Markus Ruhsam
email: m.ruhsam@rbge.ac.uk

Will Jessop

Amandine Cornille

James Renny

Rick Worrell

Crop-to-wild introgression in the European wild apple Malus sylvestris in Northern Britain

Malus sylvestris (L.) Mill, the European wild apple, is a small tree native to Europe which reaches its north-western limit in Northern Britain. It has been identified as one of the main contributors to the domesticated apple M. domestica (Suckow) Borkh. There are concerns that wild populations of this rare tree are threatened by hybridisation with M. domestica throughout Europe. We genotyped 332 trees collected in the wild from Scotland and Northern England using 14 microsatellite markers to investigate levels of introgression in wild M. sylvestris populations. Our results showed that 70 % of the samples could be considered pure M. sylvestris, 27% showed varying degrees of introgression from M. domestica and 3% were pure M. domestica. Hybrids were more frequent in areas with more intensive land use and less semi-natural woodland. About 80% of hybrids were identified as backcrosses to M. sylvestris. Areas in the Southern Highlands had the highest frequency of pure M. sylvestris trees (87%). Morphological characters such as leaf size and hairiness as well as fruit size generally used by botanists for identification of M. sylvestris in the field proved to be unreliable as only 68% of the trees could be identified accurately. Given the use of wild apple trees it seems likely that many of the trees collected from agricultural areas are planted and not the product of natural in situ hybridisation and seed dispersal events thus compounding natural hybridisation rates. Areas with high frequencies of pure M. sylvestris trees should be prioritised for conservation and possible measures are discussed to safeguard M. sylvestris as a species in Northern Britain.

Introduction

European wild apple (Malus sylvestris (L.) Mill., crab apple), a small tree in the Rosaceae family, is native across Europe and reaches its north-western distribution limit in Scotland and Norway (http://www.euforgen.org, accessed 9 July 2018). It is a light demanding species which grows naturally in gaps within or at the edge of broadleaf forests or on low competition sites such as flood plains [START_REF] Schnitzler | Wild European apple (Malus sylvestris (L.) Mill.) Population dynamics: Insight from genetics and ecology in the Rhine Valley. Priorities for a future conservation programme[END_REF]. However, it is more often associated with anthropogenic habitats such as hedges, wood pastures, semi-natural grasslands, and meadows [START_REF] Buttenschøn | Population dynamics of Malus sylvestris stands in grazed and ungrazed , semi-natural grasslands and fragmented woodlands in Mols Bjerge , Denmark[END_REF]. Wild apple is a rare tree species in Europe which is red-listed in Belgium [START_REF] Coart | Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers[END_REF], Finland and Germany [START_REF] Kik | Malus sylvestris. The IUCN Red List of Threatened Species 2011[END_REF], and part of conservation efforts in Denmark [START_REF] Graudal | A systematic approach to the conservation of genetic resources of trees and shrubs in Denmark[END_REF]. This species mainly occurs as single trees scattered throughout the landscape at low densities and for example it is estimated that only about 5500 trees are left in Germany [START_REF] Ble | BUNDESANSTALT FÜR LANDWIRTSCHAFT UND ERNÄHRUNG: Erfassung und Dokumentation genetischer Ressourcen seltener und gefährdeter Baumarten in Deutschland. Teillos 2: Wild-Apfel (Malus sylvestris) und Wild-Birne (Pyrus pyraster)[END_REF]. Malus sylvestris, like all other apple species, is outbreeding due to a self-incompatibility system [START_REF] Broothaerts | New findings in apple S-genotype analysis resolve previous confusion and request the re-numbering of some S-alleles[END_REF] and is mainly pollinated by bees [START_REF] Palmer-Jones | Observations on the pollination of apple trees (Malus sylvestris Mill.). New Zeal[END_REF]. Birds and mammals such as hares and wild pigs have been implicated in seed dispersal, however, there is evidence that domestic cattle are likely to be the main distributor of viable wild apple seeds [START_REF] Buttenschøn | Population dynamics of Malus sylvestris stands in grazed and ungrazed , semi-natural grasslands and fragmented woodlands in Mols Bjerge , Denmark[END_REF].

Recent studies have highlighted the important recent contribution of M. sylvestris to the cultivated apple genome, M. domestica (Suckow) Borkh., during the domestication history from the Central Asian progenitor M. sieversii (Ldb.) M. Roem. [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF][START_REF] Cornille | The domestication and evolutionary ecology of apples[END_REF]. It has long been recognised that wild apple and domesticated apple hybridise and that it can be very difficult to reliably identify pure wild apple from hybrids using morphological characters such as the fruit width and the hairiness of leaves [START_REF] Wagner | Zusammenstellung morphologischer Merkmale und ihrer Ausprägung zur Unterscheidung von Wild-und Kulturformen des Apfels[END_REF][START_REF] Stephan | Technical Guide Lines for Genetic Conservation and Use for Wild Apple and Pear (Malus sylvestris and Pyrus pyraster)[END_REF]. Not only has this led to discussions whether genuine wild M. sylvestris still exists today [START_REF] Henning | Morphologisch-systematische und genetische Untersuchungen an Arten und Artbastarden der Gattung Malus[END_REF][START_REF] Remmy | Untersuchungen zur Verbreitung und Morphologie des Wild-Apfels (Malus sylvestris (L.) Mill.)[END_REF][START_REF] Wagner | Zusammenstellung morphologischer Merkmale und ihrer Ausprägung zur Unterscheidung von Wild-und Kulturformen des Apfels[END_REF] but has also raised concerns about the genetic integrity of wild apple. This has recently sparked a number of studies which investigated the extent of hybridisation between the two apple species in Europe using molecular markers [START_REF] Coart | Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers[END_REF][START_REF] Coart | Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)[END_REF][START_REF] Larsen | Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)[END_REF][START_REF] Schnitzler | Wild European apple (Malus sylvestris (L.) Mill.) Population dynamics: Insight from genetics and ecology in the Rhine Valley. Priorities for a future conservation programme[END_REF][START_REF] Wagner | Hybridization and Genetic Diversity in Wild Apple (Malus sylvestris (L.) MILL) from Various Regions in Germany and from Luxembourg[END_REF][START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF][START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. Although results varied between studies the overall picture emerged that hybrids may occur at significant frequencies in wild apple populations (7% to 36%, [START_REF] Cornille | The domestication and evolutionary ecology of apples[END_REF].

