Postglacial range expansion and the role of ecological factors in driving adaptive evolution of Musa basjoo var. formosana

Genetic variation evolves during postglacial range expansion of a species and is important for adapting to varied environmental conditions. It is crucial for the future survival of a species. We investigate the nuclear DNA sequence variation to provide evidence of postglacial range expansion of Musa basjoo var. formosana, a wild banana species, and test for adaptive evolution of amplified fragment length polymorphic (AFLP) loci underlying local adaptation in association with environmental variables. Postglacial range expansion was suggested by phylogeographical analyses based on sequence variation of the second intron of copper zinc superoxide dismutase 2 gene. Two glacial refugia were inferred by the average FST parameter (mean FST of a population against the remaining populations). Using variation partitioning by redundancy analysis, we found a significant amount of explained AFLP variation attributed to environmental and spatially-structured environmental effects. By combining genome scan methods and multiple univariate logistic regression, four AFLP loci were found to be strongly associated with environmental variables, including temperature, precipitation, soil moisture, wet days, and surface coverage activity representing vegetation greenness. These environmental variables may have played various roles as ecological drivers for adaptive evolution of M. basjoo var. formosana during range expansion after the last glacial maximum.

In the Quaternary, temperature oscillations are an important historical factor influencing the current distributions of a plant species 1 . During the last glacial maximum (LGM), most plant species in the Northern hemisphere would have retreated southward toward the tropics or warmer lowland areas, and survived in refugia 2 . Taiwan is a continental island situated off the coast of the Asian mainland and lies to the south of the Ryukyu Arc and north of the Philippines Archipelago. Although remnants of glaciations at the top of some peaks along the central mountain range (CMR) were found 3 , the lowlands of Taiwan were not covered with ice but were drier and colder, and the climate changes would have confined species to refugia in low elevations 3,4 . In Taiwan, many conifers escaped to the middle elevations during the LGM 4 . These species were originally distributed at middle and low elevations and would have migrated to the lowlands. A reverse course of events occurred since the LGM 1, 2, 5 , with species that were previously confined to refugia in the south expanded polewards and lowland forests colonizing at higher elevations 1, 2, 6 . Environmental gradients can potentially act as selective drivers during postglacial range expansion and result in locally adapted variants 2 . The current distributions of species are the results of a combinatorial effect by historical events, ecological factors, and stochastic or neutral mechanisms.
Population adaptive divergence is a central issue in evolutionary biology that focuses on understanding the correlations of population genetic variation with environmental heterogeneity 7 . Studies have shown that natural population divergence is driven by variable environmental conditions and leads to the evolution of locally adapted lineages [8][9][10][11] . However, gene flow between closely related genetic lineages can be reduced by a combinatorial effect of geography and environment; and geographical isolation may play a larger role than environmental variation in shaping population structure [11][12][13] . Therefore, investigating the relative roles of geography and environment that influence genetic variation is critical to understand how environmental factors may act as selective drivers and lead to adaptive genetic variation underlying local adaptation in natural populations of a species 11,12 . Moreover, identifying environmental factors that play roles in driving adaptive divergence is particularly of interest in biological conservation and ecological restoration 14 .
In Taiwan, genetic signatures of postglacial expansion into ranges of habitats from refugia were observed in many tree species such as Cyclobalanopsis glauca 15 , Cunninghamia konishii 16 , Trochodendron aralioides 17 , Castanopsis carlesii 18 , and Cinnamomum kanehirae 6,19 . Although Taiwan spans a small range of latitude geographically, varied geographical topologies support vegetation from tropical to cool climates 20 . The dramatic topological differences combined with the influence of tropical and subtropical climates have fostered high habitat diversity and may serve as a driving force for adaptive evolution [9][10][11][12] . During the postglacial recolonization process, a species would have evolved with adaptive variation invoked by differential selection along environmental gradients occurring in species' distribution ranges.
Musa basjoo Siebold is a cold hardy banana species and is thought to be originated from southern China 21,22 and is genetically differentiated from other Musa species 22,23 . M. basjoo Siebold & Zucc. ex linuma var. formosana (Warb. ex Schum.) Ying is a variety of M. basjoo endemic to Taiwan 24 . M. basjoo var. formosana is distributed in low elevations in Taiwan 25 and has a diploid chromosome number of 2n = 22 26 . The generation time of bananas under cultivation can be up to 18 months 25 and could be longer for natural populations of M. basjoo var. formosana. Fruit from M. basjoo var. formosana is edible but has numerous large and hard seeds. This species is an important germplasm for banana breeding due to its characteristics of cold tolerance and disease resistance. For conservation of this species, it is important to identify the genetic relationships of individuals among populations and to understand the potential for evolutionary adaptation because biodiversity is increasingly threatened by human-induced anthropogenic climate change 27 .
