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. M. sylvestris that originate from five different natural populations in Saxony, Germany. Genetic analysis were performed with nuclear and chloroplast markers. We detected a high genetic diversity within the pure M. sylvestris populations but with a weak genetic structure between the M. sylvestris populations indicating an unrestricted genetic exchange between these M. sylvestris populations.The present study investigated the number of hybrids, genetic diversity, and genetic structure of 292 putative  The spatial distribution of this European crabapple ranges from South Scandinavia to the Iberian Peninsula and from the Volga to the British Isles  increases particularly in small populations  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.In our study, 22.1M. sylvestris trees sampled from five different areas within Saxony  often cultivated in Germany were included as control genotypes. The location of the captured M. sylvestris trees and the names of the 56 cultivars are available in Dryad at https://doi.org/10.5061/dryad.g79cnp5n2. Leaf material of these 56 apple cultivars was provided by the Fruit Genebank of the Institute of Breeding Research on Fruit Crops of the Julius Kühn‐Institut (JKI) at Dresden‐Pillnitz.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 y Figure  includinFresh leaf materials were collected in 2‐ml tubes and dried using silica gel according to a modified protocol of . Leaf materials were stored at room temperature until DNA isolation. LGC Genomics  performed DNA isolation and quantification using the semi‐automated high‐throughput Genespin™ platform with the company proprietary Kleargene™ extraction chemistry.2.2M. × domestica  developed for matK_dup (NCBI GenBank: AF309231), rps16 intron (NCBI GenBank: JQ391664) region and the intergenic spacer region rps16_trnQ (NCBI GenBank: AB604694) and rpl2_trnH (NCBI GenBank: JQ392141), respectively, were developed based on Malus sequence data obtained from the NCBI database  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 , for which forward primers were labeled with BMN‐5, BMN‐6, and DY751 .2.3M. sylvestris, M. × domestica or hybrids using the model‐based clustering method with STRUCTURE software version 2.3.4.  was estimated with STRUCTURE. For this, we run the program again including all pure M. sylvestris accessions with K varying from 2 to 10 with 5 runs for each K value without POPINFO model. The remaining parameters were described as above. STRUCTURE HARVESTER  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.First, samples were grouped into pure 2.4aN), effective number of alleles (eN), observed heterozygosity (oH), expected heterozygosity (eH), and number of private alleles (PA) were calculated for each nuclear SSR loci within the pure M. sylvestris individuals and the apple cultivars using the software GENALEX ver. 6.5 . Allelic richness (Ar) was calculated with the software ADZE  and the migration rate (mN) 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.An "analysis of molecular variance" (AMOVA) was performed based on nuclear and chloroplast marker data. The molecular variance (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  were assigned as pure M. sylvestris species. The remaining 37 individuals were assigned as hybrids or feral M. ×domestica . Twelve haplotypes were not observed for both species: H1, H2, H3, H5, H8, and H13 were not observed in the investigated apple cultivars whereas H4, H6, H9, H10, H14, and H15 were not found in the analyzed M. sylvestris genotypes. Only three haplotypes  were present in both M. sylvestris and M. × domestica.The combination of these different alleles resulted in 15 different haplotypes Table  Figure . In M. ×M. sylvestris and M. ×domestica . The effective gene flow was about one migrant per generation (m = N1.01). Based on cpDNA data, a great variation of 56% among pure M. sylvestris and the apple cultivars (ɸPT = 0.56 p = .001) was detected. The effective gene flow was much less than one successful migrant per generation (m = N0.39).The analysis of molecular variance (AMOVA) showed significant results for the differentiation between a Figure . Based o3.2M. sylvestris genotypes with an average number of alleles of aN = 10, an average effective number of alleles of eN = 4, a mean expected heterozygosity of eH = 0.73, and a mean allelic richness of Ar = 5.03 , previously identified by STRUCTURE as pure M. sylvestris accessions, showed a very close relationship to M. × domestica, indicating that these accessions also hybrids.In general, PCoA supported the results of the STRUCTURE analysis and showed low genetic differences between the y Figure . HoweverM. sylvestris populations with different frequencies in each population. Haplotype H3 was found in the populations LEI, VOG, and BAR, which are located in the western and eastern parts of Saxony. H13 was found in the southeastern population OEG and the northern population TOR. The both haplotypes H5 and H8 were the only private haplotypes and observed only in OEG and TOR, respectively.Based on the cpDNA data, also weak population structures were observed Figure . Of the M. × domestica  were also detected in the M. sylvestris populations with one exception. The population TOR showed only M. sylvestris‐specific haplotypes . In the both southeastern populations OEG and BAR, all three M. ×domestica shared haplotypes were observed with the highest frequency for H12   and mN = 7.07 (cpDNA) indicating random mating among each population and weak genetic structure of the populations. Mantel test resulted in a significant (p = .01) but low correlation coefficient rxy = 0.05 suggesting that the geographic distance had hardly any effect on the genetic variance in the Saxony wild apple populations.After AMOVA, the differences between the five ow Table . Based o44.1M. sylvestris and the clear identification of hybrids is one of the important prerequisites for the implementation of sustainable conservation measurements and the maintenance of pure species in natural populations. However, a clear distinguish between the European crabapple and cultivated apples is difficult because of molecular similarity between both species  but depends strongly on the cultivar . Based on this, all M. sylvestris samples and apple cultivars were clearly assigned in either a "M. sylvestris cluster" or "M. × domestica cluster." Putative M. sylvestris individuals with admixture genetic information of more than 10% of the M. × domestica gene pool were identified as hybrid (13%). This discrimination threshold (Q > 0.90) was very strict compared to other studies  in our study indicating a low historical gene flow through seed dispersal between M. × domestica and M. sylvestris. Evolutionary studies showed that large fruit development in domesticated crops such as M. ×domestica correlates with a reduction in seed number/ fruit and a loss in natural seed‐dispersal mechanism . A similar rarity of gene flow among M. sylvestris and M. × domestica in the Rhine Valley or in Finland was observed by Schnitzler et al.  than by pollen (mN = 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  are recommend. Particularly, the reintroduction of young trees and the increase of number of trees within the natural populations are recommended to reduce the risk of hybridization. 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.Therefore, the protection of existing The authors declare that they have no conflict of interest.Stefanie Reim: Conceptualization (lead); Funding acquisition ; Investigation (lead); Project administration ; Writing‐original draft (lead). Anke Proft: Data curation (supporting); Investigation ; Project administration ; Validation (supporting). Frank Lochschmidt: Data curation (supporting); Investigation (supporting); Resources (supporting). Monika Höfer: Conceptualization (supporting); Resources (supporting); Validation (supporting).Fig S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


