Abstract
Hybridization and introgression have resulted in reticulate evolution within the genus Populus. Consequently, the origin and evolutionary history of some hybrids has become blurred. P. hopeiensis and P. tomentosa are endemic to China, and there is still controversy about their origin. We employ phylogeny, Bayesian estimation of admixture, and approximate Bayesian computation to investigate their origin with 10 nuclear DNA and 6 cpDNA regions. The combined evidences firmly support the hypothesis that they are hybrids and dominated by F1s. P. hopeiensis was generated via hybridization between the paternal species P. alba and maternal species P. davidiana. Surprisingly, P. tomentosa was divided into two genetic types with different maternal parents. P. adenopoda hybridized with P. alba directly to generate the first genetic type (mb1) and hybridized with P. davidiana followed by P. alba to generate the second (mb2). In both genetic types, P. alba acted as the male parent. The maternal parent was P. adenopoda and P. davidiana for mb1 and mb2, respectively. Hybridization not only generated these hybrids but also resulted in a unidirectional gene flow from P. davidiana to P. adenopoda. The Populus species have maintained a delicate balance between their genetic integrity and gene exchange.
Similar content being viewed by others
Introduction
It has long been known that hybridization and introgression play key roles in evolution1,2. Natural interspecific hybridization has been estimated to be present in approximately 25% of all plant species3. Hybridization can trigger speciation4 but can also blur species boundaries and complicate the evolutionary history of related taxa5. Many taxonomic debates regarding the origins of various taxa have resulted from the confusing artifacts of hybridization and followed introgression6,7,8. Introgression, the movement and subsequent stable incorporation of genes from one species to the gene pool of another, is caused by hybridization and repeated backcrossing9. Thus, introgression is a special conduit for gene flow10. The spatial scale and geographic pattern of introgression are influenced by a variety of factors, including natural selection, individual dispersion distance and the place where the hybridization took place11.
Hybrid zones provide a window for the study of hybrid production and interspecific gene flow. A typical hybrid zone is a narrow region where different species meet, exchange genes, and produce hybrids12. Many hybrids and introgressive hybridization in Populus are found in these typical hybrid zones13,14. However, some hybrids jump out of the typical hybrid zone, appearing a few hundred kilometers from the nearest parent15. The geographical isolation creates conditions for production of hybrids, but also makes it difficult to distinguish them and verify their parentage. Cytonuclear discordance, which is a topological incongruity between the maternally inherited cpDNA (chloroplast DNA) and biparentally inherited nuclear DNA16,17, has received much attention within hybrid zones. Cytonuclear discordance is a useful tool to decipher the direction of introgression and whether certain species are the parental contributors to hybrids18.
Hybridization and introgression also appear to have played an essential role in diversification of Populus L. given that many members were involved in ancient hybridization events15,19,20. Species of section Leuce Duby tend not to hybridize with the other five sections of Populus (Abaso, Aigeiros, Leucoides, Tacamahaca, and Turanga), but exhibit widespread intrasectional hybridization and introgression19,21. Section Leuce itself is even thought to originate from hybridization between members of sections Leucoides and Turanga22. As such, the number of species in section Leuce remains under debate, varying from 8 to 1015,23,24. Some species are sympatric, providing many opportunities for hybridization and introgression and following some natural hybrids and/or interspecific gene flow25. Some natural hybrids, such as P. canescent (Ait.) Smith., survive by asexual reproduction26,27. The origins and evolutionary histories of some hybrid poplars have been studied extensively28,29, but are still ambiguous for some native poplars in China.
Both P. hopeiensis Hu & H. F. Chow and P. tomentosa Carr. are native to China with high quality timber24,30,31. Indeed, the origins of P. hopeiensis and P. tomentosa have been subject to much debate. Based on morphological characteristics, it is hypothesized that P. tomentosa and P. davidiana Dode are the parent species of P. hopeiensis24, but this hypothesis is rejected after reciprocal cross experimentation32. However, P. hopeiensis is suspected to have a close relationship with P. davidiana33. Despite the potential value of P. hopeiensis for the evolutionary analysis of hybridization, the genetic documentation of its putative parents is lacking.
The origin of P. tomentosa is more ambiguous; even the number of its parental species is debated. Although several workers have suggested that P. tomentosa originated from the hybridization of two taxa, varying parental species have been proposed. Bartkowiak concludes that P. tomentosa is derived from P. alba L. × P. tremula L. (♀ × ♂) based on the characters of parviflorous bracts34. In contrast, P. alba and P. davidiana are believed to act as the parents of P. tomentosa with morphological and anatomical characters35. RAPD (random amplified of polymorphic DNA) analysis suggests that P. tomentosa originated from P. alba (♀) and P. adenopoda Maxim. (♂)36. However, phylogeny of the Populus genus based on DNA sequences indicates that P. tomentosa is derived from P. davidiana (♀) and P. adenopoda (♂)33. In contrast, other researchers have suggested that P. tomentosa has more than two parental species37,38. Isoenzyme analysis indicates that three species, P. alba, P. adenopoda and P. davidiana, formed P. tomentosa, but the author unfortunately did not identify the maternal or paternal parent37. Although many studies have proposed that P. tomentosa is a natural, spontaneous hybrid, no solid evidence is available to verify its parents.
Numerous investigations have sought to clarify the origin of P. hopeiensis and P. tomentosa. Unfortunately, a dearth of studies based on gene sequence impedes the identification of their origin. Previous studies have been hampered partly by limited sampling. Indeed, nearly all the Eurasian species in section Leuce (P. alba, P. tremula, P. adenopoda, and P. davidiana) have been proposed as parent species of P. hopeiensis or P. tomentosa. In addition, close relationships occur among P. tremula, P. tremuloides Michaux, and P. grandidentata Michx15,39, making characterizing parentage challenging. Therefore, to clarify their origins, we used 392 individuals in 36 populations of 8 taxa with 10 nuclear DNA and 6 cpDNA sequences. More generally, we aimed to improve our understanding of hybridization and introgression in section Leuce and to lay the foundation for the conservation of genetic resources and breeding innovation.
Results
Polymorphic analyses and neutral test
The length of the aligned nuclear DNA sequences ranged from 388 to 808 bp, and the concatenated length of all ten nuclear loci was 5715 bp (Table S1). The aligned cpDNA sequences ranged from 945 to 2551 bp, and the concatenated length of all six cpDNA loci was 7741 bp (Table S1).
