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Detecting interspecific and geographic differentiation patterns in two interfertile oak species (Quercus petraea (Matt.) Liebl. and Q. robur L.) using small sets of microsatellite markers

Genetic analysis was carried out in order to provide insights into differentiation among populations of two interfertile oak species, Quercus petraea and Quercus robur. Gene flow between the two species, local adaptations and speciation processes in general, may leave differential molecular signatures across the genome. Three interspecific pairs of natural populations from three ecologically different regions, one in central Europe (SW Germany) and two in the Balkan Peninsula (Greece and Bulgaria) were sampled. Grouping of highly informative SSR loci was made according to the component of variation they express - interspecific or provenance specific. Species and provenance discriminant loci were characterized based on FSTs. Locus specific FSTs were tested for deviation from the neutral expectation both within and between species. Data were then treated separately in a Bayesian analysis of genetic structure. By using three species discriminant loci, high membership probability to inferred species groups was achieved. On the other hand, analysis of genetic structure based on five provenance discriminant loci was correlated with geographic region and revealed shared genetic variation between neighbouring Q. petraea and Q. robur. Small sets of highly variable nuclear SSRs were sufficient to discriminate, either between species or between provenances. Thus, an effective tool is provided for molecular identification of both species and provenances. Furthermore, data suggest that a combination of gene flow and natural selection forms these diversity patterns. Species discriminant loci might represent genome regions affected by directional selection, which maintains species identity. Provenance specific loci might represent genome regions with high interspecific gene flow and common adaptive patterns to local environmental factors.

Genetic analysis was carried out in order to provide insights into differentiation 5 among populations of two interfertile oak species, Quercus petraea and Quercus 6 robur. Gene flow between the two species, local adaptations and speciation 7 processes in general, may leave differential molecular signatures across the genome. 8 Three interspecific pairs of natural populations from three ecologically different 9 regions, one in central Europe (SW Germany) and two in the Balkan Peninsula 10 (Greece and Bulgaria) were sampled. Grouping of highly informative SSR loci was 11 made according to the component of variation they express -interspecific or 12 provenance specific. 'Species' and 'provenance discriminant' loci were characterized 13 based on F ST s. Locus specific F ST s were tested for deviation from the neutral 14 expectation both within and between species. Data were then treated separately in 15 a Bayesian analysis of genetic structure. By using three 'species discriminant' loci, 16 high membership probability to inferred species groups was achieved. On the other 17 hand, analysis of genetic structure based on five 'provenance discriminant' loci was 18 correlated with geographic region and revealed shared genetic variation between 19 neighbouring Q. petraea and Q. robur. Small sets of highly variable nuclear SSRs were 20 sufficient to discriminate, either between species or between provenances. Thus, an 21 effective tool is provided for molecular identification of both species and 22 provenances. Furthermore, data suggest that a combination of gene flow and natural 23 selection forms these diversity patterns. 'Species discriminant' loci might represent 24 genome regions affected by directional selection, which maintains species identity.