Parkia biglobosa.A good understanding of genetic variation and gene dispersal in tree populations is crucial for their sustainable management, particularly in a context of rapid environmental changes. West African Sudanian savannahs are being fragmented and degraded, partly due to expansion of crop cultivation and monocultures that reduce tree density and may impact pollinators. The population dynamics of important indigenous trees could also be affected. We investigated the influence of habitat fragmentation on patterns of genetic diversity and gene dispersal of a key Sudanian agroforestry tree species, Using 10 highly polymorphic nuclear microsatellites, we genotyped 2475 samples from reproductive trees, seedlings, and embryos in four tree populations presenting different levels of habitat fragmentation.Parkia biglobosa presented similar high genetic diversity across the four populations studied. Genetic diversity and inbreeding were similar between adults and embryo cohorts. In all four populations, the selfing rate was less than 1%. The effective number of pollen donors per tree was high (NEP~ 18–22), as was the pollen immigration rate (from 34 to 74%). Pollen dispersal was characterized by a fat‐tailed distribution with mean estimates exceeding 200 m. In three populations, stem diameter had a pronounced effect on male reproductive success. Here, the highest male reproductive success was observed in trees with a diameter at breast height between 60 and 75 cm.At the scale analyzed, fragmentation does not seem to pose limitations to gene flow in any of the sites investigated, regardless of the landscape configuration associated with the different tree stands. The study provides useful insights on the reproductive biology of an important tree species in the West African savannahs. A good understanding of genetic variation within and among populations of a species is a necessary foundation for its sustainable management, including the conservation of its genetic resources, particularly in the face of climate changes and increasing threats associated with habitat loss and fragmentation. Genetic diversity is a source of valuable traits; the main reasons for conserving it are to ensure the future adaptability of tree populations; preserve particular traits of interest for production, income, or cultural reasons; and maintain other valuable ecosystem services supporting the resilience of forests and landscapes to future environmental changes  and outcross‐pollen (outcrossing). Selfing is generally detrimental, particularly in tree species, and leads to inbreeding depression Geburek, . ReproduPesticides have been reported among the causes of observed declines in pollinators  and two from traditional agroforestry parklands with limited levels of fragmentation  and compared the two clusters for the following traits: (1) levels of genetic diversity and inbreeding; (2) mating system (i.e. outcrossing rate); (3) contemporary gene dispersals . Due to an effect of cotton cultivation on population density and pollinator abundance in CP, we expected: (1) a signal of decrease in genetic diversity and of increase in inbreeding between the adult and the embryo cohorts in CP, but no signal in NCP; (2) lower outcrossing rates in CP than in NCP; (3) lower pollen‐mediated gene flow in CP than in NCP.The present study investigated the level of genetic diversity and patterns of gene flow in an agroforestry tree species growing in landscapes with a different configuration of land uses, with variable levels of fragmentation of the suitable habitat of the tree species, depending on the degree of clearing for agriculture and cotton farming. We expected to detect differences in genetic diversity and gene flow in Parkia biglobosa (Jacq.) G.Don, commonly named African locust bean or néré (French), is a fruit tree species belonging to the Fabaceae, precisely to the mimosoid clade in the Caesalpinioideae subfamily, based on the most recent classification , where individuals of useful tree species—retained after clearing the land for agriculture—were interspersed among crops to form a so‐called parkland. The dominant tree species inside the plots was P. biglobosa, and the most‐represented associated tree species were medicinal and food trees such as Adansonia digitata, Detarium microcarpum, Diospyros mespiliformis, Tamarindus indica, Vitellaria paradoxa, and Vitex doniana. The four selected P. biglobosa populations differed in diameter structure, tree density, abundance of small and large trees, presence of regeneration  are located in areas where parklands were established in more recent times 1990s) and tree populations have never been mixed with cotton. Agriculture in these locations is a traditional farming system that is not based on pesticides use; annual crops  are cultivated in combination with a tree cover formed by useful tree species. We considered Cassou as a NCP, given that it is located 5 km from the first cotton field (the recent cotton company named Fasocotton was established in the area in 2004) and has a diameter structure that supports this classification Appendix . In each990s and Overall, DNA was isolated from seed and leaflet samples of 2475 individuals composed of 703 adult trees , 1747 embryos, and 25 seedlings from Cassou and Saki. The total genomic DNA was extracted using the DNeasy 96 Plant Kit protocol  and the manufacturer's instructions. DNA samples were genotyped using 10 nuclear microsatellite loci (nSSR) developed by Lassen et al.  and the E) following Nielsen et al. (AR) expressed as the expected number of alleles based on a minimum subsample size of k gene copies, expected heterozygosity  corrected for sample size (HE) , and individual inbreeding coefficient (F). We tested the significance of inbreeding coefficients using 10000 gene copy permutations. The presence of null alleles causes a bias in the proportion of heterozygosity and consequently affects the estimates of inbreeding coefficients within a population  per locus and cohort in each population, clustered in two groups (NCP and CP), were used for statistical analyses, using an analysis of variance procedure in R . To this purpose, we estimated the following genetic diversity variables per population and per cohort in each population using the program SPAGeDi 1.4  Nei, , observei and j based on kinship coefficient ijF  and the strength of spatial genetic structure (Sp) were quantified by Sp = bF/(F1 − 1) and Nb = (F1 − 1)/bF, where F1 is the average pairwise kinship coefficient at the first distance class and bF is the slope of the logarithm regression  and single‐locus outcrossing rate (ts) were calculated from progeny arrays using known maternal parents and the Newton–Raphson estimation procedure. Selfing rate (s) was