Abstract
Islands provide a great system to explore the processes that maintain genetic diversity and promote local adaptation. We explored the genomic diversity of the Balearic lizard Podarcis lilfordi , an endemic species characterized by numerous small insular populations with large phenotypic diversity. Using the newly available genome for this species, we characterized more than 300,000 SNPs, merging Genotype by Sequencing (GBS) data with previously published Restriction-site associated DNA sequencing (RADSeq) data, providing a dataset of 16 island populations (191 individuals) across the range of species distribution (Menorca, Mallorca, and Cabrera). Results indicate that each islet hosts a well-differentiated population (Fst=0.247±0.09), with no recent immigration/translocation events. Contrary to expectations, most populations harbor a considerable genetic diversity (mean nucleotide diversity, Pi=0.144±0.021), characterized by overall low inbreeding values (Fis<0.1). While the genetic diversity significantly decreased with decreasing islet surface, maintenance of substantial genetic diversity even in tiny islets suggests variable selection or other mechanisms that buffer genetic drift. Maximum-likelihood tree based on concatenated SNP data confirmed the existence of the two major independent lineages of Menorca and Mallorca/Cabrera. Multiple lines of evidence, including admixture and root testing, robustly placed the origin of the species in the Mallorca Island, rather than in Menorca. Outlier analysis mainly retrieved a strong signature of genome differentiation between the two major archipelagos, especially in the sexual chromosome Z. A set of proteins were target of multiple outliers and primarily associated with binding and catalytic activity, providing interesting candidates for future selection studies. This study provides the framework to explore crucial aspects of the genetic basis of phenotypic divergence and insular adaptation.
Keywords: Island, Genotype by Sequencing (GBS), Restriction-site associated DNA sequencing (RADSeq), Outlier.
Introduction
Island populations represent biological scenarios of greatest evolutionary interest due to their geographic isolation, which accelerates the process of organismal diversification (Warren et al., 2015; Whittaker et al., 2017), and their restricted scale of study, that allow close monitoring (Drake et al., 2002; Losos and Ricklefs, 2009). They also typically host a high rate of endemism, making them a global conservation priority (Manes et al., 2021; Sivaperuman et al., 2008).The Balearic Islands, encompassing the Pityusic (Ibiza and Formentera) and Gimnesic (Mallorca, Menorca, Cabrera and associated islets) islands, comprise a large number of islands that originated following a complex geological and climatic history related to the Mediterranean sea level variations during the Pleistocene (0.2- Mya) (Cuerda, 1989; Goy et al., 1997). Among the several endemic species, they host the two species of Balearic lizard (i.e., Podarcis lilfordi from the Gimnesic islands and P. pityusensis from the Pityusic islands). They are among the most extensively studied vertebrates of the archipelago, due to their remarkable phenotypic diversity (Pérez‐Cembranos et al., 2020), high population densities and reduction of anti-predatory behavior associated to the “island syndrome” (Cooper and Pérez-Mellado, 2012; Hawlena et al., 2009; Novosolov et al., 2013, Rotger et al. 2023).
P. lilfordi (Günther, A., 1874) presently occurs on 43 off-the-coast islets of Mallorca and Menorca, as well as in the Cabrera archipelago. Current distribution is the result of a vicariance process during the Mediterranean sea desiccation and subsequent reflooding (5.33 Mya) (Krijgsman et al., 1999; Terrasa et al., 2009). The disappearance of P. lilfordi from the main islands of Menorca and Mallorca dates back more than 2000 years, following the introduction of predators by humans (Alcover, 2000). The species is now confined on islets that provide an effective refuge for to date populations. Within these islets, the species evolved in a context of limited resources and absence of major predators and competitors, greatly diversifying in morphology, pigmentation (Pérez‐Cembranos et al., 2020; Rotger et al., 2021), behavior and life history traits (Salvador, A., 2009; Rotger et al., 2023). Major forces driving this diversification can be ascribed to the high intrapopulation competition (density-dependent factors) (Grant and Benton, 2000; Le Galliard et al., 2010; Massot et al., 1992), as well as intense genetic drift resulting from recurrent bottlenecks, primarily affecting the smallest islets (Bassitta et al., 2021; Charlesworth et al., 2003; Rotger et al., 2021; Terrasa et al., 2009). This context of rapid phenotypic differentiation among insular populations makes P. lilfordi an interesting vertebrate model for ecological and evolutionary studies (Camargo et al., 2010), particularly those addressing the genomic basis of local adaptation and persistence of small populations (Bassitta et al., 2021; Yang et al., 2022).
Genetic analyses of this species have been long limited to mtDNA, few nuclear genes (Brown et al., 2008; Terrasa et al., 2009) and microsatellites for few populations (Bloor et al., 2011; Rotger et al., 2021). A recent study based on single nucleotide polymorphisms (SNPs) data by Restriction Site Associated DNA Sequencing (RADSeq) (Bassitta et al., 2021) has expanded on previous knowledge on genetic diversity and phylogeographic pattern of this species, confirming the existence of two well-discriminated genetic lineages separating populations from the major archipelagos of Menorca and Mallorca/Cabrera (Brown et al., 2008; Terrasa et al., 2009. Recently, the P. lilfordi genome has been sequenced (Gomez-Garrido et al., 2023) providing the framework for a comprehensive exploration of population-level genomics of this species.
Here, we used for the first time the newly available reference genome ofP. lilfordi to map and annotate newly generated genome-wide polymorphic markers obtained by two independent sequencing methods, Genotype by Sequencing (GBS) and RADSeq. We specifically characterized SNPs of eight new populations (100 individuals) by GBS, and integrated RADSeq data of additional 10 populations (91 individuals from Bassittaet al . (2021), for a comprehensive analysis of 16 populations (two were common to both studies), spanning the main range of distribution of this species. Individual GBS and RADSeq datasets were independently analyzed as well as merged to obtain a representative set of common SNPs loci across all populations, which allowed for a robust comparative genomics within P. lilfordi .
Our main objectives were to: a) assess the level of intraspecific genetic diversity and structuring of the different populations ofP. lilfordi ; b) reconstruct the species main phylogeographic scenario of colonization of the Balearic Islands; c) identify potential signatures of genome diversification and loci underpinning the phenotypic diversity and insular adaptation of this species.
Materials and Methods