Sample size is one of the critical factors affecting the accuracy of the estimation of population genetic diversity parameters. Small sample sizes often lead to significant errors in determining the allelic richness, which is one of the most important and commonly used estimators of genetic diversity in populations. Correct estimation of allelic richness in natural populations is challenging since they often do not conform to model assumptions. Here, we introduce a simple and robust approach to estimate the genetic diversity in large natural populations based on the empirical data for finite sample sizes.We developed a non-linear regression model to infer genetic diversity estimates in large natural populations from finite sample sizes. The allelic richness values predicted by our model were in good agreement with those observed in the simulated data sets and the true allelic richness observed in the source populations. The model has been validated using simulated population genetic data sets with different evolutionary scenarios implied in the simulated populations, as well as large microsatellite and allozyme experimental data sets for four conifer species with contrasting patterns of inherent genetic diversity and mating systems. Our model was a better predictor for allelic richness in natural populations than the widely-used Ewens sampling formula, coalescent approach, and rarefaction algorithm.Our regression model was capable of accurately estimating allelic richness in natural populations regardless of the species and marker system. This regression modeling approach is free from assumptions and can be widely used for population genetic and conservation applications. Accurate estimation of genetic diversity parameters in large natural populations using finite sample sizes is one of the central issues in population and conservation genetic studies and applications. Small sample sizes can lead to significant errors in estimating the genetic diversity of the species in question. For effective genetic resource conservation, sufficient allelic richness and a minimum number of carriers for each allele must be present in the conservation population to ensure its self-sufficiency over generations, otherwise its entire purpose may be compromised if the sampling criteria are not met . This asAllelic diversity (richness) is one of the most important and commonly used estimators of genetic diversity in populations. It strongly depends on the effective population size and past evolutionary history . HoweverBased on the probability theory alone, one can calculate the sample size required to detect alleles with a certain threshold frequency ,5. Rarefme) found in an ideal population can be approximately described asIt is rarely possible to know the true number of alleles in a population unless the entire population can be analyzed, in which case the concept of "sample" is not applicable anymore ,9. TheorNe is the effective population size and μ is the mutation rate [Ne → ∞, the error in me approaches zero and the parameter θ = 4Neμ is constant. Ewens in his fundamental work [θ. Furthermore, he pointed out that the expected number of alleles E(k) for a given sample size n can be expressed aswhere ion rate . When Netal work  indicaten it can be further simplified [or, for large mplified  to(3)θ in a natural population is still complicated: i) in the expression θ = 4Neμ, there is no or very little information on the mutation rates in plant populations, plus the observed mutation rates are locus-specific, and can be confounded by selection and migration; ii) the effective population size (Ne) can be estimated by the coalescent approach [ϕ as another estimator of 4Neμ [Estimating the parameter approach ,13, but  of 4Neμ , but it Although the Ewens sampling formula and coalescent approach provide theoretical expectations for the allelic richness in a given sample, they normally assume an ideal random mating population of constant size, and without migration and selection. However, natural populations rarely conform to these and other ideal population assumptions. Selection effects may be heterogeneous in time and space, and are extremely difficult to realistically model. Random mating may be hampered by spatial genetic structure and selfing ,16. A siWe investigated several empirical data sets published for a wide variety of plant and animal species to understand the relationship between allelic richness and sample sizes. From the data published for a wide variety of organisms, our own experimental results and computer simulations, we found that the number of alleles observed in a given sample is approximately proportional to the logarithm of the sample size, and the logarithm base depends on the species and the marker system used. Based on these observations, we developed a non-linear regression model to predict the observed allelic richness in a given sample. The model could be defined as:A is the observed mean number of alleles per locus . The logarithm base βS depends on the species and the marker set used, and βn and βA are the regression coefficients for the sample size and allelic richness, respectively, which depend on the species and molecular markers used. As natural logarithms (ln) are commonly used, we replace the logarithm base to e. Thus, equation (4) can be written as follows:where ρ = 1/ln βS, so the equation (4a) can be written asTo further simplify (4a), we introduce the variable βn becomes negligible, and the equation (5) can be further simplified asThe coefficients in the regression model (5) can be empirically determined using the modified random resampling procedure and non-linear regression analysis as described below. At large sample sizes, the coefficient The empirically derived equation (5a) is similar to the modified Ewens sampling formula in equation (3).We tested the regression model (5) using (i) large empirical data sets for four conifer tree species with contrasting population genetic characteristics, and (ii) simulated population genetic data sets created using Markov-chain-based algorithm with different inherent migration and selfing rates.Pinus strobus [Picea glauca [Picea rubens , and eastern white cedar - Thuja occidentalis  were used , and AFLP (amplified fragment length polymorphism) have been used in population genetic studies, these markers are not well suited for such studies and have fallen out of favour, primarily due to their diallelic and dominant nature. Codominant SNP (single nucleotide polymorphism) markers are being used in population genetic studies. However, most of them also suffer from the limitation of being diallelic. Since the objective of the present study was to predict the allelic richness in large populations, we used microsatellite and allozyme markers for validating our model, since these markers are codominant and multiallelic.Pinus strobus and Picea glauca are predominantly outcrossing species - average multilocus outcrossing rates (tm) are 0.924, and 0.940, respectively [Picea rubens and Thuja occidentalis are mixed-mating selfing-tolerant species - tm = 0.595, and 0.635, respectively [Picea rubens and Picea glauca stands, trees were randomly selected with minimum spacing of 30-50 m between the trees to avoid the possible family structure effects. In the Pinus strobus and Thuja occidentalis stands, all mature trees within the population were sampled. The number of individuals sampled per population varied from 95 to 180  using the Gauss-Newton method implemented in the NLIN procedure in the SAS 9.1.3 statistical package . An example of the input data and SAS NLIN output, showing derivation of the regression coefficients  in (5), is provided in the Additional file The resulting allelic richness values were used to derive the estimates of θ was calculated from the allelic richness values observed in the source data sets for natural populations (Table θ was used in equation (3) to estimate the predicted allelic richness at various sample sizes. We also estimated the θ values for the source data sets obtained from natural populations using the popular maximum likelihood coalescent approach implemented in the MIGRATE 3.0 program [θ estimates were used in the equation (3) to calculate the allelic richness for various sample sizes as indicated above. Also, we estimated the predicted allelic richness values (at n = 120) in our experimental samples using the rarefaction procedure implemented in the HP-RARE 1.0 program [We also tested the simplified Ewens formula (3) as a predictor for allelic richness. First, ns Table  using th program ,23. The  program .ST for Picea rubens and Pinus strobus data sets using the GENALEX 6.1. program [Additionally, to estimate the effects of sample size on the observed genetic diversity and genetic subdivision parameters, we calculated the observed and expected heterozygosity, Shannon information index, and F program .Thuja occidentalis [n = 10,000 to represent a typical large natural plant population. The initial allele states were assigned randomly, and then populations were allowed to evolve under the above-mentioned evolutionary scenarios for 20,000 generations to yield the data set A. Then a sample of n = 200 individuals  was taken from the resulting population, and randomly replicated 50 times as described above to create the data set B of n = 10,000 individuals. Then repeated random subsampling was performed on both data sets A and B , and the allelic richness was calculated in 50 replicates as described above. Various combinations of Nm and selfing parameters used are provided in the Additional File To validate our model, we created 10 artificial data sets each containing 2 populations of 10,000 individuals, with selected combinations of inherent migration and selfing rates, using the Markov chain-based simulation algorithm implemented in the EASYPOP 2.1 program . Migratidentalis , and extdentalis . Mutatiodentalis . The popn = 120 were close to the real observed values, but these methods did not provide consistent results at larger sample sizes (see below). Coalescent approach did not provide reliable estimates for allelic richness. Although the absolute allelic richness values varied among the species and marker systems used, the model developed in this study worked equally well for both predominantly outcrossing and mixed-mating selfing-tolerant species and both types of markers. Allelic richness estimates based on θ calculated using the simplified Ewens formula (3) did not provide a good fit with the experimental data - the predicted values were outside of the 95% confidence interval  consistently overestimated or underestimated the allelic richness, depending on the species and the marker type . At the same time, the allelic richness estimates calculated by our regression model (5) were in good agreement with the experimental data sets for all four species and both marker systems used. Thus, our model was a better predictor for the allelic richness in natural populations as compared to the Ewens formula , subsampling of the pseudosimulated data sets, and other methods for four conifer species are provided in Table  formula  did not  formula  did not  formula  did not s Figure , and ove formula  did not  formula  did not ρ in the equations (5) and (5a) may be interpreted as a simplified empirical estimator for θ. The empirically derived regression coefficients βn, and βA would provide correction for possible deviations of the experimental population from the ideal population model.As mentioned above, the empirically derived equation (5a) is similar to the modified Ewens sampling formula 3). Allelic richness estimates predicted by the Ewens formula (3) significantly deviated from the empirical estimates obtained by repeated random subsampling  and the rarefaction procedure. Rarefaction estimates were close to the subsampling and regression results obtained for The proposed regression model developed in the present study has been validated by comparing the allelic richness parameters estimated by using different approaches in large Markov chain simulated populations Figure . The alln = 200 (data set B) adequately represented the major proportion of the allelic diversity existing in the entire simulated population (data set A), and pseudo-replication and subsampling apparently had little effect on allelic diversity estimates at the sample sizes below n < 100 typically used in population genetic studies. At large sample sizes (n > 500), the replicated data sets tend to underestimate the allelic richness in comparison with samples drawn from the true simulated population  alleles in a finite population would require sample sizes close to the entire number of individuals in the population.A valid concern would be that the original sample set used for the subsampling procedure may contain only a fraction of the allelic diversity present in a large natural population. Our results indicate that allelic richness estimates obtained by the model developed here in the amplified data were consistent with that actually observed in the total simulated population. The allelic diversity of various samples drawn from the entire simulated population of 10,000 individuals (data set A) was consistent with that drawn from 50-times pseudo-replicated population of 200 individuals (data set B) Figure . The repIt should be noted that existence of spatial genetic structure in a population can affect the observed allelic diversity estimate in a sample. In two of the four studied species, spatial genetic structure up to ~25 meters has been observed . Since the sampling distance normally used for population genetic studies in forest trees (30-50 m) is greater than the observed spatial genetic structure, the latter has little effect on the allelic richness estimates.