Almond cultivation has great traditional and economic relevance in Southern Italy, especially in the Apulia region, where almond trees feature an ample and ancient varietal richness. To contrast the loss of plant genetic erosion and to safeguard the available bioresources, as well as to reinforce the local production, the regional Re.Ge.Fru.P. project aimed to re-evaluate, identify, and characterize the Apulian almond germplasm that is still uncharacterized and not jet studied using a dual  approach. Collection was conducted in the regional territory of 187 among the most widespread and minor or marginalized genotypes that were molecularly fingerprinted by means of 18 nuclear microsatellites . The high number of scored alleles reflected the great level of diversification within the Apulian germplasm, as also confirmed by neighbor joining and structure analysis, that clearly distinguished different genotype clusters. The phenotypic characterization using 17 morphological and phenological descriptors mirrored the genetic results, revealing a high degree of variability. The morphological traits with the best discriminatory ability were nut ventral suture, shell softness and shape and petal color. This work emphasizes the importance of recovering the genetic variability of Apulian almond germplasm, and the need to promote added value and enhance the local agri-food economy. Prunus dulcis Mill. D.A. Webb., syn. Prunus amygdalus Batsch, syn. Amygdalus communis L.) is probably the oldest nut tree crop to be domesticated; indeed, the first signs of its cultivation appeared in literature as early as 3000 B.C. [Almond  with the aim of recovering the biodiversity of several Apulian fruit tree species, including almond, by means of various different actions, including historical investigation and cataloguing, morphological and genetic characterizations as well as ex situ conservation of local germplasm. In this context, robust methods to discriminate nonidentical individuals were essential in order to pursue some of the specified goals. The traditional methods for cultivar characterization and fruit tree identification, based on phenotypic observations and morphological descriptions, are usually coupled with the more accurate DNA-based methods. Together, they act as an extremely useful tool in breeding programs, providing a fast and indisputable fingerprint of genotypes and for the assessment of genetic relationships .Several studies were conducted to characterize different almond germplasm and evaluate the level of genetic variability within populations, using different molecular markers, such as RAPDs ,21, AFLPGiven the economic and cultural importance of almond cultivation in the Apulia region, and the high interest in contrasting plant genetic erosion, the main aims of this work were to study the genetic structure of Apulian almond germplasm and to enrich the collection of already available genetic resources, in particular safeguarding ancient and local genotypes and assessing their potential for breeding purposes.In order to evaluate the genetic diversity of the 187 Apulian almond genotypes, an in-depth molecular characterization was performed using 18 SSR markers, chosen on the basis of their dispersal map location. Capillary electrophoresis analysis produced clear genotyping profiles for all the examined loci, yielding good and reproducible amplification products within the expected allele-size ranges . A totalThe mean of the effective alleles (Ne) was 6.10, ranging from 2.23 for BPPCT014 to 11.00 for CPDCT045, slightly lower as compared to two other reports ,37. For The observed Heterozygosity (Ho) ranged from 0.335 (UDP98409) to 0.935 (CPDCT045) with a mean value of 0.711, while the expected Heterozygosity (He) varied between 0.553 for BPPCT014 to 0.909 for CPDCT045, with an average measure of 0.807 . This diMoreover, Shannon’s information Index (I) ranged from 0.926 (BPPCT014) to 2.608 (CPDCT045), with an average value of 2.052, indicating a high level of genetic diversity in the collection studied .The allelic similarity among the Apulian almond genotypes analyzed was also calculated by means of LRM estimation (pairwise relatedness), setting 0.5 as maximum value for identical genetic profiles (cases of synonymy). A complete genetic identity was observed for the cultivars “Mollesca di Ruvo”, “Troito”, “Tuono” and “Stilla” . The genMost of the examined genotypes had LRM values between 0.4 and 0.5, thus resulting unique individuals with no case of synonymy . High LMTo our knowledge, the evaluation of the LRM parameter has never previously been reported in other studies about almond genetic diversity.The neighbor-joining analysis allowed us to obtain a phylogenetic tree and to subdivide the Apulian almond population into three main clusters: G-1, G-2, and G-3 . The greThe phylogenetic clusterization of the Apulian genotypes was generally in line with the LRM values obtained, since cultivars with a high allelic similarity were found to belong to the same branch of the tree . For exaOne group of genotypes , with moderately high LRM values ranging from 0.41 to 0.44, together formed a branch of the tree in cluster G-2A. Interestingly, only four genotypes constituted cluster G-2B, namely the cultivars “Mangini”, “Sciacovelli Altamura”, “Amendolara” and “Casa Perrini”.Some cases of homonymy were discovered, since genotypes with the same name but genetically polymorphic were clearly distinguished . Among tStructure analysis allowed us to determine the genetic constitution of the Apulian almond population. Genotypes whose membership coefficient (qi) was higher than 0.6 were assigned to a defined cluster, otherwise they were considered to be of admixed ancestry. In accordance with the Evanno criterion , a stronMany cases of admixture  were evident within and among gene pools, likely as a result of hybrid origin and allele sharing among these genotypes. As an example, the cultivars “Filippo Ceo” and “Falsa Barese” were assigned to the admixed group in this study, highlighting their different allelic composition, as also confirmed by ,48. NeveThe alleles richness of the almond Apulian germplasm, confirmed by the high number of alleles scored, with a relatively congruent number of SSR loci analyzed, reflected the high level of diversification within the Apulian germplasm. This diversification was also verified and confirmed in both the neighbor-joining and structure plot that gathered the genotypes analyzed within clearly distinguishable groups.A subset of 109 cultivars was subjected to a detailed phenotypic characterization. Descriptive statistics of the analyzed traits are shown in Principle component analysis assigned most of the traits to four components which explained 46.1% of the total variation . The firBased on the first two components, the PCA plot grouped the almond cultivars according to their technological features resemblance . ProceedThe neighbor-joining clustering method utilizing all the morphological and phenological data again subdivided the Apulian almond population into three main clades with comparable dimensions . Indeed,The observed morphological variability mostly mirrored the results of the molecular characterization obtained with SSR markers, especially the M-1 clade, that was almost exclusively composed of genotypes included in the G-1 clade. Moreover, most of the genotypes with a high genetic similarity according to their LRM values were once again found to belong to the same branches of the morphological tree. For example, the high genetic similarity between the cultivars “Stilla” and “Tuono” (LRM = 0.5), “Bianchetta” and “Lunghina” (LRM = 0.49), “Del lago” and “Rachele tenera” (LRM = 0.48) also emerged from the phenotypic analysis, these genotypes being included in the same morphological subclusters.However, some exceptions and discrepancies between the morphological and the genetic analysis emerged, as also reported by , which hA total of 187 Apulian almond genotypes from different locations was considered in this study. Among them, 41 belonged to the ex situ collection of CRSFA Institute  located in Locorotondo (Bari); 23 derived from the Di.S.S.P.A  germplasm collection of the University of Bari, located in Valenzano (Bari); 43 were sampled in the experimental field of the CREA Institute  located in Bitetto (Bari), and 74 were kindly provided by local farmers from different areas in the Apulia region. In addition, 6 foreign commercial varieties provided by CRSFA were added as reference.Young leaves were collected from each plant tree and stored at −20 °C until the use. Genomic DNA was extracted according to . The qua®  and the forward primer of each couple was labelled with one of the following dyes: 6FAM™, NED™, VIC® and PET™. In order to optimize the reaction times, the same touchdown protocol was applied for all the selected SSRs, consisting of an initial denaturation at 94 °C for 5 min; 10 cycles of 94 °C for 30 s, followed by an annealing step at 55 °C for 45 s, and 1 min at 72 °C; then, 25 cycles at 94 °C for 30 s, 50 °C for 45 s, and 1 min at 72 °C; a final extension was run at 72 °C for 30 min.A set of 18 nuclear SSR was used to assess the genetic variability of the almond collection. For each SSR, information about the type of locus, repeat motif, annealing temperature, linkage group and species in which they were isolated, are reported in PCR products were prepared as described by , separathttp://biology-assets.anu.edu.au/GenAlEx accessed on 15 September 2021) [For each SSR marker, the following statistical parameters were calculated using GenALEx software v. 6.51b2 (er 2021) : number er 2021) , observeer 2021) . GenALExer 2021)  and the er 2021) . Cervus http://darwin.cirad.fr accessed on 16 September 2021) software version 6.0.010. The robustness of branches was tested using 1000 bootstraps [Genetic distance between the almond genotypes was calculated using the simple matching dissimilarity index. A weighted neighbor-joining tree  was compotstraps . In addiotstraps , which wotstraps .www.reterurale.it/flex/cm/pages/ServeBLOB.php/L/IT/IDPagina/9580 accessed on 4 October 2021). Each year, at ripening time, a sample of 100 hulled and dried fruits was taken for each genotype to study nut and kernel traits.During the years 2014 and 2015, for each genotype, 15 morphological traits and 2 phenological traits  were anaMean, minimum, maximum and coefficient of variation (CV) of coded values of traits were calculated for a representative subset of 109 cultivar. The distribution of the selected genotypes for each trait was represented in frequency histograms, based on the mean of coded values of the two years of the study. Relationships among genotypes were investigated by multivariate analysis of variance  STATISTICA .The present study made it possible to explore some aspects of almond biodiversity in Apulia region, by highlighting the hypothetical composition of the autochthonous germplasm. The combination of molecular and morphological data was proven once again to be the best choice in order to characterize the available plant resources. Microsatellites and morphological markers were successfully used to assay the genetic diversity of the Apulian almond collection and underlined a high level of diversification. All the genotypes examined were unequivocally identified, and some cases of misnaming and/or homonymy have been clarified. In particular, this aspect is extremely important and may be a prerequisite in the process of nursery material certification and official registration.Alongside the detailed characterization of the retrieved trees, the propagation, and the subsequent cultivation of some of them in appropriate collection fields could not only safeguard several genotypes, protecting them from extinction, and preserve the already known cultivars as a source of intravarietal diversity, but also allow planning of the evaluation of almonds in terms of yields and quality. The good adaption of Apulian almonds to the regional environmental conditions makes them an interesting bioresource that could be extremely useful for breeding programs and for the selection of promising, highly productive candidates that may be resilient to the climate changes underway. Moreover, the rediscovery and preservation of indigenous and putatively endangered almond cultivars, especially in marginal areas, may also have important repercussions and benefits on the local economy, further enhancing the value of the Apulian almond germplasm.Thus, the extensive genetic variability of the Apulian almond germplasm that emerged in this study indicates that these materials are an important source of genes for almond improvement and valorization. Our work offers information that may stimulate further breeding efforts to enhance the sustainability of this crop.


Favorable variation from genetic resources is anticipated to play a key role in the adaptation of crops to the increasingly unfavorable production conditions resulting from climate change FAO, . Weatherthe identification of promising germplasm bank accessions,the separation of beneficial major effect alleles from undesired linkage drag,the repackaging of polygenic variation into elite and adapted materials.Identifying promising germplasm bank accessions, which may offer single alleles with major effects or beneficial quantitative variation, often resembles looking for a needle in a haystack. In practice, it is almost never possible to phenotype a large portion of the available germplasm due to high costs, challenges with adaptation, restricted facility resources and time pressure. An informed prescreening of the available accessions will be necessary.Moreover, when accessions with putative alleles for desired traits are identified, the mission is not yet accomplished, since the beneficial variation must be integrated into elite germplasm. In the case of a simple genetic architecture such as an identified major effect gene, the novel allele can be introgressed by marker assisted backcrossing (MABC) or can be approached by gene editing. However, preceding discovery research is required to identify the genetic variation associated with the phenotypic variation. In particular, gene editing requires very precise information on the causative variation. The availability of a trait-associated marker, which may be sufficient for an application in MABC, may be insufficient for a gene editing approach. This research is resource and time consuming and carries the inherent risk of unsuccessful validation experiments due an altered effect of the allele when in combination with the genetic background of elite material.When dealing with quantitative variation, dedicated mapping experiments are not required. However, it is more difficult to bring quantitative variation into an elite background and have a product acceptable to breeders. Landraces carry many deleterious and inferior alleles which can quickly disrupt the positive linkage blocks painstakingly constructed by breeders over decades. Diminished agronomic performance makes the breeding community reluctant to include such germplasm in their elite breeding programs.Prediction approaches can help the effective use of genetic resources in two ways. First, predictions can identify the most promising candidate accessions for a certain trait, thus restricting the number of accessions to evaluate in experiments . We then discuss differences between predictive breeding approaches for genetic resources and genomic selection for elite breeding programs. We propose that research on predictive methods for genetic resources should explore approaches which are “enriched” by external information; for example, knowledge of molecular biological mechanisms, or accession “passport” data that provides information on the environmental conditions in which the accession was originally cultivated. Passport data comprising latitude, longitude, and altitude are fundamental initial information for each accession stored in the bank. The inclusion of external information may increase the power of predictive breeding approaches, especially in the context of harnessing genetic resources.In this commentary, we summarize some activities related to predictive breeding in the context of genetic