Genetic integrity is still maintained in natural populations of the indigenous wild apple species Malus sylvestris (Mill.) in Saxony as demonstrated with nuclear SSR and chloroplast DNA markers

Abstract Malus sylvestris (Mill.) is the only indigenous wild apple species in Central Europe. Agriculture, forestry, and urbanization increasingly endanger Malus sylvestris natural habitats. In addition, the risks of cross‐hybridization associated with increase in the cultivation of the domesticated apple Malus × domestica (Borkh.) threaten the genetic integrity of M. sylvestris. The present study investigated the number of hybrids, genetic diversity, and genetic structure of 292 putative M. sylvestris that originate from five different natural M. sylvestris populations in Saxony, Germany. All samples were genetically analyzed using nine nuclear microsatellite markers (ncSSR) and four maternally inherited chloroplast markers (cpDNA) along with 56 apple cultivars commonly cultivated in Saxony. Eighty‐seven percent of the wild apple accessions were identified as pure M. sylvestris. The cpDNA analysis showed six private haplotypes for M. sylvestris, whereas three haplotypes were present in M. sylvestris and M. × domestica. The analysis of molecular variance (AMOVA) resulted in a moderate (ncSSR) and great (cpDNA) variation among pure M. sylvestris and M. × domestica individuals indicating a low gene flow between both species. The genetic diversity within the pure M. sylvestris populations was high with a weak genetic structure between the M. sylvestris populations indicating an unrestricted genetic exchange between these M. sylvestris populations. The clear distinguishing of M. sylvestris and M. ×domestica confirms our expectation of the existence of pure M. sylvestris accessions in this area and supports the argument for the implementation of preservation measures to protect the M. sylvestris populations in Saxony.


| INTRODUC TI ON
The genus Malus Mill. comprises 25-47 species, depending on the taxonomic classification (Robinson et al., 2001). While several Malus species are indigenous to Asia, Malus sylvestris (Mill.) is the only indigenous wild apple species in Central Europe. The spatial distribution of this European crabapple ranges from South Scandinavia to the Iberian Peninsula and from the Volga to the British Isles (Robinson et al., 2001). Despite this vast distribution area, M. sylvestris is very rarely seen and in fact endangered in most European countries (Bitz et al., 2019;Coart et al., 2006;Höltken et al., 2014;Larsen et al., 2006;Reim et al., 2012Reim et al., , 2013Wagner et al., 2014). Its economical insignificance and the present-day intensive land usage by forestry and agriculture are the main reasons why wild apple trees have been displaced into areas with more or less unfavorable growing conditions. However, M. sylvestris trees have, except for their light requirements, low demands on environmental conditions. They are therefore able to survive in such niche areas. M. sylvestris populations are often very small and spatially distributed, which may lead to a reproductive isolation of individual populations (Reim et al., 2017;Ruhsam et al., 2019;Wagner et al., 2014). Most Malus species are self-incompatible and need compatible pollination partners to maintain the natural regeneration of the populations (Hanke et al., 2020). Insects such as bees or bumblebees mainly distribute apple pollen, and most pollination events occur in a short distance (50-100 m) around the pollinator. However, if sufficient pollination partners are missing, the distance of pollination can remarkably increase (Reim et al., 2017). Since no reproductive barriers prevent the hybridization between different species within the genus Malus, the probability of admixture with domesticated apples (Malus × domestica Borkh.) increases particularly in small populations (Korban, 1986;Larsen et al., 2006;Reim et al., 2017). Such hybridization events endanger the genetic integrity of M. sylvestris by replacing pure M. sylvestris trees with hybrids (Allendorf et al., 2001). Thus, the conservation of genetic resources must extend to include the preservation of pure M. sylvestris trees.
Knowledge of population genetic diversity and genetic structures is crucial in the identification of pure genotypes, which is integral in studies for the implementation of sustainable conservation strategies (Castiglione et al., 2010). However, in closely related species such as in Malus a clear distinguishing of species remains a challenge. Delimitation of M. sylvestris and M. ×domestica is particularly difficult because of their close relationship (Duan et al., 2017). Several morphological characteristics were described as useful for the classification of M. sylvestris like hairiness of leaf lower surface, hairiness of the flower stem; fruit size, and cover color (Wagner, 1996). However, the main disadvantage of morphological traits is their high level of variation depending on various environmental factors. For example, the intensity of hairiness, which functions as protection against evaporation, can vary during the growing season. Another example is the intensity of over color, which depends on exposure to sun light. Therefore, genetic markers are an indispensable tool for taxonomical studies of species. In conservation genetic studies, microsatellite markers or simple sequence repeats (SSRs) are still the markers of choice (Bitz et al., 2019;Coelho et al., 2018;Kaczmarczyk, 2019;Lea et al., 2018) and are useful tools for a putative identification of "hybrids" within potential wild apple populations (Koopman et al., 2007;Larsen et al., 2006). For closely related species, such as the species within the genus Malus, noncoding DNA regions of the chloroplast genome could also be an additional useful genetic tool to study the relationship among species (Fan et al., 2019;Khadivi-Khub et al., 2014;Tang et al., 2014;Volk et al., 2015). Because of their slow rate of molecular evolution and the conserved structure of their genome, chloroplast markers were developed for several important plant species (Heinze, 2007). In Malus, several intergenic spacer and introns regions in the chloroplast genome were identified and from these regions, primers successfully generated and applied for phylogenetic studies Fan et al., 2019;Khadivi-Khub et al., 2014;Tang et al., 2014;Volk et al., 2015;Xu et al., 2019).
In our study, M. sylvestris accessions from five natural populations in Saxony (Germany) were genetically investigated using nine nuclear microsatellites (ncSSR) and four chloroplast DNA (cpDNA) markers. All putative M. sylvestris individuals were analyzed with ncSSR markers in order to identify pure accessions. Pure M. sylvestris accessions and 56 M. × domestica genotypes were further analyzed with four cpDNA markers to ascertain haplotype variation within and between these species. We further estimated the genetic diversity within the M. sylvestris populations and the genetic structure among these populations in the study area. The results of this study are the basis for the implementation of conservation measures.

