trees were sampled, all in a radius of 5 m. These tree individuals had CBH > 18 cm (CBH: circumference at breast height – 1.30 m from the ground), with a straight stem without branches below 1.50 m, without a smooth or peeling cortex. In total, we sampled 72 trees distributed in 24 SUs in four valley slopes, varying from 30 to 250 m of altitude.We sampled lichens on the tree stems from 30 to 150 cm from the ground, in regular intervals of 10 cm, which represented 13 height levels, using the rubber band method (Marcelli 1992). This method consists in placing a rubber band, gradually marked in percent-coverage classes from 0 to 100% of coverage around the stem. The length of the rubber bands varied according to the stem circumference i.e., the percent coverage is always relative to the tree size. Lichen species coverage per tree was the sum of the percent coverage (standardized according to the stem circumference) and the coverage of each species in the SU corresponded to the mean value of the three sampled trees.

我们提出，基于更精细入微的性状的定性性状研究方法，或许能揭示此前被忽视的模式，尤其是结合系统发育（phylogenetic）视角时。本研究通过采样附生地衣（epiphytic lichens），并采用基于精细定性性状的功能性状研究方法——相较于地衣光共生体（photobiont）身份、皮层类型以及化学成分的分类分辨率有大幅提升——评估了环境过滤作用与系统发育限制沿大西洋雨林自然演替过程对地衣群落构建的影响。

研究发现分类学、功能与系统发育的组成、结构及多样性均发生变化。光共生体属、皮层类型、繁殖结构、繁殖体大小与保护策略等功能性状，对演替过程表现出强烈响应。冠层闭合的成熟林分存在强烈的环境过滤作用，这体现在地衣物种周转上，既限制了物种多样性，也形成了独特的功能与系统发育组成。采用精细定性性状研究方法，或可克服此类性状应用中的部分局限，并凸显此前常被忽视的关键地衣功能性状的重要性，包括光共生体中的碳浓缩机制存在性以及皮层特性。此外，本研究首次揭示了森林演替过程中的系统发育组装模式如何塑造地衣群落结构。

## 研究方法
本研究于巴西南里奥格兰德州东北部的马基内河（Maquiné）流域的森林片段中开展，地理范围为西经51°21′至50°05′、南纬29°20′至29°50′，当地气候属于亚热带湿润气候（Moreno 1961）。年平均气温介于13℃至22℃之间，年降水量约为1400至1800毫米（Hasenack and Ferraro 1989）。原生植被为大西洋雨林，属于全球最重要且物种最丰富的热带森林之一的南部分布边界（Myers et al. 2000）。该区域人口密度仅为11人/km²（IBGE 2010），主要经济活动为小规模农业（自给自足型农业），因此我们认为该区域未受到显著的人为活动干扰，不会对地衣群落造成重要影响。

地衣群落采样在4个不同样点开展，每个样点位于不同的山谷，且均包含3个不同森林演替阶段的林分：初始阶段（自然更新6~10年）、中间阶段（12~20年）与晚期阶段（40~60年）（Koch et al. 2013）。样地选择标准为：具备可靠的林分年龄信息，且可在相近海拔与气候条件范围内找到全部3个演替阶段。林分年龄通过访谈当地居民确定，仅选取在该区域居住至少40年的居民提供的信息。在每个样点的每个演替阶段中，设置2个样地单元（sampling units, SUs），总计24个样地单元。每个样地单元内随机选取3株成年树木，采样范围限定在半径5米的区域内。这些树木的胸高周长（circumference at breast height, CBH）大于18厘米，即距地面1.30米处的树干周长，树干笔直且在1.5米以下无分枝，皮层不光滑或不剥落。最终，我们在4个山谷坡地的24个样地单元中共采样72株树木，海拔范围为30~250米。

我们采用橡皮筋法（Marcelli 1992），在距地面30~150厘米的树干位置采样地衣，每10厘米设置一个高度层级，共13个高度水平。该方法是将带有0~100%覆盖度百分比分级标记的橡皮筋缠绕在树干上，橡皮筋的长度根据树干周长调整，因此覆盖度百分比始终相对于树木大小进行标准化。单棵树木的地衣物种覆盖度为各物种百分比覆盖度（按树干周长标准化后）的总和，每个样地单元的物种覆盖度为该样地内3株采样树木的平均覆盖度。