Malus sylvestris is an understudied species in Northern Britain where it occurs at the north-western limit of its distribution. Yet, there has been a long standing history of domestic apple cultivation in Scotland which can be traced back to the 12 th century [START_REF] Hayes | Ancient Orchards on the Banks of the River Tay[END_REF]. Apples were also an important commercial crop during the 19 th and 20 th century, a time when many orchards were established, especially in the Tay and Clyde valleys [START_REF] Farrar | A Clyde Valley orchards survey[END_REF]. However, the extent of crop to wild gene flow in M. sylvestris has never been assessed in the United Kingdom and although the general distribution of crab apple is well documented in the online database of British plants (https://database.bsbi.org/maps/, accessed 9 July 2018), many of these records might be questionable due the difficulty in distinguishing pure wild apple from hybrids morphologically.

This raises the question of how many crab apple trees in the Scottish landscape are really pure M.

sylvestris given the ample opportunity M. domestica and M. sylvestris have had to hybridise.

The aims of this study are therefore 1) to assess the frequency of M. sylvestris and hybrid trees in the wild 2) to assess the reliability of morphological characters used in identifying pure and hybrid samples in the field and 3) to discuss implications for the conservation of this species in Northern Britain. 1). Usually all trees encountered were sampled, but in a few woods with multiple trees, samples were selected to cover the range of morphological characteristics present.

The collected trees were allocated to one of the following seven geographical regions (Figure 1

Microsatellite markers and polymerase chain reaction (PCR) amplification

DNA was extracted using the DNeasy Plant kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. All samples were assayed for 15 microsatellite loci using the four multiplex reactions MP01, MP02, MP03 and MP04 from [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF]Table S9 and Supplementary Table S1 this publication). The suitability of these markers for population genetic analyses, i. e. their neutral and unlinked nature as well as the absence of null alleles has been demonstrated in previous studies [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF][START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF]Cornille et al., 2013a;[START_REF] Cornille | Crop-to-wild gene flow and spatial genetic structure in the closest wild relatives of the cultivated apple[END_REF]. Each multiplex PCR was carried out in volumes of 10 µl using the following protocol: 1x PCR buffer (Bioline, London, UK), 0.2 mM dNTP (Bioline, London, UK), 2.5 mM MgCl2, 0.2 µM of each forward and reverse primer in the multiplex, 1x polysorbate-20 (Tween-20) (TBT-Par), 0.05 units BioTaq (Bioline, London, UK) and 1 µL of unquantified DNA. The mixture was then cycled through the profile: 5 min at 95°C, 35 cycles of 30 sec at 94°C, 1.5 min at 55°C and 1 min at 72°C, ending with 20 min at 72°C to complete extension. All PCR reactions were performed on a Biorad Tetrad 2 thermal cycler and run on an ABI 3730 automated sequencer at the Edinburgh Genomics facility. LIZ-500 labelled internal size standard (Applied Biosystems, Foster City, California, USA) was added to each sample to size fragments. The data generated were analysed with the software GENEMAPPER, v 4.1 (Applied Biosystems, Foster City, California, USA) scoring electropherograms manually.

Morphology

We used the morphological characters described in field guides to delimit M. sylvestris from M.

domestica: the lack of hairs on the petiole and underside of the leaves, the fruit size (< 30 mm) and leaf size [START_REF] Stace | New flora of the British Isles[END_REF]. However, it has been shown that there is large morphological variability within species and that hybrid samples display a continuum of intermediate characters which makes identification based on a few characters challenging [START_REF] Wagner | Zusammenstellung morphologischer Merkmale und ihrer Ausprägung zur Unterscheidung von Wild-und Kulturformen des Apfels[END_REF]. Nevertheless, this is the reality of field work and apple trees recorded in online databases will have been identified based on this limited number of morphological characters, some of which might not have been available at the time of identification, such as fruit size. To test the reliability of such a procedure we identfied trees based on the available characters (leaf hairiness, leaf and fruit size) at the time of leaf collection for genetic analysis. Hairiness was assessed on a scale of 0-4 (0= no hairs or very few hairs on petiole only; 4 = felted hairs on petiole and all of lower leaf surface) using a schema similar to [START_REF] Tollefsrud | Registrering og genetisk karakterisering av villeple i Norge[END_REF].

Trees were recorded either as 'crab apple', 'hybrid', 'cultivated apple', 'possibly crab apple' or 'possibly hybrid'. STRUCTURE v.2.3.4 (Pritchard et al., 2000) was used to investigate the population structure and levels of admixture in the 332 field collected and 34 reference Malus samples. This software uses a Bayesian algorithm to infer the number of distinct genetic groups K by minimizing deviations from

Statistical analyses

Hardy-Weinberg and linkage equilibrium within each cluster. The analyses were carried out for K=1 to 10 using 1,000,000 MCMC iterations after a burnin of 30,000 steps and were repeated 20 times for each K, with the 'admixture' and 'correlated allele frequencies' options selected. If sampling is unbalanced, i.e. genetic clusters have widely different sample sizes, then STRUCTURE has been shown to yield poor estimates of individual ancestry and K if the default settings are used [START_REF] Wang | The computer program STRUCTURE for assigning individuals to populations: Easy to use but easier to misuse[END_REF]. We therefore followed [START_REF] Wang | The computer program STRUCTURE for assigning individuals to populations: Easy to use but easier to misuse[END_REF] recommendation and used the alternative option allowing a separate α which is a measure of the relative admixture level between populations ('Separate α for each Population' ticked). This option assumes distinct α values for the assumed K populations, and allows individuals to have their ancestry originating from the assumed K populations at K different prior probabilities. In contrast, the default setting uses a single α value for all populations and specifies that each individual has its ancestry originating from each of the assumed K populations at an equal prior probability of 1/K. We used StrAuto [START_REF] Chhatre | StrAuto: automation and parallelization of STRUCTURE analysis[END_REF], a python script, which automatically distributes STRUCTURE jobs among multiple processors reducing analysis time considerably. To identify the most likely number of distinct genetic groups K in the dataset the ΔK statistic [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF], which is based on the rate of change in the log probability of data between successive K values, as well as Pr[X|K] (the probability of obtaining the genotype data X given K) of [START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF] were calculated using the online version of the software CLUMPAK (Kopelman et al., 2015, http://clumpak.tau.ac.il). STRUCTURE output files were processed using the R package 'pophelper' [START_REF] Francis | pophelper: an R package and web app to analyse and visualize population structure[END_REF].

