Dothistroma septosporum responsible for the current outbreak of Dothistroma needle blight (DNB) on native Caledonian Scots pine (Pinus sylvestris) populations in Scotland and evaluated the role played by widespread planting of two exotic pine species in the development of the disease outbreak. We distinguished three races of D. septosporum in Scotland, one of low genetic diversity associated with introduced lodgepole pine (Pinus contorta), one of high diversity probably derived from the DNB epidemic on introduced Corsican pine (Pinus nigra subsp. laricio) in England and a third of intermediate diversity apparently endemic on Caledonian Scots pine. These races differed for both growth rate and exudate production in culture. Planting of exotic pine stands in the UK appears to have facilitated the introduction of two exotic races of D. septosporum into Scotland which now pose a threat to native Caledonian pines both directly and through potential hybridization and introgression with the endemic race. Our results indicate that both removal of exotic species from the vicinity of Caledonian pine populations and restriction of movement of planting material are required to minimize the impact of the current DNB outbreak. They also demonstrate that planting exotic species that are related to native species reduces rather than enhances the resilience of forests to pathogens.To manage emerging forest diseases and prevent their occurrence in the future, it is essential to determine the origin(s) of the pathogens involved and identify the management practices that have ultimately caused disease problems. One such practice is the widespread planting of exotic tree species within the range of related native taxa. This can lead to emerging forest disease both by facilitating introduction of exotic pathogens and by providing susceptible hosts on which epidemics of native pathogens can develop. We used microsatellite markers to determine the origins of the pathogen  Morelet responsible for a recent outbreak of Dothistroma needle blight (DNB) on pine in Scotland  of the pathogens responsible and devise appropriate control measures. If the pathogen involved is native, removal of the exotic species may be sufficient to eliminate the disease threat. However, if the pathogen is introduced, the prospects for the native species may be poor, involving death of many trees and recovery only after a prolonged period during which there is an evolution of enhanced host resistance  Maire  caused by the ascomycete D. septosporum, Scots pine P. sylvestris, comprises two distinct populations. Caledonian pines represent the remnants of native P. sylvestris populations that recolonized Scotland after the last ice age. They are confined to the Scottish Highlands and are highly fragmented, and their distribution has been reduced to less than 1% of its former area  is grown principally in Scotland where it makes up 10% of the conifer area  was first found in Britain in the 1950s in southern English nurseries on four exotic species: Corsican pine, lodgepole pine, D. septosporum population in Scotland, assess the role of the exotic Corsican and lodgepole pine species in its appearance and inform management plans for its control, particularly in the Caledonian pinewoods, we initiated a detailed analysis of the genetic structure of the pathogen across its hosts within Scotland. Recent work by Mullett, Brown, Fraser, Baden, and Tubby  Morelet outbreaks identified in disease surveys in naturally regenerated and planted forest stands (Table We performed targeted sampling of ds Table . ClassesP. sylvestris) and Corsican pine (P. nigra subsp. laricio). Three sites were sampled in 2015 (Culbin Forest (n = 39), Torrs Warren (n = 33) and Tentsmuir (n = 30)). At each site, roughly equal numbers of isolations were made from the two host species.Mixed plantations of Scots (P. contorta) stands. Three sites were sampled in 2014 (Glen Einig (n = 29), Glen Garry (n = 35), Inshriach Forest (n = 32)) and two in both 2014 and 2015 (Glen Affric (n = 21), Dundreggan (n = 39)). At each site, we made isolations from roughly equal numbers of the two hosts. In addition, we collected two lodgepole pine isolates from Strathpeffer in 2015 (n = 2).Caledonian Scots pine sites with adjacent lodgepole pine (n = 15) and Beinn Eighe (n = 23).Caledonian Scots pine sites isolated from exotic pine plantations. Two sites were sampled in 2015, Glen Tanar (n = 24), northern Scotland (n = 13) and northern England (n = 3). All other information relating to the samples and their location remains confidential.Forest nursery sites with reported disease outbreaks. Samples were isolated by Forest Research (Alice Holt) during annual forest nursery DNB surveys between 2011 and 2015. Nursery samples were classified into three categories; southern Scotland . At Caledonian pine sites, infected needles originated mostly from naturally regenerated saplings, although in some cases mature trees were sampled. A single genotype of D. septosporum from each of three North American populations of lodgepole pine  and Kispiox, Buckley Canyon))  and tissue‐lysed  prior to DNA extraction (~20 mg of lyophilized tissue). DNA extraction was performed using DNeasy Plant Mini Kit (Qiagen), following the manufacturer's guidelines. DNA for genotyping was re‐suspended in sterile distilled water (SDW) and stored at −20°C for further use.2.2.2D. septosporum were determined using species‐specific primer combinations developed by Groenewald et al.  using the adegenet package  and WSS (within sum of squares) statistics for increasing number of clusters.To infer the number of genetic clusters within Scottish populations of 2.3.2Ht) and genetic divergence (θst) between D. septosporum clusters and among populations within these clusters were calculated using the FSTAT programme  of being the product of sexual reproduction. These genotypes were then treated as clonal replicates to generate a clone‐corrected data set. Allele frequencies used in the MLGsim simulation were those estimated in the complete data set .For each cluster, the number of multilocus genotypes was found using the program MLGsim  and mean correlation among loci (rD), with significance estimated using 1,000 randomizations. In the second analysis, the proportion of locus pairs showing significant association  was computed using FSTAT. If sexual reproduction is prevalent, there will be no significant genetic differentiation between populations of opposite mating type (Ennos & Hu in prep.). Analyses were conducted both on the original and on the clone‐corrected data sets. Additionally, the extent of genetic differentiation (θmt) between the clone‐corrected mt‐1 population in the SR race and the clone‐corrected mt‐1 population in the NPR race was calculated in FSTAT.Analysis of multilocus structuring of the races was conducted in three ways. In the first analysis, the program Multilocus 1.3 Beta   from week 0 to week 8.To investigate possible genetically determined phenotypic differences between D. septosporum produces the exudate dothistromin which is known to be a virulence factor in DNB . We found LPR in all sites where isolations were made from lodgepole pine with the exception of the northern Caledonian pinewood site at Glen Einig. In some locations, such as Glen Affric and Glen Garry, LPR was the predominant race on lodgepole pine, whereas in others, such as Dundreggan, it comprised a small proportion of lodgepole pine infections  predominated in samples from more southern locations, and its frequency declined towards the north of Scotland  on lodgepole rather than Scots pine. Where it occurred at intermediate frequency in mixed plantations of Scots and Corsican pine, for example, at Tentsmuir, SR was significantly associated with Corsican pine .In contrast to LPR, the remaining two genetic clusters distinguished by the DAPC analysis were found on all three host pine species. However, they were very distinct in their geographic distributions. The first of these clusters n = 9 predominn = 209) was strongly associated with Caledonian Scots pine .The third genetic cluster recognized by the DAPC analysis n = 09 was st3.3D. septosporum races  were shared with the three individuals sampled from North American populations (Table Lodgepole pine race (LPR) was the least diverse of the three es Table . It was A = 5.50 ± 1.25 and high gene diversity Ht = 0.550. Allelic richness of SR was significantly higher in the population occupying plantations (A = 3.75) than on Caledonian pine (A = 2.64) (p = .015).In contrast, SR was characterized by the highest level of genetic variation Table . All of n = 68) and allelic richness A = 4.23 ± 1.60 were high. However, gene diversity Ht = 0.182 was much lower than in SR because allelic diversity was present predominantly at a single hypervariable locus (M). NPR showed the highest number of unique alleles (n = 22) with 16 of these found at the hypervariable locus M (Table A = 4.45) than on plantation pines (A = 3.91), this difference was not significant (p = .348).The NPR race was intermediate in genetic diversity between LPR and SR Table . 