Cinnamomum kanehirae Hayata and C. camphora (L.) Presl are important tree species in eastern Asia. The wood of C. kanehirae is in increasing demand for culturing Antrodia cinnamomea, a medicinal fungus that naturally grows inside the trunk of C. kanehirae. Putative hybrids between C. kanehirae and C. camphora were previously reported but with no scientific evidence, leading to confusion or misplanting. First, to identify the female parent of putative hybrids, the maternal inheritance InDel (insertion/deletion) markers were developed by using low-coverage sequencing. SNPs were developed by using genotyping-by-sequencing (GBS) approach in C. kanehirae, C. camphora and putative hybrids. The results indicated that the female parent of the studied hybrids was C. camphora. Eight hundred and forty of the 529,006 high-density SNPs were selected and used for analysis. Hybrids were classified as F1 (C. kanehirae × C. camphora), F2 and backcrosses. Hybridization has occurred in the human-developed area of eastern and southwestern Taiwan, and the introgression was bidirectional. For producing pure wood, buffering zones should be established around seed orchards to avoid cross-species pollination and to preserve the genetic purity of C. kanehirae. The DNA markers developed in this study will also be valuable for further wood identification, breeding and evolutionary research. Introgression may also occur when F1 hybrids backcross to one or both parental species, leading to foreign genetic material being integrated into the parent genome2. Diversification and speciation may also be caused by hybridization and introgression if offspring undergo reproductive isolation or have increased fitness. Moreover, hybridization may affect growth vigor, and as a consequence, the wood quality may change, with negative effects on parasitic species growing in the wood3. In the past, the morphological characteristics have been the only method available for classifying hybrids; however, morphological characteristics alone may not be sufficient to identify hybrids. Therefore, molecular markers provide a good tool for estimating the gene flow and genetic structure of hybrids4. Since the inheritance modes of the chloroplast/mitochondria and nuclear DNA are distinct, a combination of nuclear and chloroplast/mitochondria markers can be used to understand the complex mode of gene flow in hybrids. For instance, maternally inherited chloroplast/mitochondrial DNA markers can be used to assess the maternal parent of the hybrids and the direction of hybrid mating2.Hybridization occurs when mating between individuals from interspecific species generates viable offspring, and this process is estimated to occur in approximately 25% of all plant species5. This approach can be generalized for any plant species at a low per-sample cost, providing an incredible number of markers. GBS can be applied for genotyping the variance of individuals and is also suitable for population studies, germplasm characterization and assisted breeding in diverse organisms. InDel and SNP (single nucleotide polymorphism) markers have received increasing attention due to their many advantages, such as high polymorphism, codominance, high stability and high density. Recently, InDel and SNP markers have been used for genetic and evolutionary studies in many plant species8.Genotyping-by-sequencing (GBS) is a direct sequencing approach based on the high-throughput next-generation sequencing (NGS) of genomic subsets targeted by restriction enzymesCinnamomum is one genus in the Lauraceae family, and it is composed of approximately 250 species growing in tropical and subtropical regions in eastern Asia, Australia and the Pacific islands10. According to Huang11, there are twelve native Cinnamomum species in Taiwan. C. kanehirae is an endemic and very valuable tree species in Taiwan. Its wood is in high demand due to its unique usage for culturing the naturally parasitized fungus Antrodia cinnamomea Chang and Chou. This fungus specifically parasitizes inside of the C. kanehirae wood and has been used as a Chinese medicine known to have many pharmacological effects, with prices above 10,000 US$ per kilogram14. However, C. kanehirae wood could be wrongly identified by using only morphological methods. We previously successfully identified the wood of C. kanehirae and C. micranthum by using chloroplast and chemical markers6. Recently, it was reported that some C. kanehirae seedlings may actually be hybrids with other Cinnamomum species16. It has been reported that putative hybrids are found, though rarely, in non-natural habitats such as nurseries, seed orchards and gardens. These hybrids can cause confusion for growers, and thus, a reliable method of hybrid identification is needed. The metabolite profiles and pharmacological effects of A. cinnamomea cultivated in the C. kanehirae wood are completely different by using different wood species substrates13. In addition, according to Taiwanese regulations, all A. cinnamomea products must be obviously labeled with the culturing methods and produce processing on the package. Even in China, all of the A. cinnamomea medicine must be cultured on C. kanehirae wood. Therefore, it is very important to identify the correct C. kanehirae species and clearly differentiate it from its hybrids. However, we speculate that some morphological traits of hybrids are different from those of either C. kanehirae or C. camphora. Even the metabolite profile of A. cinnamomea cultured by using hybrid wood may be different from that using genuine C. kanehirae wood. In the aspect of management, the putative hybrids will lead to worrisome issues that may spoil pure C. kanehirae and affect the genetic pool of the natural populations of C. camphora and C. kanehirae. Moreover, hybridization may lead to genetic swamping, where hybrids overwhelm or outcompete the rarer parent species17. To avoid the loss of C. kanehirae genetic diversity and the misuse of hybrid woods, it is very important to further understand hybrids between these two species in the wild.Taiwan is located in the middle of the western Pacific Ocean and has very high biodiversity. Due to its geographical isolation, hundreds of plant species are endemic to Taiwan. C. kanehirae has improved with the recent release of the complete chloroplast genome and whole-genome sequencing data19. In the present paper, we applied GBS and a genome reference-based method for calling SNP data in 75 individuals, namely, 19 C. kanehirae, 31 C. camphora and 25 putative hybrids, in order to prove the occurrence of hybridization as well as the frequency and direction of hybridization. We also developed and validated the InDel markers by using low-coverage sequencing of interspecies and the GATK  pipeline to determine which species is the putative donor parent in cases of hybridization.Genomic research of C. kanehirae and C. camphora. The genetic analysis of these two Cinnamomum species and hybrids could provide evidence for taxonomy, practical molecular biomarkers for wood identification and recommendations for wild resource management in the future.This is the first report of SNP genotyping in Cinnamomum species: C. kanehirae (S99), C. camphora (JLH2) and C. camphora (JZS). Statistical data from this low-coverage sequencing are available in Supplementary Table C. kanehirae, we obtained the greatest number of clean bases after trimming. In total, we found 481 InDels that satisfied our filtering parameters. We validated the five longest InDels in three data sets. Finally, 7 out of 15 validated InDels (46.67%) were polymorphic in C. kanehirae and C. camphora. Among the 7 InDels, 1 had blastn matches to nuclear mRNA, 2 had blastn matches to mitochondrial sequence, 1 had blastn matches to chloroplast sequence, and 3 had no blastn matches  and the other DNA fragment is identical to C. camphora (227 bp)   and NW  populations. The genotyping of C. kanehirae and ESW C. camphora are identical (124 bp) but different from that of NW C. camphora (212 bp). This is in accordance with other studies, which observed both kinds of chloroplast sequences in C. camphora (NCBI accession numbers: MF421523 and MG021326).Low-coverage sequencing was performed in three individuals of two C. kanehirae, C. camphora and putative hybrids were 0.36–0.72, 0.39–0.95 and 0.33–0.54, respectively. Since the site of missing data could be relative to InDel or low-quality sequencing, we trimmed off the sites with missing data in all of the samples. Finally, 840 SNPs were obtained for further analysis 20. This may be due to genotyping errors, inbreeding, selection and population stratification. In genomic studies, the SNPs that are not consistent with Hardy–Weinberg equilibrium need to be removed from further analysis21. Thus, 32 SNPs were removed, and 808 SNPs were used for further analysis. The expected heterozygosity (He) was estimated in each group: 0.084 ± 0.005 (C. kanehirae), 0.076 ± 0.005 (ESW C. camphora), 0.123 ± 0.006 (NW C. camphora) and 0.143 ± 0.007 (putative hybrids). The Fis (inbreeding coefficient) values were 0.102, − 0.119, 0.118 and − 0.099 in C. kanehirae, ESW C. camphora, NW C. camphora and putative hybrids, respectively  and C. camphora (covered with a powder-like white wax). The individual putative hybrids are seedlings of fruits from the C. kanehirae. Thus, we could infer based on morphological traits that the putative mother trees are C. kanehirae and the putative male ancestor may be related to C. camphora. However, no molecular evidence was used previously to illustrate the relationship of hybridization and the level of introgression of C. camphora and C. kanehirae.In the morphological trait analysis, we collected and measured 229, 109 and 109 fruits from C. kanehirae, C. camphora and putative hybrids form three clusters. The bootstrap numbers on the nodes splitting putative hybrids, C. kanehirae and C. camphora were 1 and 0.994, which are high enough to support the clustering. This implies that the putative hybrids are genetically distinct from C. kanehirae and C. camphora. The PCoA analysis of 808 SNPs represented 83.95% (sum of 65.84% + 18.12%) of the total variance , and all of the putative hybrids were genetic composition admixtures from both species. Fifty pure individuals (19 C. kanehirae and 31 of C. camphora) had Q-values ≥ 0.9, and 25 putative hybrids had Q-values from 0.1 to 0.9. Therefore, we classified them as hybrids. When the cluster number (K) was equal to 3, the cluster including all C. camphora was divided into two clusters (green and blue). When K = 4, the genetic structure only changed in the red cluster (C. kanehirae and putative hybrids). Hybrid individuals with Q-values close to 0.5 were interpreted to be the F1 (C. kanehirae × C. camphora) and F2 (F1 × F1). Hybrid individuals with Q-values close to 0.25 or 0.75 may be interpreted as backcrossed to C. kanehirae or C. camphora (F1 × C. kanehirae or F1 × C. camphora). In the analysis using NewHybrid software, all of the posterior probabilities of C. camphora and C. kanehirae were 1  was estimated to be 2 when delta K statistics were given by the STRUCTURE Harvester26. In most angiosperms, mitochondrial DNA is maternally inherited27. The InDel markers in this study are suitable for detecting F1 hybrids but not F2 or backcross hybrids. The InDel data from the mitochondria also revealed that in these hybrids, C. kanehirae is the mother tree, and C. camphora is the donor parent. Taiwan was a possible site for the origin and differentiation of the Cinnamomum sect. camphora28. Our data also reveals that the genotypes of east and west C. camphora are different. This is consistent with the previous report that the C. camphora in eastern Taiwan is unique and genetically distinct from the C. camphora in western Taiwan and Japan29. These genotyping data are partially in accordance with previous reports. Kameyama et al. used SSR markers to calculate the genetic diversity of C. camphora from China, Japan and Taiwan, and the data showed that the C. camphora in Japan showed a high genetic diversity compared to genotypes from parts of China and Taiwan. However, the report30 lacked C. camphora individuals collected from ESW Taiwan. The topic of geographic genetic variance in C. camphora seems important for further investigation in the future.Within this study, the InDel markers were used to rapidly and successfully detect the hybrids and their parents. InDel markers are codominant and relatively abundant, and they offer easy direct