resources conducted at the International Maize and Wheat Improvement Center (SEED). For maize, more than 98% of the CIMMYT and IITA  maize collection have been genotyped. For wheat, 37 and 66%, respectively, of the CIMMYT and ICARDA  wheat collection have been genotyped . Thirteen out of 1000 screened landraces were identified as showing low susceptibility to Maize Chlorotic Mottle Virus (MCMV), the major causal component of MLN disease . This approach treats environmental variables of the sites where accessions were collected as phenotypes, and combines this information with genotypic data for the accessions in an association study. The objective is to identify genetic variation which is associated with the adaptation to certain environmental conditions  then employs the environmental data as a phenotype in the training panel to predict materials of higher value for “hands-on” evaluation. Considering the polygenic nature of many traits of interest, we are currently assessing the potential of EnvGP together with other paradigms such as crop modeling to leverage genetic resources for germplasm development.As an example addressing the process of repackaging of polygenic variation into elite and adapted materials, we cite Origin Specific Genomic Selection (OSGS; Yang et al., Germplasm bank accessions can be considered as crop “genetic insurance” for the genetic adaptation to increased abiotic and biotic stresses, in particular caused by climate change. As for other fields, “big data,” here describing the germplasm bank collections, needs innovative approaches for “data mining,” to identify and harness useful variation, and unleash its potential. We see a conceptual key in combining statistical prediction methods with additional data other than genotypes and phenotypes. Approaches of this type have been followed in genomic prediction literature, but we consider them as particularly promising when applied in the context of harnessing genetic resources. The type of data to use, and how to use it provide a large playground for the exploration of creative approaches.JM wrote the first draft and managed the edits from other authors. All authors discussed and outlined the content of the opinion and approved the published version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Molluscs are remarkably diverse and are found across nearly all ecosystems, meaning that members of this ancient animal phylum provide a powerful means to study genomic-phenotype connections in a climate change framework. Recent advances in genomic sequencing technologies and genome assembly approaches finally allow the relatively cheap and tractable assembly of high-quality mollusc genome resources. After a brief review of these issues and advances, we use a case-study approach to provide some concrete examples of phenotypic plasticity and genomic adaptation in molluscs in response to environmental factors expected to be influenced by climate change. Our goal is to use molluscs as a “common currency” to demonstrate how organismal and evolutionary biologists can use natural systems to make phenotype-genotype connections in the context of changing environments. In parallel, we emphasize the critical need to collaborate and integrate findings across taxa and disciplines in order to use new data and information to advance our understanding of mollusc biology in the context of global environmental change. We end with a brief synthetic summary of the papers inspired by the 2021 SICB Symposium “Genomic Perspectives in Comparative Physiology of Molluscs: Integration across Disciplines”. Mollusca is the second largest Metazoa phylum, representing over 90,000 extant taxa . MolluscAplysia) are used in biomedical research, while others are important agricultural pests , invasive species , or intermediate vectors of deadly human parasites   .Molluscs have gained recent attention as model species for climate change research. Global climate change is proceeding at an unprecedented rate, with major consequences for all ecosystems. A critical question in this setting whether and how natural populations will respond to global climate change and if these responses will be rapid and adequate enough for species persistence. In general, populations respond to environmental change by one or a combination of four strategies (1) shifting their range, (2) phenotypic plasticity, (3) genetic adaptation via evolution by natural selection to new conditions, and (4) persisting in the original habitat but experiencing demographic decline or extinction .Their high diversity and abundance along with presence across wide latitudinal clines and preservation in the fossil record means that molluscs can serve as a powerful indicator of environmental changes in all ecosystems . MolluscIn the following sections, we will use a case-study approach to provide some concrete examples of phenotypic plasticity and genetic adaptation in molluscs in response to global change . Our surAs is all too common in molluscs, we know comparatively little about phenotypic plasticity and genetic variation and its association—or lack thereof—with phenotypes associated with environmental stress. Most of what we do know has come from a handful of taxa that receive relatively focused attention as model organisms. We here highlight a diverse set of Molluscan taxa that provide illustrative examples of response to various environmental stresses. It is important to note that we were unable to find a substantial body of directly relevant data  from the Molluscan classes Scaphopoda, Monoplacophora, Aplacophora, or Polyplacophora also see , highligPhenotypic plasticity is defined as a situation when a genotype produces different phenotypes in response to different environmental conditions. Phenotypic plasticity is ubiquitous, and most traits are plastic  and plasticity, temperature sensitivity of metabolism, and metabolic cost of heat shock in nine species of nudibranchs collected across a thermal gradient along the northeastern Pacific coast of California. The authors reported that adaptation to relatively warm water temperatures in intertidal nudibranchs constrains plasticity to acute thermal challenge and that southern (warm adapted) species are likely most vulnerable to future warming. Climate-related range expansions were reported during relatively warm periods (between 2014 and 2017) in more than 52 eastern Pacific nudibranch species . Oysters are champions of survival in the face of harsh and dynamically changing environments in estuarine and coastal zones. These habitats experience wide temporal and spatial fluctuations of temperature and salinity, and desiccation presents a serious challenge during daily and seasonal cycles. As reviewed in 3- to 106-fold higher than those of the surrounding water, and are regarded as the most useful model for studying chemical pollution in aquatic environments  instead seem to benefit from expanding hypoxia  is only ∼20% of the control rate  genetic state as well as the adapted/evolved state in the new conditions . There aUnderstanding whether and how organismal populations can adapt to changing environmental conditions requires characterization of intraspecific genetic variation underlying organismal phenotypic variation in relevant environments because this variation provides the raw material for evolution by natural selection . To whatIt is less clear that nucleotide substitution will typically underlie evolutionary adaptation to environmental stresses in molluscs. While we again are limited to very cautious statements in light of the phylogenetically limited scope of relevant data, genomic data from bivalves like scallops  and mussP. antipodarum an especially powerful model system with respect to identifying genetic variation for various phenotypic traits  to global change. Genomic approaches can also help dissect mechanistic underpinnings by which adaptation to climate change occurs  to address genome-to-phenome research.Newly developed genomic technologies and bioinformatic approaches have opened up new opportunities for biologists to address questions of both processes and patterns of