Supplementary Data PhD Thesis

Supplementary Data associated to PhD Thesis Julia Camacho ("Adaptation along the water-depth axis in the Lake Malawi cichlid radiation: Genomic mechanisms of repeated ecological diversification"). File descriptions: CHAPTER 2: “Widespread genetic signals of visual system adaptation in deepwater cichlid fishes” This chapter has been published as: Camacho García, J. I., Malinsky, M., Joyce, D. A., Santos, M. E., Vernaz, G., Ngochera, M. J., & Svardal, H. (2025). Widespread genetic signals of visual system adaptation in deepwater cichlid fishes. Molecular Biology and Evolution, 42(7), msaf147. Supplementary Material available online: https://academic.oup.com/mbe/article/42/7/msaf147/8158605#526641506 Adapted for the thesis: Supplementary Data A1: Metadata of all samples included in this study for the analysis of eye size variation in the Lake Malawi cichlid radiation, genome-wide association and genomics analysis. Measurements of standard length (“SL”) and eye diameter (“ED”) are included for samples used in the analysis of eye size variation. Sequenced samples included in this and/or other studies have a BioSample ID associated. Supplementary Data A2: Table summarizing the number of samples per species and ecomorphological group from Supplementary Data A1 that were used in the study of eye size variation in the Lake Malawi cichlid radiation (Figure 2.1a, Supplementary Figure A1). Supplementary Data A3: Sampling and sequencing metadata of all Diplotaxodon samples used in this study, excluding RNA sequencing samples (see Supplementary Data A4). It is specified whether the samples were used in the GWAS or not (column ‘GWAS’). All samples included in this table can also be found in Supplementary Data A1. Supplementary Data A4: Metadata associated with the Diplotaxodon RNA sequencing samples used in the analysis of differential gene expression and the calculation of relative opsin expression in this study. Supplementary Data A5: Annotation of the top 0.01% outlier SNPs (N = 190) associated with relative eye size in 9 Diplotaxodon species. Includes the significance values from the likelihood ratio test, as output by GEMMA v0.98. SNPs with genome-wide significance (Bonferroni FWER < 0.05) highlighted in bold. Variants were annotated with snpEff v5.1 using the prebuilt database for the Astatotilapia calliptera reference genome fAstCal1.2 (GCA_900246225.3; GenBank assembly). The column “ENSEMBL GENE NAMES” lists gene symbols, when available, for genes with an ensemble ID. CHAPTER 3 (“The genomics of depth diversification in the benthic subradiation of Lake Malawi cichlids”) Supplementary Data B1: Samples metadata Metadata of the samples included in the study (N = 417). Species mean depth (m) and species depth groups used in the convergent score analysis (‘3.2.4 Signals of parallel evolution between clades’) are