detection by PCR and subsequent gel electrophoresis31. Regarding genetic analysis, expected heterozygosity (He) is also known as gene diversity, which is a measurement for understanding the genetic variation in a population. In this study, the He values of C. kanehirae and C. camphora are much lower than those obtained in a previous report32, although all of the previous reports now reveal that the C. kanehirae had a low level of genetic diversity in Taiwan due to the overlogging and destruction or fragmentation of the natural habitat. One of the reasons explaining the lower diversity of C. camphora may be that only the widely distributed giant trees (estimated older than 100 years old) were collected and analyzed in this study. The expected heterozygosity (He) in putative hybrids was higher than those in C. kanehirae and C. camphora. The possible reason for this result is that the genomic recombination occurs in putative hybrids and then increases the genetic diversity. The hybridization may increase the genetic diversity34. The Fis (inbreeding coefficient) is used to gauge the strength of inbreeding. Positive Fis values also suggest a significant deficiency of heterozygosity in C. kanehirae and NW C. camphora, where inbreeding leads to a decrease of genetic diversity32. The Fis of putative hybrids was negative, implying a heterozygote excess, which is possibly due to hybridization35. Fst is the level of genetic differentiation between populations and provides the basis for a measure of genetic distance. The values of Fst range from 0 to 1. A zero value indicates no population structuring or subdivision. Fst values of 0.05 < Fst < 0.15 indicate moderate differentiation, values of 0.15 < Fst < 0.25, indicate great differentiation, and values over 0.25 indicate very great differentiation36. A relatively high level of genetic differentiation was observed between the C. kanehirae and ESW C. camphora (Fst of 0.642), this implies that there is very great differentiation between C. kanehirae and C. camphora, consistent with their being taxonomically different species. A high level of genetic differentiation between putative hybrids and C. kanehirae (0.304) and NW C. camphora (0.267) was found. Hybrids had almost moderate genetic differentiation between C. camphora (0.208–0.267) and C. kanehirae (0.304). This implies that the hybrids are equally close to C. kanehirae and C. camphora (Table C. camphora (0.208) is lower than that between putative hybrids and NW C. camphora (0.267). This implies that the putative hybrids are genetically closer to ESW C. camphora than to NW C. camhora. The two C. camphora groups (NW and ESW C. camphora) also show a moderate level of differentiation from each other but high genetic diversity within C. camphora populations36. This may be due to the geographical barrier of the Central Mountain Ridge in Taiwan, and thus, the natural gene flow of C. camphora between east and west may be difficult. Most local plant species exhibit some genetic differentiation because of the Central Mountain Ridge in Taiwan37. This result is consistent with the report of Professor Naonori Hirota, in which the eastern camphor trees were found to be different from the those in western Taiwan29. In the STRUCTURE analysis  nearby TR4 did not fruit during our investigation period from 2015 to 2019. However, C. kanehirae trees may be in sparse bloom on the top layer of the trees, which we did not observe. C. camphora is mostly pollinated by insects from the order Diptera, which can carry pollen for long distances, at least 2 km, and transport viable pollen over at least 400 m41. The closest C. camphora trees are approximately 80 m away from TR4. Therefore, TR(L)i_j  have been classified as F1 and backcross (F1 × C. camphora or F1 × C. kanehirae). LC5_21 and LC5_26 were collected from a C. kanehirae tree (LC5) in the nursery, and artificial embryo culture was used to produce the seedlings. Evidence supported the natural hybridization in the individuals collected in the present experiment and was consistent with the surroundings. For instance, in the surroundings of LC5, several C. camphora trees were planted nearby, at approximately 100 m away, and the blooming times of both trees in the collected year were simultaneous. We used artificial embryo culture to produce the siblings LC5_21 and LC5_26, and other siblings  exhibit similar morphological characteristics to C. kanehirae. The hybrid individuals collected and embryo cultured from the National Museum of Natural Science (NMNS) are F2 and B × 1 or B × 0 because the collected trees there were planted very close to each other in an array along with C. kanehirae  at NMNS bloomed annually from 2014 to 2018. Thus, it is reasonable that the offspring of hybrids in NMNS are F2 and B × 1 or B × 0 (K13R_3). These data indicated that the hybridization events were spontaneous and random.Figure C. kanehirae or C. camphora) were morphologically intermediate between C. kanehirae and C. camphora. Our data implied that introgression is bidirectional in these two species, especially in human-modified areas (street trees or nursery). We know that the C. camphora and C. kanehirae rarely display synchronous blossoming in natural fields at lower elevation. Thus, a premating barrier between C. kanehirae and C. camphora is reasonable. We observed the C. kanehirae, C. camphora and hybrids bloomed synchronously in the nursery and among domesticated (street) trees. It could be possible that with the unusual climate for C. kanehirae, the premating barrier became weak, allowing some proportion of these related species to hybridize. Our data are of relevance because the C. kanehirae wood has high market value for use in culturing A. cinnamomea. Although hybrids might be useful in breeding programs and may outcompete the parent species42, the experimental evaluation of hybrid wood quality is still necessary in the future to ensure the alternative utilization of the hybrids. Therefore, from the conservation point of view, before the evaluation of the effects in the hybrid population, it is better to limit the dispersal of the hybrids in the nursery.The molecular and morphological data of hybrids in this study supported the occurrence of hybridization, which shows that the species boundaries are partly permeable. Both the STRUCTURE and the NewHybrid analyses showed that these individuals . The genetic analysis with 808 SNPs also supports the occurrence of hybridization between C. kanehirae and C. camphora. For the conservation and breeding of C. kanehirae and C. camphora, further studies are necessary to ensure the properties of hybrids, including the wood quality, adaptive ability and so on. We infer that many hybrid seedlings appear in the market and will extend to natural fields in the future. Hybrid woods may negatively affect the commercial value of pure C. kanehirae wood. Thus, we recommend that buffer zones be built around the seed orchard and nursery of C. kanehirae to avoid cross-species pollination and to preserve the purity of the species. A suitable method for the rapid molecular detection of hybrids is also necessary to preserve and control seedling production in the future.In this study, we provided useful InDels to quickly identify C. kanehirae leaves were originally collected from natural habitats all over of Taiwan. Twenty-nine C. camphora samples were collected from giant trees  in Taiwan, and two C. camphora samples were collected in Japan, while one was collected in Vietnam. Putative hybrids were collected from among street trees near the campus and museum or in the seedling nursery. The samples labeled with underscores were collected as seeds, and then, the plant was obtained by using artificial embryo culture. The method of artificial embryo culture to generate the plant from fruit trees of C. kanehirae and putative hybrids followed our previous publications44. Samples were labeled as follows: TR1_5 was a seed collected from a TR1 mother tree; K9R_1 and K9R_3 were the seeds collected from a K9R mother tree; TR4_1 and TR4_4 were seeds collected from TR4, and so forth. The DNA extraction of leaf samples followed the CTAB method45. All of the samples were collected and immediately stored in a – 80 ℃ freezer.The details of the plant samples used in this study are listed in Supplementary Table 5. The sequencing library was sequenced with the Illumina Hi-seq 4000 paired-end sequence platform  at Genomics . For low-coverage sequencing, the genomic DNAs of C. camphora and C. kanehirae were sequenced with the Illumina Mi-seq paired-end sequence platform  with a MiSeq Reagent Kit V3 (600 cycles) at the National Yang-Min University, Taiwan. The paired-end libraries were finished with Illumina TruSeq DNA PCR-free sample preparation (550 bp insert size). The adapter sequences and low-quality score of the raw reads were trimmed by the CLC quality trim tool with default parameters, and all of the trimmed reads were used for de novo assembly with the CLC genome assembler . Before InDel calling, the assembly contigs of over 1500 bp were selected for further analysis. The GATK procedure was used for InDel calling. In this procedure, we used the trimmed reads of C. kanehirae to align the assembly contigs of C. camphora (JLH2 and JZS), and the trimmed reads of C. camphora_JLH2 were aligned to contigs of C. camphora_JZS; thus, three InDel calling data sets were eventually obtained. As a result of GATK, we selected the InDel length of over 20 bp with parameters of GT (1/1) and AD (0 > 5). The primers were designed by using Primer 3 software46, and the sequences are listed in Supplementary Table DNA from the 75 plant samples was prepared for GBS and sequencing library construction following the previous report19. TASSEL v5.0 was used for genotyping summary and SNP filtering47. For obtaining real and accurate SNP sites for further analysis, we removed the SNPs that contained missing data by using TASSEL. The GenAlEx v6.5 was used to calculate observed and expected heterozygosity for each of the nuclear SNP markers. A principal coordinate analysis (PCoA), inbreeding coefficient (Fis) and level of genetic differentiation (Fst) were also calculated by using GenAlEx v6.548. The phylogenetic tree was constructed using MEGA 6.0 by the neighbor-joining (NJ) method with 1,000 bootstrap replicates49. The genetic structure was investigated using Bayesian cluster analyses with the STRUCTURE software v2.3.451. An admixture model was employed in which correlated allele frequencies were assumed and the K-value  was set from one to seven. The length of the burn-in period was set to 10,000, and the Monte Carlo Markov Chain (MCMC) model after burn-in was run for an additional 100,000 iterations. For each K, 20 replicates were run, and the best delta K was obtained by using STRUCTURE harvester online software24.The genomic reference-based method used the GATK procedure for SNP calling with only read 1 of the sequencer outputs. The accession number of the reference genome we used is GCA_003546025.1  in GenBankC. kanehirae, C. camphora and the putative hybrids. All of the pure species were validated by codominant species-specific InDel markers (JLH2_CK InDel_4). F1, F2 (F1 × F1) and backcross  hybrids were identified using the SNP genotypic data with the software STRUCTURE and NewHybrids52. The Q-value from STRUCTURE, which shows the proportion of an individual’s genome that originates from each of the K clusters, was used as a hybrid index. Thus, for the best delta K, individuals with Q-values between 0.1 and 0.9 were considered to be hybrids or backcrosses, while individuals with Q-values ≥ 0.9 were considered to be pure species. This coincides with previous studies54. NewHybrids was used to classify individuals as pure parent species, F1s, F2s or backcrosses. NewHybrids computes the posterior probabilities that an individual belongs to these different classes. For NewHybrids, we used the default genotype categories for first and second generations of crossing and ran 100,000 sweeps of five chains started from over dispersed starting values after a burn-in period of 50,000 sweeps. Jeffrey-type priors were used for the mixing proportions and allele frequencies. To ensure the successful convergence of NewHybrids, the C. kanehirae (B3) and C. camphora (JAH) were set to individual-specific prior (z option) for pure parents.For morphological trait analysis, we examined the color of the back-leaf and the fruit size in some of the Supplementary Information.


