Reproductive biology of an endangered lithophytic shrub and implications for its conservation.

BACKGROUND: Plants in cliff habitats may evolve specific reproductive strategies to cope with harsh environments, and unraveling these reproductive characteristics can improve our understanding of survival strategies and lithophyte evolution. This understanding is especially important for efforts to protect rare and endemic plants. Here, we investigated the reproductive biology of Lonicera oblata, an endangered lithophytic shrub that is scattered in highly fragmented and isolated cliff habitats of the Taihang and Yan mountains in North China. RESULTS: Flowers of L. oblata are herkogamous and protandrous, characteristics that can prevent autogamy at the single-flower level, and insects are necessary for pollination. The outcrossing index, pollen/ovule ratio, and the results of hand pollination were measured and all revealed a mixed mating system for L. oblata, that combines cross-fertilization and partial self-fertilization. The floral traits of L. oblata of zygomorphic and brightly yellowish corolla, heavy fragrance, and rich nectar, suggest an entomophilous pollination system. Sweat bees were observed as the most effective pollinators but their visiting frequencies were not high. Pollen limitation may limit the reproductive success of L. oblata. CONCLUSIONS: We determined the reproductive characteristics of L. oblata, a critically endangered species endemic to cliffs in North China, providing insight into its endangerment and suggesting conservation strategies. L. oblata has highly pollinator-dependent self-fertilization as part of a mixed mating system. Floral features such as low-flowering synchrony, asynchronous anthers dehiscence, and high duration of stigma receptivity, improve pollination efficiency in the case of low pollinator service. Our work provides reference information to understand the survival strategies and conservation of L. oblata and other lithophytes.

Phylogeny and divergence time estimation of the walnut family (Juglandaceae) based on nuclear RAD-Seq and chloroplast genome data.

The walnut family Juglandaceae was widely distributed in the Northern Hemisphere while several extant genera now exhibit intercontinental disjunctions. Recent progress in the systematics of Juglandaceae has greatly broadened our knowledge about its origin and evolution. However, there are still uncertainties about the intergeneric relationships within Juglandaceae, and discrepancies between fossil records and inferred divergence times for certain lineages were observed. In this study, well-resolved phylogenies of the Juglandaceae are reconstructed based on both the nuclear RAD-Seq and the whole chloroplast genome data. Our results support the Juglandoideae topology of (Hicoreae, (Platycaryeae, Juglandeae)) at the tribal level. Within Juglandeae, a discordant position of Pterocarya was detected between nuclear and plastid genome data, and a more likely topology (nuclear), (Juglans, (Pterocarya, Cyclocarya)), was discussed based on evidence from molecular data and fossil records. Based on carefully selected fossil calibrations, the divergence times of extant lineages were estimated and they corroborated well with fossil records (especially concerning Juglans and Pterocarya). Four sections within Juglans were strongly supported by the nuclear data. Within Juglans, the incongruent position of J. hopeiensis was recovered between the nuclear and plastid genomes. Yet the origin and evolutionary history of J. cinerea and J. hopeiensis are supported to be complicated and need further clarification. Integrative evidence from the fossil records, phylogeny and lineage divergence times shows that Juglandoideae originated in North America, and migrated to Eurasia via both the Bering and the North Atlantic land bridges. Our study shows the potential of integrative biogeographic studies for illuminating the evolutionary history of Juglandaceae.

The first complete chloroplast genome in Engelhardia sensu stricto, Engelhardia hainanensis Chen: genome characterization and its phylogenetic relationships within the family Juglandaceae.

Trees of Engelhardia are important components of subtropical and tropical forests in South-eastern Asia with great ecological and economic values. However, phylogenetic relationships within Engelhardioideae (Juglandaceae) remains obscure. In this study, we report the first complete chloroplast genome sequences of Engelhardia sensu stricto, Engelhardia hainanensis Chen, a rare species endemic in southern China. Its complete chloroplast genome is 161,574 bp in length, with a typical quadripartite structure that includes a large single-copy region of 91,158 bp, a small single-copy region of 18,790 bp, and its GC content is 35.8%. A total of 128 genes were identified, including 83 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Furthermore, a phylogenetic tree of Juglandaceae was constructed based the complete chloroplast genome sequence, which strongly support the three-subfamily classification system in Juglandaceae, and E. hainanensis was resolved sister to two Alfaropsis species. This study provides valuable genomic information for the species identification and phylogenetic study of Juglandaceae.