also assessed by NMπ software , we calculated the mean effective number of pollen donors that participate in cross‐pollination as NEP = 1/rp. We tested whether the among‐sibship‐correlated paternity is inversely correlated with the distance between mother trees using a Mantel test procedure as implemented in the zt software  was estimated with TWOGENER using as input the scale parameter of the dispersal distribution (a) as estimated with KINDIST  of maternal tree i and position  of putative pollen donor j assigned by paternity analysis. Pollen dispersal distance δp was calculated considering the Euclidean distance between the two spatial coordinates: δp(xi‐xj)2+(yi‐yj)2.A paternity analysis was then conducted to directly infer pollen dispersal using the CERVUS program ; (3) pollen immigration rates (mp), which provides an indication on the proportion of seeds pollinated by nonsampled adults ; (4) mean distance of pollen dispersal (dp); (5) shape parameter of pollen dispersal kernel (bp) ; and (6) the effect of DBH on male reproductive success. For each population, analyses were run on two data sets: (1) all adults from the plot only (used to estimate mp); (2) all adults from the plots and the transects .Characteristics of the pollen dispersal kernel were then evaluated using the neighborhood model as implemented in NMπ. This method allows estimating through a maximum likelihood approach: (1) genotyping error rates per locus; (2) selfing rate  and its diameter at breast height (DBH). To this aim, we used a generalized linear model (GLM) with negative binomial distribution and log‐link. Covariates were the DBH, its quadratic term and the interaction with the respective tree population. The DBH has been centered to its sample mean value (48.7).rp, NEP, δp) between CP and NCP, we performed a bootstrap procedure for independent samples using SPSS as above based on pollen dispersal data inferred per maternal trees in each population from KINDIST and CERVUS.To test differences in mean values of pollen dispersal  only in the embryo and mature tree cohorts from Saki, probably in relation to the presence of null alleles  and inbreeding (F) between NCP and CP were not significant  in all studied populations  in Cassou and for the two first distance classes in Walley (0–200 and 201–400 m). The pairwise kinship coefficients decreased with distance and became nonsignificantly different from zero at 100 m in Cassou (8.3 adults/ha) and 300 m in Walley (4.7 adults/ha), indicating that spatially close individuals are related at these respective distances. Overall, the strength of SGS in the four studied populations was very weak  and Walley (CP) Fig. . The SGStm − ts), that varied from 0.03 to 0.05 without significant differences between CP and NCP  and the shape parameter affecting the fatness of the tail of the dispersal distribution (b) were similar in all populations except Vouza. This result indicates that pollen dispersal events occur at shorter distances in this population . The estimate of pollen pool differentiation in the maternal trees was low and similar (Φft = 0.03–0.04) between NCP and CP. The proportion of individuals that participate in pollination (DEP/D) varied from 51%  to 96% (Vouza). The average within‐sibship‐correlated paternity was also low and relatively similar between NCP and CP . The average pollen dispersal distance estimated through indirect analyses based on the spatial structure of pollen pools was 81–242 m and 132–259 m in NCP and CP, respectively , suggesting the presence of a self‐incompatibility system in the species  was the highest in Saki (the higher‐density population), with 74% of pollination events coming from pollen donors located outside of the plot , whereas Cassou corresponds to another (CWA3)  and similar to those found in other P. biglobosa populations from the central part of West Africa  was relatively weak and equivalent in all populations. SGS was calculated based on the spatial distribution of genetic diversity of adult trees and is indicative of a predominantly outcrossing species. It is important to note that in Walley and Saki populations, most adult trees were already in place before the start of cotton cultivation, so the effect of cotton cultivation on SGS cannot be evaluated in these populations. It would have been interesting to compare patterns of SGS in older trees (those present before cotton cultivation started) and younger trees (those established after the start of cotton cultivation), but sample sizes would be too small.The strength of the fine‐scale SGS , with little selfing  and with insignificant inbreeding. Previous studies based on allozyme markers and a controlled pollination experiment showed that P. biglobosa is self‐compatible with a selfing rate of 5%  was found in a drier site with a tree population density 20 times lower than in our site with lowest tree density (0.26 trees/ha), which seems to suggest that selfing does not occur until a minimum threshold in population density is reached. On the other hand, in a humid site previously studied, selfing increased from 2 to 19% when both bats and honey bees were excluded  according to the pollen structure model in POLDISP, which roughly corresponds to estimation using MLTR (NEP ~18–22). We obtained a similar range of pollen dispersal distances using either indirect (KINDIST: δp ~82–259 m) or direct (CERVUS: δp ~220–270 m) analyses , the consequences on population reproductive dynamics cannot be demonstrated. Moreover, male reproductive success was the highest for trees with DBH 60–75 cm in three of the four populations , landscape fragmentation, and the mechanism of self‐incompatibility.Our study suggests that, in the study sites investigated, the reduced density of We note again that the regeneration of two CP seems to be affected because we could not find seedlings in these populations. If the same trend is confirmed, these populations will be strongly affected in upcoming years. We should better understand the reproductive biology of this very important tree species exposed to significant land‐use changes.Given ongoing threats to agroforestry trees in the Sudanian region of West Africa, in situ and ex situ conservation strategies need to be developed. For ex situ strategies, it should be considered that all seeds in a pod are full siblings  of the four Parkia biglobosa populations.Click here for additional data file.APPENDIX S2. Spatial distribution of Parkia biglobosa showing location of individual trees in all study sites.Click here for additional data file.APPENDIX S3. Genetic diversity and inbreeding coefficient in each of the four populations and per locus based on 2468 genotypes of Parkia biglobosa.Click here for additional data file.APPENDIX S4. Parameters of spatial genetic structure, mating system, and pollen dispersals in the four Parkia biglobosa populations investigated.Click here for additional data file.APPENDIX S5. Parameters of spatial genetic structure, mating system, and pollen dispersals in the four Parkia biglobosa populations investigated.Click here for additional data file.APPENDIX S6. Male reproductive success rate: relationship between the number of times an individual contributed to pollination of collected seeds (y‐axis), as estimated by paternity analysis, and its diameter at breast height , in each Parkia biglobosa population.Click here for additional data file.