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'Provenance specific' loci might represent genome regions with high interspecific 26 gene flow and common adaptive patterns to local environmental factors. 27 28 29 KEY WORDS: Microsatellites, genetic differentiation, genetic structure, genetic 30 introgression, 'species discriminant loci', 'provenance discriminant loci'. 31 32 Since Darwinian times, oaks have served as a model genus for studying evolutionary 3 processes and speciation. High adaptability and high levels of interspecific gene flow 4 were among the features which significantly contributed to the genesis of several 5 hundreds of species, subspecies and ecotypes (Kleinschmit, 1993;Petit et al., 2004;6 Stebbins, 1950). Species identification and characterization have often been a 7 challenge since interspecific barriers are very weak. In recent years, a growing 8 number of studies support the notion that differentiation is restricted to some 9 limited genomic hot-spots, while the rest of the genetic information remains shared 10 between related taxa (Bodénès et al., 1997b;Curtu et al., 2007a; Muir and 11 Schlötterer, 2005). 12 13 Q. petraea and Q. robur are the most important oak species in central Europe, both 14 economically and ecologically. They are interfertile and have widely overlapping 15 distribution range. Within this range, Q. robur grows on humid soils with high 16 nutrient availability, while Q. petraea rather occupies dry and more acidic sites 17 (Rushton, 1979). However, the two species often occur in sympatry and hybridization 18 under natural conditions is common (Bacilieri et  differentiation within and between the two species using eight interspecific pairs of 39 neighboring stands located across Europe. Interestingly, they described one locus 40 (B11-1500) exhibiting extremely low interspecific differentiation between 1 neighbouring populations, while differentiation among populations from different 2 geographic locations was significant. A combination of interspecific gene flow and 3 adaptation to heterogeneous site conditions may account for this result. 4 5 In our study, we aimed to capture the genetic imprints of the aforementioned 6 evolutionary processes. By choosing neighbouring stands of Q. petraea and Q. robur 7 from three different regions along an ecological gradient we aimed to observe the 8 differential effects of gene flow and local adaptations on several genomic loci. We 9 aimed to partition the loci according to the component of genetic variation they 10 include. First, loci with consistently high interspecific differentiation may represent 11 genomic regions which reflect species' ecological requirements and the respective 12 adaptive profiles. Second, loci which vary strongly among regions, but show low 13 interspecific differentiation at the local scale may indicate adaptations to the 14 regional conditions, which are common between the two species and transferable 15 through gene flow. shown in Figure 1. In order to estimate aridity we used both Thornthwaite's 39 precipitation effectiveness index (Thornthwaite, 1931) and De Martonne's aridity 40 index (De Martonne, 1926), which are based on precipitation and mean annual 1 temperatures. A thorough discussion about the use of climatic parameters and 2 indices in plant geography is included in Tuhkanen (1980). The respective formulas 3 were used to calculate values for each stand: (1) PE= Σ n=1 12 [115*[p n /(t n -10)] 10/9 ], 4 where PE= Thornthwaite's precipitation effectiveness index, p n = mean monthly 5 precipitation in inches, T= mean monthly temperature in °F, (2) H= P/(T+10), where 6 H= de Martonne's aridity index, P= mean annual precipitation in mm, T= mean 7 annual temperature in °C. The latter index can be used both for annual or seasonal 8 data (Tuhkanen, 1980). We made the respective calculations by using means of the 9 months July-September in order to estimate summer drought. In all cases, lower 10 values indicate increased aridity. For the calculations we used climatic data from 11 meteorological stations in the vicinity of each stand for the period 1961-1990. In 12 general, data show an increasing aridity towards south, especially concerning the 13 summer months ( Scoring errors tested, included large allele drop-off and error due to stuttering. In 40 the first case, large-sized alleles are poorly amplified during PCR, thus leading to 1 homozygote excess of small-sized alleles. Scoring errors due to stuttering result in a 2 deficiency of heterozygote individuals differing by a single repeat. Finally, in the case 3 of 'null alleles', there is an overall significant homozygote excess. One thousand 4 randomizations of alleles within each locus and population were applied for the 5 tests. Frequencies of such 'null alleles' were calculated according to Van Oosterhout 6 et al. (2006). When tests supported presence of null alleles in more than two 7 populations, loci were removed from further analyses, since they could lead to an 8 overestimation of the homozygosity in a population. 9 10 Subsequently, we calculated population diversity measures. Observed and expected 11 heterozygosity were calculated using the GenAlEx 6.1 software (Peakall and Smouse, 12 2006). We additionally used the FSTAT software (Goudet, 1995) to calculate allelic 13 richness after rarefaction for each population and locus, which is a diversity measure 14 independent of the population size (Petit et al., 1998 thus be assigned to more than one subpopulation, given that Q. petraea and Q. 22 robur are interfertile (Falush et al. 2003). Furthermore, we used the correlated allele 23 frequency model. For the main data analysis we applied 100,000 updates of the 24 Markov chain and 100,000 MCMC iterations. We ran our data for K=1…6. Ten runs 25 were performed for each K. Maximum posterior probability (lnP(D)) and minimum 26 deviation between runs were used to infer ΔΚ, a statistic based on the rate of change 27 of lnP(D) between successive K values and its variation among runs. ΔΚ corresponds 28 to the second order rate of change of lnP(D) for a given value of K and more 29 accurately detects the uppermost hierarchical level of structure, providing an 30 accurate estimation of population clustering (Evanno et al., 2005). 31 32 33 3. RESULTS 34 35 3.1. Population diversity and differentiation 36 37 High allelic richness and heterozygosity were generally observed in our samples. and between 4.29 and 28.08 among populations of Q. robur (Tables 2, 3). Significant 2 inbreeding coefficients (F IS ) due to homozygote excess were observed in several 3 cases. This was mostly due to 'null alleles', as supported by the results from the 4 Microchecker software. There was no evidence about large allele drop-out or scoring 5 errors due to stuttering. Loci MSQ4, QM50-3M, QpZAG46, QrZAG39, 73 and 75 were 6 rejected from further analyses, since test for 'null alleles' was significant in more 7 than two populations. 'Null allele' frequency at these loci varied between 5 and 33% 8 ( Tables 2, 3 The 14 remaining loci presented generally non-significant F IS values with a few local 18 departures. Three loci exhibited high frequency of one allele and very low diversity in 19 one species, whereas they remained highly variable in the other (Figure 1) among populations of the same species for these loci. Therefore, henceforth we call 32 these loci 'species discriminant'. Allele frequency diagrams indicate that high levels 33 of interspecific differentiation are due to a high degree fixation of a specific allele in 34 one species, while diverse frequency patterns are kept in the other (Figure 2).