In this study, the second intron of copper zinc superoxide dismutase 2 gene (Cu/Zn SOD2) was sequenced for 46 individuals from eight populations (Table 1 and Fig. 1) to characterize the postglacial recolonization event. Moreover, amplified fragment length polymorphism (AFLP) 28 was also used to survey genetic variation of 112 individuals to examine the genetic relationships of individuals from different populations and test for association of AFLP loci with environmental variables underlying local adaptation driven by environmental gradients. We used variation partitioning based on redundancy analysis (RDA) 29 to assess the relative influences of geographical and ecological isolation contributes to AFLP variation. F ST outliers were identified using genome scan methods. In light of understanding local adaptations associated with environmental gradients, multiple univariate logistic regression was used to correlate environmental variables with AFLP loci that have potentially evolved under selection. We hypothesized that frequencies of AFLP outliers may display correlations with environmental gradients underlying local adaptation because the distribution of M. basjoo var. formosana in different geographical regions represents habitat environmental heterogeneities across the CMR. The main goals of this study were to: (i) demonstrate postglacial expansion of M. basjoo var. formosana; and (ii) find evidence for potential adaptive evolution associated with environmental gradients.

Results
Nucleotide diversity, population differentiation, and test for postglacial expansion based on the second intron sequences of Cu/Zn SOD2. We obtained 92 sequences of the second intron of Cu/ Zn SOD2 with an aligned length of 1264 bp. No sign of recombination was found examined within populations. However, one non-significant recombination event (P = 0.876) was found between nucleotide sites 15 and 510 when total samples were analyzed. In addition, 2 of 46 samples collected from populations Shouka and Wufeng were heterozygotes and one single-base pair indel was found in the aligned sequences. Sixteen haplotypes were identified in the 92 sequences analyzed (Table 1 and Fig. 2). The number of haplotypes ranged from 1 to 5 for each population. The most common haplotype (haplotype I) was found in all populations examined and the second most common haplotype (haplotype II) was found in four populations (populations Shanmai, Shitou, Shouka, and Wulai) ( Table 1 and Fig. 2). Populations Shitou and Wufeng had the greatest number of haplotypes and the population Sandimen had only one haplotype (the most common haplotype I). Although mismatch distribution of the frequency of pairwise differences among haplotypes did not fit tightly with a population expansion model (Kolmogorov-Smirnov test, P < 0.001, Supplementary Fig. S1), the haplotype network displayed "star-like" phylogeny in two separate groups indicating population expansion and subdivision (Fig. 2). Population pairwise F ST values calculated from Cu/Zn SOD2 second intron sequence variation was mostly low (Supplementary Table S1) and averaged 0.053. The southern population Sandimen showed the highest mean pairwise F ST (=0.154) against the remaining populations (Fig. 3), which may have been the southern glacial refugium for M. basjoo var. formosana. Table 1 presents the descriptive statistics of nucleotide diversity of the second intron of Cu/Zn SOD2 include haplotype diversity (Hd) and nucleotide diversity θ S and θ π . Hd ranged between 0 (population Sandimen) and 0.848 (population Shitou), θ π ranged between 0 (population Sandimen) and 0.00173 (population Beishi), and θ S ranged between 0 (population Sandimen) and 0.00168 (population Wufeng). The levels of Hd, θ π , and θ S were not correlated with the number of sequences (sample size) via Pearson's correlation test (Hd, r = −0.497, P = 0.209; θ π , r = −0.383, P = 0.349; and θ S , r = −0.469, P = 0.240).