DnaSP v540 was used to analyze polymorphic and test neutrality of variation. All taxa had generally high nucleotide diversity in the nuclear loci (Table S2), ranging from 0.00281 (P. grandidentata) to 0.00802 (P. adenopoda). The overall nucleotide diversity of cpDNA was lower than that of nuclear DNA, ranging from 0.0001 (P. grandidentata) to 0.00103 (P. davidiana).
Some of the nuclear loci significantly departed from neutrality based on Tajima’s D, Fu and Li’s D* and Fu and Li’s F*, such as locus 6 in P. tremuloides and locus YLT24 in P. tremula (Table S2). Conversely, our MLHKA analysis only supported a significant difference between the neutral model and the selection model at locus 6 of P. tremuloides (Table S3).
Phylogenetic analyses
We firstly recovered ten individual nuclear phylogenetic trees of section Leuce. In these individual gene trees, it was found that most of the P. hopeiensis were clustered together with P. alba, P. davidiana and P. tomentosa, whilst the most P. tomentosa were clustered together with P. alba, P. davidiana, P. adenopoda and P. hopeiensis. Some P. adenopoda were clustered together with P. davidiana (Fig. S1).
Phylogenies of both concatenated nuclear DNA and cpDNA datasets were well resolved (Figs 1 and S2). Section Leuce formed a highly supported (Posterior probability, PP = 1) clade with P. grandidentata sister to the remaining taxa in the concatenated nuclear phylogenetic tree. The others fell into two major clades (clade A and B). Within clade A, some P. tomentosa sequences grouped with P. adenopoda in clade A1 with high support (PP = 1) and these were sister to clade A2. The other P. tomentosa sequences, P. hopeiensis and P. alba, formed the well supported clade A2 (PP = 0.96). Although P. tomentosa was located in different clades, for any sample of P. tomentosa, one of its two sequences is located in clade A1. The other sequence is located in clade A2. Clade B was composed of P. davidiana, P. tremula, P. tremuloides and a few P. adenopoda sequences that were lowly resolved. Although sequences from the same species did not always group together, clusters formed by sequences from different species were poorly supported (Fig. S2).
Three cpDNA trees also had approximately the total same topology (Figs 1, S3 and S4). The positions of eight taxa were identical across trees with minor differences only noted in the terminal positions of some haplotypes within a taxon. Similar to the nuclear DNA phylogeny, section Leuce was monophyletic with high support across all cpDNA phylogenetic analyses (Bootstrap, BS = 100, Fig. 1). The analysis also revealed that P. grandidentata diverged first with clade C and clade D, forming a sister relationship. The moderately supported group C (BS = 87) was composed of P. adenopoda, P. alba, and the major haplotypes of P. tomentosa (named by genetic types P. tomentosa mb1; Table 1, Fig. 1). P. tomentosa mb1 (15 haplotypes, 217 individuals) first grouped with P. adenopoda, and they were sister to P. alba (BS = 87). Clade D was also recovered with weak support (BS = 65) and contained P. hopeiensis, P. davidiana, P. tremula, P. tremuloides, and the remainder of the haplotypes of P. tomentosa (P. tomentosa mb2; Fig. 1). P. tremuloides was sister to others in this group. P. tomentosa mb2 (4 haplotypes, 18 individuals) clustered with P. hopeiensis and P. davidiana (BS = 89) with P. tremula sister to these with good support (BS = 94).
Genetic structure analyses based on ten nuclear DNA
Clustering analysis for nuclear DNA was conducted using principal component analysis (PCA) in GENALEX 6.5 to detect complex patterns of genetic structure41. The PCA based on genetic distance demonstrated that P. hopeiensis was located between P. davidiana and P. alba with closer affinity with the later. Whilst P. tomentosa was located among P. adenopoda, P. alba and P. davidiana, with a closer affinity with the first two. It is worth noting that P. adenopoda is divided into two clusters (A and B), cluster B is located between cluster A and P. davidiana. Cluster B is composed of those individuals which appear to have undergone introgression with P. davidiana (discussed below; Fig. 2).
To assign the individuals to the populations and estimate potential admixture, STRUCTURE42 was used to estimate the overall genetic structure of all taxa with 10 nuclear loci. STRUCTURE analysis indicated that all populations likely fell into three genetic clusters at the optimal number of modelled populations (K = 3, usepopinfo = 1) (Fig. S5A,B). At K = 3, P. adenopoda fell into one cluster with four individuals (three from Hubei and one from Guizhou) mixed by other species. P. alba fell into another cluster, and P. davidiana, P. grandidentata, P. tremuloides and P. tremula fell into the third cluster (Fig. 3A). P. hopeiensis were a mixture of two clusters (Fig. 3A). P. tomentosa could be split into two genetic types (P. tomentosa mb1 and mb2) along the division recovered in the cpDNA phylogenies. Most individuals of P. tomentosa mb1 were a mixture of P. adenopoda and P. alba, and P. tomentosa mb2 was a mixture of all three clusters (Fig. 3A). It is notable that four individuals assigned to P. tomentosa mb1 based on our phylogenetic analysis (three individuals from Shaanxi and one from Hebei) were shown to be a mixture of all three clusters (Fig. 3A). Although the genetic structure of P. hopeiensis and P. tomentosa were unclear at K = 3, genetic affinities were clearer at higher values of K combined with phylogeny results. The clustering patterns at K = 4 and K = 5 indicated that P. alba and P. davidiana contributed genetic material to P. hopeiensis, whereas P. davidiana contributed to P. tomentosa mb2 and some individuals of P. tomentosa mb1. Finally, the mosaic individuals of P. adenopoda were admixed with P. davidiana (named admixed P. adenopoda for convenience). The most likely number of clusters was k = 2 when the STRUCTURE analysis was conducted without location information (usepopinfo = 0) (Fig. S5C,D). The cluster results generated without location information were very similar to those generated with location information (Fig. S6).