Oryctolagus cuniculus) is declining in large parts of Europe but populations in some German cities remained so far unaffected by this decline. The question arises of how urbanization affects patterns of population genetic variation and differentiation in German rabbit populations, as urban habitat fragmentation may result in altered meta-population dynamics. To address this question, we used microsatellite markers to genotype rabbit populations occurring along a rural-to-urban gradient in and around the city of Frankfurt, Germany. We found no effect of urbanization on allelic richness. However, the observed heterozygosity was significantly higher in urban than rural populations and also the inbreeding coefficients were lower, most likely reflecting the small population sizes and possibly on-going loss of genetic diversity in structurally impoverished rural areas. Global FST and G′ST-values suggest moderate but significant differentiation between populations. Multiple matrix regression with randomization ascribed this differentiation to isolation-by-environment rather than isolation-by-distance. Analyses of migration rates revealed asymmetrical gene flow, which was higher from rural into urban populations than vice versa and may again reflect intensified agricultural land-use practices in rural areas. We discuss that populations inhabiting urban areas will likely play an important role in the future distribution of European rabbits.The European rabbit ( Urbanization has been identified as one of the major global threats to biodiversity in the Anthropocene3 and a major driver of evolutionary change4.As cities expand worldwide, urban areas will double in size, cover approximately 10% of Earth’s landmass, and host around 5 billion people by 20306. Only organisms that are able to cope with (or adapt to) the novel set of abiotic and biotic conditions colonize urban ecosystems7. Understanding how the expansion of cities—as a major component of global environmental change—affects the distribution of biodiversity as well as evolutionary change within species has become increasingly important for the conservation of biodiversity on Earth8. At the same time, cities can be viewed as unintended ‘large-scale experiments’8, providing scientists with the unique opportunity to elucidate general principles of ecological dynamics and study evolutionary processes.Urbanization leads to the destruction of natural habitats and creates new, highly fragmented landscapes with increased impervious surfaces, as well as air, water, light and noise pollution9. Two widely studied evolutionary processes in urban wildlife populations are genetic drift and gene flow4. The strength of genetic drift is predicted to increase with increasing urbanization4. Most of the wildlife in cities occurs within the few remnant semi-natural patches and/or other human-made green spaces such as parks, botanical and private gardens4. These areas are likely to harbour small and isolated populations, surrounded by strong barriers to dispersal . Empirical evidence for reduced dispersal and gene flow among urban populations comes from studies on various animal and plant species4. This is true even over short geographic distances and for otherwise very mobile species such as song sparrows (Melospiza melodia)10, wren-tits (Chamaea fasciata)11 and red foxes (Vulpes vulpes)12. Isolation of populations, coupled with small population sizes, will exacerbate the effects of genetic drift and can result in reduced genetic diversity within and greater genetic differentiation among urban populations, as has been shown for a number of species, including the white-footed mouse (Peromyscus leucopus)13, the yellow toadflax (Linaria vulgaris)14 and the fire salamander 15.Using molecular markers and spatial ecological tools, a number of studies addressed the question of how evolutionary processes—including novel mutations, genetic drift, gene flow, and selection—operate in cities18. For instance, increased urbanization does not reduce gene flow nor genetic diversity in the downy yellow viole (Viola pubescens)19, orchid bees (Euglossa viridissima and E. dilemma)20, the spotted salamander (Ambystoma maculatum)21, the west coast ctenotus 22, the ornate box turtle (Terrapene ornata)23, the red-tailed bumblebee (Bombus lapidarius)24 and the Eurasian tree sparrow (Passer montanus)25. This is not surprising, as the effects of urbanization are likely dependent on the form and intensity of land use, the spatial scale of investigation, and especially ecological attributes of the studied taxonomic group . Urban habitats can sometimes harbour large populations of certain species and do not always hinder, but might rather facilitate gene flow. Green spaces within the urban ecosystem may promote gene flow28 and may act as sources of genetic diversity29. Furthermore, while rivers and roads in cities represent barriers to some species31 they may function as corridors for dispersal and gene flow in others33.Nonetheless, some animal and plant species are thriving in cities and do not seem to follow the aforementioned patterns of altered meta-population dynamics and reduced genetic variability34. Under isolation-by-distance (IBD), geographic  distances predict genetic differentiation, as the likelihood of dispersal decreases when the distance between populations increases36. Moreover, the extent of gene flow can be a correlate of environmental (not geographic) distances between populations, resulting in patterns of isolation-by-environment (IBE)38. IBE may result from various mechanisms, including selection and local adaptation, as well as non-random dispersal and gene flow, and could be as common as IBD across environmental gradients35. In a study of white-footed mouse populations in the New York City metropolitan area, Munshi-South et al.13 used IBD and IBE modelling to demonstrate that urbanization  drives genetic differentiation to a greater extent than geographic distances .Several factors—including landscape processes—can lead to a reduction of gene flow and thus, genetic differentiation between populationsOryctolagus cuniculus) occurring in and around the city of Frankfurt am Main in Germany. European rabbits are native to the Iberian Peninsula39, from where they reached Western Europe since Roman times40. While European rabbits are considered a multifunctional keystone species in their native range, crucial in maintaining the organization and diversity of local ecological communities41, they are also one of the most successful invasive mammals world-wide, often with dramatic negative effects on local biodiversity, ecosystems, and the economy44. Currently, populations within the species’ native range (and throughout Europe) are dramatically declining45, most likely due to habitat loss as a result of intensified agricultural practices in combination with diseases like myxomatosis and rabbit calicivirus, as well as human-induced