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Allopolyploidy: An Underestimated Driver in Juniperus Evolution

Perla Farhat
Sonja Siljak-Yakovlev
Najat Takvorian
Magda Bou Dagher Kharrat
1,5 and
Thierry Robert
Laboratoire Biodiversité et Génomique Fonctionnelle, Faculté des Sciences, Université Saint-Joseph, Campus Sciences et Technologies, Mar Roukos, Mkalles, BP, 1514 Riad el Solh, Beirut 1107 2050, Lebanon
Ecologie Systématique Evolution, Univ. Paris-Sud, CNRS, AgroParisTech, Université Paris-Saclay, 91190 Gif-sur-Yvette, France
CEITEC—Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
Faculté des Sciences et Ingénierie, Sorbonne Université, UFR 927, 4 Place Jussieu, 75252 Paris, France
European Forest Institute, Mediterranean, Sant Pau Art Nouveau Site, St. Antoni M. Claret, 167, 08025 Barcelona, Spain
Authors to whom correspondence should be addressed.
Life 2023, 13(7), 1479;
Submission received: 8 May 2023 / Revised: 16 June 2023 / Accepted: 26 June 2023 / Published: 30 June 2023
(This article belongs to the Special Issue Genome Evolution Mechanism of Plant Polyploids)


Allopolyploidy is considered as a principal driver that shaped angiosperms’ evolution in terms of diversification and speciation. Despite the unexpected high frequency of polyploidy that was recently discovered in the coniferous genus Juniperus, little is known about the origin of these polyploid taxa. Here, we conducted the first study devoted to deciphering the origin of the only hexaploid taxon in Juniperus along with four of its closely related tetraploid taxa using AFLP markers with four primers combinations. Phylogenetic analysis revealed that the 10 studied species belong to 2 major clusters. J. foetidissima appeared to be more related to J. thurifera, J. sabina, and J. chinensis. The Bayesian clustering analysis showing a slight variation in genetic admixture between the studied populations of J. foetidissima, suggesting an allopolyploid origin of this species involving J. thurifera and J. sabina lineages, although a purely autopolyploidy origin of both J. thurifera and J. foetidissima cannot be ruled out. The admixed genetic pattern revealed for J. seravschanica showed that the tetraploid cytotypes of this species originated from allopolyploidy, whereas no clear evidence of hybridization in the origin of the tetraploid J. thurifera and J. chinensis was detected. This study provides first insights into the polyploidy origin of the Sabina section and highlights the potential implication of allopolyploidy in the evolution of the genus Juniperus. Further analyses are needed for a more in-depth understanding of the evolutionary scenarios that produced the observed genetic patterns.

1. Introduction

Natural hybridization and polyploidy have long been considered as partners in plant evolution and promoters of biodiversity [1]. Allopolyploidy is when genome duplication involves the genomes from diverged species through hybridization, inducing diversification and speciation. Indeed, allopolyploidy is a common pathway for hybrid speciation [2,3,4]. It has been well documented in angiosperms, yet overlooked in gymnosperms (except Ephedra L. [5,6]). Recently, an unexpected frequency of polyploid species (15% of natural taxa) was determined in the coniferous genus Juniperus L. It was suggested that at least ten polyploidy events have occurred in the course of Juniperus evolution [7]. All polyploid Juniperus taxa are distributed in the old world and mainly in the Mediterranean region [7]. Juniperus is the most diversified genus in the Cupressaceae family and the second in the conifers after Pinus L., with approximately 75 species widely distributed in the northern hemisphere from sea level until the tree line, except for one species. Only Juniperus procera Hochst. ex Endl. is restricted to the Southern Hemisphere [8]. This monophyletic genus is divided into 3 monophyletic sections, Caryocedrus (1 species, Juniperus drupaceae Labill.), Juniperus (13 species), and Sabina (61 species) [8]. All polyploidy cases in this genus have been observed in the Sabina section [7,9,10,11,12]. This section includes diploid species (2n = 2x = 22), tetraploid species (2n = 4x = 44), and just one hexaploid Juniperus foetidissima Willd. (2n = 6x = 66) [7,9,10,11,13]. Interestingly, J. foetidissima is the only identified hexaploid conifer found in the Mediterranean region [7] and only the second identified in the whole world, the first one being Sequoia sempervirens (D.Don) Endl. [14].
Juniperus foetidissima is a dioecious, conical tree reaching up to 20 m in height. Its scale leaves are thick (ca. 1.5 mm) comparing to Juniperus closely related species.. The seed cones, globose, 7–12 mm, maturing in 2 years, commonly contain just 1 or 2 seeds. This species is distributed in the eastern Mediterranean region, mainly on rocky high mountains. It is found on the Balkans Mountains in Albania, Greece, North Macedonia, on Caucasus Mountains, Turkey, Syria, Lebanon, southwestern Turkmenistan, Iran’s high mountains, and in an isolated population on Crimea [8,15]. The origin of J. foetidissima, and especially whether it is an allopolyploid or an autopolyploid, is still unclear. Molecular phylogenetic inferences have placed J. foetidissima in the third major clade (corresponding to the Sabina section) on the sister branch of the tetraploid species J. thurifera L. [8]. Juniperus foetidissima appears to be closely related to J. excelsa M. Bieb., J. polycarpos K. Koch., and J. seravschanica Kom. [8]. This suggests that the ancestors of some of these species could have been involved in the origin of J. foetidissima, if it was an allopolyploid. Nevertheless, this phylogenetic tree of Juniperus was built using ITS (nuclear region) and four chloroplast regions. These markers have been shown to be efficient for phylogenetic reconstructions. However, due to their relatively low polymorphism compared with other markers such as AFLP, their ability to resolve relationships between closely related species is limited [16]. The AFLP technique can generate a large number of biallelic markers, which are sampled in approximately the entire genome. They usually display strong phylogenetic signals among closely related species [17,18,19,20,21].
The first aim of this study was to decipher the origin of the hexaploid J. foetidissima by using AFLP markers. Juniperus species of Sabina section were selected for this study based on their belonging to the same clade of J. foetidissima in the phylogenetic tree [8] and based on their close geographical distribution near this species. Recently, evidence of current or past events of interspecific hybridization has been revealed between J. sabina and J. thurifera lineages [22,23,24]. Therefore, our second aim was to investigate the signals of genetic admixture and phylogenetic relationships between the chosen species in this study using AFLP markers.