To further investigate these hypothesized clusters, we performed four hierarchical analyses. We first analyzed P. hopeiensis, P. alba, and P. davidiana alone. Our results indicated that the most likely number of clusters was K = 2 (Fig. S5E,F). In addition, P. alba and P. davidiana clustered in distinct groups with high probability (Fig. 3B). P. hopeiensis was admixed and assigned to both clusters with moderate probability. P. hopeiensis was assigned to a discrete cluster at K = 3 (Fig. 3B). We next analyzed P. tomentosa mb1, P. alba, and P. adenopoda alone. In this analysis, we found that P. tomentosa mb1 was a mixture of P. alba and P. adenopoda at optimal cluster K = 2 but was recovered as a distinct cluster at K = 3 (Figs 3C and S5G,H). To investigate the precise genetic composition of P. tomentosa mb2, we analyzed P. alba, P. davidiana, and ‘unmixed’ P. adenopoda. At optimal cluster K = 3, P. alba, P. davidiana and ‘unmixed’ P. adenopoda were recovered as separate clusters, whereas P. tomentosa mb2 was a mixture of all three (Figs 3D and S5I,J). At K = 4, P. tomentosa mb2 was mixed with 2 clusters, including P. alba. At K = 5, P. tomentosa mb2 was recovered as a distinct cluster (Figs 3D and S5I,J). Finally, we analyzed P. adenopoda and P. davidiana alone. We found that at K = 2 (Fig. S5L,M), some individuals of P. adenopoda showed mixing from P. davidiana, and these individuals did not form a distinct cluster at K = 3(Fig. 3E).
Classification analysis
Based on the genotype posterior probability, Newhybrids v1.143 was used to identify and characterize the hybrids in the admixed populations. Newhybrids analysis indicated that all samples could be confidently assigned to a particular genotype class. This analysis recovered all specimens of P. alba and P. davidiana as pure parents with high support (posterior probability (PP) >99; Fig. 4A). Most specimens of P. hopeiensis were classified as F1s; only two individuals were classified as F2s (Fig. 4A). Similar results were obtained when analyzing the putative parents of P. tomentosa mb1 (Fig. 4B). All specimens of P. alba and P. adenopoda were recovered as pure parents, whereas all specimens of P. tomentosa mb1 were classified as F1s. This finding also indicated the hybrid origin of P. hopeiensis and P. tomentosa mb1.
Demographic estimates using Bayesian approximation
To further investigate the origin pattern of P. hopeiensis and P. tomentosa (mb1 and mb2), seven alternative scenarios (Figs 5 and 6) were summarized and tested with DIYABC V 2.1.044. In the analysis of P. hopeiensis, scenario 5 exhibited the highest support (PP = 0.9700; 95% confidence interval: 0.9445–0.9912; Table 2), suggesting that P. alba and P. davidiana hybridized and generated P. hopeiensis (Fig. 5).
In our analysis of P. tomentosa mb1, scenario 5 was again the most well supported model (PP = 0.9780; 95% confidence interval: 0.99972–1.0000; Table 2). This scenario suggests that P. tomentosa mb1 originated from the hybridization of P. alba and P. adenopoda (Fig. 5). Our analyses of P. tomentosa mb2 indicated that scenario 6 was the most likely (PP = 0.6753; 95% confidence interval: 0.5934–0.9538; Table 2). These results suggest that P. davidiana hybridized with P. adenopoda first, then hybridized with P. alba and generated P. tomentosa mb2 (Fig. 6).
Discussion
Our results suggested that the genetic relationships among taxa in section Leuce were more complex than expected. Phylogenetic analyses show that P. hopeiensis clusters with P. davidiana, P. alba and some P. tomentosa in our nuclear DNA phylogeny and with P. davidiana in cpDNA phylogeny (Figs 1, S1 and S2). The change of phylogenetic location implies a hybrid origin45. P. hopeiensis is a mosaic of P. alba and P. davidiana at K = 2, but it is assigned to a unique cluster at K = 3 (Fig. 3B), which is strongly indicative of hybrid origin, moreover, this is emerging as a common pattern in many other hybrids46. DIYABC simulation analyses also suggest that P. hopeiensis is generated by hybridization between P. davidiana and P. alba. Indeed, it is believed that P. hopeiensis has obvious lineage of P. davidiana due to the morphological similarities between them, and it is likely formed by asexual propagation47. It is reasonable to believe that P. davidiana served as the female parent of P. hopeiensis since they are sister groups in the cpDNA phylogenetic tree (Fig. 1).
Our NewHybrids analysis suggests that almost all individuals of P. hopeiensis we sampled are F1s, only two are F2s, and no post F2 hybrids are found (Fig. 4A). The progeny of P. hopeiensis, obtained by crossing experiments, are morphologically variable, which seems to prove this point47. In fact, many hybridizations within genus Populus are also limited to F1s48. For instance, the great majority individuals of P. × canescens are F1 hybrids between P. alba and P. tremula29. Here, it seems plausible that advanced generations P. hopeiensis would occur naturally given the presence of some F2s and especially because we observe several large individuals of P. hopeiensis that are old enough to produce advanced generation hybrids. It is also surprising that no individual is identified as the product of the backcrossing of P. hopeiensis with either parent species. The absence of post-F1 hybrids may be resulted from hybrid sterility and F2 breakdown49.
Our analysis proves that P. tomentosa is also a hybrid. A concatenated nuclear phylogenetic tree was recovered with statistical phasing of alleles from original direct sequences. For any individual of P. tomentosa, one of its haplotype sequences is clustered with P. adenopoda, and the other is grouped with P. hopeiensis and P. alba in the nuclear phylogenetic tree (Fig. S2). This topology is the same as that of hybrid origin of P. tomentosa. Two distinct genetic types of P. tomentosa (mb1 and mb2) are identified in the cpDNA tree where mb1 clusters with P. adenopoda, and mb2 clusters with P. davidiana and P. hopeiensis (Fig. 1). Therefore, phylogenetic analyses imply P. tomentosa exhibits close affinity with P. hopeiensis, P. alba, P. adenopoda and P. davidiana. It is not surprising that P. tomentosa has a close relationship with P. hopeiensis, considering that P. hopeiensis is a hybrid of P. alba and P. davidiana. Clustering results of PCA also suggest that P. tomentosa is related to P. adenopoda, P. alba and P. davidiana, and has a closer affinity with the first two (Fig. 2). P. tomentosa could be split into two genetic types (mb1 and mb2) along the division recovered in the cpDNA phylogenies (Fig. 3A). P. tomentosa mb1 is a mixture of P. adenopoda and P. alba (Fig. 3A, K = 3; Fig. 3C, K = 2), and it is recovered as a distinct population isolated from P. alba and P. adenopoda at K = 3 (Fig. 3C). This result strongly suggests that P. tomentosa mb1 is a natural hybrid between P. adenopoda and P. alba. P. adenopoda is the female parent, because P. tomentosa mb1 first clusters with P. adenopoda in the cpDNA tree, and without doubt, P. alba is the paternal parent. DIYABC also provides further evidence for a hybrid origin of P. tomentosa mb1 (Table 2, Fig. 5). Although we detect a small amount of genetic material from P. davidiana in four individuals of P. tomentosa mb1 (Fig. 3A), we hypothesize that this material is contributed by admixed individuals of P. adenopoda through hybridization with P. alba to generate P. tomentosa mb1(discussed below).
