Intraspecific divergence in a coastal plant, Euphorbia jolkinii, at a major biogeographic boundary in East Asia.

PREMISE: Quaternary climatic fluctuations and long-distance seed dispersal across the sea are critical factors affecting the distribution of coastal plants, but the spatiotemporal nature of population expansion and distribution change of East Asian coastal plants during this period are rarely examined. To explore this process, we investigated the genome-wide phylogenetic patterns of Euphorbia jolkinii Boiss. (Euphorbiaceae), which grows widely on littoral areas of Japan, Korea, and Taiwan. METHODS: We used plastome sequences and genome-wide single nucleotide polymorphisms in samples across the species range to reveal phylogeographic patterns and spatiotemporal distributional changes. We conducted ecological niche modeling for the present and the last glacial maximum (LGM). RESULTS: Genetic differentiation was observed between the northern and southern populations of E. jolkinii, separated by the major biogeographic boundary, the Tokara Gap. These two groups of populations differentiated during the glacial period and subsequently intermingled in the intermorainic areas of the central Ryukyu Islands after the LGM. Ecological niche models suggested that the potential range of E. jolkinii was restricted to southern Kyushu; however, it was widespread in the southern Ryukyu Islands and Taiwan during the LGM. CONCLUSIONS: This study provides evidence of genetic differentiation among coastal plant populations separated by the prominent biogeographical boundary. Although coastal plants are typically expected to maintain population connectivity through sea-drifted seed dispersal, our findings suggest that genetic differences may arise because of a combination of limited gene flow and changes in climate during the glacial period.

Salvia guidongensis sp. nov.: unraveling a critical evolutionary link in East Asian Salvia from Central China integrating morphology, phylogeny, and plastid genomics.

Introduction: Salvia L., representing the largest genus within the mint family, is noted for its global distribution of approximately 1000 species, with East Asia, and particularly China, recognized as a critical center of diversity for the genus. Methods: Our research was conducted through extensive fieldwork in Guidong County, Hunan Province, China, where we identified a previously undescribed species of Salvia. The identification process involved detailed morphological observations, phylogenetic analyses, and plastid genomics. Results: The newly discovered species, Salvia guidongensis, exhibits unique characteristics not commonly observed in the East Asian lineage of Salvia, including dual floral colors within natural populations-either pale purple or pale yellow. Morphologically, while it shares similarities with members of sect. Glutinaria, S. guidongensis is distinct in its floral morphology, stature, and specific foliar traits. Phylogenetic analysis places S. guidongensis in a unique clade within the East Asian lineage of Salvia, suggesting it may serve as an important evolutionary link. Additionally, we explored the plastome features of S. guidongensis, comparing them with those of closely related species. Discussion: The discovery of S. guidongensis not only entriches the taxonomic tapestry of Salvia but also provides critical insights into the biogeography and evolutionary pathways of the genus in East Asia. By integrating morphological and molecular data, we validate the novel status of S. guidongensis and highlight its significance in bridging taxonomic and evolutionary gaps within Sect. Glutinaria of Salvia.

Phylogenomics and morphological evolution of the mega-diverse genus Artemisia (Asteraceae: Anthemideae): implications for its circumscription and infrageneric taxonomy.

BACKGROUND AND AIMS: Artemisia is a mega-diverse genus consisting of ~400 species. Despite its medicinal importance and ecological significance, a well-resolved phylogeny for global Artemisia, a natural generic delimitation and infrageneric taxonomy remain missing, owing to the obstructions from limited taxon sampling and insufficient information on DNA markers. Its morphological characters, such as capitulum, life form and leaf, show marked variations and are widely used in its infrageneric taxonomy. However, their evolution within Artemisia is poorly understood. Here, we aimed to reconstruct a well-resolved phylogeny for global Artemisia via a phylogenomic approach, to infer the evolutionary patterns of its key morphological characters and to update its circumscription and infrageneric taxonomy. METHODS: We sampled 228 species (258 samples) of Artemisia and its allies from both fresh and herbarium collections, covering all the subgenera and its main geographical areas, and conducted a phylogenomic analysis based on nuclear single nucleotide polymorphisms (SNPs) obtained from genome skimming data. Based on the phylogenetic framework, we inferred the possible evolutionary patterns of six key morphological characters widely used in its previous taxonomy. KEY RESULTS: The genus Kaschgaria was revealed to be nested in Artemisia with strong support. A well-resolved phylogeny of Artemisia consisting of eight highly supported clades was recovered, two of which were identified for the first time. Most of the previously recognized subgenera were not supported as monophyletic. Evolutionary inferences based on the six morphological characters showed that different states of these characters originated independently more than once. CONCLUSIONS: The circumscription of Artemisia is enlarged to include the genus Kaschgaria. The morphological characters traditionally used for the infrageneric taxonomy of Artemisia do not match the new phylogenetic tree. They experienced a more complex evolutionary history than previously thought. We propose a revised infrageneric taxonomy of the newly circumscribed Artemisia, with eight recognized subgenera to accommodate the new results.