The chromosome-scale genome of Kobresia myosuroides sheds light on karyotype evolution and recent diversification of a dominant herb group on the Qinghai-Tibet Plateau.

Kobresia species are common in meadows on the Qinghai-Tibet Plateau. They are important food resources for local livestock, and serve a critical foundation for ecosystem integration. Genetic resources of Kobresia species are scarce. Here, we generated a chromosome-level genome assembly for K. myosuroides (Cyperaceae), using PacBio long-reads, Illumina short-reads, and Hi-C technology. The final assembly had a total size of 399.9 Mb with a contig N50 value of 11.9 Mb. The Hi-C result supported a 29 pseudomolecules model which was in consistent with cytological results. A total of 185.5 Mb (44.89% of the genome) transposable elements were detected, and 26,748 protein-coding genes were predicted. Comparative analysis revealed that Kobresia plants have experienced recent diversification events during the late Miocene to Pliocene. Karyotypes analysis indicated that the fission and fusion of chromosomes have been a major driver of speciation, which complied with the lack of whole-genome duplication (WGD) in K. myosuroides genome. Generally, this high-quality reference genome provides insights into the evolution of alpine sedges, and may be helpful to endemic forage improvement and alpine ecosystem preservation.

Phylogeny of Celastrus L. (Celastraceae) inferred from two nuclear and three plastid markers.

This is the first comprehensive molecular investigation of the genus Celastrus L. Phylogenetic relationships within the genus were assessed based on sequences of two nuclear (ETS, ITS) and three plastid (psbA-trnH, rpl16 and trnL-F) regions using the Bayesian inference and the maximum parsimony methods. Our results show that Celastrus, together with Tripterygium, formed a maximal supported clade. Within the cluster, Celastrus is composed of a basal clade and a core Celastrus clade, and the latter is consisted of six subclades. Relationships among species are more influenced by latitude than continental distribution patterns. The cauline cyme and lunate seeds are distinct characters to one of the maximal supported subclades. Their close relationship, similar geographical pattern and habitat imply that C. flagellaris may be a potential invasive species threatening C. scandens in North America. Celastrus leiocarpus, C. oblanceifolius and C. rugosus are confirmed as synonyms of C. punctatus, C. aculeatus and C. glaucophyllus, respectively. Discordance between the molecular data and previous morphology-based subgeneric classifications are noted. More works are needed to clarify the relationship between Celastrus and Tripterygium and the species within Celastrus.

Genomic Data Reveals Profound Genetic Structure and Multiple Glacial Refugia in Lonicera oblata (Caprifoliaceae), a Threatened Montane Shrub Endemic to North China.

Characterizing genetic diversity and structure and identifying conservation units are both crucial for the conservation and management of threatened species. The development of high-throughput sequencing technology provides exciting opportunities for conservation genetics. Here, we employed the powerful SuperGBS method to identify 33, 758 high-quality single-nucleotide polymorphisms (SNP) from 134 individuals of a critically endangered montane shrub endemic to North China, Lonicera oblata. A low level of genetic diversity and a high degree of genetic differentiation among populations were observed based on the SNP data. Both principal component and phylogenetic analyses detected seven clusters, which correspond exactly to the seven geographic populations. Under the optimal K = 7, Admixture suggested the combination of the two small and geographically neighboring populations in the Taihang Mountains, Dongling Mountains, and Lijiazhuang, while the division of the big population of Jiankou Great Wall in the Yan Mountains into two clusters. High population genetic diversity and a large number of private alleles were detected in the four large populations, while low diversity and non-private alleles were observed for the remaining three small populations, implying the importance of these large populations as conservation units in priority. Demographic history inference suggested two drastic contractions of population size events that occurred after the Middle Pleistocene Transition and the Last Glacial Maximum, respectively. Combining our previous ecological niche modeling results with the present genomic data, there was a possible presence of glacial refugia in the Taihang and Yan Mountains, North China. This study provides valuable data for the conservation and management of L. oblata and broadens the understanding of the high biodiversity in the Taihang and Yan Mountains.

