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