2. Materials and Methods

2.1. Plant Material

The selected taxa included in this study were: J. chinensis L., J. excelsa, J. foetidissima, J. phoenicea L., J. polycarpos, J. procera, J. sabina var. sabina L., J. sabina var. balkanensis R. P. Adams and A. Tashev., J. seravschanica, J. thurifera, and J. turcomanica B. Fedtsch. (Table 1). Juniperus phoenicea was chosen as an outgroup.
In total, 147 Juniperus samples (fresh or dried leaves) were analyzed (details are presented in Table 1). All dried leaves samples were provided in silica gel from Baylor University Herbarium (BAYLU), except for samples of J. thurifera from the French Alps, which were provided dried from the herbaria of the National Alpine Botanical Conservatory (CBNA).

2.2. DNA Extraction

Tot section al genomic DNA was extracted via the cetyltrimethyl ammonium bromide (CTAB) method [26], according to the modifications for conifers described by Bou Dagher-Kharrat [18]. Approximately 30 mg of dried leaves was ground in a 2% CTAB solution (1.4 M NaCl, 20 mM EDTA, and 100 mM Tris-HCl; pH 8.0; 2% CTAB; and 2% polyvinylpyrrolidone (PVP)).

2.3. Amplified Fragment Length Polymorphism (AFLP) Analysis

The AFLP method used is based on the standard protocol [27]. To improve the reliability of AFLP data, 34 replicates of AFLP profiles were generated from independent restriction digests of the same DNA (repetition type 1) and from different DNA extractions of the same sample (repetition type 2) (Table 2). Replicates covered around 23% of the entire samples of the dataset. Additionally, 8 repetitions of the negative control (H2O) were included. Genomic DNA (250 ng) was digested at 37 °C for 2 h 30 min. The digestion reaction contained 5 units of EcoRI and 5 units of Tru1I restriction enzymes in 1X ligase buffer (Fermentas MBI, Burlington, ON, Canada) in a final volume of 25 μL. Then, restriction enzymes were inactivated at 70 °C for 20 min. The ligation reaction mix contained 5 pmol EcoRI adaptors and 50 pmol Tru1I adaptors, 1X ligase buffer (Fermentas MBI), 1 unit of T4 DNA ligase (Fermentas MBI), and the entire digestion product (25 μL) in a final volume of 50 μL. Ligation was performed at 20 °C for 2 h 30 min. Digested and ligated DNA was diluted 10X with TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA, pH 8.0). Pre-selective amplification was carried out using a primer pair complementary to each restriction site plus one specific nucleotide at the 3′ end of each primer (Table 3). Polymerase chain reaction (PCR) was carried out in a final volume of 50 μL. In each reaction, 5 μL of diluted restriction-ligation products was added to 10 pmol of each primer, 0.2 mM of each dNTP, 2.5 mM of MgCl2, 1X Taq DNA polymerase buffer without MgCl2, and 1 unit of Taq DNA polymerase (MP Biomedicals, Santa Ana, CA, USA). The pre-selective amplification protocol involved initial denaturation at 94 °C for 1 min, followed by 20 cycles at 94 °C for 30 s, 56 °C at 1 min, 72 °C at 1 min, and a final extension at 72 °C for 5 min. To verify the pre-selective amplification success, 7 μL of the PCR products was electrophoresed on 2% agarose gel stained with GelRed at 100 V for 1 h. The pre-selective amplified PCR products were diluted 50X with ddH2O. Selective amplifications using four primers combination were performed using three labeled EcoRI primers (two primers labeled with FAM and one with PET (Table 3)) and four unlabeled Tru1I (Table 3). Selective primers were similar to the pre-selective ones with the addition of two nucleotides randomly chosen at the 3′ end of the sequences (Table 3). The selective PCR contained, for each reaction, 4 μL of the diluted pre-selective PCR product, 1X of Dream Taq buffer, 0.5 mM of MgCl2, 0.2 mM of each dNTPs, 0.2 µM of each selective primer, and 1 unit of DreamTaq polymerase (Thermo Fisher Scientific, Waltham, MA, USA) in a final volume of 25 μL. The PCR protocol was initial denaturation at 94 °C for 1 min, 10 cycles at 94 °C for 1 min, annealing at 65 °C to 56 °C (touchdown of 1 °C per cycle) for 1 min, 72 °C for 1 min 30 s, followed by 23 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min, and a final extension at 72 °C for 3 min. To check the selective PCR reactions success, 7 μL of the PCR products was electrophoresed on 2% agarose and gel stained with GelRed at 100 V for 1 h.

2.4. AFLP Scoring

AFLP products along with GS-500 LIZ size standard were run on a capillary sequencer (Applied Biosystems® 3730XL, San Francisco, CA, USA) at GENTYANE Platform-INRA (Clermont-Ferrand, France). AFLP migration profiles were analyzed using GeneMapper v.5 (Thermo Fisher Scientific). The default AFLP settings of GeneMapper v.5 software for allele parameters, peak detection algorithm, peak quality, and quality flags were used. Fragment sizes between 50 bp and 500 bp were taken into consideration in the analysis. The allele’s bins were scored as “0” if the peak height was ≤50, “1” if the peak height was ≥100, and by a “check” for the bins with the peak heights between 50 and 100. Manual analysis of the bin was performed based on the control individuals. Each peak that was present in the negative controls was discarded from the following analysis. Secondly, for each allele, genotyping error based on repetition type 1 (repetition from same DNA and independent restriction digests) and repetition type 2 (repetition from the same sample and different DNA extractions) was estimated using the Bonin error rate [28]. Loci that showed an error rate less than 10% were taken into consideration for further analyses. All loci that showed an error rate more than 10% were discarded. Then, peaks patterns that were scored as “check” were visually examined in GeneMapper and manually edited. All loci displaying singletons were also removed from the analyses.

