A key evolutionary step determining osmoregulatory ability for freshwater colonisation in early life stages of fish

Colonisation of freshwater habitats by marine animals is a remarkable evolutionary event that has enriched biodiversity in freshwater ecosystems. The acquisition of tolerance to hypotonic stress during early life stages is presumed to be essential for their successful freshwater colonisation, but very little empirical evidence has been obtained to support this idea. This study aimed to comprehend the evolutionary changes in osmoregulatory mechanisms that enhance larval freshwater tolerance in amphidromous fishes, which typically spend their larval period in marine (ancestral) habitats and the rest of their life history stages in freshwater (derived) habitats. We compared the life history patterns and changes in larval survivorship and gene expression depending on salinity among three congeneric marine-originated amphidromous goby species (Gymnogobius), which had been suggested to differ in their larval dependence on freshwater habitats. An otolith microchemical analysis and laboratory-rearing experiment confirmed the presence of freshwater residents only in G. urotaenia and higher larval survivorship of this species in the freshwater condition than in the obligate amphidromous G. petschiliensis and G. opperiens. Larval whole-body transcriptome analysis revealed that G. urotaenia from both amphidromous and freshwater-resident populations exhibited the greatest differences in expression levels of several osmoregulatory genes, including aqp3, which is critical for water discharge from their body during early fish development. The present results consistently support the importance of enhanced freshwater tolerance and osmoregulatory plasticity in larval fish to establish freshwater forms, and further identify key candidate genes for larval freshwater adaptation and colonisation in the goby group.

Methods
A) Person-Period data for survival analysis.xlsx
This dataset was used for a survival time analysis to evaluate larval tolerance to freshwater in the three Gymnogobius species, one of which has freshwater resident populations. In this analysis, to compare the survivorship between the freshwater and seawater groups, Cox proportional hazard models were established for each species using the coxph function in the survival R package version 3.2-13. In addition, to directly illustrate interspecific variations in the freshwater tolerance and test whether freshwater tolerant species also exhibit high survival in seawater (i.e. exhibit physiological plasticity), the survival in each salinity group was compared among the species by establishing the Cox proportional hazard models with the explanatory variable being species.

B) Data for osmoregulatory gene expression analysis.xlsx
This dataset was used to compare the magnitude of expression changes of important osmoregulatory genes in larval fish between the Gymnogobius species with and without freshwater resident populations. The target genes for this analysis were selected by identifying differentially expressed genes (DEGs) between freshwater and seawater conditions, identifying osmoregulatory DEGs using the Gene Ontology (GO) term lists, and further extracting those which have been demonstrated to be responsible for osmoregulation in teleosts through an exhaustive review of published researches. The differences in the magnitude were tested by investigating the significance of the interaction effect between the salinity and presence of freshwater forms in the species in LMMs using the lmer function.

海洋动物对淡水生境的拓殖，是极具代表性的演化事件，极大提升了淡水生态系统的生物多样性。学界普遍认为，幼体阶段获得低渗应激耐受性，是海洋动物成功拓殖淡水的必要条件，但目前支撑这一假说的实证依据极为匮乏。本研究旨在阐明两生洄游鱼类中，提升幼体淡水耐受性的渗透调节机制演化变化；这类鱼类通常将幼体阶段置于海洋（祖先生境）中度过，其余生命阶段则栖息于淡水（衍生生境）。本研究选取3种同属且起源于海洋的两生洄游虾虎鱼（裸头虾虎属，Gymnogobius）——这些物种被报道在幼体对淡水生境的依赖程度上存在差异，通过它们比较了生活史模式、幼体存活率随盐度变化的特征，以及基因表达谱。耳石微化学分析与室内繁育实验证实，仅在尾纹裸头虾虎（G. urotaenia）中存在淡水定居种群，且该物种在淡水环境下的幼体存活率，显著高于专性两生洄游的佩氏裸头虾虎（G. petschiliensis）与奥氏裸头虾虎（G. opperiens）。全幼体转录组分析显示，无论是两生洄游种群还是淡水定居种群的尾纹裸头虾虎，其多个渗透调节基因的表达水平差异最为显著，其中包括水通道蛋白3（aqp3）——该基因在鱼类早期发育过程中对体内水分排出至关重要。本研究结果一致证实，提升幼体淡水耐受性与渗透调节可塑性，是鱼类形成淡水定居种群的关键；同时本研究还筛选出了虾虎鱼类群中，与幼体淡水适应及拓殖相关的关键候选基因。

## 方法
A) 生存分析个体时段数据集.xlsx
本数据集用于生存时间分析，以评估3种裸头虾虎属（Gymnogobius）物种的幼体淡水耐受性，其中1种存在淡水定居种群。本分析中，为比较淡水组与海水组的存活率，研究人员针对每个物种，基于生存R包（survival R package）3.2-13版本中的coxph函数，构建了Cox比例风险模型。此外，为直观展示不同物种间淡水耐受性的差异，并验证淡水耐受物种是否同样能在海水环境中保持高存活率（即具备生理可塑性），研究人员以物种作为解释变量构建Cox比例风险模型，以此比较各盐度组中不同物种的存活率。

B) 渗透调节基因表达分析数据集.xlsx
本数据集用于比较存在与不存在淡水定居种群的裸头虾虎属物种之间，幼体重要渗透调节基因的表达变化幅度。本分析的靶基因筛选流程为：首先鉴定淡水与海水环境下的差异表达基因（DEGs），再通过基因本体（GO）术语列表筛选出渗透调节相关差异表达基因，最后通过全面梳理已发表文献，提取出已被证实参与硬骨鱼类渗透调节的基因。本研究通过线性混合模型（LMMs），利用lmer函数检验盐度与物种是否存在淡水定居种群这两个因素的交互效应显著性，以此比较不同物种间基因表达变化幅度的差异。