organismal change. How can we understand the functional context of such “big” data within the intact organism, and how does genomic variation contribute to phenotype? Our symposium is especially novel from the perspective of bringing together the organismal biologists, ecophysiologists, and genomicists/bioinformaticians that are needed to provide a qualitative step forward in understanding the biology and ecology and predicting the future of one of the most important animal groups alive today. Presentations, discussions, and syntheses focused on topics including overviews of the biological and genomic diversity of molluscan life, technological progress towards highly contiguous molluscan genomes, challenges in assembling molluscan genomes, and how genome-scale processes underpin organismal physiology and interact with ecological and evolutionary processes over multiple spatiotemporal scales can be found throughout the special issue that our symposium inspired.This special issue presents a series of papers addressing mollusc genome-to-phenome responses in the context of global climate change. Connecting genotypes and genomic variation to functional and ecological consequences demands tools and concepts from a diverse set of fields including molecular biology, physiology, quantitative genetics, ecology, and evolutionary biology. This type of integrated approach will help to identify and decouple genetic vs. plastic underpinnings of ecologically relevant functional variation and characterize the ecological consequences of that variation. Our goal for the symposium was to bring together a transdisciplinary set of experts in mollusc biology to provide an unprecedented opportunity for knowledge exchange, discussion, and catalysis of new partnerships. The talks in our symposium featured a wide range of ecologically important concepts and traits including but not limited to immune function and symbiosis to mitochondrial performance and host-parasite interactions, and are united by their use or application of genomic techniques and resources.Ghiselli et al. provide a comprehensive review of one of most striking features of bivalve molluscs: their peculiar mitochondrial genome biology. As Ghiselli et al. describe, bivalves have facultatively anaerobic mitochondria that allow them to survive prolonged periods of anoxia/hypoxia. Molluscs also exhibit the only known and evolutionarily stable exception to the strictly maternal inheritance of mitochondria, so-called doubly uniparental inheritance, now described in 100+ molluscan taxa to date. In this review, the authors highlight recent works studying mitochondrial biology in bivalves at the genomic and physiological level and stress that an integrated approach and collaborative relationships are the only possible ways to succeed in connecting mitochondrial genome-to-phenome relationships in bivalves.Crassostrea virginica) to identify adaptive genetic variation for tolerance to low salinity in two populations from the Gulf of Mexico. The authors compared allele frequencies at 152 salinity-associated genes for larval families pre- and post-low salinity exposure and used these data to demonstrate evidence for purging of deleterious alleles at the larval stage in C. virginica. This study also revealed standing genetic variation for salinity tolerance and demonstrated increases in allele frequencies at multiple loci following selection, indicating a polygenic basis for adaptive responses to low salinity but also suggesting that some components of tolerance are genotype specific.Griffiths et al. used a single-generation selection experiment and pooled sequencing of larvae from the eastern oyster (Phyllaplysia taylori) populations under four temperature-salinity scenarios in a laboratory acclimation experiment. Acclimation to warmer conditions significantly increased critical thermal minima, while low-salinity conditions resulted in high mortality. Individuals that survived the low-salinity treatments were able to respond to temperature and salinity stresses more rapidly than individuals acclimatized to saltier conditions, though the most rapid response time for the low-salinity acclimatized sea hares was at a higher temperature than the individuals acclimatized to saltier conditions. Together, these results led the authors to conclude that acclimation to climate change-induced warming will likely present challenges with respect to the ability of these sea hares to weather existing and predicted cold extremes and precipitation events.Tanner et al. measured thermal plasticity in two central California eelgrass sea hare (Vibrio vulnificus) across four populations of Crassostrea virginica along the North Carolina and Virginia coast. The authors observed significant genetic structure with respect to the distribution of mitochondrial cytochrome oxidase subunit I (COI) haplotypes between locations. The expression of stress-response genes including toll-like receptors, mannose receptor, defensin, and the complement gene Cq3 was specific to locations as well as to the stressors involved. Altogether, these data hint at a complex relationship between genotypes, phenotypes, and stress responses, with indirect evidence for both plasticity and genetic variation for stress responses.Furr et al. explored genetic structure and physiological responses to hypoxia and immune stress challenge , to identify regions of the genome in bobtail squids that are under selection in squid lineages that maintain symbioses with bioluminescent bacteria , setting the stage to identify genes instrumental in the evolution of these mutualistic associations. This study also provided new genomic resources that will be useful for comparative work in cephalopods and beyond.Heath-Heckman and Nishiguchi used newly generated genomic sequence data from four bobtail squid taxa (Our symposium and symposium papers illuminate the power and utility of molluscs as model organisms in a variety of contexts and how organismal and evolutionary biologists can leverage these fascinating organisms to generate new insights into phenotype–genotype connections. These connections are especially relevant and important in the context of anthropogenic change. We believe that we have made a strong case that molluscs can be applied to characterize the potential of and limits to plastic and evolutionary change in response to these planetary consequences of human activities. We also have emphasized the central role that cross-disciplinary collaboration and integration will play in the achievement of these goals.


During the Neolithic expansion, cattle accompanied humans and spread from their domestication centres to colonize the ancient world. In addition, European cattle occasionally intermingled with both indicine cattle and local aurochs resulting in an exclusive pattern of genetic diversity. Among the most ancient European cattle are breeds that belong to the so-called Podolian trunk, the history of which is still not well established. Here, we used genome-wide single nucleotide polymorphism (SNP) data on 806 individuals belonging to 36 breeds to reconstruct the origin and diversification of Podolian cattle and to provide a reliable scenario of the European colonization, through an approximate Bayesian computation random forest (ABC-RF) approach.Our results indicate that European Podolian cattle display higher values of genetic diversity indices than both African taurine and Asian indicine breeds. Clustering analyses show that Podolian breeds share close genomic relationships, which suggests a likely common genetic ancestry. Among the simulated and tested scenarios of the colonization of Europe from taurine cattle, the greatest support was obtained for the model assuming at least two waves of diffusion. Time estimates are in line with an early migration from the domestication centre of non-Podolian taurine breeds followed by a secondary migration of Podolian breeds. The best fitting model also suggests that the Italian Podolian breeds are the result of admixture between different genomic pools.This comprehensive dataset that includes most of the autochthonous