Genetic Structure of the Liriope muscari Polyploid Complex and the Possibility of Its Genetic Disturbance in Japan.

Anthropogenic activities, such as the movement of plants through greening, can result in genetic disturbance that can interfere with local adaptation in wild populations. Although research is underway to prevent genetic disturbance associated with greening, genetic disturbance of intraspecific polyploidy, which is estimated to be present in 24% of vascular plants, has not been well studied. Liriope muscari is a polyploid complex with known diploid (2n = 36), tetraploid (2n = 72), and hexaploid (2n = 108) forms. The plants of this species tolerate dry and hot conditions and are therefore frequently used for greening and gardening. However, the distribution of this polyploid in Japan, its genetic structure, and genetic disturbance are not known. In this study, we investigated the polyploidy distribution and genetic structure in naturally distributed L. muscari in Japan using chloroplast DNA (cpDNA) haplotypes and nuclear DNA (nDNA). Commercially produced individuals were also studied and compared with natural populations to assess any genetic disturbance of the ploidy complex in this species. Chromosome counts, cpDNA, and nDNA results showed three genetically and cytologically distinct groups in Japan: first, a tetraploid group in mainland Japan; second, a hexaploid group in the Ryukyu Islands; and third, a diploid and tetraploid group in the Ryukyu Islands. Significant isolation by distance was also detected within the three groups (p = 0.001). Genetic disturbance due to greening and gardening should be avoided among the three groups. Genetic disturbance can be reduced by using individuals derived from natural populations that are close to the sites used for greening and gardening. For commercially produced individuals, genetic disturbance is unlikely in the Kanto region, an area of high usage, while genetic disturbance is thought possible in the Ryukyu Islands.

First record of non-flying mammalian contributors to pollination in a tropical montane forest in Asia.

This study aims to identify the flower visitors of Mucuna thailandica (Fabaceae), endemic plant species in montane forests in Thailand, to determine their potential pollinators. The genus Mucuna produces papilionaceous flowers and has an explosive flower-opening step. Explosive opening rapidly exposes stamens and pistil from keel petals and releases pollen. The flower of this species depends completely on animals to perform this step, essential for pollination success. Using a camera trap survey, we revealed that non-flying mammals, such as squirrels (Callosciurus sp.) and masked palm civets (Paguma larvata), opened flowers explosively. Thus, these mammals contribute to the pollination of M. thailandica. This is the first report of non-flying mammals contributing to pollination in montane forests in tropical Asia.

Analyses of Plastome Sequences Improve Phylogenetic Resolution and Provide New Insight Into the Evolutionary History of Asian Sonerileae/Dissochaeteae.

Sonerileae/Dissochaeteae (Melastomataceae) comprises ca. 50 genera, two thirds of which occur in Southeast Asia. Phylogenetic relationships within this clade remain largely unclear, which hampers our understanding of its origin, evolution, and biogeography. Here, we explored the use of chloroplast genomes in phylogenetic reconstruction of Sonerileae/Dissochaeteae, by sampling 138 species and 23 genera in this clade. A total of 151 complete plastid genomes were assembled for this study. Plastid genomic data provided better support for the backbone of the Sonerileae/Dissochaeteae phylogeny, and also for relationships among most closely related species, but failed to resolve the short internodes likely resulted from rapid radiation. Trees inferred from plastid genome and nrITS sequences were largely congruent regarding the major lineages of Sonerileae/Dissochaeteae. The present analyses recovered 15 major lineages well recognized in both nrITS and plastid phylogeny. Molecular dating and biogeographical analyses indicated a South American origin for Sonerileae/Dissochaeteae during late Eocene (stem age: 34.78 Mya). Two dispersal events from South America to the Old World were detected in late Eocene (33.96 Mya) and Mid Oligocene (28.33 Mya) respectively. The core Asian clade began to diversify around early Miocene in Indo-Burma and dispersed subsequently to Malesia and Sino-Japanese regions, possibly promoted by global temperature changes and East Asian monsoon activity. Our analyses supported previous hypothesis that Medinilla reached Madagascar by transoceanic dispersal in Miocene. In addition, generic limits of some genera concerned were discussed.

