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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., This work was supported by the National Natural Science Foundation of China (grant nos. 31870179, 31570204, 31270237 and J1310002), the International Partnership Program (grant no. 151853KYSB20190027), Sino-Africa Joint Research Center (grant no. SAJC201614), Key technology projects of Jiangxi Province's major scientific and technological research and development project (grant no. 20223AAF01007), Survey of Wildlife Resources in Key Areas of Tibet (grant no. ZL202203601) and National Plant Specimen Resource Center (grant no. E0117G1001) of the Chinese Academy of Sciences, Key Project at Central Government Level: The Ability Establishment of Sustainable Use of Valuable Chinese Medicine Resources (grant no. 2060302) and Project of the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences (grant no. AAAA-A21-121011290024-5)., INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Conclusions SUPPLEMENTARY DATA FUNDING ACKNOWLEDGEMENTS CONFLICT OF INTEREST LITERATURE CITED Supplementary data

The earliest colonization of oceanic islands by Homo sapiens occurred about 50,000–30,000 years ago in the tropical and temperate waters of the western Pacific, yet how this was achieved remains un...

A taxonomic re-examination of Sedum boninense, which is endemic to the Muko-jima and Chichi-jima groups of the Bonin Islands, Japan, was carried out. Morphological and physiological comparisons revealed differences between plants from the Muko-jima and Chichi-jima groups in the shape and size of leaves and underground bulb scale leaves, plant habit, habitat and flowering season. Molecular phylogenetic analysis using the ITS region revealed that the plants from respective island groups formed sister clades. Therefore, the plant from Muko-jima group is described as a new species of S. mukojimense.

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 kōzo 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 kōzo 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 kōzo. 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, kōzo appears to have a single origin and the possibility of a Korean origin cannot be ruled out.

Fourteen flavonols, four flavones and six isoflavones were isolated from the aerial parts of two Japanese Glycine species, G. tabacina and G. koidzumii, and the leaves of Taiwanese G. max subsp. formosana. Of their flavonoids, twelve flavonols were identified as kaempferol 3-O-sophoroside (1), 3-O-rutinoside (2), 3-O-robinobioside (3) and 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside] (4), quercetin 3-O-gentiobioside (5), 3-O-glucoside (6), 3-O-galactoside (7), 3-O-rutinoside (8), 3-O-robinobioside (9) and 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside] (10), and isorhamnetin 3-O-rutinoside (11) and 3-O-robinobioside (12). Other two flavonols were characterized as isorhamnetin 3-O-rhamnosylrhamnosylglucoside (13) and 3-O-rhamnosylrhamnosylgalactoside (14). Four flavones and six isoflavones were estimated as schaftoside (15), apigenin 6,8-di-C-arabinoside (16), luteolin 7-O-glucoside (17) and chrysoeriol 7-O-glucoside (18), and daidzein 7-O-glucoside (19), 4'-O-glucoside (20) and 7-O-xylosylglucoside (21), genistein 7-O-glucoside (22) and 4'-O-glucoside (23), and 3'-O-methylorobol 7-O-glucoside (24). Although flavonoid composition of G. tabacina and G. koidzumii was similar to each other, that of G. max subsp. formosana was different with those of two Japanese Glycine species described above. Flavonoids of their Glycine species were reported for the first time except for those of G. tabacina., 論文

Background and AimsThe 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.MethodsWe 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 ResultsPhylogenomic 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.ConclusionsThe 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.