θ, but in our opinion, the robustness of the subsequent results far outweighs the expenses associated with running a small pilot study.The logarithmic nature of the relationship between allelic richness and sample size holds true regardless of the organism and marker system used. In addition to our own data sets for conifer tree species, we observed this relationship in a number of other studies published for various taxa, e.g. -32. In tOur approach takes into account possible deviations from the ideal population model occurring in such complex systems as natural forest tree populations, where long distance gene flow, population bottlenecks, selection, varying mating systems, and overlapping generations are the norm. One of the other advantages of our model over the coalescent approach is that it does not require high computation resources.n = 200. Our simulation study and experimental results indicate that it is unlikely that a sample of 200 individuals would capture all alleles in a real natural population. The bivariate linear regression model used in [The minimum sample size for population genetics and conservation studies has been a hotly debated topic. Although it is usually desired to capture 90-95% of allelic diversity, it is often not feasible, as the true number of alleles in the population is rarely known. A recent study by Gapare and Aitken  claims t used in  may not  used in .n  were illustrated using red spruce (Picea rubens) as an example. The observed and expected heterozygosities were generally insensitive to the sample size (data not shown), which corresponds well with the previously published data [ST , and stabilized after certain n (n = 60...90 in our case). Small allele frequency fluctuations have little effects on the observed FST values. As discussed above, at large n, the observed allelic richness increases primarily by low frequency alleles that have little effect on the observed genetic differentiation parameters. Thus, n = 60 to 90 appears to be the optimum sample size for common population genetic purposes.For most population genetic studies, an adequate sample size would be the one that allows for reliable estimation and comparison of genetic diversity and genetic subdivision parameters among populations. The effects of sample size on other observed population genetic parameters .Click here for fileAllelic richness estimated by repeated random resampling in simulated population genetic data with various combinations of migration and selfing rates.Click here for fileAllelic richness predictions for individual populations of all four species based on our regression model (5), Ewens formula and coalescent approach. The population names are provided in Table θ calculated from the empirical source data set. Coalescent - allelic richness predicted by equation (3), θ estimated by coalescent from the empirical source data.Click here for file


Genetic estimates of effective population size often generate surprising results, including dramatically low ratios of effective population size to census size. This is particularly true for many marine species, and this effect has been associated with hypotheses of "sweepstakes" reproduction and selective hitchhiking.Here we show that in advective environments such as oceans and rivers, the mean asymmetric transport of passively dispersed reproductive propagules will act to limit the effective population size in species with a drifting developmental stage. As advection increases, effective population size becomes decoupled from census size as the persistence of novel genetic lineages is restricted to those that arise in a small upstream portion of the species domain.This result leads to predictions about the maintenance of diversity in advective systems, and complements the "sweepstakes" hypothesis and other hypotheses proposed to explain cases of low allelic diversity in species with high fecundity. We describe the spatial extent of the species domain in which novel allelic diversity will be retained, thus determining how large an appropriately placed marine reserve must be to allow the persistence of endemic allelic diversity. To do so, we define a "retentive population" as a demographically stable group of individuals that can persist without immigration from outside its domain. This definition is equivalent to that of Booke .e, Nallele will not satisfy (1) – and alleles are not retained – if Ladv is non-zero. Allelic diversity in downstream regions will be set by the allelic composition of migrants from upstream. So, in an advective environment the evolution of allelic diversity in the entire population will be governed by the allelic diversity in the retentive population, and the Ne for the entire population should approach the census population of the retentive population given by eq. (2). However, heterogeneity in abiotic  as well as biotic  factors that result in an interruption or severe reduction of larval transport from upstream may lead to the formation of additional retention zones within a species' geographic range that harbor additional diversity [Downstream of the retentive population that defines Niversity ,13. As dadv downstream, with standard deviation of Ldiff with a Gaussian dispersal kernel [To test these ideas, we use a simple numerical model, similar to those used by Pringle and colleagues ,13. In ol kernel  or with adv is zero; in the other it is 4 km/generation to the right. In both, the stochastic component of larval transport Ldiff is 10 km/generation. In the case with no mean larval transport, all genetic diversity is retained. However, when there is mean larval transport, only the upstream allele persists and the other alleles are lost downstream, for only the upstream allele begins in the retentive region that lies within Lreten = 25 km of the upstream edge of the domain.To illustrate how advection reduces genetic diversity in a population, two domains are initialized with five different alleles each in different parts of the domain in the numerical model figure . In one diff is fixed to 100 km and Ladv is varied from 0 to 116 km. There is a mutation rate μ = 10-3 such that larvae randomly carry a new allele with this frequency . In these model runs, Nallele is uniform in the interior and small near the edges when Ladv is zero, but as Ladv increases, Nallele becomes largest near the upstream edge of the species domain, and is one in the interior of the model domain   in the retention zone that lies within Lreten from the upstream edge of the domain, and does not elsewhere. In the model, time to fixation or extinction of all novel alleles is tracked as a function of their origin. As discussed above, enhanced reproductive success within a distance Lreten from the upstream edge allows novel alleles to persist longer in the upstream retentive population, for a time appropriate to Ne as given by Eq. (1), while those in downstream regions are lost much more quickly  figure , we findy figure . The regs figure , as prede as a function of Ladv and Ldiff, we calculate the inbreeding effective population size Ne [e [e from the average fixation time of 100 model runs. In Figure domain = 103, 4 × 103, and 1.6 × 104 km. In each domain, we fix Ldiff to 200 km, and vary Ladv from 0 to 110km, and compare the estimated Ne from (2) to the estimation from fixation time in an upstream region of the model Lreten in size. Once there is fixation in this upstream region, the allele fixes rapidly in the rest of the species domain in approximately Ldomain/Ladv generations. When the size of the domain is less than Lreten in extent, Ne is limited to the population census size  captures the variability of Ne with Ladv very well, capturing the several order of magnitude decline in Ne with increasing Ladv. As mentioned above, the estimate of Ne from (2) is, for most values of Ladv, very much smaller than – and not dependent upon – the census population size. When the model is re-run with Laplace's dispersal kernel, the results shown in Figure To determine N size Ne  given thze Ne [e , and so e is intended to reflect the number of individuals that contribute to the evolutionary potential of a species [i.e. a "nest-site" model [The concept of "effective population size" is typically used as a numerical trait of a population more than as a descriptor of biological reality . But Ne  species ; in adve species . Other r species , i.e. a e" model . In these in natural populations has recently been focused on a number of demographic causes for reduced Ne/N ratios [eμ (or Neμ for haploid markers), in an advective environment most of these are more quickly lost due to advection (a time in generations of about Ldomain/Ladv) than to stochastic genetic drift  Ne will be more disassociated from actual census size than for populations less affected by advection.A growing body of literature attempts to link patterns of genetic diversity with patterns of biodiversity, for the purposes of elucidating the mechanisms underlying broad-scale biogeographic structure and for conservation-focused predictions ,32. Howeacross populations of low-dispersal species, recent isotropic descriptions of metapopulation structure [e. Metapopulation structure in general may bias the measurement of Ne and gene flow measures [To test this prediction, one might imagine comparing species with very different dispersal strategies, or comparing the same or similar species in two locations with different dispersal conditions. However, while classical population genetics predicts elevated diversity tructure  show thameasures , and Folmeasures  suggestsmeasures -38, one measures , and in measures .e/N ratios observed are for species with broad dispersal potential in regions where ocean currents would be expected to generate a large Ladv [e and actual census size. In the end, any mechanism that increases the variance in reproductive success among individuals, whether due to stochastic, biological, or spatial processes, will reduce genetic variation in a species [e/N ratios  if thers (e.g., . Our worThis work was developed as equal-authorship collaboration between JPW, who conceived of the study and drafted the manuscript, and JMP who developed the numerical simulations and scalings and coordinated the results, and helped to draft the manuscript. All authors read and approved the final manuscript.