An important economic reason for the loss of local breeds is that they tend to be less productive, and hence having less market value than commercial breeds. Nevertheless, local breeds often have irreplaceable values, genetically and sociologically. In the breeding programs with local breeds, it is crucial to balance the selection for genetic gain and the maintaining of genetic diversity. These two objectives are often conflicting, and finding the optimal point of the trade-off has been a challenge for breeders. Genomic selection (GS) provides a revolutionary tool for the genetic improvement of farm animals. At the same time, it can increase inbreeding and produce a more rapid depletion of genetic variability of the selected traits in future generations. Optimum-contribution selection (OCS) represents an approach to maximize genetic gain while constraining inbreeding within a targeted range. In the present study, 515 Ningxiang pigs were genotyped with the Illumina Porcine SNP60 array or the GeneSeek Genomic Profiler Porcine 50K array. The Ningxiang pigs were found to be highly inbred at the genomic level. Average locus-wise inbreeding coefficients were 0.41 and 0.37 for the two SNP arrays used, whereas genomic inbreeding coefficients based on runs of homozygosity were 0.24 and 0.25, respectively. Simulated phenotypic data were used to assess the utility of genomic OCS (GOCS) in comparison with GS without inbreeding control. GOCS was conducted under two scenarios, selecting sires only (GOCS_S) or selecting sires and dams (GOCS_SD), while kinships were constrained on selected parents. The genetic gain for average daily body weight gain (ADG) per generation was between 18.99 and 20.55 g with GOCS_S, and between 23.20 and 28.92 with GOCS_SD, and it varied from 25.38 to 48.38 g under GS without controlling inbreeding. While the rate of genetic gain per generation obtained using GS was substantially larger than that obtained by the two scenarios of genomic OCS in the beginning generations of selection, the difference in the genetic gain of ADG between GS and GOCS reduced quickly in latter generations. At generation ten, the difference in the realized rates of genetic gain between GS and GOCS_SD diminished and ended up with even a slightly higher genetic gain with GOCS_SD, due to the rapid loss of genetic variance with GS and fixation of causative genes. The rate of inbreeding was mostly maintained below 5% per generation with genomic OCS, whereas it increased to between 10.5% and 15.3% per generation with GS. Therefore, genomic OCS appears to be a sustainable strategy for the genetic improvement of local breeds such as Ningxiang pigs, but keeping mind that a variety of GOCS methods exist and the optimal forms remain to be exploited further. The past century has witnessed the development of a few intensively selected commercial breeds (and lines) in farm animals and the challenge that many more local breeds had been lost or are endangered ,2. The lGenomic selection (GS) has been a revolutionary method for the genetic improvement of farm animals ,6. GS caOptimum contribution selection consists of a variety of algorithms and methods. Generally speaking, OCS maximizes genetic gain under a predefined genomic inbreeding rate by calculating the optimal contribution of all selected candidates to the next generation through a constrained optimization. Like pedigree OCS, genomic OCS has been refined considerably to accommodate operational breeding constraints, such as restricting the number of individuals contributing to the next generation and imposing upper or lower limits on how much an individual contributes. Some other forms of genomic mating and relevant computation strategies are also worth mentioning hereafter. Meuwissen  proposedIn pig breeding, there were a limited number of simulation studies that applied traditional OCS, such as Gourdine et al.  and DagnDNA was isolated from ear or hair samples, collected from two sets of Ningxiang pigs. The NXP set 1 consisted of 212 pigs, which were raised in the conservation region of Ningxiang pigs and genotyped with an Illumina Porcine SNP60 Version 2 array 68,528 SNPs). The NXP set 2 has 303 pigs, which were raised in Ningxiang Pig Farm of Dalong Livestock Technology Co., Ltd. and genotyped with a GeneSeek Genomic Profiler (GGP) Porcine 50K Version 1 array . These two populations were historically genetically correlated but have diverged from each other in the past decades due to closed breeding within each population. The number of genotyped SNPs and their minor allele frequencies by chromosome (chromosomes 1–18 and X) based on the Sscrofa10.2 assembly for the two sets of pigs are listed in  SNPs. Thi and j, denoted by fij, is the probability that two alleles randomly chosen from both individuals were IBD. The inbreeding of an individual, say i, is the probability that two alleles carried by this individual at a given locus are IBD. Kinship coefficients were calculated with two methods: 1) locus-wise (LW-kinship) or 2) as the proportion of the genome included in runs of homozygosity (ROH-kinship), which are contiguous regions of the genome where an individual is homozygous across all sites. ROH arises when two copies of an ancestral haplotype are brought together in an individual. Hence, that haplotype would be autozygous, i.e., homozygous by descent. In the present study, ROH is defined through a genomic scan for a minimum of 20 SNPs with zero heterozygous genotypes and zero missing genotypes, also subject to a maximum marker spacing of 100Kb between two neighboring SNPs within ROH.The kinship between two individuals M is the number of SNPs on the GGP 50K SNP array, pk and qk are the frequencies of the two alleles at locus k at the current generation, and Following Toro et al. , we compLk is the length of the k-th shared IBD segment measured over homologous a of individual i and homologous b of individual j, and T is the total length of the genome.ROH-kinship coefficients were computed based on segments of identity-by-status shared between pairs of individuals. These segments are potentially ROHs in the offspring of each mating pair . That isFt+1) deviated from that in the previous generation (Ft), expressed relative to what remained to be non-inbred at generation t [Similar to the computing of kinship coefficients, the inbreeding coefficients were also computed locus-wise and based on ROH, respectively. The inbreeding coefficient of an individual can be viewed as the self ancestry with itself, which equal to ration t :ΔF=Ft+1j of sex i, was simulated as follows:aj was the animal’s true additive genetic value, sexi was the sex effect, and eij was the residual term. The true breeding value of an individual was computed to be the sum of the simulated QTL effects for that animal. The distribution of true breeding values for the 303 Ningxiang pigs is shown in h2 = 0.28, mimicking the heritability estimate from a previous study [cov = 2kij, and kij was the estimated genomic kinship coefficient between animals i and j.Phenotypes of average daily body weight gain (ADG) were simulated for the 303 Ningxiang pigs  with the GGP 50K SNP genotypes. Briefly, 2,000 SNPs, each with MAF > 0.10 were randomly assigned to be QTLs affecting ADG. The QTL additive genetic effects were simulated following a Gamma distribution, where the shape parameter was 0.4 and the scale parameter was 1.66 , and theus study . The sexui was the EBV of ADG of i-th individual, which was estimated using the model (4), xij was the genotype of the j-th SNP for the i-th individual, which was coded numerically as 0 (AA), 1 (AB), and 2 (BB) respectively, bj was the association effects for the j-th SNP, and ϵj was an error term. Then, the genomic-estimated breeding value (GEBV) of each animal was taken to be the sum of estimated SNP effects corresponding to all the SNP genotypes that this animal had.Before the implementation of genomic OCS and genomic selection, SNP effects on EBV of ADG were estimated using ridge-regression BLUP  based onX is the centered SNP genotype matrix for all breeding candidates and c be a vector of proportional contribution of individuals to the next generation. Then, the inbreeding and coancestry for a given choice of c is defined as:In the present study, the 303 animals were treated as founders. Let  markers , and c bFurther, let ci≥0 for candidate i and N was the total number of candidates. For each sex, we had Ns was the number of animals of a specific sex. The objective was to maximize genetic gain, subject to the restriction that the maximum kinship was no greater than an upper bound of kinship, set up as follows:Ne was the effective population size, and L was the generation interval. The optimal c that maximized (7) was obtained by maximizing the following Lagrangian objective function [λ0 and λ were the Lagrangian multipliers (λ was a vector of length 2), Q was a known incidence matrix for the sex of candidates, and r was a vector of 0.5’s of length 2. This problem can be solved either using Lagrangian multipliers [In the above, the restrictions on the weights were function :H=c′b−λtipliers  or usingtipliers . Thus, gLong-term selection on simulated average daily gain was conducted for ten generations using genomic OCS and genomic selection, respectively, with the set of 303 Ningxiang pigs used as the base population. The purpose was to evaluate the utility of using genomic OCS as a sustainable selection strategy for Ningxiang pigs. For genomic selection, ten sires and 50 dams with the largest GEBV of ADG were selected and mated randomly in each generation. Genomic OCS was conducted under two scenarios. In the first scenario (denoted by GOCS_S), the top 10 sires with the largest contribution rates were selected, and each chosen sire was mated to 5 dams, which had the smallest kinships with this sire. This was to mimic the real situations in which the highly-intensive selection is not desirable, but minimizing kinships in parents (and hence inbreeding in progenies) is of more relevance. Thus, we limited selection for genetic gain on sires only. However, OCS requires constraining the average coancestry of selected parents, which otherwise is not met if each sire is randomly mated with equal probability to each unselected dam. Hence, minimizing kinships between selected parents in the present study was achieved by mating each chosen sire to five dams, which had the smallest kinships with this sire. In the second scenario (denoted by GOCS_SD), GOCS was implemented more closely following Sonesson et al. , in whic−8 per nucleotide was simulated for all chromosomes, regardless of if the locus is causative or non-causative. For each type of mating and selection, long term selection on simulated phenotypes was duplicated stochastically 100 times. Simulation and genomic OCS were conducted using an internal R package called GMT, developed at Neogen GeneSeek.Progeny genotypes were simulated using a stochastic procedure, given the parental haplotypes. The SNP map was derived from the porcine genomic assembly 10.2 (Sscrofa10.2), and the gametes with recombination were produced by drawing a number of crossovers from a Poisson distribution with the mean being 1 per Morgan. The position of crossovers was taken at random without inference. A mutation rate of 1 × 10Average LW-inbreeding coefficients were 0.41 and 0.37 for the two sets