mortality45. Rabbit populations in several German cities appeared to be largely unaffected by this decline and were reported to reach high densities48. Urban habitat diversity and the increased availability of suitable sites for burrow construction in parks and gardens are among the hypothesized drivers underlying this pattern49. However, a vast decrease in hunting bag records within German cities such as Frankfurt indicate that also urban rabbit populations are affected by the overall population decline . The European rabbit is considered as an ‘urban adaptor’—a species that may utilize human resources and survive in human-dominated areas, but does not necessarily receive an added benefit from living with humans17.Here, we examine whether and how urbanization influences genetic diversity, dispersal/gene flow, and genetic differentiation in populations of the European rabbit . Second, we predicted that both geographic distances (IBD) and environmental distances (IBE) predict the amount of gene flow, which should decrease with increasing geographic and environmental distances.Based on previous studiesth century50. Frankfurt is the largest city in the state of Hesse and the fifth-largest city in Germany51. About 736,000 people live in the city’s administrative boundaries and about 2.4 million in its functional urban area, with an average density of 3,000 inhabitants per square kilometer51. Nonetheless, Frankfurt is considered a ‘green city’ since more than 50% of the area within the city limits are protected green areas .According to city archive records, rabbit populations have been established in Frankfurt am Main at least since the 1930s (Stadtarchiv Frankfurt). In Römer-Büchner (1827) we found indications that rabbits were present close to the city of Frankfurt am Main already in the 19We chose our eight study sites along a rural-to-urban gradient and included two parks in the city centre  that are surrounded by a high density of roads and human infrastructure, three parks located at the former periphery of the administrative district in Frankfurt with a medium density of roads and human infrastructure , and three adjacent rural areas with a low density of roads and human infrastructure . Additionally, we estimated the intensity of disturbance by humans  and leashed or unleashed dogs during the main activity period of the rabbits at dawn and dusk. Locations to quantify disturbance were randomly selected for each study site using the ArcMap Random Point Generator in ESRI ArcGIS 10 . The appropriate number of transect belts within study sites was determined in relation to the size of the area. These random locations were used as starting points to draw a virtual transect line of 25 m towards the North. All pedestrians, bikers and dogs crossing this transect line were counted for three minutes every 30 min. In total, 20 counts per site were performed on five consecutive days. Furthermore, we quantified the proportion of artificial ground cover  within a radius of 500 m from each of our study sites using a geographic information system (ArcGIS 10). We log‐transformed our data and subjected all variables to a principal component analysis in SPSS v. 13.0 for windows. The first principal component explained 80.87% of the variance and was used as a metric of the ‘degree of urbanity’49.Since the degree of urbanization does not necessarily decrease continuously towards the outskirts of a cityWe extracted high molecular weight DNA using the Qiagen DNeasy Blood & Tissue Kit® following the manufacturer’s instructions . We stored DNA samples at −20 °C until PCR-amplification at ten previously described microsatellite loci , observed (Ho) and expected heterozygosities (HE), as well as the inbreeding coefficient (FIS) and pairwise FST and G′ST-values58 between all eight sampling sites using 1,000 bootstrap iterations.We inspected our microsatellite data for the presence of null-alleles, large allele drop-out, and mis-scoring due to ‘stutter-bands’ using MICROCHECKER v. 2.2.359. BOTTLENECK compares the observed heterozygosity at each locus with the expected heterozygosity assuming mutation-drift equilibrium using a Wilcoxon signed-rank test. An excess in heterozygosity suggests recent population contraction (bottleneck) and heterozygosity deficit suggests recent population expansion. We used a stepwise mutation model (SMM) and a two-phase model  followed by 10,000 simulation iterations.To investigate whether a given population has undergone a recent bottleneck we used the BOTTLENECK v.1.2.02 softwareK-means clustering implemented in the find.clusters function within the R60 package adegenet61. This function first transforms the data using a principal component analysis and then it runs successive K-means clustering with increasing number of clusters (K). We performed the analysis without prior information on group membership of individuals and we assess the optimal number of groups using the Bayesian information criterion (BIC). To assess the level of admixture within sites, we used discriminant analysis of principal components (DAPC)62, implemented in the R package adegenet61. This approach transforms multilocus genotype data using principal component analysis to derive uncorrelated variables that serve as input for discriminant analysis. In the assessment of population structure, the discriminant analysis aims to maximize among-group variation and minimize within-group variation. In contrast to Bayesian clustering methods, DAPC does not require a population genetic model (Hardy–Weinberg or gametic equilibrium) and performs better at handling hierarchical structure or cline variation caused by isolation-by-distance63. The analysis was performed both with and without prior information on individual populations. When group priors were used, we used a barplot (compoplot command in adegenet) to visualize membership of individuals to different clusters.To infer the number of genetic clusters we used m) between populations, we used BayesAss v. 1.364, which uses a Bayesian approach and MCMC sampling. Migration rate estimates in BayesAss are based on the proportion of individuals in each population that are assigned to other populations with high probability, which can be detected within a few generations. BayesAss allows deviation from Hardy–Weinberg equilibrium but assumes linkage equilibrium, small migration rates and that (sub)population allele frequencies are unaffected by recent genetic drift or migration64. We ran BayesAss with 10 million iterations, a sampling frequency of 2,000 and a burn-in of 10%. All other settings were left as default. Ten independent runs with different random seeds were performed to assess MCMC convergence and evaluate the consistency of the results.To estimate recent migration rates ], log-transformed geographic distances, and environmental distances (pairwise differences of the ‘urbanity index’) using multiple matrix regression with randomization