Plant species with extremely small populations conservation program: achieving Kunming-Montreal global biodiversity targets.

Conservation programs for plant species with extremely small populations (PSESP) have been successfully implemented for several decades in China. Here we highlight how their inclusion in several national conservation policies helps meet targets of the Kunming-Montreal Global Biodiversity Framework (KMGBF) and show how lessons from these programs can be applied more widely.

Chromosome-level genome assembly of the threatened resource plant Cinnamomum chago.

Cinnamomum chago is a tree species endemic to Yunnan province, China, with potential economic value, phylogenetic importance, and conservation priority. We assembled the genome of C. chago using multiple sequencing technologies, resulting in a high-quality, chromosomal-level genome with annotation information. The assembled genome size is approximately 1.06 Gb, with a contig N50 length of 92.10 Mb. About 99.92% of the assembled sequences could be anchored to 12 pseudo-chromosomes, with only one gap, and 63.73% of the assembled genome consists of repeat sequences. In total, 30,497 genes were recognized according to annotation, including 28,681 protein-coding genes. This high-quality chromosome-level assembly and annotation of C. chago will assist us in the conservation and utilization of this valuable resource, while also providing crucial data for studying the evolutionary relationships within the Cinnamomum genus, offering opportunities for further research and exploration of its diverse applications.

Chromosome-scale genomes of Quercus sichourensis and Quercus rex provide insights into the evolution and adaptation of Fagaceae.

The Fagaceae, a plant family with a wide distribution and diverse adaptability, has garnered significant interest as a subject of study in plant speciation and adaptation. Meanwhile, certain Fagaceae species are regarded as highly valuable wood resources due to the exceptional quality of their wood. In this study, we present two high-quality, chromosome-scale genome sequences for Quercus sichourensis (848.75 Mb) and Quercus rex (883.46 Mb). Comparative genomics analysis reveals that the difference in the number of plant disease resistance genes and the nonsynonymous and synonymous substitution ratio (Ka/Ks) of protein-coding genes among Fagaceae species are related to different environmental adaptations. Interestingly, most genes related to starch synthesis in the investigated Quercoideae species are located on a single chromosome, as compared to the outgroup species, Fagus sylvatica. Furthermore, resequencing and population analysis of Q. sichourensis and Q. rex reveal that Q. sichourensis has lower genetic diversity and higher deleterious mutations compared to Q. rex. The high-quality, chromosome-level genomes and the population genomic analysis of the critically endangered Q. sichourensis and Q. rex will provide an invaluable resource as well as insights for future study in these two species, even the genus Quercus, to facilitate their conservation.

Comparison of Rhizosphere Bacterial Communities of Pinus squamata, a Plant Species with Extremely Small Populations (PSESP) in Different Conservation Sites.

Pinus squamata is one of the most threatened conifer species in the world. It is endemic to northeastern Yunnan Province, China, and has been prioritized as a Plant Species with Extremely Small Populations (PSESP). The integrated study of soil properties and rhizosphere bacteria can assist conservation to understand the required conditions for the protection and survival of rare and endangered species. However, differences between the rhizospheric bacterial communities found in the soil surrounding P. squamata at different conservation sites remain unclear. In this study, Samples were collected from wild, ex situ, and reintroduced sites. Bacterial communities in different conservation sites of P. squamata rhizosphere soils were compared using Illumina sequencing. The soil physicochemical properties were determined, the relationships between the bacterial communities and soil physicochemical factors were analyzed, and the potential bacterial ecological functions were predicted. The reintroduced site Qiaojia (RQ) had the highest richness and diversity of bacterial community. Actinobacteria, Proteobacteria, and Acidobacteriota were the dominant phyla, and Bradyrhizobium, Mycobacterium, Acidothermus were the most abundant genera. Samples were scattered (R = 0.93, p = 0.001), indicating significant difference between the different conservation sites. The abundance of Mycobacterium differed between sites (0.01 < p ≤ 0.05), and the relative abundances of Bradyrhizobium and Acidothermus differed significantly among different sites (0.001 < p ≤ 0.01). Soil total potassium (TK) and available nitrogen (AN) were the main factors driving bacterial community at the phylum level (0.01 < p ≤ 0.05). This study generated the first insights into the diversity, compositions, and potential functions of bacterial communities associated with the rhizosphere soils of P. squamata in different conservation sites and provides a foundation to assess the effect of conservation based on bacterial diversity and plant growth-promoting rhizobacteria (PGPR) to guide future research into the conservation of P. squamata.