Thaumetopoea pityocampa/T. wilkinsoni complex, Notodontidae), a forest pest occurring around the Mediterranean Basin and in southern Europe. This species is responding to the current climate change by rapid natural range expansion and can also be accidentally transported by humans. Our aim was to assess if Quaternary climatic oscillations had a different effect across the species' range and to determine if genetic footprints of contemporary processes can be identified in areas of recent introduction.Quaternary climatic oscillations had dramatic effects on species evolution. In northern latitudes, populations had to survive the coldest periods in refugial areas and recurrently colonized northern regions during interglacials. Such a history usually results in a loss of genetic diversity. Populations that did not experience glaciations, in contrast, probably maintained most of their ancestral genetic diversity. These characteristics dramatically affected the present-day distribution of genetic diversity and may influence the ability of species to cope with the current global changes. We conducted a range-wide study of mitochondrial genetic diversity in the pine processionary moth  where highly differentiated, endemic haplotypes were found. Recent introductions were typified by the existence of closely-related haplotypes in geographically distant populations, which is difficult to detect in most of Europe because of a lack of overall genetic structure.In regions that were not prone to marked glaciations, recent moth introductions/expansions could be detected due to the existence of a strong spatial genetic structure. In contrast, in regions that experienced the most intense Quaternary climatic oscillations, the natural populations are not genetically structured, and contemporary patterns of population expansion remain undetected. The contemporary distribution of genetic diversity cannot be understood without studying how organisms responded to climate over geological times. Many terrestrial species are today responding to the contemporary global warming , and the intense ,4. Fores intense -10. The  intense ).Following Pinho and collaborators , we can i.e. species occurring at higher latitudes, were selected for increased vagility and generalism. Dispersal-related traits should have been optimized during the northward progression because high mobility provided an elevated fitness within populations that were tracking a moving habitat. In the same way, generalists  had a smaller risk of their niche disappearing. Over evolutionary times, the selective pressures are likely to have changed, with dispersion and generalism favoured during interglacials, and less so during glacial periods when the species were restricted to suitable refugia.Going further, Dynesius and Jansson  have preThe effects of differential intensities of glaciations on the evolution of the species, described above, are expected for mainland species for which the tracking of acceptable environments through migration was possible. The situation was drastically different for species or populations on islands situated beyond dispersal range, for which any change had to be endured locally, either by altitudinal shifts or by the evolution of local adaptations. Moreover, smaller effective population sizes could have resulted in loss of genetic diversity due to genetic drift. In this case, evolution on islands may have been more rapid than the rate of change on continents , and islSpecies or populations that experienced marked climatic oscillations in the past can be seen as a selected assemblage of geographically mobile and latitudinally-independent organisms that are likely to be best adapted for the future climate changes, unless human activity precludes such an option . Yet, coThaumetopoea pityocampa/wilkinsoni species complex), which develops mainly on pine species (Pinus spp.). It is a serious forest pest as it can cause heavy defoliations of pines in Mediterranean countries. T. pityocampa has a typical winter larval development [Thaumetopoea pityocampa and T. wilkinsoni. The differentiation between these two species was recently shown [T. wilkinsoni populations in the near East was confirmed [Here, we present a range-wide genetic study of a circum-Mediterranean insect taxon: the winter pine processionary moth . The new haplotypes found in the present study have been deposited in GenBank (accession numbers GQ507373 to GQ507422). A total of 67 combined (COI-COII) haplotypes (ht) were found. The selected model of evolution was the General Time Reversible model with gamma distributed heterogeneity of rates (GTR gamma). Interestingly, Bayes factors (BF) indicated a much stronger fit for this model when a clock was assumed than when branch lengths were unconstrained . This was confirmed when the performance of models was assessed with the Bollback approach [We obtained 34 COI and 51 COII haplotypes. Among these, 14 COI and 21 COII haplotypes were known from either Salvato et al.  or SimonT. pityocampa - wilkinsoni complex was structured in three strongly supported clades  and North Algeria . The third clade comprised 24 European ht, from Spain and Portugal to Greece (with the notable exception of Crete), together with the 7 ht found in Morocco and South Algeria. It will hereafter be referred to as the 'pityocampa clade'. The main nodes were dated by Bayesian inference using a Yule prior and the estimates are given on the phylogenetic tree , while the separation of the pityocampa vs. ENA clades was dated back to 6.7 Myrs (4.9 - 8.6). The age of the most recent common ancestor (MRCA) of the wilkinsoni clade was estimated to 5.3 Myrs (3.7 - 7.1) while that of the ENA clade was ca. 3.1 Myrs (2.1 - 4.3) and that of the pityocampa clade was estimated to 2.3 Myrs (1.6 - 3.1).The haplotype composition of each sampled population is given in Additional file s Figure . A firstlkinsoni  togetherwilkinsoni clade, 4 distinct sub-clades were found with very high support values  the Iberian Peninsula, (ii) North Morocco, (iii) South Morocco & South Algeria and (iv) the Rest of Europe. Interestingly, 16 individuals sampled in Spain and 2 individuals from western Turkey had "Rest of Europe" haplotypes ; tMRCAs obtained with an exponential prior are reported only for this group (Table Time of most recent ancestor (tMRCA) of each sub-clade was estimated by Bayesian inference including all the individuals of a given group and assuming a priori either a constant population size or an exponential growth. However, the rate of exponential growth resulted to be positive, with an associated 95% confidence interval excluding 0, for the Rest of Europe only , and ht 4N, 12R and 12K that each occurred in two populations , with one main ht shared by ca. 74% of sampled individuals and all other ht diverging from it by only one or two mutations. Haplotype 1G was restricted to central and southern Italy. Interestingly, all other haplotypes in Europe were rare, shared by 6 individuals at most and usually endemic to one population. In the Iberian Peninsula, ht 29BA was found in 57% of individuals and in all populations but Gibraltar and two southern sites. None of the other sub-clades in the pityocampa group showed a star shape.Haplotype networks were reconstructed for each of the 3 main clades, and haplotype distributions were mapped  as the sister group of the ENA and pityocampa clades. Determining the taxonomic status of the clusters identified here is beyond the scope of the present study, and would need complementary data such as nuclear markers and morphological data. For this reason, we will hereafter mention three clades  without further discussion of their taxonomic level.The pine processionary moth is currently understood to consist of a species complex containing two taxa, namely lkinsoni ,17. Surpe Figure , and theThaumetopoea pityocampa and T. wilkinsoni was estimated to 4.5 - 5.2 Myrs [T. pityocampa (as the population from Spain contained only European haplotypes rather than Iberian ones) and one Turkish population of T. wilkinsoni were analyzed. In the study presented here, a thorough sampling of populations  and a Bayesian approach taking into account the gamma-distributed heterogeneity of rates, allowed us to obtain a different estimate for the age of the main evolutionary events. In particular, the split between the wilkinsoni and the pityocampa-ENA clades was dated on average to 7.5 Myrs, with a confidence interval of 5.8 - 9.3 Myrs, which could correspond to the full opening of the Aegean Trench ca. 9 Myrs ago [wilkinsoni clade, the estimates of node ages we obtained were very similar to estimates obtained previously using codon-partitioned models [wilkinsoni haplotypes was dated back to about 5.3 Myrs, which corresponds to the Messinian salinity crisis and the time when the Mediterranean Sea was at its lowest level, thus making the colonization of islands easier [Another striking result is that the species complex is ancient, and predates the Quaternary by a few million years. In a previous study, the divergence between 5.2 Myrs . That reMyrs ago . Interesd models . While ws easier . Node ags easier .pityocampa and the ENA clades was unexpected, and cannot be explained by classical barriers to gene flow such as mountain ranges or fragmentation of suitable habitats. Similar patterns of East-West genetic differentiation have occasionally been found in North Africa for other organisms [pityocampa and the ENA clades some 6-7 Myrs ago.The differentiation between the rganisms -27, but rganisms . Moreoverganisms . Finallyrganisms  that perT. pityocampa/wilkinsoni complex date from the end of the Miocene, all clades also predate the Quaternary. Each of the identified clades thus experienced the Quaternary climatic oscillations after they split from a common ancestor, and the impact of ice ages can easily be compared between these closely-related clusters.If the main divergences within the Each of the three identified clades showed a strong phylogeographical structure, and was composed of 3, 4 or 5 well-differentiated sub-clades. With the notable exception of the Rest of Europe (see below), each sub-clade was restricted to a rather narrow geographical region. Interestingly, a vast majority of haplotypes (54 out of 67) were endemic to one single population, and only five were found in three or more populations. Thus, the pine processionary moth exhibits an extreme spatial structure and a highly reduced mitochondrial female gene flow even on a regional scale, even though results based solely on a mitochondrial marker should be interpreted with caution. Over most of the distribution range, the actual dispersal of the females is thus highly limited. The main barriers to gene flow are sea straits, mountain