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Conversely, for another set of five loci we observed higher F ST values among 37 populations of the same species, than between species in general (Table 4) frequencies at this locus reveals that allele "153" occurs in high frequencies in both 2 species in the two German populations, while its frequency decreases towards 3 Greece irrespective of the species (Figure 3). Moreover, frequency gradients for this 4 and for other alleles can be observed along the three areas and patterns are mostly 5 shared between the two species for a specific sampling region. Loci with similar 6 behaviour 7 were QpZAG1/5, 15, 110 and QrZAG87. Hereafter, we call all five loci with higher 8 intraspecific than interspecific differentiation 'provenance discriminant'. 9 10 Subsequently, we tested the aforementioned F ST -values against the evolutionary 11 neutral expectation within and between species. The observed F ST value exceeded 12 the neutral expectation significantly in only one case. In particular, locus QrZAG96 13 possessed a probability of 0.999 of being equal or larger than the expected value 14 when we compared the two species (Table 5). Two more loci, QrZAG30 and 15 QrZAG112 displayed high P-values between species (0.952 and 0.935 respectively) 16 although not being outliers. No F ST -outliers with P-values higher than 0.975 were 17 found at the intraspecific level (by testing F ST -values among populations within 18 species). In two cases -QpZAG9 in Q. petraea and QrZAG104 in Q. robur -19 differentiation was lower than the expected under selectively neutral conditions 20 with P-values being less than 0.025. Low differentiated loci were more common at 21 the interspecific level. P-values lower than 0.025 were found at several loci (Table 5). 22 All five 'provenance discriminant' loci were less differentiated than expected under 23 selective neutral conditions. At the same time, these loci were more differentiated 24 within species with P-values exceeding 0. 8  Initially, all loci were jointly used for calculating proportions of membership to 35 assumed K subpopulations. The rate of change of data posterior probability (lnP(D)) 36 between successive runs was maximum for K= 2, giving the highest posterior 37 probability of K (Figure 4). Two homogenous subpopulations, corresponding to the 38 two species, were detected, when we set K= 2. Proportions of membership of each 39 one of the original populations to the modeled subpopulations were high and varied 40 between 0.977 and 0.988 for the Q. petraea populations and between 0.970 and 1 0.990 for Q. robur ( Figure 5). In the whole sample (288 individuals) five trees 2 presented individual membership proportions to an inferred cluster lower than 0.85 3 (two Q. petraea and two Q. robur from Bulgaria, and one Q. robur from Germany). 4 Three designated Q. robur and seven designated Q. petraea individuals showed 5 slightly admixed genotypes (membership proportion to the respective cluster 6 between 0.85 and 0.95; results not shown). The respective number for Q. petraea 7 was seven. By setting K= 3, clustering pattern was not consistent among the ten runs 8 performed. For K= 4, within species structure was observed for both Q. petraea and 9 Q. robur. The two German populations were genetically distinct (proportion of 10 membership to separate groups 0.962 and 0.944 respectively; Figure 6). The Greek 11 and the Bulgarian Q. petraea populations were classified in the same cluster 12 (proportion of memberships 0.968 and 0.946 respectively). Similarly, in Q. robur the 13 Greek and the Bulgarian populations were sorted to the same cluster (proportion of 14 memberships 0.967 and 0.918 respectively). Interestingly, the statistic ΔΚ presented 15 a secondary peak for K= 4, supporting an additional structure among provenances 16 (Figure 4). By setting K≥5 we were not able to receive interpretable results.

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Second, we analyzed population structure using the 'species discriminant' loci 19 QrZAG30 The number of such individuals varied between 16 and 17 in Q. petraea and between 28 four and six in Q. robur, with the results differing slightly among runs. For K= 3 and 29 K= 4 structures were weaker, but generally followed the same patterns as when all 30 markers were used (results not shown). Posterior probability as revealed by ΔΚ was 31 again highest for K= 2 (Figure 4).

