Phylogeographic breaks and how to find them: An empirical attempt at separating vicariance from isolation by distance in a lizard with restricted dispersal

Abstract

Aim: Discontinuity in the distribution of genetic diversity (often based on mtDNA) is usually interpreted as evidence for phylogeographic breaks, underlying vicariant units. However, misleading signal of phylogeographic break can arise in the absence of barrier to gene flow, under mechanisms of isolation by distance (IBD). How and under which conditions phylogeographic breaks can be safely differentiated from populations evolving under IBD remain unclear. Here, we use multi-locus sequence data from a widely distributed lizard species to address these questions in an empirical setting. Location: Morocco. Taxon: Spiny-footed lizard (Acanthodactylus erythrurus), Squamata: Lacertidae. Methods: Using 323 samples from 40 localities, we identified genetic discontinuities within A. erythrurus based on a mitochondrial fragment and nine nuclear markers independently. Using the nuclear markers, we then applied linear regression models to investigate whether genetic divergence could be explained by geographical distances alone, or barriers to gene flow (real phylogeographic breaks). Result: A. erythrurus is characterized by an important mitochondrial diversity, with 11 strongly supported phylogeographic lineages with a crown age of 8.5 Mya. Nuclear markers, however, yielded weak phylogenetic support for these lineages. Using clustering methods based on genotypes at nine nuclear loci, we identified phylogeographic clusters that were partly discordant with the mtDNA lineages. Tests of IBD delimited at least four groups of populations separated by barriers to gene flow, but unambiguous separation of vicariance from IBD remained challenging in several cases. Main conclusions: The genetic diversity of A. erythrurus originates from a mix of IBD and vicariance, which were difficult to distinguish, and resulted in similar levels of mitochondrial differentiation. These results highlight that phylogeographic breaks inferred from mitochondrial data should be further investigated using multi-locus data and explicit testing to rule out alternative processes generating discontinuities in mitochondrial diversity, including IBD. We identified four groups of populations within A. erythrurus, separated by barriers to gene flow, but even using nine independent nuclear makers the power of our approach was limited, and further investigation using genome-wide data will be required to resolve the phylogeographic history of this species.

Combining RADseq and contact zone analysis to decipher cryptic diversification in reptiles: Insights from the Spiny‐footed Lizard (Reptilia, Lacertidae)

“…A total of 35 individuals from the A. erythrurus complex, kept in the collection of the «Biogeography and Ecology of Vertebrates» team in Montpellier (CNRS & Ecole Pratique des Hautes Etudes, BEV-EPHE, UMR 5175-CEFE Montpellier) and already included in Rancilhac et al (2022), were selected for this study (Table 1). These individuals were sampled during dedicated field trips in Morocco between 2004 and 2011 at 16 separate locations (Figure 1).…”

“…Sampled locations cover two previously identified genetically and morphologically distinct lineages, Rif (Rif Massif and Northern Middle Atlas to Lake Ifrah) and Mid-Atl (Middle-Atlas south of Lake Ifrah; Bons & Geniez, 1995;Miralles et al, 2020). However, it should be noted that uncertainty remains about the ranges of these lineages since the identity of many other Moroccan and Algerian populations is unclear (Beddek et al, 2018;Miralles et al, 2020;Rancilhac et al, 2022).…”

“…To increase diversity within the library and reduce the number of phasing errors, which can be responsible for reduced sequence quality (Elshire et al, 2011), a set of 32 MIDs of 6 and 7 bp was used. The set was constructed to maximize the balance between nucleotides at each position and the MIDs differed by at least three nucleotides in order to reduce Beddek et al, 2018;Miralles et al, 2020;Rancilhac et al, 2022). The square and diamond symbols correspond to the outgroup A. montanus and A. lacrymae, respectively.…”

“…They are characterized by deep mitochondrial divergences (c. 7.5 million years ago [Mya]), whereas nDNA analysis failed to recover them as reciprocally monophyletic (Rancilhac et al, 2022). Furthermore, statistical testing of IBD did not unambiguously support that these two groups are separated by a barrier to gene flow (Rancilhac et al, 2022). While these analyses are limited by the restricted dataset employed and sampling gaps, the contrast between mitochondrial and nuclear phylogeographic patterns is puzzling, and the existence of barriers to gene flow between the two groups remains to be confirmed.…”

“…Consequently, RADseq‐based investigations of contact zones have been conducted in many systems to refine species boundaries (Caeiro‐Dias, Brelsford, et al, 2021; Caeiro‐Dias, Rocha, et al, 2021; Dufresnes et al, 2019; Dufresnes, Brelsford, et al, 2021; Dufresnes & Martínez‐Solano, 2020). In this study, we use this approach to investigate a secondary contact zone of a Moroccan lizard, where previous multilocus analyses suggested low levels of admixture (Rancilhac et al, 2022).…”