included in columns ‘mean_depth’ and ‘sp_group’, respectively. Species groups abbreviations: DD = deep-living deep benthic, SD = shallow-living deep benthic, SS = shallow-living shallow benthic, DS = deep-living shallow benthic. Supplementary Data B2: GWAS results Genome-wide single-nucleotide polymorphisms (SNP) associated with depth (705 SNPs; Bonferroni FWER < 0.05). Includes the significance values from Wald, likelihood ratio and score tests, as output by GEMMA v0.98.3. Variant annotation by snpEff v5.2.1 shown in columns ‘ANN[*].GENE’ and ‘ANN[*].EFFECT’. Note that SNPs may have more than one annotation. Supplementary Data B3: Genotype-phenotype regression results Results of binomial Generalized Linear Models for GWAS outlier SNPs with habitat depth as the predictor variable. Supplementary Data B4: Derived allele presence at GWAS outliers across radiation Overrepresentation test results for allele-sharing patterns across the Malawi cichlid radiation. This analysis tested whether the observed presence of derived alleles at GWAS outlier SNPs across clades deviated from the null expectation derived from genome-wide allele-sharing patterns. Specifically, it evaluated whether certain clade combinations contained derived alleles more often than expected by chance. The first column contains 7-bit allele-sharing patterns where each bit corresponds to the presence (1) or absence (0) of the allele in a clade. Clade order, from left to right: (1) Shallow benthic, (2) Rhamphochromis, (3) Utaka, (4) Deep benthic, (5) Diplotaxodon, (6) Astatotilapia calliptera, (7) Mbuna. Allele sharing can be read as (see also table below): 0000000: derived allele absent in all clades 1111111: derived allele present in all clades 1001000: derived allele present in shallow benthic and deep benthic 0001100: derived allele present in deep benthic and Diplotaxodon .... Position in string 1 2 3 4 5 6 7 Clade Shallow benthic Rhamphochromis Utaka Deep benthic Diplotaxodon AstCal Mbuna 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 0 0 1 0 0 0 0 0 0 1 1 0 0 CHAPTER 4 (“Genomic patterns of parallel adaptation among deepwater cichlid lineages of Lake Malawi”) Supplementary Data C1: Samples metadata Metadata of the samples included in the study (N = 608). Eye size group classification for Diplotaxodon samples (‘smalleye’, ‘bigeye’) are included in the column ‘Eye_size_group’. Supplementary Data C2: Convergence score outlier SNPs Outlier SNPs from convergence score analysis (cutoff 99.9th percentile; score > 0.18; N = 1,463 SNPs), including chromosome, position and allele frequency per clade (columns ‘Deep’, ‘Shallow’, ‘Dbigeye’, and ‘Dsmalleye’, denoting Deep benthic, Shallow benthic, Diplotaxodon ‘bigeye’ and Diplotaxodon ‘smalleye’, respectively). Per SNP, it is noted whether they fall within defined 50 kb candidate windows (see methods), whether they overlap