2.5. AFLP Data Analysis

The proportion of polymorphic fragments (PLPs) for each primer combination was determined using aflp-survey 1.0 software [29]. Pairwise dissimilarity indices between individuals were calculated according to Nei and Li [30]. A neighbor joining (NJ) tree was created using PAUP v.4 based on the matrix of individuals dissimilarities. A heuristic search for the best NJ tree was conducted under the optimality criterion distance (minimum evolution (ME)) with 1000 bootstrap replicates. The starting seed was automatically generated. All characters had equal weight, and the branch-swapping algorithm followed tree bisection–reconnection (TBR). The builtNJ tree was unrooted and visualized using Interactive Tree Of Life (iTOL) v5 [31].
Because the occurrence of current or past events of interspecific hybridization has recently been documented between J. sabina and J. thurifera [23,24] or has involved an ancestral lineage of these species [22,32,33], we decided to also look at signals of genetic admixture between the species within the Sabina section represented in our sample. To achieve this goal, a Bayesian clustering approach was carried out on the basis of the AFLP fragments on the whole dataset using tess software v. 2.3.1 [34]. This procedure allowed us to infer the number (K) of unobserved ancestral genetic clusters that best explained the sample genetic structure. When the model with admixture was chosen, the proportion of individual genomes originating from each potential ancestral genetic clusters (individual ancestry coefficients (q)) was estimated. In this case, the model took into account the possibility of historical events of genetic admixture of clusters. The statistical method implemented in tess is spatially explicit in the sense that the admixture model assumes individual genetic relatedness are spatially auto correlated in a continuous manner (isolation by distance).
A first set of 140 runs (without admixture and correlated allele frequencies model) was carried out, varying K from 2 to 15 (10 runs per value of K) in order to estimate the best K values using the deviance information criterion (DIC) as a statistical variable for model choice (Figure S1). MCMCs were carried out using a burn-in period of 70,000, followed by 120,000 iterations. The best values of K were chosen as those for which the plate with the smallest deviance information criterion (DIC) values was reached. Fifty new runs were performed for each of those K values using the admixture (CAR) model.
In order to identify potential different solutions for each K value (due to potential multimodality in the posterior distributions of the q values) and to manage the label switching among replicates in the tess outputs, clumpp v1.1.2 was used with the Greedy option [35]. The probability of each solution was estimated based on their frequency among 50 runs. Plots representing the individual genomes ancestries to the inferred ancestral genetic clusters were created using the online Structure Plot v.2.0 application. Because Bayesian clustering analyses on genotyping data always generate a background of spurious genome admixture in each individual, and because this background was mostly less than 10% in most individuals belonging to diploid species, individual ancestry coefficients less than 10% were not considered as an indication of interspecific genome admixture.

3. Results

3.1. Polymorphism of AFLP Markers between Juniperus Species

In total, the 4 primer combinations generated 1022 loci used for the analyses. The proportion of polymorphic loci (PLPs) differed between species and primer combinations (Table 4). The PLP ranged from 38.4% (for J. polycarpos) to 68.5% (for J. chinensis) in the primer combination EcoRI-1/Tru1I-1. In the primer combination EcoRI-1/Tru1I-3, the PLP ranged from 36.2% (for J. thurifera) to 58.5% (for J. chinensis). The EcoRI-2/Tru1I-4 primer combination showed the highest PLP among all the primer combinations, with 73.1% (for J. chinensis); the lower PLP in this primer combination was 43.8% (for J. thurifera). The primer combination EcoRI-4/Tru1I-2 showed a PLP ranging from 31.2% (for J. turcomanica) to 59.9% (in J. chinensis). Interestingly, J. chinensis showed the highest PLP for all primer combinations despite the low sample size analyzed (only nine individuals).
This observation deserves to be explored on the basis of a more representative sampling scheme.

3.2. Phylogenetic Relationships among Juniperus Taxa

The NJ phylogenetic tree was supported with high bootstraps on all nodes and provided a similar pattern to that of Bayesian clustering. This showed that all individuals of the same species clustered together except in three cases (Figure 1). Firstly, two individuals of J. excelsa from the Mrebbine population in north Lebanon belonged to the same evolutionary lineage of all J. polycarpos individuals. The second case concerned all J. turcomanica (=J. polycarpos var. turcomanica) individuals that were intermixed with J. polycarpos. The third case was observed for two individuals of J. polycarpos from the population Wadi El Njass in Lebanon collected in this study. These individuals clustered closely to the J. seravschanica lineage (Figure 1).
By taking J. phoenicea as an outgroup, the NJ tree revealed that all studied taxa belong to two major lineages. The first one contains all individuals of J. chinensis, J. foetidissima, J. Sabina, and J. thurifera. The second lineage includes all individuals of J. excelsa, J. polycarpos, J. procera, J. seravschanica, and J. turcomanica. In both major lineages, each species is clearly separated from the others, except for J. polycarpos, J. turcomanica, and J. seravschanica. The latter species show unclear phylogenetic boundaries, despite the high number of AFLP markers studied. The current topology of the phylogenetic tree suggests that one or several ancestral lineages of J. thurifera, J. chinensis, and J. sabina could have contributed to the origin of the hexaploid J. foetidissima.