STRUCTURE analysis indicates that the genetic material of P. tomentosa mb2 is a mosaic of various other groups of taxa (Fig. 3A): P. alba, P. adenopoda, and P. davidiana; P. alba, P. davidiana, and P. tomentosa mb1; or P. alba, P. adenopoda, and P. hopeiensis. Therefore, five taxa (P. alba, P. adenopoda, P. davidiana, P. tomentosa mb1, and P. hopeiensis) are potential parents of P. tomentosa mb2. P. tomentosa mb2 first clusters with P. davidiana in cpDNA phylogeny (Fig. 1), which indicates that P. davidiana is most likely to serve as the maternal parent in single cross or in either cross of trihybrid cross. Therefore, possible combinations (♀ × ♂) are P. davidiana × (P. adenopoda × P. alba) (same as P. davidiana × P. tomentosa mb1); (P. davidiana × P. adenopoda) × P. alba; (P. davidiana × P. alba) × P. adenopoda (same as P. hopeiensis × P. adenopoda); and P. davidiana × (P. alba × P. adenopoda). DIYABC analysis suggest that trihybridization (P. davidiana × P. adenopoda) × P. alba is the most probable pattern (Table 2, Fig. 6). Subsequent hierarchical STRUCTURE analyses also support these three as potential parent species (Fig. 3A,D). Importantly, these are the parents proposed by Song37. Dickmann also speculated that P. tomentosa was a signal cross hybrid or a trihybrid and that its parents may have been either P. alba and P. adenopoda or P. alba, P. adenopoda, and P. tremula (note that, in this work, P. davidiana is referred to as a geographic variety of P. tremula)50. Although natural three-way hybrids have been reported, they are very rare to our knowledge13,20. For example, only a single trihybrid individual is detected across the three species P. deltoides, P. nigra, and P. balsamifera13. We identified 18 individuals as trihybrids, which represents 7.7% of all P. tomentosa specimens examined (Table 1). This rarity may be caused by the difficulty of trihybridization.
All P. tomentosa mb1 examined are F1 hybrids (Fig. 4B), explaining why P. tomentosa fertility is low. We predict that P. tomentosa had originated at least twice and in multiple regions because two types with different parents existed in different regions. P. tomentosa mb1 and mb2 with different origins and different genetic characteristics are likely to be perpetuated by asexual propagation35. In fact, asexual propagation is a very common in Populus27,51. Together, P. tomentosa mb1 and mb2 increase the diversity of P. tomentosa and have caused many debates about its origins.
Four specimens that we identify as P. adenopoda based on overall morphology (three from Hubei and one from Guizhou) exhibit several morphological similarities to P. davidiana, including oval leaves that are not glandular punctate. These individuals are clustered together with P. davidiana in nuclear phylogenetic tree and locate between P. davidiana and other P. adenopoda individuals in PCA analysis, suggesting their close relationship with P. davidiana (Figs 2 and S2). STRUCTURE analyses indicate that these individuals are a mixture of P. davidiana, which is similar to results that might be expected of a hybrid (Fig. 3A). We postulate that this genetic pattern is the result from introgression by P. davidiana. This hypothesis is strongly supported by the STRUCTURE analysis, which demonstrates that the admixed individuals of P. adenopoda display a mosaic cluster pattern at K = 2 and K = 3 (Fig. 3E). We would expect that a true hybrid would fall into a single cluster at higher K values (inferred from Gompert, et al.46). Single copy and neutral nuclear DNA markers have previously been used to refute alterative hypotheses, such as convergence and symplesiomorphy51. Indeed, introgression of varying degrees has been demonstrated in 82 genera of angiosperms, including Populus and Salix9. Finally, we observe this admixture in only a few specimens of P. adenopoda, indicating that gene flow between the species is restricted to a very small area. Heiser termed this pattern ‘localized introgression’52. This finding is consistent with the geographic scale of introgression, which is dependent on the geographical location of hybridization and the dispersal ability of hybridized offspring11. Species that are incompletely genetically isolated may exchange genes uni- or bidirectionally53. It is worth noting that the gene flow is unidirectional from P. davidiana to P. adenopoda. Such asymmetric gene flow has previously been demonstrated to be common in Salicaceae25,54. For example, asymmetric introgression has been detected from P. fremontii Wats. (section Aigeiros) to P. angustifolia James (section Tacamahaca)55. Although the causes of unidirectional gene flow remain unclear, contributing factors may include incongruent flowering times;56 species abundance effects within the hybrid zone25,57; species biases, where only those hybrids having a particular maternal species are viable58; epistatic interactions13; and heterogametic sex determination59. Our field investigations indicate that P. adenopoda and P. davidiana have similar flowering times (March to April) and are present in similar numbers in adjacent areas. In addition, both phylogeny and STURCTURE analyses indicate that either P. adenopoda or P. davidiana could act as female parents for P. tomentosa. Thus, epistatic interactions and heterogametic sex determination might be the key for unidirectional gene flow in poplar, but this hypothesis requires more testing.
Materials and Methods
Poplar taxa and individuals
We selected P. hopeiensis, P. tomentosa, P. alba, P. adenopoda, P. davidiana, P. tremula, P. tremuloides, and P. grandidentata as the objects of this study. We performed range-wide sampling of representative populations from 2010 to 2016 (392 individuals in 36 populations of 8 taxa, detailed sampling information is listed in Table S4, S5). Although triploids have been found in P. tomentosa, we did not collect them based on our previous records60. Conspecific specimens collected from the same geographic location were grouped as ‘populations’, for convenience, even though some ‘populations’ included only a few individuals. P. lasiocarpa Oliv. (section Leucoides) was selected as an outgroup based on previous work33. Fresh leaves of all selected trees were collected and stored in silica gel.
