Increase of genetic diversity indicates ecological opportunities in recurrent-fire landscapes for wall lizards

Socioeconomic and climatic factors are modifying fire regimes with an increase of fire frequency and extension. Unfortunately, the effects of recurrent fires on biological processes that ultimately affect the genetic diversity of animal populations are mostly unknown. We examined genetic patterns of diversity in the wall lizard Podarcis guadarramae in northern Portugal, one of the European regions with the highest percentage of burnt land. This species is a small saxicolous lizard as it inhabits natural outcrops and artificial stone walls, likely in recurrent-fire landscapes. We genotyped nine microsatellites from ten populations selected according to a gradient in fire recurrence, and compared genetic diversity indexes and demographic patterns among them. At the population level, we hypothesize that a high level of mortality and population bottlenecks are expected to reduce genetic heterozygosity in sampled localities affected by recurrent fires. Alternatively, genetic signatures are expected to be absent whether fire did not cause high mortality. Regardless of levels of mortality, we expect a gain in genetic diversity whether recurrent fires facilitate lizard dispersal and migration due to the increased quality of the habitat for wall lizards. At the regional level, we examine whether a recurrent fire regime may disrupt the spatial structure of populations. Our results showed an increase in genetic diversity in recurrently burnt populations, and a decline in longer-unburnt populations. We did not detect bottleneck effects in repeatedly-burnt populations. High genetic diversity in recurrent fire populations suggests a high dispersion rate between adjacent metapopulations and perhaps immigration from outside the fire boundary. At the regional level, lizard populations show low differentiation and weak genetic structure, suggesting no effects of fire. This study confirms field-based censuses showing that recurrent-fire regimes give ecological opportunities to wall lizards that benefit from habitat openness.

Socioeconomic and climatic factors are modifying fire regimes with an increase of fire frequency and extension. Unfortunately, the effects of recurrent fires on biological processes that ultimately affect the genetic diversity of animal populations are mostly unknown. We examined genetic patterns of diversity in the wall lizard Podarcis guadarramae in northern Portugal, one of the European regions with the highest percentage of burnt land. This species is a small saxicolous lizard as it inhabits natural outcrops and artificial stone walls, likely in recurrent-fire landscapes. We genotyped nine microsatellites from ten populations selected according to a gradient in fire recurrence, and compared genetic diversity indexes and demographic patterns among them. At the population level, we hypothesize that a high level of mortality and population bottlenecks are expected to reduce genetic heterozygosity in sampled localities affected by recurrent fires. Alternatively, genetic signatures are expected to be absent whether fire did not cause high mortality. Regardless of levels of mortality, we expect a gain in genetic diversity whether recurrent fires facilitate lizard dispersal and migration due to the increased quality of the habitat for wall lizards. At the regional level, we examine whether a recurrent fire regime may disrupt the spatial structure of populations. Our results showed an increase in genetic diversity in recurrently burnt populations, and a decline in longer-unburnt populations. We did not detect bottleneck effects in repeatedly-burnt populations. High genetic diversity in recurrent fire populations suggests a high dispersion rate between adjacent metapopulations and perhaps immigration from outside the fire boundary. At the regional level, lizard populations show low differentiation and weak genetic structure, suggesting no effects of fire. This study confirms field-based censuses showing that recurrent-fire regimes give ecological opportunities to wall lizards that benefit from habitat openness.
Wildfires have played a determining role in shaping the evolution and function of many ecosystems around the world 1,2 . More than a local ecological disturbance, fire is a global ecosystem process 2 , with approximately one third of the land mass experiencing intensive burning 3 . In the last decades, fire-prone regions as the western Mediterranean are experiencing a major shift in the fire regime with an increase of fire frequency and extension 4 . The main driver of this shift is socioeconomic, i.e. increased amount of fuel due to rural abandonment 4,5 , in parallel to a global climate warming 6,7 . Shifts in particular fire-regimes can have devastating impacts on the sustainability of many ecosystem components 8 .