analysis (MMRR65) in the ecodist66 package in R using 1,000 permutations. Additionally, we tested for an association between IBD, IBE and migration rate estimates between populations. MMRR has been shown to be robust towards a wide range of dispersal rates and may be preferable to Mantel tests because of more appropriate Type I error rates, and because it ranks variables in terms of their relative effects on genetic distance65. To preserve the directional information in the BayesAss migration rates, we ran separate MMRR models on both top and bottom halves of the pairwise matrices  and isolation-by-environment (IBE) as an estimate of the impact of urbanization on population differentiation. To this end, we correlated genetic distances [linearized es Table . In all lme467. We additionally used linear modelling (LM) to test for differences in migration rates between alternative migration routes . For this analysis all sites within the city where treated as ‘urban’ and geographic distances between sites served as a covariate. Model assumptions were checked visually. Unless noted otherwise, all statistical analyses were performed in R v. 3.0.260.We explored the relationship between the urbanity index and rarefied allelic richness as well as inbreeding coefficients using linear mixed-effects models (LMM). We tested the relationship between the urbanity index and expected and observed heterozygosities by fitting generalized linear mixed-effects models (GLMM) with binomial error structure. This allowed us to control for differences in variability among the examined microsatellite loci by modelling ‘locus ID’ as a random effect. All mixed models were implemented in R using the package All experimental procedures described here were in accordance with the current laws on animal experimentation in Germany and the European Union and approved by licensed hunters .Ho) per locus ranging from 0.36 to 0.71 and expected heterozygosities (HE) ranging from 0.55 to 0.76 (Supplementary Table sat12) showed evidence for null alleles and was therefore excluded from subsequent analyses. Significant linkage disequilibrium (LD) was found between 7L1F1 and each of the following loci: 7L1B10, 7L5A4, sat7, 6L3B4, and sat12. Consequently, we also excluded the 7L1F1 locus from subsequent analyses.The number of alleles at each of the ten examined microsatellite loci ranged from 3 to 6, with observed heterozygosities (AR), based on rarefaction, was 2.725 ± 0.132 within our entire sample  and across loci, while the mean observed heterozygosity (HO) was 0.553 ± 0.050, and the mean expected heterozygosity (HE) 0.555 ± 0.038 (Table p < 0.05). We found a significant relationship between the urbanity index and HO  and FIS , and HO increased, while FIS decreased from rural over suburban towards urban populations  and AR  were not statistically significant. We did not find evidence for genetic bottlenecks in any of our populations  allelic richness  supported the idea that populations were significantly differentiated. The global multilocus FST-value indicated moderate differentiation across populations , as did the global G′ST . Most population pairs showed moderate to high genetic differentiation  that represented more than 95% of the total variance were retained. The lowest BIC-value (115.70) corresponded to K = 8 genetically distinct clusters  for all populations Table . The higp = 0.868; Fig. p = 0.542). We did, however, find evidence for isolation-by-environment (IBE), as pairwise environmental distances (based on the urbanity index) was a significant predictor of genetic distance , as the effect of geographic distance on genetic differentiation was not statistically significant  did not explain migration rates estimated from BayesAss  were thriving in urban areas have not yet been conducted. Here, by assessing allele length polymorphisms of ten nuclear microsatellite markers and employing a set of population genetic analyses, we investigate the genetic diversity, population genetic structure, and recent migration among O. cuniculus populations across a rural-to-urban gradient in Frankfurt am Main in Germany. Observed heterozygosity (HO) increased, while the inbreeding coefficient (FIS) decreased with increasing levels of urbanization . Our microsatellite data also revealed significant genetic structure among the studied populations, and models assuming isolation-by-environment (IBE)—based on urbanization effects—explained a greater proportion of both pairwise genetic differentiation (FST) and migration rates (m) than models assuming isolation-by-distance (IBD). We argue that rabbit populations in rural and urban areas derived from the same ancestral populations that were present as early as 182750 and successfully colonized the inner city in 1930 (Stadtarchiv Frankfurt am Main). This migration pattern is still observable today, since we found higher migration rates from rural into urban populations than vice versa.Despite the rapid decline of European rabbit populations across Europe4. By contrast, urbanization in the metropolitan area of Frankfurt and the associated typical changes in landscape structure and habitat types  seem to have a neutral or even a positive effect on the genetic diversity of European rabbit populations, as our analyses revealed higher heterozygosity and reduced inbreeding with increasing urbanization. However, our results are not surprising for an ‘urban adaptor’—such as the European rabbit—that during our sampling period occurred at high population densities in German cities49. Habitat heterogeneity, increased availability of resources, and low densities (or absence) of predators and competitors are among the hypothesized factors explaining the successful colonisation of cities by European rabbits49. Our results are in line with a previous study on a small mammalian ‘urban adaptor’, the white-footed mouse (Peromyscus leucopus), in New York City68. Munshi-South & Kharchenko68 found high genetic diversity, measured as heterozygosity, in urban populations, most likely explained by the high population densities in urban forest fragments.Several studies documented a loss of heterozygosity and reduced allelic richness at microsatellite loci in animal and plant populations inhabiting urban areas69. Previous studies reported high population densities in scrublands and areas characterized by interspersed patches of natural vegetation and crops70. Such landscapes provide a combination of both food and refuge for rabbits39. However, rural landscapes in Europe are becoming increasingly less structurally-complex and functionally-diverse due to a general homogenisation of agro-ecosystems and fragmentation of remaining patches of less intensely used areas71. This trend leads to the widespread loss of important habitats and the necessary landscape configuration required by European rabbits72 and is likely to be a major cause of the on-going decline of rabbit populations in