DNA extraction, polymerase chain reaction (PCR), and sequencing
Total DNA was isolated from collected leaves with a Plant Genomic DNA Kit DP320 (Tiangen, Beijing, China). The integrity of all DNA was tested with 1% agarose gel electrophoresis.
We used six cpDNA primer pairs and ten single-copy nuclear DNA primers for PCR and sequencing (Table S6). Four of the cpDNA primers (trnk, psbM-trnDGTC, rpoB-trnCGCA, and atpI-atpH) were modified from Demesure, et al.61 and Shaw, et al.62; the remaining two (YLT9, YLT24) were from Wang, et al.63. Nine of the nuclear DNA primers were modified from Du, et al.39.
PCR amplifications were performed in a reaction volume of 30 μL, containing 3 μL 10 × PCR Buffer, 0.12 mM dNTPs, 0.75 U Taq DNA polymerase, 0.2 μM of each primer, and 30 ng of genomic DNA. PCR conditions were described by Wang et al.63. PCR products were sequenced directly with the amplification primers after purification with a TIANgel Midi Purification Kit (Tiangen, Beijing, China). Bidirectional sequencing was used if the length of sequences was greater than 800 bp. When a clear sequence was not obtained, PCR products were cloned with the pGEM-TEasy Vector System II (Promega, Madison, USA). Then, six to ten positive clones were randomly selected and sequenced with M13. Sequences generated were deposited in GenBank (accessions numbers MF512193-MF521199 and MG202418-MG203618). All the sequences were aligned and refined with Bioedit64. The phase program with default algorithm in software DnaSP v5 was used to phase the alleles of nuclear DNA, with the clone sequences of P. tomentosa as the known sequence40,65. In this process, degenerate bases of the original sequence will be diverged. The diverged haplotype sequences then were used for subsequent analyses.
Neutral test and genetic diversity analyses
DnaSP v540 was used to calculate nucleotide diversity (π)66, Watterson’s parameter (θw)67, the number of segregating sites (S), and the number of haplotype (Nh) for each loci of all taxa.
To test the neutrality of variation, we used DnaDP v540 to calculate Tajima’s D68 as well as Fu and Li’s D* and F*69. For loci that all indices calculated by DnaSP were significant, we used MLHKA70 to further judge whether they departed from neutrality. That is, the maximum likelihood (ML) ratio between the average ML value of a given locus in neutrality and the average ML value of them in nonneutrality was calculated with 3 independent runs of 100,000 sweeps each. Then, a chi-square test was performed (p < 0.05 was considered significant).
Phylogenetic analyses
We used seven tests in RDP3 to assess for potential recombination events for nuclear DNA71. The nonrecombined fragments were trimmed and further analyzed. We used the simple indel coding method in GapCoder to code all DNA indels generated after alignment72. Jmodel test 2.1.473 was used to decide the best nucleotide substitution model for all loci under the Akaike Information Criterion. Given cpDNA is maternally inherited and conserved, we combined all six cpDNA loci for phylogenetic analysis. Conversely, phylogenetic analyses of the combined nuclear DNA were performed under a partition scheme (ten data subsets: partitioned by ten loci) by using the models determined by Jmodeltest for of each locus.
PAUP* 4.0b10*74 was used to conduct a maximum parsimony (MP) analysis for cpDNA. An MP heuristic search performed with 1000 replicates of random taxon addition with tree bisection and reconnection (TBR) branch swapping, without steepest descent, and with unordered, equally weighted characters. To assess topological robustness, we performed 1000 bootstrap replicates with the same options. We used RAxML75 to analyze ML for cpDNA using two sets of 1000 rapid bootstrap replicates. ML was also performed employing IQ-TREE-1.6.6 using separate models for nuclear DNA with 1000 ultrafast bootstrap approximation (UFBoot)76. We also analyzed nuclear and chloroplast DNA with Bayesian inference in MrBayes v3.2.177. Four independent MCMC analyses were run for 3,000,000 or 100,000,000 generations each with sampling every 1000 generations. We assumed that the dataset had reached convergence when the average standard deviation of split frequencies was less than 0.01. 25% of the samples were discarded as burn-in. All trees and edge support values were visualized in Figtree v1.4.078.
Genetic structure
To detect complex patterns of genetic structure, clustering analysis for nuclear DNA was conducted with principal component analysis (PCA) in GENALEX 6.541. Phased nuclear DNA sequences were used for PCA analysis.
We first used STRUCTURE v2.3.342 to assign the eight taxa to several different genetic clusters (K) based on ten nuclear DNA loci and determine the potential parents of hybrids. Then, three hierarchical STRUTURE analyses with only hybrids and their potential parents included were run again to further verify the previous inference. We also analyzed mechanisms of gene exchange between P. adenopoda and P. davidiana with STRUTURE separately. In this process, we divided P. adenopoda into two groups depending on whether it was introgressed by P. davidiana. An admixture model with correlated allele frequencies between populations was used. Considering the relationship between P. adenopoda and P. davidiana presented in the phylogenetic tree, we used the location information (usepopinfo = 1) to detect migrant individuals in the first structure analysis (including eight taxa) but not in the later hierarchical analyses. Moreover, we also run structure without location information (usepopinfo = 0) for eight taxa. We performed ten independent runs for each possible value of K from one to ten or five with a burn-in of 100,000 followed by 200,000 MCMC iterations. The most likely value of K based on the negative natural log likelihood of the data (LnP(K)) and ∆K 79 was calculated using STRUCTURE HARVESTER80. CLUMPAK81 was used to create and visualize population bar plots. To avoid cluster departure caused by differences in quantity among different populations, only haplotypes were used in analyses.
Identification and classification of hybrids
We used Newhybrids v1.143 to classify the genotype of hybrids and their parents based on posterior probability. For P. hopeiensis and its parent species, the six genotype classes used were pure parent P. alba (Phal), pure parent P. davidiana (Phda), F1 generation, F2 generation, backcross with P. alba (Bhal), and backcross with P. davidiana (Bhda). Only P. tomentosa mb1 and its parent species were analyzed given that Newhybrids v1.1 limits the allowed number of parents. The six genotype classes used for P. tomentosa were pure parent P. alba (Ptal), pure parent P. adenopoda (Ptad), F1 generation, F2 generation, backcross with P. alba (Btal), and backcross with P. adenopoda (Btad). We performed two replicate runs with a burn-in of 1,000,000 iterations followed by 1,500,000 sweeps in Jeffreys-like and uniform prior respectively. Default genotype categories were chosen. Together, the z and s options were also used to stipulate that some P. alba and P. davidiana or P. adenopoda and P. alba standard samples are of pure origin but preventing them from affecting the estimation.