| Study site, sampling, and DNA isolation
The study was carried out in the federal state of Saxony, in Southeast Germany. Saxony comprises an area of 18,413 square kilometers, and hills and mountains mainly compose the landscape. Two hundred and ninety-two potential M. sylvestris trees sampled from five different areas within Saxony (Figure 1) formed the basis of this study.
The mapped trees were mainly found in sparse forests, along forest edges, stonewalls, or other open landscape structures. The tree density within the populations was low in most cases with partly large distances between the single trees. The trees of some population such as in the East Ore Mountains (OEG) were distributed over several square meters. Compact populations with more than 50 individuals in a close spatial network only existed in the Leipziger Auwald (LEI) and Bahretal (BAR). Seven percent of the sampled trees were located in a nature reserve. The remaining 93% were located in unprotected areas. The selection of trees was based on several morphological characters described by Wagner (1996)  Fresh leaf materials were collected in 2-ml tubes and dried using silica gel according to a modified protocol of (Chase & Hills, F I G U R E 1 Origin, number of sampled M. sylvestris trees and number of hybrids in five natural populations in Saxony, Germany TA B L E 1 Chloroplast DNA (cpDNA) and nuclear SSR (ncSSR) markers used in this study.
Multiplex PCR was performed using the Type-It kit (Qiagen, Germany) according to the manufacturer's protocol with three primer pairs per PCR in a total volume of 10 µl. PCR fragments were analyzed on a CEQ 8000XL Genetic Analyzer System (Beckman Coulter, Germany), for which forward primers were labeled with BMN-5, BMN-6, and DY751 (Biomers, Germany).

| Hybridization and population structure analysis
First, samples were grouped into pure M. sylvestris, M. × domestica or hybrids using the model-based clustering method with STRUCTURE software version 2.3.4. (Pritchard et al., 2000). In order to improve the accuracy of the inference, the analysis was The remaining parameters were described as above. STRUCTURE HARVESTER (Earl & Vonholdt, 2012) was used for detecting the most likely value for K based on Evanno's ΔK method (Evanno et al., 2005). To examine the genetic structure of M. sylvestris, a principal coordinate analysis (PCoA) was performed based on the distance matrix data set of the 255 pure M. sylvestris accessions and the 56 reference apple cultivars in GeneAlex ver. 6.5 with 1,000 random permutations.

| Diversity analysis
The mean number of alleles by locus (N a ), effective number of alleles  (Szpiech et al., 2008) using the rarefaction method to correct differences in population sizes (Kalinowki, 2005). Because of the small sample size of the TOR and VOG populations, the Ar was weighted to fourteen individuals.
The allele frequencies of the chloroplast DNA markers were compared among the M. sylvestris individuals and the apple cultivars.
An "analysis of molecular variance" (AMOVA) was performed based on nuclear and chloroplast marker data. The molecular variance (ɸ PT, an analogue of F st ) and the migration rate (N m ) were calculated among the pure M. sylvestris genotypes and the apple cultivars as well as among the different M. sylvestris populations using GENALEX ver. 6.5.
The correspondence (rxy) between geographic and genetic distance was performed by Mantel test with statistical testing by 9,999 permutations using the software GENALEX ver. 6.5 (Mantel, 1967).