Characterization of the complete chloroplast genome of a subalpine deciduous shrub Lonicera angustifolia var. myrtillus (Caprifoliaceae).

The genus Lonicera (Caprifoliaceae) is of great economical significance. It has been taxonomically studied frequently in history, while phylogenetic relationships intra the genus are still obscure. Here, we reported the first species complete chloroplast genome sequence in the section Isoxylosteum, Lonicera angustifolia var. myrtillus. It is 156,222 bp in length, comprising a large single-copy (LSC) region of 89,838 bp, a small single-copy (SSC) region of 19,211 bp, and a pair of inverted repeats (IRs) of 23,509 bp. In L. angustifolia var. myrtillus chloroplast genome, a total of 114 functional genes were identified, with an overall GC content of 38.4%. The phylogenetic relationships of Lonicera based on maximum-likelihood (ML) showed that L. angustifolia var. myrtillus is most closely related to L. nervosa in section Isika. Our study contributes to the molecular phylogenetic studies of Lonicera and Caprifoliaceae.

Plastid phylogenomics and biogeography of the medicinal plant lineage Hyoscyameae (Solanaceae).

The cosmopolitan family Solanaceae, which originated and first diversified in South America, is economically important. The tribe Hyoscyameae is one of the three clades in Solanaceae that occurs outside of the New World; Hyoscyameae genera are distributed mainly in Europe and Asia, and have centers of species diversity in the Qinghai-Tibet Plateau and adjacent regions. Although many phylogenetic studies have focused on Solanaceae, the phylogenetic relationships within the tribe Hyoscyameae and its biogeographic history remain obscure. In this study, we reconstructed the phylogeny of Hyoscyameae based on whole chloroplast genome data, and estimated lineage divergence times according to the newly reported fruit fossil from the Eocene Patagonia, Physalis infinemundi, the earliest known fossil of Solanaceae. We reconstructed a robust phylogeny of Hyoscyameae that reveals the berry fruit-type Atropa is sister to the six capsule-bearing genera (Hyoscyameae sensu stricto), Atropanthe is sister to the clade (Scopolia, Physochlaina, Przewalskia), and together they are sister to the robustly supported Anisodus-Hyoscyamus clade. The stem age of Hyoscyameae was inferred to be in the Eocene (47.11 Ma, 95% HPD: 36.75-57.86 Ma), and the crown ages of Hyoscyameae sensu stricto were estimated as the early Miocene (22.52 Ma, 95% HPD: 15.19-30.53 Ma), which shows a close correlation with the rapid uplift of the Qinghai-Tibet Plateau at the Paleogene/Neogene boundary. Our results provide insights into the phylogenetic relationships and the history of the biogeographic diversification of the tribe Hyoscyameae, as well as plant diversification on the Qinghai-Tibet Plateau.

The complete chloroplast genome of Symphoricarpos orbiculatus (Caprifoliaceae), an important ornamental plant.

Symphoricarpos orbiculatus is an important landscape and ornamental plant. In this study, we report the complete chloroplast genome sequences of S. orbiculatus. The complete chloroplast genome of S. orbiculatus was 156,044 bp in length. The genome has a typical quadripartite structure, including a large single-copy (LSC) region of 88,756 bp, a small single-copy (SSC) region of 19,130 bp, and two inverted repeat (IR) regions of 24,079 bp each. Overall, the GC content was 38.4%. In the genome, it was identified to comprise130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. This study provides valuable information for molecular phylogenetic study of Caprifoliaceae and is significant for variety development of Symphoricarpos.

The complete chloroplast genome of Physochlaina physaloides (Solanaceae), an important medicinal plant.