91% of  M Table . AlthougD. septosporum races were strongly genetically differentiated from each other with overall θst = 0.558, p < .01. LPR diverged the most from the other two races, showing pairwise θst = 0.8037 (p < .05) with NPR and θst = 0.5119 (p < .05) with SR. The other two races, NPR and SR, were genetically closer, with a moderate but still significant level of divergence θst = 0.3997 (p < .05).The three st = 0.075, p > .05) among the major populations scored . For SR, there was low but significant genetic differentiation  among the plantation and nursery populations  . In NPR, there was low but significant genetic divergence among Caledonian pine populations  but much higher genetic differentiation among plantation populations θst = 0.318 (p < .01) (Table st = 0.040), while mean pairwise differentiation between Caledonian pine populations and the two plantation populations located further south was much higher   Table . SR also1) Table . Mean pa6) Table .3.4IA = 0.842, rD = 0.176, p = .003) and clone‐corrected data sets , as expected if LPR reproduces asexually.Lodgepole pine race (LPR) comprised only five multilocus genotypes (MLGs), among which one was a clonal MLG Table . All of n = 58, 11 of which were clonal MLGs , which suggests the possibility of sexual reproduction within SR populations. There was however significant linkage disequilibrium among loci present in the original  and clone‐corrected data sets . We also found a significant amount of genetic divergence between the two mating type populations , rejecting the hypothesis of random mating within SR populations. The results are compatible with SR being predominantly asexual with low levels of sexual reproduction.Southern race (SR) was characterized by the highest number of MLGs Gs Table . Mating n = 51 were present in the NPR population with a relatively small number of clonal MLGs (n = 5)  and clone‐corrected data sets . Although both mating type variants (mt‐1 and mt‐2) were present in NPR populations, the frequency of individuals bearing the mt‐1 allele was limited to only 3% of the population. Genetic differentiation was high and significant between the two mating type populations within NPR . The mating system of NPR is therefore likely to be predominantly asexual. Genotypes possessing the mt‐1 variant were noticeably more genetically variable than those possessing mt‐2 .A moderate number of MLGs 5) Table . Within 3.5F2,6 = 20.40, p = .002), and significant interaction between race and temperature for this character  . The three D. septosporum races showed different temperature optima for growth. LPR exhibited the slowest growth at all three temperatures tested, NPR was the fastest growing race at 10 and 22.5°C, while SR exhibited the fastest growth at 17.5°C.We found significant differences in mycelial growth rate among the races (1) Table   and a significant race x temperature interaction   Table . LPR pro9) Table b.4D. septosporum population in Scotland was able to assign individuals to the three major genetic groups previously recognized by Mullett et al.  is the most genetically variable of the three races implying a large effective population size as expected in an epidemic population. Equality of mating type frequencies and low (though significant) correlation among markers together with limited differentiation across mating types suggests that SR may practise a low frequency of sexual reproduction. Therefore, some long‐distance dispersal by ascospores could occur in this race. Given the preference of SR for Corsican pine, a predominantly southern distribution, and near absence from Caledonian pine, SR is likely to have originated via recent dispersal from the epidemic of DNB on Corsican pine in England. Transfer of SR to northern plantations in Scotland via infected nursery stock is suggested firstly by the prevalence of SR on nursery material from south Scotland. In addition, the genetic composition of the SR population found on southern nursery samples is very similar to that at Tentsmuir and Culbin Forest (low genetic differentiation), a result that would be expected if infected nursery stock from southern nurseries was the source of SR at these northern sites.D. septosporum races, NPR, is strongly associated with Caledonian pine in our study . NPR was also isolated from lodgepole pine adjacent to Caledonian pine stands and was predominant in northern Scottish nurseries. In plantations, the frequency of NPR declined from north to south and data from Mullett et al. , overall gene diversity is low. The ratio of mating types in NPR is very highly skewed, and there is a strong allelic correlation among markers and genetic differentiation between mating types, all suggesting that NPR reproduces asexually. Neither low genetic diversity nor asexual reproduction are generally considered to be attributes of endemic populations. On the other hand, NPR possesses 22 alleles that are not found in the most closely related race SR. Such race specific alleles can only have accumulated by mutation if the races have been isolated for a considerable number of generations.D. septosporum arrived in Scotland with pine populations colonizing from continental Europe after the last glaciation  appears to have become infected by the endemic NPR race of D. septosporum to Caledonian pine. The presence of susceptible Corsican pine throughout Britain has facilitated the introduction of race SR from continental Europe, while planted lodgepole pine has brought with it a second exotic race of D. septosporum LPR from North America. The two exotic races are now sympatric with race NPR which prior to their arrival was not causing any significant damage. What are the risks posed by these novel introductions?Our scenario implies that the planting of related exotic species has significantly altered the biodiversity threat posed by Analysis of the outcome of natural inoculations by the SR race present at Torrs Warren showed high genetic variation for susceptibility both within and among populations of Caledonian pine  than from mt‐1 isolates in the SR race . A possible explanation is that the mt‐1 mating types within NPR are actually hybrids between the SR and NPR races. Analysis of the genomic constitution of these isolates, now underway, will provide further evidence for or against this hypothesis (Stukenbrock, Tentative evidence for hybridization between NPR and SR comes from an analysis of the small number of NPR isolates from Caledonian pine that carry the mt‐1 allele. Genetic differentiation of these isolates from mt‐2 isolates of the NPR mating type is much larger (θPossible steps to reduce the spread of SR into Caledonian pine include a moratorium on movement of nursery stock of either Scots or lodgepole pine into Caledonian pinewood sites, and removal of plantations of either Scots, lodgepole or Corsican pine from areas within the range of sexual spore dispersal. If the SR race nevertheless becomes established within Caledonian pine populations, control of DNB would be best achieved by promoting management of the pinewoods for natural regeneration (Cavers & Cottrell, Heterobasidion (Garbelotto, Guglielmo, Mascheretti, Croucher, & Gonthier, Cryphonectria (Dutech, Fabreguettes, Capdevielle, & Robin, Hymenoschyphus (Gross et al., Overall, our results demonstrate the power of adopting a forensic forest pathology approach in which population genetic analysis of molecular markers is used to unravel the origins and subsequent evolution of emerging forest pathogens. The approach has already provided previously inaccessible information on the introduction pathways and subsequent behaviour of forest pathogens in genera such as From a wider forest policy viewpoint, our analysis of DNB in Scotland provides a clear illustration of the dangers of establishing plantations of exotic tree taxa that are related to and share pathogens with native tree species (Burgess & Wingfield, MJP, CR, PNH and RAE were all involved in the design and performance of the research together with data collection, analysis and interpretation. MJP and RAE wrote the manuscript with significant input from CR and PNH.https://doi.org/10.5061/dryad.7p5s7.Data available from the Dryad Digital Repository:  Click here for additional data file.


Quercus austrocochinchinensis and Q. kerrii  are a pair of closely related species in the Indo-China area. Morphological intermediates of the two species have been reported in this region. In this study, we used AFLP, SSR and two key leaf morphological diagnostic traits to study the two Q. austrocochinchinensis populations, two pure Q. kerrii and two putative hybrid populations in China. Rates of individual admixture were examined using the Bayesian clustering programs STRUCTURE and NewHybrids, with no a priori species assignment. In total, we obtained 151 SSR alleles and 781 polymorphic loci of AFLP markers. Population differentiation inferred by SSR and AFLP was incoherent with recognized species boundaries. Bayesian admixture analyses and principal coordinate analysis identified more hybrids and backcrossed individuals than morphological intermediates in the populations. SSR inferred a wide genetic assimilation in Q. austrocochinchinensis, except for subpopulation D2 in the core area of Xi-Shuang-Ban-Na Nature Reserve (XSBN). However, AFLP recognized more Q. austrocochinchinensis purebreds than SSR. Analysis using NewHybrids on AFLP data indicated that these hybridized individuals were few F2 and predominantly backcrosses with both parental species. All these evidences indicate the formation of a hybrid swarm at XSBN where the two species co-exist. Both AFLP and SSR recognized that the core protected area of XSBN (D2) has a high percentage of Q. austrocochinchinensis purebreds and a unique germplasm. The Hainan population and the other subpopulations of XSBN of the species might have lost their genetic integrity. Our results revealed a clear genetic differentiation in the populations and subpopulations of Q. austrocochinchinensis and ongoing introgression between Q. austrocochinchinensis and Q. kerrii at the disturbed contact areas. Combining the results from genetic and morphological analyses, the conservation of subpopulation D2 should be prioritized. Conservation and restoration of the integrity of tropical ravine rainforest is an important long-term goal for the successful conservation of Q. austrocochinchinensis. The fine-scale landscape might play an essential role in shaping the spatial patterns of hybridization. Further studies are needed to evaluate these patterns and dynamics.Natural introgression can cause negative effects where rare species experience genetic assimilation and invade by their abundant congeners.  