Allopatric Lineage Divergence of the East Asian Endemic Herb Conandron ramondioides Inferred from Low-Copy Nuclear and Plastid Markers.

The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant, Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan-Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan-Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82-3.86), followed by divergence between the Taiwan-Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45-1.3). Furthermore, corolla traits (floral lobe length to tube length ratios) correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species' biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary.

The complete chloroplast genome of a coastal plant, Euphorbia jolkinii (Euphorbiaceae).

The complete chloroplast genome sequence of a coastal plant, Euphorbia jolkinii Boiss. (Euphorbiaceae), was determined. The chloroplast genome was 162,854 bp in length, consisting of a large single copy region (90,726 bp), a small single copy region (18,422 bp), and two inverted repeats (26,853 bp). The chloroplast genome contained 115 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, four unique rRNA genes, and one pseudogene, rps16. GC content of the whole chloroplast genome was 35.6%. The phylogenetic analysis showed a close relationship between E. jolkinii and E. pekinensis Rupr. The sequence data would provide useful information to understand the evolutionary process of E. jolkinii.

Plastome phylogenomics of Allaeanthus, Broussonetia and Malaisia (Dorstenieae, Moraceae) and the origin of B. × kazinoki.

Species of Broussonetia have been essential in the development of papermaking technology. In Japan and Korea, a hybrid between B. monoica and B. papyrifera (= B. × kazinoki) known as kozo and daknamu is still the major source of raw materials for making traditional paper washi and hanji, respectively. Despite their cultural and practical significance, however, the origin and taxonomy of kozo and daknamu remain controversial. Additionally, the long-held generic concept of Broussonetia s.l., which included Sect. Allaeanthus and Sect. Broussonetia, was challenged as phylogenetic analyses showed Malaisia is sister to the latter section. To re-examine the taxonomic proposition that recognizes Allaeanthus, Broussonetia, and Malaisia (i.e., Broussonetia alliance), plastome and nuclear ribosomal DNA (nrDNA) sequences of six species of the alliance were assembled. Characterized by the canonical quadripartite structure, genome alignments and contents of the six plastomes (160,121-162,594 bp) are highly conserved, except for the pseudogenization and/or loss of the rpl22 gene. Relationships of the Broussonetia alliance are identical between plastome and nrDNA trees, supporting the maintenance of Malaisia and the resurrection of Allaeanthus. The phylogenomic relationships also indicate that the monoecy in B. monoica is a derived state, possibly resulting from hybridization between the dioecious B. kaempferi (♀) and B. papyrifera (♂). Based on the hypervariable ndhF-rpl32 intergenic spacer selected by sliding window analysis, phylogeographic analysis indicates that B. monoica is the sole maternal parent of B. × kazinoki and that daknamu carries multiple haplotypes, while only one haplotype was detected in kozo. Because hybridizations between B. monoica and B. papyrifera are unidirectional and have occurred rarely in nature, our data suggest that daknamu might have originated via deliberate hybrid breeding selected for making hanji in Korea. On the contrary, kozo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Sedum formosanum subsp. miyakojimense (Crassulaceae), a new subspecies from Miyako-jima Island of the Ryukyu Islands, Japan.

We re-examined the taxonomic status of plants treated as Sedum formosanum (Crassulaceae) from Miyako-jima Island of the Ryukyu Islands, Japan, using morphological comparison and molecular phylogenetic analyses with related species. In morphology, plants from Miyako-jima Island bore a close resemblance to the other plants of S. formosanum, but differed in being perennial, polycarpic, and having lateral axillary branches. Molecular analyses based on ITS of nrDNA and six regions of cpDNA sequencing indicated that the Miyako-jima plants formed a distinct subclade. This subclade was part of a polytomy with three other subclades comprising nine taxa endemic to Taiwan and S. formosanum from other areas, including the type locality. Therefore, we propose and describe the Miyako-jima plants as a new subspecies, Sedum formosanum subsp. miyakojimense.

Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data.

BACKGROUND AND AIMS: The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. METHODS: We first compared the performance of phylogenetic analysis based on the plastid matK and trnL-F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. KEY RESULTS: Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0-84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. CONCLUSIONS: The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Unique parallel radiations of high-mountainous species of the genus Sedum (Crassulaceae) on the continental island of Taiwan.

We explored the temporal and spatial diversification of the plant genus Sedum L. (Crassulaceae) in Taiwan based on molecular analysis of nrITS and cpDNA sequences from East Asian Sedum members. Our phylogenetic and ancestral area reconstruction analysis showed that Taiwanese Sedum comprised two lineages that independently migrated from Japan and Eastern China. Furthermore, the genetic distances among species in these two clades were smaller than those of other East Asian Sedum clades, and the Taiwanese members of each clade occupy extremely varied habitats with similar niches in high-mountain regions. These data indicate that species diversification occurred in parallel in the two Taiwanese Sedum lineages, and that these parallel radiations could have occurred within the small continental island of Taiwan. Moreover, the estimated time of divergence for Taiwanese Sedum indicates that the two radiations might have been correlated to the formation of mountains in Taiwan during the early Pleistocene. We suggest that these parallel radiations may be attributable to the geographical dynamics of Taiwan and specific biological features of Sedum that allow them to adapt to new ecological niches.