ranges , or desert regions where hosts are lacking (Libya).i.e. to the Early- or Mid- Pleistocene. One could suggest that local ecological pressures recurrently acted to reinforce and maintain the genetic structures whenever gene flow had been interrupted. As migration is very limited and cannot counteract the effects of drift, genetic differentiation then simply increases with time, leading to divergent lineages in different regions. Ecological factors involved in differentiation include reproductive phenology, which can prevent mating by shifting adult emergence periods in different populations, or local adaptation to host characteristics, which, it has been proved, can lead to complete mortality in translocated larvae [Within-clade structures were all dated back to at least 1.3 Myrs Figure , i.e. tod larvae . A more th century except for remote relictual stands ,31,329,3o et al.  for a dePinus sylvestris [Europe (except the Iberian Peninsula that harbours a specific sub-clade) is characterized by a major haplotype that occurs from the Atlantic coast to the Greek islands and even along the Turkish border. Moreover, the Rest of Europe sub-clade had the star-shape that is typical for populations expanding after a demographic bottleneck , and thelvestris , which sAndricus gall wasps developing on oaks, see [With the exception of Lebanon and Israel where the moth settled and expanded only recently (see below), our results show contrasting patterns of evolution during the Quaternary in the different regions of the moth's distribution range, corresponding to our expectations. In particular, populations occurring in the highest latitudes exhibit a radically different genetic footprint to that of all other sub-clades. If moth populations in the vast majority of the distribution range are characterized by a strong spatial genetic structure, a high number of endemic haplotypes and a restricted geographical range for each identified sub-clade, the patterns in the Rest of Europe are completely the opposite. In this European region, overall genetic diversity is low; spatial genetic structure is limited as a consequence of the large distribution of the major haplotype 1A; and this single sub-clade is distributed over one half of the total distribution area of the species complex. Moreover, signs of recent expansion were detected only in the European sub-clade, that is, in the region where glacial cycles were probably most intense. As for most European species, endemic haplotypes and some genetic variability can still be detected in plausible refugial areas near the Pyrenees, in Italy and in the Balkans ,8,13. Inaks, see ,6,32).pityocampa clade, the Cretan ht form the sister-group of all other sub-clades within the wilkinsoni clade, and the highly isolated moths of Cyrenaica (Libya) are most divergent in the ENA clade. Moreover, the second most differentiated group in the wilkinsoni clade is the Cypriot cluster. Each of the island lineages thus diverged from the corresponding sub-clade a long time ago (from 5.3 Myrs for the Cretan haplotypes to 1.8 Myrs for Cyprus). On the other hand, the most recent common ancestors for each island are much more recent (0.38 Myrs in Crete and 0.15 Myrs in Cyprus for example). Hence, it is not possible at this point to determine when exactly the colonization of each island (or isolated place) occurred, and for how long the moths have been isolated from the continent. However, even if we consider only the estimated age of the MRCA . Given the natural spatial genetic structure in these regions, the recent modifications in moth distributions due to global changes are actually easy to track. The populations discussed above all likely originated from the closest natural stand, and could be the result of non-assisted moth expansion (but a better sampling in Algeria is needed to confirm this). The mitochondrial marker we used here would also be useful to identify between-subclades female gene flow, but a nuclear marker is necessary to track male exchanges. In most of Europe, however, where the populations are not genetically structured in space and where overall genetic diversity is low, probably as a consequence of Quaternary history, one cannot distinguish recent and historical events, as contemporary expansions  result des, see ,19.Pinus pinaster) occur naturally and exhibit a high degree of local genetic diversity [The patterns are somewhat different for islands. Some harbour populations of moths that are genetically very close, or even similar, to their closest continental neighbours. This is not surprising for islands that are located very close to the continent, like most Greek islands or Sicily, that can probably be recurrently colonized from mainland sources. A similar result was found, for example, for rodents . In contiversity . In contWe conducted a range-wide study of genetic diversity in a species complex occurring across regions in which Quaternary oscillations differed in intensity - or were absent. We have clearly shown that the sub-clade distributed over Europe had a phylogeographical pattern typical for species that experienced marked glaciation cycles. Refugial areas, where genetic diversity was retained and where endemic haplotypes were found, were identified in Italy, in the Alps and in the Balkans. All other populations were characterized by the occurrence of one main haplotype and by a strong reduction in genetic diversity, as is expected in regions that were rapidly re-colonized by a limited number of migrants when climatic conditions improved. We have ecological evidence that the moth populations are currently experiencing an expansion due to global change (both climate warming and host plantations). However, in the temperate regions of Europe, the natural populations are not genetically structured in space. The contemporary patterns are thus indistinguishable from historical ones as they also consist in progressions of the most widely distributed haplotypes. In contrast, all other sub-clades occur in limited ranges and are strongly structured in space, as is expected in regions that did not experience Quaternary cycles of glaciations. In these areas, genetic diversity has been retained in most populations, and each haplotype is usually found in only one population. The genetic signatures of recent moth introductions/expansions in these regions can be easily detected: recent expansions are characterized by the loss of genetic diversity across whole regions (e.g. Lebanon and Israel), and recent introductions are typified by the existence of closely related haplotypes in geographically distant populations. A strong differentiation is also expected for island populations if the island colonization occurred naturally in geological times. Thus, the occurrence (or not) of a significant 'natural' genetic structure of populations will determine whether or not recent expansions or introductions can be detected in the genetic data.Complementary data based on polymorphic nuclear sequences would now be useful to compare biparental and maternally inherited markers, and to detect how male dispersal may have influenced the global evolutionary history of the species. Finally, our findings could be interesting for pest control as individuals present in different clades or sub-clades may have evolved different ecological characteristics , which can affect pest management strategies. Phenotypic traits should now be measured within each phylogenetic clade and sub-clade and compared between regions to test this hypothesis.Thaumetopoea pityocampa and T. wilkinsoni were collected in 51 different locations from 16 countries in Europe and around the Mediterranean Basin. In addition, data from the 9 populations studied in Salvato et al. [Eggs and larvae of o et al.  and the DNA was extracted using a salting-out procedure . Two mitA partition homogeneity test was performed for the COI and COII fragments using Paup*4b10 . The tesModel selection was performed using a Bayesian framework, through comparison of Bayes factors . In addi6 generations, incrementally heated with T = 0.15. Convergence was assessed by computing the potential scale reduction factor with sump in MrBayes. Differences between Bayes factors obtained from the different models tested, calculated as twice the difference in the logarithm of harmonic means of likelihoods, were compared with reference values from Kass and Raftery [For Bayes factors calculation, likelihoods for a given model were estimated using MrBayes v3.1.2 , and har Raftery .For model performance assessment we chose as discrepancy variable the multinomial test statistics . PosteriRelationships between haplotypes and molecular dating were estimated by Bayesian inference of phylogeny using Beast v1.4.8 . The modFor the most recent nodes, demographic Bayesian analyses were performed separately for each of the identified sub-clades using Beast and including all the sequences of a given group. Assumptions and settings were the same as above, except that coalescent priors of constant size and of exponential growth were used instead of Yule priors, and that two MCMC runs of 100 million steps were performed. tMRCAs of recent sub-clades were estimated assuming a 2% divergence, and must therefore be interpreted as the maximum age for a given sub-clade .The phylogenetic reconstructions allowed us to identify three highly supported monophyletic clades within which a statistical parsimony network was computed using TCS v1.21 . Such a ut [t the expansion time in number of generations, and u the mutation rate per sequence and per generation. This last value was calculated using the relationship u = 2 μk, with μ the mutation rate per nucleotide and k the length of the sequence in nucleotides. The 2% pairwise sequence divergence defined by DeSalle [Gene diversity H and nucleotide diversity per site π were calculated within populations and within previously identified sub-clades. To infer whether each sub-clade has experienced recent population expansions, Tajima's D and Fu's Fs statistics were calculated and tested with DnaSP 4.10 . Mismatcut , τ being DeSalle  was usedCK, LZ and MS analyzed the data, AB, JR and AR planned the research, MS and PS performed the research, CK, LZ and AB wrote the paper and revised the manuscript. All authors read and approved the final manuscript.Sampling sites, geographic coordinates, host pine, collector and haplotype composition of each locality. The number in brackets after each haplotype name is the number of individuals with that haplotype. Codes refer to the localities shown in Figure Pinus brutia; PH: P. halepensis; PM: Pinus mugo/uncinata; PN: Pinus nigra; PP: P. pinaster; PR: Pinus radiata; PS: P. sylvestris; CA: Cedrus atlantica, CD: Cedrus deodara.Click here for file