The islands of Southeast Asia comprise one of the most geologically and biogeographically complex areas in the world and are a centre of exceptional floristic diversity, harbouring 45,000 species of flowering plants. Cyrtandra, with over 800 species of herbs and shrubs, is the largest genus in the family Gesneriaceae and is one of the most emblematic and species-rich genera of the Malesian rainforest understorey. The high number of species and tendency to narrow endemism make Cyrtandra an ideal genus for examining biogeographic patterns. We sampled 128 Cyrtandra taxa from key localities across Southeast Asia to evaluate the geo-temporal patterns and evolutionary dynamics of this clade. One nuclear and four chloroplast regions were used for phylogenetic reconstruction, molecular dating, and ancestral range estimation. Results from the dating analysis suggest that the great diversity of Cyrtandra seen in the Malesian region results from a recent radiation, with most speciation taking place in the last five million years. Borneo was recovered as the most likely ancestral range of the genus, with the current distribution of species resulting from a west to east migration across Malesia that corresponds with island emergence and mountain building. Lastly, our investigation into the biogeographic history of the genus indicates high levels of floristic exchange between the islands on the Sunda shelf and the important role of the Philippines as a stepping stone to Wallacea and New Guinea. These patterns underlie much of the plant diversity in the region and form an emerging paradigm in Southeast Asian plant biogeography.

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.

In northeast Asia, substantial portion of the floras, including endangered species, are shared among its component countries in the continental, peninsula, and island parts largely through Quaternary migration. To effectively conserve nationally endangered plants in Northeast Asia, transnational conservation studies are vitally needed. Lychnis wilfordii (Caryophyllaceae) has disjunct distribution in Russian Far East (Primorsky Krai), northeast China (Jilin), Korea (Gangwon-do) and Japan (Hokkaido, Aomori, Nagano), surrounding the sea, and this is designated as an endangered species in Japan and Korea. Population genetic and molecular dating analyses were conducted 1) to elucidate geographic genetic structure covering the species range, 2) to test possible scenarios of migration, and 3) to develop logical plans for effective conservation. Population genetic analyses indicated the continent and peninsula parts (north and south Primorsky Krai, Jilin, and Gangwon-do) had higher genetic diversity compared to those in the Japanese Archipelago (Hokkaido and Nagano). Five genetically distinct groups were recognized, namely, Nagano, Gangwon-do, Jilin, north and south Primorsky Krai plus Aomori, and Hokkaido. Genetic distance between Hokkaido and Nagano was larger than between Hokkaido and north Primorsky Krai, and between Nagano and Gangwon-do, crossing national borders and the natural barrier of the sea. Considering these results, L. wilfordii likely migrated from the Asian continent to the Japanese Archipelago using two routes: north route from Russian Far East to Hokkaido and Aomori, and south route from the Korean Peninsula to Nagano. Based on molecular dating, migration from the continent to the islands likely occurred from the middle Pleistocene to the Holocene. For effective conservation of L. wilfordii, Hokkaido and Nagano populations should be distinguished as different evolutionary significant units, although these two regions belong to the same country, because Hokkaido and Nagano populations are at the different ends of the two migratory routes based on the migration scenario.

The fresh leaves of Hillebrandia sandwicensis and 126 Begonia taxa were chemotaxonomically surveyed for flavonoids. Of their taxa, H. sandwicensis and 119 species, one variety and three hybrids were analyzed for flavonoids for the first time. Ten flavonols and eleven C-glycosylflavones were isolated and characterized as quercetin 3-O-rutinoside (1), kaempferol 3-O-rutinoside (2), isorhamnetin 3-O-rutinoside (3), quercetin 3-O-glucoside (4), quercetin 3-methyl ether 7-O-rhamnosylglucoside (5), quercetin 3,3'-dimethyl ether 7-O-rhamnosylglucoside (6), quercetin glycoside (13), quercetin glycoside (acylated) (14), kaempferol glycoside (17) and quercetin 3-O-rhamnoside (18) as flavonols, and isovitexin (7), vitexin (8), isoorientin (9), orientin (10), luteolin 6-C-pentoside (11), luteolin 8-C-pentoside (12), schaftoside (15), isoschaftoside (16), chrysoeriol 6,8-di-C-pentoside (19), apigenin 6,8-di-C-arabinoside (20) and isovitexin 2''-O-glucoside (21) as C-glycosylflavones. Quercetin 3-O-rutinoside (1) alone was isolated from H. sandwicensis endemic to Hawaii. Major flavonoids of almost Begonia species was also 1. Begonia species were divided into two chemotypes, i.e. flavonol containing type and C-glycosylflavone containing type. Of 14 section of the Begonia, almost species of many section, i.e. sect. Augustia, Coelocentrum, Doratometra, Leprosae, Loasibegonia, Monopteron and Ruizoperonia, were flavonol types. On the other hand, C-glycosyflavone type was comparatively most in sect. Platycentrum.