The 108 individuals were sampled in five populations, A–E .Q. kerrii and Q. austrocochinchinensis and even to assign their hybrids  . The PCR reactions were performed in 20 μl reaction volume containing 10 μl TIANGEN PCR Master Mix , 0.3 μl/L of each primer (10 mM), and 20 ng genomic DNA; PCR reactions were performed as follows: 5 min initial denaturation at 94°C, 35 cycles of 40 s at 94°C, 30 s at 55°C, 1 min elongation at 72°C, and 7 min extension at 72°C. Finally, Gene-Scan-500 LIZ size standard (Applied Biosystem™) was added to all the samples before loading on an automated sequencer ABI 3730 (Applied Biosystem™). The final step was performed by a private professional commercial lab .Total genomic DNA for each sampled individual was extracted from the silica gel-dried leaf tissue using the modified CTAB method . The digestion mixtures were incubated at 37°C for 3 h and the digested mixture was then incubated at 70°C for 10 min to denature the enzymes. Subsequently, 4 μL of digested DNA were added to 16 μL of ligation mix containing 2 U T4 DNA ligase (New England BioLabs), 5 pmol EcoRI, and 50 pmol MseI adaptor. The mixture was incubated at 10°C for 14 h and then denatured at 70°C for 10 min. The ligated DNA samples were diluted 5-fold with double sterile water. Pre-selective amplification reactions were carried out using EcoRI-A (5′-GACTGCGTACCAATTCA-3′) and MseI-C (5′-GATGAGTCCTGAGTAAC-3′) in a 50 μL volume containing 1.5 mmol/L MgCl2, 200 μmol/L of each dNTP, 1.25 μmol/L of each primer, and 0.6 U rTaq DNA polymerase  under the following cycle: 3 min at 72°C, 30 cycles of 30 s denaturing at 94°C, 30 s annealing at 56°C, 1 min extension at 72°C, and a final extension for 5 min at 72°C. After a 1:20 dilution of pre-selective PCR products, seven primer combinations were performed in selective amplification . The EcoRI primers were fluorescently labeled with 6-FAM. These primer pairs were chosen because they generated clear and fewer bands (thus decreasing the risk of fragment non-homology) with sufficient variability in preliminary tests. Selective PCRs were carried out in a 20 μL volume containing 2.5 mmol/L MgCl2, 200 μmol/L of each dNTP, 1.25 μmol/L of each primer, and 0.2 U of rTaq DNA polymerase  and under the following cycle: 3 min at 94°C, 9 cycles of 40 s at 94°C, 30 s at 65–57°C touchdown (reducing the temperature at 1°C per cycle), 15 min at 72°C, 20 cycles of 40 s at 94°C, 30 s at 56°C, 1.5 min at 72°C, and a final extension for 7 min at 60°C. The PCR products were 10-fold diluted and mixed with Gene-Scan-500 LIZ size standard (Applied Biosystem™); products from each primer combination were loaded separately on an automated sequencer ABI 3730 (Applied Biosystem™) by the same commercial service provider mentioned above.The AFLP method was performed following the protocol by Vos et al. , with miRaw data of SSR and AFLP samples were collected and analyzed using GeneMarker®v2.2.0. The samples with low quality of size calibrations or peaks were excluded from allele calling. The allele sizes of SSR were read manually and MicroChecker v 2.2.3 , Observed and Expected Heterozygosity (HO and HE), were performed by GenAlEx version 6.5  was estimated as (2N/(2N-1)) * HE. Fixation Index (FIS), or inbreeding coefficient was estimated as (HE–HO)/HE , allele frequency distribution, Shannon's Information Index , and differentiation statistics were calculated using the AFLP-SURV v. 1.0 software  and HT = 1/2(p1+p2)*(q1+q2), with q = 1-p. For SSR markers, FST for each loci was calculated using GenePop v. 4.2 , log10 PO > 1.0 (strong), log10 PO > 1.5 (very strong), and log10PO > 2.0 (decisive support for accepting a model) , program BayeScan v. 2.1  generated from STRUCTURE was used to classify individuals into purebred and hybrids, using a threshold q-value of = 0.1, where samples with q-values < 0.1 or > 0.9 were classified as purebred and those with q-values between 0.1 and 0.9 as hybrids, including F1 and backcrosses , we also used the program InStruct . All the 11 SSR loci were analyzed using this software. For AFLP data, NewHybrids will assign the individuals more accurately, using the loci with high differentiation at interspecies level. Therefore, we filtered the AFLP loci according to FST. We applied three approaches to compare the results obtained by NewHybrids, using 100 loci with highest FST value at interspecies level, 279 loci (FST ≥ 0.1), and 450 AFLP loci (FST ≥ 0.03), respectively, for analysis. A burn-in period of 7,500 MCMC repetitions was defined and 10,000 iteration were run thereafter.The NewHybrids software v. 1.1 beta, employing a Bayesian analysis  aims to visualize similarities or dissimilarities of individual data based on a distance matrix. This method is also an alternative algorithm without any assumptions about the population genetic model. For AFLP and SSR data, the pairwise Euclidian distance matrix was constructed, and the first two principal co-ordinates were visualized by GenAlEx v. 6.5  and LAR = 0.78 (0.23–1.36). Of the sampled Q. austrocochinchinensis individuals, only the samples from D2 and E were totally separated from Q. kerrii samples. They had larger LR and LAR values compared to other populations, with an average of LR = 3.71 (2.79–5.32) and LAR = 2.06 (1.50–3.20). Morphological variation was higher in Q. austrocochinchinensis individuals than in Q. kerrii specimens, with standard deviations (σ) for Q. austrocochinchinensis and Q. kerrii of 0.72 and 0.25 for LR and 0.64 and 0.25 for LAR, respectively.Morphological measurements showed that the two species could be distinguished using a combination of two ratio values, which were leaf length-to-width ratio (LR) and leaf apex length-to-width ratio (LAR) Figure . We founQ. kerrii, with an average of LR = 2.52 (2.22–2.74) and LAR = 0.82 (0.47–1.13). However, the putative hybrids of population C had a sharper leaf apex with LAR = 1.54 (0.86–2.27), although the leaves were as broad as those of Q. kerrii with LR = 2.77 (2.35–3.47). In addition, there were seven Q. austrocochinchinensis individuals from D3 and D6 with leaf morphological traits similar to those of Q. kerrii.The putative hybrids of D5 within the hybrid zone had morphological values similar to HO) and Expected Heterozygosity (HE)  across all loci in Q. austrochichinensis was 0.706 and higher than that in Q. kerrii with 0.595. Species-specific alleles were found at several loci, especially rare alleles restricted to Q. austrocochinchinensis . Heterozygote deficiency was found at all loci except Qk17611 and QpZAG36 (p < 0.01). The estimation of genetic differentiation between Q. austrocochinchinensis and Q. kerrii was low across all SSR loci (FST = 0.138). The differentiation among population within species was also low for both species, with FST values of 0.130 and 0.042 for Q. austrocochinchinensis and Q. kerrii, respectively.In total, 151 alleles were obtained from the 11 SSR loci used in this study. Some SSRs were highly variable, e.g., CG371, QpZAG16, and QpZAG110, containing more than 20 alleles. These SSRs also had higher Observed Heterozygosity  or the whole sample level (Ht), were very similar  (Table FST = 0.0246 (p < 0.01) for Q. austrocochinchinensis and FST = 0.0667 (p < 0.01) for Q. kerrii. The FST distribution of 781 AFLP alleles followed an L-shaped curve, which suggested that most of the alleles  had lower FST values (FST < 0.1). Only a few alleles exhibited higher FST values  than FST . By using BayeScan, 3 out of 781 AFLP loci (0.38%) were identified as outlier loci under directional selection at a threshold of log10 PO > 2 (posterior probabilities higher than 0.99)  Table . Similars Figure . The PST) Figure .K) of STRUCTURE analysis can be inferred by the peak of ΔK. We found that K = 2 was the optimal K value for both AFLP and SSR . The two morphological intermediate putative hybrid populations C and D5 contained a high proportion of Q. austrocochinchinensis germplasm . Among the individuals identified as Q. austrocochinchinensis, in populations D1, D2, and E, the mean proportion of Q. austrocochinchinensis germplasm was 89.1% , but mean proportion was considerably lower in D3 with 34.5% (1.1–71.3%). Given that we considered individuals with q-values between 0.6 and 0.9 as backcrosses, we did not identify any backcrossed individuals based on SSRs. However, for AFLP, 25 out of 108 (23.1%) samples were recognized as backcrosses.The most likely number of clusters , except for one individual (Ca21). The remaining individuals identified as Q. austrocochinchinensis in D1, D3, D6, and E were assigned to either Q. kerrii or backcrosses to Q. kerrii, as well as the morphological intermediates individuals in Population C and D5. Among the 36 individuals identified as Q. kerrii, most were assigned to Q. kerrii, with mediate to high probabilities, two  were assigned to backcrosses to Q. kerrii, with probabilities of 0.935 and 0.802, respectively  Nature Reserve in Yunnan province and Ba-Wang-Ling (BWL) Nature Reserve in Hainan Province , located at contact zone subpopulation D1, is F1. NewHybrids analysis is more sensitive to assign the genotype to different genetic categories and detected a general presence of backcrosses to Q. kerrii, both in morphologically identified Q. kerrii and Q. austrocochinchinensis and the intermediate populations, but no F1, F2, or backcrosses to Q. austrocochinchinensis. The SSR results indicate unidirectional