Anopheles gambiae species complex are the primary vectors of human malaria in sub-Saharan Africa. Many host genes have been shown to affect Plasmodium development in the mosquito, and so are expected to engage in an evolutionary arms race with the pathogen. However, there is little conclusive evidence that any of these mosquito genes evolve rapidly, or show other signatures of adaptive evolution.Mosquitoes of the An. gambiae species complex in both East and West Africa.Three serine protease inhibitors have previously been identified as candidate immune system genes mediating mosquito-Plasmodium interaction, and serine protease inhibitors have been identified as hot-spots of adaptive evolution in other taxa. Population-genetic tests for selection, including a recent multi-gene extension of the McDonald-Kreitman test, were applied to 16 serine protease inhibitors and 16 other genes sampled from the Serine protease inhibitors were found to show a marginally significant trend towards higher levels of amino acid diversity than other genes, and display extensive genetic structuring associated with the 2La chromosomal inversion. However, although serpins are candidate targets for strong parasite-mediated selection, no evidence was found for rapid adaptive evolution in these genes.An. gambiae complex can present special problems for the application of standard population-genetic tests for selection, and this may explain the failure of this study to detect selection acting on serine protease inhibitors. The pitfalls of uncritically applying these tests in this species complex are highlighted, and the future prospects for detecting selection acting on the An. gambiae genome are discussed.It is well known that phylogenetic and population history in the  Plasmodium parasites, Anopheles mosquitoes are a central component of the Malaria crisis. Consequently, there has been a substantial effort to identify the genes involved in the mosquito immune response against Plasmodium, including studies to identify genes associated with variation in vector competence . As expLP , in thes . An. gambiae and An. arabiensis share a more recent common ancestor that either does with An. melas, the lower differentiation between An. gambiae and An. arabiensis could also be explained (at least in part) by differences in effective population size within the complex. Since πs is an estimator of 4Neμ, differences in neutral diversity imply that An. gambiae and An. arabiensis have larger effective population sizes than An. merus, consistent both with their wider geographic range, and with potentially higher levels of shared ancestral polymorphism . Diversity in An. gambiae and An. arabiensis is similar to that seen in African populations of Drosophila simulans and D. melanogaster  . Thus, y) [e.g. ], and asnogaster , i.e. wenogaster ].M and S molecular forms of An. gambiae is a major focus of ongoing research .D was negative), consistent either with population growth, or with weak selection against some synonymous variants. In contrast, however, the data presented here also identify a strong skew toward intermediate frequency alleles in An. gambiae population KY . One potential explanation for this is that population KY is admixed or contains cryptic population structure, for example as would be the case if two divergent lineages of S-form are coexisting there. Alternatively, a positive Tajima's D could also result from a recent decrease in effective population size.Most populations showed a slight skew in the allele-frequency spectrum toward low-frequency variants .An. gambiae population KY, considerable differentiation was identified between 2La and 2L+a homozygotes around the SRPN1, 2 and 3 cluster . For these loci KST = 0.25 between inversion states, which is actually higher than the overall differentiation between An. gambiae and An. arabiensis, and twice as high as the differentiation between M-form and S-form An. gambiae. No such differentiation was seen for other loci on chromosome arm 2L, indicating that this is strongly associated with the inversion. Although chromosomal inversions are in general expected to suppress recombination, especially near breakpoints, this is unlikely to lead to extreme or long-term differentiation unless maintained by selection . In . In An. on . HoweveAnopheles population genetic data? One possibility is to use approaches based solely on within-population diversity to identify recent selective sweeps or regions of elevated polymorphism, and this will work for some loci , thoughe Figure  and prevsquitoes  may holdThe authors declare that they have no competing interests.DJO selected the loci, designed the primers, and performed all PCR, sequencing and analysis. JJW developed the likelihood-based methods for inferring the proportion of adaptive substitutions, and provided statistical support. TJL conceived the project, and all authors contributed to the final manuscript.PCR primers. Locus names, identifiers, genomic locations and PCR primer sequences.Click here for fileCorrelations in genetic diversity between species. The correlation in genetic diversity for loci sampled from Anopheles gambiae and Anopheles arabiensis.Click here for fileGenetic diversity and differentiation. Sheet 1: genetic diversity at synonymous and non-synonymous sites, and Tajima's D statistic for synonymous sites, for all loci and populations. Sheet 2: genetic differentiation  between populations for all loci.Click here for fileMcDonald-Kreitman data. Sample sizes, analysed gene lengths, polymorphisms and fixed differences for each locus, for each pair of speciesClick here for file


Recurrent climatic oscillations have produced dramatic changes in species distributions. This process has been proposed to be a major evolutionary force, shaping many life history traits of species, and to govern global patterns of biodiversity at different scales. During range expansions selection may favor the evolution of higher dispersal, and symbiotic interactions may be affected. It has been argued that a weakness of climate fluctuation-driven range dynamics at equatorial latitudes has facilitated the persistence there of more specialized species and interactions. However, how much the biology and ecology of species is changed by range dynamics has seldom been investigated, particularly in equatorial regions.We studied a three-species symbiosis endemic to coastal equatorial rainforests in Cameroon, where the impact of range dynamics is supposed to be limited, comprised of two species-specific obligate mutualists –an ant-plant and its protective ant– and a species-specific ant parasite of this mutualism. We combined analyses of within-species genetic diversity and of phenotypic variation in a transect at the southern range limit of this ant-plant system. All three species present congruent genetic signatures of recent gradual southward expansion, a result compatible with available regional paleoclimatic data. As predicted, this expansion has been accompanied by the evolution of more dispersive traits in the two ant species. In contrast, we detected no evidence of change in lifetime reproductive strategy in the tree, nor in its investment in food resources provided to its symbiotic ants.Despite the decreasing investment in protective workers and the increasing investment in dispersing females by both the mutualistic and the parasitic ant species, there was no evidence of destabilization of the symbiosis at the colonization front. To our knowledge, we provide here the first evidence at equatorial latitudes that biological traits associated with dispersal are affected by the range expansion dynamics of a set of interacting species. Variation in parameters of the Earth's orbit around the sun has led to recurrent dramatic climatic shifts throughout its history Frangula alnus young populations of central Europe differed from old populations in Spain in lifetime reproductive strategy, fruit morphology, fruit ripening phenology and, as a consequence, in their associated guild of seed dispersers Within species, vagility  is predicted to be favored during range expansion episodes Another predicted consequence of ORD is reduced quantitative importance of co-evolutionary processes. Indeed, different species may expand at different rates, limiting opportunities for continued interactions. Hence strong ORD limits opportunities for long term co-evolution Studies of ORD and its biological consequences within species have focused on temperate-zone species Leonardoxa africana subsp. africana is an ant-plant, which provides its mutualistic ant Petalomyrmex phylax with nest sites in its hollow internodes and feeds it nectar. A numerous worker force continuously patrols and efficiently protects young leaves against insect herbivores Cataulacus mckeyi is an ant that may replace P. phylax and that is parasitic on this mutualism. It feeds on the nectar, uses the hollow internodes but has much smaller numbers of patrolling workers, which confer little or no protection against herbivores We investigate here ORD and its influence on traits associated with dispersal in a narrowly endemic three-species symbiosis of African equatorial forests. The tree EH, variance of allele size, V, or both . Indeed, FST values between populations located less than two kilometers apart often revealed significant genetic differentiation, and this for the three species. Finally, a significant genetic pattern of Isolation By Distance (IBD) was detected for each of the three taxa . Thus, overall differentiation among populations was strong and congruent in all three species, even at short spatial distances, suggesting spatially limited dispersal.Genetic differentiation among populations was marked for each of the three taxa. The global multilocus T≪1/μ and to become apparent for T≥1/μP. phylax (out of 12), and only one for L. a. africana (out of nine) and for C. mckeyi (out of 10) presented statistics of population differentiation based on allele size, RST, significantly higher from those based on allele identity, FST. Divergence among populations is thus probably recent, as stepwise mutation at microsatellite loci contributed to differentiation between populations for only five out of 31 loci. These loci were used to estimate upper bounds of the confidence intervals for dating colonization of the transect, and the other loci were used to estimate the lower bounds. Estimates of the time of divergence in numbers of generations, T, were of the same order of magnitude for the three taxa: 968≤T≪3,104 for P. phylax, 712≤T≪2,503 for C. mckeyi, and 619≤T≪2,774 for their host-plant. Age estimates of the cline, expressed in numbers of generations, are rather similar, although the plant presents much longer generation time than