top 5% fdM windows (candidate regions for excess allele sharing between Diplotaxodon ‘bigeye’ and Deep benthic; ‘fdM_overlap’), the number of overlapping fdM windows (‘n_fdM_win’) and max fdM (‘max_fdM’). As above, variants were annotated using snpEff v5.2.1. Supplementary Data C3: Candidate selection windows (XP-EHH) Candidate windows for divergent selection between shallow and deep benthic species, based on genome-wide cross-population extended haplotype homozygosity (XP-EHH) scores averaged across four shallow-deep benthic comparisons (see Supplementary Fig. C4). Windows with maximum absolute XP-EHH peaks exceeding the empirical 95th percentile (P < 0.05) are included. Per window, it is included: number of overlapping convergence score outliers above Q0.999 (‘n_outliers_parallel’) and Q0.9995 thresholds (‘n_outliers_parallel_str’), proportion of extreme positive XP-EHH scores (top 1% of the positive tail of the genome-wide XP-EHH distribution; ‘prop_extreme_xpehh’), mean XP-EHH across the window (‘mean_xpehh’), maximum |XP-EHH| across the window (‘maxabs_xpehh’), the number of SNPs (‘nsnps’) and the sign of the XP-EHH peak (‘sign_peak_xpehh’) where 1 is positive (putative deep benthic sweep) and -1 is negative (putative shallow benthic sweep). Supplementary Data C4: Dinvestigate results Dinvestigate 50 SNP windows with fdM values within the top positive 5% genome-wide fdM distribution (N = 2,846 windows). fdM and associated statistics (D, f_d, d_f) are shown (see https://github.com/millanek/Dsuite). Gene information (fAstCal1.2.99) was added for windows overlapping the coding sequence of a gene using pybedtools v0.10.0. Supplementary Data C5: Biological function classification candidate genes Zebrafish (Danio rerio) GO terms used for assigning candidate genes in Table 4.1 to functional categories. GO terms extracted from the AmiGO2 database and include both specific terms (‘GO term(s)’) as well as their parent terms (‘GO parent term(s)’). From these, simplified functional categories were assigned when possible (e.g., specific, non-general, informative terms were found).与朱莉娅·卡马乔（Julia Camacho）博士论文《马拉维湖慈鲷辐射类群沿水深梯度的适应：重复生态分化的基因组机制》相关的补充数据。 文件说明： 第二章：“深水慈鲷视觉系统适应的广泛遗传信号” 本章节已正式发表： Camacho García, J. I., Malinsky, M., Joyce, D. A., Santos, M. E., Vernaz, G., Ngochera, M. J., & Svardal, H. (2025). Widespread genetic signals of visual system adaptation in deepwater cichlid fishes. 分子生物学与进化（Molecular Biology and Evolution）, 42(7), msaf147. 在线补充材料：https://academic.oup.com/mbe/article/42/7/msaf147/8158605#526641506 为本论文改编的补充数据： 补充数据A1： 本研究中用于马拉维湖慈鲷辐射类群眼尺寸变异分析、全基因组关联分析及基因组学研究的所有样本的元数据。包含用于眼尺寸变异分析的样本的标准长度（"SL"）与眼径（"ED"）测量数据。本研究及/或其他研究中已测序的样本均关联有生物样本标识符（BioSample ID）。 补充数据A2： 汇总了补充数据A1中，用于马拉维湖慈鲷辐射类群眼尺寸变异研究的各物种及生态形态类群的样本数量（对应图2.1a、补充图A1）。 补充数据A3： 本研究中使用的所有双棘丽体鲷（Diplotaxodon）样本的采样与测序元数据，不含RNA测序样本（详见补充数据A4）。表格中注明了样本是否用于全基因组关联分析（Genome-Wide Association Study, GWAS，列"GWAS"）。