Approximate Bayesian computation
To derive a detailed origin pattern of P. hopeiensis and P. tomentosa, we compared plausible scenarios using approximate Bayesian computation (ABC) in DIYABC V 2.1.044 using ten nuclear loci. Based on the results of our phylogenetic and STRUCTURE analyses, we only analyzed hybrids and their putative parental species with ABC.
We designed seven scenarios in three categories to explain the possible alternative origin pattern of P. hopeiensis and P. tomentosa mb1 (Fig. 5). Category 1 (scenario 1) modeled an ancestral split into three populations at time t0. Category 2 (scenarios 2, 3, and 4) modeled an ancestral split into two lineages at time t0 followed by a further diversification event in one of the lineages at time t1. Category 3 (scenarios 5, 6, and 7) was modeled as the split of two species at time t0 followed by hybridization and the generation of a new lineage at time t1.
Seven alternative scenarios were designed to explain the origins of P. tomentosa mb2 (Fig. 6): (1) P. tomentosa mb2 branched off from an ancestor directly; (2) P. tomentosa mb2 descended from P. davidiana; (3) P. tomentosa mb2 descended from the ancestor of P. adenopoda and P. alba; (4) P. tomentosa mb2 was the product of hybridization between P. davidiana and the ancestor of P. adenopoda and P. alba; (5) P. adenopoda hybridized with P. alba and then further hybridized with P. davidiana to generate P. tomentosa mb2; (6) P. adenopoda hybridized with P. davidiana and then further hybridized with P. alba to generate P. tomentosa mb2; and (7) P. adenopoda hybridized with P. alba and then further hybridized with P. davidiana to generate P. tomentosa mb2.
Historical models, genetic data and summary statistics parameters were listed in Table S7. To test these alternate theories, we ran 1,000,000 simulations for each scenario and selected the 1% of the simulated data closest to the observed data used to assess the posterior probabilities of all scenarios with logistic regression44.
Data Availability
DNA sequences: Genbank accessions: MF512193-MF521199 and MG202418-MG203618.
References
Rieseberg, L. H. Hybrid origins of plant species. Annual Review of Ecology and Systematics 28, 359–389, https://doi.org/10.1146/annurev.ecolsys.28.1.359 (1997).
Barton, N. H. The role of hybridization in evolution. Molecular Ecology 10, 551–568, https://doi.org/10.1046/j.1365-294x.2001.01216.x (2001).
Mallet, J. Hybridization as an invasion of the genome. Trends in Ecology &. Evolution 20, 229–237, https://doi.org/10.1016/j.tree.2005.02.010 (2005).
Mallet, J. Hybrid speciation. Nature 446, 279–283, https://doi.org/10.1038/nature05706 (2007).
Buerkle, M. A. et al. Hybridization and speciation. Journal of Evolutionary Biology 26, 229–246, https://doi.org/10.1111/j.1420-9101.2012.02599.x. (2013).
Mcfadden, C. S. & Hutchinson, M. B. Molecular evidence for the hybrid origin of species in the soft coral genus Alcyonium (Cnidaria: Anthozoa: Octocorallia). Molecular Ecology 13, 1495–1505, https://doi.org/10.1111/j.1365-294X.2004.02167.x (2004).
Petit, R. J., Bodénès, C., Ducousso, A., Roussel, G. & Kremer, A. Hybridization as a mechanism of invasion in oaks. New Phytologist 161, 151–164, https://doi.org/10.1046/j.1469-8137.2003.00944.x (2004).
Schulte, K. et al. Detection of recent hybridization between sympatric Chilean Puya species (Bromeliaceae) using AFLP markers and reconstruction of complex relationships. Molecular Phylogenetics and Evolution 57, 1105–1119, https://doi.org/10.1016/j.ympev.2010.09.001 (2010).
Rieseberg, L. H. & Wendel, J. F. In Hybrid zones and the evolutionary process (ed Richard Gerald Harrison) 70-109 (Oxford University Press 1993).
Schemske, D. W. & Morgan, M. T. The evolutionary significance of hybridization in Eucalyptus. Evolution 44, 2151–2152, https://doi.org/10.2307/2409622 (1990).
Harrison, R. G. & Larson, E. L. Hybridization, Introgression, and the Nature of Species Boundaries. Journal of Heredity 105, 795–809, https://doi.org/10.1093/jhered/esu033 (2014).
Barton, N. H. & Hewitt, G. M. Analysis of hybrid zones. Annual review of Ecology and Systematics 16, 113–148, https://doi.org/10.1146/annurev.es.16.110185.000553 (1985).
Thompson, S. L., Lamothe, M., Meirmans, P. G., Perinet, P. & Isabel, N. Repeated unidirectional introgression towards Populus balsamifera in contact zones of exotic and native poplars. Molecular Ecology 19, 132–145, https://doi.org/10.1111/j.1365-294X.2009.04442.x (2010).
Jiang, D. et al. Genetic origin and composition of a natural hybrid poplar Populus × jrtyschensis from two distantly related species. Bmc Plant Biology 16, 89, https://doi.org/10.1186/s12870-016-0776-6 (2016).
Eckenwalder, J. E. In Biology of Populus and its Implications for Management and Conservation (ed H. D. Bradshaw R. F. Stettler, Jr., P. E. Heilman, and T. M. Hinckley) 7–32 (NRC Research Press, National Research Council of Canada 1996).
Song, B. H., Wang, X. Q., Wang, X. R., Ding, K. Y. & Hong, D. Y. Cytoplasmic composition in Pinus densata and population establishment of the diploid hybrid pine. Molecular Ecology 12, 2995, https://doi.org/10.1046/j.1365-294X.2003.01962.x (2003).
Huang, D. I., Hefer, C. A., Natalia, K., Douglas, C. J. & Cronk, Q. C. B. Whole plastome sequencing reveals deep plastid divergence and cytonuclear discordance between closely related balsam poplars, Populus balsamifera and P. trichocarpa (Salicaceae). New Phytologist 204, 693–703, https://doi.org/10.1111/nph.12956 (2015).