cattle breeds belonging to the so-called Podolian trunk allowed us not only to shed light onto the origin and diversification of this group of cattle, but also to gain new insights into the diffusion of European cattle. The most well-supported scenario of colonization points to two main waves of migrations: with one that occurred alongside with the Neolithic human expansion and gave rise to the non-Podolian taurine breeds, and a more recent one that favoured the diffusion of European Podolian. In this process, we highlight the importance of both the Mediterranean and Danube routes in promoting European cattle colonization. Moreover, we identified admixture as a driver of diversification in Italy, which could represent a melting pot for Podolian cattle.The online version contains supplementary material available at 10.1186/s12711-021-00639-w. Bos primigenius primigenius and Bos primigenius namadicus which were widespread across Africa and Eurasia during Middle Pleistocene. They diverged approximately 250,000 years before present (YBP) and were independently domesticated giving rise to the current Bos taurus taurus (taurine) and Bos taurus indicus (zebuine) subspecies, respectively [B.t. taurus) and about 8000 YBP in the Indus Valley (B.t. indicus), cattle followed colonization routes in parallel with the Neolithic human expansions [Bos primigenius present in both Europe and Africa [B. t. indicus, European B. t. taurus and African B. t. taurus) with different levels of genetic admixture among them [The tangled evolutionary history of cattle stems from two main wild aurochs subspecies, ectively , 2. Sincpansions , 4. Indepansions . The wesd Africa , 8, althd Africa . Therefod Africa –12 and id Africa , whereasd Africa –20. On td Africa . On the ong them , 22.B. t. taurus origin. However, an indicine component was observed in diverse breeds, such as the Turkish Grey and some Italian breeds that belong to the so-called Podolian trunk  and show evidence of mixed B. t. taurus and B. t. indicus ancestries [The European cattle gene pool is mainly of cestries –24.Cattle breeds belonging to the so-called Podolian trunk, also known as grey steppe cattle (hereafter referred to as “Podolian”) are distributed across continental Europe and the Mediterranean basin, and represent a fundamental resource and income for traditional farming thanks to local products. In general, these breeds, compared with other European cattle breeds, still display many ancestral features such as long horns, sexual dimorphism, longevity, and are well adapted to the local climatic and environmental conditions. However, their history and origin are still not well established, although several hypotheses have been proposed to explain the origin of Podolian cattle. Analysis of mitochondrial DNA data suggests an Anatolian and South-Western Asian origin of Central Italian Podolian breeds, docked to Italy via the sea route, probably during the Etruscan period , 25. AltTo disentangle this complex history, we used genome-wide SNP data to specifically: (i) reconstruct the origin and diversification of the Podolian cattle breeds by comparing them with other European taurine, African taurine and Asian indicine cattle breeds, (ii) better understand the genomic relationships within Podolian cattle breeds, and (iii) provide the most plausible scenario of European colonization, through an approximate Bayesian computation random forest (ABC-RF) approach.We used a set of 806 individuals belonging to 36 cattle breeds genotyped with the Illumina BovineSNP50 Table  and (seeo) and expected (He) heterozygosity and average MAF were estimated using PLINK and trends in effective population size (Ne) based on linkage disequilibrium (LD) were estimated using the SNeP v1.1 software [SNP genotypes were obtained from previous studies , 26–32, software .2 higher than 0.1 within 50-SNP windows in 10 steps, thus obtaining a final dataset consisting of 17,096 SNPs (17 K).To reduce the number of SNPs in LD, we pruned the dataset using the --indep-pairwise flag in PLINK by removing SNPs with an rTo explore the genetic differentiation of the whole dataset, the pairwise genetic relationships were estimated using a matrix of genome-wide identity-by-state (IBS) genetic distances calculated by PLINK and plotted in a multidimensional scaling (MDS) plot. To assess reticulated relationships between populations, pairwise Reynolds genetic distances were estimated with the ARLEQUIN  softwareK ancestral populations using a cross-validation approach (CV = 10) for K values ranging from 2 to 23. Then, the R package BITE [membercoef.circos function.ADMIXTURE v 1.2  was usedage BITE  was usedoptM function in the R package OptM .Finally, we used the software TREEMIX  to explopcadapt v.4.1.0 [K = 5) was evaluated using the graphical ‘scree plot’ approach [p-values of the Mahalanobis distances were used as the test statistics to detect outlier SNPs. The distribution of the observed p-values was checked against the expected p-values using a Q-Q plot. The q-value procedure implemented in the qvalue R package was performed by setting a cut-off of 0.1% as false discovery rate threshold for detecting outlier SNPs that were likely under selection. Thus, 314 SNPs were removed from the dataset. In addition, to reduce the computational burden, the dataset was further pruned for LD using the --indep flag with an r2 threshold of 1.2% resulting in 8440 (8 K) SNPs being retained. To assess if this pruned dataset still contained a sufficient amount of information regarding genetic structure, we performed a principal component analysis (PCA) as implemented in pcadapt and compared results to those obtained using the complete dataset.To ensure that our dataset contained only putatively neutral loci, we used the package  v.4.1.0  to identapproach  and p-vaPossible colonization routes of the Podolian cattle were evaluated in an Approximate Bayesian Computation (ABC) framework using a tree-based classification method as implemented in the DIYABC random forest software . This meFor this analysis, breeds were grouped based on both MDS and TREEMIX results and two populations per group were selected. This process aimed at avoiding outliers or topological inconsistencies and at assessing whether the outcomes could be related to breed choice rather than reflecting their demographic history. In doing so, we did not consider breeds with strong inbreeding values or low genetic diversity levels, to retain an adequate amount of ancestry information to be incorporated at the coalescence. Simulations were then performed by grouping individuals into five main populations according to the main geographic areas .To model our scenarios we assumed that: (i) all extant breeds had a common ancestor which gave rise to indicine and taurine lineages; (ii) the taurine domestication took place in the Fertile Crescent between 3600 and 7800 generations ago ; and ii. The remTo have an internal time calibration point, we incorporated a uniform prior distribution, with lower and upper bounds at 3600 and 7800 generations, respectively, to account for the size reduction that occurred during domestication in the Fertile Crescent  and expected (He) heterozygosity, minor allele frequencies (MAF) and effective population size (Ne) are in Table o and He. Regarding Ne, we observed low values for Asian indicine breeds and some Podolian breeds such as the Calvana, Maremmana, Serbian Podolsko, Croatian Podolian, Bulgarian Grey and Romanian Grey, with the latter being represented, in our dataset, by only four individuals.Genetic indices including observed , the indicine component shows a higher penetration, reflecting an East–West cline which has almost completely disappeared in the non-Podolian European taurine breeds such as Holstein, Angus, Piedmontese, Tyrolean Grey, Gascon and Modenese  is revealed among the Gascon, Tyrolean Grey, Piedmontese and Modenese as well as among the Asian indicine and African taurine breeds  are highly related, whereas the South-Eastern Mediterranean Podolian and the Balkan and Central European Podolian breeds form a single cluster, with genetic relationships