| Genetic differentiation between M. sylvestris and M. × domestica
Of 292  Based on cpDNA data, a great variation of 56% among pure M.
The effective gene flow was much less than one successful migrant per generation (N m = 0.39).

| High genetic diversity and weak genetic structure between the M. sylvestris populations
All ncSSR markers showed reproducible results with one or two am-  After AMOVA, the differences between the five M. sylvestris populations were significant, but low (Table 3) (Coart et al., 2003;Höltken et al., 2014;Schnitzler et al., 2014), and therefore, the frequency of hybrids is slightly higher in our population but corresponds well to the morphological characteristics of the sampled M. sylvestris accessions. These results indicate that a genetic differentiation of both species based on ncSSR is still possible although nuclear genome markers disclose less genetic differences in closely related species (Korotkova et al., 2014;Zheng et al., 2014).

| Haplotype variation between M. sylvestris and M. × domestica
In closely related species such as apple, the investigation of phylogenetic relationships based on different cell genomes is recommended (Nikiforova et al., 2013). Chloroplast DNA is maternally inherited and hybridization events are limited to gene flow by seeds which results in a higher level of genetic differentiation among species or populations (Ennos, 1994;Korotkova et al., 2014;Shizuka et al., 2015).
In our study, 20% of the detected haplotypes were common in M. sylvestris and M. × domestica suggesting that these haplotypes are not species specific. Cross-species shared chloroplast haplotypes were observed for several perennial species such as birch, eucalyptus and willows (Fogelqvist et al., 2015;Nevill et al., 2014;Palme et al., 2004). Within the genus Malus, shared haplotypes were also identified in studies including numerous apple individuals of different cultivars and species (Savolainen et al., 1995;Volk et al., 2015). M. × domestica is known to be an admixed species from a number of progenitor species such as M. sieversii, M. orientalis, M. sylvestris, and M. prunifolia (Cornille et al., 2013;Robinson et al., 2001;Velasco et al., 2010;Volk et al., 2015). Thus, shared haplotypes may reflect the historical introgression between different Malus species due to gene flow or retention of ancestral polymorphisms (Koopman et al., 2007;Nevill et al., 2014;Volk et al., 2015).

| Gene flow between the M. sylvestris populations in Saxony
Fragmented and spatially isolated populations of rare species such as M. sylvestris are often expected to have reduced genetic diversity and strong genetic structure because of a restricted gene flow (Bacles & Jump, 2011;Kramer et al., 2008;Pierce et al., 2017).
However, despite the fragmented distribution of M. sylvestris in the study area, the results of our study indicate a high genetic diversity and a weak genetic structure between the single populations.
Other studies also documented that particularly long-lived plants such as M. sylvestris can maintain connectivity even in highly fragmented populations through extensive gene flow via pollen and/ or seeds (Feurtey et al., 2017;Gonzalez-Varo et al., 2019;Lowe et al., 2016;Ozawa et al., 2013;Plue & Cousins, 2018;Schnitzler et al., 2014;Wang et al., 2014). The comparison of nuclear and chloroplast data allows the separation of the impact of pollen and seed-mediated gene flow between populations (Ennos, 1994). The N m -value in our study indicated a high historical genetic exchange with a higher number of migrants by seeds (N m = 7.07) than by pollen (N m = 4.14.) between the M. sylvestris populations. Apple seeds are distributed by numerous wild animals such as mammals or birds, feed on the apple fruit. These animals travel over long distances and probably accounting for the large apple-dispersal capacities (Schnitzler et al., 2014). In addition, humans dispersed wild apple fruits over long distances in recent times (Spengler, 2019). As a result, in the past genetic exchange by seeds between population occurred and even one single hybridization event retained within a lineage because the chloroplast genome is uniparental inherited (Currat et al., 2008).
Additionally, wild apple populations probably compensated their spatial isolation by higher pollen dispersal distances. Single located individuals can act as so-called stepping stones and bridge larger distances between groups of trees (Albaladejo et al., 2012;Kramer et al., 2008 introgressed wild apples were observed in our study, the implementation of sustainable conservation strategies seems to be necessary for a long-term preservation of this rare species.  An additional conservation of pure M. sylvestris accessions ex situ can be also meaningful. That can be implemented by the storage of seeds in a GenBank or the establishment of seed orchards.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available