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Third, we conducted analysis using loci QpZAG1/5, 15, 110, QrZAG87 and 101 which 34 exhibited higher differentiation within species than between the two species. Results 35 appeared to be remarkably different than in the former two cases. For K= 2, the 36 detected genetic structures did not correspond to the two species, but more to the 37 different geographic regions. Both Q. petraea and Q. robur from Germany displayed 38 relatively high proportions of membership to the same subpopulation (0.942 and 39 0.940; Figure 5). Respectively, Q. petraea and Q. robur from Greece could be 40 classified to the same inferred "Balkan" cluster with values between 0.928 and 1 0.908. The population of Q. petraea from Bulgaria showed also high affinity to the 2 same cluster (0.938), while Bulgarian Q. robur was more admixed and showed a 3 membership proportion of 0.717 to the same cluster. A higher degree of admixture 4 was also detected from the analysis of individual probabilities. On average, thirty 5 nine individuals possessed admixed genotypes with membership proportions below 6 0.85 (results not shown). Results for K≥3 were not informative ( Figure 6). Posterior 7 data probability was highest for K=2, suggesting that these five loci share common 8 geographic structures between the two species, while interspecific differentiation is 9 not detectable (Figure 4). 10 11 12 4. DISCUSSION 13 14 Interspecific and intraspecific differentiation patterns in the closely related and 15 interfertile Q. petraea and Q. robur were investigated using a highly informative set 16 of microsatellite loci. We were able to discriminate clearly between the two species 17 using populations from different regions -refugial and non-refugial. Furthermore, 18 genetic structures within species were revealed from our data, providing evidence 19 about provenance differentiation and local common structures between the two 20 species, based on a group of 'provenance discriminant' loci. By partitioning our set of 21 loci we could detect distinct diversity patterns for specific groups of loci. This might 22 reflect the differential effects of speciation and local adaptation processes across the 23 genome of both species.

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Observation of the genetic structures with use of 14 microsatellite loci distinctly 26 separated our samples to the species categories. Additional substructures were 27 revealed within the two main species groups. However, splitting into main species 28 structures and regional substructures was the result of the differential contribution 29 of 'species' and 'provenance discriminant' loci. Partitioning of our marker set gave 30 insights into the aforementioned differentiation components.

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A common feature of the three 'species discriminant' loci is the high frequency of 33 one allele in only one of the two species, resulting to high interspecific F ST values.

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Fixation of alleles favoured by selection and variability reduction at neighbouring 35 hitchhiked genome areas is well recognized as the result of directional selection for a 36 specific adaptive trait (Andolfatto 2001;Futuyma, 1998). Allele fixation can also be 37 the result of genetic drift. However, diversity reduction due to genetic drift affects 38 the genome rather uniformly (Schlötterer 2002). In our case, allele "135" at locus 39 QrZAG96 shows over 80% frequency in all Q. robur populations compared to 2.1% 40 over all Q. petraea populations, where the locus remains highly variable. On the 1 other hand, loci QrZAG30 and 112 display high frequencies of a specific allele in Q. 2 petraea remaining variable in Q. robur. In general, selection for one adaptive trait 3 leads to hitchhiking, thus affecting the allele frequencies of neighbouring neutral 4 marker loci in a wider genome area (Andolfatto, 2001). Interestingly, the F ST -outlier 5 locus QrZAG96 resides within a genomic area associated with a morphological QTL 6 (petiole length as a ratio of the total leaf and petiole length), which is strongly 7 discriminative between Q. petraea and Q. robur as well, causing reduction of heterozygosity and high frequencies of specific alleles. 10 At locus QrZAG30 allele "170" was the most frequent in Q. petraea (56.7%), while it 11 did not occur in any of the Q. robur individuals. We point out that frequency patterns 12 of our 'species discriminant' loci are highly consistent throughout our study area, 13 although ecological conditions and historical events are significantly different 14 between the regions examined. The three loci apparently represent genome areas 15 accounting for maintaining species integrity.

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The aforementioned results agree with the findings of many previous studies 18 supporting that differentiation between Q. petraea and Q. robur is restricted to 19 some specific genome regions, while, in contrast, the rest of the genome is three markers for carrying out Structure analysis in our study has been proved to be 23 very effective in order to distinguish between species. Membership proportion to 24 inferred groups corresponding to species using only the three 'species discriminant' 25 microsatellite loci (QrZAG30, 96 and 112) was almost as high as the respective by 26 using all of fourteen loci.