To assess the frequency of pure and hybrid apple trees in our dataset we considered two approaches. The first approach used the estimated individual membership coefficients from STRUCTURE [START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF]. These are the proportions P for each of the identified (K) genetic clusters that have contributed to an individual's genomic makeup [START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF].

There is no objective threshold for P which can be used to define pure and hybrid samples but a value ranging between 0.05 to 0.2 has been generally applied in the literature [START_REF] Larsen | Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)[END_REF][START_REF] Vähä | Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci[END_REF][START_REF] Starr | Asymmetric hybridization and gene flow between Joshua trees (Agavaceae: Yucca) reflect differences in pollinator host specificity[END_REF][START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. A higher threshold can result in the misclassification of hybrids as pure and a lower threshold might have the opposite effect and identify a sample as hybrid when it is actually pure. In previous Malus studies values of P ≤0.1 [START_REF] Cornille | Crop-to-wild gene flow and spatial genetic structure in the closest wild relatives of the cultivated apple[END_REF][START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF], P ≤0.15 [START_REF] Larsen | Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)[END_REF] and P ≤0.2 [START_REF] Coart | Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)[END_REF][START_REF] Wagner | Hybridization and Genetic Diversity in Wild Apple (Malus sylvestris (L.) MILL) from Various Regions in Germany and from Luxembourg[END_REF][START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF] have been used to classify pure individuals. We therefore calculated the percentage of pure and hybrid samples using all of the above thresholds, resulting in three scenarios. In a conservative scenario only samples with a membership coefficient for the M.

domestica gene pool P dom ≤0.1 were considered to be pure M. sylvestris trees. Samples with 0.10<P dom <0.45 were classed as backcrosses to M. sylvestris, with 0.45<P dom <0.55 as F1, with 0.55<P dom <0.90 as backcrosses to M. domestica and with P dom ≥0.90 as pure M. domestica. In the two other less stringent scenarios samples with P dom ≤0.15 and P dom ≤0.2 were considered to be pure M.

sylvestris and samples with P dom ≥0.85 and P dom ≥0.80 as pure M. domestica. The threshold for the other classes stayed the same apart from the backcross boundaries being adjusted to reflect the altered threshold for purity. The second approach employed the software NEWHYBRIDS v1.1 [START_REF] Anderson | A model-based method for identifying species hybrids using multilocus genetic data[END_REF], which uses a Bayesian algorithm to estimate the probability of individuals belonging to any one of six distinct classes (pure parental, F1, F2 and backcross categories). We allocated individuals to a specific class if they had a posterior probability ≥ 55% of belonging to this class. However, if the probability was split more or less equally between two classes, e. g. 45% backcross to M. sylvestris and 55% F1 then the individual status was left undecided. The analysis was carried out with the default parameters, Jeffreys priors, a burn-in phase of 100,000 steps and 500,000 sweeps. No prior information about pure parental samples was included.

To investigate the population structure of pure M. sylvestris samples in Scotland a second STRUCTURE analysis with the same parameters specified above was carried out on a subset of the data. The dataset only included pure M. sylvestris samples i.e. samples which had a membership coefficient for the M. domestica gene pool P dom ≤0.1 in the analysis using the full dataset.

Standard genetic diversity estimates and population differentiation were calculated for all pure M. sylvestris populations (P dom ≤0.1 ) with more than ten pure individuals using the R package 'diveRsity' [START_REF] Keenan | diveRsity : An R package for the estimation and exploration of population genetics parameters and their associated errors[END_REF] and GenAlEx v6.5 [START_REF] Peakall | GenALEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update[END_REF]. To test for isolation-by-distance a Mantel test was carried out between the geographic and genetic distance matrix of pure samples in GenAlEx v6.5 using 9999 permutations [START_REF] Peakall | GenALEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update[END_REF].

The full dataset was also subjected to a multivariate analysis (Principal Coordinate Analysis -PCO) in GenAlEx v6.5 [START_REF] Peakall | GenALEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update[END_REF] using the pairwise genetic distances and the 'standardised covariance' option.

To assess the reliability of morphology in identifying apple trees correctly, field and genetic identification using the P dom ≤0.1 threshold were compared for each sample.

Results

Genetic structure

Microsatellite locus CH04e05 in MP02 was difficult to score and was excluded from further analyses which are therefore based on 14 loci only. The STRUCTURE analysis using the full dataset (n=366) indicated that, depending on the evaluation statistic used, the most likely number of genetic groups was either K=2 [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF] or K=3 [START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF] (Supplementary Figure S1). For [START_REF] Wang | The computer program STRUCTURE for assigning individuals to populations: Easy to use but easier to misuse[END_REF]. This is the case in our dataset where the number of reference samples is much smaller than the number of samples collected in the field. Secondly, K=3 differentiated between a M. domestica and M. baccata -M. hupehensis -M. orientalis group which is biologically a more meaningful grouping than an undifferentiated group containing five different Malus species. This is also more consistent with [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF] analysis of a much larger dataset which yielded distinct groups for each of their reference Malus species (M. baccata, M. sieversii, M. orientalis, M. domestica). The reason why the different reference Malus species did not fall into their own respective clusters in our dataset is probably due to the very small number of samples in each reference group (n=2 to 5 per species), and the existence of considerable levels of admixture and ancestral polymorphism which often clusters M. bacccata and M. hupehensis as well as M. domestica and M. sieversii together [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF][START_REF] Duan | Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement[END_REF]. Thirdly, the K=3 analysis resulted in the same genetic clusters and individual membership coefficients in all 20 repeat runs whereas results for K>3 produced up to four alternative ('minor') modes suggesting conflicting signals in these clustering solutions (Supplementary Figure S2).

To assess levels of introgression in Northern British populations of M. sylvestris we combined the estimated membership coefficients coming from the M. domestica and M. baccata -M. hupehensis -M. orientalis group to obtain a figure for the total contribution of genetic clusters other than M.

sylvestris in each individual. As individual contributions from the M. baccata -M. hupehensis -M.

orientalis group were well below 1% for the vast majority of samples (Figure 2) we subsequently refer to this combined figure as M. domestica contribution.