3.3. Admixture Patterns between Juniperus Taxa

The Bayesian clustering analysis provided K = 5, 6, and 7 ancestral genetic clusters as the best fits for the present data. However, when six and seven genetic clusters were considered, one and two “empty” clusters, respectively, were clearly identified (clusters with only very low or null coancestry values across the whole sample;). We therefore focused only on K = 5 as the best and more realistic number of ancestral genetic clusters. Three solutions for K = 5 (Figure 2 and Figure S2) were identified on the basis of 50 runs. The most likely solution (Figure 2) held the highest probability (50%), followed by the less likely solutions A (36%) and B (14%) (Figure S2). In addition, the most likely solution (Figure 2) was the only one that displayed five truly distinct genetic groups, because solutions A and B each displayed one “empty” genetic group, with the fifth one being ghosted as part of the genetic identity of J. chinensis (genetic cluster represented by the yellow in Figure S2). For the aforementioned reasons, we based our interpretation in this study mainly on the most likely solution presented in Figure 2.
The Bayesian clustering therefore revealed that J. phoenicea clearly belongs to a separate genetic cluster. Juniperus polycarpos, J. turcomanica, and J. seravschanica were overwhelmingly assigned to the same cluster. However, individuals from J. seravschanica showed an admixed genome composition between the J. polycarpos cluster and the same cluster as J. thurifera and J. foetidissima. Both varieties of J. sabina, i.e., J. sabina var. sabina (2n = 2x) and J. sabina var. balkanensis (2n = 4x), belong to the same genetic cluster with no admixture pattern, except for two individuals of J. sabina var. balkanensis, which presented a slight level of admixture with the genetic cluster of J. foetidissima and J. thurifera.
Juniperus excelsa and J. procera belong to the same genetic cluster. Remarkably, three individuals of J. excelsa were purely assigned to two different genetic clusters, two individuals to the J. polycarpos, cluster and the remaining one to the J. thurifera and J. foetidissima cluster. Additionally, a third J. excelsa individual displayed a strong signal of genome coancestry with the J. sabina cluster.
Juniperus chinensis showed a clear trend of ancestry with the J. sabina cluster, in accordance with their evolutionary proximity shown on Figure 1. However, it also displayed a complex mosaic pattern of coancestry with the genetic clusters of J. excelsa/J. procera and J. phoenicea.
The hexaploid J. foetidissima and the tetraploid J. thurifera were clearly assigned to the same genetic cluster. This was also very clear in solution B (only four genetic clusters, see above) and only partly confirmed in solution A (Figure S2).
In the most likely solution (Figure 2), J. thurifera showed a slight signal of genetic admixture, with two individuals displaying genome coancestry (21% and 22%) with the same genetic cluster as J. sabina (a trend that was again more obvious in solution A but not in solution B). Similarly, these two genetic clusters were shown in the genetic ancestry of two J. foetidissima individuals from Greece (18% and 36%). In contrast, the population of J. foetidissima from Lebanon did not show any admixture signals in its ancestry.

4. Discussion

AFLP markers have been shown to display strong phylogenetic signals among closely related species [17,18,19,20,21]. Homoplasy or comigration of DNA fragments generated from different loci [29] is a potential challenge to the phylogenetic reconstruction based on AFLP markers. However, it was demonstrated to be a minor issue for studies dealing with closely related species [16], because homoplasy is expected to be less frequent. Additionally, the dominant nature of AFLP markers (the absence of a given amplified fragment being recessive) was also pointed out as a potential problem in assessing the genetic distance between a polyploid and each of its parents because the level of genotyping uncertainty increases with the ploidy level [36]. However, as noted by these authors, this should not inhibit the detection of hybridizations. Numerous studies have confirmed the power of AFLP markers in detecting interspecific hybridization (e.g., [23,24,37,38,39]). Altogether, the 1022 loci produced in this study allowed a powerful discrimination between the studied species and the identification of several individuals that could be considered as progenies of interspecific hybridization.

4.1. Genetic Delimitation of Diploid Juniperus Taxa

Globally, the Bayesian clustering approach and phylogenetic analysis produce largely convergent results. Among the studied taxa, J. phoenicea, J. excelsa, J. procera, J. polycarpos, and J. turcomanica are exclusively diploid [7]. The results presented above showed that J. phoenicea is clearly an independent evolutionary lineage, consistent with available phylogenetic analysis inferring this species to the base of all ca. 60 species of the Sabina section [8].
Our data also showed that Juniperus excelsa and J. procera are genetically strongly related to each other, a result that is congruent with previous phylogenetic analyses based on ITS and four chloroplast genes [7,8]. Unexpectedly, two individuals of J. excelsa from the Mrebbine population in north Lebanon belong to the same genetic group as J. polycarpos in the Bayesian clustering and were located within the cluster of J. polycarpos in the NJ tree, indicating a possible misidentification of these individuals. Due to the morphological uniformity between the two species, J. polycarpos was discovered in Lebanon only few years ago based on genetic investigations [8,25,40,41]. Indeed, the separation between J. polycarpos and J. excelsa has been demonstrated solely based on microsatellite (SSR), nrDNA (ITS), and chloroplast sequences [40,41] and never on the basis of morphological traits. Therefore, along with the mentioned markers, the AFLP markers provided a very clear and strong separation between these two species and represent a future promising cost-effective method for a heuristic geographic screening of these taxa. On the other hand, the AFLP markers failed to differentiate between J. polycarpos and J. turcomanica (=J. polycarpos var. turcomanica). In the present study, both species belonged to the same cluster without a clear delimitation between them (Figure 1 and Figure 2). Congruently, previous studies using a few nuclear (low copy genes and ITS) and four chloroplast sequences [41,42] showed no clear separation between these two taxa. Therefore, based on the current and aforementioned studies, we propose considering J. polycarpos and J. turcomanica as a species complex. Deep phylogenomic analyses must be conducted to obtained a clear understanding of this complex species, notably using NGS techniques such as GBS [43] and Hyb-Seq [44].