of Ningxiang pigs genotyped with the Illumina PorcineSNP60 or the GGP Porcine 50K, respectively. The corresponding 95% confidence intervals were between 0.32 and 0.51 in NXP set 1, and between 0.25 and 0.46 in NXP set 2. The average LW-inbreeding coefficient in NXP set 1 was slightly larger than that in NXP set 2. There was evidence that, based on genomic-estimated kinship coefficients, animals in the first set were more closely related to each other than those in the second set . This diAverage LW-inbreeding in these Ningxiang pigs was roughly comparable to the genetic diversity of two closed Iberian pig lines  but consAverage ROH-inbreeding coefficients were estimated to be 0.24 and 0.25, respectively, in the two sets of Ningxiang pigs. The 95% confidence intervals of ROH-inbreeding were approximately between 0.16 and 0.35 in both datasets. Overall, ROH-inbreeding coefficients were smaller and more consistent between datasets than LW-inbreeding coefficients. It is generally acknowledged that ROH-inbreeding provides a more accurate measure of IBD than LW-inbreeding ,41 becauInbreeding is associated with a wide range of genetic phenomena, which include a decrease in genetic diversity of finite populations, a decrease in effective population size, genetic drift, changes in population structure, deviations from Hardy-Weinberg equilibrium, and decreases in population means . Often, In the NXP set 2, genomic selection led to a high rate of genetic gain in ADG in the first two generations . ConversIn the two scenarios of genomic OCS, the rate of inbreeding per generation was controlled to be less than 5% for each of the ten generations of selection under scenario 1 , and mosArguably, genomic OCS is becoming a well-established method that can be used in many practical animal breeding applications. Nevertheless, improvements are also emerging. In genomic OCS, a measure of genetic gain is often taken to be the total expected breeding value of the progeny. Still, it carries no information about the variability of breeding values among full-sibs. Akdemir and Sanchez  introducGenomic OCS provides a well-established approach to balancing genetic gain and the loss of genetic diversity. The present results investigated the performance of GOCS in Ningxiang pigs with observed genotypes and simulated phenotypes of average daily body weight gain. Two scenarios of genomic OCS were examined, in comparison with GS without controlling inbreeding. Scenario No. 1 (GOCS_S) presented a modified version of genomic OCS for indigenous breeds such as Ningxiang pigs, in which selection for genetic gain operated on sires only but constraining kinships on both sires and dams. This type of non-random mating arrangement was a necessary step with scenario No. 1. In genomic OCS under scenario No. 2 (GOCS_SD), both sires and dams with the largest contribution rates were selected, and the kinships between selected parents were constrained. The present results supported the utility of genomic OCS for the breeding of Ningxiang pigs because it can maintain a sustainable long-term genetic gain while controlling the rate of inbreeding within a desirable range for most of the generations of selection. The present study involved only a single trait, namely, average daily gain. In a real situation, multiple traits of economic importance will need to be considered, which could include, for example, growth rate, intramuscular fat, and reproductive performance. Meanwhile, future considerations may also include investigating alternative, more cost-effective forms of genomic mating or genomic OCS for sustainable genetic improvement of local breeds .


Habitat fragmentation is a major extinction driver. Despite dramatically increasing fragmentation across the globe, its specific impacts on population connectivity across species with differing life histories remain difficult to characterize, let alone quantify. Here, we investigate patterns of population connectivity in six songbird species from Singapore, a highly fragmented tropical rainforest island. Using massive panels of genome‐wide single nucleotide polymorphisms across dozens of samples per species, we examined population genetic diversity, inbreeding, gene flow and connectivity among species along a spectrum of ecological specificities. We found a higher resilience to habitat fragmentation in edge‐tolerant and forest‐canopy species as compared to forest‐dependent understorey insectivores. The latter exhibited levels of genetic diversity up to three times lower in Singapore than in populations from contiguous forest elsewhere. Using dense genomic and geographic sampling, we identified individual barriers such as reservoirs that effectively minimize gene flow in sensitive understorey birds, revealing that terrestrial forest species may exhibit levels of sensitivity to fragmentation far greater than previously expected. This study provides a blueprint for conservation genomics at small scales with a view to identifying preferred locations for habitat corridors, flagging candidate populations for restocking with translocated individuals and improving the design of future reserves. The gloIn fragmented landscapes, subpopulations may become completely isolated from one another depending on the distance among patches, the suitability of the intervening habitat matrix and the species’ dispersal capability. A lack of gene flow and connectivity among subpopulations increases the probability of inbreeding and stochastically induced loss of genetic diversity, with a potentially detrimental impact on population fitness, as inbreeding may lead to the expression of deleterious genetic effects and low genetic diversity may hamper adaptation to environmental change  in combination with multiple population‐genomic approaches, we investigated how life history and ecological requirements affect a species’ response to fragmentation on a heavily urbanized tropical rainforest island.22.1Pellorneum malaccense (Short‐tailed Babbler) and Cyanoderma erythropterum (Chestnut‐winged Babbler)) and two edge‐tolerant species (Turdinus abbotti (Abbott's Babbler) and Mixornis gularis (Striped Tit‐babbler)) (Table Pycnonotus simplex (Cream‐vented Bulbul)) and one edge‐tolerant species (Pycnonotus plumosus (Olive‐winged Bulbul)). Mist‐netting and sampling in Singapore were conducted in accordance with Institutional Animal Care and Use Committee regulations (B13‐4738/ B16‐0572) (permit NP/RP13‐019‐2). We also collected blood of P. malaccense and C. erythropterum from Danum Valley  for comparisons of genetic diversity ) Table . Moreovety Table  adheringty Table  and S2. 2.2P. plumosus samples were extracted with an Exgene Clinic SV kit (GeneAll Biotechnology) and were sequenced in one additional library by a different provider  on a 100 bp paired‐end run on an Illumina HiSeq 2000 platform. This library was prepared following Tan et al.’s  following the manufacturer's protocols for blood or tissue. We prepared five libraries following Ng et al.’s  double‐det al.’s  protocolet al.’s .2.3M. gularis and P. plumosus) with “process_radtags” in STACKS 1.42 . We randomly subsampled individuals from Singapore to ensure equal representation of samples for compared localities and further controlled for sampling area size between compared localities.To compare genomic diversity of the fragmented population from Singapore to populations from contiguous blocks of unfragmented forests elsewhere, we created an additional set of unlinked SNPs for each species  were run using 50,000 burn‐in steps and 100,000 Markov chain Monte Carlo samples. We identified the number of genetic clusters which best partitions the data using Evanno, Regnaut, and Goudet’s  an individual can have. Individuals that have the smallest genetic distance are connected according to a threshold that varies with k. The level of genetic differentiation between identified clusters for each species was assessed by computing Nei's pairwise FST and its significance with the R package adegenet 1.3‐1  as implemented in R to investigate potential barriers to or conduits for gene flow  in the forest‐dependent m Figure  and S2. P. simplex as well as the edge‐tolerant T. abbotti, M. gularis and P. plumosus displayed no spatial clustering across the main island of Singapore .Pairwise Fs Figure  by reveas Figure . Corresp3.4C. erythropterum and P. malaccense . No significant barriers were found in P. simplex (data not shown).Areas of high genetic dissimilarity, corresponding to areas where genomic outliers (possible immigrants) were sampled, were observed in s Figure . After rs Figure . A sampls Figure . Similars Figure . In thisC. erythropterum and P. malaccense, respectively, indicating poor dispersal capability  and two frugivorous bulbuls (one forest‐dependent and one edge‐tolerant) across a heavily fragmented and urbanized tropical forest landscape in Singapore. The strength of our data set is that it compares the genomic signatures of more widespread generalist species with those of habitat specialists of a formerly widespread distribution that have – by now – become restricted to the last big forest reserve of the island. While populations of the forest‐dependent 4.1M. gularis, P. plumosus and P. simplex appears comparable between Singapore Island and contiguous forest blocks of a similar size in Sarawak and Vietnam  than for the Central Catchment in Singapore – despite the comparable size of Singapore's Central Catchment and the area in Danum Valley where the samples had been obtained. This dramatic reduction of population genetic diversity in Singapore's fragmented landscape is consistent with results obtained from mitochondrial analysis of P. malaccense  was shown to be a relatively poor disperser  most closely approach each other. The area in question happens to be near the only site where forests north and south of the main dividing reservoir are connected along a narrow nexus, delimited on the west by a large highway , but also those currently considered safe, such as C. erythropterum, flagging them for regional conservation concern. Additionally, we find that even edge‐tolerant and seemingly well‐connected species, such as M. gularis, are likely impacted by fragmentation in the longer term (see also Tan et al., Genome‐wide markers allowed us to identify extremely fine‐scale geographic barriers to connectivity and to characterize dispersal capability. Using a panel of six songbird species, some landscape features such as reservoirs and man‐made structures were shown to drastically compromise connectivity in understorey insectivores, underscoring the usefulness of genomic tools in understanding the impact of fragmentation on population connectivity. Importantly, we demonstrate that these landscape barriers have adverse impacts on connectivity not only on IUCN‐listed species such as Barriers affecting understorey insectivorous birds undoubtedly impact other forest biota such as reptiles, amphibians and invertebrates that are known to be even more sensitive to habitat fragmentation (Keinath et al., 5What causes the continuing local extinction of species even in well‐protected areas such as Singapore's long‐established reserve network? Even in scenarios where total protected forest area remains stable, species loss will sometimes continue, as has been shown for Singapore Yong, . This st Click here for additional data file.