Mckinnon, G. E., Steane, D. A., Potts, B. M. & Vaillancourt, R. E. Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae). American Journal of Botany 86, 1038–1046, https://doi.org/10.2307/2656621 (1999).
Willing, R. & Pryor, L. Interspecific hybridisation in poplar. Theoretical and Applied Genetics 47, 141–151, https://doi.org/10.1007/BF00274943 (1976).
Floate, K. D. Extent and patterns of hybridization among the three species of Populus that constitute the riparian forest of southern Alberta, Canada. Canadian Journal of Botany 82, 253–264, https://doi.org/10.1139/b03-135 (2004).
Lexer, C., Fay, M., Joseph, J., Nica, M. S. & Heinze, B. Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression. Molecular ecology 14, 1045–1057, https://doi.org/10.1111/j.1365-294X.2005.02469.x (2005).
Du, S. Mocecular phylogeny of genus Populus L. and biogeography of three aspen species. PhD thesis, Chinese Academy of Forestry (2014).
Zsuffa, L. In Proceedings 14th meeting of the Canadian Tree Improvement Association, Part 2 Vol. 14 107–123 (Canadian Forestry Service, Fredericton NB 1975).
Fang, Z., Zhao, S. & Skvortsov, A. K. In Flora of China (eds Zhengyi Wu & Peter H. Raven.) 139–274 (Science Press 1999).
Fossati, T. et al. Development of molecular markers to assess the level of introgression of Populus tremula into P. alba natural populations. Plant Breeding 123, 382–385, https://doi.org/10.1111/j.1439-0523.2004.00979.x (2004).
He, C. et al. Clonal reproduction and natural variation of Populus canescens patches. Tree Physiology 30, 1383–1390, https://doi.org/10.1093/treephys/tpq083 (2010).
Mitton, J. B. & Grant, M. C. Genetic variation and the natural history of quaking aspen. Bioscience 46, 25–31, https://doi.org/10.2307/1312652 (1996).
Van Loo, M., Joseph, J. A., Heinze, B., Fay, M. F. & Lexer, C. Clonality and spatial genetic structure in Populus × canescens and its sympatric backcross parent P. alba in a Central European hybrid zone. New Phytologist 177, 506–516, https://doi.org/10.1111/j.1469-8137.2007.02266.x (2008).
Rajora, O. & Dancik, B. Genetic characterization and relationships of Populus alba, P. tremula, and P. x canescens, and their clones. Theoretical and Applied Genetics 84, 291–298, https://doi.org/10.1007/BF00229485 (1992).
Zhou, H. & Hu, X. Familiar trees of Hopei. 25–39 (Peiking Jingsheng biological survey 1934).
Cariere, E. Populus tomentosa Carr. Revue Horticole 1867, 340 (1867).
Genetic breeding lab Research Institute of Forestry Chinese Academy of Forestry. The Populus genus. 92 (China Forestry Publishing House 1959).
Wang, Z. et al. Phylogeny reconstruction and hybrid analysis of populus (salicaceae) based on nucleotide sequences of multiple single-copy nuclear genes and plastid fragments. PloS one 9, e103645, https://doi.org/10.1371/journal.pone.0103645 (2014).
Bartkowiak, S. Przysadki kwiatowe u topól sekcji Leuce Duby. Arboretum Kórnickie 3, 221–236 (1958).
Zhang, T. Studies on the Floral Variafion of Populus tomentosa Clones and Their Populations. Journal of Northwest Forestry College 10, 43–47 (1995).
Li, K., Huang, M. & Wang, M. Study on Origin of Populus tomentosa Carr. Acta Phytotaxonomica Sinica 35, 24–31 (1997).
Song, L. et al. Quantitive Classification on Isoperoxidase for 35 Species of Sect.Populus from Henan. Journal of Hebei Forestry College 11, 7–11 (1996).
He, C. Study of Genetic Diversity and Origin of Populus tomentosa, Beijing Forestry University, (2005).
Du, S. et al. Multilocus analysis of nucleotide variation and speciation in three closely related Populus (Salicaceae) species. Molecular ecology 24, 4994–5005, https://doi.org/10.1111/mec.13368 (2015).
Librado, P. & Rozas, J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452, https://doi.org/10.1093/bioinformatics/btp187 (2009).
Peakall, R. & Smouse, P. E. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28, 2537–2539, https://doi.org/10.1093/bioinformatics/bts460 (2012).
Pritchard, J. K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. Genetics 155, 945–959 (2000).
Anderson, E. & Thompson, E. A model-based method for identifying species hybrids using multilocus genetic data. Genetics 160, 1217–1229 (2002).
Cornuet, J.-M. et al. DIYABCv2. 0: a software to make approximate Bayesian computation inferences about population history using single nucleotide polymorphism, DNA sequence and microsatellite data. Bioinformatics 30, 1187–1189, https://doi.org/10.1093/bioinformatics/btt763 (2014).
Rieseberg, L. H. & Carney, S. E. Plant hybridization. New Phytologist 140, 599–624, https://doi.org/10.1046/j.1469-8137.1998.00315.x (1998).
Gompert, Z., Fordyce, J. A., Forister, M. L., Shapiro, A. M. & Nice, C. C. Homoploid hybrid speciation in an extreme habitat. Science 314, 1923–1925, https://doi.org/10.1126/science.1135875 (2006).
Sun, X., Liu, R., Tao, Y., Li, Y. & Kang, X. Studies on Investigation and Use of Gene Resource about Populus hopeiensis in Gansu. Journal of Gansu Agricultural University 25, 14–20 (1990).
Eckenwalder, J. E. Natural intersectional hybridization between North American species of Populus (Salicaceae) in sections Aigeiros and Tacamahaca. II. Taxonomy. Canadian Journal of Botany 62, 325–335, https://doi.org/10.1139/b84-051 (1984).
Nason, J. D., Ellstrand, N. C. & Arnold, M. L. Patterns of hybridization and introgression in populations of oaks, manzanitas, and irises. American Journal of Botany 79, 101–111, https://doi.org/10.2307/2445203 (1992).
Dickmann, D. I. In Poplar culture in north America (ed P. B. Cavers) 1–42 (NRC Research Press 2001).
Rieseberg, L. H. & Brunsfeld, S. J. In Molecular systematics of plants (eds Pamela S. Soltis, Douglas E. Soltis, & Jeff J. Doyle) 151–176 (Springer 1992).