Fire shapes community composition for many taxonomic groups especially due to its major effect on the habitat structure 9 . Understanding the emergent genetic patterns of diversity resulting from habitat disturbance is critical in predicting how species will adapt to and persist in changing environments. However, the role of fire-regime as a driver of the patterns and distribution of genetic diversity is poorly understood 10,11 . Due to their mobility, the response of animals to fire is complex 12 , and the lack of information about the genetic patterns resulting from responses of organisms to fire is especially relevant 13 . To understand this issue, one must consider that the demographic impact of fire may depend on particular life history traits, such as dispersal ability, environmental requirements 14 , and resistance to fire 15 . The complexity builds up when both mortality and recruitment interact to determine the consequences of fire on within-population genetic diversity 11 . On the one hand, high mortality translates into population bottleneck, thereby reducing effective population sizes and producing a random loss of genetic diversity 16 . On the other hand, post-fire population recovery can stem from either survivors or through colonizers from adjacent unburnt areas 17 , which may also have an impact on the distribution patterns of genetic diversity. The loss of allelic diversity will be low if there is either a high survival rate 18 or if recruitment comes from multiple sources 19,20 . Post-fire population dynamics, such as genetic drift and/or migration, can affect genetic differentiation between populations 21,22 . Conversely, if post-fire migration is facilitated between populations, increased gene flow can reduce genetic differentiation 23,24 . This is the case in open-habitat or early colonizer species, which increase their local density and genetic variation in recently burnt sites 19,20 . In contrast, genetic diversity in long-unburnt specialists may decrease due to habitat fragmentation patterns imposed by fire 25 .
In this study, we have investigated the effects of a recurrent fire regime on the genetic diversity and spatial structure of the wall lizard Podarcis guadarramae 26 in northern Portugal. Wildfire is one of the most important agents of landscape change in Portugal 27 . The study area, in particular, is one of the European regions with the highest percentage of burnt land due to a combination of socioeconomic (continuous surface of highly flammable eucalyptus and pine forests) and climate (high rainfall rate episodes followed by dry periods) factors. Podarcis guadarramae is a small-size saxicolous wall lizard 28 that uses rocks, trunks, and bare ground for thermoregulation, foraging and shelter 29 . In northern Portugal, this lizard has a patchy distribution with populations located in natural open rocky outcrops and artificial stone walls surrounding agriculture fields 26 . The forested and bush matrix isolates lizard populations and hampers inter-population contact due to the low dispersion ability of this species through vegetated areas. Wildfire reduces vegetation cover, increases the extent of open outcrops, and allows lizard dispersion among patches with favourable (open) habitats. These life-history traits can explain the positive population responses of P. guadarramae to fire observed in northern Portugal 30,31 . However, the demographic processes (e.g. mortality, survival, migration) underlying the positive response of wall lizards to fire are unknown. For this reason, we have collected lizard samples in ten populations selected according to a gradient of disturbance level (i.e. number of fires and time since the last fire). The objective of this study was to examine how recurrent fire regimes affect: i) at the population level, the genetic diversity of each lizard population sampled, and ii) at the regional level, the geographic/genetic structure of the sampled P. guadarramae populations. Emergent genetic patterns of diversity can help to understand demographic processes linked to fire recurrence and habitat use. Based on P. guadarramae life-history traits, we have addressed the following hypotheses.
At the population level, if fire causes high mortality rate, wall lizard populations located on rocky outcrops that have experienced higher number of fires are expected to exhibit genetic signatures consistent with recent demographic bottlenecks and low genetic diversity. Alternatively, such genetic signatures are expected to be absent (i.e. no diversity loss) when fire does not cause severe mortality. Moreover, if recurrent fires improve habitat quality and facilitate lizard dispersal and migration, genetic diversity gains are expected regardless of lizard mortality caused by fire. At the regional level, we hypothesize that fire can shape population structure due to the expected fire impacts over the genetic diversity and ultimately over population differentiation. Accordingly, we expect fire to disrupt the spatial structure of populations by decreasing the naturally expected correlation between genetic and geographic distances.