rural parts of Europe39. The rural sites sampled for our present study are prime examples of homogenized and structurally impoverished open landscapes. In a previous study that included the same study sites, Ziege et al.46 found considerably lower population densities in our rural  compared to the urban  and suburban study areas . The small remnant populations in rural areas are likely prone to loss of genetic diversity due to drift and inbreeding.The distribution and abundance of rural rabbit populations are determined to a considerable extent by habitat heterogeneity, such as the proportion and type of canopy cover, availability of shrubs, and the general plant species compositionF-statistics) among all sampled populations revealed deviation from panmixia, and all urban populations were genetically differentiated from each other. This was also supported by the K-means clustering and discriminant analysis of principal components (DAPC), in which eight genetically distinct clusters were identified with relatively high assignment probabilities for our predefined populations . However, studies on Australian O. cuniculus populations demonstrated the capability of this species to move more than 20 km75. A recent study on home ranges of seven females and six males in two of the study sites included here, the suburban site RP (Rebstockpark) and the urban site Friedberger Anlage (belonging to IGG), revealed exceedingly small home ranges, with a ranging radius of less than 50 m, at both sites . The results of our present study, therefore, support the general notion of limited dispersal behaviour in rabbits74. Relatively higher dispersal at low population densities could explain the comparatively lower genetic differentiation and higher migration rates among our rural study populations, as well as the asymmetric  migration rates from rural into urban populations.European rabbits change their dispersal behaviour depending on local ecological conditions. Dispersal increases at low population densities, while philopatry prevails when population densities are high, whereby males show more dispersal than femalesMultiple matrix regression with randomization (MMRR) revealed that isolation-by-environment (IBE)—based on differences in the degree of urbanization differences among our study sites—but not isolation-by-distance (IBD) explained the observed population genetic differentiation and variation in migration rates among populations.76 that uncovered a high prevalence of IBE in natural systems. The patterns of IBE observed here can be explained by the action of natural selection in urban environments and/or non-random mating or migration associated with urbanization. The absence of IBD indicates that genetic patterns across our study populations are unlikely to be the product of drift and dispersal limitation alone. Reduced representation genome sequencing  across several independent, replicated urbanization gradients spanning the distribution range of European rabbits will be necessary to robustly assess genome-wide genetic diversity at thousands of SNPs and to partition genetic diversity into neutral and adaptive diversity. Such an approach will also allow identifying both key demographic parameters  and candidate loci under selection in urban populations.Overall, our results suggest that urbanization has played an important role in shaping the spatial distribution of genetic variation of European rabbit populations in and around Frankfurt am Main. Our results are in line with meta-analyses78. This highlights the need for empirical data on the influence of cities on the ecology and evolution of species that live side-by-side with humans, which could provide the basis for future conservation and management plans. The results presented here are in line with previous studies suggesting that urban habitats can support high populations of European rabbits49. Nonetheless, we also identified urbanization as a strong driver of genetic differentiation. The substantial genetic structure observed in urban populations, in combination with high genetic diversity, likely reflects high population densities but limited dispersal abilities in urbanized areas, or simply reduced dispersal behaviour. Habitat corridors that promote the connectivity of green areas both within cities and to the rural outskirts might enhance gene flow and help maintain the genetic diversity of urban populations, which may become vital in the future conservation of this species.The demographic changes and transformation of land-use characterising the Anthropocene will likely continue for the next decades49. A major limitation of our present study, however, results from the small number of neutral microsatellite markers employed in our study. Another potential caveat is that the population genetic patterns observed here could be due to released or escaped domesticated rabbits interbreeding with wild European rabbits. However, we argue that this is highly unlikely to happen for two main reasons: first, survival and reproduction rates of released domestic rabbits are considerably lower than those of wild rabbits, as domestic rabbits are more susceptible to predation79. Second, during our field work (2010–2015), we observed only one individual with a fur colouration characteristic of domestic rabbits amongst hundreds of wild rabbits that were monitored49. This individual was easy to distinguish from wild rabbits as it was considerably smaller, with an entirely black fur. If different levels of interbreeding between pet and wild rabbits would be the main driver behind the patterns described in our present study, this should visibly affect phenotypes; e.g. hybrid individuals should differ in size , behaviour (tamer than wild rabbits), and especially fur coloration (with black coloration being dominant in crossbreeds)81. Based on these criteria, none of the studied populations showed any indication of interbreeding.Our findings are largely consistent with previous studies on population monitoring of European rabbits in the region82. Finally, we found significant genetic differentiation between all studied populations, making it unlikely that repeated translocations between them occurred.Finally, we cannot exclude the possibility that rabbits were  relocated within our study region. Although relocation of European rabbits in the federal state of Hessia is forbidden by law, some hunters might still have moved individuals between populations so as to enhance their hunting grounds. Relocations could have occurred between urban donor and rural recipient populations. Nonetheless, we argue that potential relocations had a minor influence on the observed population genetic patterns due to the rarity of such events. Moreover, translocation success is low owing to the strong territorial behaviour and matrilineal social organization of European rabbitsTable S1; Table S2; Table S3; Table S4; Table S5; Table S6a,b; Table S7.