Glyphoglossus yunnanensis (Microhylidae), and two alternative hypotheses were tested: That is, the geographical distribution of genetic variation was determined by either the contemporary drainage basin or historical drainage basins.With the late Cenozoic uplift of the Qinghai–Tibetan Plateau (QTP), drainage of the southeastern edge of the QTP changed significantly. However, the impact of this dramatic change on the geographical distribution and genetic diversity of endemic organisms is still poorly understood. Here, we examined the geographical patterns of genetic variation in the Yunnan small narrow‐mouthed frog, The Mountains of southwest China.Analyses were based on 417 specimens collected from across the distribution of the species. We reconstructed the genealogy (Bayesian and maximum parsimony methods) and assessed demographic history based on DNA sequencing data from mitochondrial and nuclear markers. We also mapped the genetic diversity and estimated the divergence times by a relaxed clock model.G. yunnanensis were almost concordant with those of Nanorana yunnanensis.The species has maintained a relatively stable population size without recent population expansion. Four major maternal lineages were identified with good support, one representing a possible cryptic species and the other three showing further subdivision. The distribution of these deeply differentiated lineages/sublineages corresponded well to geographical regions. The secondary contact zones and phylogeographic breaks in distinct lineages of G. yunnanensis was shaped by historical drainage patterns. Concordance in phylogeographic patterns may suggest a shared response to common hydrogeological history and also might indicate that there was more contribution of the drainage history than ecological or life‐history traits in structuring genetic variation between these two disparate codistributed taxa G. yunnanensis and N. yunnanensis.Lineage division conformed to the hypothesis of drainage system evolution, that is, the phylogeographic pattern of  G. yunnanensis was shaped by historical drainage patterns. Concordance in phylogeographic patterns from different taxa G. yunnanensis and N. yunnanensis may suggest a shared response to common hydrogeological history.Lineage division conformed to the hypothesis of drainage system evolution, that is, the phylogeographic pattern of  These mIncreasing evidence has shown that the rearrangement of drainage systems has played a key role in shaping current geographical patterns of genetic and species diversity of aquatic organisms in many parts of the world   , adjacent northern Vietnam, and presumably in adjacent Laos  and cytochrome b (CYTB), were amplified for all individuals. Mitochondrial ribosomal subunit 16S rRNA and nuclear gene tyrosinase (Tyr) were amplified only for a small subset of individuals  belonging to different lineages identified by preliminary analysis. References for primers are given in Supporting Information Table Using the proteinase K/sodium dodecyl sulfate (SDS) method . Plasmids carrying the PCR fragment were then extracted and sequenced. For each PCR product, at least four clones were randomly selected, with one then sequenced.Nuclear gene clonal sequencing was carried out for individuals that contained more than one ambiguous site and failed to infer alleles using PHASE v2.1.1  and maximum parsimony (MP) were implemented using DATA I. The best‐fit models of sequence evolution were determined using ModelTest 3.7  per million years (Ma) for CYTB based on evolutionary rates commonly suggested for anurans  tree based on uncorrected 2.6h) and nucleotide diversity (π) were estimated for overall and for each population with more than five samples, respectively. Geographical partitioning of genetic diversity was inferred via two approaches. First, the G. yunnanensis populations were grouped using spatial analysis of molecular variation (SAMOVA)   analyses , E (eastern), and C  Figures  and 2. LLineages C and E co‐occurred in populations 4 and 5 Figure . LineageLineage LD diverged from the remaining ingroups about 5.98 Ma . Lineage W diverged about 3.10 Ma (late Pliocene) , and lineage E diverged from lineage C about 2.48 Ma . The most recent common ancestor of lineage W was 1.77 Ma (early Pleistocene) , with lineages E1–E2 diverging about 1.16 Ma (early Pleistocene) . In lineage C, lineage C1 first diverged about 1.82 Ma (early Pleistocene) , whereas lineages C2–C4 became isolated from each other about 1.44 Ma   of groups (from three to nine)  Table . For thes Figure . Localits Figure . Furtherp < .01; Table FCT value (0.7186). In contrast, grouping of contemporary drainage basins did not adequately explain the distribution of genetic diversity across the study (FCT = 0.3033).The AMOVA results revealed significant geographical structure in the mtDNA genetic variation at all hierarchical levels examined p < .0; Table 1FST estimates ranged from 0.00 to 1.00, and analysis of genetic differentiations showed strong evidence for correspondence between geographical area and lineage as well as between geographical subdivision and sublineage. Genetic differentiations between geographical zones and between subdivisions were significantly high    roughly corresponds to the late Cenozoic extrusion uplift of the CYP (2 ~ 4 Ma)  in the Jinsha River drainage basin Figures  Figure , which rTerminalia franchetii  . This further indicates that the evolution of drainage basins in southwest China has had significant impact on the contemporary geographical distribution of G. yunnanensis genetic variation.Like the matriline patterns of 5G. yunnanensis and N. yunnanensis occurs in many areas. For example, in the CYP, localities 4, 5, and 17 from this study largely correspond to N. yunnanensis sample sites 32, 55–56, and 70 (Zhang, Chen, et al., Sympatric distribution of distinct lineages in 6G. yunnanensis. We found that historical drainage systems are responsible for the divergence between lineages/sublineages. The frog diverged prior to Pleistocene climatic change and experienced a stable demographic history thereafter. Sympatric distributions of distinct lineages resulted from secondary contact, and these areas are major genetic reservoirs and require priority protection. The phylogeographic patterns of G. yunnanensis are almost in concordance with those of N. yunnanensis, thus showing a shared response to hydrogeological history. Comparative phylogeographic studies of other endemic taxa in southwest China are now required to estimate whether their population genetic structures still preserve evidence of these late Cenozoic changes in palaeohydrology.Our study documents the genetic structure within None declared.DRZ, DQR, and HX conceived the ideas; HH and DRZ carried out fieldwork, with active assistance from XQS, SL, JSW, and SQY. DRZ performed laboratory and data analyses, and drafted the manuscript. GHY helped to revise the manuscript and gave constructive advice. All authors contributed to and approved the manuscript. Click here for additional data file.