The STRUCTURE analysis revealed considerable levels of introgression in the trees collected throughout Scotland and Northern England (Figure 2, Supplementary Table S2). We have used three thresholds for the permissible proportion of M. domestica P dom (P dom ≤ 0.1, 0.15 and 0.2) in a pure M.

sylvestris sample. This resulted in somewhat differing proportions for pure and hybrid classes (Table 1) and generated uncertainty for a few individuals regarding their 'true' class. However, it did not affect the overall picture and the interpretation is the same regardless of the threshold used. Both the STRUCTURE and NEWHYBRIDS results indicated that although 70 to 80% of the samples collected in the field were likely to be pure M. sylvestris, between 16 to 27% of the samples showed varying degrees of introgression from M. domestica (Figure 2, Table 1). Samples of hybrid origin were mainly identified as backcrosses to M. sylvestris (12%-21%) whereas F1 (~3%), F2 (0.3%) and backcrosses to M. domestica (1%-2%) were rare. There was generally good agreement between the two different methods used to classify hybrid and pure samples (Table 1). A small percentage of samples (4%)

could not be confidently allocated to a single category by NEWHYBRIDS as they shared a more or less equal probability to be either a backcross to M. sylvestris or a pure M. sylvestris (3.7%) or an F1 (0.3%) or F2 (0.3%) individual (Table 1).

The PCO analysis revealed a relatively well defined cluster of reference individuals consisting of M.

domestica and reference samples for the other four Malus species (M. baccata, M. hupehensis, M.

orientalis and M. sieversii) which was well separated from a pure M. sylvestris group (Figure 3). The space between these two groups was filled by hybrids which intermingled to a large extent with the pure M. sylvestris group and to a much lesser extent with the M. domestica cloud. Relatively little of the total variation in the dataset was explained by axis 1 (6.5%) and axis 2 (3.6%).

Geographic variation of introgression rates

The proportion of introgressed trees varied considerably between geographical regions. Using the more conservative threshold of P dom ≤0.1 for pure M. sylvestris samples, the West Coast, Aberdeenshire and the Central Belt were identified as regions with more than 50% of the trees showing introgression. In contrast, regions with ≤ 25% introgressed trees were the Lake District, Dumfries and the Southern Highlands (Figure 4A). The average contribution of the M. domestica genepool to the identified hybrids over all regions was 27% and reached its highest value with 34% on the West Coast (Figure 4B). Interestingly, three trees from the West Coast and two from the Trossachs-Loch Lomond area (SH) had contributions of >0.9 from the M. baccata -M. hupehensis -M. orientalis genetic cluster (Figure 2, Supplementary Table S2).

To assess the population structure within pure M. sylvestris populations (n=227) we used a pruned data set including only samples which were estimated to have a membership coefficient for the M.

domestica gene pool P dom ≤0.1 in the first analysis. The analysis did not reveal any population structure within Scotland and Northern England as all samples had more or less equal proportions from K genetic groups for each K from 2 to 6 (Supplementary Figure S3).

Genetic diversity estimates of pure M. sylvestris populations

Four out of the seven geographic regions had more than ten pure (P dom ≤0.1) M. sylvestris trees and were used for genetic diversity estimates. Genetic diversity (H e ) and allelic richness (A r ) were generally high in all populations with no evidence for inbreeding (F is not significantly different from zero) (Table 2). Overall population differentiation was significant but very low (G st =0.018±0.003; D est =0.066±0.013). Equally, pairwise population differentiation was very low but significant with the highest differentiation displayed between the Lake District and the Central Belt (G st =0.012;

Supplementary Table S3). The Mantel test revealed a very weak isolation-by-distance pattern (Rxy=0.05, P=0.004) with hardly any of the variation explained by a linear relationship (R 2 =0.003).

This suggests that there is no pattern of isolation-by-distance in Scottish crab apple, i. e. geographic proximity of trees does not correlate with closer genetic relatedness.

Reliability of identification based on morphological characters taken in the field

A total of 239 trees were used to test the reliability of morphological characters recorded in the field to assess whether trees were either pure or of hybrid origin. Field identifications were compared to the genetic identifications using a threshold of P dom ≤0.1 for pure M. sylvestris samples and P dom >0.1 for hybrid samples. 58% (n=139) of the samples were correctly identified in the field using morphology (Table 3) whereas 25% (n=59) were identified incorrectly. A total of 17% (n=41) of the samples could not be identified with confidence in the field and were tentatively allocated to either 'possibly crab apple' or 'possibly hybrid'. This 'informed' guess turned out to be correct in 58% (n=24) of the cases and incorrect in 42% (n=17). Over all 68% (n=163) of trees were correctly identified (Table 3).

Discussion

A number of studies have investigated levels of hybridisation between the European wild crab apple (Malus sylvestris) and the domesticated apple (M. domestica) on different geographical scales ranging from regional [START_REF] Schnitzler | Wild European apple (Malus sylvestris (L.) Mill.) Population dynamics: Insight from genetics and ecology in the Rhine Valley. Priorities for a future conservation programme[END_REF][START_REF] Wagner | Hybridization and Genetic Diversity in Wild Apple (Malus sylvestris (L.) MILL) from Various Regions in Germany and from Luxembourg[END_REF] to country [START_REF] Coart | Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers[END_REF][START_REF] Coart | Chloroplast diversity in the genus Malus: new insights into the relationship between the European wild apple (Malus sylvestris (L.) Mill.) and the domesticated apple (Malus domestica Borkh.)[END_REF][START_REF] Larsen | Hybridization and genetic variation in Danish populations of European crab apple (Malus sylvestris)[END_REF][START_REF] Wagner | Hybridization and Genetic Diversity in Wild Apple (Malus sylvestris (L.) MILL) from Various Regions in Germany and from Luxembourg[END_REF][START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF][START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF] and European wide [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF][START_REF] Cornille | Crop-to-wild gene flow and spatial genetic structure in the closest wild relatives of the cultivated apple[END_REF][START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF]. However, no study has ever focused on M.

sylvestris in the United Kingdom where reliable distribution data are rare and nothing is known about the extent of introgression in this enigmatic tree. One aim of this study was to investigate the frequency of hybrids in wild Northern British Malus sylvestris populations which occur in the UK at the north-western edge of their natural distribution range.