Results
From the initial battery of nine microsatellite loci genotyped, one (Ph17) showed presence of null alleles in seven out of 10 populations (Supplementary material Table S1) and for this reason it was excluded from further analyses. Five out of the remaining eight loci showed evidence for the presence of null alleles and/or stuttering (Supplementary material Table S1); however, these patterns affected only a minority of populations. Despite the existence of null alleles, no deviations from either HWE or LE were found in the eight loci, and therefore we opted not to exclude any other loci.
Mean number of alleles per locus was 7.73 (±0.67 s.d.; range 5-19 alleles). Overall, the expected heterozygosity (H E ) of the eight loci showed moderate values, ranging from 0.663 to 0.732 (average 0.694 ± 0.019), while the observed heterozygosity averaged 0.647 (±0.026). The allelic richness (A R ) per population ranged from 5.922 to 7.763, whereas the mean number of alleles (N A ) per population varied from 6.125 to 8.625 (Table 1). Four of the populations sampled had private alleles, especially those populations subjected to recurrent fire-regimes (Table 1). A R , N A and H E diversity indices showed a general pattern of increasing diversity values from unburnt towards burnt populations, with significant differences detected for three out of five pairs, suggesting that fire changed the pattern of genetic diversity. When pools of populations with the similar condition (unburnt vs. repeatedly burnt) were compared, a clear difference was reported for N A and P A (Table 1). Similarly, there was a trend of increasing F IS towards the repeatedly burnt populations detected for all pairs, although pairwise differences within each pair was only significant for one population ( Table 1).
All genetic diversity measures (A R , N A , H E) ) and F IS were positively correlated with the number of fires that occurred at each population ( Almost all population pairs showed significant, although low, allele-frequency-based differentiation (Supplementary material Table S4). Significant pairwise F ST values were low (0.015-0.088). With exception of Moledo pair, all remaining locations showed no significant differentiation regarding the pairs of populations. AMOVA results revealed that all levels of genetic differentiation were significant ( Table 2). Most of the genetic variation was found within populations (96.2%); only a small but significant percentage was attributed to the variation among the five population pairs (2.17%), and to between pairs of populations within each pair (1.63%), which corroborates the fact that almost all pairwise F ST values were significant.
www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ The Mantel test showed a significant correlation between genetic distance (Supplementary material Table S5) and log-transformed geographical distance (r M = 0.547; P < 0.0001; 999 permutations; Fig. 2B), meaning that the observed differences were most likely a result of spatial structure and isolation by distance and not fire history. This result can be visualized in the PCoA (the two coordinate axes together explained 71.98% of the total genetic variation) since it mirrors the paired organized spatial location of sampled populations (Fig. 2).
Bottleneck test results were not robust to changes in the assumptions regarding mutation models (Table 3). M-ratio values were generally high across populations, suggesting low general evidence for bottlenecks. Nevertheless, there were five localities (notably including both unburnt and burnt populations) in which M-ratios fall below the highest Mc threshold which corresponds to the assumptions of the strict SMM (which is unlikely to fit most microsatellites) and 4Neµ = 5 (see Supplementary material Table S6). Only two unburnt populations (Leonte and Póvoa de Lanhoso) showed M-ratios lower than the Mc values calculated relaxing the assumptions of the strict SMM, but these values are still quite high and hence not robust to small changes in model assumptions. With respect to the heterozygosity excess test, results ranged from significant heterozygosity excess in all populations (when assuming the IAM) to no evidence of bottleneck in any population (when assuming the SMM). When fitting the TPM, the evidence varied along the proportion of single step mutations included in the model. Assuming the default parameters incorporated in BOTTLENECK (70% of mutations fitting the SMM, with a variance of 30%), only two unburnt localities showed significant heterozygote excess, as suggested by the Wilcoxon test. In summary, comparing the various tests, only the unburnt population located at Leonte showed consistent evidence for the occurrence of a bottleneck. Even though results are not robust to changes in the mutation model, it is clear that the evidence is not larger for bottlenecks in repeatedly burnt, when compared to unburnt, localities.