the ants. We suggest that because more polymorphic loci, presenting higher mutation rates than average, were selected during marker development, the calculated upper bounds of the ranges may be overestimates. Whether the homogeneity in the estimates results from a non-random choice of highly polymorphic microsatellites during marker development requires further studies.As we had three repeats of the same history, we inferred a rough estimate of the age of the cline using the difference in behavior among microsatellite markers. The effect of stepwise mutations on population divergence at a locus is predicted to be negligible for a divergence time Overall, the combination of clinal loss of genetic diversity coupled with a genetic pattern of IBD and with recent divergence times between populations for the plant as well as its associated ants, strongly support the hypothesis that the whole system experienced a progressive and relatively recent range expansion towards the south.We investigated variation of phenotypic traits implicated in species interactions that are likely to be selected during range expansion. For the plant we analyzed size at first reproduction and number of foliar nectaries. For the ants we analyzed investment in reproduction versus investment in colony survival, temporal dynamics of occupation of nest sites and variation in traits correlated with capacity to found new colonies.Leonardoxa a. africana showed no evidence of a shortened juvenile period towards the south .The host plant he south . Indeed,he south  but did P = 0.948; rS = 0.021, N = 12 populations). Hence there was no evidence for a clinal variation in food resources available to ants . Thus, this component of investment of trees in production of food for the mutualists seemed to be independent of geographic location along the north/south transect. Hence, we detected no evidence of change in the host plant's phenotype in response to the colonization process.The mean number of observed nectaries per leaf varied little among populations (from 11 to 14) and did not show clinal variation along the transect , but failed to detect clinal variation of turnover of inhabitants in P. phylax inhabited trees  and BOU (among the northernmost) were more thoroughly analyzed. Estimates of relative dry weight of adult females, but not that of adult males or workers, differed markedly between these two extreme populations  . Thus, in P. phylax, size and weight of sexual females increased southward and translated into higher capacity to found new colonies.In addition to variation at the colony level, ulations . The diftion see  increaseons) see , and wasP. phylax and C. mckeyi inhabited trees over five plant patches sampled from one to three years , and the second one followed an arid episode centered 3,000–2,500 years BP. The data on C. mckeyi with an estimate of divergence time along the transect ≪2,500 generations, and a colony mortality of at least 25% per year  along the transect, from populations exhibiting secondarily polygynous mature colonies in the northern part of the study area to populations in the south, only 86 km distant, presenting almost exclusively monogynous colonies P. phylax mostly results from lack of queen dispersal associated with intra-nidal mating, whereas monogyny occurs through outbred queens dispersing and founding new colonies independently from the assistance of workers P. phylax –along with its associated mating system– correlates with a historical process corresponding to a progressive colonization of coastal southern Cameroon. Gyny is one of several life history traits classically associated with dispersal in ants P. phylax queens and a component of their capacity to found new colonies. It is noteworthy that clinal variation in body size was expressed in females but not in males and workers, which do not participate in foundation of new colonies. We note that the effect of wing load ( = body mass per unit wing area) variation on dispersal capability of P. phylax queens needs further study  suggest that they will not flower before reaching tens of years in age.The northern part of the current distribution of L. a. africana, and the population EBO (near the village Ebodjé) is the southernmost known population of L. a. africana. Despite the abundance of the plant and its ant occupants in the vicinity of Ebodjé, neither field nor herbarium research led to the discovery of more southerly populations of L. a. africana. EBO was thus considered to represent the probable front of colonization of the system, and in the following all statistical treatments involving geographic distance along the transect are based on geographic distance (in km) from the southernmost documented occurrence of the three species (population EBO). Unless stated otherwise, to assess the existence of clinal variation of each measure, we tested for a correlation between the spatial distance to EBO and the mean values per population using Spearman rank correlation coefficient (rS). Correlations were tested using the software Analyse-it version 2.12  or the VassarStats web site for statistical computation (http://faculty.vassar.edu/lowry/corr_rank.html).For this study 29 populations, constituting a roughly north-south transect, were sampled for genetic studies, phenotypic studies or both  sampled in 2000–2007, and individual genotypes were inferred using 9 markers amplified following Léotard et al. C. mckeyi was analyzed for 206 diploid individuals from 14 populations  sampled in 2000–2002, and genotypic data from 10 markers were already available for this sample P. phylax was analyzed for 475 diploid individuals from 21 populations  sampled in 2000–2002, and genotypic data from 12 markers were already available for this sample We investigated genetic variation in host plant, parasitic ant and mutualistic ant using nuclear microsatellite markers: (i) Genetic variation in HEVV returns to equilibrium much more slowly than HE. Thus V is expected to enable detection of older (in terms of numbers of generations) colonization processes than is HEnp, the number of polymorphic loci; na, the mean number of observed alleles; and A, the mean allelic richness using the rarefaction method to provide unbiased estimates corrected for unequal sample size Using the software SPAGeDi 1.1 FST values following Weir and Cockerham FST/(1−FST) on pairwise ln-geographical distances For each species, genotypic population differentiation was analyzed for each pair of populations and over all populations by estimating RST = FST) for a divergence time T≪1/μ generations and will become apparent (RST>FST) for T≥1/μ generations. First, the mutation rate at locus i was estimated as −4Vi was the observed average variance of allele size at locus i over all populations and FST) and based on allele size (RST) were compared following Hardy et al. RST and FST to compute a rough lower bound of the time elapsed since the divergence of populations, and we used the average rate of mutation of the other loci to compute a rough upper bound of this time.In an attempt to produce a rough estimate of the age of the cline, we tried to estimate the mutation rate for each locus and then used the hypothesis of Hardy et al. Leonardoxa reached sexual maturity at progressively smaller size when getting closer to the postulated colonization front we measured, for a total of 402 trees in six populations (41 to 126 trees per population), the diameter at the trunk base (in mm) and estimated the total height (in m). We then scored the stage of maturity  of each individual. Indeed Leonardoxa a. africana is cauliflorous and the inflorescences leave detectable scars on the trunk, so that each tree could be categorized as immature versus mature. The relationship between the stage of maturity and geographical distance to the colonization front, controlling for the effect of tree size , was assessed using multiple logistic regression analysis with the program SYSTAT 9. Size of sampled trees and geographical distance were slightly correlated , so we included an interaction term in the statistical model. For each population sample separately we then used multiple logistic regressions to estimate the size at which half the trees had reached sexual maturity and tested whether these estimates varied clinally.Some north-temperate plants are known to reproduce earlier in life in sites in the northern part of their range, far from glacial refugia P. phylax in 12 populations (10–30 trees per population) in 2000–2002. L. a. africana possesses paripinnate leaves with four to eight leaflets. For this analysis missing leaflets were thus considered as bearing no nectaries. This analysis provides an estimate of this component of actual resources per leaf provided by the trees to the ants. In a separate analysis of the dataset, we quantified the mean number of nectaries produced per basal leaflet and we considered missing leaflets as missing data. This analysis provides an estimate of the potential, ontogenetic, investment of the plants in production of food per leaf for ant colonies. We only used the basal leaflets because the number of nectaries varies according to leaflet position, basal leaflets bear the greatest number of nectaries, and basal leaflets are less often missing than those in other positions.To assess spatial variation in a main component of food resources provided by the plants to ant colonies, we counted the number of nectaries per leaf for 1,293 leaves (4–11 leaves per tree) from a total of 161 trees occupied by P. phylax and 75 colonies from 10 populations of C. mckeyi were entirely collected. In P. phylax, colonies become effectively sexually mature when reaching 1,000 workers C. mckeyi the limit was set at 20 workers P. phylax and the number of nymphs plus the number of young, not yet fully melanized imagos for C. mckeyi. Counting nymphs is intrinsically easier and in principle should yield a more precise estimate of investment, but numbers of nymphs were so low in C. mckeyi colonies that too much stochastic noise was added. Therefore the not yet fully melanized imagos  were very carefully counted, thus allowing the reduction of statistical noise in C. mckeyi.It is predicted that, along a colonization front, organisms should invest more in reproduction and hence dispersal at the expense of survival. In both ant species we tested for variation among populations in the production by colonies of new workers, gynes and males. Eighty-six colonies from 13 populations for 10). The log-transformed variables were number of adult workers, number of young workers, , . To control for potential collinearity effects in multiple regressions, we verified that the explanatory variables –number of adult workers and geographic distance– were not correlated