H′ = 1.40) > pod (H′ = 1.062) > plant (H′ = 0.64) > leaf (H′ = 0.565), while the diversity of the quantitative traits of each organ was ranked as seed (H′ = 2.021) > pod (H′ = 1.989) > phenology (H′ = 1.941) > plant (H′ = 1.646). In the cluster analysis, 115 accessions were clustered into four groups. The accessions in the first and fourth group were characteristic of the Andean gene pool, while the accessions in the second group were characteristic of the Mesoamerican gene pool. However, the accessions of the third group possessed the combined characteristics of both gene pools and were thus classified as introgressed type. Four principal components represented 40.30% of the morphological diversity, with the first principal component representing the traits associated with plant growth; the second principal component representing flower characteristics; the third principal component being composed of seed characters; and the fourth principal component representing pod characteristics. The common bean germplasm resources are widely distributed in Chongqing, and the introgressed types are more prevalent in this region than elsewhere, with a rich genetic diversity and high utilization value in genetic breeding.In this study, the genetic diversity of 115 common bean germplasm resources collected from 27 counties in Chongqing over 3 years (2015–2017) was assessed. The results showed that the genetic diversity of the common bean germplasm resources was high, with an average diversity index of 1.447. The diversity of the qualitative traits of each organ was ranked as seed ( Phaseolus vulgaris L.) is one of the most important legumes and is high in protein, low in fat, and rich in vitamins and dietary fiber. The regular consumption of common bean can reduce coronary heart disease, type II diabetes, and cancer  . There wAgronomic traits were investigated at the experimental base in Wu long  at 1280 m above sea level. The average annual temperature was 14.8°C in 2017 and 2018, and the soil is sandy loam with good drainage. The experimental design was a randomized complete block with three repetitions. The experimental plots consisted of six rows with a planting distance of 0.75 m between the rows and 0.25 m within the rows, and with two seeds sown per planting station. The agronomic practices were the same for all plots and are typically used by most farmers.Phaseolus vulgaris L.) (Twenty-nine morpho-agronomic characters were measured for the 115 accessions based on the Descriptors and Date Standard for Common Bean (aris L.) . HypocotThe 13 quantitative traits in this study included agronomic and morphological traits. All of the recording standards and methods of the traits are listed in X) and standard deviation (s) of each trait were calculated. The coefficient of variation (CV) was used to evaluate the degree of phenotypic variation of each trait as follows: CV = s/X. Each quantitative trait of all the accessions was assigned to classes ranging from class 1: [Xi < (X − 2s)] to class 10: [Xi > (X + 2s)]. Each 0.5s was assigned as a class, where Xi was the value in class i. The Shannon–Weaver index (H′) was used to calculate the genetic diversity index , and the average value  were visualized by plotting the scores of the first and second principal components in two-dimensional space. For the cluster analysis, all the traits of each accession were standardized, and the Euclidian distances were calculated using the unweighted group average linkage method (UPGMA).According to the analysis of the 13 quantitative traits and 16 qualitative traits, the common bean germplasm resources in Chongqing are rich in genetic diversity.H′ = 2.021) > pod (H′ = 1.989) > phenological (H′ = 1.941) > plant (H′ = 1.646).The quantitative traits are shown in H′ = 2.083) > SW (H′ = 2.022) > HSW (H′ = 2.013) > LWS (H′ = 1.968). The length/width of seed (LWS) of 61.7% of the accessions was at the middle level, indicating that most seeds had a more oval SS. In addition, 87% of the accessions were large to medium seeds, while only 13% were small seeds, suggesting that consumers in Chongqing prefer large and oval seeds.For the seeds, the genetic diversity index was ranked: SL (H′ = 2.066) > NP (H′ = 1.997) > PL (H′ = 1.950) > PW (H′ = 1.943). More than 50% of NP and NSP were medium, and the numbers at the high level were significantly higher than that at the low level, which indicated that most of the accessions have the potential to achieve high yield.For the pods, the genetic diversity index was ranked as: NSP (H′ = 2.023) > DF (H′ = 1.858). The materials of the short growth period were 20.9%, while those of the long growth period were only 14.02%.For the phenological traits, the genetic diversity index was ranked as: DM (H′ = 1.972) > NS (H′ = 1.760) > PH (H′ = 1.206). Due to differences in GH, the variation coefficient of PH was the highest, while the diversity index was the lowest. In addition, more than half of the accessions were prostrate.In terms of the plant traits, the genetic diversity index was ranked as: NB (H′ = 1.40) > pod (H′ = 1.062) > plant (H′ = 0.64) > leaf (H′ = 0.565).The qualitative traits are shown in H′ = 0.692) > HP (H′ = 0.646) > ST (H′ = 0.615) > GH (H′ = 0.607). The HP was mainly green, accounting for 65.2% of the accessions, while the remainder were purple. The GH was mainly prostrate type. There were more determinate pods than indeterminate pods in PHA, which indicated that farmers in Chongqing have a preference for prostrate-type common bean.In terms of the plant traits, the order of the genetic diversity index was ranked as: PHA  > CW (H′ = 1.252). Both the CS and the CW were predominantly white.For the flower traits, the genetic diversity index was ranked as follows: CS (H′ = 1.932) > PC (H′ = 1.187) > PS (H′ = 0.679) > SPA (H′ = 0.448). The PC was mainly light brown, whereas only six accessions were least speckled. Most accessions belonged to the short flat strip type, while only four accessions were of the long round stick-shaped type. In terms of the shape of pod apex (SPA), 83.5% had an acute apex, while the remainder had an obtuse apex. PS was mainly characterized by tiny protrusions.For the pod traits, the genetic diversity index was as follows: SP (H′ = 1.836) > SCC (H′ = 1.814) > CSSC (H′ = 1.283) > SSC (H′ = 1.055) > HC (H′ = 1.012). The SS of most accessions was kidney-shaped, with 29 accessions. The majority of accessions (50) had a striped SCC, which indicated that the farmers in Chongqing have a preference for both the striped seed coats and large seeds.The seed traits and genetic diversity were ranked as follows: SS  contained 34 accessions, and the agronomic characteristics were as follows: the DM was 70.2 days, which was the shortest of the four groups. The HP was mainly green. The GH of the accessions in Pop I was erect. The ST was mainly normal, and the PHA was mainly determinate, with a few indeterminate accessions. The CS was predominantly light purple, while the CW was mainly white. The NP was the lowest among the four groups (7.32 pods). The SP was mainly short flat strips, while the SS was largely long ellipse. Most of the seeds had punctiform pink stripes. The LWS was the largest, indicating that the seeds of Pop I were the most elongated among the four groups. The HSW was 38.66 g.The second group (Pop II) contained 35 accessions, and the agronomic characteristics were as follows: the DM was the longest among the four groups at 79.24 days. The HP was mainly green, and the GH of the accessions was prostrate. The PHA of the accessions was mainly indeterminate, with a few that were determinate. The CS and CW were mainly light purple. The NP was the highest among the four groups, with 10.34 pods. The pods of most of the accessions were sickle-shaped, while SS was mainly kidney-shaped. There were no stripes on the seeds of the accessions in Pop II, and the SCC was mainly white. The HSW was 34.87 g.The third group (Pop III) contained 24 accessions, and the main agronomic traits were as follows: the DM was 77.6 days. The HP was mainly purple, and the GH of the accessions in this group was prostrate. The PHA of the accessions was mainly indeterminate. The CS and CW were mainly light purple and purple. The LS of Pop III was mainly rhombic ovate, which is unique among the four groups. The SP was mainly round curved stick-shaped, and the SS was mainly kidney-shaped, and most seeds had punctiform black stripes. The HSW was 33.03 g.The fourth group (Pop IV) contained 22 accessions, and the main agronomic traits were as follows: the DM was 78.64 days. The GH of the accessions in Pop IV was mainly prostrate, and the PHA of the accessions was mainly indeterminate, with a few that were determinate. The CS and CW were mainly pink. The SP was mainly short flat strip with oval seeds, and most of the seeds possessed red punctiform speckles. The HSW was 40.38 g, and the seeds of Pop IV were the largest and roundest of the four groups.The above results indicate that the characteristics of these four groups are quite different. The accessions of Pop I were more similar to the germplasm resources from the Andean gene pool due to the erect GH, white flowers, pink speckled seed coats, and large seeds. The accessions of Pop IV were more similar to the germplasm resources from the Peru (P) race in the Andean gene pool, due to the prostrate GH and larger and oval seeds. We thus classified the accessions of Pop I and Pop IV as being derived from the germplasm resources of the Andean gene pool. The accessions of Pop II were more similar to the germplasm resources of the Mesoamerican gene pool, as the seeds were small and free of speckles, and the seed coat was black, yellow, and white. Therefore, the accessions from Pop II were identified as being derived from the germplasm resources of the Mesoamerican gene pool. The accessions of Pop III were mainly prostrate, the seed coat was striped and medium in size, the flowers were mostly colorful, and there was both an indeterminate and determinate PHA, thus combining the characteristics of the Andean gene pool and the Mesoamerican gene bank. Therefore, Pop III was classified as introgressed.The results of the Principal component analysis (PCA)  of the 2Based on the cluster analysis, the first and second principal component scores of each of the 29 phenotypic traits were used to construct a two-dimensional scatter plot , and theCombined with the results of the cluster analysis, there were 52 accessions derived from the Andean gene pool, which was the most widely distributed gene pool in Chongqing, accounting for 45.2%. The accessions derived from the Mesoamerican gene pool accounted for 34.8%, while the introgressed-type accessions accounted for 20%. The accessions derived from the Mesoamerican gene pool were more widely distributed than the other germplasm types in ecological region I. However, natural ecological regions II, III, and IV were the opposite, and the accessions derived from the Andean gene pool were more widely distributed. The distribution of introgressed-type accessions differed among the four ecological regions, and the proportion of introgressed-type accessions in ecological region III and IV was greater than that in the other ecological regions.H′ = 1.344), Sichuan (H′ = 1.390), and Yunnan (H′ = 1.330) provinces, and lower than that in Guizhou (H′ = 1.499) and Shanxi (H′ = 1.450) provinces (The results of the diversity analysis showed that the average diversity index of the common bean germplasm resources in Chongqing was 1.447, which is higher than that in Shaanxi (rovinces .Among the four natural ecological regions in Chongqing, ecological region II has great variation in longitude, latitude, and elevation, and thus the diversity index of the accessions in this ecological zone was the highest at 1.737, followed by the accessions from ecological region IV, ecological region III, and ecological region I . The resThe common bean germplasm resources in Honduras, Nicaragua, and Mexico have a short DF ; howeverDiversity analysis of common bean germplasm resources in Slovenia and its vicinity, the Iberian Peninsula, and central Africa has confirmed the existence of gene introgression between the two gene pools . Gene inThe distribution of common bean germplasm resources depends on the unique agricultural production preferences of farmers, and it has been suggested that seed selection is closely related to seed size and color . The divThe genetic background of the introgressed-type accessions was between the Andean gene pool and the Mesoamerican gene pool, providing new material and a bridge between the two gene pools for breeders. This is of great significance for improving the yield, quality, and disease resistance in common bean cultivation . BreederAll datasets generated for this study are included in the article/supplementary material.CD was responsible for the study design. HC and QW performed to collect all germplasm resources. JZ, XZ, and PW analyzed the data. JW and JL contributed to the writing of the manuscript. 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.