In continental island biogeography, geologically age-old straits have been considered to be the most likely barriers in determining geographical patterns of speciation/genetic differentiation among islands. Straits with similar ages may have had different influences if geographical width across the straits had fluctuated differently in the past. However, this issue has seldom been discussed. We explored it by studying the Viola iwagawae-tashiroi species complex in the Ryukyu Archipelago, Japan. The archipelago is divided into three island groups, the northern, central, and southern Ryukyus, by two old straits called the Tokara and Kerama gaps, which are roughly of the same age. Bayesian and maximum parsimony phylogenetic analyses of section Plagiostigma and Bayesian molecular dating using multiple calibration points were conducted based on nuclear ribosomal DNA internal transcribed spacer (ITS) sequences. To reveal the detailed genealogy of the species complex, statistical parsimony networks were estimated separately for the ITS and chloroplast DNA (cpDNA) sequences and a calibrated multispecies coalescent tree based on both ITS and cpDNA sequences was constructed. Results suggest that the V. iwagawae-tashiroi species complex originated in the Ryukyu Archipelago when this region formed part of the East Asian continental margin or was formed by a few larger islands and that the complex was already distributed across the archipelago by the late Pliocene–early Pleistocene. Divergence time estimations suggest that the Kerama Gap has been a long-term effective barrier to dispersal preventing gene flow across it, whereas recent dispersal over the Tokara Gap was inferred. These contrasting results are likely explained by the fact that the minimum geographical width across the straits is geohistorically and currently greater over the Kerama Gap than at the Tokara Gap.

Equatorward contraction and re-expansion during and after the last glacial maximum has been reported for temperate plants, but little is known about latitudinal shifts of tropical plants in insular Asia. We used molecular methods to test the post-glacial migration hypothesis for Begonia fenicis at the northern limit of tropical insular Asia, namely, the northern Philippines, southern Taiwan and the southern Ryukyus of Japan. Based on internal transcribed spacer sequences, analyses of phylogeographical structure suggested long-term survival of island populations and negated recent northward migration; demographic analyses indicated spatial expansion in the early Pleistocene and subsequent stable demography. Based on nuclear microsatellites, Bayesian clustering and other spatial analyses indicated inter-island differentiation, and coalescent analysis of migration indicated the lack of inter-island migration during and after the last glacial maximum. Our results refuted the postglacial migration hypothesis for B. fenicis and suggest in situ survival through the last glacial maximum. The stable historical distribution and demography are probably attributable to the Kuroshio Current bringing warm and humid air, which washed these islands even during the glacial periods. Additionally, because these are low islands, absence of competition with montane plants descending to the lowlands during cooler periods may have mitigated the influence of temperature lowering and facilitated their survival. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 305–325.

Molecular phylogenetic analyses using ITS sequences were used to reconsider the taxonomic validity of Bredia okinawensis, B. yaeyamensis, and B. sinensis as an independent genus Tashiroea. The result showed that the three species formed a different phylogenetic lineage from other species of Bredia including its type species. We suggest that B. okinawensis, B. yaeyamensis, and B. sinensis should be treated as T. okinawensis, T. yaeyamensis, and T. sinensis, respectively, following Ito and Matsumura (1899) and Diels (1924).