introgression from Q. kerrii to Q. austrocochinchinensis.Overall, our study revealed that there were fewer 1 and F2 hybrids and backcrosses. STRUCTURE inferred the existence of F1 hybrids and backcrosses, whereas NewHybrids inferred absences of F1, but predominant backcrosses. STURECTURE is more efficient to evaluate the presence of hybrids in wild populations, whereas NewHybrids algorithm explicitly searches for hybrid and parental classes with assumption of two parental classes, which generally showed higher assignments accuracy than STRUCTURE  contain historical and ongoing gene flow between the two species.For the AFLP data set, both Bayesian clustering approaches used (implemented in STRUCTURE and NewHybrids) detected an unexpected high number of backcrosses and hybrid genotypes. The threshold values and loci used on assigning individuals to different genetic categories are different in STRUCTURE and NewHybrids. Therefore, the two methods provided different percentages on FQ. kerrii, mainly in terms of the shallow cupule, persistent trichomes on the leaf abaxial surface, and a relatively thick bark. However, their sharp leaf apex and leaf margin teeth more resemble Q. austrochochinensis  might have been already genetically swamped. The same situation might also have occurred in population E in Hainan, as SSR indicated that no Q. austrocochinchinensis purebreds exist, and the AFLP inferred co-existence of Q. austrocochinchinensis purebreds and its backcrosses.A hybrid swarm is often characterized by a wide spectrum of phenotypic variation, the existence of backcrosses, and high genetic variation , which are distant from all the known populations of Q. austrocochinchinensis. Interspecific gene flow is a widespread and ongoing process among oaks, especially in species with close genetic relationship  have low FST values (FST < 0.1). The results of BayeScan also suggested that only three loci (0.38%) are potentially under selection among the 10 populations. Therefore, most of the AFLP markers are selectively neutral. However, SSR markers may have been subjected to more selective pressure. Morgante et al. , the molecular method detected more hybrids than expected. Moreover, individuals with one parental morphological characteristic may be identified as another parental purebred, e.g., a part of trees from population D3. There may be two explanations for these discrepancies. First, trees with clear parental characteristics could in fact be FQ. austrocochinchinensis in appearance were all located in the national nature reserve, where canopy density is usually high. The stellate and fused fascicular trichomes and dense stomata, as well as the thick leaf cuticles are important features in seasonal dry areas with strong sun light (Mediterranean-type) climate . BayeScan analysis indicated most AFLP loci were selection neutral, but almost all the SSR loci violated HWE assumption. Therefore, AFLP result is more reliable to infer the gene flow between the two species. Recently simulation model study demonstrated when outercrossing rate between the two parental species is different, hybridization can facilitate invasions of the species with high outcrossing rate, even without enhancing local adaption (Mesgaran et al., Q. austrocochinchinensis at middle to low elevation of tropical Asia, the ongoing hybrid swarm and the predominance of Q. kerrii at co-occur region, Q. austrocochinchinensis faces critical extinction risks both in terms of lost habitat and genetic assimilation. Furthermore, our results indicate that only subpopulation D2 maintains its genetic integrity, as the most pure individuals of Q. austrocochinchinensis are still purebreds. The other subpopulations in XSBN Nature Reserve and adjacent areas formed a hybrid swarm. Once introgression is detected in an endangered species, protective intervention measures should be adopted immediately to prevent species integrity loss. Otherwise, all subpopulations in the reserve could become hybrid swarms.Our data indicates ongoing and historical introgression in the two in situ conservation of species genetic resource can be realized by establishing nature reserves. According to our results, the most pure Q. austrocochinchinensis population D2 has the highest forest canopy density and lowest human disturbance. Although it is difficult to prove the correlation between ecological integrity and hybrid degree in this case study, we hypothesize that it is important for in situ conservation to maintain the existing ecological balance of the habitat of population D2. To avoid further asymmetrical introgressions, forest landscape restoration is also essential for the hybrid Q. austrocochinchinensis populations. Secondly, considering the abundance of Q. kerrii and the efficient pollen dispersal abilities of oaks, ex situ conservation of pure Q. austrocochinchinensis should also be considered. Although we cannot claim that Q. austrocochinchinensis homozygous individuals still exist, molecular markers and leaf morphological features all identified D2 as the most pure Q. austrocochinchinensis population. Therefore, we suggest that the D2 population area should be designated as the priority conservation zone.Firstly, Q. austrocochinchinensis in China is experiencing introgression from Q. kerrii. The incoherent genetic structure inferred by AFLP and SSR, as well as morphological traits, might be due to the different selective pressures. The extinction risk of Q. austrocochinchinensis is higher than previously expected, as much less Q. austrocochinchinensis purebreds were detected based on molecular markers; in addition, the species faces ongoing hybrid swarm with Q. kerrii and habitat loss in tropical Asia. The subpopulation D2 of Q. austrocochinchinensis in the core area of the XSBN Nature Reserve, with unique germplasm and vulnerable to disturbance, should be prioritized for protection. The habitat with high forest canopy density and humidity may act on shaping the genetic structure of the two species at the contact zone. Further studies using high throughput molecular markers and coupling the environmental parameters at fine scale to study the genetic diversity patterns at the co-occurring area of the two species and scan more Q. kerrii purebred populations can provide a better understanding on the dynamics of the hybrid zone and determine the underlying genes involved in local adaptation.This study suggests that MA and MD designed the study, wrote, and revised the manuscript; MD was responsible for the manuscript submission. SZ and MA performed the experiments; XJ assisted to analyze the data; MD and YS collected the plant materials.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.


Optimum contribution selection (OCS) is effective for increasing genetic gain, controlling the rate of inbreeding and enables maintenance of genetic diversity. However, this diversity may be caused by high migrant contributions (MC) in the population due to introgression of genetic material from other breeds, which can threaten the conservation of small local populations. Therefore, breeding objectives should not only focus on increasing genetic gains but also on maintaining genetic originality and diversity of native alleles. This study aimed at investigating whether OCS was improved by including MC and modified kinships that account for breed origin of alleles. Three objective functions were considered for minimizing kinship, minimizing MC and maximizing genetic gain in the offspring generation, and we investigated their effects on German Angler and Vorderwald cattle.In most scenarios, the results were similar for Angler and Vorderwald cattle. A significant positive correlation between MC and estimated breeding values of the selection candidates was observed for both breeds, thus traditional OCS would increase MC. Optimization was performed under the condition that the rate of inbreeding did not exceed 1% and at least 30% of the maximum progress was achieved for all other criteria. Although traditional OCS provided the highest breeding values under restriction of classical kinship, the magnitude of MC in the progeny generation was not controlled. When MC were constrained or minimized, the kinship at native alleles increased compared to the reference scenario. Thus, in addition to constraining MC, constraining kinship at native alleles is required to ensure that native genetic diversity is maintained. When kinship at native alleles was constrained, the classical kinship was automatically lowered in most cases and more sires were selected. However, the average breeding value in the next generation was also lower than that obtained with traditional OCS.For local breeds with historical introgressions, current breeding programs should focus on increasing genetic gain and controlling inbreeding, as well as maintaining the genetic originality of the breeds and the diversity of native alleles via the inclusion of MC and kinship at native alleles in the OCS process.The online version of this article (doi:10.1186/s12711-017-0320-7) contains supplementary material, which is available to authorized users. In recent decades, the widespread use of artificial insemination and other reproductive technologies has resulted in substantial genetic gains in livestock populations. However, another consequence is that only a limited number of animals with high estimated breeding values (EBV) have been intensively used in breeding programs, which can result in increasing rates of inbreeding to undesired levels. A high rate of inbreeding not only leads to considerable reduction in genetic variation but also more deleterious recessive alleles become homozygous, which may threaten the entire future of the population . Thus, tCrossbreeding