Extent of hybridisation between M. sylvestris and M. domestica

The results from both the NEWHYBRIDS and STRUCTURE (P dom ≤0. et al., 2014). Although caution is necessary when comparing hybridisation rates from different studies due to differences in marker numbers, samples and applied thresholds, Scottish levels of hybridisation appear to be comparable to the ones found in other European countries. However, there were clear differences in the extent of hybridisation between regions within Scotland. Strongholds for pure M. sylvestris trees are clearly the Southern Highlands where only 13% of the samples turned out to be of hybrid origin.

This was closely followed by Dumfriesshire and the English Lake District with 25% hybrids. Most of these areas are characterised by rugged terrain, a relatively high proportion of woodland of natural origin and are likely to have relatively low numbers of cultivated apple trees and orchards. In contrast, the Central Belt is the most densely populated area in Scotland which is characterised by low lying fertile agricultural land and a higher incidence of woodland and trees of planted origin. An orchard survey of the Forth Valley [START_REF] Hayes | Survey of Traditional Orchards in the Forth Valley[END_REF] and Clyde Valley (Ironside [START_REF] Farrar | A Clyde Valley orchards survey[END_REF], which are part of this region, listed 67 and 63 orchards respectively, many of which have grown or grow cultivated apples. It is therefore not surprising that ~ 50% of the assayed samples are of hybrid origin in the Central Belt. Our results indicate similar hybrid frequencies in Aberdeenshire, along the West Coast and in parts of the Northern Highlands, however, these are based on much smaller sample sizes. This suggests that hybrid trees are more common in regions where apple cultivation is more prevalent (and has been for many centuries), and where trees (including crab apples) are typically more likely to have been planted rather than regenerated naturally.

Interestingly, five trees (two from the Southern Highlands and three from the West Coast) had more than 90% of their genetic make up derived from the M. baccata-M. hupehensis-M. orientalis genetic group. These are likely to be either M. baccata trees which are sometimes planted as ornamental trees (Stace, 2010, p. 200) and have been recorded from the West Coast (https://database.bsbi.org/maps/?taxonid=2cd4p9h.asm, accessed 9 March 2017) or M. hupehensis trees which have been planted by, amongst others, the Forestry Commission, as a component of a mix of trees used for landscaping purposes (Peter Quelch, pers. comm.). Although such ornamental trees will contribute to the pollen pool which M. sylvestris trees are exposed to, cross-pollination with subsequent hybrid establishment is likely to be a very rare event as all of the assayed hybrid samples had much less than 10% contribution from this genetic group (Figure 2).

Hybrid classes detected in samples from Northern Britain

Our data indicate that the most successful hybridisation events are backcrosses to M. sylvestris (crosses between a pure M. sylvestris and an F1 or later generation hybrid) as ~20% of all samples and about 80% of all hybrids fall into this category. In comparison, only 3% of the samples (or about 15% of all hybrids) were classified to be F1 (i. e. the mating between a pure M. sylvestris and a pure M. domestica tree) regardless of analysis. The generation of F1 with the subsequent successful establishment of the hybrid seed seems to be a relatively rare event. Backcrosses to M. domestica and F2 seem to be even rarer, as only 1-2% and 0.3%, respectively, were classified as such.

A few studies have investigated pollen dispersal distances of apple trees, and show that the maximum distance can vary considerably from as short as 300 m [START_REF] Larsen | Pollen mediated gene flow in a native population of Malus sylvestris and its implications for contemporary gene conservation management[END_REF] to as far as 4 km [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF] and 11 km [START_REF] Reim | Pollen movement in a Malus sylvestris population and conclusions for conservation measures[END_REF]. [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF] reported that although about 5% of the pollination events occurred between trees more than 1 km apart, 75% occurred at distances of less than 100 m and 25% even below 15 m. Interestingly, pollination distances decreased with the level of M. domestica ancestry of the father tree so that trees genetically closer to M. sylvestris had higher pollen dispersal distances than more cultivated apple trees [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF] also showed that the number of hybridization events increased with decreasing tree density, suggesting that when fewer crab apple trees are close together pollinators have to fly larger distances which presumably increases the chance of encountering domesticated apple trees.

Our results indicate that a large proportion of hybridisation events in the wild were facilitated by M.

sylvestris pollen as 80% of all detected hybrids are likely to be backcrosses to M. sylvestris. This is somewhat unexpected regarding the rare occurrence of F1. One explanation for this could be that cultivated apple trees are rare and therefore do not contribute significantly to the pollen pool.

However, this is unlikely as 44% of the samples collected in the Central Belt, where a substantial number of orchards exist, were classified as backcrosses to M. sylvestris. An alternative explanation is that a large proportion of the samples might not necessarily be the product of a natural hybridisation event which occurred in situ but instead are planted specimens of unknown origin. In the densely populated Central Belt, a landscape which has also been heavily modified by agriculture and human activities, very little natural habitat is left. Indeed, the vast majority of samples was collected along country roads, field borders and hedges. Only a few samples came from semi-natural looking habitats, which also showed clear signs of human influence. It is therefore very likely that the samples collected in these areas were planted and are derived from nursery stock of unknown origin. A recent study investigating the genetic integrity of crab apple trees sold by nurseries reported that all samples sold as wild crab apples from commercial seed companies had substantial levels of M. domestica ancestry [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. It is therefore possible that the high frequency of backcrosses to M.

sylvestris in certain areas of Scotland is not necessarily a sign of natural hybridisation events in these areas but may be partly a reflection of the purity of the crab apple stock sold by nurseries.