consistent with their geographic native area. All other non-Podolian taurine breeds are at a basal position of the tree Fig. b. The opThe 8 K dataset carried a level of information on the genetic structure of the analysed breeds that was similar to that of the larger dataset . Indeed, 250,000–300,000 years is a rather short time to allow complete genome segregation as confirmed by the presence in the British wild aurochs of the same genomic component that is found at a high frequency in the modern indicine breeds  and Italian (K = 5) Podolian breeds  breed is genetically more similar to the Italian Podolian breeds, in particular with Maremmana and Podolica Italiana, than to the Balkan Podolian breeds, which confirms a previous result obtained with microsatellite markers  and is n K = 5, the first breeds that separated were Calvana and Chianina, the ADMIXTURE analysis showed the extensive occurrence of a shared genetic component among the Italian Podolian breeds until K = 10. The divergence of other Italian Podolian breeds appeared at higher values of K, such as for Romangnola (K = 14) and Modicana (K = 15). Branch lengths and genetic indices seem to suggest that, in particular, the Calvana breed is under strong genetic drift and has a low genetic variability. Furthermore, the tree-based graph reconstruction places the two Sicilian breeds at a basal position with respect to all the other Italian Podolian breeds , which is a plausible time window corresponding to a first Early Neolithic farmer expansion. From Anatolia, the human population expanded through two well-defined streams, along the Danube  with several waves of migrants into this region and along the Mediterranean coast , 53. SucAmong the competing historical models, we found support for the admixed origin of the Italian Podolian breeds with a contribution of both Balkan and South-East Podolian gene pools. This result suggests that both the Danube  and sea (Mediterranean) routes have played a central role in this secondary migration. In this context, the date of our estimates of the time of admixture is about 1975 YBP (95% CI 402–5132 YBP). Although an Anatolian origin of the Etruscans is still controversial and seems to be denied by the analysis of ancient human DNA , a sea rIn this study, we used a comprehensive dataset including most of the autochthonous cattle breeds belonging to the so-called Podolian trunk to shed light into the origin and diversification of this group of European cattle. Podolian cattle are a good model to investigate historical routes of colonization in Europe, since they share ancestral traits pointing at a common origin. They also encompass an important fraction of the diversity of the European autochthonous breeds, which represents a fundamental resource and income for traditional farming thanks to local products. Considering the mtDNA signature, there is growing evidence of a decrease in genetic diversity as the distance to the domestication centre increases. However, several local breeds especially from the Italian Peninsula, show levels of genetic diversity that are similar to those observed in autochthonous breeds from the domestication centre, and intriguing explanations have been advanced to disclose this pattern. Our genomic analysis is in line with such a scenario that indicates a notable geographic cline from South-Western Asia to Europe. Furthermore, we also find a marked genomic diversity that localizes within the Italian Peninsula. The presented ABC framework allowed us to test different scenarios of colonization that highlight a complex evolutionary history of European taurine cattle. According to our coalescent and demographic reconstruction, European taurine experienced two principal migration waves, i.e. an early migration which gave rise to the non-Podolian taurine breeds followed by a secondary migration, which was probably prompted by increasing trade that favoured the diffusion of European Podolian. In addition, the most well-supported scenario suggests admixture events as an important driver in shaping the genetic diversity of Podolian cattle, which partially explains the marked genomic diversity observed in the Italian Peninsula. Finally, the role of hybridization with local aurochs’ populations still remains a pending question that needs further investigation.Additional file 1: Table S1. Name of the breeds, breed codes, sample size (N), sub-species, continent and geographic origin, and source of genotyping data.Additional file 2: Figure S1. All modelled scenarios for colonization tested in the ABC framework. Description: In all the tested scenarios, we assumed that taurine and indicine cattle separated first. Subsequent reduction in effective population size was modelled to take the two independent domestication events that occurred in the Fertile Crescent and the Indus Valley into account. From these known evolutionary events, two sets of scenarios were built. The first three scenarios mirror to two different waves of migration, an early Neolithic migration involving non-Podolian taurine cattle and a secondary migration involving Podolian cattle after their genetic admixture with indicine cattle that occurred in South-Western Asia. On the opposite, the other three scenarios reflect a single taurine diffusion that occurred after the admixture event between indicine and taurine. Within each set of scenarios, we drew different hypotheses of colonization. A Mediterranean route (Scenarios 1 and 4) in which the Italian Podolian breeds mainly derived from the South-Eastern Mediterranean region and introduced via sea trade. To model this scenario, we assumed that from an ancestral population of size N3 located in the South-Eastern Mediterranean region, a first colonization prompted the split between the Balkan and Central Europe breeds, while a subsequent split, driven by sea routes, led to the formation of the Italian Podolian breeds. In this latter separation, we incorporated a reduction in effective population size to accommodate a founder effect as expected when an population introduced by sea trade starts spreading from few individuals. A Balkan route (Scenario 2 and 5) in which we assumed a terrestrial model of colonization, that therefore we modelled from an ancestral population of size N3 located in South-Eastern Mediterranean, a first split gave rise to Balkan and Continental Podolian breeds while a subsequent divergence event led to the origin of the Podolian cattle in Italy. In this model, we do not constrain priors on effective population size in order to simulate a long-term dispersal as a consequence of a terrestrial expansion. Finally, the admixture scenario simulates an equal contribution of the two routes followed by admixture events leading to the formation of the Italian Podolian breeds.Additional file 3: Table S2. Set of priors used to model the scenarios in the ABC framework.Additional file 4: Figure S2. Genetic diversity indices: observed and expected heterozygosity (Ho and He), effective population size (Ne) and minor allele frequencies (MAF) calculated for each breed. Asian indicine (blue), African taurine (yellow), European Podolian (orange), European non-Podolian (red).Additional file 5: Figure S3. Cross-validation plot of the admixture analysis for all values of K (number of clusters) ranging from 2 to 23.Additional file 6: Figure S4. Admixture analysis plot in a circular fashion with all values of K (number of clusters) ranging from 2 to 23.Additional file 7: Figure S5. Increment in the log likelihood for the complete dataset for all tested migration events, calculated by using the optM function in the R package OptM.Additional file 8: Figure S6. Increment in the log likelihood for the reduced (European taurine and the Guelmoise) dataset for all tested migration events, calculated by using the optM function in the R package OptM.Additional file 9: Figure S7. Principal component analysis (PCA) for the two datasets used (17 K and 8 K).Additional file 10: Figure S8. Projection on a single LDA axis in the model-grouping approach (a) and on the first two LDA axes in the six scenarios separately (b).