Discussion
Our study design (a gradient of recurrent-fire histories) provided the opportunity to perform tests to enlighten which genetic patterns and processes are driven by the occurrence of recurrent wildfires, and to extend the knowledge in this research field that is currently lacking information 11,13 . We demonstrated emergent genetic patterns of diversity resulting from demographic responses to fire by the wall lizard P. guadarramae at population level (i. e. genetic diversity indexes), but not at regional level (i. e. relationship between genetic and geographic distances).  Table 2. Summary results of analysis of molecular variance (AMOVA) within and among locations of Podarcis guadarramae sampled. SS -Sum of squares; VC -Variance component; * P = 0.003; ** P = 0.000. www.nature.com/scientificreports www.nature.com/scientificreports/ Due to its environmental requirements of open outcrops 32 , P. guadarramae benefits from recurrent burned landscapes dominated by granitic rocks where lizard populations achieve high effective population sizes 30,33 . The positive response of P. guadarramae to fire in northern Portugal 30,31 showed that recurrent-burnt landscapes give ecological opportunities to this wall lizard that seem to be related with the increase of genetic variability in the repeatedly burnt populations.
Wildfires alter the patterns of genetic diversity in P. guadarramae. Population recovery following major disturbances such as wildfires is likely a reflection of two non-mutually exclusive biological processes: i) abundance of survivors; and ii) post-fire recruitment (e.g. local reproduction or immigration) 13,16,17 . Some studies have documented a short-term post-fire decline of fire-sensitive species 34 , although studies on vertebrates have shown that fires rarely cause complete mortality and that residual populations may remain in situ in burnt areas 16,18 , specifically cliff-and rock-specialist species for which habitat is not destroyed by fire 34 . Biological processes, such as mortality, survival, and post-fire recolonization can affect genetic diversity in contrasting ways: 11 reductions in heterozygosity and allelic richness resulting from population bottlenecks 22 ; increases in genetic diversity and reduced regional genetic differentiation resulting from high immigration rates 19,20 ; and intermediate values of unchanged genetic diversity when mortality is low or is compensated by immigration 18 .
Analyses of genetic diversity helped us to unravel how recurrent fires affect the population dynamics of P. guadarramae. Our results clearly showed that fires altered the genetic diversity of wall lizards. Our first expectation was that fires could alter patterns of genetic diversity in burnt populations by decreasing it through successive bottlenecks and founder effects. These results have been found in populations of butterflies 25 and lizards 23 and have been further attributed to bottlenecks in several taxa such as mammals 35 , birds 22 , and lizards 36 . Consequently, burnt populations would become more differentiated from unburnt populations by means of genetic drift. However, our results did not meet this expectation. Interestingly, we obtained the opposite pattern: significantly increasing allelic diversity with number of fires, and conversely, a significant decreasing of allelic diversity with time since the last fire. This trend was valid for the overall correlation between fire and genetic diversity parameters, and also, in general, to local pairwise comparisons.
We suggest that the pattern of higher genetic diversity (mean number of alleles, allelic richness, private alleles, expected heterozygosity) found in repeatedly burnt populations of P. guadarramae can be explained by a combination of both high survival rate, mobility, immigration, and a post-fire increase in the carrying capacity of disturbed habitats.