Sorbus genus remains not fully recognized. We analysed the genetic and morphological variability of several Sorbus species including native (Sorbus aria), foreign (S. intermedia) and potentially hybrid (S. carpatica) individuals from the Polish Carpathian range. Patterns of variation at 13 nuclear microsatellite loci show hybridization between the tested species and confirm the existence of the hybrid form S. carpatica. Biometric analysis on leaves, based of 10 metric features and three parameters, identified several characters for preliminary taxonomic classification; however, none of them could be used as a fully diagnostic marker for faultless annotation of S. intermedia and S. carpatica. The genetic structure analysis indicated complex patterns of population differentiation and its diverse origin. The results allow assessment of genetic variation and identification of parental species participating in hybridization. This knowledge will advance the management of genetic diversity and development of conservation strategies for efficient maintenance of the unique protected ecosystem.Hybridization and introgression are important processes influencing the genetic diversity and evolution of species. These processes are of particular importance in protected areas, where they can lead to the formation of hybrids between native and foreign species and may ultimately result in the loss of parental species from their natural range. Despite their importance, the contribution of hybridization and introgression to genetic diversity in  Sorbus genus. The phenomenon of interspecific hybridization between native and foreign species is frequently a consequence of human activity. It may lead to increased diversity of plants but also it may significantly reduce genetic variation if invasive genotypes of natural species participate in the process. We report a preliminary study of the hybrid taxon occurring in natural and protected areas of Tatra National Park in Poland. Variation patterns at nuclear microsatellite loci were used to distinguish a hybrid form of Sorbus carpatica from morphologically very similar S. intermedia.Hybridization, polyploidization and apomixis are important processes which affect the taxonomic complexity of  Hybridization is based on the transmission of alleles between species that may skew their frequency distribution in the population as expected under equilibrium either by the increase of polyploid levels or by introgression , located in the Polish part of Carpathians originates mainly from the concerns about the genetic influence of foreign species, which may spread and interbreed with native species. Within the TNP, there are several stands of S. intermedia which were introduced artificially in the past or are a result of ornithochoria from trees located in the nearby urban greenery  according to the manufacturer’s instructions. The extraction efficiency was analysed by 1 % agarose gel electrophoresis and the quantity was checked using NanoDrop Life (Thermo Scientific). DNA extracts were diluted with nuclease-free water (A&A Biotechnology) to a concentration of 10–20 ng/µL for genetic analysis.Sorbus species was carried out using 13 nuclear microsatellite markers (nSSR—nuclear Simple Sequence Repeats)  depending on the locus and Na), Simpson index defined as 1 − D (Hexp). The following interspecies genetic parameters were calculated: MLG, number of multilocus genotypes, which was a unique combination of alleles in all the loci for the individual; eMLG, the number of expected MLG at the smallest sample size ≥ 10 based on rarefaction. The Stoddart and Taylor index  was performed  or clonally from root suckers. As the way of propagation may be related to the polyploidy of organisms, it was assumed that the imbalance of conjunctions occurs when alleles in two or more loci coexist more frequently than is predicted based on their frequency. The association index estimated based on the frequency of genotypes for randomly crossed populations is 0. Any statistically significant deviation from the expected zero would therefore suggest clonal reproduction. In this analysis, the significance was tested based on 999 permutations conducted in the R poppr package (The genetic structure of the examined s (DAPC) . In thiss (DAPC) . The anas (DAPC)  and the M), minimum and maximum values , standard deviation (SD) and coefficient of variation (CV) were calculated for the morphometric features. The significance was checked using non-parametric tests. The results of Shapiro–Wilk and Levene’s test led to the use of ANOVA test of Kruskal–Wallis rank. To assess the grouping factors, the analysis of principal components (PCA) for standardized data was applied. The statistical analysis was performed using Excel (2013) and Statistica 13.3 software.Ten metric features and three parameters calculated from the obtained measurements were used for the morphometric analysis . The parSorbus population. The number of observed alleles at different loci ranged from 3 to 15, with an average of 8.5 , corresponding to the examined species: S. aria, S. carpatica and S. intermedia , and additionally trees C13, C14 and C20, representing originally S. carpatica and J11 initially assigned as S. intermedia . When analysing a larger number of clusters (K = 6), the S. aria group was divided into three smaller groups. This division may have originated from the large genetic distance of S. aria individuals, high variability and non-panmictic reproduction  spatially distributed on four narrow branches indicating their greatest genetic distance. On the same branches, we found some of the S. carpatica individuals (green circles) and S. intermedia (blue circles) wrongly annotated in morphological analysis. Individuals of S. intermedia constituted a single group with connections with other individuals, initially erroneously assigned morphologically as representatives of S. carpatica. In MSN analysis, the hybrids were divided into two groups, which were in line with the results of the dendrogram for K = 6.To validate the initial genetic assignment of ividuals , we perfividuals . The netS. aria , S. intermedia  and S. carpatica hybrids . For S. aria, 27 different genotypes were identified for the 27 examined trees. For S. carpatica and S. intermedia the number of genotypes was lower  and lowest for S. intermedia (Hexp = 0.481). Based on the association index (ṝd = 0.0689), interbreeding was found to be the main reproduction strategy for S. aria. In contrast, S. carpatica had an association index significantly different from zero, suggesting that it mainly utilizes clonal reproduction , with the smallest average circumference (LP), which is the effect of shallow serration of leaf blades of this species. The leaves of S. intermedia had the longest and thickest petioles  and the deepest leaf blade serration (LP). The leaves of S. carpatica were characterized by the smallest parameters of leaf  and petiole size . The highest intraspecies variability (above 20 %) was observed for such parameters as: LA, LP, LW2, PL and PA, especially for S. aria and S. intermedia. Leaf area (LA), maximum leaf blade width (LW) and leaf blade width at 50 % of the length (LW1) differentiated most S. carpatica from S. aria and S. intermedia based on Kruskal–Wallis test following ANOVA (S. intermedia from the other two species. Sorbus aria differed from S. carpatica and S. intermedia by the angle between the main nerve and a point on the edge of the leaf blade located at 10 % of the leaf length (LA1) and the width of the leaf blade at 90 % of its length (LW2). However, biometric analysis of leaves did not allow us to identify diagnostic markers for faultless delimitation of the Sorbus species.Leaf morphology showed some differences between the specimens , but no ng ANOVA . The cir[see  ], had the greatest contribution to explaining the observed variability [see  ]. The first component showed a negative correlation with all analysed features, except for the angle between the main nerve and a point on the edge of the leaf blade located at 10 % of the leaf length (LA1). This means that the samples located in the figure on the right side of the x-axis were characterized by low average values of almost all analysed leaf parameters. The first principal component was influenced by the maximum width of the leaf blade (LW), the width of the leaf blade measured at 50 % of the leaf blade length (LW1), the area (LA) and length of the leaf blade (LL) and the width of the leaf blade measured at 90 % of the leaf blade length (LW2). The second component was influenced by features related to the angle between the main nerve and the point on the edge of the leaf blade located at 10 % and 90 % of the leaf length (LA1 and LA2) and the width-to-length ratio of the leaf blade (LW/LL). The PCA did not show the grouping of individuals from a particular species in relation to the analysed factors.The PCA was used to establish if leaf features can be used to assign the taxa. Principal component analysis showed the existence of four factors meeting the Kaiser criterion, with own values greater than 1, which together explained 93.52 % of the variance. The first component, which explained 52.85 % of the variance, and the second one, which explained 19.72 % of the variance Sorbus genus within the TNP. In our analysis, we have studied the native, hybrid and foreign Sorbus species. The genetic analysis allowed for discrimination of S. aria and S. intermedia individuals and indicated the existence of their potential hybrid form S. carpatica. Patterns of variability at nuclear microsatellite loci confirmed that S. aria in the TNP is diploid. The observed population genetics parameters for S. aria in the Tatra Mountains are similar to these of individuals from the Iberian Peninsula, where the selected loci also had two alleles . This high genetic variability, together with the cross-propagation system, confirmed by our study, is characteristic mainly for diploid Sorbus species  has appeared in the Tatra Mountains relatively recently . SA01, SA06, SA07, MSS5, SA02, SA08, CH01h01, CH01h10, MS14h03, CH02C09, SA09, SA14, SA19.1 – locus name, 1 – 13 – locus number. Light blue rectangle - the occurrence of two alleles in the specific individual and locus, dark-blue rectangle - the occurrence of three alleles in the specific individual and locus, white rectangle – missing data. Figure S1. The specific numerical distribution of the observed alleles in the studied loci for tested Sorbus genera. A1-23, C1-20, J1-30 – species adherence and number of individual . Doted lines were showing the direction of the total variance of PCA. Figure S3. The grouping of species based on principal component analysis (PCA). Abbreviations of morphological features according to Sorbus aria, I – Sorbus intermedia, C – Sorbus carpatica. The left and bottom axes belong to the vectors of primary variables and the top and right axes belong to the scores of the samples (dots).Figure S2. Scatterplot of the DAPC of genetic differentiation data for tested Sorbus species. The diagram showed the first two principal components of the DAPC using species adherence as prior clusters. Individuals representing different Sorbus species (after identification on the field) were shown by different colors .The data set and