Flavones and anthocyanins were isolated from the leaves and flowers of 14 Ajuga taxa (Lamiaceae), which are all native or naturalized in Japan. Of 13 flavones obtained from the leaves, 11 were characterized as apigenin, luteolin, 6-hydroxyluteolin and acacetin glycosides. Ten flavones were isolated from the flowers. Ten anthocyanins were isolated from the flowers. Six of these anthocyanins were identified as acylated delphinidin glycosides and four were shown to be acylated cyanidin glycosides. Japanese Ajuga taxa were chemotaxonomically discussed by their distribution patterns, especially foliar flavonoids.

An antitropical distribution represents an intriguing disjunction, in which a given species or sister lineages occupy regions north and south of the tropics but are absent from the intervening areas. Solenogyne mikadoi endemic to the Ryukyu Archipelago is regarded as an Australian element. Testing the phylogenetic relationship with Australian congeners and discussing the onset timing and causes of the disjunction would potentially enhance the understanding of antitropical distribution. A nuclear ribosomal DNA phylogeny was reconstructed using Bayesian and most parsimonious criteria with allied genera. Solenogyne was monophyletic and clustered with Lagenophora huegelii endemic to Australia, indicating the antitropical distribution and Australian origin of Solenogyne. Multispecies coalescent analysis based on nuclear ribosomal DNA and chloroplast DNA indicated the divergence of S. mikadoi and Australian congeners in the Plio-Pleistocene. Phylogenetic network analyses suggested that the ancestral lineage of S. mikadoi first colonized the southernmost island in the archipelago and then dispersed northward. The migration to the archipelago likely followed the flourishing of Solenogyne in open vegetation communities that radiated in south-eastern Australia during the late Pliocene. This disjunction might arise through long-distance dispersal across the tropics or, alternatively, through extinction in the tropics as a result of unsuitably high temperatures during climate oscillation and/or competitions from diverse tropical flora surviving since the early Tertiary.

Comparison of leaf morphometric variation and phylogenetic analyses based on nrITS and partial trnC-D cpDNA sequences were conducted for samples of Lysionotus pauciflorus sensu lato collected from China, Japan and Taiwan. In the morphometric comparison, leaves of plants from Japan proper and those from Taiwan proper were not significantly distinguishable by their leaf length to width ratio. In phylogenetic analyses, monophyly of L. pauciflorus s. l. samples was strongly supported, and Japanese and Taiwanese samples fell into a clade separate from Chinese samples. In the Japan–Taiwan clade, plants from Japan proper formed a sister clade to those from Okinawa Island (Japan) and Taiwan. The present results suggest that (1) the L. pauciflorus s. l. samples from China, Japan and Taiwan must be treated as a single species; (2) L. pauciflorus s. l. originated in China; (3) a single dispersal event, dispersal via a land-bridge during low sea levels in a Pleistocene glacial maximum, from China to Taiwan and Japan is proposed; (4) three oversea dispersal events, probably through anemochory, to Japan proper, across the Tokara Gap, to Okinawa islands crossing the Kerama Gap, and from Taiwan to Lanyu Island are proposed. The study gives further insight into the biogeographical dynamics between the Ryukyus and surrounding areas.

1. Subject and sourceThegenusCassytha(Lauraceae)isaparasiticherb,whichcontainsca.20speciesandisdistributedinPacificRimcountries,mainly Australia, Africa, America and Japan (Mabberley, 1997). The leaves are degenerated and almost assimilate into thestem, which has small elliptical haustoria (Weber, 1981). Three Cassytha taxa, Cassytha filiformis L. var. filiformis, C. filiformisvar. duripraticola Hatus. and Cassytha pergracilis Hatus., are distributed in Japan, and the latter two are listed as endangeredplants by the Ministry of Environment, Japan. However, their taxonomic treatment underwent various changes (Hatusima,1971, 1975, 1978; Hatusima and Amano, 1994). Based on the specimens in Australia, Hatusima and Amano (1994) andHatusima (1978) treated C. filiformis var. duripraticola as Cassytha pubescens R. Br., and C. pergracilis as Cassytha glabella R.Br. However, it was difficult to classify them with only morphological characters. In this study, we describe the flavonoidsfrom the aerial parts of six Cassytha taxa and chemotaxonomically discuss them. The Cassytha species examined in this studywere collected in the sites described in Table 1. Voucher specimens were deposited in the Herbarium of National Museum ofNature and Science, Japan (TNS).2. Previous workPhylogeneticanalysesof thefamilyLauraceaeincludingCassytha,CassythaciliolataNees,C.filiformisandC.pubescenshavebeen carried out (Rohwer, 2000; Chanderbali et al., 2001). As chemical components in Cassytha species, many aporphine