The likelihood of immediate survival of an individual during a fire will be influenced by the severity of the fire, its distance to potential refuges and behavioural mechanisms the organisms may use to avoid direct flames and heat 16,37 . Refuges enhance immediate survival during a fire event but can also facilitate the post-fire persistence of individuals and populations within the burnt landscape by providing resources in the short-(food, shelter) or long-term (resident habitat) from a series of disjunct isolated populations to a metapopulation, and to a patchy population linked by frequent movements 16,17,38,39 .
The response of P. guadarramae to fire has been demonstrated in several demographic studies in the region 30,31 . Physiological experiments 40,41 and habitat preference studies have highlighted that this lizard copes very well with post-fire habitat conditions. These studies and our personal observations in recently burnt spots support the increased dispersal ability of individuals in post-fire habitats that brings new diversity (and increase allelic richness and heterozygosity). Increased dispersal activity in recently burnt habitats was the cause of increased genetic diversity in the agamid Amphibolurus norrisi in Australia 42   www.nature.com/scientificreports www.nature.com/scientificreports/ model based on the habitat preferences of each species, by examining the direction of gene flow (i.e. immigration into or out of preferred habitat). The burnt area indeed received influx of individuals from open-habitat species, increasing genetic diversity. In other taxonomic groups with higher dispersal capability, a post-fire increase in genetic diversity was also reported; e.g. the mountain brushtail possum Trichosurus cunnunghami 43 , and the Gran Canaria chaffinch Fringilla teydea polatzeki 18 . A combination of life history traits (e.g. dispersal abilities), and habitat characteristics (fire-adapted vegetation, use of unaffected refuges by fire) could have contributed to increase genetic diversity.
Our results suggest that refuges are preponderant both in the survival rate of wall lizards as well as for colonizers. Podarcis guadarramae is a highly saxicolous lizard and it is associated with rocky habitats 28,44 . Likewise, its preference for these habitats with a naturally high number of crevices and burrows, where the lizard could take refuge during the fire events, may allow a considerable level of survival among animals (see a similar result for the saxicolous gecko Tarentola mauritanica 34 ), allowing the retention of a large fraction of the pre-fire genetic diversity. This fact is in line with the lack of evidence for severe bottlenecks in burnt populations.
Moreover, given that fire clears vegetation and exposes previously unsuitable rocky outcrops, unoccupied habitats represent a new ecological opportunity for wall lizards. This not only supports an increase in the local effective population size, but also allows the dispersal of individuals between metapopulations, and potentially, recruitment and settlement of migrants from outside the affected area. Both mobility and migration can positively increase genetic diversity at the population level. Our results shown an increase in F IS in recently burnt localities, which may be in line with Wahlund effects caused by recent migrants from different gene pools 45 . We acknowledge that this hypothesis cannot be corroborated by the absence of pre-fire genetic data nor movement data. However, the negative correlation between genetic diversity and the time since the last fire was indirect evidence of the mobility/migration lizard pattern in burnt populations. Indeed, long-unburnt localities showed reduced genetic diversity of lizard populations likely by vegetation re-growth that reduces lizard mobility and isolates small populations in small rocky spots. The incorporation of knowledge of both direct, in particular the demographic and mobility processes 13 , and indirect effects (shift in habitat structure) of fire is important to understand the underlying processes behind the patterns of genetic diversity found 11 . Wildfires did not alter the patterns of geographic differentiation in P. guadarramae. The observed general increments of genetic differentiation according to geographic distance (isolation by distance) and not to fire history, suggested limited gene flow among all P. guadarramae populations. This result configures a classical metapopulation scenario, especially when individuals show low dispersal ability, like the lizard case 46 , and matches findings from closely related species (P. bocagei and P. carbonelli) also inhabiting western Iberia, in which, similarly low or slightly higher levels of population differentiation were reported across their distribution areas, yet