in this dataset . In multiple regressions, the partial regression coefficient (yib) denotes the regression coefficient of each explanatory variable on the dependent variable, while removing the effect of all other explanatory variables as though they were kept constant. The standardized coefficient (yi′b) gives the rate of change in standard deviation units of the dependent variable per one standard deviation unit of explanatory variable, and allows direct comparison of the effects of the different variables independently of measurement units.The relationship between colony productivity and geographical distance to the colonization front, controlling for the effect of colony size , was assessed using multiple regression analysis with the program SYSTAT 9 P. phylax, by C. mckeyi or by both species), we used an information matrix derived from recapture histories of nesting sites and the multi-state option in the software MARK The temporal dynamics of occupation of nest sites were investigated by monitoring a total of 1,151 permanently tagged trees in six populations (85 to 442 trees per population) over three to seven years. To describe population-level dynamics of the different occupancy states  and the dry weight of entire individuals. For the five morphometric estimates, we collected data from a total of 1,036 adult female sexuals (10–376 per population) from a total of 256 colonies (2–53 per population) and 21 populations sampled in 1997–2003 and stored in 95% ethanol  from 10 colonies in population EBO (Ebodjé) and from 9 colonies in population BOU (N'kolobondé), two geographically extreme populations along the transect. These 270 individuals were removed from ethanol six months after collection, dried for 4 h at 40°C and weighed individually. Storage in ethanol dissolves some body constituents, such as lipids, so that these data are clearly underestimates but provide relative within-caste estimates of dry weight for these two populations. To test for differences in these relative estimates between these two populations, we performed nested analyses of variance for each caste separately with colony (within population) and population as factors explaining weight variation.P. phylax, colony foundation is initiated by a winged female that disperses from her natal tree, searches for a vacant host-plant, digs a hole in a young twig, removes the pith from its inside, enters the domatium and blocks the entrance hole. The new queen thus confined within a domatium rears the first brood in isolation by claustral founding. To simulate claustral founding, a total of 146 winged females were collected inside colonies between March 30 and April 8, 2002 from ten populations   see . Single Table S1The study populations: location and measures of genetic diversity. Statistics summarizing within-population genetic variation over 9 microsatellite loci for 19 populations of Leonardoxa a. africana, 12 loci for 21 populations of Petalomyrmex phylax, and 10 loci for 14 populations of Cataulacus mckeyi. Distance is the map distance (in km) from the southern edge of the range of the system. N is the number of diploid individuals genotyped per population; np, number of polymorphic loci; na, mean number of observed alleles; A, mean allelic richness; HE, mean expected heterozygosity; and V, mean variance of allele size. The two last rows indicate the means of values over all populations and the correlation between population means and the map distance from the southernmost limit of the range . Data for P phylax are from Dalecky et al. (0.01 MB PDF)Click here for additional data file.Table S2Size at which half the trees had reached sexual maturity. Estimated values [and 95% confidence interval] across trees per population and over all individuals of basal trunk diameter and tree height at which the probability of flowering is 0.50 based on logistic regressions. Populations are arranged in descending order according to their geographical distance from the southernmost known limit of the range of the system (these distances are given in (0.00 MB PDF)Click here for additional data file.Table S3Tree investment in the feeding of ants. Mean (±standard deviation) values across trees per population of number of (1) observed nectaries per leaf and of (2) produced nectaries per basal leaflet. Populations are arranged in descending order according to their geographical distance from the southernmost known limit of the range of the system (these distances are given in (0.00 MB PDF)Click here for additional data file.Table S4Temporal dynamics of occupation of nest sites by ants. Maximum-likelihood estimates [and 95% confidence interval] of annual survival probabilities of host-plant occupancy for Petalomyrmex phylax and Cataulacus mckeyi in six populations ordered from north to south (computed using Mark software). Only the probabilities of continued occupancy of a tree by the same species are presented here. The last row indicates the correlation between population estimates and the spatial distance from the southernmost limit of the range .(0.00 MB PDF)Click here for additional data file.Table S5Geographic variation in P. phylax queen size and potential founding capacity. Within population average (mean±standard deviation) of size and survival of alate female sexuals of Petalomyrmex phylax from 21 populations: head length (HL), head width (HW), partial forewing length (PWL), forewing width (WW), total forewing length (TWL) (mm), dry weight (DW) (in mg), and survival at 60 days under claustral foundation conditions (S60 days). N is the number of individuals, n is the number of colonies. Populations are arranged in descending order according to their map distance from the southernmost known limit of the range of P. phylax. The last row indicates the correlation between population means and the spatial distance from the southernmost limit of the range .(0.01 MB PDF)Click here for additional data file.Table S6Petalomyrmex are larger in the south. Comparison of estimated dry weight (mg) of adult individuals between the two geographically extreme populations  according to caste; descriptive statistics and results of ANOVAs. ns: not significant, *: P<0.05, **: P<0.01, ***: P<0.001).Alate female (0.04 MB RTF)Click here for additional data file.


Mirounga leonina, remains were found along the Victoria Land Coast (VLC) in the Ross Sea, Antarctica, 2,500 km from the nearest extant breeding site on Macquarie Island (MQ). This habitat was released after retreat of the grounded ice sheet in the Ross Sea Embayment 7,500–8,000 cal YBP, and is within the range of modern foraging excursions from the MQ colony. Using ancient mtDNA and coalescent models, we tracked the population dynamics of the now extinct VLC colony and the connectivity between this and extant breeding sites. We found a clear expansion signal in the VLC population ∼8,000 YBP, followed by directional migration away from VLC and the loss of diversity at ∼1,000 YBP, when sea ice is thought to have expanded. Our data suggest that VLC seals came initially from MQ and that some returned there once the VLC habitat was lost, ∼7,000 years later. We track the founder-extinction dynamics of a population from inception to extinction in the context of Holocene climate change and present evidence that an unexpectedly diverse, differentiated breeding population was founded from a distant source population soon after habitat became available.Environmental change drives demographic and evolutionary processes that determine diversity within and among species. Tracking these processes during periods of change reveals mechanisms for the establishment of populations and provides predictive data on response to potential future impacts, including those caused by anthropogenic climate change. Here we show how a highly mobile marine species responded to the gain and loss of new breeding habitat. Southern elephant seal,  In order to understand how biodiversity is generated and maintained over time, we need to understand the process by which populations form and diverge. Natural variation within species is typically partitioned among populations, which sometimes forms the basis for speciation events. One mechanism for the establishment of novel variation at the population level is through a response to emerging habitat. Here we use data from ancient DNA to show how elephant seal populations responded when new breeding habitat was gained and then lost over the course of approximately 7,000 years. We show that the seals quickly took advantage of newly available breeding habitat, far from the nearest extant breeding site, and that a highly diverse and genetically differentiated population was established over a matter of generations. The key factors were likely the abundant local food resource and extensive physical habitat that allowed rapid expansion after the initial founder event and a tendency for females to return to annual breeding sites in this species. Tracking the founder-extinction dynamics of historical populations provides insight into the likely implications of future environmental change. This is an important tool in our efforts to mitigate the impact of human-induced change. Populations can respond to changing habitats by adapting , moving , by adjusting population size, or some combination of the above. Both natural selection and genetic drift can shape populations as they evolve in this context. It is unusual for there to be an opportunity to track population dynamics and genetics during extended periods of environmental change, but a now extinct population of elephant seals from the Victoria Land Coast (VLC) in the Ross Sea, Antarctica provides such an opportunity. Here we investigate the demographic and population genetic consequences of emerging breeding habitat suitable to this species in an environment rich in resources, and the impact of the eventual loss of that habitat.Open beach along the VLC was probably released around 8,000–7,500 YBP, based on the grounding-line retreat of the West Antarctic ice sheet Southern elephant seals have a circumpolar distribution, breeding on sub-Antarctic (north of the Antarctic convergence) and Antarctic (south of the Antarctic convergence) islands . They arA large number of exceptionally well-preserved skin samples and mummified remains, including pups, were collected from raised Holocene beaches along the VLC, adjacent to the Ross Sea , from a We utilise these data to track the founder-extinction dynamics of an ephemeral population from inception to extinction in the context of Holocene climate change. We test the hypothesis that the release of new habitat in a highly productive environment can lead to the rapid founding and expansion of a new population, and investigate the consequences with respect to genetic diversity and gene flow among populations. We further investigate the consequences of the loss of this habitat on genetic diversity and connectivity.