The chromosome numbers of four species of Ochrosia sensu lato (Apocynaceae) were reported: those of Neisosperma glomerata (2n=22) and Neisosperma citrodora (2n=23) were documented here for the first time, while those of Neisosperma oppositifolia (2n=22) and Ochrosia coccinea (2n=22) verified the previous counts.

Chromosomal fluorescent in situ hybridization (FISH) and PCR fragment intensity analysis were used to determine the number and sizes of 45S nuclear ribosomal DNA (nrDNA) arrays and their inheritance in two Streptocarpus species, Streptocarpus rexii and Streptocarpus dunnii. A strong correlation between 45S nrDNA signals and PCR band intensity was observed. They revealed two loci in both species, with variation in nrDNA copy number within arrays: S. rexii had relatively homogeneous loci, while one individual of S. dunnii was heterozygous at the main locus, possessing a large and a small nrDNA array, and homozygous at the second locus, which was very small. This small nrDNA array was not PCR detectable and explains the non‐Mendelian results in some backcrosses. Our results indicated that nrDNA sequences in Streptocarpus were highly homogenized and that the inheritance of nrDNA was additive without any detectable recombination. PCR segregation of 45S nrDNA markers in backcross plants supported the hypothesis...

We investigated the hybrid origin of × Crepidiastrixeris denticulato-platyphylla using RAPDs and ITS sequence data. The putative parents Paraixeris denticulata and Crepidiastrum platyphyllum represent separate species, irrespective of geographical origin. The occurrence of species specific RAPD markers from P. denticulata and C. platyphyllum in × C. denticulato-platyphylla established unambiguously a hybrid origin between the two taxa. This was in line with the occurrence of a combination of morphological characters such as plant habit and floret numbers. The parent taxa differed from each other by 7 nucleotide substitutions and 2 indel events in the ITS region. The hybrids showed sequence additivity and most likely represent F1 plants, with the exception of two plants which were of possible F2 origin, possessing either the ITS sequences of one parent only, or one predominant ITS type. The hybrids occurred in two out of three localities where the parents occurred sympatrically. This fact, together with the short life-span of the plants, suggests that × C. denticulato-platyphylla exists as a result of repeated, frequent hybridization between the parent species. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society, 2006, 151, 333–343.

Comparisons of corolla morphology were made between plants of Conandron ramondioides (Gesneriaceae) from 23 localities in Japan and Taiwan. Lobe-length to tube-length ratios were found to correlate with geographical distribution and support the recognition of two previously described varieties: var. ramondioides in mainland Japan (lobe length less than twice tube length) and var. taiwanensis from Iriomote Island (Japan) and Taiwan (lobe length more than three times tube length). Mitotic chromosomes were also observed but no differences were seen between accessions, all of which had 2n=32.

Somatic chromosomes of four species of Ceratozamia, C. hildae, C. kuesteriana, C. mexicana and C. norstogii, and Stangeria eriopus, were observed and compared by the fluorescence in situ hybridization method using 5S ribosomal (rDNA) probes. The four Ceratozamia species and S. eriopus showed the same chromosome number of 2n = 16, and had similar karyotypes, comprising 12 metacentric (m), two submetacentric (sm) chromosomes and two telocentric (t) chromosomes. The four Ceratozamia species exhibited a proximal 5S rDNA site in the interstitial region of two m chromosomes. Stangeria eriopus exhibited a distal 5S rDNA site in the interstitial region of two m chromosomes, which probably indicates that the two genera differ in chromosome structure by at least one paracentric inversion. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 145, 499–504.

