Fungal and algal lichen symbionts show different transcriptional expression patterns in two climate zones

In the lichen symbiosis, the fungal and algal partners constitute a closely integrated system. The combination of fungal and algal partners changes along climate gradients in many species, and is expected to be adaptive. However, the functional mechanisms behind this symbiosis-mediated environmental adaptation are unknown. We investigate which transcriptional profiles are associated with specific fungal-algal symbiont pairings found in lichens from high-elevation (Lower Supratemperate) and low-elevation (Lower Mesomediterranean) sites at two extremes of a climatic gradient on Mt. Limbara, Sardinia. Using laboratory-acclimatized thalli, we find that lichen fungal and algal symbionts show variable expression profiles between high- and low-elevation individuals: circadian- and temperature-associated genes for fungi and light-responsive genes for algae show climate-specific patterns. High- and low-elevation individuals differentially express sugar transporters in both symbionts, pointing to symmetrical and climate-dependent sugar transport mechanisms between them. A light pulse treatment identified asymmetries between fungal and algal light response, with high- and low-elevation fungal symbionts but only low-elevation algal symbionts showing a response. Together, these results tie previously-observed genomic variation along climatic gradients in a lichen species to functional differences in transcription for the fungal and algal symbionts, contributing to our understanding of environmental specialization and niche-specific partner combinations in lichens.

Methods
Lichen thalli were sampled in the field (Sardinia, Italy) and dried before transport to Frankfurt, Germany. Dried individuals were split into two pieces (LL and DD) and then rehydrated within 3 days of field collection and allowed to acclimatize over 3 days in a plant growth chamber (12h light/12h dark cycles, 60 μmol photons m-2 s-1 at 16° C).

After 3 days, samples were exposed to 24 h of total darkness before LL group was additionally exposed to 20 minutes of light (60 μmol photons m-2 s-1). All samples were then harvested into liquid nitrogen.

After tissue harvesting from lichen thalli (150 mg), RNA was extracted with TRI Reagent (Zymo Research Europe GmbH, Freiburg, DE) according to the manufacturer’s instructions. The extracted RNA was then sent to Novogene (Cambridge, UK) for library construction, quality control and 150 bp paired-end sequencing with NovaSeq. All sequence information can be found on the European Nucleotide Archive (ENA) under project accession number PRJEB72275.

在地衣共生体系中，真菌与藻类共生伙伴构成了紧密整合的生命系统。诸多地衣物种中，真菌-藻类的组合会随气候梯度发生变化，且被认为具有适应性意义。然而，这种共生介导的环境适应性背后的功能机制仍未明确。本研究聚焦于撒丁岛林巴拉山（Mt. Limbara）气候梯度两端的高海拔（下超温带Lower Supratemperate）与低海拔（下地中海亚热带Lower Mesomediterranean）生境中的地衣，探究其特定真菌-藻类共生配对所对应的转录谱（transcriptional profiles）特征。本研究利用实验室驯化的地衣体开展实验，结果显示，高海拔与低海拔地衣的真菌、藻类共生伙伴的表达谱存在显著差异：真菌的昼夜节律相关基因与温度相关基因、藻类的光响应基因均呈现出气候特异性表达模式。两类共生体在高、低海拔个体中均差异表达糖转运蛋白（sugar transporters），表明二者间存在对称且依赖于气候的糖转运机制。光脉冲（light pulse）处理实验揭示了真菌与藻类光响应的不对称性：高、低海拔的真菌共生体均产生光响应，而仅低海拔的藻类共生体出现光响应。综上，本研究将此前观测到的地衣物种沿气候梯度的基因组变异，与真菌、藻类共生体的转录功能差异关联起来，有助于加深我们对地衣的环境特化与生态位特异性共生伙伴组合的理解。

实验方法
地衣体采自野外（意大利撒丁岛），经干燥后运至德国法兰克福。干燥样本被分为LL与DD两组，共两份，在野外采样后3天内进行复水，并于植物生长箱中驯化3天（光照12h/黑暗12h循环，光照强度60 μmol光子·m⁻²·s⁻¹，温度16℃）。
驯化3天后，所有样本先经历24h全黑暗处理，随后LL组额外接受20分钟光照（60 μmol光子·m⁻²·s⁻¹）。所有样本均置于液氮中速冻收获。
称取150 mg地衣体组织，使用TRI Reagent（德国弗莱堡Zymo Research Europe GmbH）按照制造商说明书提取RNA。提取的RNA送往英国剑桥的Novogene公司进行文库构建、质量控制及NovaSeq平台150 bp双端测序。所有序列数据可在欧洲核苷酸档案馆（European Nucleotide Archive, ENA）下以项目登录号PRJEB72275获取。