In this study we assessed the level of genetic introgression between red foxes bred on fur farms in Poland and the native wild population. We also evaluated the impact of a geographic barrier and isolation by distance on gene flow between two isolated subpopulations of the native red fox and their genetic differentiation. Nuclear and mitochondrial DNA was collected from a total of 308 individuals (200 farm and 108 wild red foxes) to study non-native allele flow from farm into wild red fox populations. Genetic structure analyses performed using 24 autosomal microsatellites showed two genetic clusters as being the most probable number of distinct populations. No strong admixture signals between farm and wild red foxes were detected, and significant genetic differentiation was identified between the two groups. This was also apparent from the mtDNA analysis. None of the concatenated haplotypes detected in farm foxes was found in wild animals. The consequence of this was that the haplotype network displayed two genetically distinct groups: farm foxes were completely separated from native ones. Neither the River Vistula nor isolation by distance had a significant impact on gene flow between the separated wild red fox subpopulations. The results of our research indicate a low probability of genetic introgression between farm and native red foxes, and no threat to the genetic integrity of this species. Many wild animal populations are endangered by genetic introgression from domesticated animals that have escaped from farms ,5,6,7,8.The distributions of species are altered and disturbed by humans in many ways, ranging from transfer of animals between continents to conversion and destruction of habitats. The introduction of non-native species from intercontinental translocations is particularly harmful to native populations and is considered one of the main causes of biodiversity decline ,19.Vulpes vulpes) is currently not an endangered species . HoweveConcern, ,22,23.The wild population of the red fox in Poland, estimated at 200,000–250,000 individuals , is partFarm foxes reflect many generations of selective breeding for a number of traits important for fur breeders. This predisposes them to success in human-altered environments . TherefoThere have been few studies of genetic introgression between farm red foxes and their wild counterparts. As the main research aim, genetic introgression was investigated in North America by Sacks et al. , LounsbeBoth farm and wild red foxes belong to the same species; there is no reproductive isolation between them. However, the Eurasian wild red fox belongs to the Holarctic clade, while the farm red fox, of North American ancestry, belongs to the Nearctic clade ,32. AccoThe main objective of this study was to investigate the genetic introgression of red foxes bred on fur farms in Poland into the wild native red fox population, and to assess its level and spread within the country. We also evaluated the impact of a geographic barrier (the River Vistula) and isolation by distance on gene flow between two native red fox subpopulations, isolated from each other by the river, and their genetic differentiation.n = 200, 85 males and 115 females, denoted FARM) were obtained from two fur farms (n1 = 120 and n2 = 80) located in western Poland, whereas those from wild foxes  came from 16 locations evenly distributed across the study area . To ensure the lack of any relationship between the farm animals, their pedigrees, available in farm documentation, were meticulously analysed. The map with sampling sites is shown in The study area covered two regions of Poland, about 500 km apart, and separated by the River Vistula (Wisła). A total of 308 tissue samples (tongue slivers) were used in this study. The samples from unrelated farm foxes (n = 308) were genotyped at 24 autosomal microsatellite loci  using previously published primers [Genomic DNA was extracted according to the protocols used in the ARK Genomics Lab (currently Edinburgh Genomics) . The tis primers ,37,38,39Mitochondrial DNA was extracted using the Sherlock AX kit . Eighty nine samples (48 from farm foxes and 41 from wild foxes) were used to study maternal gene flow from farm to native populations.Two regions of mtDNA were amplified: a 878-bp segment of the cytochrome b gene and a 443-bp segment of the D-loop. The PCR mixtures were prepared using a DreamTaq Master Mix kit  according to the manufacturer’s protocol. A new set of primers for both sequences, designed by the authors of this study, as well as the thermal cycle conditions for both markers were described by Zatoń-Dobrowolska et al. . The mtDThe PCR products were purified and sequenced using an ABI BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and ABI 3730 capillary sequencer (Applied Biosystems) for cytochrome b (sequenced from primer VVGluF3) and D-loop (sequenced from primer VVCRR2). The whole lengths of sequences of the two regions combined into concatenated haplotypes were used to investigate mitochondrial lineages.O) and expected (HE) heterozygosity, the inbreeding coefficient (FIS) and the population genetic differentiation (FST) were estimated using the hierfstat package in R [pegas package [R) and null allele frequencies were calculated using the PopGenReport package [Initially, we estimated basic parameters of genetic diversity for both red fox populations . The observed (Hage in R . The num package . Allelic package .F-statistics were used to estimate gene flow between WILD-SW and WILD-SE [IS and FST was verified with the diveRsity package [mantel.randtest function [FST estimates (FST/(1 − FST), were used as a measure of genetic distance [FST-matrix. The barrier matrix contained 1 if the samples were separated by the River Vistula and 0 if they were not. The matrix of geographic distance contained Euclidean distances (in km) between localities. The Mantel test was applied to study the association (significance of correlation) between the genetic differentiation matrix and the other two matrices. The hierarchical analysis of molecular variance (AMOVA), using the poppr package [Conventional  WILD-SE . The sta package . The Manfunction ,48, was distance . The mat package ,51, was  package ,53. The snmf included in LEA was used to estimate the individual admixture coefficients from the genotypic matrix, and then to provide least-squares estimates of ancestry proportions  best explaining the genotypic data.To evaluate the level of admixture between farm and wild foxes  the Bayesian clustering approach implemented in Structure v. 2.3.4 ,55 was up.function in R [For all assignments, 95% credibility intervals were estimated to assess confidence in the assignment of individuals. This was done using the ion in R . These aadegenet package in R [To identify genetic clusters in the studied red fox populations, principal component analysis (PCA) for microsatellites was performed with the age in R . This apage in R .pegas package in R [After reading, editing and chromatogram quality-checking , the seqage in R . The nomage in R  were appR) estimated over all loci was 4.22 for FARM, 13.33 for WILD-SW and 13.87 for WILD-SE. Observed heterozygosity (HO) estimated over all loci was the lowest for FARM (0.47), while WILD-SW and WILD-SE had higher and comparable HO . The same applied to expected heterozygosity (HE): higher values of HE over all loci were estimated for WILD-SW and WILD-SE , whereas in FARM it was 0.48.The genetic diversity parameters estimated for FARM, WILD-SW and WILD-SE are shown in IS) estimated over all loci were significant (p < 0.05) for all three red fox populations studied (IS was estimated for FARM (0.18), whereas WILD-SW and WILD-SE had lower and comparable values of FIS . The FIS values estimated per locus were significant (p < 0.05) for two loci in FARM, four loci in WILD-SW and six loci in WILD-SE, indicating deviations from Hardy–Weinberg equilibrium.The inbreeding coefficients  indicate much higher genetic diversity among the wild foxes (both WILD-SW and WILD-SE) in comparison to the farm foxes.The genetic diversity parameters  revealed that a very small part of the total variance (0.45%) was caused by variability between populations, 6.98% was caused by variability between localities within populations, while as much as 92.57% was caused by variability within localities.The genetic structure analysis indicated two genetic clusters as being the most probable number of genetically distinct populations . The DelThe 95% credibility intervals of