Heiser, C. B. Introgression re-examined. Botanical Review 39, 347–366, https://doi.org/10.1007/BF02859160 (1973).
Petit, R. J. & Excoffier, L. Gene flow and species delimitation. Trends in Ecology &. Evolution 24, 386–393, https://doi.org/10.1016/j.tree.2009.02.011 (2009).
Brunsfeld, S. J. Systematics and evolution in Salix section Longifoliae PhD thesis, Washington State University (1990).
Keim, P., Paige, K., Whitham, T. G. & Lark, K. Genetic analysis of an interspecific hybrid swarm of Populus: occurrence of unidirectional introgression. Genetics 123, 557–565 (1989).
Ren, G. et al. Genetic divergence, range expansion and possible homoploid hybrid speciation among pine species in Northeast China. Heredity 108, 552–562, https://doi.org/10.1038/hdy.2011.123 (2012).
Lepais, O. et al. Species relative abundance and direction of introgression in oaks. Molecular Ecology 18, 2228–2242, https://doi.org/10.1111/j.1365-294X.2009.04137.x (2009).
Currat, M., Ruedi, M., Petit, R. J. & Excoffier, L. The Hidden Side of Invasions: Massive Introgression by Local Genes. Evolution 62, 1908–1920, https://doi.org/10.1111/j.1558-5646.2008.00413.x (2008).
Yin, T. et al. Genome structure and emerging evidence of an incipient sex chromosome in Populus. Genome research 18, 422–430, https://doi.org/10.1101/gr.7076308 (2008).
Feng-ying, B. et al. Ploidy level and contrast analysis of the traits for superior trees of Populus tomentosa Carr. in gene pool. Journal of Beijing Forestry University 37, 113–119, https://doi.org/10.13332/j.1000-1522.20140247 (2015).
Demesure, B., Sodzi, N. & Petit, R. J. A set of universal primers for amplification of polymorphic non‐coding regions of mitochondrial and chloroplast DNA in plants. Molecular Ecology 4, 129–134, https://doi.org/10.1111/j.1365-294X.1995.tb00201.x (1995).
Shaw, J., Lickey, E. B., Schilling, E. E. & Small, R. L. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. American Journal of Botany 94, 275–288, https://doi.org/10.3732/ajb.94.3.275 (2007).
Wang, D., Wang, Z., Du, S. & Zhang, J. Phylogeny of section Leuce (Populus, Salicaceae) inferred from 34 chloroplast DNA fragments. Biochemical Systematics and Ecology 63, 212–217, https://doi.org/10.1016/j.bse.2015.09.020 (2015).
Hall, T. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium series 41, 95–98 (1999).
Stephens, M., Smith, N. J. & Donnelly, P. A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics 68, 978–989, https://doi.org/10.1086/319501 (2001).
Tajima, F. Evolutionary relationship of DNA sequences in finite populations. Genetics 105, 437–460 (1983).
Watterson, G. On the number of segregating sites in genetical models without recombination. Theoretical population biology 7, 256–276, https://doi.org/10.1016/0040-5809(75)90020-9 (1975).
Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585–595 (1989).
Fu, Y.-X. & Li, W.-H. Statistical tests of neutrality of mutations. Genetics 133, 693–709 (1993).
Wright, S. I. & Charlesworth, B. The HKA test revisited a maximum-likelihood-ratio test of the standard neutral model. Genetics 168, 1071–1076, https://doi.org/10.1534/genetics.104.026500 (2004).
Martin, D. P. et al. RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26, 2462–2463, https://doi.org/10.1093/bioinformatics/btq467 (2010).
Young, N. D. & Healy, J. GapCoder automates the use of indel characters in phylogenetic analysis. Bmc Bioinformatics 4, 6, https://doi.org/10.1186/1471-2105-4-6 (2003).
Posada, D. jModelTest: phylogenetic model averaging. Molecular biology and evolution 25, 1253–1256, https://doi.org/10.1093/molbev/msn083 (2008).
Swofford, D. L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b10. (Sinauer Associates 2003).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313, https://doi.org/10.1093/bioinformatics/btu033 (2014).
Lam-Tung, N., Schmidt, H. A., Arndt, V. H. & Quang, M. B. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology &. Evolution 32, 268–274, https://doi.org/10.1093/molbev/msu300 (2015).
Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574, https://doi.org/10.1093/bioinformatics/btg180 (2003).
Rambaut, A. Molecular evolution, phylogenetics and epidemiology Figtree v1.4, Available from http://tree.bio.ed.ac.uk/software/figtree (2012).
Evanno, G., Regnaut, S. & Goudet, J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular ecology 14, 2611–2620, https://doi.org/10.1111/j.1365-294X.2005.02553.x (2005).
Earl, D. A. & VonHoldt, B. M. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conservation genetics resources 4, 359–361, https://doi.org/10.1007/s12686-011-9548-7 (2012).
Kopelman, N. M., Mayzel, J., Jakobsson, M., Rosenberg, N. A. & Mayrose, I. CLUMPAK: a program for identifying clustering modes and packaging population structure inferences across K. Molecular ecology resources 15, 1179–1191, https://doi.org/10.1111/1755-0998.12387 (2015).
Acknowledgements
We sincerely thank Dr. Pingdong Zhang from the Beijing Forestry University, as well as Dr. Yanfei Zeng and Dr. Aiguo Duan for collecting the specimens and extracting DNA. We also thank Dr. Wenting Wang from the Northwest University for Nationalities for her advice on data analysis. This work was supported by the Fundamental Research Funds of Chinese Academy of Forestry [No. CAFYBB2017ZX001–1], and the grants of the National Natural Science Foundation of China [No. 31470665].
Author information
Authors and Affiliations
Contributions
J.Z. and Z.W. conceived and designed the study, D.W. and X.K. performed the experiments, D.W. analyzed the data, and wrote the manuscript. All authors contributed to the final version of the paper, and all authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Competing Interests
The authors declare no competing interests.
Additional information
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Wang, D., Wang, Z., Kang, X. et al. Genetic analysis of admixture and hybrid patterns of Populus hopeiensis and P. tomentosa. Sci Rep 9, 4821 (2019). https://doi.org/10.1038/s41598-019-41320-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-019-41320-z
This article is cited by
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.