本表格中的所有样本均可在补充数据A1中找到。 补充数据A4： 本研究中用于差异基因表达分析及视蛋白相对表达量计算的双棘丽体鲷RNA测序样本的元数据。 补充数据A5： 针对9个双棘丽体鲷物种中与相对眼尺寸相关的前0.01%极端单核苷酸多态性（Single Nucleotide Polymorphism, SNP）（共190个）的注释。包含由GEMMA v0.98输出的似然比检验显著性值。基因组水平显著性（邦费罗尼家族式错误率<0.05，Bonferroni FWER < 0.05）的SNP以粗体标注。使用基于卡氏丽体鲷（Astatotilapia calliptera）参考基因组fAstCal1.2（GCA_900246225.3；GenBank组装版本）的预建数据库，通过snpEff v5.1对变异进行注释。"ENSEMBL GENE NAMES"列列出了具有Ensembl ID的基因的基因符号（如可获取）。 第三章：“马拉维湖底栖慈鲷亚辐射类群的深度分化基因组学” 补充数据B1：样本元数据 本研究纳入的417个样本的元数据。 物种平均水深（m）及用于趋同得分分析的物种深度分组（详见3.2.4"支系间平行进化信号"）分别对应列"mean_depth"与"sp_group"。物种分组缩写：DD=深水栖居深底栖类群，SD=浅水栖居深底栖类群，SS=浅水栖居浅底栖类群，DS=深水栖居浅底栖类群。 补充数据B2：全基因组关联分析结果 与水深相关的全基因组单核苷酸多态性（Single Nucleotide Polymorphism, SNP，共705个SNP；邦费罗尼家族式错误率<0.05）。包含由GEMMA v0.98.3输出的Wald检验、似然比检验及得分检验的显著性值。通过snpEff v5.2.1完成变异注释，对应列"ANN[*].GENE"与"ANN[*].EFFECT"。请注意，单个SNP可能存在多个注释结果。 补充数据B3：基因型-表型回归分析结果 以栖息水深为预测变量的全基因组关联分析极端SNP的二项式广义线性模型分析结果。 补充数据B4：辐射类群中全基因组关联分析极端位点的衍生等位基因分布 马拉维湖慈鲷辐射类群中等位基因共享模式的过度富集检验结果。本分析旨在检验全基因组关联分析极端SNP位点上的衍生等位基因在各支系中的观测分布是否偏离基于全基因组等位基因共享模式的零假设，具体而言，本分析评估了特定支系组合携带衍生等位基因的频率是否显著高于随机预期。 第一列为7位二进制等位基因共享编码，每一位对应一个支系中等位基因的存在（1）或缺失（0）。支系顺序从左至右依次为： (1) 浅底栖类群，(2) 喙嘴慈鲷属（Rhamphochromis），(3) 乌塔卡慈鲷（Utaka），(4) 深底栖类群，(5) 双棘丽体鲷属（Diplotaxodon），(6) 卡氏丽体鲷（Astatotilapia calliptera），(7) 姆布纳慈鲷（Mbuna）。 等位基因共享模式可解读如下（详见下表）： 0000000：所有支系均无衍生等位基因 1111111：所有支系均携带衍生等位基因 1001000：浅底栖类群与深底栖类群携带衍生等位基因 0001100：深底栖类群与双棘丽体鲷属携带衍生等位基因 …… 字符串位置 1 2 3 4 5 6 7 支系 浅底栖类群 喙嘴慈鲷属 乌塔卡慈鲷 深底栖类群 双棘丽体鲷属 卡氏丽体鲷 姆布纳慈鲷 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 0 0 1 0 0 0 0 0 0 1 1 0 0 第四章：“马拉维湖深水慈鲷支系间平行适应的基因组模式” 补充数据C1：样本元数据 本研究纳入的608个样本的元数据。双棘丽体鲷样本的眼尺寸分组分类（"smalleye"=小眼型，"bigeye"=大眼型）对应列"Eye_size_group"。 补充数据C2：趋同得分分析极端SNP 趋同得分分析得到的极端SNP（筛选阈值为第99.9百分位；得分>0.18；共1463个SNP），包含每条染色体的位置及各支系的等位基因频率（列"Deep""Shallow""Dbigeye""Dsmalleye"，分别对应深底栖类群、浅底栖类群、双棘丽体鲷"大眼型"及双棘丽体鲷"小眼型"）。对于每个SNP，标注其是否落在预设的50kb候选窗口内（详见方法）、是否与前5%fdM窗口重叠（fdM_overlap，指双棘丽体鲷"大眼型"与深底栖类群间等位基因共享过剩的候选区域）、重叠的fdM窗口数量（n_fdM_win）及最大fdM值（max_fdM）。 与前文一致，本部分变异通过snpEff v5.2.1完成注释。 补充数据C3：候选选择窗口（XP-EHH） 基于四项浅-深底栖类群比较的基因组水平交叉群体扩展单倍型纯合性（Cross-Population Extended Haplotype Homozygosity, XP-EHH）平均分得到的浅底栖与深底栖类群间的趋异选择候选窗口（详见补充图C4）。纳入最大绝对XP-EHH峰值超过经验第95百分位（P<0.05）的窗口。 每个窗口包含以下信息：落在Q0.999和Q0.9995阈值以上的趋同得分极端SNP数量（n_outliers_parallel和n_outliers_parallel_str）、极端正向XP-EHH得分的比例（即基因组水平XP-EHH分布正向尾部前1%的得分，prop_extreme_xpehh）、窗口内平均XP-EHH值（mean_xpehh）、窗口内最大绝对XP-EHH值（maxabs_xpehh）、窗口内单核苷酸多态性数量（nsnps）及XP-EHH峰值符号（sign_peak_xpehh，1为正向，对应推定的深底栖类群选择性清除；-1为负向，对应推定的浅底栖类群选择性清除）。 补充数据C4：Dinvestigate分析结果 包含2846个fdM值处于基因组水平正向fdM分布前5%范围内的50个SNP窗口的Dinvestigate分析结果。展示fdM及相关统计量（D、f_d、d_f）（详见https://github.com/millanek/Dsuite）。 使用pybedtools v0.10.0为与卡氏丽体鲷参考基因组fAstCal1.2.99的基因编码序列重叠的窗口添加基因注释信息。 补充数据C5：候选基因的生物学功能分类 用于将表4.1中的候选基因归类至功能类别的斑马鱼（Danio rerio）基因本体（Gene Ontology, GO）术语。GO术语从AmiGO2数据库中提取，包含特异性术语（"GO term(s)"）及其父级术语（"GO parent term(s)"）。在此基础上，尽可能将候选基因归类至简化的功能类别（例如，优先选择特异性、非泛化且具有信息价值的术语）。