Epigenetic and genetic population structure is coupled in a marine invertebrate
Katherine Silliman, Laura H Spencer, Steven B Roberts

1. General O. lurida methylation characteristics

Supplemental Figure 1: Frequency distribution of % methylation in O. lurida prior to filtering (left) and after filtering for loci with at minimum 5x coverage (right).

Feature % of all CpGs in genome % of all methylated loci Ratio of % meth:%CpG
Exon 3.99% 14.71% 3.69
Intron 13.94% 19.76% 1.42
5’ flanking region (-2kb) 3.91% 4.64% 1.19
3’ flanking region (+2kb) 3.92% 4.66% 1.19
Transposable Elements 14.93% 13.83% 0.93
Unknown genome regions 56.01% 32.25% 0.58

Supplemental Figure 2: Top: Table showing the % of all CpG loci and methylated loci that overlap with each genomic feature. Bottom: A correlation plot, showing residuals from chi-squared tests of homogeneity on the distribution of methylated loci and CpG loci that intersect with features. Blue=positive associations, red=negative associations, and size of circles represents the absolute value of each correlation coefficient.

Supplemental Figure 3: Gene body methylation patterns. A) relative location of methylation within gene bodies in Ostrea lurida. For this analysis methylated loci were designated as those with >50% methylation when averaged across all MBD18 samples, B) percent methylation across a gene body averaged across all MBD18 samples, and C) variation in percent methylation across a gene body, across all MBD18 samples.

Supplemental Table 1: Biological functions that are enriched in the Ostrea lurida genome. Also available in .csv format, methylated-loci-enriched-BP.csv

GO Term Biological Process PValue FDR GO slim
GO:0007067 mitotic nuclear division 0 0.37 cell cycle and proliferation
GO:0007049 cell cycle 0 0.66 cell cycle and proliferation
GO:0051321 meiotic cell cycle 0.05 1 cell cycle and proliferation
GO:0000082 G1/S transition of mitotic cell cycle 0.07 1 cell cycle and proliferation
GO:0000086 G2/M transition of mitotic cell cycle 0.09 1 cell cycle and proliferation
GO:0007067 mitotic nuclear division 0 0.37 cell organization and biogenesis
GO:0016569 covalent chromatin modification 0.01 1 cell organization and biogenesis
GO:0060271 cilium morphogenesis 0.01 1 cell organization and biogenesis
GO:0042384 cilium assembly 0.01 1 cell organization and biogenesis
GO:0007030 Golgi organization 0.02 1 cell organization and biogenesis
GO:0030030 cell projection organization 0.05 1 cell organization and biogenesis
GO:0007005 mitochondrion organization 0.05 1 cell organization and biogenesis
GO:0001822 kidney development 0.02 1 developmental processes
GO:0001843 neural tube closure 0.07 1 developmental processes
GO:0006281 DNA repair 0 0.08 DNA metabolism
GO:0006260 DNA replication 0 0.37 DNA metabolism
GO:0006310 DNA recombination 0.04 1 DNA metabolism
GO:0006302 double-strand break repair 0.08 1 DNA metabolism
GO:0006284 base-excision repair 0.09 1 DNA metabolism
GO:0051301 cell division 0 0.12 other biological processes
GO:0007018 microtubule-based movement 0.02 1 other biological processes
GO:0042254 ribosome biogenesis 0.05 1 other biological processes
GO:0016032 viral process 0.05 1 other biological processes
GO:0043547 positive regulation of GTPase activity 0.05 1 other biological processes
GO:0000910 cytokinesis 0.06 1 other biological processes
GO:0032092 positive regulation of protein binding 0.08 1 other biological processes
GO:0008104 protein localization 0.09 1 other biological processes
GO:0031047 gene silencing by RNA 0.01 1 other metabolic processes
GO:0006511 ubiquitin-dependent protein catabolic process 0 0.12 protein metabolism
GO:0006412 translation 0 0.37 protein metabolism
GO:0050821 protein stabilization 0.01 1 protein metabolism
GO:0006468 protein phosphorylation 0.01 1 protein metabolism
GO:0006413 translational initiation 0.03 1 protein metabolism
GO:0000209 protein polyubiquitination 0.06 1 protein metabolism
GO:0042787 protein ubiquitination involved in ubiquitin-dependent protein catabolic process 0.08 1 protein metabolism
GO:0016567 protein ubiquitination 0.09 1 protein metabolism
GO:0000398 mRNA splicing, via spliceosome 0 0.53 RNA metabolism
GO:0006397 mRNA processing 0 1 RNA metabolism
GO:0006364 rRNA processing 0.01 1 RNA metabolism
GO:0006396 RNA processing 0.01 1 RNA metabolism
GO:0008380 RNA splicing 0.02 1 RNA metabolism
GO:0006366 transcription from RNA polymerase II promoter 0.05 1 RNA metabolism
GO:0008033 tRNA processing 0.06 1 RNA metabolism
GO:0006355 regulation of transcription, DNA-templated 0.09 1 RNA metabolism
GO:0006974 cellular response to DNA damage stimulus 0 0.05 stress response
GO:0006281 DNA repair 0 0.08 stress response
GO:0006302 double-strand break repair 0.08 1 stress response
GO:0006284 base-excision repair 0.09 1 stress response
GO:0015031 protein transport 0 0.23 transport
GO:0006886 intracellular protein transport 0 0.81 transport
GO:0006888 ER to Golgi vesicle-mediated transport 0 0.96 transport
GO:0016192 vesicle-mediated transport 0.02 1 transport
GO:0006406 mRNA export from nucleus 0.04 1 transport
GO:0006606 protein import into nucleus 0.09 1 transport
GO:0042147 retrograde transport, endosome to Golgi 0.1 1 transport
GO:0098609 cell-cell adhesion 0.04 1 NA
GO:0070936 protein K48-linked ubiquitination 0.07 1 NA
GO:0003341 cilium movement 0.07 1 NA