farm foxes overlapped the 100% assignment to the FARM genetic cluster in all individuals. The same was observed for wild foxes—all 95% credibility intervals overlapped the 100% assignment to the WILD (WILD-SW + WILD-SE) genetic cluster in all cases. This indicates with high probability that there is no admixture between farm and wild red foxes.PCA carried out to show relationships between multi-locus genotypes in two-dimensional space  revealedST) estimated over all loci showed a significant genetic differentiation between FARM and WILD-SW , and FARM and WILD-SE , but a non-significant genetic differentiation between WILD-SW and WILD-SE (FST = 0.004).The fixation index . Isolation by distance of both subpopulations also had no significant effect on their genetic differentiation .The Vistula (the largest river in Poland), separating WILD-SW and WILD-SE, did not significantly limit gene flow between these populations. The standard Mantel test done for microsatellite markers indicates a non-significant correlation between genetic differentiation and the presence of this geographic barrier  found in FARM, WILD-SW and WILD-SE, as well as the haplotype network indicating relationships between haplotypes of FARM, WILD-SW and WILD-SE, are shown in n = 17), followed by FOX12-FOX32 (n = 8), and FOX14-FOX34 and FOX9-FOX33 (for both n = 7). In WILD-SW and WILD-SE the most numerous haplotype was FOX29-D408 , followed by RF01-FOX36  and FOX27-D09 .The most numerous haplotype in FARM was FOX9-FOX30  taken from three groups of red foxes: farm foxes, wild foxes living in the area where these farms were located, and wild foxes from an area several hundred km distant from the first two and isolated by a geographic barrier (the River Vistula). We assumed that if the flow of non-native alleles from fox farms into the wild population of the red fox was not prevented, admixture analysis should show successively higher levels of introgression with increasing proximity to the locations of fur farms. Otherwise, admixed red foxes should not be observed at all or only in the immediate vicinity of the farms.R, H0, HE are much lower than in native individuals), and WILD-SW and WILD-SE . Indeed, the cross-entropy criterion estimated when analysing the genetic structure of wild foxes (using the LEA package) as well as PCA performed for WILD-SW and WILD-SE combined, clearly indicated one genetic cluster (ST) between FARM and both WILD-SW and WILD-SE; and (3) no genetic differentiation between WILD-SW and WILD-SE. Furthermore, the Mantel test showed that the Vistula, the longest river in Poland and the ninth-longest river in Europe , with sandy islands in the course of the river, was not a geographic barrier effectively preventing gene flow between WILD-SW and WILD-SE (this is probably one of the reasons for the genetic homogeneity of native red foxes). Therefore, it can be assumed that the Vistula would not be an effective barrier to the flow of non-native alleles from WILD-SW (if they were admixed with FARM) or farm escapees to WILD-SE. Neither was any signal of isolation by distance found, which confirms the unrestricted gene flow between WILD-SW and WILD-SE.The results of our research are consistent and indicate no or very limited genetic introgression between farm and native red foxes in Poland. Both the analysis of the genetic structure based on microsatellite markers (using the Structure package and PCA) as well as the comparison of haplotypes between FARM, WILD-SW and WILD-SE (haplotype network) indicate the coexistence of two genetic clusters (wild and farm red foxes) with no or very little admixture of non-native alleles. Additional support for this observation is provided by: (1) genetic diversity parameters estimated for the studied red fox populations. Even if they are not measures of genetic differentiation, they may suggest a genetic distinctiveness between FARM  sigNyctereutes procyonoides): the Central European cluster and the North Eastern European cluster. These two groups were found to be separated by the River Vistula, indicating limited movements of the host species  across the river. According to Stojak and Tarnowska [Despite the fact that our study does not indicate that the River Vistula is an important geographic barrier to gene flow between the studied subpopulations of the red fox, some authors ,70 suggearnowska , howeverGenetic introgression is more often observed when the ranges of closely related species overlap , or one In red foxes, males and females have different dispersal dynamics. Males migrate at higher rates and for longer distances, whereas females are philopatric . TherefoThe geography of admixture between native and non-native individuals is also an important issue when investigating introgression. In the situation where fox farms are scattered throughout the country (as in our research), introgression is not limited to distribution margins, as in the wave model, but is rather caused and accelerated by hybridization spots within the range of a native species . This shV. v. macroura) populations in Colorado. They reported that native haplotypes were predominant in mountainous areas, while non-native haplotypes were more common in more urbanized and lowland areas. They also confirmed higher male-mediated gene flow. Poland, compared to other European countries and the USA, is a relatively poorly urbanized. Most of its territory is rural or non-urbanized. Additionally, Poland is a rather lowland country with mountain ranges located in the south of the country. Fox farms are located in agricultural and poorly urbanized areas, usually in close proximity to forests and open areas, far from cities and large human clusters. Therefore, we believe that these landscape conditions do not play a significant role in limiting potential genetic introgression between farm and wild red foxes .American researchers  studied In our previous research , we idenDespite the fact that we did not detect strong signals of admixture between farm and wild foxes, Horecka et al. , who invST = 0.14, p < 0.001) between farm and wild foxes, indicating little gene flow between the two groups of foxes. Admixture and principal component analyses supported the clear separation of farm and wild red foxes. The authors concluded that gene flow from the Newfoundland red fox farm to the native population had caused little, if any, admixture. However, they suggested that the apparent admixture could have reflected a type I error, which was expected to occur in the sample on the basis of chance alone.Signals of admixture between farm and native red foxes on the island of Newfoundland (Canada), where a large fox farm has been operating for more than 30 years, were evaluated by Lounsberry et al. . They coV. v. patwin) and introduced, phylogenetically divergent red foxes resulting from a century of domestication. They found that hybridization was primarily restricted to a narrow zone where the two populations came into contact. The authors speculated that native foxes rejected introduced individuals. They also hypothesized that the monogamous mating system of red foxes could enhance their resistance to hybridization. According to Rheindt and Edwards [One of the possible reasons for the very limited non-native introgression from farm to wild foxes could be the monogamous mating system of red foxes. This was suggested by Sacks et al. ), who st Edwards , a rare Our research indicates that the genetic integrity of the native red fox population in Poland is currently not threatened by an inflow of non-native alleles from farm foxes. We found that the native red fox population, though divided by the River Vistula, in fact comprise one single genetic cluster without or very little admixture of non-native alleles. Given the relatively large scale of our study, we may be justified in stating that it does appear to reflect the real picture of genetic introgression between farm and wild red foxes in the country. Nevertheless, even though we did not detect strong signals of admixture between farm and native red foxes, the genetic structure of the Polish red fox population needs to be monitored so that relevant protective measures can be implemented as needed. This will allow wildlife organizations and fur breeders (1) to develop a model for effectively controlling the inflow of non-native alleles to the Polish red fox population, and (2) to avoid a scenario when the cause of, and then the solution to the problem is sought only when the species is already at risk.