has been demonstrated to be an efficient method to reduce the threat of inbreeding depression and increase the level of genetic diversity . In addioptiSel.Optimum contribution selection (OCS) is a selection method that is effective at achieving a balance between rate of inbreeding and genetic gain. This selection process maximizes genetic gain in the next generation while constraining the rate of inbreeding via restriction of relatedness among offspring –6. The sOCS is efficient for controlling the level of kinship among progeny and the rate of inbreeding in future generations and can ultimately maintain genetic diversity , 19, 20.Instead of focusing on genetic gain and rate of inbreeding only, a reasonable breeding objective would be to also include recovery of genetic originality by reducing migrant contributions (MC). The diversity of native alleles may also be important for conservation. Thus, to conserve breeds with historical migrations, Wellmann et al.  recommenOur aim was to investigate whether including MC and modified kinship matrices that account for breed origin of alleles as additional constraints in OCS can improve breeding programs in local breeds. Both conservation progress and genetic gain were evaluated. The following scenarios based on different objective functions were considered: (1) maximizing the diversity of native alleles while restricting MC and/or the average breeding value of the progeny generation at desired levels; (2) minimizing MC while restricting the loss of diversity of native alleles and/or the average breeding value of the progeny generation at desired levels; and (3) maximizing the average breeding value of the progeny generation while restricting MC and/or the loss of diversity of native alleles at desired levels. The traditional pedigree-based kinship was constrained in all optimization scenarios.Data from two local German cattle breeds, Angler and Vorderwald, were analyzed. The Angler breed is mainly located in the northern part of Germany and represents a dual-purpose breed, although the primary emphasis is on milk production. With the introduction of other breeds to improve milk yield, the Angler breed has experienced a considerable amount of migrant breed introgressions . The AngSelection candidates were chosen among animals that were classified as purebred in the herdbook in order to compute their optimum contributions with different approaches. Sires that had progeny born in 2005 and 2006 were set as male selection candidates and selected males were mated to 1000 randomly chosen dams, which are called female selection candidates. For the Angler breed, 1199 selection candidates were available and 15,370 animals were involved in the pedigree that included all selection candidates and their ancestors. For the Vorderwald breed, 1123 selection candidates were available and 12,934 animals were involved in the pedigree. For a better comparison of results between the two breeds, EBV were normalized across all selection candidates of each breed, with a mean of 0 and a standard deviation of 1.c with individual genetic contributions. The genetic contribution The output of the optimum contribution selection procedure is a vector Four kinships that are involved in the calculation of the OCS procedure were applied. The diversity parameters described in  are comp,…,φD in . The rel,…,φD in .), was restricted in all scenarios. For breeds with historical migrations and foreign introgressions, Wellmann et al. [The classic kinship n et al.  proposedNote that this is equal to the probability that both alleles are IBD and native plus the probability that at least one allele is from a migrant.Kinship matrix X and Y at a locus that are randomly chosen from the offspring population are IBD, given that both descended from native founders  and the output from a truncation selection scenario (TS). In scenario REF all selection candidates were used as parents and had equal contributions to the offspring generation. For endangered breeds, an effective population size . When the restriction on average EBV was set to 0.516, the average kinship REF scenario (0.926). Similar results were obtained for Vorderwald cattle. When the upper bound for REF scenario (0.852).Table minfC.A and minfC.A.MC. After adding an EBV constraint of 0.516, REF scenario (0.527). For Vorderwald cattle, the minimum average minfC.A.MC.EBV, REF scenario (0.380).Results when minimizing REF scenario (0.049). For Vorderwald cattle, when REF scenario (0.072).When the kinship at native alleles, es Table ; in AnglminMC.A.EBV increased to 0.622 and REF scenario (0.722). Results were similar for Vorderwald cattle. The minimum MC achieved in the next generation was 0.527 when the upper bound for REF scenario (0.605).Table REF scenario achieved the lowest average EBV in the offspring generation. This value was not zero because male and female selection candidates had different mean EBV. For Angler cattle, scenario maxEBV.A achieved a higher EBV (1.226 vs. 1.184) than the TS scenario, although the average kinship REF scenario (0.211). Similar results were observed for the Vorderwald cattle population. Scenario maxEBV.A achieved a similar EBV as the TS scenario (1.164 vs. 1.161) but the average kinship REF scenario (0.287).Results for maximizing the average EBV in the progeny generation under various constraints are in Table The number of selected sires with non-zero genetic contributions was calculated in each scenario, as well as the standard deviation of the genetic contribution of all male selection candidates. Among all scenarios, TS selected the smallest number of sires. Adding a constraint on For the breeding schemes of the two breeds considered in this study, two conflicts must be addressed: (1) the conflict between increasing genetic gain while managing inbreeding and (2) the conflict between maintaining genetic diversity while controlling loss of genetic uniqueness. The purpose of this study was to determine whether OCS with additional constraints that involve modified kinship matrices and MC was more efficient at conserving genetic diversity and originality while also ensuring genetic improvement than traditional OCS. Using data on German Angler and Vorderwald cattle, various scenarios were compared. Both breeds have been frequently crossed with high-yielding breeds to improve performance. We found that diversity of native alleles decreased more rapidly in Vorderwald cattle than in Angler cattle, whereas MC was higher in Angler cattle. The consequences of the scenarios were similar for both breeds. Compared to traditional OCS, constraining kinship maxEBV.A. Compared to the TS scenario, average EBV was higher in the traditional OCS scenario for both breeds, while the average relatedness was lower. Probably, the average EBV in TS was smaller because the TS scenario assumed equal contributions for selected sires, whereas OCS optimizes their contributions. Because MC and EBV were positively correlated, traditional OCS increased the average MC, which is undesirable when the aim is to conserve the genetic originality of local breeds.Traditional OCS achieved the highest average EBV in the progeny generation among all scenarios with a restriction on rate of inbreeding, which, in our study, is represented by scenario Both n et al.  reportedAmong all the scenarios, TS used the smallest number of sires and resulted in the highest average genetic contribution of selected sires. Including kinship entclass1pt{minimaScenarios with optimizations of both male and female contributions were also evaluated (results not shown), using the same calculation methods to obtain the constraint value settings. For all scenarios and both breeds, the constraint settings were stricter than in the scenarios that optimized male contributions. The performance of all scenarios improved when both male and female selection were optimized, which is consistent with Sánchez-Molano et al. , who useREF scenario when MC was constrained or minimized. Hence, the individuals with the lowest MC may not carry some native alleles that are still present in individuals with higher MC. Thus, in this case, constraining Previous OCS approaches for maximizing genetic gain while limiting rate of inbreeding did not consider MC. Introgression of migrant breed alleles must be managed to maintain genetic uniqueness and conserve local breeds. As expected, the average EBV obtained with and without MC as a constraint showed that controlling MC restricts increases in genetic gain. Interestingly, kinship at native alleles increased compared to the However, maximum genetic gains can only be achieved by allowing for the introgression of foreign genetic material. Therefore, the two main purposes in a breeding program, i.e. conserving local breeds and improving genetic gain, are contradictory and must be balanced by the breeding organization. In this study, we set the proportion of breeding progress to be achieved at Because of advances in molecular genetics, genome-wide dense marker genotype data are increasingly available, even for some endangered breeds and have shown promise in capturing genetic variation due to Mendelian sampling . The appMaintaining genetic originality is essential for conserving local breeds. It was shown that using an OCS approach as developed in this study can effectively maintain the diversity of native alleles and genetic originality, while ensuring genetic improvement. The most promising approach involved the inclusion of additional constraints for migrant contributions and kinship at native alleles Additional file 1: Table S1. Threshold settings for all parameters for the Angler and Vorderwald populations.Additional file 2: Table S2. Correlation between OC and EBV and between OC and MC.