with a clear correspondence to geography ( Concluding remarks. Due to their particular life-history traits (small size, low mobility, ectothermy, low reproductive recruitment), reptiles are important organisms to examine in their responses to fire at different complementary approach levels, i. e. demographic, genetic, ecologic. Given the saxicolous preferences of P. guadarramae, all the population trends suggest that this lizard benefits from recurrent fires. Microhabitat preference and post-fire dispersal seem to be traits that help this lizard to resist fire and persist afterwards. Mobility is an attribute that makes fauna a challenging group to examine their responses to fire 12 . However, data on this trait needs to be integrated with other biological traits to capture all relevant information in predicting how reptile populations respond to recurrent disturbances 50 . Integrative approaches are necessary to forecast biodiversity patterns in future scenarios of shifts in fire regime.  Fig. 3). The populations that are the object of this study correspond to the north-western subspecies, P. g. lusitanicus. It is a saxicolous and small (50-70 mm adult snout-to-vent length) diurnal lacertid lizard 28 with Atlantic ecological affinities 53 . Its depressed body shape and flattened skull facilitate entrance into narrow, irregular crevices. Individuals aggregate around favourable open areas with rock crevices, artificial walls, and isolated big blocks 33 . To test how the repeated fire regime that characterizes the study area affected genetic variation in the wall lizard P. guadarramae, we selected ten locations based on a digital national cartography of burnt areas from 1975 to 2013 (http://www.icnf.pt/portal/florestas/dfci/inc/info-geo). These locations followed a gradient of recurrent fires and time-since-fire periods. Burnt locations had 5.8 fires on average (range 2-8 fires) during the period 1975-2013 (Supplementary material Table S7. Moreover, locations were selected following a paired-population sampling scheme, each pair composed with locations with contrasting fire histories (from unburnt to repeatedly burnt). Pairs of locations were located on average at 4.9 km distance between each (range 2.0-12.5 km per location; Fig. 3). This sampling design avoids spatial pseudo-replication in fire regimes, and minimizes the potential effect of spatial structuring to mask the effect of recurrent fires on the genetic diversity.

Study species: the wall lizard
Sampling was conducted between July-October 2014. Except one recurrently burnt population, at least 20 individuals were sampled from each population. Individuals were captured using a noose or by hand. Sampling syntopic individuals was avoided as well as sampling juveniles. The tail tip of each individual was collected and stored in 96% ethanol. All individuals were then released back to their collection site. The experiments were performed in accordance with general guidelines and regulations of live vertebrates. The experimental protocol was approved by the Instituto da Conservação da Natureza e das Florestas (ICNF) from the Portuguese Government that provided the permit for sampling lizards in northern Portugal (number 578/2014/CAPT).  www.nature.com/scientificreports www.nature.com/scientificreports/ Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD). Bonferroni corrections were applied due to multiple tests 69 . With the final data set, we performed three types of analyses: i) population genetics; ii) population differentiation; and iii) bottleneck tests.
Analysis of genetic diversity. Using Arlequin, we calculated allele frequencies, expected (H E ) and observed (H O ) heterozygosities, mean number of alleles (N A ), and number of private alleles (P A ) for each population. We also calculated allelic richness (A R ; calculated using a rarefaction methodology considering the lowest number of individuals genotyped in a locality sensu El Mousadik & Petit 70 ) and inbreeding coefficient F IS 71 with FSTAT v2.9.3.2 72 .
Differences in genetic diversity indexes (N A , P A , H E , and F IS ) between each population pair, and between burnt and unburnt populations as a whole were explored using a permutation approach with 999 permutations. These analyses were performed with Python 2.7 scripts (available from the authors upon request).
We further explored the relationship between genetic diversity indices (N A , A R , H E , F IS ) and fire history variables (number of fires, time since the last fire) by using simple linear regression in STATISTICA v8.0 73 . Due to the independence of each fire event, these analyses disregard the paired sampling design and focus on an overall comparison of fire-history and genetic diversity among all ten populations sampled.

Analysis of population differentiation.
