Koh Nakamura

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.

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae).

UNLABELLED PREMISE OF THE STUDY The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. • METHODS AND RESULTS Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. • CONCLUSIONS The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.

Developing DNA barcodes for species identification in Podophylloideae (Berberidaceae)

Species of Podophyllum, Dysosma, Sinopodophyllum, and Diphylleia, genera from Podophylloideae of Berberidaceae, have long been used in traditional herbal medicine in East Asia and/or North America. Accurate identification of the species of these four genera is crucial to their medicinal uses. In this study, we tested the utility of nine barcodes (matK, rbcL, atpH‐atpI, rpl32‐trnLUAG, rps18‐clpp, trnL‐trnF, trnL‐ndhJ, trnS‐trnfM, and internal transcribed spacer (ITS)) to discriminate different species of Podophylloideae. Thirty‐six individuals representing 12 species of Podophylloideae were collected from different locations in China, Japan, and North America. We assessed the feasibility of amplification and sequencing of all markers, examined the levels of the barcoding gap based on DNA sequence divergence between ranges of intra‐ and interspecific variation using pairwise distances, and further evaluated successful identifications using each barcode by similarity‐based and tree‐based methods. Results showed that nine barcodes, except rps18‐clpp, have a high level of primer universality and sequencing success. As a single barcode, ITS has the most variable sites, greater intra‐ and interspecific divergences, and the highest species discrimination rate (83%), followed by matK which has moderate variation and also high species discrimination rates. However, these species can also be discriminated by ITS alone, except Dysosma versipellis (Hance) M. Cheng ex T. S. Ying and D. pleiantha (Hance) Woodson. The combination of ITS + matK did not improve species resolution over ITS alone. Thus, we propose that ITS may be used as a sole region for identification of most species in Podophylloideae. The failure of ITS to distinguish D. versipellis and D. pleiantha is likely attributed to incomplete lineage sorting due to recent divergence of the two species.

Development of microsatellite markers in heterostylous Hedyotis chrysotricha (Rubiaceae)

PREMISE OF THE STUDY Microsatellite primers were developed for a heterostylous herb, Hedyotis chrysotricha to investigate the effect of habitat fragmentation on its genetic diversity and population structure. METHODS AND RESULTS Twelve primer sets were developed and their polymorphisms were tested on 47 individuals from two island populations of H. chrysotricha in Thousand Island Lake of China. The number of alleles per locus ranged from five to 10, with an average of seven alleles. Expected heterozygosity per locus ranged from 0.284 to 0.821 and observed heterozygosity ranged from 0.191 to 0.851. CONCLUSIONS We showed that all of the 12 microsatellite markers developed for H. chrysotricha are polymorphic within populations, which should provide a powerful tool for assessing population structure and genetic diversity across fragmented and continuous populations, and for studying the genetic effects of habitat fragmentation on this species.

Antitropical distribution of Lobelia species (Campanulaceae) between the Ryukyu Archipelago of Japan and Oceania as indicated by molecular data

We tested the antitropical distribution of Lobelia loochooensis, endemic to the Ryukyu Archipelago of Japan, and its putative sister species of the same section Hypsela in Oceania (Australia and New Zealand). Molecular phylogenetic analyses based on cpDNA sequences were conducted for 41 Lobelia species of 11 sections including all the species of sect. Hypsela investigated in Asia and Malesia and 32 species of 16 allied genera of family Campanulaceae, because the genus Lobelia is known to be polyphyletic. In the result, L. loochooensis and an Australian endemic L. fluviatilis formed a clade, and this clade was sister to a clade of four New Zealand endemics: L. carens, L. fatiscens, L. fugax and L. ionantha. These two clades were nested in a clade with two other Australian congeners. We conclude that: (1) the lineage of L. loochooensis and the five Lobelia species occurring in Oceania demonstrate an antitropical distribution pattern; and (2) L. loochooensis has likely originated from a dispersal event from Australia to the Ryukyu Archipelago.

Isolation of compound microsatellite markers in Begonia fenicis (Begoniaceae) endemic to East and Southeast Asian islands.