4.2. Potential Origin of the Tetraploid Junipers

Juniperus sabina, J. chinensis, and J. seravschanica have two cytotypes (2n = 2x and 4x) [7], whereas J. thurifera was the only studied species that is exclusively tetraploid. In this study, we analyzed two varieties of J. Sabina: the diploid J. sabina var. sabina (population: Austria, Alps) and the tetraploid J. sabina var. balkanensis (populations: Bosnia-Herzegovina and Croatia). Both varieties clustered in the same branch in the NJ tree and belonged to the same genetic group in the Bayesian clustering analysis. Only very few events of admixture were found for the tetraploid variety, and this result could be interpreted as a signal of shared ancestral polymorphism. Recently, J. sabina var. balkanensis has been described as a hybrid between the diploid variety of J. sabina and the tetraploid J. thurifera, since it holds the nuclear nrDNA (ITS) sequence of J. sabina var. sabina and the chloroplast haplotype (based on four genes) of J. thurifera [22,32,33]. These studies failed to uncover any evidence of a contribution of J. thurifera to the nuclear marker (ITS) of J. sabina var. balkanensis, in agreement with the AFLP in the present study despite the large number of loci. Therefore, it is likely that, after the ancient hybridization between a female J. sabina and a male J. thurifera, at least most of parental genetic nuclear material from J. thurifera was removed in the lineage of J. sabina var. balkanensis. This could have been achieved by several generations of backcross to the female J. Sabina, as described in one of the parsimonious hypothetical polyploidization pathways presented by Farhat et al. [12]. However, meiotic disorders may have helped in accelerating this process of elimination of one parental genetic material from the genome of the interspecific hybrid. Recently, interspecific hybridization was detected between the J. sabina (2n = 2x) and J. thurifera (2n = 4x) present in sympatric populations in France (Alps) and Spain (in two populations), giving rise to triploid and possibly tetraploid individuals [23,24]. Interestingly, male triploid hybrids examined in the French Alps population showed well-conformed and potentially fertile pollen [23], which might reflect the fertility of the triploid hybrids. Consequently, the presence of fertile triploid hybrids might engender a better establishment of the polyploid toward a more stable ploidy level. These latter findings provide more support for the hybrid origin of the tetraploid J. sabina var. balkanensis involving the parental species J. thurifera and J. sabina.
Juniperus chinensis showed a complex genetic pattern. However, interpretation of this pattern should be very cautious. It could be partly due to the fingerprint of shared ancestral polymorphism and/or to uncertainties in ancestry inferences because of the constrained and limited number of ancestral genetic cluster of the solutions retained after Bayesian clustering (five or four genetic clusters in this analysis). Nevertheless, our results strongly underline the strong coancestry of J. chinensis individuals with J. sabina individuals. In addition, J. chinensis was located in the NJ tree on the sister branch of J. sabina. This is compatible with a previous analysis showing that J. sabina and J. chinensis are phylogenetically closer to each other than the remaining taxa examined in our study [8]. Additionally, the complex admixed genetic pattern of J. chinensis found in this study might be the consequence of plausible introgression events from neighboring taxa. Juniperus chinensis is currently distributed in China and Japan, far from all the species analyzed in this study, except for J. sabina var. sabina, which has a wide distribution reaching China (Figure S3) [8]. Therefore, if the observed complex genetic pattern in J. chinensis was produced by introgression events, it might implicate historically dated events during which the geographical distribution of the studied species was possibly closer than that observed currently.
The revealed pattern of genetic admixture of the genome of most J. seravschanica individuals between the genetic groups of J. polycarpos/J. turcomanica and J. foetidissima/J. thurifera suggests that this species is a hybrid. This result is in accordance with the phylogenetic closeness previously revealed between J. seravschanica and J. foetidissima [8]. Moreover, it has lately been suggested based on nrDNA (ITS) and four chloroplast regions that J. seravschanica obtained the chloroplasts from an ancestor of J. foetidissima/J. thurifera through an ancient chloroplast capture [45], meaning that hybridization has occurred in the past between these two lineages (J. seravschanica and J. foetidissima/J. thurifera). Interestingly, the studied populations of J. seravschanica were previously identified as tetraploid [7]. Therefore, the AFLP data suggest that tetraploid individuals of J. seravschanica issued from an interspecific hybridization event, and the inferred admixed status of the genome of some of J. seravschanica individuals may testify to their allopolyploid nature. Based on the admixed pattern revealed in this study and the chloroplast capture analysis of Adams [45], we propose that the J. polycarpos/J. turcomanica lineage has contributed maternal parent and the J. foetidissima/J. thurifera lineage has contributed the paternal parent of tetraploid J. seravschanica. In addition, the strong admixture level shown by a few J. seravschanica individuals (ca. 50% of the admixture) (Figure 2) may also be the consequence of still ongoing genetic introgression. Indeed, the current geographical distributions of J. foetidissima, J. polycarpos, J. turcomanica, and J. seravschanica are currently overlapping. More in-depth analysis using large and genome-wide DNA sequence data should be conducted with a more extensive and more geographically representative sample to look further into these hypotheses. Especially, the sympatry zones of J. seravschanica with parental species candidates would be of prime interest from this perspective. In contrast, J. thurifera is currently very distant from the aforementioned species (Figure S3). Therefore, if we assume that the introgression event leading to the formation of J. seravschanica was recent, the geographical distribution of these species supports J. foetidissima rather than J. thurifera as the paternal parent. In this potential scenario on the origin of tetraploid J. seravschanica, the fertilization between a reduced gamete from J. foetidissima (n = 3x) and a reduced gamete from the diploid J. polycarpos/J. turcomanica (n = x) would directly produce tetraploid progeny.
Juniperus thurifera belongs to the same genetic cluster as J. foetidissima without a significant admixture pattern, with the exception of two individuals from Greece, in which a large part of the genome (>20%) was assigned to the J. sabina cluster (Figure 2). A similar case was previously found in a population from the French Alps, where J. thurifera and J. sabina were present in sympatry and where triploid hybrids producing well-conformed pollen grains were discovered [23]. In this mixed population, individuals morphologically identified as J. thurifera were genetically found to have an admixed genetic pattern between J. thurifera and J. sabina. Therefore, Farhat et al. [23] designated these individuals as tetraploid progenies of hybrids originating from the triploid hybrids between J. thurifera and J. sabina. It is therefore possible that J. thurifera individuals showing a slightly admixed genetic pattern with J. sabina in the present study are also the result of genetic introgression from J. sabina. Previous phylogenetic analyses showed that J. thurifera and J. foetidissima were separated by sister branches [7,8], which is not the case in our NJ phylogenetic tree because J. thurifera appears to be closer to J. sabina (Figure 1). The existence among our studied samples of J. thurifera individuals genetically introgressed by genetic material from J. sabina may account for this discrepancy. Altogether, our results show no clear evidence of hybridization in the origin of the tetraploid J. thurifera but confirm that J. thurifera harbors signs of genetic introgression from J. sabina in its genome due to the existence of recent gene flow between the species.