Transcriptome data analysis provides insights into the conservation of Michelia lacei, a plant species with extremely small populations distributed in Yunnan province, China.

BACKGROUND: Michelia lacei W.W.Smith (Magnoliaceae), was classified as a Plant Species with Extremely Small Populations (PSESP) by the Yunnan Provincial Government in both action plans of 2012 and 2021. This evergreen tree is known for its high ornamental and scientific value, but it faces significant threats due to its extremely small population size and narrow geographical distribution. The study aims to understand the genetic structure, diversity, and demographic history of this species to inform its conservation strategies. RESULTS: The analysis of transcriptome data from 64 individuals across seven populations of M. lacei identified three distinct genetic clusters and generated 104,616 single-nucleotide polymorphisms (SNPs). The KM ex-situ population, originating from Longling County, exhibited unique genetic features, suggesting limited gene flow. The genetic diversity was substantial, with significant differences between populations, particularly between the KM lineage and the OTHER lineage. Demographic history inferred from the data indicated population experienced three significant population declines during glaciations, followed by periods of recovery. We estimated the effective population size (Ne) of the KM and OTHER lineages 1,000 years ago were 85,851 and 416,622, respectively. Gene flow analysis suggested past gene flow between populations, but the KM ex-situ population showed no recent gene flow. A total of 805 outlier SNPs, associated with four environmental factors, suggest potential local adaptation and showcase the species' adaptive potential. Particularly, the BZ displayed 515 adaptive loci, highlighting its strong potential for adaptation within this group. CONCLUSIONS: The comprehensive genomic analysis of M. lacei provides valuable insights into its genetic background and highlights the urgent need for conservation efforts. The study underscores the importance of ex-situ conservation methods, such as seed collection and vegetative propagation, to safeguard genetic diversity and promote population restoration. The preservation of populations like MC and BZ is crucial for maintaining the species' genetic diversity. In-situ conservation measures, including the establishment of in-situ conservation sites and community engagement, are essential to enhance protection awareness and ensure the long-term survival of this threatened plant species.

Transcriptome data reveals the conservation genetics of Cypripedium forrestii, a plant species with extremely small populations endemic to Yunnan, China.

The Cypripedium forrestii is an orchid species with extremely small populations (PSESP) in Yunnan, China. C. forrestii is range-restricted and less-studied than many orchid species, and it is exposed to various threats to its survival. We investigated its potential habitats and collected 52 samples from eight locations, as well as two outgroup species for reference. We developed genetic markers (SNPs) for C. forrestii based on transcriptome sequencing (RNA-seq) data, and analyzed the genetic diversity, population structure, gene flow and demographic history of C. forrestii in detail. C. forrestii is a taxonomically independent species to protect. We found that the genetic diversity of C. forrestii was very low (1.7e-4) compared with other endangered species. We identified three genetic clusters, and several populations with distinct genetic backgrounds. Most genetic diversity was found within sampling sites (87.87%) and genetic clusters (91.39%). Gene flow has been greatly limited over the most recent generations, probably due to geographical distance, historical climate change and habitat fragmentation. We also detected a severe bottleneck event brought about by the recent population constraints. These factors, together with its reproductive characteristics, contribute to the population fragmentation and low genetic diversity of C. forrestii. Based on our findings, we suggest an integrative conservation strategy to protect and recover the genetic diversity of C. forrestii and a further comprehensive study of its ecological traits in the future.

The chromosome-scale genome of Magnolia sinica (Magnoliaceae) provides insights into the conservation of plant species with extremely small populations (PSESP).

Magnolia sinica (Magnoliaceae) is a highly threatened tree endemic to southeast Yunnan, China. In this study, we generated for the first time a high-quality chromosome-scale genome sequence from M. sinica, by combining Illumina and ONT data with Hi-C scaffolding methods. The final assembled genome size of M. sinica was 1.84 Gb, with a contig N50 of ca. 45 Mb and scaffold N50 of 92 Mb. Identified repeats constituted approximately 57% of the genome, and 43,473 protein-coding genes were predicted. Phylogenetic analysis shows that the magnolias form a sister clade with the eudicots and the order Ceratophyllales, while the monocots are sister to the other core angiosperms. In our study, a total of 21 individuals from the 5 remnant populations of M. sinica, as well as 22 specimens belonging to 8 related Magnoliaceae species, were resequenced. The results showed that M. sinica had higher genetic diversity (θw = 0.01126 and θπ = 0.01158) than other related species in the Magnoliaceae. However, population structure analysis suggested that the genetic differentiation among the 5 M. sinica populations was very low. Analyses of the demographic history of the species using different models consistently revealed that 2 bottleneck events occurred. The contemporary effective population size of M. sinica was estimated to be 10.9. The different patterns of genetic loads (inbreeding and numbers of deleterious mutations) suggested constructive strategies for the conservation of these 5 different populations of M. sinica. Overall, this high-quality genome will be a valuable genomic resource for conservation of M. sinica.