It is noteworthy that five out of the 17 (~30%) reference samples included for M. domestica had contributions of P syl ≥10% from the M. sylvestris genome ranging from 13% < P syl < 32%. If collected in the wild, these samples would have been regarded as backcrosses to M. domestica. However, these five individuals belong to well known cultivars ('Red Falstaff', 'Early Julyan', 'Alderman', 'pomme de fer' and 'Corte pendu') which might indicate that the samples identified as backcrosses to M. domestica or F1 are in fact M. domestica cultivars. [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF] have highlighted that many cultivars including commercial varieties such as Granny Smith can have large admixture proportions from the M. sylvestris genepool. For example, the English apple cultivar 'Cox Orange Pippin' has a 55% M. sylvestris contribution [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF], Table S2) and would therefore be classified as F1

on the basis of admixture proportions. The Cox is the UK's most popular dessert apple and accounts for about 42% of the UK's apple production (https://quadram.ac.uk/spotlight/apple-facts/, accessed 10 May 2018). This opens up the possibility of an alternative interpretation where some hybrid samples collected from the wild are not necessarily the product of a recent hybridisation event but could actually be 'escaped' cultivars which have grown from the seed of an apple core carelessly thrown out of a car or discarded at the wayside. Interestingly, about 88% (7/8) of the samples which had a higher than 50% posterior probability to be an F1 in the NEWHYBRIDS analysis were collected either along roads (n=5), on the bank of an abandoned railway line (n=1) or next to a foot path (n=1).

No population genetic structure in pure M. sylvestris populations

The STRUCTURE analysis of a pruned data set where all trees with a domesticated membership coefficient of P Dom >10% had been removed did not reveal any population structure. This is also apparent in the very low genetic differentiation between all populations as well as in pairwise population comparisons and the lack of an isolation-by-distance pattern. This suggests that crab apple pollen is effectively dispersed over long distances in Northern Britain which is supported by pollination studies reporting dispersal distances of up to 11 km [START_REF] Reim | Pollen movement in a Malus sylvestris population and conclusions for conservation measures[END_REF] but might also partly be due to human mediated dispersal. The lack of a significant population structure is also consistent with previous studies. Although [START_REF] Cornille | Anthropogenic and natural drivers of gene flow in a temperate wild fruit tree: A basis for conservation and breeding programs in apples[END_REF] detected five different genetic clusters in wild apple on a European scale, population structure within each of these populations or on a smaller regional scale is generally weak or non-existent (Cornille et al., 2013a;[START_REF] Schnitzler | Wild European apple (Malus sylvestris (L.) Mill.) Population dynamics: Insight from genetics and ecology in the Rhine Valley. Priorities for a future conservation programme[END_REF][START_REF] Wagner | Hybridization and Genetic Diversity in Wild Apple (Malus sylvestris (L.) MILL) from Various Regions in Germany and from Luxembourg[END_REF].

Is morphology a useful indicator to distinguish between pure and hybrid samples?

Only 68% of our field identifications turned out to be correct. This highlights the difficulty in reliably distinguishing between pure wild apple and hybrid samples solely based on morphology.

Morphological characters which have been used in the literature to delimit pure M. sylvestris are the lack of hairs on the petiole and underside of the leaves, the fruit size (< 30 mm) and leaf size [START_REF] Wagner | Zusammenstellung morphologischer Merkmale und ihrer Ausprägung zur Unterscheidung von Wild-und Kulturformen des Apfels[END_REF][START_REF] Stephan | Technical Guide Lines for Genetic Conservation and Use for Wild Apple and Pear (Malus sylvestris and Pyrus pyraster)[END_REF]. However, it has been shown that there is large morphological variability within species and that hybrid samples display a continuum of intermediate characters [START_REF] Wagner | Zusammenstellung morphologischer Merkmale und ihrer Ausprägung zur Unterscheidung von Wild-und Kulturformen des Apfels[END_REF]. As noted by [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF] even cultivars can have large M. sylvestris contributions which will have a compounding effect on the morphology and these discriminating characters. This is clearly echoed by our results as some trees which were identified as crab apple in the field turned out to be domestic apple genetically. In some cases a sample was identified as a domestic apple when it was a crab apple genetically. This suggests that identifications based on morphology need to be treated with caution.

However, there is also reason to be cautious about viewing genetic data as the silver bullet for all apple identification problems. Some of the cultivars assayed by [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF] had very high contributions from M. sylvestris -in the case of the Russian cultivar 'borovitsky', which is better known as 'Duchess of Oldenburg' in Britain [START_REF] Smith | National Apple Register of the United Kingdom[END_REF], to the point that it would have been classified as pure M. sylvestris genetically but more likley as a hybrid or domesticated apple morphologically due to the larger fruit size (P syl =0.933, Table S2 in [START_REF] Cornille | New Insight into the History of Domesticated Apple: Secondary Contribution of the European Wild Apple to the Genome of Cultivated Varieties[END_REF]).

This clearly highlights that there is no foolproof way to obtain accurate identification of trees either in the field or in the lab alone. It therefore seems that a combination of field assessment and genetic data will provide the most reliable basis for the identification of an unknown sample.

Implications for conservation

There is evidence for a long standing history of apple cultivation in Scotland which can be traced back to the 12 th century [START_REF] Hayes | Ancient Orchards on the Banks of the River Tay[END_REF]. Many orchards were founded throughout the centuries with a peak period of fruit growing in the early 20th century [START_REF] Farrar | A Clyde Valley orchards survey[END_REF]. This clearly shows that there has been ample opportunity for hybridisation between wild apple and domesticated apple. Wild apple trees have also been planted as alternative pollinators for eating and cooking apples and the fruits have been used for animal fodder as well as cider and jelly making (Cox and Beaton, 2002, p. 101).

These planted trees might have been raised from local stock in the past but nowadays are more likely to be of unknown origin with a high chance of being introgressed by M. domestica [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. It is also worth mentioning that the term 'crab apple' does not necessarily refer to the European wild apple M. sylvestris but is generally used for any Malus species producing small fruits to distinguish it from the cultivated apple. Nurseries offer a large number of crab apple varieties many of which have nothing to do with the European wild apple M. sylvestris and often are of obscure parentage [START_REF] Fiala | Flowering Crabapples: The Genus Malus[END_REF]. In some instances their origin is well documented as in the case of the popular crab apple Malus 'Evereste' which is a hybrid between Malus domestica 'Rome Beauty'

x Malus floribunda clone 821 [START_REF] Decourtye | Evereste', un nouveau pommier d'ornement[END_REF]. This array of crab apple varieties is confusing for customers and might result in the unintentional planting of exotic instead of native wild apple trees. It is possible that the five samples identified as belonging to the 'M. baccata group' in the STRUCTURE analysis could be an example for this.