Recircumscription of Bredia and resurrection of Tashiroea (Sonerileae, Melastomataceae) with description of a new species T.villosa.

Bredia (Melastomataceae) is an Asian genus that extends from central and southern mainland China to Taiwan and the Ryukyu islands. Molecular phylogenetic analyses reveal that the type of Bredia is nested in a clade of 20 species, while Tashiroea, a genus previously synonymized in Bredia, falls in another distantly related clade of 10 species. Our morphological survey shows that the two clades can be distinguished by several diagnostic features including leaf indumentum, texture, leaf surface sculpture under SEM, presence/absence of yellowish uniseriate trichomes, and capsule morphology. Based on molecular and morphological evidence, Bredia is recircumscribed and Tashiroea is resurrected. Description and a list of species are provided for the two genera with the description of a new species, T.villosa.

The pollination system of the widely distributed mammal-pollinated Mucuna macrocarpa (Fabaceae) in the tropics.

Although the pollinators of some plant species differ across regions, only a few mammal-pollinated plant species have regional pollinator differences in Asia. Mucuna macrocarpa (Fabaceae) is pollinated by squirrels, flying foxes, and macaques in subtropical and temperate islands. In this study, the pollination system of M. macrocarpa was identified in tropical Asia, where the genus originally diversified. This species requires "explosive opening" of the flower, where the wing petals must be pressed down and the banner petal pushed upward to fully expose the stamens and pistil. A bagging experiment showed that fruits did not develop in inflorescences (n = 66) with unopened flowers, whereas fruits developed in 68.7% of inflorescences (n = 131) with opened flowers. This indicated that the explosive opening is needed for the species to reproduce. Four potential pollinator mammals were identified by a video camera-trap survey, and 78.3% and 60.1% of monitored inflorescences (n = 138) were opened by gray-bellied squirrels (Callosciurus caniceps) and Finlayson's squirrels (C. finlaysonii), respectively, even though more than 10 mammal species visited flowers. Nectar was surrounded by the calyx, and the volume and sugar concentration of secreted nectar did not change during the day. This nectar secretion pattern is similar to those reported by previous studies in other regions. These results showed that the main pollinators of M. macrocarpa in the tropics are squirrels. However, the species' nectar secretion pattern is not specifically adapted to this particular pollinator. Pollinators of M. macrocarpa differ throughout the distribution range based on the fauna present, but there might not have been no distinctive changes in the attractive traits that accompanied these changes in pollinators.

Differentiation of germination characteristics in Scutellaria rubropunctata (Lamiaceae) associated with adaptation to rheophytic habitats in the subtropical Ryukyu Islands of Japan.

Seed germination characteristics of a facultative rheophyte Scutellaria rubropunctata, a perennial skullcap endemic to the subtropical Ryukyu Islands of Japan, were investigated at temperatures of 15 °C, 20 °C, 25 °C, and 30 °C. The rheophytic type of S. rubropunctata showed relatively simultaneous germination times and reached high germination rates under all temperatures tested. This characteristic may allow it to establish quickly without being influenced by temperature, giving a survival advantage in rheophytic habitats by avoiding seeds being washed away by the rising water. In contrast, the final germination rate of the terrestrial type of S. rubropunctata, as well as that of the congener S. indica var. parvifolia, was highest at 15 °C and declined drastically at 30 °C. Seeds of both of these plants that remained ungerminated at higher temperatures germinated soon after the temperature was changed to 15 °C, indicating that the germination suppression was a state of enforced dormancy that was released after transfer to the optimal temperature. These characteristics suggest an asynchronous germination which is thought to reduce the risk of disturbance in fluctuating environments. Our study may be the first to demonstrate the differentiation of seed germination characteristics in relation to adaptations to a rheophytic habitat. Because rheophytes occupy such a harsh environment under strong selective pressure, the adaptive differentiation of seed germination characteristics may occur in many other rheophytes.