Somatic chromosomes of Stangeria eriopus (Stangeriaceae, Cycadales) were investigated by fluorescent in situ hybridization (FISH) using an 18S ribosomal DNA (rDNA) probe. Stangeria eriopus showed a chromosome number of 2n = 16 with a karyotype of 12 median-, 2 subterminal-, and 2 terminal-centromeric chromosomes. FISH study of S. eriopus revealed 16 signals made up of rDNA sites located on the terminal regions of the long arms of the 7 median- and 2 subterminal-centromeric chromosomes, on terminal region of the short arm of the 1 median-centromeric chromosome, on the terminal regions of the long and the short arms of 1 median- and 2 terminal-centromeric chromosomes. This result suggests that, not only karyomorphologically but also molecular-cytologically, the genus Stangeria may be more closely related to the genus Ceratozamia than the genus Bowenia or the genus Microcycas previously hypothesized.

We compared Sedum formosanum with related species using morphological traits and molecular phylogenetic analysis of nrITS sequences. Morphological comparisons revealed that the plants historically treated as S . formosanum in the Danjo Islands of Japan had 4-merous flower; 8 stamens; narrow triangular sepals of equal size; horizontal carpels when matured; and an irregular branching form. These traits differed from those of S . formosanum in other regions, which has 5-merous flowers; 10 stamens, thick spatulate sepals of unequal size; erect carpels when matured; and a trichotomous branching form. Phylogenetic analysis indicated that plants known as S . formosanum in the Danjo Islands were sister group to S . tetractinum , which are endemic to China and belong to a different clade than S . formosanum , which are found in other regions. Based on the present morphological comparisons and phylogenetical analyses, we describe plants from the Danjo Islands as a new species, S. danjoense , which is distinct from S . formosanum .

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.

Somatic chromosomes at mitotic metaphase of two species and two undescribed populations of Bowenia, and Stangeria eriopus, which were classified in Stangeriaceae, Cycadales, were compared using the standard aceto-orcein staining method. All Bowenia taxa showed a chromosome number of 2n = 18, while S. eriopus showed a chromosome number of 2n = 16. The chromosome number of 2n = 18 in B. ‘Kuranda’ is reported for the first time. The present karyotype analysis indicates that B. ‘Kuranda’ and another undescribed taxon, B. ‘Tinaroo’, are cytotaxonomically closer to B. spectabilis than B. serrulata, and that the karyotype of Stangeria is unlikely to have been derived from that of Bowenia by a simple chromosomal change such as centromeric fission and deletion.

Mitotic chromosome of two cultivars of Colocasia esculenta var. aquatilis and five cultivars of C. esculenta var. esculenta was investigated by the standard aceto-orcein staining method and the fluorescent in situ hybridization with ribosomal DNA probe (rDNA). One cultivar of C. esculenta var. aquatilis and three cultivars of C. esculenta var. esculenta showed the chromosome number of 2n=28, and exhibited an rDNA site on two chromosomes in a mitotic complement. On the other hand, one cultivar of C. esculenta var. aquatilis and two cultivars of C. esculenta var. esculenta showed the chromosome number of 2n=42, and exhibited an rDNA site on three chromosomes in a mitotic complement. These results supported the basic chromosome number of x=14 in C. esculenta.

Orcein staining, differential staining with CMA and DAPI, and FISH with an rDNA probe were used to compare somatic chromosomes ofCeratozamia mexicana andMicrocycas calocoma. CMA-positive dots and hybridization signals appeared on chromosomes at early interphase and mitotic prophase, but in significantly different number in the two species. InCeratozamia mexicana, the CMA-positive and DAPI-negative bands and the hybridization signal were located at the terminal region of the long arm of three median-centromeric chromosomes, the terminal region of the short arm of two median-centromeric chromosomes and the terminal region of the long arm of two subterminal-centromeric chromosomes. InMicrocycas calocoma, they were located at the pericentric region of two median-centromeric chromosomes. These chromosome data suggested thatMicrocycas has no simple Robertsonian relationship toCeratozamia.