All this highlights the difficulty of assessing how much M. sylvestris growing in Scotland can be considered 'wild', i. e. has not been planted. This is certainly true for densely populated lowland areas and other locations where agriculture has had a major impact on the landscape. In the Central Belt and Aberdeenshire for example many of the trees which turned out to be pure M. sylvestris grow in places with obvious human presence and interference such as hedges, next to roads, along fences of small semi-natural woodlands, pastures, and field edges. Given the value this species has had for humans it is hard to imagine that these individuals which sometimes seem to be many decades old are the remnants of a natural population. The origin of these trees is probably threefold. Firstly, in rare cases they could be the product of a natural dispersal event; or, more likely, they are planted trees which were either raised from seeds collected from local trees in the countryside (planted or natural)

or from a commercial seed orchard.

In contrast, in upland areas such as the Loch Lomond National Park in the Southern Highlands, parts of Dumfries and Galloway and the Lake District, where 75-87% of the collected samples can be considered pure, many of the wild apple trees grow in seemingly natural or semi-natural habitat with little reason to suspect deliberate planting. The component of hybrid trees in these areas is probably increasing only slowly due to the high frequency of pure wild apple trees and the lower frequency of domesticated apples. These populations should therefore be of priority conservation concern in order to safeguard M. sylvestris as a species in Northern Britain. They could be used as sources for the establishment of seed orchards with certified pure M. sylvestris trees. This would be a timely conservation measure considering the difficulty to obtain unadulterated material from commercial nurseries [START_REF] Feurtey | Crop-to-wild gene flow and its fitness consequences for a wild fruit tree: towards a comprehensive conservation strategy of the wild apple in Europe[END_REF]. Orchards should have high tree densities and be sited at some distance from known M. domestica populations to minimise the potential of hybridisation with cultivated apple [START_REF] Reim | Pollen movement in a Malus sylvestris population and conclusions for conservation measures[END_REF]. Stock from such a seed orchard, or from controlled wild collections in the uplands would serve as reliable sources of pure wild apple, which could either be used for re-introductions in areas where the occurrence of truly wild M. sylvestris is doubtful or to support regeneration more widely. Despite the prolific fruit production of many crab apple trees we have seen very little in terms of natural regeneration in the field. This is concerning as wild apple trees are apparently not particularly long lived trees (80-100 years, [START_REF] Stephan | Technical Guide Lines for Genetic Conservation and Use for Wild Apple and Pear (Malus sylvestris and Pyrus pyraster)[END_REF] and a substantial number seem to be of fairly old age in the wild. Another approach to encourage regeneration is to introduce managed low intensity grazing by cows or horses. It has been shown that cattle and horses are effective dispersers of wild apple seeds, which readily germinate in dung patches and hoof marks.

Successful regeneration could then be encouraged by managed grazing in subsequent years (Buttenschøn andButtenschøn, 1985, 1999). Deer might also be contributing to the seed dispersal of wild apple trees.

We also encountered a few wild apple trees in forests with no fruit set and poor vitality which was probably due to the disadvantageous light conditions. As M. sylvestris has high light requirements, pruning or thinning of nearby trees might be an inexpensive and effective measure to improve the local light condition for a particular crab apple tree resulting in increased vitality and flowering [START_REF] Stephan | Technical Guide Lines for Genetic Conservation and Use for Wild Apple and Pear (Malus sylvestris and Pyrus pyraster)[END_REF].

Conclusion

Our data suggest that there is a significant amount of hybridisation between crab and domesticated apple in Northern Britain. Given the use of wild apple trees and their association with man-made habitats it seems likely that many of the hybrid (and pure) samples collected from lowland agricultural areas are actually planted trees and not the product of a natural hybridisation event with subsequent seed dispersal and establishment. The overall hybridisation rate reported in this study is therefore likely to be the result of natural processes as well as human activities, and is probably be an overestimation. In areas with a high frequency of pure wild apple trees the component of hybrid trees is probably increasing only slowly, and basic conservation measures should be effective in safeguarding M. sylvestris as a species in the long term. To estimate levels of natural hybridisation between wild and domesticated apple in the wild, genotyping of seedlings collected from pure M. sylvestris trees in different habitats is required. This would allow the potential threat of introgression to be evaluated more accurately. domestica genepool ≥0.9) and in (B) the total number of hybrid samples (P syl < 0.9).

Table and Figure captions

Table 1. Introgression rates of field collected Malus trees in Scotland and Northern England inferred using STRUCTURE and NEWHYBRIDS. For the STRUCTURE results three different thresholds for

  ): Lake District (LD, n=24), Dumfries (DF, n=89), Central Belt (CB, n=66), Southern Highlands (SH, n=122), West Coast (WC, n=9), Aberdeenshire (AB, n=9) and Northern Highlands (NH, n=13, including one sample from Shetland). The delimitation of these regions is broadly based on geographical features and the intensity of land usage. The regions of the Lake District (LD), Dumfries (DF), Southern and Northern Highlands (SH and NH, respectively) are mainly upland, characterised by less intense agricultural usage, relatively high incidence of semi-natural woodland and lower population densities.In contrast, the areas where samples were collected in the Central Belt (CB), Aberdeenshire (AB) and the West Coast (WC) are mainly low lying with little remaining semi-natural woodland and more intense agricultural land use and/or higher population densities.A total of 366 silica dried leaf samples were assayed including 332 trees collected throughout Scotland and adjacent parts of Northern England 34 trees served as reference samples for six Malus species including 24 samples from the living apple collection of the Royal Botanic Garden Edinburgh (Malus domestica (n=11), M. baccata (L.) Borkh. (n=5), M. hupehensis (Pamp.) Rehd. (n=3), M. orientalis Uglitzk. (n=2) and M. sieversii (n=3)) as well as 10 samples from France which were used in Cornille et al. (2015) and were shown to be genetically pure M. domestica (n=6) and M. sylvestris (n=4).

Figure 1 .

 1 Figure 1. Sampling locations and broad geographical regions of 332 Malus trees collected in the field in Northern Britain. Lake District (LD, n=24), Dumfries (DF, n=89), Central Belt (CB, n=66), Southern Highlands (n=122), West Coast (WC, n=9), Aberdeenshire (AB, n=9) and Northern Highlands (NH, n=13, including one sample from Shetland).

Figure 2 .