Secale cereale L.) is a cereal crop of major importance in many parts of Europe and rye breeders are presently very concerned with the restrict pool of rye genetic resources available. Such narrowing of rye genetic diversity results from the presence of “Petkus” pool in most modern rye varieties as well as “Petkus” × “Carsten” heterotic pool in hybrid rye breeding programs. Previous studies on rye's genetic diversity revealed moreover a common genetic background on landraces (ex situ) and cultivars, regardless of breeding level or geographical origin. Thus evaluation of in situ populations is of utmost importance to unveil “on farm” diversity, which is largely undervalued. Here, we perform the first comprehensive assessment of rye's genetic diversity and population structuring using cultivars, ex situ landraces along a comprehensive sampling of in situ accessions from Portugal, through a molecular-directed analysis using SSRs markers. Rye genetic diversity and population structure analysis does not present any geographical trend but disclosed marked differences between genetic backgrounds of in situ accessions and those of cultivars/ex situ collections. Such genetic distinctiveness of in situ accessions highlights their unexplored potential as new genetic resources, which can be used to boost rye breeding strategies and the production of new varieties. Overall, our study successfully demonstrates the high prospective impact of comparing genetic diversity and structure of cultivars, ex situ, and in situ samples in ascertaining the status of plant genetic resources (PGR).Rye ( Secale cereale L.) belongs to the Triticeae tribe, along with other economically important cereals such as wheat and barley. Much controversy about the taxonomy of the Secale genus remains, despite the large number of studies performed , Secale comprises four species, namely S. cereale L., S. sylvestre Host, S. vavilovii Grossh., and S. strictum (Presl.) Presl. (syn. S. montanum Guss). Within S. cereale, eight subspecies are recognized: afghanicum (Vavilov) K. Hammer, ancestrale Zhuk., dighoricum Vavilov, forma unranked rigidum Antropov and Antropova, segetale Zhuk., tetraploidum Kobyl, tsitsinii Kobyl and cereale L. (the only cultivated). Within S. strictum, there are five subspecies: africanum (Stapf) K. Hammer, anatolicum (Boiss.) K. Hammer, ciliatoglume (Boiss.) K. Hammer, kuprijanovii (Grossh.) K. Hammer and strictum .Rye  as well as through the introgression of rye chromatin in wheat varieties, particularly by the short arm of chromosome 1R (1RS), as a source of genes for agronomic and resistant improvement  conservation, it has long been recognized that effective strategies need to integrate in situ and ex situ approaches  landraces conserved ex situ and in situ indicated significant genetic differentiation in ex situ subpopulations as well as loss of alleles, gain of new alleles, and reduction of rare alleles with an increase of common alleles  and cultivars, which is indicative of a common genetic background, regardless of breeding level or geographical origin  were selected from different geographic regions. The panel consist of eight ex situ accessions from gene banks, nine cultivars , along with 11 in situ accessions collected “on farm” in 2014 from Northeast Portugal  and Nordic Genetic Resource Center . Rye accessions were grouped and mentioned hereforth as cultivars, ex situ, and in situ/on farm accessions. Cultivars are those resulting from modern rye breeding, in situ are collections held in farmers' fields and ex situ are those collections detained in gene banks or botanical gardens. Both ex situ and in situ accessions collectively will be referred as landraces. The 11 in situ Portuguese accessions were collected from local farms on Northern region , while for Portuguese in situ collections 16 individuals were used, for accounting within-population diversity. Genomic DNA (gDNA) was extracted from young leaves using the cetyltrimethylammonium bromide (CTAB) procedure adapted from Thomas et al. . These values were computed, as described in Chapuis and Estoup (Na), private alleles, inbreeding coefficient , observed (HO), and expected (HE) heterozygosity. Deviations from Hardy–Weinberg equilibrium (HWE) were assessed for each locus-population combination and linkage disequilibrium (LD) to determine the extent of distortion from independent segregation of loci using GenePop v4.5 ] by applying a sequential Bonferroni correction  and FST-values using the ENA method ) matrixes were obtained in GenoDive 2.0b27 , and the mean correlation coefficient (RXY) over 999 random permutations as implemented in GenALEx 6.5.To detect isolation-by-distance (IBD) effects, Population structure was addressed using three approaches: (i) estimating relations among populations using genetic distances; (ii) hierarchical genetic analysis by AMOVA; and (iii) individual-based clustering.DC, Cavalli-Sforza and Edwards, DCINA), and Nei's distance  was characterized using Analysis of Molecular Variance  as groups, and the second, narrowed to landraces using ex situ and in situ accessions alone. In each AMOVA, the total variance was partitioned into components to account for differences between two defined groups , differences among populations within those groups (Vb), differences among individuals within populations (Vc). Variance components  were used to calculate the fixation indices  according to Weir and Cockerham  scores, maintaining default parameters and retaining all principal components (PCs). Cross validation using the xvalDapc function was pursued to determine the optimal number of PCs to retain in the Discriminant Analysis (DA).DAPC was implemented in R using Jombart, . The funp < 0.05); while after sequential Bonferroni correction only four loci  displayed significant deviations, matching 11 of the 406 locus-population combinations (Supplementary Table F) estimates were observed across several loci, exceptions for the EST-SSRs SCM152 (0.060), and SCM63 (0.017) and in the gSSR SCM39  accessions, results showed that in landraces the mean allele number is higher with 4.4 in all SSRs, 4.8 with gSSRs, and 4.4 in EST-SSRs, against the values in cultivars , but similar values to those obtained for cultivars.Overall, a total of 122 alleles were detected in the 285 individuals analyzed Table  being nogSSRs = 0.73) than in EST-SSRs (PICEST-SSRs = 0.59). Similar PIC-values were observed in cultivars, ranging from 0.366 (SCM63) to 0.695 (SCM138), and in landrace accessions . In both cultivars and landraces, gSSRs revealed to be more polymorphic than EST-SSRs. In our 14-loci dataset, observed heterozygosity (HO) varied from 0.51 (SCM166) to 0.85 (SCM86) with a mean of 0.68  compared to the expressed loci .Overall, Polymorphic Information Content (PIC) values ranged from 0.39 (SCM66) to 0.85 (SCM138) with a mean value of 0.67 Table . Average68 Table ; and expF  exhibits values from −1 to +1. Values close to zero are expected under random mating, while substantial positive values indicate inbreeding or undetected null alleles. Negative values denote excess of heterozygosity, due to negative assortative mating, or selection for heterozygotes. Overall, negative F-values were observed across most accessions (Table F was encountered in six Portuguese in situ accessions (SECCE1–SECCE6), ranging from 0.002 (SECCE5) to 0.049  was used as a measure of the extent of genetic differentiation among subpopulations, with values ranging from 0 (no differentiation) to 1 (high differentiation). Mean pairwise FST was 0.136  indicating an overall low level of population's differentiation (Supplementary Table FST < 0.250), while the remaining 35 pairwise populations displayed high genetic differentiation (pairwise FST ranged from 0.251 to 0.418). The presence of null alleles has not caused a significant overestimation of the level of population differentiation, as low population differentiation was also depicted with S. strictum), with 747 pairwise populations showing low to moderate genetic differentiation  effects were addressed. D and DCINA genetic distances  without any link with other rye accessions and the other Portuguese accessions placed within the same clade as cultivars and ex situ accessions.UPGMA and NJ trees were built using Nei's s Figure , displayD distance, Figure DCINA Supplementary Figure Conversely to UPGMA-derived dendograms, the two genetic distance algorithms produced very dissimilar NJ-generated trees (Nei's ex situ and in situ accessions), AMOVA results showed that molecular variation was mainly (86.84%) found within accessions, whereas variation among accessions within groups explained 8.86% and variance among groups represents only 4.30% of the total genetic variability , rather than among groups (2.86%) or within groups (7.49%). In both cases, a high molecular variation was found within accessions, as expected for a cross-pollinated species, as previously detected in other rye studies , determined K = 2 as the most likely model, following Evanno et al.  = L(K) for each value of K (data not shown). Particularly, K-models above 10 revealed high standard deviations of log-likelihood along low values following ΔK ad hoc statistics, thus showing that these cluster assignment are not reliable to describe our dataset. Though, this exploratory STRUCTURE results prompted us to subsequently constraint runs to 10 possible clusters (K = 1–10). This analysis assigned K = 2 as the optimal number of groups based on ΔK, with K = 3 also displaying high ΔK-values , along with the ex situ collections; whereas in situ are grouped essentially in a second cluster (pink cluster) together with S. strictum , excluding “Sved” and “Riodeva” grouped in a different cluster , as in K = 2, with few or even no admixture. In situ populations SECCE7 to SECCE11 are assembled in the green cluster (C2), together with “Sved” and “Riodeva” ex situ accessions, with the occurrence of other admixed in situ accessions (SECCE1–SECCE6), which belong to the blue cluster , thus reflecting a common genetic diversity with the rye crop wild relative that might be linked to preservation of an ancient diversity resulting from the low diversification rate of Portuguese accessions.Exploratory STRUCTURE run considering the biggest range of clusters conceivable  score was selected, predicting a K = 3  and 2 Discriminant Functions (explaining 92.9% of cumulative variance), for inferring the 3 genetic clusters. When displaying loading plots from both discriminant functions, one can determine which variables  contributed the most for the three-clustering assembly. As such, alleles 177 (SCM138), 369 (SCM152), 245 (SCM166) from DF1, and alleles 147 and 150 from SCM164 together with 182 (SCM75), 171 (SCM138), and 194 (SCM166) from DF2 are responsible for most of the genetic variation explaining the three genetic cluster assignment . Analysis of K = 2 from DAPC , capturing 60% of cumulative variance, and with only 1 Discriminant Function for describing the 2 genetic clusters (data not shown). Following this cluster assignment, two different genetic backgrounds of Portuguese in situ accessions can be depicted: one group (SECCE7–SECCE11) that shares