Similar karyotypes of 2 n =22 in Cycas circinalis, C. media var. basaltica, C. revoluta var. revoluta, C. revoluta var. taiwaniana and C. siamensis were compared with each other by using the CMA and DAPI fluorescent staining methods. Their four largest submedian-centromeric chromosomes each had a CMA band at the terminal region in common. Their 12 terminal-centromeric chromosomes commonly displayed CMA bands at the terminal region and the pericentric region. Two of the 12 terminal-centromeric chromosomes carried a CMA band somewhere in the interstitial region of the long arm. C. circinalis alone showed it at a relative position closer to the centromere. The other taxa showed it at a relative position near the terminal region. All of the chromosomes exhibited the DAPI dot at the centromeric region.

The chromosomes of five species and one variety of Cycas studied showed commonly the morphological characteristics of the complex chromocenter type at early interphase and the interstitial type at mitotic prophase. All of the taxa had the common chromosome number of 2n=22 and very similar karyotypes to each other; C. media var. basaltica and C. siamensis showed the chromosome number of 2n=22 for the first time.

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.

Aim Phylogeographical patterns in the Ryukyu Archipelago have been explained primarily by landbridge formation and the opening of two straits in the Pliocene, namely the Tokara and Kerama gaps. These old straits have been considered to be the barriers most likely to determine genetic boundaries. To test this, we conducted a molecular analysis of the herb Ophiorrhiza japonica. We discuss the causes of and processes involved in its phylogeographical structure and explore aspects of island separation other than the duration of the straits to explain genetic boundaries at the gaps. Location Ryukyu Archipelago, Japan. Methods Plants were collected from 40 localities in the archipelago and vicinity. Non-coding regions of chloroplast DNA were sequenced. The genealogical relationships among haplotypes were estimated using a statistical parsimony network. To examine the phylogeographical structure, we compared two parameters of population differentiation, namely GST and NST, and conducted correlation analysis of genetic and geographical distances. Genetic boundaries were identified using Monmonier’s maximum difference algorithm. To test vicariance–dispersal hypotheses, that is, vicariance after migration via the Pliocene landbridge or over-sea dispersal in the Pleistocene, molecular dating analysis was conducted. Results A statistical parsimony network revealed that the haplotypes from the Ryukyu Archipelago and northwards coalesce to one ancestral haplotype in Taiwan. A clear phylogeographical structure was observed: plants within the same population and populations in geographical proximity were phylogenetically close. A genetic boundary was recognized across the Kerama Gap, but not across the Tokara Gap. Dating analysis suggested that population divergence across the Kerama Gap occurred in the early to late Pleistocene. Main conclusions The statistical parsimony network suggests migration from Taiwan and northward range expansion in the archipelago. Based on the divergence time, over-sea dispersal in the Pleistocene is likely, although migration via a Pliocene landbridge is not totally rejected. Negligible genetic differentiation across the Tokara Gap suggests recent over-sea dispersal, possibly facilitated by the small geographical width of the gap. Conversely, the large genetic differentiation across the Kerama Gap is probably explained by the large geographical distance across it. The past splitting of a landbridge would have had a significant influence on population differentiation after a certain geographical distance was reached.

Phytochemical characters of the plants that have been treated as Disporum luzoniense (Merrill & Merritt 1910) or Disporopsis fuscopicta (Jessop 1979) in the Philippines were compared with those of Disporum kawakamii and Disporopsis fuscopicta in Taiwan. The present phytochemical study revealed that Disporum kawakamii had luteolin, apigenin and chrysoeriol as free state, while these flavonoids were not detected in the Philippine plant and Disporopsis fuscopicta from Taiwan. Moreover, flavone O-glycosides were isolated from Disporum kawakamii, while flavone C-glycoside was isolated from the Philippine plant and Disporopsis fuscopicta from Taiwan. In conclusion, the present study suggests that the Philippine plant is chemotaxonomically related to Disporopsis and this agrees with the taxonomic treatment of Jessop (1979).