To test for genetic differentiation among all populations pairs, pairwise F ST values were calculated. A hierarchical population structure was evaluated through an analysis of molecular variance (AMOVA) grouping by pairs of location in order to test the null hypothesis that genetic variation was not associated with spatial structure. Both analyses were performed in ARLEQUIN using 10,000 permutations.
Geographic distances between populations were calculated from the pairwise Euclidean distance according to the latitude and longitude of each location with Geographic Distance Matrix Generator, version 1.2.3 (Ersts, American Museum of Natural History, Center for Biodiversity and Conservation, http://biodiversityinformatics. amnh.org/open_source/gdmg). We tested for isolation by distance by regressing log transformed geographic distances (in km) against Slatkin's linearized F ST (F ST /(1 − F ST )). Statistical significance was examined by Mantel test with 999 permutations in PASSaGE 74 . A Principal Coordinate Analysis (PCoA) was computed in GenAlex 6.5 75,76 to visualize patterns of genetic differentiation among populations.
Bottleneck tests. Bottlenecks (such as those expected to derive from the occurrence of wildfires) can be detected by taking into account patterns of microsatellite allele size distribution and/or frequency data [77][78][79] . To evaluate the hypothesis that wildfires caused a significant reduction in population sizes in lizard populations, we employed two commonly used methods to detect population bottlenecks: Garza and Williamson's (2001) M-ratio, and a test for heterozygosity excess developed by Cornuet & Luikart 77 . This test is based on the principle that, when populations experience a sudden decrease in population size, the number of alleles decreases faster than heterozygosity. Hence, a transient excess in heterozygosity can be detected in bottlenecked populations when compared to mutation-drift equilibrium expectations. We used the software BOTTLENECK v. 1.2 80 to evaluate such heterozygosity excess. Because the expected patterns of variation vary extensively depending on the mutation model assumed, the test of the robustness of the inferences was performed under the infinite allele model (IAM, Kimura & Crow 81 ), the strict stepwise mutation model (SMM, Ohta & Kimura 82 ), and under a wide range of two-phase mutation model (TPM, Di Rienzo et al. 83 ) parameters. BOTTLENECK provided several tools to compare hypothesis; we chose the Wilcoxon's test, since it has been shown to be the most powerful, especially for data sets with a small number of loci such as in our case 80 .
The M-ratio test quantifies gaps in the distribution of microsatellite allele sizes. Under a bottleneck, the number of alleles k is expected to decrease faster than allele range r. Values of M = k/r lower than expected given equilibrium expectations may thus be indicative of a bottleneck. We used the R package strataG 84 to perform the calculation of M and the script "Critical_M" (obtained at https://swfsc.noaa.gov/textblock.aspx-?Division=FED&id=3298) was used to determine the "Critical M" (Mc) value. This program calculates the M-ratio from simulated data sets under equilibrium for a given set of relevant parameter values and returns the value representing the fifth percentile of this distribution. We calculated Mc based on the sample size and the number of loci specific to our study. We also used a range of reasonable parameter values for the pre-bottleneck population mutation rate (from 5 to 20) and for the microsatellite mutation model parameters, including both the strict SMM as well as allowing for larger size mutations in varying proportions (see Supplementary material for details).
Both types of tests rely up to a great extent on the mutation model assumed for microsatellite loci. Except in the case of the IAM, the mutation models typically used for microsatellite data do not tolerate the existence of alleles resulting from insertions and deletions in the flanking regions. An observation of allele sizes in our case revealed the existence of a few alleles not compatible with strict repeat-number mutations. Therefore, to accommodate our data to the models under which these hypotheses were tested, we performed the analyses described above in a reduced data set resulting from the removal of the loci that, in each population, deviated from the expected repeat number pattern. This resulted in data sets containing six loci in all localities, except Santo Tirso -burnt, where five loci were retained.

Data Availability
All data generated or analysed during this study are included in this published article (and its Supplementary  Information files).