Naemorhedus caudatus (subfamily Caprinae), a vulnerable and protected species designated by IUCN and CITES, has sharply been declining in the population size and is now becoming critically endangered in South Korea. This species has been conserved as a natural monument by the Korean Cultural Heritage Administration since 1968. In this study, using 78 fecal DNA samples with a non-invasive genetic approach, we assessed the genetic integrity and individual identification-based population size for the goral population from Seoraksan National Park representing the largest wild population in Korea. Using the successfully isolated 38 fecal DNA, phylogeographic and population genetic analyses were performed with mitochondrial DNA control region (CR) sequences and nine microsatellite loci. We found seven CR haplotypes, of which five were unique to the Seoraksan population, considering previously determined haplotypes in Korean populations. The Seoraksan population showed higher haplotype diversity (0.777 ± 0.062) and mean number of alleles (4.67 ± 1.563) relative to southern populations in Korea reported from previous studies, with no signal of a population bottleneck. Microsatellite-based individual identification estimate based on probability of identity (PID) indicated a population size of ≥30 in this population. Altogether, we suggest that for future management efforts of this species in the Seoraksan National Park, conserving its genetic integrity as an ‘endemic’ lineage, and curbing a decrease in its number through mitigating habitat destruction might be key to secure the population for the long term.The long-tailed goral   In a prNe) Yang . Only a Ne) Yang . This nab (cyt b) -based phylogeography  and birds using a non-invasive genetic approach to individual identification  and reverse (ArtR: 5-TCGACTGGYTGKCCTCCAATTC-3) primers, which were designed for the long-tailed goral (N. caudatus) based on four sequences of phylogenetically related species  of Artiodactyla available from GenBank database . PCR cycling conditions comprised an initial denaturation phase at 94°C for 5 min, followed by 35 cycles of 94°C for 20 s, 54°C for 30 s, and 72°C for 30 s, followed by a terminal extension phase at 72°C for 12 min. Only 43 of the samples were successfully amplified for the mtDNA cyt b gene (472 bp). By using BLAST , we confirmed that the obtained cyt b sequences matched with N. caudatus with the highest ident value (99.9%).A total of 78 fecal samples were collected from two localities (Jangsoodae and Namseorak) at Seoraksan National Park in South Korea between 2015 and 2016 mainly during a winter season (CR region (592 bp) was amplified with the Cytb up and NH16129 primers  were aligned using Clustal W in BIOEDIT v7.2.5  and polymorphic sites (NP), haplotype diversity (h) and nucleotide diversity (π) were estimated using ARLEQUIN v3.5.1 (Excoffier and Lischer http://ncbi.nlm.nih.gov/nuccore).The mtDNA 2.5 Hall  and manu2.5 Hall  with sev2.5 Hall ; the 25 N. caudatus , which were designed for the long-tailed goral NA), observed (HO) and expected (HE) heterozygosity and inbreeding coefficient (FIS) were estimated using GENEPOP v.4.0  and linkage disequilibrium (LD) tests for genotypes among pairs of nine loci were calculated in GENEPOP using a Bonferroni correction.The number of alleles across loci  and PID for siblings  in GIMLET v1.3.3  model in BOTTLENECK v1.2.02  detected in this study. Only two haplotypes  found in this study were identical to the previously determined haplotypes, while the remaining five haplotypes have never been detected before, suggesting they are unique to the Seoraksan National Park region  of N. caudatus from Seoraksan National Park, seven haplotypes were detected with 89 polymorphic sites including 76 indel mutation  was shared by 13 individuals (43.3%), and the next most frequent haplotype (H5) had 16.7% of the total sampled individuals. The five haplotypes (H1-H5) were found to be unique haplotypes (i.e. private haplotypes) that can be only observed in Seoraksan National Park.For the mutation . EstimatNA across the loci was 4.67 ± 1.563, and HE and HOwere 0.559 ± 0.127 and 0.502 ± 0.106, respectively  were 0.199, ranging from 0.000 (SY58) to 0.439 (SY84B), suggesting very low to moderate levels of probability of null alleles. Based on multi-locus tests for HWE expectations, the possibility of non-random mating (e.g. inbreeding) could not be rejected (P = 0.011). Tests of genotypic LD showed no significant allelic association between the nine loci (P > 0.05) [except SY76 versus SY3A and SY12A (P < 0.05)] after the Bonferroni correction.For nine microsatellite loci, ectively . The FISA BOTTLENECK analysis of nine microsatellite genotypes showed that the expected number of loci were five for a deficiency of heterozygosity and four for an excess. Allelic frequency distribution showed a normal L-shaped distribution with a probability of 0.320. Therefore, null hypothesis could not be rejected with a Wilcoxon sign rank test, suggesting the study population is unlikely to have experienced a recent genetic bottleneck.ID: 2.0 × 10−4; PIDsib: 2.4 × 10−2) (The nine loci genotyped for 38 non-invasive samples showed the extremely low levels of probability of identity (P × 10−2) , suggest × 10−2) . Seven o × 10−2) .Table 2ID < 0.001). Unexpectedly, however, the nine loci genotypes from the fecal samples could not 100% match with the CR haplotypes from the same individuals. In fact, two samples of S3 had different CR haplotypes, H1 and H4, respectively, suggesting they represent genetically different individuals. Therefore, we finally determined at least 30 Korean goral individuals in Seoraksan National Park. Ne estimate for the Seoraksan population was only a median of 10.2 with 95% confidence interval of 6.6–16.2.Assuming that identical genotypes for all the nine loci analyzed were considered as the same individuals, we found five cases of identical nine loci genotypes among 38 fecal samples: two fecal samples (S1 in red); two (S2 in yellow); two (S3 in green); four (S4 in blue); four (S5 in purple)  and previous . However, further investigation of phylogeographic relationships in this species, including recently published dataset containing longer sequences of 1099 bp obtained from populations of Injae, Gosung, Yanggu and Hwacheon located in the northern parts of South Korea  and southern (0.609) parts of South Korea documented in a recent study that found 22 CR haplotypes from 66 N. caudatus individuals  of genetically defined individual gorals in Seoraksan National Park is lower than would be expected. In a combination of feces tracking and camera traps, the population size in this region was found to range from 160 to 251 individuals  alleles, and etc. Ideally, using genome-wide scans or mtDNA and microsatellites as genetic markers, population genetic (genomic) assessments with non-invasive genetic approach using a much larger number of fecal DNA samples should be used to more accurately identify the number of individuals within populations and also determine the level of gene flow among populations in order to help to design the effective management strategies.Based on the results of this study, we suggest that future conservation management efforts of endangered long-tailed goral of the Seoraksan population should involve maintaining the ‘endemic lineage ’ to prevent the loss of its genetic integrity (Antognazza et al.