Molecular phylogenetic analyses, morphological observations, and nomenclatural studies were carried out to investigate the taxonomic status of Portulaca boninensis, endemic species from the Bonin (Ogasawara) Islands (Japan). The results addressed controversy between the widely naturalized P. boninensis and P. pilosa, indicating that they are phylogenetically and morphologically distinct. Furthermore, P. boninensis was showed to be conspecific to P. psammotropha which is until now recorded in southern China, Taiwan, and the northern Philippines, but not in the Ryukyus. The name of P. psammotropha was lectotypified on a specimen preserved at K. Based on phylogeography, P. psammotropha likely migrated to the Oceanic Bonin Islands oversea by sea-current dispersal or by exo- and/or end ozoochory through migratory birds without passing through Ryukyus.

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.

The chromosomal sites of 5S rDNA, 45S rDNA and Arabidopsis-iypt telomere sequence repeats characterize individual species and population constitutions in chrysanthemums and cycads and those of Sau3A in spruces. The sites of the telomere sequence repeats in the cycad chromosomes occur not only in the usual terminal regions of chromosome-arms as small dots, but also in several centromeric to proximal regions as relatively large signals. Large signals of the 278 bp DNA segment with respect to Cycas revoluta involved with pCrT7-4. The Y-chromosome of Cycas revoluta was distinct by lack of the large signal of pCrT7-4 and the Arabidopsis-type telomere sequence repeats. A highly conserved chromosomal location of these genes lead to speculation on the karyotype evolution and species property of the chrysanthemum and cycad groups.

Our molecular phylogeographic analyses based on the nrDNA ITS and cpDNA trnLF of Sedum tosaense populations in the Shikoku District, Japan and Jeju Island, Korea suggested a disjunct distribution. Plants of S. tosaense from the two regions comprised a well-supported clade consisted of plants from Kochi (Shikoku District) and those from Jeju Island; we estimated a divergence time of 0.61 Ma between the Kochi and Jeju populations based on the ITS and partial trnLF. We conclude that: 1) S. tosaense has a disjunct distribution between Kochi and Jeju Island, and 2) plants of this species might have dispersed between Kochi and Jeju Island over water, but not via a land bridge, which flooded before subclade divergence.

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 with 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.

The absence of the cosmopolitan genus Limonium Miller (1754: no pagination) (Plumbaginaceae) in the mega-diverse flora of the Malesian region (comprising Brunei Darussalam, Indonesia, Malaysia, Papua New Guinea, the Philippines, and Singapore; Merrill 1923, Ridley 1923, Steenis 1949, Backer & Bakhuizen van den Brink Jr. 1965, Balgooy 1993, Coode et al. 1996, Balgooy 2001, Conn et al. 2004, Chong et al. 2009, Pelser et al. 2011) has intrigued taxonomists and biogeographers since Steenis (1949) noted this. During a field survey as part of the project on floristic and phylogenetic biogeography in the island chain of the Philippines, Taiwan, and southern Japan, we found a population of Limonium in the northern Philippines (Batan Islands) that represents the first record of the genus in the Malesian region. Batan Islands, comprising 10 small (≤ 83.1 km2) oceanic islands, is the northernmost tip of the Malesian region, being ca. 190 km north of Luzon Island of the Philippines and ca. 140 km southeast of Taiwan Island. From the geological location, the present finding suggests that a more extensive survey in Batan Islands may add some more East Asian temperate genera to the flora of the Malesian region, although two enumerations of the early and mid 20th century provide us baseline knowledge of the flora of Batan Islands (Merrill 1908, Hatusima 1966).