CV) can be attributed to the environment or genetics under natural conditions. Here, we examined 260 individuals from 13 locations of the rare (semi‐)dry calcareous grassland species Trifolium montanum L. in terms of iFDCV, within‐habitat heterogeneity, and genetic diversity. The iFDCV was assessed by measuring functional traits . Abiotic within‐habitat heterogeneity was derived from altitude, slope exposure, slope, leaf area index, soil depth, and further soil factors. Based on microsatellites, we calculated expected heterozygosity (He) because it best‐explained, among other indices, iFDCV. We performed multiple linear regression models quantifying relationships among iFDCV, abiotic within‐habitat heterogeneity and genetic diversity, and also between separate functional traits and abiotic within‐habitat heterogeneity or genetic diversity. We found that abiotic within‐habitat heterogeneity influenced iFDCV twice as strong compared to genetic diversity. Both aspects together explained 77% of variation in iFDCV . The majority of functional traits  were related to abiotic habitat conditions indicating responses to environmental heterogeneity. In contrast, only morphology‐related functional traits  were related to genetics. Our results suggest that both within‐habitat heterogeneity and genetic diversity affect iFDCV and are thus crucial to consider when aiming to understand or predict changes of plant species performance under changing environmental conditions.Intraspecific trait variation (ITV), based on available genetic diversity, is one of the major means plant populations can respond to environmental variability. The study of functional trait variation and diversity has become popular in ecological research, for example, as a proxy for plant performance influencing fitness. Up to now, it is unclear which aspects of intraspecific functional trait variation  can be attributed to the environment or to genetics under natural and present environmental conditions. Here, we used data from an extensive field study  on the (semi-)dry calcareous grassland species Trifolium montanum (mountain clover). We demonstrated that abiotic within-habitat heterogeneity had twice as much impact as genetic diversity on ITV explaining together 77% of variation. Phenotypic plasticity, that is, the phenotypic variation expressed by a single genotype under different environmental conditions . Investigations are based on 260 individuals from 13 Central European populations of the rare (semi‐)dry calcareous grassland species 22.1Trifolium montanum L. (Fabaceae) is a perennial herb of extensively used, calcareous grasslands distributed in Europe and Western Russia , and thus, the majority of mating events occur on small distances  and from May to June 2016 (Germany).From each location, we collected 20 individuals of a 2.3As traits change with the season  with Pocket PEA, preparing leaves according to the manufacturer's instructions  is related to the efficiency of PS II electron transport and indicates abiotic and/or biotic stress due to photoinhibition  per individual [g]. We determined the weight of two fresh leaves per individual, conducted leaf scans, and calculated leaf area (LA) using the “LeafTraits” R package vers. 1.0 . RH and AGB are related to competitive ability  as guard cell length [µm] * guard cell width  were determined as a measure of all potentially exchangeable cations in total (CECpot), for sodium (CECNa), potassium (CECK), calcium (CECCa), and magnesium (CECMg). Soil reaction was obtained by using soil suspension mixed with deionized water  and potassium chloride (pHKCl). Furthermore, soil contents were analyzed concerning organic carbon (Corg) [%], lime (Canorg × 8.3 = CaCO3) [%], nitrogen (N) [%], plant‐available phosphor (P) [mg/100 g], and plant‐available potassium (K) [mg/100 g]. All soil analyses were conducted in the soil laboratory of the Thüringer Landesanstalt für Landwirtschaft und Ländlichen Raum (TLLLR) following standardized protocols. Mean annual temperature (Ta) and mean annual precipitation (Pa) were interpolated for study locations using ArcMap  and data from WorldClim 1.4 global climate database from 1960 to 1990 .Per replicate, we assessed GPS coordinates and altitude [m.a.s.l.] , slope exposure [°] and slope [°] . In addition, LAI  and soil depth [cm] were measured five and ten times, respectively. We also took five random soil samples per replicate , and 1.5 µl of undiluted template DNA. PCR cycling was performed using primer‐specific annealing temperatures (ta): 65°C (ats002 and ats032), 51°C (ats006), 58°C (ats029), 56°C (Tm10 and Tm12), 55°C (Tm21 and Tm24), and 61°C (Tm16). We carried out locus‐specific touchdown programs to increase PCR specificity  to (ta) to (ta − 5°C)/ 45 s, extension 72°C/45 s]; (c) 9× (IRD 700) and 15× (IRD 800) ; 15× (d) ; and (e) final extension 72°C/30 min. All PCRs were stored at 4°C. We combined differently labeled PCR products for electrophoretic analyses of fragment lengths using a LI‐COR Long Readir 4200 .We isolated DNA of sampled individuals from approximately 20–25 mg dry leaf material using a modified CTAB protocol  instead of the coefficient of variation (R2). This has the benefit of avoiding model overfitting in R2 calculation  were performed with R vers. 3.6.0 . We also checked collinearity among traits, that is, when two or more traits were highly correlated  due to non‐normal distribution of data. The r package “corrgram” vers. 1.13  were below ~│.7│, we did not exclude particular functional traits  in functional traits, we excluded all records  from the dataset with a distance of >4 standard deviations from the mean of all individuals  within a species  is appropriate for studying environmental or genetic effects on ITV.ITV  can be calculated in different manners. Functional trait dissimilarity among or within species is used to calculate FD via trait distance matrices and dendrograms , AGB (CVAGB), LA (CVLA), SLA (CVSLA), LDMC (CVLDMC), Fv/Fm (CVFv/Fm), PI (CVPI), SPS (CVSPS), and SPI  due to non‐normal distribution of data. The r package “corrgram” vers. 1.13 , and CECMg (rSP = .50), and one variable due to an almost complete absence of variation , slope exposure (CVslope exposure), slope (CVslope), leaf area index (CVLAI), soil depth (CVsoil depth), potential soil cation‐exchange capacity (CVCECpot), pH (CVpH), soil nitrogen content (CVN), soil phosphor content (CVP), and soil potassium content  with CLUMPP vers. 1.1.2 . Population size of T. montanum was positively related to genetic diversity , and varying genetic diversity is needed to examine its effect on iFDCV.We scored microsatellite fragments with an internal size standard. We proved the scoring procedure at least three times and removed ambiguous results. Analyses were conducted for all individuals characterized by at least four microsatellite loci resulting in a final sample size of A) and mean values of private allelic richness (PAp), observed (Ho) and expected heterozygosity (He), and Shannon´s diversity index (I), using GenAlEx vers. 6.503  are saturated within populations/habitats, we used an r script .To examine whether iFD2.6.5CV is related to within‐habitat heterogeneity and genetic diversity (and to assess which specific genetic diversity index best explains iFDCV), we employed a multiple linear regression model with iFDCV as the dependent variable, and HD and genetic diversity indices NA, PAp, Ho, He, and I as explaining, independent variables. To fulfill statistical assumptions, we standardized independent variables to zero mean for unit variance (z‐transformation). We conducted linear regression model simplification with the standard backward selection approach by always excluding the least significant variable (p > .1) until the final minimal adequate model was attained  as explaining, independent variables  compared to the other indices  as genetic diversity .To assess whether iFDCV and HD or GD  within linear regression models, we calculated Moran's I  comprised only six to 11% significant p values on average with different increment settings across linear regression models indicating only very weak spatial autocorrelation. Hence, we did not consider models accounting for spatial autocorrelation.To test for spatial autocorrelation among populations/habitats  to achieve normal distribution. We used the rcorr function within the r package “hmisc” vers. 4.2‐0  and genetic diversity , accounting for 67.42% (p < .001) and 32.58% (p < .05) of explained variation, respectively .The results of the multiple linear regression model demonstrated that iFDCV and HD, and iFDCV and GD were also statistically significant  whereas a linear, though weaker, relationship was found between iFDCV and GD .Individual relationships between iFDt Figure : The iFDT. montanum populations were not significantly related in our study . We observed saturation of all diversity variables. For iFDCV, HD and GD, the curves began to saturate between 5–10, 4–5, and 10–15 samples, respectively  and HD than between functional traits  and GD. CVRH is strongest related to HD (R2 = .73), followed by CVSLA (R2 = .39), CVFv/Fm (R2 = .39), CVPI (R2 = .39), CVAGB (R2 = .37), and CVLDMC (R2 = .28). With GD, CVLA (R2 = .34), CVAGB (R2 = .32), and CVRH (R2 = .29) exhibited significant relationships of similar strength.Most functional traits were significantly positively related to HD and some to GD  and CVslope . Many traits were positively correlated to CV of soil nutrients, that is, CVN (CVRH and CVAGB), CVP (CVSLA and CVLDMC), and CVK (CVFv/Fm and CVPI). No significant trait CV correlations were found to CValtitude, CVLAI, CVsoil depth, CVCECpot, and CVpH. Correlations between trait CVs and environmental CVs revealed widely positive coefficients (~71%).We found  significant positive correlations between functional traits and abiotic environmental factors chosen to calculate HD Figure : Some tr4CV) of T. montanum can be attributed to a high extent (77%) to both abiotic within‐habitat heterogeneity and population genetic diversity under natural environmental conditions  was predominantly correlated to variation of slope characteristics and soil factors . High soil potassium content was mainly found in CaCO3‐rich soils, which T. montanum prefers . In general, several functional traits may respond simultaneously to environmental heterogeneity in T. montanum habitats. Intraspecific functional trait variation captured by iFDCV is probably the response of populations to increased abiotic and biotic environmental differences within their habitats physiology‐related traits  and genetic diversity within species  best‐explained iFDCV. He is based on allelic structure, represents genotype and allele frequencies, and is less sensitive to population history , we did not observe a significant effect. Several reasons can explain this result. Large phenotypic plasticity of traits can enable individuals to inhabit different environmental niches potentially shielding them from natural selection  due to sheep and goat grazing (transhumance). Connectivity would have allowed for gene flow among them  indicating an association between environment and genetics (and trait variation) within habitats of a species  characterized by large population sizes , genetic diversity, and iFDCV. More environmental replicates would have potentially led to higher within‐habitat heterogeneity estimates additionally strengthening positive relationships among iFDCV, within‐habitat heterogeneity, and genetic diversity.Relations between iFDy Figure  are repoCV, HD, and GD) is an important feature. Unsaturated variables can bias relationships, and can lead to false results and conclusions. In T. montanum, at least five to 10 samples were sufficient to saturate iFDCV within populations  environmental changes  and provides empirical evidence to encourage the incorporation of intraspecific functional trait variation into interspecific comparisons ; Investigation ; Methodology ; Software ; Visualization ; Writing‐original draft ; Writing‐review & editing . Kathleen Prinz: Conceptualization (lead); Methodology ; Project administration ; Supervision ; Writing‐original draft ; Writing‐review & editing . Frank Hellwig: Conceptualization (lead); Project administration ; Writing‐original draft (supporting); Writing‐review & editing (supporting). Christine Römermann: Conceptualization (lead); Formal analysis ; Investigation ; Methodology ; Project administration ; Supervision ; Writing‐original draft ; Writing‐review & editing . The study was designed by K.P., F.H., and C.R. K.K. sampled and analyzed the data Statistical data analyses were supported by K.P. and C.R. K.K. wrote the manuscript with equal contributions from K.P., F.H., and C.R.Supplementary MaterialClick here for additional data file.