 2 Figure 2. Barplot of STRUCTURE membership coefficients inferred with 14 nSSRs for all assayedMalus trees (n=366, six species) for K=3, the most likely number of genetic clusters according to the method by[START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF]. Red (dark grey in print version) represents the M. domestica cluster, blue (light grey in print version) the M. sylvestris cluster and black the 'M. baccata -M. hupehensis -M. orientalis' cluster. Grey header boxes highlight the included reference Malus species (Domestica -M. domestica; Baccata -M. baccata; Hu -M. hupehensis; Or -M. orientalis; Si -M. sieversii; Syl -M. sylvestris) and sampling locations.

Figure 3 .

 3 Figure3. Principal Coordinate Analysis (PCO) of all assayed Malus samples using 14 nSSRs (n=366). Individuals are coloured according to the K=3 STRUCTURE groupings. 'M. domestica group'…M. domestica reference samples plus individuals with genomic M. domestica membership coefficient ≥0.9. '"M. baccata" group'…samples with M. baccata/M. hupehensis/M. orientalis membership coefficient ≥0.9. 'M. sylvestris group'…samples with M. sylvestris membership coefficient ≥0.9. 'Hybrid'…introgressed M. sylvestris samples with 0.1<P dom < 0.9.

Figure 4 .

 4 Figure 4. A. Proportion of field collected samples which were either pure Malus sylvestris (proportion of M. sylvestris genepool P syl ≥0.9, blue; light grey in print version) or of hybrid origin (P syl < 0.9, red; dark grey in print version) using STRUCTURE results for K=3. B. Mean proportion of M. sylvestris (blue) and cumulative proportions of M. domestica and 'M.baccata group' genetic contributions ('cultivated genome'; red) in hybrid samples. LD -Lake District, DF -Dumfries, CB -Central Belt, SH -Southern Highlands, WC -West Coast, AB -Aberdeenshire and NH -Northern Highlands. 'n' in (A) denotes the total number of samples excluding pure M. domestica samples (proportion of M.

  Our fieldwork comprised both locating trees and attempting to develop a representative sample as the project progressed. Sampling was carried out based on field records in the BSBI online atlas for

	76	Methods Sampling and plant material
		Malus sylvestris s. l. (https://database.bsbi.org/maps/, accessed 9 March 2017) and the Forestry
		Commission Native Woodland Survey of Scotland
		(https://scotland.forestry.gov.uk/supporting/strategy-policy-guidance/native-woodland-survey-of-
		scotland-nwss, accessed 9 March 2017). Additionally, samples were located via professional contacts
		and by searching likely habitats encountered during field work. However, time and budget constraints
		meant that it was impossible to search the whole of Northern Britain comprehensively although most
		areas were covered (Figure
		6

  K=2, a M. sylvestris group and a group containing cultivated samples and the other four reference Malus species were differentiated. For K=3, a M. sylvestris and a M. domestica/ M. sieversii group were retrieved, as well as a group containing the other three Malus species (M. hupehensis, M.

baccata and M. orientalis). We considered K=3 to be the most likely number of groups in our dataset for the following reasons.

Firstly, it has been shown that overall

Pritchard et al.'s (2000)

method is more accurate than

[START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF]

especially if sampling is unbalanced, i.e. the inferred genetic groups have unequal sample sizes

Acknowledgements

Special thanks go to Robin Buxton, Colin Hughes, Bob Stubbs and Andrew Fairbairn. We are grateful to the many people who located trees for us or sent in leaves for sampling; especially Mary-Ann Smythe, Richard Cunningham, Crystal Maw, Neil Mackenzie, Joyce Gilbert, Iain Brodie, Paul Harvey and John Parrot. We also thank the editor and two anonymous reviewers for their comments which improved an earlier version of this manuscript.

Funding

This work was supported by the Patsy Wood Trust; the Scottish Forestry Trust and Sustainable Forestry SCIO; the Falkland Stewardship Trust; and the Forestry Commission Scotland via the Native Woodland Survey of Scotland. The Royal Botanic Garden Edinburgh is supported by the Scottish Government's Rural and Environment Science and Analytical Services Division.

Conflicts of interest

None declared the contributions from other than parental groups have been applied to call a sample pure (P≤0.10/0.15/0.20; upper, middle and lower row, respectively). NEWHYBRIDS assignments are based on a posterior probability ≥ 55% of belonging to a certain class. 'Pc dom ' denotes the combined contributions from the M. domestica and M. hupehensis -M. baccata -M. orientalis group; 'P dom ' is the contribution from the M. domestica group; 'Syl' pure M. sylvestris; 'BX-Syl' backcross to M. sylvestris; F1/F2 first/second generation hybrid, respectively; 'BX-Dom' backcross to M. domestica; 'Dom' pure M. domestica. 'Syl/BX-Syl', 'F1/BX-Syl' and 'F2/BX-Syl' denote the percentage of samples in the NEWHYBRIDS output which had more or less equal chances of belonging to either of the two categories.

Table 2. Genetic diversity estimates of pure M. sylvestris populations with more than ten pure individuals (based on the STRUCTURE results for K=3 and a P dom ≤0.1 threshold for pure individuals).

'n' number of samples; 'H o ' and 'H e ' observed and expected heterozygosity, respectively; 'F is ' inbreeding coefficient; 'A r ' allelic richness estimated by rarefaction using the lowest sample size of n=17.

Table 3. Reliability of morphological field assessments judged against genetic data using a threshold of P dom ≤0.1 for pure M. sylvestris and P dom >0.1 for hybrid samples.

SUPPLEMENTARY MATERIAL

Supplementary Figure S1. Statistics of the STRUCTURE analysis over 20 runs for each K=2 to 10.

(A) Mean estimated ln probability L(K) of data (mean ± SD) (B) Rate of change of the likelihood distribution (mean ± SD) (C) ∆K method for most likely K according to [START_REF] Evanno | Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study[END_REF] (D) Pr [X|K] method for most likely K according to [START_REF] Pritchard | Inference of Population Structure Using Multilocus Genotype Data[END_REF]. Supplementary Figure S2. STRUCTURE results for K=2 to 10 using the full dataset (n=366). Only major modes are shown. Number of minor modes in square brackets. Reference samples of the following species: 1…M. baccata, 2…M. hupehensis, 3…M. orientalis, 4…M. sieversii, 5…M. sylvestris Supplementary Figure S3. STRUCTURE results for K=2 to 6 using only pure M. sylvestris samples (n=227, P dom ≤ 0.1). Only major modes are shown. Number of minor modes in square brackets.