Further characterization of foliar flavonoids in Crossostephium chinense and their geographic variation.

Foliar flavonoids of Crossostephium chinense in Japan and Taiwan were isolated and further characterized. Eighteen flavonoid aglycones, luteolin, apigenin, hispidulin, chrysoeriol, 5,7,4'-trihydroxy-6,3',5'-trimethoxyflavone, jaceosidin, cilsimaritin, quercetin 3-methyl ether, axillarin, chrysosplenol-D, cirsiliol, apometzgerin, 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone, luteolin 3',4'-dimethyl ether, cirsilineol, eupatilin, nepetin and 5,7,3',4'-tetrahydroxy-6,5'-dimethoxyflavone, were identified by UV, 1H and 13C NMR spectroscopic, LC-MS and HPLC comparisons w ith authentic samples. The compounds existed on the leaf surface. Four flavonoid glycosides, quercetin 3,7-di-O-glucoside, quercetin 3-O-rutinoside, luteolin 7-O-glucoside and apigenin 7-O-rutinoside, were also isolated as the intracellular flavonoids. It was shown by HPLC survey that variation of the species' flavonoids occurs among the collection sites.

Extreme habitats that emerged in the Pleistocene triggered divergence of weedy Youngia (Asteraceae) in Taiwan.

Weeds with broad distributions and large morphological variation are challenging for systematists and evolutionarily intriguing because their intensive dispersal would likely prevent local morphological differentiation. Study on weeds will help to understand divergence in plants unlikely to be affected by geographical and ecological barriers. We studied Youngia japonica based on nrDNA and cpDNA sequences. This is a widespread native in Asia and invasive worldwide; nevertheless, three subspecies (japonica, longiflora, and formosana) and an undescribed variant occur in Taiwan. Bayesian and the most parsimonious phylogenies revealed that subspecies longiflora is a different linage and independently arrived in Taiwan during the Pleistocene via land connection to the Asian Continent. Bayesian time estimation suggested that Youngia in Taiwan diverged in the lower Pleistocene or more recently. Extreme habitats that emerged in the Pleistocene, i.e., cold mountain ranges for subspecies formosana and xeric, raised coral reefs for the undescribed Youngia variant probably had triggered the divergence. Components of Youngia in Taiwan are not monophyletic; a coalescent-based test suggested incomplete lineage sorting. Nevertheless, the samples within each taxon share unique morphological features suggesting a common gene pool and each taxon has different dominant ITS and/or cpDNA types; these conditions suggest ongoing process toward monophyly via coalescent processes and support the delimitation of intraspecific taxa.

Apigenin di- and trirhamnoside from Asplenium normale in Malaysia.

Two new flavone rhamnosides, apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside and apigenin 7-O-alpha-L-rhamnopyranosyl-(1-->4)-O-alpha-L-rhamnopyranoside-4'-O-alpha-L-rhamnopyranoside were isolated from the fronds of Asplenium normale D. Don, together with two known C-glycosylflavones, vicenin-2 and lucenin-2. The chemical structures of the isolated glycosides were established by UV, LC-MS, characterization of acid hydrolysates, and 1H and 13C NMR spectroscopy.

Evidence of three parallel evolutions of leaf dwarfism and phytogeography in Lysimachia sect. Nummularia in Japan and Taiwan.

Phylogenetic analyses were performed using nrITS variations of eight species including one variety of the genus Lysimachia sect. Nummularia in Japan and Taiwan, with another 34 Lysimachia species and Glaux maritime obtained from a DNA database. Among the four dwarf-leaf taxa in Japan and Taiwan, L. japonica var. minutissima of Japan and two Taiwanese species were placed into two different clades; and L. liukiuensis of Japan, was positioned outside of these two clades. These results suggest that three independent dwarfism events generated the four dwarf-leaf taxa; and that there were likely multiple migration events from China to Japan and Taiwan.

Phylogenetic systematics of the monotypic genus Hayataella (Rubiaceae) endemic to Taiwan.

Hayataella (Rubiaceae) is a monotypic genus endemic to Taiwan that comprises H. michelloides. In recent years, Hayataella was considered to be synonymous with Ophiorrhiza; however, no specific data have been reported, and the systematic treatment of Hayataella has been unclear. To elucidate the systematic treatment of Hayataella, molecular phylogenetic analyses based on ITS of nrDNA, atpB-rbcL, and trnK/matK of cpDNA were conducted with four Ophiorrhiza species. In the Bayesian and the most parsimonious trees, H. michelloides was included in the Ophiorrhiza clade. The monotypic status of Hayataella is, therefore, not considered appropriate, and the combination Ophiorrhiza michelloides (Masam.) H. S. Lo is supported.