Conservation genetics and potential geographic distribution modeling of Corybas taliensis, a small 'sky Island' orchid species in China.

BACKGROUND: Corybas taliensis is an endemic species of sky islands in China. Its habitat is fragile and unstable, and it is likely that the species is threatened. However, it is difficult to determine the conservation priority or unit without knowing the genetic background and the overall distribution of this species. In this study, we used double digest restriction-site associated DNA-sequencing (ddRAD-seq) to investigate the conservation genomics of C. taliensis. At the same time, we modeled the extent of suitable habitat for C. taliensis in present and future (2030 and 2090) habitat using the maximum-entropy (MaxEnt) model. RESULTS: The results suggested that the related C. fanjingshanensis belongs to C. taliensis and should not be considered a separate species. All the sampling locations were divided into three genetic groups: the Sichuan & Guizhou population (SG population), the Hengduan Mountains population (HD population) and Himalayan population (HM population), and we found that there was complex gene flow between the sampling locations of HD population. MT was distinct genetically from the other sampling locations due to the unique environment in Motuo. The genetic diversity (π, He) of C. taliensis was relatively high, but its contemporary effective population size (Ne) was small. C. taliensis might be currently affected by inbreeding depression, although its large population density may be able to reduce the effect of this. The predicted areas of suitable habitat currently found in higher mountains will not change significantly in the future, and these suitable habitats are predicted to spread to other higher mountains under future climate change. However, suitable habitat in relatively low altitude areas may disappear in the future. This suggests that C. taliensis will be caught in a 'summit trap' in low altitude areas, however, in contrast, the high altitude of the Himalaya and the Hengduan Mountains are predicted to act as 'biological refuges' for C. taliensis in the future. CONCLUSIONS: These results not only provide a new understanding of the genetic background and potential resource distribution of C. taliensis, but also lay the foundation for its conservation and management.

Seed Germination and Seedling Growth Influenced by Genetic Features and Drought Tolerance in a Critically Endangered Maple.

Understanding the adaptation of plant species will help us develop effective breeding programs, guide the collection of germplasm, and improve the success of population restoration projects for threatened species. Genetic features correlate with species adaptation. Acer yangbiense is a critically endangered plant species with extremely small populations (PSESP). However, no information was available on its seed germination and seedling growth in populations with different genetic characteristics. In this study, we investigated seed germination and compared the performance of 566 seedlings in 10 maternal half-sib families cultivated in Kunming Botanical Garden. The results showed that A. yangbiense seeds required an average of 44 days to start germinating, with a 50% germination rate estimated to take about 47-76 days, indicating slow and irregular germination. There is a trade-off between the growth and survival in A. yangbiense seedlings, with fast growth coming at the cost of low survival. Groups that were able to recover from a recent bottleneck consistently had higher relative growth rates. High genetic diversity and low levels of inbreeding are likely to be responsible for their improved survival during drought conditions and rapid growth under optimal environmental conditions. Our results suggest that maternal genetic traits might be used as indicators for conservation and population restoration. These findings provide us with new information that could be applied to support ex situ conservation and reintroduction of threatened species.

A haplotype-resolved genome assembly of Rhododendron vialii based on PacBio HiFi reads and Hi-C data.

Rhododendron vialii (subgen. Azaleastrum) is an evergreen shrub with high ornamental value. This species has been listed as a plant species with extremely small populations (PSESP) for urgent protection by China's Yunnan provincial government in 2021, due to anthropogenic habitat fragmentation. However, limited genomic resources hinder scientifically understanding of genetic threats that the species is currently facing. In this study, we assembled a high-quality haplotype-resolved genome of R. vialii based on PacBio HiFi long reads and Hi-C reads. The assembly contains two haploid genomes with sizes 532.73 Mb and 521.98 Mb, with contig N50 length of 35.67 Mb and 34.70 Mb, respectively. About 99.92% of the assembled sequences could be anchored to 26 pseudochromosomes, and 14 gapless assembled chromosomes were included in this assembly. Additionally, 60,926 protein-coding genes were identified, of which 93.82% were functionally annotated. This is the first reported genome of R. vialii, and hopefully it will lay the foundations for further research into the conservation genomics and horticultural domestication of this ornamentally important species.