2. Differential methylation analysis among populations

Supplemental Figure 4*: Heat map of loci that are differentially methylated (DMLs) between the two populations.

Supplemental Figure 5: PCA of methylation data using DMLs only

Supplemental Figure 6: The % difference between each population’s mean methylation for differentially methylated loci, which highlights that both populations have a similar number of hyper/hypo-methylated loci. Loci are sorted by % methylation difference.

Supplemental Table 2: GO terms and GO Slim terms of biological functions enriched in DMGs & DMLs. EASE scores shown. FDR for all terms is 1.0. Also available in .csv format, DMG-DML-EnrichedBP.csv

Go Slim Term Function DMG DML
cell adhesion GO:0007155 cell adhesion 0.012
cell adhesion GO:0007156 homophilic cell adhesion via plasma membrane adhesion molecules 0.026 0.00091
cell adhesion GO:0016339 calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules 0.083
cell organization and biogenesis GO:0007015 actin filament organization 0.014
cell organization and biogenesis GO:0048675 axon extension 0.047
cell organization and biogenesis GO:0007411 axon guidance 0.063
cell organization and biogenesis GO:0000904 cell morphogenesis involved in differentiation 0.095
cell organization and biogenesis GO:0030198 extracellular matrix organization 0.033
cell organization and biogenesis GO:0032836 glomerular basement membrane development 0.046
cell organization and biogenesis GO:0007040 lysosome organization 0.047
cell organization and biogenesis GO:0031023 microtubule organizing center organization 0.095
cell organization and biogenesis GO:0051259 protein oligomerization 0.098
cell organization and biogenesis GO:0048841 regulation of axon extension involved in axon guidance 0.033
cell organization and biogenesis GO:0045214 sarcomere organization 0.069 0.019
cell organization and biogenesis GO:0055003 cardiac myofibril assembly 0.082
cell organization and biogenesis GO:0032438 melanosome organization 0.021
cell organization and biogenesis GO:0045332 phospholipid translocation 0.0058
death GO:0008625 extrinsic apoptotic signaling pathway via death domain receptors 0.095
developmental processes GO:0048675 axon extension 0.047
developmental processes GO:0007411 axon guidance 0.063
developmental processes GO:0032836 glomerular basement membrane development 0.046
developmental processes GO:0048841 regulation of axon extension involved in axon guidance 0.033
developmental processes GO:0045214 sarcomere organization 0.069 0.019
developmental processes GO:0007423 sensory organ development 0.095
developmental processes GO:0055003 cardiac myofibril assembly 0.082
developmental processes GO:0060429 epithelium development 0.071
developmental processes GO:0040027 negative regulation of vulval development 0.082
developmental processes GO:0021942 radial glia guided migration of Purkinje cell 0.082
developmental processes GO:0050767 regulation of neurogenesis 0.0058
developmental processes GO:0060438 trachea development 0.082
developmental processes GO:0001570 vasculogenesis 0.047
other biological processes GO:0016477 cell migration 0.029 0.032