4.3. Insights into the Origin and Hypothetical Hexaploidy Pathway in J. foetidisima

Despite the rarity of high ploidy levels in conifers, J. foetidissima was determined as a hexaploid species [7], which is the highest ploidy level reported in this group. Juniperus foetidissima and J. thurifera showed a clear genome assignment to the same ancestral genetic cluster, different from all remaining species except for J. seravschanica (see above). This observation and the fact that J. thurifera is tetraploid suggest that the ancestor of J. foetidissima belongs to the lineage of J. thurifera. Interestingly, most of the studied individuals of J. foetidissima from Greece presented a significant part of their genome assigned to the genetic group of J. sabina, similar to J. thurifera but in contrast to the individuals from Lebanon.
For a better understanding of the possible origins of J. foetidissima based on the currently available data, we illustrate four simplified schematic pathways in Figure 3 summarizing four hypothetical parsimonious pathways of this hexaploid formation. One of the hypotheses is that J. sabina and J. thurifera were both involved in the origin of J. foetidissima. This hypothesis is supported by the existence of interspecific hybrids between those two species reported in several populations in France and Spain [23,24]. Interestingly, in these populations, the ploidy level of hybrids was either triploid or tetraploid. Both triploid and tetraploid hybrids may have been involved in the genesis of the hexaploid lineage through several pathways (Figure 3, pathways I, II, and III). The first pathway could be the union between a 2x gamete from the tetraploid J. thurifera ancestor and an x gamete of the diploid J. Sabina ancestor that gave rise to triploid progeny (Figure 3, pathway I). A whole-genome duplication (WGD) in triploid hybrids allows us to restore cytogenetic stability and full fertility and is therefore selectively advantageous (Figure 3, pathway I). According to this pathway, the expected genomic contribution of J. sabina to the hexaploid genome of J. foetidissima would be one-third at the beginning of the process, which is in accordance with the admixed genome pattern of only a few individuals from Greece in our study. However, different mechanisms such as high rates of nonhomologous association, homologous recombination, homologous chromosome replacement, and bias in chromosomal segregation may produce diverse genetic combination in the progenies of newly formed polyploid individuals with variable contribution of genomes from both parental species (e.g., [46]). Additionally, selection can then favor or eliminate certain combinations, or drift processes and the founder effect along pathways of dispersal can lead to the random fixation of certain genetic combinations. All those processes could have happened in the case of the evolution of J. foetidissima in the eastern part of the Mediterranean region.
Alternative pathways are illustrated in Figure 3 (pathways II and III), following possibly similar scenarios as discovered in the French Alps and Spain [23,24]. The triploid generated after the cross between ancestors of J. thurifera (n = 2x) and J. sabina (n = x) would, in turn, produce gametes with three ploidy levels: n = x, 2x, and 3x. It is very unlikely that the triploid gamete would interfere in this scenario with the ancestor of J. thurifera (2n = 4x) because it would have resulted in pentaploid progenies. Therefore, we discard this possibility. In the first case, the cross of the (n = x) gamete of the neo-triploid hybrid with (n = 2x) of J. thurifera ancestor would produce another triploid hybrid that would duplicate its genome through a WGD reaching the hexaploid level (Figure 3, pathway II). In the second case, the diploid gamete (n = 2x) of the neo-triploid hybrid might unify with the J. thurifera ancestor (n = 2x), giving rise to tetraploid level progenies, as found in the French Alps (Figure 3, pathway III). A further hybridization between the triploid and tetraploid progenies (2n = 3x and 2n = 4x) may give rise to triploid hybrids (among other ploidy levels), followed by a WGD reaching the hexaploid level. In both pathways II and III, the original contribution of the genome of J. sabina to the genome of J. foetidissima may widely vary from zero to one-third.
On the other hand, if the hybridization between the ancestors of J. thurifera and J. sabina happened before the WGD in the J. thurifera lineage, the only scenario to reach a hexaploid level after hybridization would be through the contribution of “unreduced gametes” in one of the parents. This scenario would generate triploid hybrids followed by WGD. However, this hypothesis is not very plausible because the polyploidy event in J. thurifera was already estimated to be very old (paleo-tetraploid) based on cytological evidence [11].
All of the hypotheses discussed above suggest the diploid J. Sabina ancestor as one of the contributors to the J. foetidissima genome. The alternative possible hypothesis would involve both lineages of the tetraploid J. thurifera and the tetraploid J. sabina var. balkanensis (Figure 3, pathway IV). In this hypothesis, the fertilization between an unreduced gamete of J. thurifera (n = 4x) and a reduced gamete of the tetraploid J. sabina var. balkanensis (n = 2x) would directly result in hexaploid progenies. The expected original genetic contribution of J. sabina var. balkanensis in the hexaploid progeny would thus be one-third, in agreement with the admixed pattern shown in some individuals of J. foetidissima from Greece. The same mechanisms discussed above and the removal of most or all of the contribution of the J. sabina genome (pathway I) would result in the observed genetic pattern of J. foetidissima from Lebanon. There are three arguments in favor of this hypothesis, which may give it an advantage over the previously proposed origins. First, on the genetic level, the expected and observed genetic patterns of J. foetidissima (Greece) are congruent as explained above. Second, the meiotic level showed the possibility of producing well-formed (potentially fertile) unreduced pollen in J. thurifera from the French Alps [23]. The third is the geographical distribution of the parental species. Currently, the geographical ranges of the parental lineages J. thurifera and J. sabina var. balkanensis do not overlap. Nevertheless, the fact that J. sabina var. balkanensis originated from a hybridization between the diploid J. sabina and the tetraploid J. thurifera (Figure S3) implies that during the previous period, they would have had overlapped geographical distributions.
In our study, we focused on identifying the potential lineages of J. foetidissima, which we narrowed down to J. thurifera and J. sabina, including two of its varieties: J. sabina var. sabina (2n = 2x) and J. sabina var. balkanensis (2n = 4x). However, to gain a more comprehensive understanding of the origin of J. foetidissima, further evidence and insights are necessary. Ideally, a broader range of populations would provide informative genetic patterns and reveal the influence of the J. sabina lineage in the genome of J. foetidissima. It is important to determine if the genetic patterns of these populations are comparable to those found in the populations studied in Lebanon and Greece or if a potential third lineage could complete the understanding of the origin and evolution of the hexaploid J. foetidissima. It would also be plausible that the common ancestor of J. thurifera/J. foetidissima entered the WGD events in an autopolyploidy scenario, giving rise to two lineages, the first of which was the tetraploid “Thurifera” lineage that conquered the western part of Europe and the second was the hexaploid “Foetidissima” that occupied areas in the east. The key point to better identify the most probable polyploidy scenarios is to estimate the time period of the WDG events in J. thurifera and J. foetidissima. A genomic approach with complete sequencing is also clearly needed but is still highly challenging because of the very large size of the genomes of these species and the need to take into account, through sampling, the biogeographic variations in genomic diversity and possibly introgression patterns, the first drafts of which we obtained as a result. A genotyping-by-sequencing approach could be a fruitful intermediate method. Coalescence modeling based on large sequence data may help to infer the likelihoods and relative dating of major events of species admixtures that we hypothesized

Supplementary Materials

The following are available online at, Figure S1. Plot of the deviance information criterion (DIC) indicating the best genetic group (K) values for the studied AFLP markers data. Figure S2. Plot of the Bayesian clustering analysis under the less likely solutions A and B under the genetic group K = 5. Figure S3. Geographical distribution of the studied Juniperus taxa extracted from (Adams 2014 and Farhat et al., 2019, [7,8]).