The chromosome-level genome assembly and genes involved in biosynthesis of nervonic acid of Malania oleifera.

Nervonic acid (C24:1 Δ15, NA) is a very long-chain monounsaturated fatty acid, a clinically indispensable resource in maintaining the brain and nerve cells development and regeneration. Till now, NA has been found in 38 plant species, among which the garlic-fruit tree (Malania oleifera) has been evaluated to be the best candidate for NA production. Here, we generated a high-quality chromosome-scale assembly of M. oleifera employing PacBio long-read, short-read Illumina as well as Hi-C sequencing data. The genome assembly consisted of 1.5 Gb with a contig N50 of ~4.9 Mb and a scaffold N50 of ~112.6 Mb. ~98.2% of the assembly was anchored into 13 pseudo-chromosomes. It contains ~1123 Mb repeat sequences, and 27,638 protein-coding genes, 568 tRNAs, 230 rRNAs and 352 other non-coding RNAs. Additionally, we documented candidate genes involved in NA biosynthesis including 20 KCSs, 4 KCRs, 1 HCD and 1 ECR, and profiled the expression patterns of these genes in developing seeds. The high-quality assembly of the genome provides insights into the genome evolution of the M. oleifera genome and candidate genes involved in NA biosynthesis in the seeds of this important woody tree.

Applying image clustering to phylogenetic analysis: A trial.

•Molecular phylogenetic analysis can be supplemented by image clustering analysis that uses pretrained machine learning tools.

Deciphering complex reticulate evolution of Asian Buddleja (Scrophulariaceae): insights into the taxonomy and speciation of polyploid taxa in the Sino-Himalayan region.

BACKGROUND AND AIMS: Species of the genus Buddleja in Asia are mainly distributed in the Sino-Himalayan region and form a challenging taxonomic group, with extensive hybridization and polyploidization. A phylogenetic approach to unravelling the history of reticulation in this lineage will deepen our understanding of the speciation in biodiversity hotspots. METHODS: For this study, we obtained 80 accessions representing all the species in the Asian Buddleja clade, and the ploidy level of each taxon was determined by flow cytometry analyses. Whole plastid genomes, nuclear ribosomal DNA, single nucleotide polymorphisms and a large number of low-copy nuclear genes assembled from genome skimming data were used to investigate the reticulate evolutionary history of Asian Buddleja. Complex cytonuclear conflicts were detected through a comparison of plastid and species trees. Gene tree incongruence was also analysed to detect any reticulate events in the history of this lineage. KEY RESULTS: Six hybridization events were detected, which are able to explain the cytonuclear conflict in Asian Buddleja. Furthermore, PhyloNet analysis combining species ploidy data indicated several allopolyploid speciation events. A strongly supported species tree inferred from a large number of low-copy nuclear genes not only corrected some earlier misinterpretations, but also indicated that there are many Asian Buddleja species that have been lumped mistakenly. Divergent time estimation shows two periods of rapid diversification (8-10 and 0-3 Mya) in the Asian Buddleja clade, which might coincide with the final uplift of the Hengduan Mountains and Quaternary climate fluctuations, respectively. CONCLUSIONS: This study presents a well-supported phylogenetic backbone for the Asian Buddleja species, elucidates their complex and reticulate evolutionary history and suggests that tectonic activity, climate fluctuations, polyploidization and hybridization together promoted the diversification of this lineage.

The complete chloroplast genome of Acer Pubipetiolatum var. pingpienense (Sapindaceae).

The genus Acer is widespread throughout the northern temperate zone, and many species within the genus are of ecological and economical importance. Here we report the newly sequenced chloroplast genome of Acer pubipetiolatum var. pingpienense. This chloroplast genome has a total length of 156,730 bp, and contains a pair of inverted repeats (IRs, 26,743 bp), a large single-copy (LSC) region of 71,582 bp and a small single-copy (SSC) region of 18,092 bp. Phylogenetic analysis suggests that A. pubipetiolatum var. pingpienense is closely related to A. laevigatum, and both fall into Section Palmata. The complete A. pubipetiolatum var. pingpienense chloroplast genome will provide an important genetic resource for future research into the conservation and evolution of this genus. Our findings also suggest that further research is necessary to elucidate the phylogenetic relationships between plant species within this genus.