Neutrality tests based on Tajima's D 30 and Fu's Fs 31 revealed non-significant negative values in many populations and even positive values were observed ( Table 2). In contrast, for pooled samples, significant negative values in Tajima's D (=−1.6544, P = 0.015) and Fu's Fs (=−6.8767, P = 0.008) were found. Moreover, non-significant raggedness index (rg) 32 was found for all individual populations and the pooled samples (rg = 0.0697, P = 0.84) ( Table 2). Significant values of the sum of square deviations (SSD) statistic were found for several populations (populations Guanhu, Shitou, Shouka, and Wulai) and R 2 index 33 was non-significant for all individual populations (Table 2). Nevertheless, non-significant SSD (=01976, P = 0.19) and significant R 2 index (=0.0416, P = 0.04) were found for pooled samples, indicating spatial range expansions 34 . The mean demographic expansion factor (τ) 35 , representing the time since the beginning of an expansion, was 1.6167 (0.30225-8.57544, 95% confidence intervals (CIs)). The time at which the expansion event took place was dated with a mean of 21317 (3985-113072,   Table S2). The proportion of AFLP polymorphic loci ranged from 50.7% (population Wulai) to 75.4% (population Shanmai) with an average value of 63.1% ( Table 2). The level of Nei's genetic diversity (H E ) 38 averaged 0.276 and ranged from 0.231 (population Shouka) to 0.336 (population Wufeng) ( Table 2). The mean H E was 0.276.
In AFLP data, the northern population Wulai had the largest average pairwise F ST against the remaining populations (mean F ST = 0.274, Fig. 2 and Supplementary Table S1). In the HICKORY analysis 39 Table S4). When the population Wulai was excluded (because it was highly differentiated from other populations based on DAPC analysis; see result in the following section, Fig. 4), AMOVA revealed a Φ ST value of 0.1308 (P < 0.001). AMOVA Φ ST value based on the three DAPC clusters was 0.2091 (P < 0.001).
In the STRUCTURE analysis 41 , the maximal ΔK value (change in the log probability) occur at K = 2 ( Supplementary Fig. S2). However, the highest mean log likelihood (LnP(D)) was obtained when K = 8. In the LEA analysis 42 , the minimal cross-entropy was lowest when K = 7 ( Supplementary Fig. S3). However, analysis based on discriminant analysis of principal components (DAPC) 43 revealed three genetic clusters with the first two linear discriminants described 74.48% of the total AFLP genetic variation (Fig. 3). DAPC results provided prominent phylogenetic breaks among the clusters. In addition, the values of symmetric similarity coefficient 44 (SSC) were higher when K = 2, 3, and 4 (0.995, 0.986, and 0.983, respectively) and lower when K ≥ 5 ( Supplementary Fig. S4). Therefore, K = 3 could be the most probable genetic clustering scenario according to the results of DAPC, STRUCURE, and LEA analyses ( Fig. 3 and Supplementary Fig. S5).
Effect of environmental variables on AFLP variation among populations. Seven environmental variables, including annual mean temperature (BIO1), annual precipitation (BIO12), mean wind speed (WS mean ), normalized difference vegetation index (NDVI), soil pH, soil moisture index (TMI) 45 (Table 3). Samβada analysis found seven AFLP loci strongly correlated with environmental variables (Table 3). We considered four AFLP loci (P1_17, P3_23, P3_24, and P6_12) as adaptive loci potentially evolved under selection because they were identified by either neutrality test method and correlated strongly with environmental variables. Four AFLP loci that identified as outliers by DFDIST or BAYESCAN and correlated strongly with environmental variables, logistic regression plots were drawn (Fig. 5). In the Samβada analysis, significant positive relationships were found for locus P1_17 with soil moisture and wet days (pseudo-R 2 = 0.251, P < 0.0001; pseudo-R 2 = 0.106, P = 0.0025, respectively) and locus P3_24 with annual mean temperature and NDVI (pseudo-R 2 = 0.094, P = 0.00897; pseudo-R 2 = 0.105, P = 0.00897, respectively). Significant negative relationships were found between locus P3_23 and NDVI (pseudo-R 2 = 0.115, P = 0.0056) and between locus P6_12 and annual mean temperature (pseudo-R 2 = 0.101, P = 0.00368). Samβada analysis reported several types of pseudo-R 2 values, we adopted the Nagelkerke pseudo-R 2 because its calculation is based on log likelihoods rather than on residual deviance and scaled approximately from 0 to 1 equivalent to the unadjusted R 2 in linear regression 49 .