Floral traits of mammal-pollinated Mucuna macrocarpa (Fabaceae): Implications for generalist-like pollination systems.

Floral traits are adapted by plants to attract pollinators. Some of those plants that have different pollinators in different regions adapt to each pollinator in each region to maximize their pollination success. Mucuna macrocarpa (Fabaceae) limits the pollinators using its floral structure and is pollinated by different mammals in different regions. Here, we examine the relationships between floral traits of M. macrocarpa and the external morphology of mammalian pollinators in different regions of its distribution. Field surveys were conducted on Kyushu and Okinawajima Island in Japan, and in Taiwan, where the main pollinators are the Japanese macaque Macaca fuscata, Ryukyu flying fox Pteropus dasymallus, and red-bellied squirrel Callosciurus erythraeus, respectively. We measured the floral shapes, nectar secretion patterns, sugar components, and external morphology of the pollinators. Results showed that floral shape was slightly different among regions and that flower sizes were not correlated with the external morphology of the pollinators. Volume and sugar rate of nectar were not significantly different among the three regions and did not change throughout the day in any of the regions. However, nectar concentration was higher in Kyushu than in the other two regions. These results suggest that the floral traits of M. macrocarpa are not adapted to each pollinator in each region. Although this plant limits the number of pollinators using its flower structure, it has not adapted to specific mammals and may attract several species of mammals. Such generalist-like pollination system might have evolved in the Old World.

Population Dynamics and the Effects of Temperature on the Eggs of the Seawater-dispersed Stick Insect Megacrania tsudai (Phasmida: Phasmatidae).

Shun Kobayashi, Ryota Usui, Kouta Nomoto, Mineyuki Ushirokita, Tetsuo Denda, and Masako Izawa (2016) Although the stick insect Megacrania tsudai cannot fly, its eggs are dispersed by seawater, which allows the species to distribute itself widely via the ocean. The life history of this non-volant insect that establishes settlements by egg dispersal remains poorly understood. We aimed to clarify the population dynamics and effects of temperature on the eggs of M. tsudai through field observation in near the northern limits of the distribution and laboratory experiments. In the wild, all instars appeared over a period of several months, with a larger proportion of first instars emerging from December to March, and a greater number of adults being observed from June to August. Laboratory experiments showed that the hatching rate of M. tsudai was not different between conditions of 25 and 30°C, whereas it was significantly lower at 20°C. The egg-development period was shorter when eggs were maintained under high temperature conditions, and longer for eggs kept at 20°C. The effective cumulative temperature was estimated as 1561.5-2000.0°C, and the developmental zero was estimated as 12.2-13.9°C. The peak appearance of the first instar in the laboratory, which was used to obtain an estimate for the effective cumulative temperature and peak appearance of adults in the wild, was not entirely accurate because the peak appearance of the first instars in the wild was not consistently observed when the estimation indicated that they should appear. Megacrania tsudai is a univoltine near the northern limit of its distribution, and temperature has a strong effect on its egg development. Our estimation of population dynamics by laboratory experiments did not exactly predict what was observed in the field and it may be controlled by other factors. However, our findings indicate that the limiting factor of the distribution of this species is likely the effect of temperature on egg hatching.

Breakdown of distyly in a tetraploid variety of Ophiorrhiza japonica (Rubiaceae) and its phylogenetic analysis.

