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“EPIgenetic impacts on early-stage SPECiation and adaptive radiation”

Periodic Reporting for period 1 - EpiSpec (“EPIgenetic impacts on early-stage SPECiation and adaptive radiation”)

Reporting period: 2015-04-01 to 2017-03-31

Epigenetics is the study of functionally relevant changes to heritable characteristics that occur without changes to the underlying nucleotide sequence. DNA methylation, the most well-studied type of epigenetic variation, comprises the addition of a methyl group (CH3) to cytosine. This modification has been shown to play an important role in regulating gene transcription, and has been implicated in clinical studies of human disease. However, few studies to date have focused on the ecological/evolutionary impact of epigenetic variation in wild populations.
Phenotypic plasticity is the ability of a single genotype to produce multiple phenotypes in response to cues from the environment. It has long been considered important on short time scales for buffering individuals against environmental fluctuations and colonizing novel habitats. However, the longer-term role of plasticity in evolution has been unclear, in particular how it may help during the evolution of new species (speciation).
DNA methylation, through changes in gene regulation, is thought to underpin much of phenotypic plasticity. The presence or absence of methylation at a gene locus has been shown to be induced by specific changes to the organism’s environment and there is increasing evidence that environmentally induced methylation can be passed onto offspring. This transgenerational maintenance of environmentally induced methylation would allow evolutionary forces such as natural selection to shape patterns of epigenetic variation and therefore could facilitate the transition between ephemeral individual plasticity and longer-term (genetically based) adaptive evolution.

One key prediction for an active role of environmentally-induced DNA methylation in facilitating speciation and associated ecological divergence is that patterns of phenotypic plasticity found in the ancestral species shape the trajectories of evolutionary and ecological diversification exhibited by newly evolved species within a radiating lineage: the “flexible stem hypothesis”. To date, there have been very few published studies that empirically investigate epigenetic variation and its association with speciation and ecological diversification. Speciation epigenomics therefore remains a fundamental missing link in speciation research.

Study System

This project has focussed on the endemic haplochromine cichlid fishes of crater lake Massoko, Tanzania. Lake Massoko contains two “ecomorphs” of Astatotilapia that are in the process of diverging into new species. There is a benthic blue ecomorph (the “deep” ecomorph) that is found from 15-25m, and a littoral yellow ecomorph found mostly from 0-10m (the “shallow” ecomorph). Phylogenetic analyses have confirmed that these species diverged in-situ from a riverine ancestor: Astatotilapia calliptera, still found in the rivers around Massoko (see Figure 1).

In addition to colour and depth habitat, these ecomorphs have diverged in many other characteristics including head, body and oral jaw shape, pharyngeal (throat) jaw morphology and diet. The Lake Massoko Astatotilapia radiation offers the rare opportunity to simultaneously characterize and compare epigenetic variation across the genome of the ancestral riverine species with that of the radiating descendent species. Therefore, this study system gives a unique evolutionary window to study the impact of DNA methylation on early-stage adaptive divergence and speciation.

Project Objectives

In project EpiSpec, we have addressed the following scientific questions: 1) Do eco-morphological differences correlate with epigenetic differentiation during adaptive radiation? This has helped to inform us if epigenetic differentiation is important in cichlid fish speciation. 2) Do genomic regions exhibiting divergence between new ecomorphs/species have disproportionate levels of epigenetic marks? This will provide a test of whether the formation of “speciation islands” could follow epigenetic inheritance of plastic phenotypic characteristics. 3) Do genomic regions undergoing divergence in the new adaptive radiation have disproportionately high levels of epigenetic marks in populations of the ancestral species? This will provide a test of whether epigenetic variance in ancestral populations could provide a basis for phenotypic divergence when given ecological opportunity.
Successful field seasons were carried out in August 2015 and April 2016 with collaborators from the University of Bangor (G. Turner; A. Tyers) and the Tanzania Fisheries Research Institute (TAFIRI). Individuals sampled for EpiSpec were also included in a parallel project conducting a genome wide association study (GWAS) of the Massoko fishes, currently being undertaken by collaborators in Bangor University (G. Turner, A. Tyers), Cambridge University (E. Miska) and the Sanger Institute (R. Durbin).

The proposed EpiSpec laboratory methods were enhanced to utilise the latest advances in high-throughput sequencing technology: reduced-representation bisulfite sequencing (RRBS). The new design involved sequencing 12 individuals from each ecomorph (shallow, deep, riverine). Liver tissue was chosen as it plays a key role in homeostasis and metabolism. Following optimization of DNA extractions, library preparation and sequencing were highly successful. Average sequencing coverage was high: 11.15 % to 43.62 % (mean = 24.48 %).

These aligned data have enabled us to quantify patterns in methylomic variation in the liver between the Lake Massoko populations in relation to the ancestral riverine population (Objective 1). Principal Component Analysis (PCA) of methylation at the nucleotide level has revealed these populations have strikingly different liver methylation profiles. Overall, the deep ecomorph appears to show the most distinct liver methylomes, the Massoko shallow and the ancestral riverine ecomorphs being more similar (see Figure 2). These data provide strong evidence that changes to methylation have taken place alongside genomic changes during adaptation to novel environments, as would be predicted if epigenetic variation played a key role in early-stage ecological speciation and adaptive radiation. We are now testing approaches to identify and annotate differentially methylated regions, and assessing how methylation load relates to the extent of genomic differentiation observed among populations (Objectives 2 & 3).
The results of EpiSpec have already increased our knowledge of levels of methylation variation in wild populations, and enabled tests of its importance in driving adaptive divergence into novel habitats and facilitating long-term speciation and adaptive radiation. We are currently in the early stages of drafting a manuscript on the system. The overall objective for this manuscript, when published, is that it forms a foundation for future research in the nascent discipline of speciation epigenomics. Alongside publications, the research findings will be presented at international scientific conferences over the next year.

Project EpiSpec was represented at Bristol Bright Night 2015, where it was presented to a spectrum of the general public. The activity succeeded in its key aims of demonstrating the broad range of scientific research ongoing within Bristol, and communicating the benefits of the EU as an active funder of research in the UK.
Figure 2: Project reuslts
Figure 1: Overview of the Geography and Natural History of the study system