other biological processes GO:0007281 germ cell development 0.048
other biological processes GO:0019915 lipid storage 0.043
other biological processes GO:0048477 oogenesis 0.046
other biological processes GO:0040008 regulation of growth 0.032
other biological processes GO:0042254 ribosome biogenesis 0.092
other biological processes GO:0019233 sensory perception of pain 0.016
other biological processes GO:0006879 cellular iron ion homeostasis 0.047
other biological processes GO:0050982 detection of mechanical stimulus 0.082
other biological processes GO:0050801 ion homeostasis 0.082
other biological processes GO:0007017 microtubule-based process 0.082
other biological processes GO:0032465 regulation of cytokinesis 0.057
other biological processes GO:0006941 striated muscle contraction 0.082
other metabolic processes GO:0042157 lipoprotein metabolic process 0.095
other metabolic processes GO:0010508 positive regulation of autophagy 0.063
other metabolic processes GO:0010923 negative regulation of phosphatase activity 0.028
protein metabolism GO:0031398 positive regulation of protein ubiquitition 0.098 0.093
protein metabolism GO:0016567 protein ubiquitition 0.0033
protein metabolism GO:0042787 protein ubiquitition involved in ubiquitin-dependent protein catabolic process 0.003
signal transduction GO:0046426 negative regulation of JAK-STAT cascade 0.071
stress response GO:0042594 response to starvation 0.03
transport GO:0006895 Golgi to endosome transport 0.043 0.019
transport GO:0034220 ion transmembrane transport 0.086 0.00049
transport GO:0008333 endosome to lysosome transport 0.083
transport GO:0045332 phospholipid translocation 0.0058
NA GO:0044331 cell-cell adhesion mediated by cadherin 0.033 0.071
NA GO:0072015 glomerular visceral epithelial cell development 0.095
NA GO:0086010 membrane depolarization during action potential 0.095
NA GO:1903955 positive regulation of protein targeting to mitochondrion 0.0082
NA GO:0038061 NIK/NF-kappaB sigling 0.071
NA GO:0090175 regulation of establishment of plar polarity 0.082

3. Genetic Structure

# Add admixture figure here

Supplemental Figure 7: Admixture plot for 114 individuals at K=2 (determined to be the best K using the Evanno method), based on 3,724 SNPs.

4. Epigenetic and genetic comparision

# add "DMLs (PC1) vs filtered SNPs (PC1)" figure here 
# add "Pst vs Fst, 2kbslop all genes w/ meth data, 237 genes" figure here
# combine into multiplot and display

Supplemental Figure 8: Correlation of genetic PC1 vs PC1 of DMLs: Pearson’s r = 0.896 p-value < 1E10-9 and Spearman’s ρ = 0.81, p-value < 1E10-4).

5. mQTL analysis

Supplemental Figure 9: Comparison of mQTL results that intersect with the following genomic features: exon, intron, promoter region (within 2kb of the 5’ end of a gene), gene region (genes plus 2kb upstream and downstream), transposable element, and unknown region of genome. A) SNPs designated as either local (red, within 50kb) or distant (orange) mQTLs, compared with the background (blue) of all SNPs used in the mQTL analysis. B) CpGs associated with either local (red) or distant (orange) mQTLs, compared to the background (blue) of all CpGs used in the mQTL