Genomic Data Reveals Population Genetic and Demographic History of Magnolia fistulosa (Magnoliaceae), a Plant Species With Extremely Small Populations in Yunnan Province, China.

Elucidating the genetic background of threatened species is fundamental to their management and conservation, and investigating the demographic history of these species is helpful in the determination of the threats facing them. The woody species of the genus Magnolia (Magnoliaceae) have high economic, scientific and ecological values. Although nearly half of all Magnolia species have been evaluated as threatened, to date there has been no population genetic study employing Next Generation Sequencing (NGS) technology in this genus. In the present study, we investigate the conservation genomics of Magnolia fistulosa, a threatened species endemic to the limestone area along the Sino-Vietnamese border, using a double digest restriction-site-associated DNA-sequencing (ddRAD-seq) approach. To increase the reliability of our statistical inferences, we employed two approaches, Stacks and ipyrad, for SNP calling. A total of 15,272 and 18,960, respectively, putatively neutral SNPs were generated by Stacks and ipyrad. Relatively high genetic diversity and large population divergence were detected in M. fistulosa. Although higher absolute values were calculated using the ipyrad data set, the two data sets showed the same trends in genetic diversity (π, H e), population differentiation (F ST) and inbreeding coefficients (F IS). A change in the effective population size of M. fistulosa within the last 1 Ma was detected, including a population decline about 0.5-0.8 Ma ago, a bottleneck event about 0.2-0.3 Ma ago, population fluctuations during the last glacial stage, and the recovery of effective population size after the last glacial maximum. Our findings not only lay the foundation for the future conservation of this species, but also provide new insights into the evolutionary history of the genus Magnolia in southeastern Yunnan, China.

Demographic history and identification of threats revealed by population genomic analysis provide insights into conservation for an endangered maple.

Recent advancements in whole genome sequencing techniques capable of covering nearly all the nucleotide variations of a genome would make it possible to set up a conservation framework for threatened plants at the genomic level. Here we applied a whole genome resequencing approach to obtain genome-wide data from 105 individuals sampled from the 10 currently known extant populations of Acer yangbiense, an endangered species with fragmented habitats and restricted distribution in Yunnan, China. To inform meaningful conservation action, we investigated what factors might have contributed to the formation of its extremely small population sizes and what threats it currently suffers at a genomic level. Our results revealed that A. yangbiense has low genetic diversity and comprises different numbers of genetic groups based on neutral (seven) and selected loci (13), with frequent gene flow between populations. Repeated bottleneck events, particularly the most recent one occurring within ~10,000 years before present, which decreased its effective population size (Ne ) < 200, and severe habitat fragmentation resulting from anthropogenic activities as well as a biased gender ratio of mature individuals in its natural habitat, might have together contributed to the currently fragmented and endangered status of A. yangbiense. The species has suffered from inbreeding and deleterious mutation load, both of which varied among populations but had similar patterns; that is, populations with higher FROH (frequency of runs of homozygosity) always carried a larger number of deleterious mutations in the homozygous state than in populations with lower FROH. In addition, based on our genetic differentiation results, and the distribution patterns of homozygous deleterious mutations in individuals, we recommend certain conservation actions regarding the genetic rescue of A. yangbiense. Overall, our study provides meaningful insights into the conservation genetics and a framework for the further conservation for the endangered A. yangbiense.

Genome-wide analysis of butterfly bush (Buddleja alternifolia) in three uplands provides insights into biogeography, demography and speciation.

Understanding processes that generate and maintain large disjunctions within plant species can provide valuable insights into plant diversity and speciation. The butterfly bush Buddleja alternifolia has an unusual disjunct distribution, occurring in the Himalaya, Hengduan Mountains (HDM) and the Loess Plateau (LP) in China. We generated a high-quality, chromosome-level genome assembly of B. alternifolia, the first within the family Scrophulariaceae. Whole-genome re-sequencing data from 48 populations plus morphological and petal colour reflectance data covering its full distribution range were collected. Three distinct genetic lineages of B. alternifolia were uncovered, corresponding to Himalayan, HDM and LP populations, with the last also differentiated morphologically and phenologically, indicating occurrence of allopatric speciation likely to be facilitated by geographic isolation and divergent adaptation to distinct ecological niches. Moreover, speciation with gene flow between populations from either side of a mountain barrier could be under way within LP. The current disjunctions within B. alternifolia might result from vicariance of a once widespread distribution, followed by several past contraction and expansion events, possibly linked to climate fluctuations promoted by the Kunlun-Yellow river tectonic movement. Several adaptive genes are likely to be either uniformly or diversely selected among regions, providing a footprint of local adaptations. These findings provide new insights into plant biogeography, adaptation and different processes of allopatric speciation.