We examined the floral morph of tetraploid Ophiorrhiza japonica Blume var. amamiana Hatus. and diploid O. japonica var. japonica to elucidate the association of distyly and ploidy levels. Chloroplast DNA phylogeny was reconstructed to determine the number of tetraploidization events and floral morph shifts in O. japonica. All individuals of O. japonica var. amamiana proved to be long-homostylous, whereas O. japonica var. japonica was distylous with typical long- and short-styled flowers. Distyly is related to the ploidy level. The bagging treatment of flowers indicated that O. japonica var. amamiana is self-compatible and potentially automatically self-pollinating. In cpDNA sequencing analysis, no haplotype was shared between the two varieties. The cpDNA haplotype network displayed the monophyly of O. japonica var. amamiana, suggesting a single origin of this variety. Hence, both tetraploidization and the breakdown of distyly to homostyly in O. japonica var. amamiana likely occurred just once. Because O. japonica var. amamiana having the morphological and cytological entity is recognized as a single lineage and clearly separated from O. japonica var. japonica, this variety can be considered to be a distinct species. We therefore propose to raise O. japonica var. amamiana to the rank of species.

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.

The leaf morphologies of the subtropical rheophyte Solenogyne mikadoi and its temperate relative S. bellioides (Asteraceae) are affected differently by plant hormones and their biosynthesis inhibitors.

Solenogyne mikadoi is a subtropical rheophyte endemic to the Ryukyu Archipelago that develops rosette leaves 2-3 cm in diameter. In contrast, the other three species of this genus all occur in temperate grasslands of Australia and develop rosette leaves about 10 cm in diameter. To examine the involvement of the plant hormones gibberellin and brassinosteroid in the adaptive dwarfism of S. mikadoi, we compared the effects of GA(3) and brassinolide, and their biosynthesis inhibitors on the morphology of the first leaves of S. mikadoi and its temperate relative S. bellioides. In S. mikadoi, one-directional (lengthwise) leaf elongation was strongly facilitated by the application of GA(3) and suppressed by a gibberellin-biosynthetic inhibitor, uniconazole-P, while leaf width (transverse) expansion was insensitive to and was never facilitated by any of the compounds used. Conversely, in S. bellioides, brassinolide facilitated both the elongation and expansion of leaves, while a brassinosteroid-specific biosynthesis inhibitor, brassinazole220, suppressed both. One-directional leaf elongation caused by the reduced sensitivity to brassinolide in S. mikadoi and brassinolide-dependent two-dimensional leaf expansion in S. bellioides both appear to be adaptations to their respective habitats: S. mikadoi has narrow leaves resistant to flowing water, whereas S. bellioides has broad leaves capable of harnessing sufficient light and water in temperate grasslands.

Cytogeography of Ixeris nakazonei (Asteraceae, Lactuceae) in the Ryukyu Archipelago of Japan and Taiwan.

The cytogeographical structures of Ixeris nakazonei, a putative hybrid between I. debilis (6x) and I. repens (2x), were investigated in the Ryukyu Archipelago and Taiwan. In the Ryukyus, I. debilis occurs on Miyakojima Island of the southern Ryukyus and northward, while I. repens occurs on all islands except for Iriomotejima and Yonagunijima Islands. I. nakazonei, comprises six polyploid cytotypes, 3x, 4x, 5x, 6x, 7x and 8x, based on x=8. Four cytotypes from 3x to 6x occur in the central Ryukyus, while four cytotypes from 5x to 8x occur in the southern Ryukyus. The higher polyploids of I. nakazonei tend to be distributed in the more southerly area. Tetraploids of I. nakazonei always co-occur with I. debilis and I. repens, supporting the hybrid origin of this cytotype. Considering the chromosome number, octoploids, which predominate in the southern Ryukyus and Taiwan, may have derived directly from hybridization between I. debilis and I. repens. Odd-numbered polyploids of I. nakazonei, 3x, 5x and 7x, are relatively rare. Their chromosome numbers indicate that triploids and heptaploids are hybrids between the tetraploid of I. nakazonei and I. repens, and between the octoploid of I. nakazonei and I. debilis, respectively. Pentaploids of I. nakazonei in the central and southern Ryukyus are, respectively, hybrids between the tetraploid of I. nakazonei and I. debilis and between the octoploid of I. nakazonei and I. repens, indicating that pentaploids of I. nakazonei have at least two independent origins.