Quantifying CpG variants in a pied flycatcher population

Natal dispersal is a key life-history trait determining fitness and driving population dynamics, genetic structure and species distributions. Despite existing evidence that not all phenotypes are equally likely to successfully establish in new areas, the mechanistic underpinnings of natal dispersal remain poorly understood. The propensity to disperse into a new environment can be favored by a high degree of phenotypic plasticity which facilitates local adaptation and may be achieved via epigenetic mechanisms, which modify gene expression and enable rapid phenotypic changes. Epigenetic processes occur in particular genomic regions - DNA methylation on CpG sites in vertebrates -, and thus individual genomes may differ in their capacity to be modified epigenetically. This “Epigenetic potential” (EP) may represent the range of phenotypic plasticity attainable by an individual, and be a key determinant of successful settlement in novel areas. We investigated the association between EP – quantified as the number of genome-wide CpG variants – and natal dispersal propensity in a long-term study population of Pied flycatchers (Ficedula hypoleuca) monitored since colonization of a new habitat 35 years ago. We tested this association at three levels, comparing EP between: i) individuals dispersing between and within habitat patches; ii) immigrants to the population and locally-born individuals; and iii) individuals from first (comprising colonizers or their direct descendants) and later generations of the population (consisting of locally-born individuals, which did not show natal dispersal). Results show a significant, positive association between EP and dispersal propensity in comparisons i) and iii), but not ii). Furthermore, CpG variants were non-randomly distributed across the genome, suggesting species- and/or population-specific CpGs being more frequent in promoters and exons. Our findings point to EP playing a role in dispersal propensity at spatial and temporal scales, supporting the idea that epigenetically-driven phenotypic plasticity facilitates dispersal and environmental coping in free-living birds.

Methods
Samples were collected in population of pied flycatchers (Ficedula hypoleuca) breeding in nest boxes in central Spain (La Hiruela, 41°04’ N, 3°27’ W; 1250 masl), and monitored since 1984. Total genomic DNA was extracted from blood samples using a standard ammonium acetate protocol, and diluted to a working concentration of 25 ng/μL.  Besides, on an additional set of samples from 1998 (used in one of the group comparisons), DNA had already been extracted from whole blood using Chelex resin-based extraction.

Whole genome sequencing was performed by Novogene Europe in two sequencing runs, where paired-end Illumina next-generation sequencing (150bp) was carried out on an Illumina NovaSeq 6000 sequencer. Libraries were prepared using either the Novogene NGS DNA Library Prep Set (Cat No.PT004) at Novogene, or small-scale library prep (1/10th standard volumes) for Illumina DNA Prep with IDT index sets, using the Mosquito aliquoting system at the DeepSeq Facility at the University of Nottingham, UK. Both library preparation methods have previously been validated and shown to have comparable sequencing outcomes in another passerine species, the house sparrow (M. Ravinet, unpubl. data).

Genome sequences were aligned against the collared flycatcher (Ficedula albicollis) reference genome (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000247815.1/ ; accession AGTO00000000.2), the closest relative species with a reference genome available. In order to map reads to the collared flycatcher reference genome, we used a custom genotyping pipeline developed in Nextflow (https://github.com/markravinet/genotyping_pipeline). The pipeline is based on a previously used protocol and is designed to go from raw reads to sequence alignment, genotyping and variant filtering in several simple, reproducible steps. Following variant calling, we quantified CpG polymorphism across the genomes of all individuals.