Discussion
In this study, we surveyed sequence variation in the second intron of Cu/Zn SOD2 and AFLP variation to investigate whether the postglacial range expansion occurred in M. basjoo var. formosana and adaptive evolution in association with the environmental gradients of contemporary populations in this species. Sequence variation in the second intron of Cu/Zn SOD2 suggested range expansion since the LGM. A vast amount of unaccounted variation was observed, which is typical for RDA multivariate analysis 50 . In addition, a considerable amount of unexplained variation could also be attributed to random processes triggered by ecological drift and dispersal, non-spatially structured biological, and/or unmeasured environmental differences 11,51 . In addition, the natural landscapes of Taiwan have been dramatically altered by humans, particularly in the lowlands, which may have played a role in influencing genetic variation surveyed in M. basjoo var. formosana. Nevertheless, a significant amount of AFLP variation can be explained by the environmental variables. Local adaptation in M. basjoo var. formosana populations was suggested by the AFLP loci that were potentially evolved under selection in association with environmental variables, including annual mean temperature, annual precipitation, surface coverage activity (NDVI), soil moisture, and number of days with > 0.1 mm of rain per month (wet days). Glaciers have had a serious impact on the current distribution of plant species and most subtropical species have retreated toward the tropics or warmer lowland areas 2 . The discrepancy in detecting population expansion for individual populations could be because of either population reduction, population subdivision, a recent bottleneck, or migration 52 . Our data revealed that the mismatch distribution for the frequency of pairwise differences among haplotypes did not fit tightly with a population in the expansion model (Kolmogorov-Smirnov test, P < 0.001, Supplementary Fig. S1), which indicates diminishing populations or structured sizes of M. basjoo var. formosana 53,54 . In un-subdivided populations, molecular signature characteristics of sudden expansions might not be observed, even when populations had expanded by several orders of magnitude after the LGM 55 . Although no consistent evidence for population growth was found for individual populations, our results showed evidence for historical spatial range expansion in pooled samples because of significant negative values of neutrality test statistics (Tajima's D = −1.6544, P = 0.015; Fu's Fs = −6.8767, P = 0.008) ( Table 2). Moreover, the hypothesis of historical spatial range expansion could not be rejected in pooled samples based on non-significant small rg value (rg = 0.0697, P = 0.84) 32 , non-significant SSD (SSD = 00096, P = 0.81) 56 , and significant R 2 (R 2 = 0.0416, P = 0.04) 33 (Table 2).
Based on the average F ST value for each population in comparison with the remaining populations, glacial refugia of plant species have been inferred in many plant species in Taiwan. The most divergent populations can be found located in the north-central and south (particularly in the southeastern part) parts of Taiwan for tree species 6 . Populations served as refugia in the north-central part of Taiwan were found for tree species, including T. aralioides 17 19 and Q. glauca 59 based on nuclear DNA variation. Our results in M. basjoo var. formosana reflect recolonization events after the latest glacial period that probably originated from the southern population Sandimen due to its highest mean pairwise F ST (=0.154) against the remaining populations based on sequence variation of the second intron of Cu/Zn SOD2 (Fig. 3). However, the population Wulai had the highest average F ST based on AFLP variation (Fig. 3) and clearly distinguished from other populations according to the DAPC genetic clustering analysis (Fig. 4), suggesting that the population Wulai may have been the northern glacial refugium for M. basjoo var. formosana.
In comparison with other angiosperm species that occurred in Taiwan, M. basjoo var. formosana had lower nucleotide diversity in the Cu/Zn SOD2 second intron (Hd = 0.628, θ π = 0.00119, and θ S = 0.00280) compared to introns of chalcone synthase (Chs) and leafy (Lfy) genes in Ci. kanehirae (Chs: Hd = 0.841, θ π = 0.00716, and θ S = 0.00371; Lfy: Hd = 0.895, θ π = 0.00479, and θ S = 0.00805) 19 and intron of glyceraldehyde-3-phosphate dehydrogenase gene in Q. glacuca (Hd = 0.840 and θ π = 0.00500) 59 . However, these comparisons may not be appropriate based on only one gene and the gene sequences compared between species were different. Nevertheless, the level of sequence nucleotide diversity reflects the past demographic events in natural populations. Although populations Sandimen and Wulai may have been the glacial refugia for M. basjoo var. formosana, populations Beishi, Shitou, and Wufeng located in north and central Taiwan across the CMR had higher levels of nucleotide diversity (Table 1). These results suggested that populations Beishi, Shitou, and Wufeng may be the melting pots of diversity 5 . However, the migration routes may have occurred mostly northward from the southern refugium (population Sandimen) and between adjacent populations except for the population Wulai; and scarcely southward from the northern refugium (population Wulai). This is because the population Wulai had a high average F ST (=0.274) in comparison with all other populations based on AFLP variation (Supplementary Table S1) and individuals of the population Wulai are clearly differentiated from individuals of all other populations (Fig. 4). Compared to other populations, the population Wulai is identified as potential refugium located in higher elevation because the rugged geographic topology might serve as an insular area for the survival of this population during the LGM.