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The same genomic pattern of differentiation occurs in many examples of interfertile 29 species from the plant and the animal kingdoms. Selection against migrants at 30 specific QTLs hampers the exchange of genetic information at these loci and 31 hitchhiked areas around them, where F ST outlier loci can be found. Wang et al.

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(1997) and Nagy (1997) present results from hybrid zones among hybridizing 33 Artemisia and Gilia subspecies, respectively, which are adapted to divergent 34 environments and where hybrids demonstrate limited adaptability. Via and West 35 (2008) described the same patterns in two partially reproductively isolated host 36 races of pea aphids and revealed that hitchhiked areas can be extended up to more 37 than 10cM. On the other hand, it has been shown that the rest of the genome of 38 interfertile species can be susceptible to free exchange of neutral or mutually 39 beneficial allelic variants through gene flow between them leading to common 1 adaptations ( Barton and Gale, 1993).
In our study, use of loci that displayed high intraspecific differentiation for Bayesian 4 analysis demonstrated common structures between neighbouring populations of the 5 two species. Clusters derived from five 'provenance discriminant' loci corresponded 6 to the German and the Balkan provenances and no matching of the clusters to the 7 two species could be observed. Differential adaptation to geography and ecological 8 gradients might have contributed to the formation of common genetic structures 9 between Q. petraea and Q. robur. Variation clines in oak have been described among 10 others for bud burst, growth and stem form, and frost hardiness (Kremer et al., 11 2002). Similar variation patterns in adaptive genes have also been described in forest 12 trees. For instance, Ingvarsson et al. (2006)  from ecologically equivalent areas of other refugia would shed more light to this 39 aspect. A further perspective is to extend the study using more marker loci in order 1 to understand better the genomic architecture within and between the two species. 2 3 4 5. CONCLUSIONS 5 6 In conclusion, by using 14 nuclear microsatellite loci we were able to provide diverse 7 information about differentiation patterns within and between Q. petraea and Q. 8 robur using populations from Central Europe and the Balkans along an ecological 9 gradient with increasing aridity towards south. The inclusion of the refugial area of 10 the Balkan Peninsula, which harbours significant levels of biodiversity, is considered 11 as an additional asset of this research. Restrictive use of two groups of loci -'species 12 discriminant' and 'provenance discriminant' -allowed discrimination of both species 13 and provenances. would be necessary to provide results with higher resolution and higher 26 probabilities. The remaining six SSR loci seem to express "permeable" genome 27 regions subjected to interspecific gene flow, which is supported by the high degree 28 of individual and population admixture.

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Population studies that utilize adaptive marker loci (e.g. SNPs; Scotti-Saintagne et al., 31 2004a) have had a limited impact to the description of selection and adaptation 32 processes in natural populations of Q. petraea and Q. robur. Highly variable nuclear 33 microsatellite loci remain a very useful tool for studying genetic diversity and 34 structure, as well as for gathering information about adaptive processes. Our study 35 points towards the use of sets of microsatellite loci as effective tools in provenance 36 and species identification, which reduce both time and cost of the analysis. Use of 37 such 'species' and 'provenance discriminant' markers with high resolution and a 38 probable adaptive background makes a significant contribution to management and 39 conservation of the two species. Finally, focusing on microsatellite loci with 40 differential frequency patterns could lead to a targeted study of adaptive loci, aiming 1 to elucidate the underlying evolutionary mechanisms in natural populations.
We owe special thanks to Radko Babakov, Nour Alhammoud and Barbara Madariaga 7 for their contribution to the samplings and laboratory analyses. We gratefully 8 acknowledge the assistance of Dimitris Tampakiotis  These loci exhibit high interspecific differentiation. For markers QrZAG30 and 112 3 alleles "170" and "85" occur with high frequencies in Q. petraea (56.7 % and 85.9 % 4 respectively), while Q. robur shows a more diverse pattern. Allele QrZAG30 is 5 completely absent from Q. robur. Conversely, at QrZAG96 allele "135" tends to be 6 fixed in Q. robur (frequency 81.3%) while it occurs with a much lower frequency in Q. 7 petraea (2.1%). 8 9 1 Fig. 3 -Allele frequency at locus QrZAG101. 2 Allele frequencies correspond more to the provenance than to the species. For 3 example, allele "153" occurs with high frequency in both species in Germany, while 4 this frequency decreases in Bulgaria and Greece.