The Influence of Environmental Variation on the Genetic Structure of a Poison Frog Distributed Across Continuous Amazonian Rainforest

Biogeographic barriers such as rivers have been shown to shape spatial patterns of biodiversity in the Amazon basin, yet relatively little is known about the distribution of genetic variation across continuous rainforest. Here, we characterize the genetic structure of the brilliant-thighed poison frog (Allobates femoralis) across an 880-km-long transect along the Purus-Madeira interfluve south of the Amazon river, based on 64 individuals genotyped at 7609 single-nucleotide polymorphism loci. A population tree and clustering analyses revealed 4 distinct genetic groups, one of which was strongly divergent. These genetic groups were concomitant with femoral spot coloration differences, which was intermediate within a zone of admixture between two of the groups. The location of these genetic groups did not consistently correspond to current ecological transitions between major forest types. A multimodel approach to quantify the relative influence of isolation-by-geographic distance (IBD) and isolation-by-environmental resistance (IBR) nevertheless revealed that, in addition to a strong signal of IBD, spatial genetic differentiation was explained by IBR primarily linked to dry season intensity (r2 = 8.4%) and canopy cover (r2 = 6.4%). We show significant phylogenetic divergence in the absence of obvious biogeographical barriers and that finer-scaled measures of genetic structure show patterns that are associated with environmental variables also known to predict the density of A. femoralis. Methods Extraction of DNA and single-nucleotide polymorphism (SNP) discovery was carried out at Diversity Arrays Technology sequencing Pty. Ltd. (DArTseq) facility (Canberra, Australia; more detail, please see the Supplementary Information in the article). A modified double-digest restriction-site associated DNA sequencing protocol was performed on libraries prepared using a combination of Pstl-Hpall restriction enzymes. The Pstl enzyme adaptor also contained an Illumina adaptor sequence, a primer sequence and a variable-length barcode. The Hpall adaptor contained an Illumina flow cell attachment and overhang sequence. Following enzymatic digestion, fragments were amplified and sequenced on an Illumina HiSeq2500. DNA sequences were aligned via BLAST using the Nanorana parkeri reference genome. To check for contamination, sequences were also blasted to bacterial and fungal genomes (NCBI). Usage Notes This data set was filtered for missing data using the filter_dart function of the R package RADIATOR v. 0.010. Only individuals and loci with ≥95% SNPs geno- typed were retained. SNPs were also screened for allele coverage, with any SNPs displaying a local and global minor allele frequency threshold of less than 1% removed from the dataset. In cases where multiple SNPs were found within the same read, only one locus was retained (chosen randomly per RAD tag) to avoid statistical bias from physical linkage. Two samples from M14 had <95% of loci genotyped and were removed, which resulted in 64 individuals from 13 populations genotyped at 10 275 SNPs (please see the article Table S2 for summary of filtering steps).

诸如河流类的生物地理屏障已被证实可塑造亚马逊盆地的生物多样性空间格局，但目前学界对连续热带雨林内的遗传变异分布特征仍知之甚少。本研究基于亚马逊河南部普鲁斯-马代拉河间地沿线880公里样带内64个个体的7609个单核苷酸多态性（single-nucleotide polymorphism, SNP）位点基因分型数据，解析亮股箭毒蛙（Allobates femoralis）的遗传结构。种群树与聚类分析结果显示存在4个独立的遗传类群，其中1个类群的遗传分化程度极高。这些遗传类群与个体的股斑体色差异高度对应，其中两个类群的杂交过渡区内的个体，其股斑体色呈两个类群的中间型。这些遗传类群的分布边界并未与当前主要森林类型间的生态过渡带完全吻合。本研究采用多模型方法量化地理距离隔离（isolation-by-geographic distance, IBD）与环境阻力隔离（isolation-by-environmental resistance, IBR）的相对影响，结果显示：除显著的IBD信号外，空间遗传分化还主要由与旱季强度（r²=8.4%）和冠层覆盖度（r²=6.4%）相关的IBR所解释。本研究证实，即便在无明显生物地理屏障的生境中，仍存在显著的系统发育分化；且更精细尺度的遗传结构分析结果显示，其遗传格局与已知可预测亮股箭毒蛙种群密度的环境变量显著相关。

## 实验方法
DNA提取与单核苷酸多态性发掘工作在澳大利亚堪培拉的多样性阵列技术测序（Diversity Arrays Technology sequencing, DArTseq）平台完成，详细流程请参见本文附录补充材料。本研究针对采用Pstl-Hpall限制性内切酶组合制备的测序文库，使用经改良的双酶切限制性位点相关DNA测序方案。Pstl接头同时包含Illumina接头序列、引物序列与可变长度的条形码。Hpall接头则包含Illumina测序流动槽结合序列与突出端序列。酶切完成后，对DNA片段进行扩增，并在Illumina HiSeq2500平台上完成测序。研究采用BLAST算法，以高原倭蛙（Nanorana parkeri）基因组为参考序列对DNA序列进行比对。为检测样本污染，研究还将序列比对至NCBI数据库中的细菌与真菌基因组。

## 使用说明
本数据集采用R包RADIATOR v.0.010中的filter_dart函数进行缺失数据过滤，仅保留基因分型完成率≥95%的个体与位点。同时对SNP位点进行等位基因覆盖度筛选，将本地与全局次要等位基因频率低于1%的SNP从数据集中剔除。若同一条测序读段中存在多个SNP位点，则仅保留一个位点（按RAD标签随机选取），以避免物理连锁带来的统计偏差。有2份采自M14的样本基因分型完成率低于95%，被予以剔除；最终得到来自13个种群的64个个体的10275个SNP位点分型数据（过滤流程汇总详见本文表S2）。