Author Contributions

T.R., S.S.-Y. and M.B.D.K. conceived the project; P.F. conducted the AFLP experiments; P.F. and T.R. performed the genetic analyses; M.B.D.K., S.S.-Y. and T.R.: funding acquisition; P.F. wrote the first draft of the manuscript; P.F., N.T., M.B.D.K., S.S.-Y. and T.R.; edited and approved the manuscript. All authors have read and agreed to the published version of the manuscript.


The work was supported by the National Council for Scientific Research-Lebanon (CNRS-FS90), the International Relations of Paris-Saclay University, and the Saint-Joseph University Research Council (CR-USJ) (FS-111).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.


The authors thank the GENTYANE Platform at the INRA Research Unit “Génétique, Diversité et Ecophysiologie des Céréales” (UMR 1095), Clermont–Ferrand (France), for AFLP genotyping on the ABI sequencer. We thank Bouchra Douaihy from the Lebanese University, Lebanon, for providing the DNA of Juniperus polycarpos from Lebanon. We also thank Bozo Frajman and Peter Schönswetter from the Department of Botany, University of Innsbruck, Austria; Faruk Bogunic from Faculty of Forestry, University of Sarajevo, Bosnia and Herzegovina; and Luc Garraud from the Conservatoire Botanique National Alpin, Domaine de Charance, Gap, France, for their support with plant material.