allelic diversity with cultivars and ex situ material in the blue cluster, and other group (SECCE1–SECCE6) grouped with S. strictum in the pink cluster, thus showing a common genetic diversity background with wild rye  rye's genetic diversity do not follow a geographical/spatial trend (ii) in situ accessions display similar genetic diversity than cultivars/ex situ collections yet with a different genetic background and (iii) exists an unexplored genetic diversity on in situ accessions which can represent an effective alternative to increment rye heterotic pool in future breeding programs.In this study, 28 rye accessions screened with SSRs included nine international rye cultivars, eight worldwide O) obtained in our study  with a mean of 0.68, is higher than those reported for 9 Latvian rye accessions using 9 genomic loci  to 0.84 (SCM9). Furthermore, a recent study using 32 SSRs in 14 rye accessions  likely due to a higher number of rye populations screened (n = 29).Twenty-nine rye accessions were first genotyped with 16 SSRs, and after SSRs quality assessment 14 markers were further used for subsequent genetic diversity analysis .PIC-values obtained for SSRs used were high (average PIC = 0.67) which indicates their high informativeness, which was predictable since the SSRs markers were not selected randomly, but based on the previous performance analyses . Overall, cultivars displayed lower mean allele number 3) than landraces (4), but similar genetic diversity was observed as denoted by levels of observed heterozygosity  than in situ (5) although showing similar heterozygosity  suggests a high gene flow between accessions, which may be attributed to the wind-pollinated reproduction of rye allied with its outcrossing habit. One can therefore depict that rye-breeding system plays an important role in driving the generally observed high diversity within-accessions. Outcrossing plant species tend to have higher genetic variation within-populations, whereas selfing species or species with a mixed mating system are often genetically less variable  were expected, which is in agreement with a recent rye study using SNPs , which may be suggestive of high gene flow between in situ populations from Northeast Portugal and/or an evidence of a single recent genetic source.AMOVA showed that the majority of the genetic diversity lies within rye accessions with only little additional diversity present among groups or within groups. The large proportion of diversity found within accessions for the two types of groupings  or not (K = 3). Population structuring of cultivars with ex situ collections is further reinforced by the UPGMA analysis, which also highlights a highly supported clade consisting of in situ collections with the rye CWR. Altogether, our data provides evidences of cultivars and ex situ collections harboring a different genetic diversity in contrast to in situ accessions. As ex situ accessions offers a static genetic snapshot, reflecting a population's adaptation to environmental conditions where they were collected, and considering that cultivars are a result of a controlled breeding process, a common gene pool can be depicted between cultivars and ex situ accessions here analyzed from eight European regions. Particularly, in situ populations screened in our study are originated from the region of excellence for rye production in Portugal, with diversity being preserved under farmer management. A recent study using dominant markers in three regional populations from the Northern Portugal shows the clustering of Portuguese populations in a different set than other rye cultivars, i.e., “Imperial,” “Dankowskie Zlote,” and the Portuguese cultivar “Alvão”  analysis. Interestingly, one cultivar (“Kungs II”) and one ex situ accession (“R780Spain”) originated from similar geographical regions as “Sved” and “Riodeva,” respectively, displayed a distinct genetic diversity from the former accessions and with the Portuguese in situ populations, thus highlighting the genetic distinctiveness of such rye accessions.Northeast Portuguese landraces have always been of great importance for local farmers, yet few studies have been performed for addressing its genetic diversity and population structure  along with rye CWR, highlighting a hidden diversity on Portuguese rye gene pool yet to be uncovered.“Sved” accession is a Swedish rye landrace that clustered with another accession  in a way distinct from all other Scandinavian and European rye landraces  constitute panmictic populations harboring high levels of genetic variation in their genetic build-up , Agenda 21, and the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), emphasizing the importance of on farm conservation as an essential component of sustainable agriculture. By assessing in situ genetic diversity one can determine which landraces may hold “new” genetic variation that could be useful to supplement crop cultivars diversity, in traits with agronomical importance . Such genetic diversity is generally concentrated in centers of diversity as well as on farm conditions since landraces structure and dynamics result from both natural and human selection (Gepts, Conservation of PGR through n Gepts, . Such riin situ genetic reserves and, besides ex situ conservation at national and international gene banks, an inventory periodically updated should be pursued to monitor on farm maintenance of landrace diversity.On farm conservation of local landraces, particularly those found in our study, reflects a specific case, since we studied landraces not listed at both national and international gene banks. As such, we believe that the first intervention toward a conservation protocol is to implement a new national seed policy on landraces, which usually favors only varieties that are distinct, uniform and stable, discouraging the use of more heterogeneous, variable landraces. In agricultural systems as the one practiced in Northern Portugal rye fields, farmers typically save seed from one season to the next and may share seed with other farmers, being seed sourcing embedded in well-structured traditional systems with rules and expectations based on family and local social networks (Veteläinen et al., ex situ and in situ samples, along rye cultivars thus highlighting in situ collections from Northeast Portugal as new genetic resources being distinct genotypes to those reported for rye ex situ and cultivars. Identification of alleles/genes underlying such distinctive diversity would be of utmost importance for determining their usefulness for incorporating future rye breeding programs and to additionally propose on farm conservation policies at national level.Overall, our study successfully illustrates the significance of comparing the genetic diversity and structure of FM, AM, HO, and WV designed the research. FM carried out molecular work. FM, PV, and AB analyzed the data. FM wrote the manuscript and all authors improved upon versions. All authors read and approved the final manuscript.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.


We estimated genetic diversity and population demography by analyzing the ancient mitochondrial DNA sequences alongside those from modern giant pandas, as well as from other members of the bear family (Ursidae). Phylogenetic analyses show that one of the ancient haplotypes is sister to all sampled modern pandas and the second ancient individual is nested among the modern haplotypes, suggesting that genetic diversity may indeed have been higher earlier during the Holocene. Bayesian skyline plot analysis supports this view and indicates a slight decline in female effective population size starting around 6000 years B.P., followed by a recovery around 2000 years ago. Therefore, while the genetic diversity of the giant panda has been affected by recent habitat contraction, it still harbors substantial genetic diversity. Moreover, while its still low population numbers require continued conservation efforts, there seem to be no immediate threats from the perspective of genetic evolutionary potential.The giant panda was widely distributed in China and south-eastern Asia during the middle to late Pleistocene, prior to its habitat becoming rapidly reduced in the Holocene. While conservation reserves have been established and population numbers of the giant panda have recently increased, the interpretation of its genetic diversity remains controversial. Previous analyses, surprisingly, have indicated relatively high levels of genetic diversity raising issues concerning the efficiency and usefulness of reintroducing individuals from captive populations. However, due to a lack of DNA data from fossil specimens, it is unknown whether genetic diversity was even higher prior to the most recent population decline. We amplified complete cyt Ailuropoda melanoleuca) is an iconic species for world wildlife conservation. Fossil findings indicate that the ancestors of this species probably originated in the late Miocene in the south-west of China, expanded their habitat range during the early Pleistocene, and began to contract in habitat range during the late Pleistocene [The giant panda  and human activities  have played important roles in decreasing the genetic diversity and population size of megafauna ,20,21,22Several previous studies have examined the genetic diversity and relationships of extant giant panda populations using both mitochondrial and nuclear data. Early studies using proteins , the mitb) and 12s rRNA genes, partial 16s rRNA and ND1 genes, and the control region (CR) from two middle Holocene giant panda specimens collected from south-western China. We use these data to investigate the genetic diversity and demographic history of giant panda populations, and discuss the mechanisms behind the giant panda’s range contraction and population size reduction.Although a recent study confirmed the potential for genetic data recovery from ancient giant panda remains , ancientElephas maximus, Bos gaurus, Dicerorhinus sumatraensis, Equus yunnanesis, Cervus unicolor, and several other species [Two samples of Holocene sub-fossil giant pandas  were collected from a natural sinkhole, 41–60 m deep from the surface, at Jiangdong Hill, Tengchong County in Yunnan Province, south-western China . The sam species ,35,36,37E. coli DH5α. White transformants obtained from LB plates were screened by PCR with the M13 primer pair. For each fragment, a minimum of eight clones, four from each of two independent primary amplifications, were sequenced at Nanjing Genscript Ltd. Company on an ABI 3700 sequencer. When consistent differences were found between the two independent amplifications due to sequence errors, most likely resulting from template damage, a third amplification was performed to determine which sequence was reproducible [Ancient DNA extractions and polymerase chain reactions (PCRs) were set up in an ancient DNA facility at China University of Geosciences (Wuhan), in a building physically separated from post-PCR facilities. Ancient DNA was extracted from approximately 250 mg bone powder following a silica method optimized for ancient DNA extraction . Extractoducible .Parts of the ancient DNA sequences