Physochlaina is an important perennial herbaceous genus with significant medicinal value, while the phylogeny of Physochlaina and tribe Hyoscyameae is not well resolved yet. In this study, we report the complete chloroplast genome sequences of Ph. physaloides, its complete chloroplast genome is 156,413 bp in length, which is a typical quadripartite structure that includes a large single-copy region of 86,659 bp, a small single-copy region of 18,012 bp, and its GC content was 37.7%. A total of 132 genes were identified, including 87 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Furthermore, a phylogenetic tree of the tribe Hyoscyameae was constructed based the complete chloroplast genome sequence, and a new topology of the tribe was obtained. This study provides valuable genetic information for the conservation and utilization of Ph. physaloides and also provide the potential for better understanding of the phylogeny of Hyoscyameae and Solanaceae.

The complete chloroplast genome of Lonicera oblata, a critically endangered species endemic to North China.

Lonicera oblata, a critically endangered species endemic to North China with about 30 wild individuals, has long been ignored for conservation since its publication because of little attention on its living situation. In this study, we characterized the complete chloroplast (cp) genome of L. oblata. The cp genome was 155,481 bp in length, included a large single-copy (LSC) region of 89,139 bp, a small single-copy (SSC) region of 18,676 bp, and two inverted repeat (IR) regions of 23,833 bp each. The genome contains 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic position of L. oblata was also investigated based on cp genome phylogeny of Lonicera representatives. This study is valuable for molecular phylogenetic study and conservation of Lonicera and related taxa.

Molecular Analysis of Chinese Celastrus and Tripterygium and Implications in Medicinal and Pharmacological Studies.

Celastrus and Tripterygium species, which are used in traditional Chinese medicine, have attracted much attention due to their anti-tumor promoting and neuroprotective activities, in addition to their applications in autoimmune disorders. However, systematic relationships between them and among species are unclear, and it may disturb their further medicinal utilization. In the present study, the molecular analysis of combined chloroplast and nuclear markers of all Chinese Celastrus and Tripterygium was performed, and clear inter- and intra-genus relationships were presented. The result suggests that Tripterygium constitute a natural monophyletic clade within Celastrus with strong support value. Fruit and seed type are better than inflorescence in subgeneric classification. Chinese Celastrus are classified for three sections: Sect. Sempervirentes (Maxim.) CY Cheng & TC Kao, Sect. Lunatus XY Mu & ZX Zhang, sect. nov., and Sect. Ellipticus XY Mu & ZX Zhang, sect. nov. The phylogenetic data was consistent with their chemical components reported previously. Owing to the close relationship, several evergreen Celastrus species are recommended for chemical and pharmacological studies. Our results also provide reference for molecular identification of Chinese Celastrus and Tripterygium.

Unveiling the Identity of Wenwan Walnuts and Phylogenetic Relationships of Asian Juglans Species Using Restriction Site-Associated DNA-Sequencing.

Juglans species have considerable ecological and economic value worldwide. In China, Wenwan walnuts have been collected by aristocrats and noblemen for more than 2000 years. As a diversity center of Asian Juglans, five species are widely distributed in China. The most famous of these is Mahetao (J. hopeiensis), which is an uncharacterized species that is mostly cultivated. Wild J. hopeiensis individuals are very rare and are endemic to Hebei Province. Because of the minimal variations in previously used molecular markers and the heterogeneity between chloroplast and nuclear genomes, determining the phylogenetic relationships among the Juglans species has been challenging, and has hindered subsequent evolutionary inferences. In this study, we collected enough materials for both cultivated and wild Mahetao to construct well-resolved phylogenetic trees for Asian Juglans species. We used a high-throughput genome-wide restriction site-associated DNA sequencing method. Consequently, the identity of J. hopeiensis has been clearly resolved. Our results indicate that J. hopeiensis is a hybrid of J. regia and J. mandshurica. However, J. hopeiensis, J. regia and J. sigillata should be considered as a single species from section Juglans. Additionally, J. ailantifolia, J. cathayensis, and J. mandshurica likely represent one species from section Cardiocaryon according to morphological and molecular studies. These results are supported by population structure analysis and morphological comparison. We propose that J. hopeiensis trees growing in the wild should be conserved because of the economic value of their nuts. These trees may be of particular importance to impoverished communities. Furthermore, they may serve as a valuable genetic resource relevant for enhancing the production of edible walnuts. The 2b-RAD method is a viable option for future phylogenetic studies of Juglans species as well as other plant species.