−7 (−9–2.06×10−6) substitutions per site per year (s.s.yr−1). This rate is in agreement with estimates obtained from various other aDNA datasets −7 s.s.yr−1 calculated over a similar timeframe  from Adelie penguin aDNA −7 s.s.yr−1) Ancient DNA extractions were successful for approximately 90% of samples trialled. From 223 successfully sequenced ancient VLC sample extractions, 177 haplotypes were identified, whereas from 49 MQ samples, 16 haplotypes were identified , all metρ value estimates for that clade. Two of these were sampled from the VLC site, while one was sampled from MQ. Coalescent theory predicts that ancestral haplotypes will be basal and/or central within the haplogroup ρ value estimates identify MQ as the most likely source of the VLC samples . One possible interpretation would be increasing contact between VLC and MQ. These significant φST values also show that VLC must have become an independent breeding population, and was not merely or predominantly a geographically distant moulting haul-out site. Results using an isolation with migration model Recent back migration to MQ can be estimated by examining for the presence of MQ lineages that are most likely to have evolved at VLC , that isNe), provided more equivocal results. There is only a slight indication of expansion when VLC is considered on its own . Only the VLC>1,000 YBP and MQ samples had non-overlapping distributions for π according to the analysis in DNASP , and much lower estimates for MQ and the putative ancestor population ; Ancestral: 13,263 ) ) . This ma22,827)) . Calcula22,827)) .Effective management and mitigation of the impact of climate change on biodiversity requires an understanding of how populations are likely to respond to the consequent gain and loss of critical habitat. Predictive models One of our key hypotheses was that a highly mobile species can respond to distant habitat gain and loss through migration. All potential breeding habitat on the Antarctic mainland for this species was likely under ice during the last glacial period, so the VLC population must have been established sometime after the last glacial maximum. Geological evidence suggests that the timing of this would have been possible around 8,000 YBP We therefore investigated this aspect of the relationship between VLC and MQ in considerable detail. One possible interpretation is that both VLC and MQ were derived historically from a third population. However, all major extant populations were sampled for this study, and none are possible candidates due to their genetic distance from the VLC. Therefore, this hypothetical population would have to be extinct today. The only known possible candidate would be the population from the north-western coast of Tasmania, extinct since prehistoric times If both VLC and MQ had been derived as founders from an unsampled historical population, both should show evidence for expansion, though only VLC does . From the phylogenies, you would expect to see some MQ haplotypes as derived tips within the network, and this pattern is clearly evident. Furthermore, these are primarily derived from the more recent VLC samples  or population expansion (such as Tajima's D) A further consideration with IM is the assumption that the two populations being compared were sampled at the same point in time. While this is not the case for comparisons between VLC and MQ, the chronological results from this analysis are nevertheless consistent with those obtained using other methods and with the geologic record. This also applies to other analyses that involve the incorporation of different chronologies into estimates of population differentiation (such as φST data (for example) show that MQ and VLC are differentiated. However, empirical data and simulation studies suggest skyline plots are robust to violations of assumptions of panmixia Further support for the proposed pattern of demographic expansion and contractions at VLC comes from the BSP profile shown in Ne was lower . Again, this likely reflects a large  and if some level of gene flow was maintained . The more recent, shared TMRCA among the other populations is perhaps more unexpected see . One posST values throughout the relevant time period, the distinct mismatch distribution profile, and IM results for time of splitting). The genetic data clearly distinguishes the VLC population from a site used exclusively for moulting, which could have represented the temporary assemblage of seals from one or more separate breeding populations. The implication is that discovery of emergent habitat was facilitated by long-distance foraging excursions, and that some level of continued dispersal between sites allowed the return of a proportion of the founder population to the source colony, when the new habitat was lost many generations later. This type of long distance networking may be possible for other species of conservation concern at times of rapid environmental change. In contrast, species with a more local distribution and range may be less able to cope. For example, Arctic foxes from midlatitude Europe went extinct during the Pleistocene when available habitat contracted Taken together, our results indicate that new habitat was quickly exploited, and that the founder population became independent of the distant source population , 10 mM Tris-HCL, and 50 µl Proteinase K, at 50°C overnight. Digests were centrifuged at 13,000 g for 1 min, the aqueous phase was removed, and added to 5 volumes of PB Buffer (QIAgen) and vortexed. This solution was passed in 700 µl quantities through QIAquick spin filter columns (QIAgen), centrifuged at 13,000 g for 1 min, and the flow-through discarded. DNA was then twice washed with 700 µl of Buffer PE (QIAgen), centrifuged as before, and the flow through discarded. The filter column was placed in a sterile 1.5 ml tube, and DNA was eluted by applying 35 µl of Buffer EB (QIAgen) to the column. This was then centrifuged at 13,000 g for 1 min. DNA samples were stored at −20°C until required. DNA extractions were performed in batches of 20 samples with 1–2 blanks (no tissue) per batch.Taq Hi-Fidelity, 1×buffer, 1.5 mM MgCl2 , 0.2 µM each primer and 200 µM each dNTP. BSA was found to be inhibitory, possibly due the presence of hairs in the elephant seal skin samples 5′-GCTGACATTCTACTTAAACT-3′ & SESanc3r 5′-ATGTACATGCTTATATGCAT-3′; SESmdbf 5′-AGCCCTATGTATATCCTGCATT-3′ & SESmdbr 5′-CAGTATAGAAACCCCCACATGA-3′. Purified PCR products were sequenced at Macrogen (S. Korea) and at the University of Durham. There was no variation among replicates for a given sample. During cloning and sequencing, no evidence of nuclear copies was detected, while one sample displayed evidence of damage and was removed from further analyses, consistent with the generally excellent preservation of these ‘freeze-dried’ Antarctic samples.PCR reactions (25 µl volumes) contained: 2 µl extract, 1 U Platinum Radiocarbon dating of 223 ancient VLC samples was by Accelerator Mass Spectrometry (AMS) at the NOSAMS laboratory at Woods Hole Oceanographic Institution and at the University of Arizona. All dates were calibrated using the CALIB n = 49; Marion Island n = 48) or data incorporated from earlier studies using Genbank sequences  5′-TGACATCCATCCCCCTTATT-3′ and SESMR: 5′-TGTGTG ATCATGGGCTGATT-3′) were designed to amplify a 440 bp fragment encompassing the entire aDNA target fragment.Modern southern elephant seal samples were analysed after completion of all aDNA work (Macquarie Island http://www.fluxus-engineering.com) ρ value estimates ρ value estimates were then calculated between ancestral VLC haplotypes and the seven modern population haplogroups, representing all major extant breeding colonies . An implicit assumption in these analyses is that no alternate potential source population  has gone to extinction, and thus remains unsampled in the current study.Phylogenies Bayesian coalescent analysis using Markov Chain Monte Carlo (MCMC) was implemented in the program BEAST v. 1.4.8 and v. 1.5.b To ensure the substitution rate estimate was not merely an artefact of the dates provided, i.e. that the aDNA sequences used in this study were rate informative, these analyses were repeated three times by randomly re-assigning calendar ages to sequences. A recent re-analysis NASP v. 10.4.9 S over the average number of pairwise nucleotide differences k describes the ‘expansion coefficient’ NASP was also used to compare genetic diversity estimates for nucleotide diversity. We used 1,000 coalescent simulations to derive 95% confidence intervals for these estimates, in order to assess whether the distributions overlapped (with non-overlapping distributions providing a conservative estimate of significant differentiation). Tajima's DFsFs is less conservative than Tajima's D, and is expected to be more sensitive to large population expansions . Mismatch distributions ST  using Rarefactor . An Isolation-with-Migration model was implemented in IM Ne estimates . Radiocarbon dates were converted to calendar years.(1.49 MB TIF)Click here for additional data file.Figure S2Isolation-with-migration (IM) output of population genetic parameters for the Macquarie Island (MQ), Victoria Land Coast (VLC) and Ancestral “populations.” See (8.75 MB TIF)Click here for additional data file.Figure S3BEAST output of Time-to-Most-Recent-Common-Ancestor (TMRCA) estimates (YBP). For: all modern populations grouped (besides Macquarie Island) (blue), Macquarie Island (MQ - yellow), and Victoria Land Coast (VLC - red).(1.49 MB TIF)Click here for additional data file.Figure S4BEAST output of southern elephant seal substitution rate estimated from 223 randomized radiocarbon dated samples from Victoria Land Coast (VLC). Radiocarbon dates were converted to calendar years. Dates were randomly assigned to sequences to assess whether the non-randomized ages and sequences were rate informative. These analyses were repeated three times, and all randomized rate estimates were highly skewed to zero.(1.99 MB TIF)Click here for additional data file.Table S1Sample location and age.(0.32 MB DOC)Click here for additional data file.Table S2Isolation-with-migration (IM) output of population genetic parameters for the Macquarie Island (1), Victoria Land Coast (2) and Ancestral (a) “populations.” See (0.04 MB DOC)Click here for additional data file.Table S3ST = pairwise estimate between MQ and VLC “subset.” % TMRCA = subset age interval (%) relative to the time-to-most-recent-common-ancestor of the VLC sample .Parameter comparisons against chronology subsets for VLC. Φ(0.03 MB DOC)Click here for additional data file.