were replicated at the Australian Centre for Ancient DNA (ACAD) at the University of Adelaide .https://blast.ncbi.nlm.nih.gov/Blast.cgi, default settings) in order to validate the data. The newly determined DNA sequences were deposited in GenBank .Each fragment obtained was compared to the sequences available in GenBank using NCBI’s online Nucleotide BLAST  as an outgroup for sequence alignment and further phylogenetic analyses , accession number SRA053353 . We usedanalyses .ingressus, spelaeus and eremus, which may represent different subspecies or species [We subsampled four species or species groups from the aforementioned 254 individuals, which were giant panda, brown bear, Late Pleistocene cave bears, and polar bear, that had a sufficient number of sequences sampled across their respective geographic distributions to compare their genetic diversity. The cave bear sample included sequences assigned to the taxa  species . It shouCanis lupus familiaris served as an outgroup to root the tree. The D-loop sequences were not used for this analysis due to the possibility of saturation and ambiguous alignment of divergent intergeneric sequences. Optimal alignment partitions were selected from all possible combinations of 12s, 16s and the individual codon positions of the protein coding cytb and ND1 genes under the Bayesian Information Criterion using PartitionFinder v1.1.1 [Phylogenetic relationships among the 54 sampled giant panda sequences, as well as their position within the larger Ursid phylogeny, were inferred by maximum likelihood in RAxML-HPC v.8  on the Cr v1.1.1  with theTo further compare ancient giant panda and previously analyzed extant giant panda haplotypes, we aligned our newly obtained D-loop sequences against the 655 bp D-loop data set from previous studies ,30. We uChanges in female effective population size over the timespan of the mitochondrial phylogeny were inferred by Bayesian skyline plot analysis  in BEASTb and ND1 gene sequences was estimated by aligning sequences from seven living and two extinct representatives of the Ursidae to the sequence of a modern giant panda and computing a time-calibrated phylogeny. Four node calibration priors based on fossils and other evidence were applied, following the approach used in a recent investigation of the Ursidae [A uniform prior on the basal divergence of the Ursidae with an upper limit of 12 million years, representing the age of a well-documented fossil representative of the Ailuropodinae, and a lower limit of 20 million years based on a previous molecular dating study.Plionarctos.A uniform prior on the divergence of the Tremarctinae and Ursinae clades of 7 to 14 million years, based on the fossil Tremarctine bear Ursus minimus.A uniform prior on the basal divergence of the Ursinae of 4.3 to 6 million years, based on reported ages of A uniform prior on the divergence of brown and polar bears of 0.48 to 1.1 million years, based on previous studies. This prior was applied to the common ancestor of a polar bear and a Finnish brown bear, likely representing the initial divergence of these respective species, rather than more recent gene flow events.The substitution rate for the panda lineage averaged across the 12s, 16s, cyt Ursidae . The mon Ursidae  and refeAiluropoda lineage was extracted from the posterior sample of trees using TreeStat v1.8.2 and assessed in Tracer.Optimal partitions and substitution models were selected as described for the maximum likelihood phylogenetic analysis, considering all substitution models available in BEAUti v.1.8.2 (part of the BEAST v1.8.2 distribution ). A lognAiluropoda lineage estimated by the calibrated Ursidae analysis, optimal substitution models were selected for the partitions selected for that analysis with the addition of the D-loop as a separate partition. A single relaxed-clock model was then used for the 12s, 16s, cytb and ND1 partitions, with a normal prior applied to the per lineage substitution rate approximating the posterior estimate generated by the calibrated Ursidae analysis. A separate, unlinked relaxed-clock model was specified for the D-loop, with the substitution rate estimated within an uninformative, uniform prior between 0 and 2 × 10−7 substitutions per site per year. Default exponential priors were used for the ucld.stdev parameters of both relaxed-clock models. The MCMC chain was run as described previously, and the maximum clade credibility tree was extracted and annotated with relevant summary statistics using TreeAnnotator, with node heights scaled to the median of the posterior sample. The Bayesian skyline reconstruction was generated in Tracer.Demographic analysis of the 54 sampled giant panda sequences was carried out using a piecewise constant coalescent Bayesian Skyline model with 10 groups. The giant panda D-loop sequences were included in this analysis as they could be aligned unambiguously and substantial saturation is unlikely at the population level. As we failed to recover several sections of the D-loop from the ancient samples, all homologous positions of the D-loop alignment containing missing data were removed. To facilitate the application of the substitution rate for the b (1140 bp) and 12s rRNA (966 bp) genes, partial 16s rRNA , and partial ND1  genes, and D-loop sequences (1052 bp for both specimens) (We amplified 67 overlapping fragments (size range was 53–159 bp excluding primers) from the two Holocene samples. The fragments were used to build the complete cytecimens) . IdenticAverage pairwise substitutions for brown bear haplotypes were highly variable and included by far the highest diversity values observed for any group. Late Pleistocene European cave bears, despite covering several tens of thousands of years, showed less variance in mean substitutions across replicates, with values falling within those observed for brown bears. Polar bear haplotypes were highly similar with little apparent variation introduced by sampling. Diversity estimates for giant panda haplotypes were generally lower than observed for brown bears, always lower than observed for Late Pleistocene European cave bears, but always higher than observed for polar bears .b, and ND1 sequences. The optimal data partitions selected by PartitionFinder are shown in Ailuropoda mitochondrial lineage as sister to all other sampled ursid lineages with strong bootstrap support  retrieved a network profile of 42 haplotypes GH 1–42, , with thAiluropoda substitution rate averaged across the 12s, 16s, cytb and ND1 genes, with a median value of 3.3954 × 10−8 substitutions/site/year. Based on this result we applied a normal prior on the per lineage substitution with a mean value of 3.3954 × 10−8 substitutions/site/year and standard deviation of 7.705 × 10−9 substitutions/site/year to the corresponding partitions in order to calibrate the Bayesian skyline analysis.Partitions and substitution models used in the BEAST analyses are shown in Congruent with the maximum likelihood analysis, the maximum clade credibility tree generated by Bayesian skyline analysis returned the sequence of the ancient sample 97001 as sister to all other sampled panda haplotypes with a posterior clade probability 0.72 . Many reThe applied tip and substitution-rate calibrations returned a median coalescence time for all sampled haplotypes of ~62,000 years . The median estimated coalescence time for sampled modern haplotypes is ~39,000 years . The Bayesian skyline reconstruction showed a decline in female effective population size around 6000 years ago, followed by population recovery during the last 2000 years .Large-scale environmental alteration due to influences such as climate change or human activities has caused many formerly widespread species to survive in small fractions of their original range. These range contractions have often been accompanied by a loss of intraspecific genetic diversity due to the reduction of effective population size and the extinction of regional genetic lineages . Giant pb, and ND1 fragments (4054 bp)) to be 36. However, this increases to 71 when individual No. 97001 is also included. The giant panda probably disappeared from Yunnan Province approximately 5000 years ago as the dates of our samples represent the latest record of its appearance in this area. The extinction of this population appears to have resulted in a loss of distinct mitochondrial lineages as seen in other species [The mid-Holocene giant panda analysed in this study represent two new haplotypes in the network profile based on a 655 bp D-loop dataset . While o species ,33. A lo species ,67. In c species . However species , but wit species , one shoHowever, despite the deep divergence of one of the fossil individuals and although neither of the two ancient haplotypes have been found in modern giant panda populations, the high haplotype diversity observed suggests that the range losses for giant pandas, such as the local extinction at the sampling site in this study, and recent reduction in wild population size have not resulted in genetic impoverishment during the past 8000 years. Historical distribution data show that as recent as the sixteenth to nineteenth centuries, giant pandas were still widely distributed in south-western China, including Hunan, Hubei, Sichuan, Shaanxi, and Gansu Provinces ,72. ThisAlthough modern giant panda populations display moderate genetic diversity, a previous study suggested that the giant panda population experienced two recent bottlenecks at ~0.2 million years and ~20,000 years ago, respectively . HoweverBoth global climate change and human activities have been cited as key factors shaping giant panda population demography ,74. The 13C time series of the C. mulieensis remains and mixed plant cellulose of the Hongyuan peat in south-western China reflects that the Indian Ocean summer monsoon precipitation declined during 4200–4000 years B.P. [18O record of the Dunde ice core from Qilian Mountain in western China suggested that the lowest temperature was detected in that area in the same period. This period was also the time when Dayu regulated floodwater in southern China according to historical records, suggesting high precipitation during this cold period [The reduction in female effective population size from 6000 to 2000 years B.P. in our skyline plot does coincide with the appearance of a cold period in south-western China . It has ars B.P. . The δ18d period . The nord period . At LianWhile it seems that the giant panda individuals in this study fell into the sinkhole while the area was still richly forested, the fact that their bones had been washed into their positions, and that the soil deposits in the floor in the sinkhole revealed flooding events and soil erosion, could suggest local deforestation by humans . Later oThe mitochondrial sequence data set used here has already provided clues with regard to population size and genetic diversity through time. Larger sequence datasets, such as the whole genome sequencing of Holocene giant panda individuals, will likely allow further identification of the extent of genetic diversity loss and hopefully reveal key factors involved in the contraction of giant panda population and range sizes.