AFLP   to the low degree of inbreeding revealed by the HICKORY analysis (Supplementary Table S3). Long life span and predominant outcrossing by animal pollinators may account for high AFLP diversity in wild bananas 61,64 . The potential build-up of genetic variation during the course of expansion since the LGM, particularly under climate change conditions, is important for species' survival facing global climate change 27 . M. basjoo var. formosana harbors a substantial amount of AFLP variation and is an important resource for populations to adapt to changing environmental conditions under natural selection. In M. basjoo var. formosana, pure environmental and spatially structured environmental factors explained a significant amount of AFLP variation (Supplementary Table S6), which suggests that environments play important roles in influencing genetic variation of this species. Environmental variables such as temperature, precipitation, surface coverage activity, soil moisture, and wet days are the most important ecological drivers influencing genetic variation of M. basjoo var. formosana and correlated strongly with four AFLP loci based on multiple univariate logistic regression analysis (Table 3 and Fig. 5), indicating fitness-related change in AFLP variation. Temperature and precipitation are commonly found to be the ecological drivers strongly correlated with adaptive AFLP variation for many plant species that occur in Taiwan, such as R. oldhamii 11 , Keteleeria davidiana var. formosana 9 , and Salix species 12 . Temperature and precipitation appeared to be common ecological drivers for adaptive AFLP variation in natural populations of various plant species 8,[65][66][67][68] .
Soil properties can explain a non-negligible proportion of the spatial distribution of tree species 69 and influence genetic variation among populations of tree species [70][71][72] . Soil moisture is associated with allozyme genotypes at the glycerate dehydrogenase locus and may play an important role in the adaptation of Pinus edulis 70 ; and with AFLP loci such as in Fagus sylvatica 71 and Eperua falcate 72 . NDVI is a measure of surface coverage activity indicating the level of vegetation greenness and acts as a proxy for a biotic competitive environment. NDVI, estimated with the moderate resolution imaging spectroradiometer, has been shown to be linear with the fraction of absorbed photosynthetically active radiation (fPAR) 73 , which was not retained as explanatory variable in this study (Supplementary Methods). NDVI and/or fPAR can be influential factors acting on the genetic variation of a species in response to interactions with other species in a local ecological community 74 ; and has been shown to be correlated with population adaptive divergence 12 and adaptive divergence between species 12,13 .
Understanding environmental variables acting as ecological drivers and playing roles in shaping the contemporary gene pool structure of M. basjoo var. formosana from experienced postglacial range expansion is important. Our results suggest that there are two glacial refugia located in the northern and southern part of the M. basjoo var. formosana distribution range. Postglacial expansion confronting ecological discontinuity may have triggered the evolution of environmentally associated AFLP variation underlying local adaptation. Although a vast amount of AFLP variation was attributed to residual effects, a significant amount of explained variation attributed to environmental effects was found. In conclusion, environmental variables include temperature, precipitation, soil moisture, surface coverage activity, and wet days may have been the most important ecological drivers for adaptive evolution of postglacial expanded M. basjoo var. formosana populations, and have played roles in shaping the current distributions of this species.

Sampling.
A total of 112 individuals from eight populations of M. basjoo var. formosana were collected ( Fig. 1; Tables 1 and 2). All samples were subjected to AFLP genotyping and 46 were used to obtain the Cu/Zn SOD2 second intron sequences.
An initial screening found no variation in introns of Cu/Zn SOD1 and Cu/Zn SOD2 except the second intron of Cu/Zn SOD2 in several samples from different populations. Specific CSD2-F1 (5′-CTCCACTGGTAAACCCTCG-3′) and CSD2-R1 (5′-GAGGTCCTGCATGACAACAAG-3′) primers were used to amplify Cu/Zn SOD2 second intron sequences of 46 individuals in PCR reactions with 6 min at 94 °C for 6 min, followed by 30 cycles of 30 sec at 94 °C, 30 sec at 57 °C, 1.5 min at 72 °C, and 5 min holding at 72 °C. The sequential PCR fragments were ligated into pGEM-T vector, transformed into E. coli, and double sequenced using M13 primers and haplotype sequences deposited (GenBank accessions: KX688826~KX688841).