Chromosome-level genome assembly and population genetic analysis of a critically endangered rhododendron provide insights into its conservation.

Rhododendrons are woody plants, famous throughout the world as having high horticultural value. However, many wild species are currently threatened with extinction. Here, we report for the first time a high-quality, chromosome-level genome of Rhododendron griersonianum, which has contributed to approximately 10% of all horticultural rhododendron varieties but which in its wild form has been evaluated as critically endangered. The final genome assembly, which has a contig N50 size of approximately 34 M and a total length of 677 M, is the highest-quality genome sequenced within the genus to date, in part due to its low heterozygosity (0.18%). Identified repeats constitute approximately 57% of the genome, and 38 280 protein-coding genes were predicted with high support. We further resequenced 31 individuals of R. griersonianum as well as 30 individuals of its widespread relative R. delavayi, and performed additional conservation genomic analysis. The results showed that R. griersonianum had lower genetic diversity (θ = 2.58e-3; π = 1.94e-3) when compared not only to R. delavayi (θ = 11.61e-3, π = 12.97e-3), but also to most other woody plants. Furthermore, three severe genetic bottlenecks were detected using both the Stairway plot and fastsimcoal2 analysis, which are thought to have occurred in the late Middle Pleistocene and the Last Glacial Maximum (LGM) period. After these bottlenecks, R. griersonianum recovered and maintained a constant effective population size (>25 000) until now. Intriguingly, R. griersonianum has accumulated significantly more deleterious mutations in the homozygous state than R. delavayi, and several deleterious mutations (e.g., in genes involved in the response to heat stress) are likely to have harmed the adaptation of this plant to its surroundings. This high-quality, chromosome-level genome and the population genomic analysis of the critically endangered R. griersonianum will provide an invaluable resource as well as insights for future study in this species to facilitate conservation and in the genus Rhododendron in general.

Impatiens wutaishanensis (Balsaminaceae), a new species from Southeast Yunnan, China.

Impatiens wutaishanensis R.L. Liao & Lei Cai, a new species from Southeast Yunnan, China, is here described and illustrated. This new species is most similar to Impatiens parvisepala S.X. Yu & Y.T. Hou in its racemose inflorescences, its four lateral sepals, the leaf arrangement, and in having yellow flowers. However, it differs in the height of the plants, the length of the petiole, the size and shape of the leaf blade, the shape of the spur, and the number of flowers in each inflorescence. A detailed description, color photographs, and a provisional IUCN red list assessment are provided, and its geographical distribution, ecology, and morphological relationship with relevant similar species are discussed.

Natural hybridization between two butterfly bushes in Tibet: dominance of F1 hybrids promotes strong reproductive isolation.

BACKGROUND: It has been recognized that a certain amount of habitat disturbance is a facilitating factor for the occurrence of natural hybridization, yet to date we are unaware of any studies exploring hybridization and reproductive barriers in those plants preferentially occupying disturbed habitats. Buddleja plants (also called butterfly bush) generally do grow in disturbed habitats, and several species with hybrid origin have been proposed, based solely on morphological evidence. RESULTS: In the present study, we test the hypothesis that B. × wardii is of natural hybridization origin in two sympatric populations of three taxa including B. × wardii and its parents (B. alternifolia and B. crispa) plus 4 referenced parental populations, using four nuclear genes and three chloroplast intergenic spacers, as well as with 10 morphological characters. Our results suggest that at both sites B. × wardii is likely to be a hybrid between B. alternifolia and B. crispa, and moreover, we confirm that most of the hybrids examined are F1s. That these plants are F1s is further supported by morphology, as no transgressive characters were detected. B. crispa was found to be the maternal parent in the Bahe (BH) population, from cpDNA evidence. However, in the Taji (TJ) population, the direction of hybridization was difficult to establish due to the shared cpDNA haplotypes between B. alternifolia and B. crispa, however we still predicted a similar unidirectional hybridization pattern due to results from cross-specific pollination treatments which supported the "SI × SC rule". CONCLUSIONS: The presence of mainly F1 hybrids can successfully impede gene flow and thus maintain species boundaries in parental species in a typical distribution of Buddleja, i.e. in disturbed habitats.