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).