PREMISE OF THE STUDY We developed compound microsatellite markers for Begonia fenicis, a species endemic to eastern and southeastern Asian islands, to investigate geographical genetic structure. METHODS AND RESULTS Using the compound microsatellite marker technique, 21 markers were developed and six polymorphic markers were characterized for samples from four islands in Taiwan and southern Japan. The number of alleles per locus ranged from two to six (mean = 4.33). Observed and expected heterozygosities were 0.125-0.725 (0.383) and 0.498-0.719 (0.641), and polymorphic information content was 0.371-0.664 (0.567). The interspecific transferability of the 21 markers was evaluated for eight species of the section Diploclinium from the Philippines; 15 markers were successfully amplified in one to eight species. CONCLUSIONS These results indicate the utility of the six microsatellite markers in B. fenicis to investigate geographical genetic structure. The transferable markers are potentially useful for other species of the section.

Correction of Confusions Regarding the Identity and Synonymy of Youngia (Asteraceae: Tribe Cichorieae) in Taiwan

Abstract Youngia japonica, the type species for its genus, has the widest distribution in the genus and is invasive worldwide. Recent molecular phylogenetic analysis supported recognition of three ecologically differentiated entities: Y. japonica subsp. formosana, Y. japonica subsp. japonica, and an undescribed morphological variant thought to be a new subspecies in Taiwan. These are the only infraspecific taxa currently recognized in this morphologically variable species. Use of proper names for these taxa and related species is important for future revision of Y. japonica in other regions, for nomenclature and biogeography of the genus, and for activities to control its invasiveness. Careful examination of the holotype of Y. japonica subsp. formosana and relevant literature revealed that the name has long been misapplied to Youngia in mountain ranges in Taiwan; it should be applied to the morphological variant growing on littoral, raised coral reefs in southwestern Taiwan. The misidentified entity is here described as Youngia japonica subsp. monticola. In addition, Y. taiwaniana was treated as a synonym of Y. longiflora (??? Y. japonica subsp. longiflora) in the Flora of Taiwan, but the holotype has achenes with a long beak and does not belong to Youngia. It is here treated as a synonym of Ixeridium laevigatum. A key to Youngia in Taiwan is provided.

Cassytha pubescens and C. glabella (Lauraceae) are not disjunctly distributed between Australia and the Ryukyu Archipelago of Japan: evidence from morphological and molecular data

Abstract. Morphological comparisons and molecular phylogenetic analyses were conducted to resolve taxonomic confusion in Cassytha glabella and C. pubescens, both of which were first described from Australia and subsequently considered to be disjunctly distributed between Australia and the Ryukyu Archipelago of Japan. In the morphological comparisons, plants considered as C. pubescens in the Ryukyus differ from C. pubescens in Australia in the presence or absence of hairs on the petals, and those considered as C. glabella in the Ryukyus differ from the C. glabella in Australia in bract and peduncle morphology. The molecular analyses indicated that plants attributed to C. pubescens in the Ryukyus were not closely related to C. pubescens in Australia, and were nested in a clade of populations of a Pan-Western Pacific species C. filiformis. Plants attributed to C. glabella in the Ryukyus were distantly related to C. glabella in Australia. We concluded that plants considered as C. pubescens and C. glabella in the Ryukyus are to be respectively treated as C. filiformis and the Ryukyu endemic species C. pergracilis.

Phylogeny and biogeography of the Viola iwagawae - tashiroi species complex (Violaceae, section Plagiostigma ) endemic to the Ryukyu Archipelago, Japan

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.

The impacts of historical barriers on floristic patterns of plant groups with different dispersal abilities in the Ryukyu Archipelago, Japan

The effects of historical barriers in biogeographical patterns are expected to persist differently depending on dispersal abilities of organisms. We tested two hypotheses that plant groups with different dispersal abilities display different floristic patterns, and that historical barriers can explain floristic differentiation patterns in plants with low dispersal ability but not in plants with higher dispersal ability, in the seed plant flora of the Ryukyu Archipelago. This area is biogeographically interesting because several similar floristic differentiation patterns have been proposed, all of which are primarily explained by two historical barriers, the Tokara Tectonic Strait (Tokara Gap) and the Kerama Gap, which arose during the formation of the islands. We calculated floristic dissimilarity distance among 26 islands based on data sets for three dispersal-ability classes. Clustering analyses based on the floristic dissimilarity distance generated similar floristic patterns regardless of dispersal-ability class. We propose that because the landscape resistance is so strong that migration of plants is severely restricted regardless of their dispersal abilities, the similar floristic differentiation patterns are generated. Multivariate regression analyses using Mantel’s randomization test indicated that floristic differentiations among islands were explained by the both effects of the historical barriers and geographic distance in all dispersal-ability classes. Significance of the historical barriers is not determined by the plant dispersal abilities but presumably by the spatial distribution of the islands, stochastic dispersals, and time since the formation of the barriers.