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. NJ phylogenetic distance, unrooted tree including ten Juniperus species. Species abbreviations: JEX: J. excelsa, JPR: J. procera, JSE: J. seravschanica, JTU: J. turcomanica, JPO: J. polycarpos, JPH: J. phoenicea, JFO: J. foetidissima, JTH: J. thurifera, JCH: J. chinensis, and JSA: J. sabina (JSA1, 2, and 3 belong to J. sabina var. Sabina, and the remaining samples belong to J. sabina var. balkanensis). Red asterisk “*” indicates individuals not clustered with other individuals of the same species. Black asterisk “*” designates 100 bootstraps on the node. Lineages colors are the same as those used in the Bayesian inference.
Figure 1. NJ phylogenetic distance, unrooted tree including ten Juniperus species. Species abbreviations: JEX: J. excelsa, JPR: J. procera, JSE: J. seravschanica, JTU: J. turcomanica, JPO: J. polycarpos, JPH: J. phoenicea, JFO: J. foetidissima, JTH: J. thurifera, JCH: J. chinensis, and JSA: J. sabina (JSA1, 2, and 3 belong to J. sabina var. Sabina, and the remaining samples belong to J. sabina var. balkanensis). Red asterisk “*” indicates individuals not clustered with other individuals of the same species. Black asterisk “*” designates 100 bootstraps on the node. Lineages colors are the same as those used in the Bayesian inference.
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Figure 2. Plot of the Bayesian clustering analysis under the most likely solution, displaying five genetic groups (K = 5). Each vertical line represents one individual. Species abbreviations: JEX: J. excelsa, JPR: J. procera, JSE: J. seravschanica, JTU: J. turcomanica, JPO: J. polycarpos, JPH: J. phoenicea, JFO-G: J. foetidissima from Greece, JFO-L: J. foetidissima from Lebanon, JTH: J. thurifera, JCH: J. chinensis, and JSA-A: J. sabina var. sabina, and JSA-B: J. sabina var. balkanensis. Below, the ploidy level(s) reported for each taxon [7] is/are represented by a circle colored in green, blue, and dark-red for diploid, tetraploid, and hexaploid levels, respectively.
Figure 2. Plot of the Bayesian clustering analysis under the most likely solution, displaying five genetic groups (K = 5). Each vertical line represents one individual. Species abbreviations: JEX: J. excelsa, JPR: J. procera, JSE: J. seravschanica, JTU: J. turcomanica, JPO: J. polycarpos, JPH: J. phoenicea, JFO-G: J. foetidissima from Greece, JFO-L: J. foetidissima from Lebanon, JTH: J. thurifera, JCH: J. chinensis, and JSA-A: J. sabina var. sabina, and JSA-B: J. sabina var. balkanensis. Below, the ploidy level(s) reported for each taxon [7] is/are represented by a circle colored in green, blue, and dark-red for diploid, tetraploid, and hexaploid levels, respectively.
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Figure 3. Simplified schematic representation of four hypothetical parsimonious pathways for the formation of the hexaploid J. foetidissima.
Figure 3. Simplified schematic representation of four hypothetical parsimonious pathways for the formation of the hexaploid J. foetidissima.
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Table 1. Details on the studied populations of Juniperus species. *, material collected and used in the Ph.D. research of Bouchra Douaihy [25]. The ploidy level of each taxon was extracted from Farhat et al. [7]. The ploidy level appended by “i”, “ii”, or “iii” is the exact samples measured by Farhat et al. [7,12,23].
Table 1. Details on the studied populations of Juniperus species. *, material collected and used in the Ph.D. research of Bouchra Douaihy [25]. The ploidy level of each taxon was extracted from Farhat et al. [7]. The ploidy level appended by “i”, “ii”, or “iii” is the exact samples measured by Farhat et al. [7,12,23].
TaxonPopulationSomatic Ploidy LevelCountryFresh/Dried LeavesNumber of IndividualsGPS Coordinates
Juniperus excelsaAfqa *2xLebanonFresh4N 34°4′25″,
E 35°54′20″
Barqa *LebanonFresh1N 34°11′48″,
E 36°8′15″
Devas Mountain, Agios Georgious ForestGreeceFresh1N 39°45′0.4716″,
E 19°40′59.8944″
Qammoua forest *LebanonFresh5N 34°29′34″,
E 36°15′14″
MrebbineLebanonFresh2N 34°34′4.008″,
E 36°7′33.996″
Nebha, Alkedam, Wadi ghned, ArsalLebanonFresh2N 34°11′19.7952″,
E 36°12′57.6684″
J. chinensisJapan4xiJapanDried3N 35°01.44′,
E 138°47.30′
Lanzhou2x, 4xChinaDried4N 36°6′0″,
E 103°43′59.88″
XianChinaDried2N 34°08′50.2”,
E 109°34′41.3”
J. foetidissimaDevas Mountain, Agios Georgious Forest6xiGreeceFresh9N 39°45′0.4716″,
E 19°40′59.8944″
HermelLebanonFresh14N 34°19′8.7528″,
E 36°15′29.7792″
Qammoua forestLebanonFresh3N 34° 30′ 44.388″,
E 36°16′30.36″
J. phoeniceaBaćina2xCroatiaFresh9N 43°05′12.6”,
E 17°22′36.2”
J. polycarposArsal *2xLebanonFresh17N 34°4′57″,
E 36°28′33.996″
Azerbaijan2xiAzerbaijanDried2N 40°44′41.064″,
E 47°35′19.14″
Wadi El Njass *2xLebanonFresh8N 34°19′49″,
E 36°3′16″
Wadi El NjassLebanonFresh8N 34°20′48″,
E 36°5′17″
J. proceraEthiopia2xiEthiopiaDried4N 9°1′59.9988″,
E 38°23′60″
near Abha2xSaudi ArabiaDried4N 18°16′59.988″,
E 42°21′0″
J. sabina var. sabinaAustria, Alps2xiiAustriaDried3N 46°56′6″,
E 11°2′20.4″
J. sabina var. balkanensisMts. Cvrsnica and Cabulja4xiiBosnia-HerzegovinaFresh7N 43°34′18.084″,
E 17°30′39.888″
Biokovo Mts.CroatiaFresh7N 43°19′31.1808″,
E 17°2′57.0228″
J. seravschanicaKazakhstan4xiKazakhstanDried2N 42°24′31.788″,
E 70°28′30″
KazakhstanKazakhstanDried3N 42°10′46.2″,
E 70°20′0.816″
Oman2x, 4xOmanDried3N 23°7′41.016″,
E 57°36′9.288″
PakistanPakistanDried2N 30°13′0.012″,
E 67°5′60″
J. thuriferaAlps4xiiiFranceDried5N 44°43′7.986″,
E 6°36′13.0392″
Monegros region4xiSpainFresh3N 41°36′19.3428″,
W 0°15′7.092″
Southern Iberian central rangeSpainFresh4N 40° 0′ 14.4936″,
W 5°1′11.82″
J. turcomanicaTurkmenistan2xiTurkmenistanDried2N 38°25′7.212″,
E 56° 58′ 48″
Shahmirzad2xIranDried1N 35°50′54.996″,
E 53°26′24.216″
BajgirnaIranDried1N 37°25′9.804″,
E 58°32′0.204″
BaladaeIranDried1N 36°14′34.404″,
E 51°50′20.4″
FasaIranDried1N 29°9′57.816″,
E 53°40′7.788″
Table 2. Number of replicates for each selected species from: repetition type 1 (repetition from same DNA and independent restriction digests) and repetition type 2 (repetition from the same sample and different DNA extractions).
Table 2. Number of replicates for each selected species from: repetition type 1 (repetition from same DNA and independent restriction digests) and repetition type 2 (repetition from the same sample and different DNA extractions).
SpeciesReplicate Number
of Repetition Type 1
Replicate Number
of Repetition Type 2
J. excelsa12
J. polycarpos36
J. foetidissima112
J. seravschanica10
J. turcomanica10
J. chinensis10
J. procera10
J. sabina12
J. thurifera10
J. phoenicea10
Table 3. Details of the AFLP adapters and primers used in this study.
Table 3. Details of the AFLP adapters and primers used in this study.
CODEPrimer (5′-> 3′)LengthGC (%)TmLabel
AdapterEcoRI-adapter LCTCGTAGACTGCGTACC1758.855.2No label
EcoRI-adapter SAATTGGTACGCAGTC1546.745.1No label
Tru1I-adapter LGACGATGAGTCCTGAG1656.351.7No label
Tru1I-adapter STACTCAGGACTCAT1442.940No label
PreamplificationEcoRI-PAACTGCGTACCAATTCA1643.846.6No label
Tru1I-1GATGAGTCCTGAGTAACTA1942.152.4No label
Tru1I-2GATGAGTCCTGAGTAACTG1947.454.5No label
Tru1I-3GATGAGTCCTGAGTAACAG1947.454.5No label
Tru1I-4GATGAGTCCTGAGTAACTT1942.152.4No label
Amplification primers combinationsEcoRI-1/Tru1I-1
Table 4. Proportion (PLP) of polymorphic loci (%) generated by AFLP from the four primer combinations of the studied Juniperus species. The red asterisk (*) represents the highest PLP in a primer combination, and the black asterisk (*) represents the lowest PLP.
Table 4. Proportion (PLP) of polymorphic loci (%) generated by AFLP from the four primer combinations of the studied Juniperus species. The red asterisk (*) represents the highest PLP in a primer combination, and the black asterisk (*) represents the lowest PLP.
J. chinensis68.5 *58.5 *73.1 *59.9 *
J. excelsa46.848.160.644.2
J. foetidissima50.942.248.541.6
J. phoenicea39.142.549.837.2
J. polycarpos38.4 *36.950.832
J. procera40.438.750.832
J. sabina49.
J. seravschanica56.84656.643.9
J. thurifera44.236.2 *43.8 *32.7
J. turcomanica39.937.654.531.2 *
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Farhat, P.; Siljak-Yakovlev, S.; Takvorian, N.; Bou Dagher Kharrat, M.; Robert, T. Allopolyploidy: An Underestimated Driver in Juniperus Evolution. Life 2023, 13, 1479.

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Farhat P, Siljak-Yakovlev S, Takvorian N, Bou Dagher Kharrat M, Robert T. Allopolyploidy: An Underestimated Driver in Juniperus Evolution. Life. 2023; 13(7):1479.

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Farhat, Perla, Sonja Siljak-Yakovlev, Najat Takvorian, Magda Bou Dagher Kharrat, and Thierry Robert. 2023. "Allopolyploidy: An Underestimated Driver in Juniperus Evolution" Life 13, no. 7: 1479.

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