Sequence alignment, nucleotide diversity, haplotype, and demography. Sequences were aligned with Clustal X 77 and nucleotide diversity θ π and θ S , haplotype diversity (Hd), R 2 index, and pairwise mismatch distribution estimated using DnaSP v5.0 78 . θ π and θ S were calculated based on the average pairwise number of differences between sequences and the number of segregating sites per sequence, respectively. Population pairwise F ST , raggedness (rg) index of observed mismatch distribution, and neutrality statistics (Tajima's D and Fu's Fs) were estimated using Arlequin v3.5 79 . Moreover, goodness-of-fit of the observed mismatch distribution to that expected under the spatial expansion model was tested using the sum of square deviations (SSD) statistic with Arlequin. We generated a haplotype network with the R "pegas" package 80,81 .
Neutrality statistics are nearly zero in constant-size populations, whereas a significant negative value and significant positive value reflect processes such as population subdivision or recent bottlenecks. Positive R 2 value and significance of coalescent simulation against neutral model indicate population expansion. The raggedness index is a measure that quantifies the smoothness of the observed mismatch distribution and small rg values represent a population that has experienced sudden expansion 32 . A significant SSD value indicates departure from spatial expansion 34 .
To estimate the time since the beginning of an expansion, we used t = τ/2μk, where t is the time elapsed between initial and current population sizes, τ is the estimated number of generations since the expansion, μ is the mutation rate, and k is the sequence length. A mutation rate of 1.5% per 10 6 year per site was used 19 . Demographic expansion factor (τ) was estimated using Arlequin. We assumed a mean generation time of 2 years for M. basjoo var. formosana.
Initial detection of genetic structure of M. basjoo var. formosana was carried out with two assignment test methods: Bayesian clustering 41 and sparse non-negative factorization (sNMF) 42 methods. Bayesian clustering method adapted for dominant markers implemented in STRUCTURE 41 was used to estimate an individual's probability of belonging to a homogeneous cluster (K populations). An admixture model was adopted and tested with ten runs, for K ranging from 1 to 9, with 10 6 iterations and 10 5 burn-in steps. R package "pophelper" 86 was used to summarize mean log likelihood (LnP(D)), change in the log probability (ΔK) 87 , and symmetric similarity coefficient (SSC) 88 to evaluate the fit of different clustering scenarios. Genetic assignment of individuals was also inferred for K values ranging from 1 to 9 based on sNMF algorithm and least-squares optimization with the "LEA" package of R 42 . The settings for the LEA analysis were: regularization parameter = 100, iterations = 200, and repetitions = 10 with other arguments set to defaults, and the best K evaluated with the means of minimal cross-entropy.
The relationships between individuals and populations of M. basjoo var. formosana were also visualized based on the discriminant analysis of principal components (DAPC) using the R "adegenet" package 43,89 . DAPC first performed a principal component analysis (PCA), and an optimal number of PCs was estimated and retained for further discriminant analysis.
Effect of environmental variables on AFLP variation. Redundancy analysis (RDA) was used to partition AFLP variation and explained by environmental and geographical variables using the R "vegan" package 90 and significance tested with 9,999 permutations. Proportions of AFLP genetic variation (adjusted R 2 ) explained by pure environmental variables, geographically structured environmental variables, pure geographical variables, and residual components were estimated 29 . The longitude and latitude of sample localities were used as geographical variables.
AFLP outlier detection and association with environmental variables. Two F ST outlier detection methods (DFDIST and BAYESCAN) were used to identify AFLP loci that potentially evolved under selection. DFDIST implemented in Mcheza software 46 estimated allele frequencies based on the Bayesian approach 82 and the highest and lowest 30% of the initial F ST were removed for calculating the mean F ST . Outliers were identified by observed F ST and H E compared to simulated neutral distributions generated using 10 5 iterations of coalescent simulations. Loci with unusually high F ST values at the 95% confidence level by applying a false discovery rate of 5% were considered potentially evolved under selection. BAYESCAN uses a Bayesian likelihood approach via a reversible-jump Markov Chain Monte Carlo algorithm in comparing the selection versus neutrality model to identify AFLP loci that are potentially evolved under selection 47 . Posterior odds (PO), the ratio of posterior probabilities of selection over neutrality, was estimated with the settings of a sample size of 50,000 and thinning interval of 20 among 10 6 iterations, following 100 pilot runs of 50,000 iterations. When an AFLP locus with log 10 (PO) > 1.5 91 was considered to have strong evidence for selection. Samβada 48 was further used to evaluate the associations between frequencies of AFLP loci and values of environmental variables using logistic regression model; and significant fit was identified based on the Wald and likelihood ratio tests with Bonferroni correction at P < 0.01. Given the relationships between values of environmental variables and frequencies of AFLP outlier loci, logistic regression plots were depicted.