Development and characterization of nuclear microsatellite markers in Aphananthe aspera (Thunb.) Planch. (Cannabaceae)

Nuclear microsatellite markers were developed for Aphananthe aspera (Thunb.) Planch. (Cannabaceae), a deciduous canopy tree species distributed in East Asia, to evaluate the genetic diversity and genetic structure of A. aspera populations in remnant forest fragments in urbanized areas of Japan. A total of 94 primer pairs were designed based on genomic sequence data. Of the 25 primer pairs which showed clear microsatellite peaks, 20 pairs showed allelic polymorphisms in 57 individuals collected from two distant populations. The length of PCR products ranged from 120 to 482 bp, and expected heterozygosity for the 20 microsatellite markers ranged from 0.017 to 0.768. These newly developed simple sequence repeat markers will be used in population genetic studies of A. aspera to evaluate genetic diversity and the extent of genetic isolation of the fragmented populations in urban areas

琉球列島奄美大島の絶滅危惧植物アマミヒイラギモチ（モチノキ科）の植物体サイズと葉の二型性

The tree Ilex dimorphophylla Koidz.（Aquifoliaceae）is a vulnerable species that is endemic to the summit of Mt. Yuwan on Amamioshima Island, Japan. This plant hows characteristic leaf dimorphism, which increases its horticultural value, and many individuals have been illegally removed from the natural habitat. We examined the umber of shoots, leaf morphology and plant size of the species in the natural habitat. Based on field observation for two years, we found 84 shoots, of which 32（38.1%）had entire mature leaves, whereas 52（61.9%）had only serrate juvenile leaves. Majority of the examined shoots surveyed fell into the smallest size classes : < 100 cm in eight（ca. 45%）and < 25 mm in diameter at the trunk base（ca. 60%）. Leaf dimorphism was clearly demarcated in trees at 130―210 cm in height and 15―27 mm in diameter at the trunk base.アマミヒイラギモチ（Ilex dimorphophylla Koidz.）は奄美大島の湯湾岳山頂付近に発達した風衝林内に生育するモチノキ科の樹木である。本種における樹木サイズと葉の二型性について自生地で調査した。84 シュートの生育を確認し，そのうちの32 シュートが全縁の成熟葉を，残りの52 シュートが鋭い鋸歯のある幼葉のみをもっていた。葉の二型性は樹木高で約130―210 cm，根元直径で15―27mm を境にして分かれていることが明らかになった

日本におけるハマダイコンの葉毛密度の地理的変異

Understanding the geographic variation of leaf trichome in wild radish accessions would help to elucidate how morphological variation can be molded by natural selection. Here, we investigated the geographic variation of leaf trichomes in wild radish populations across Japan covering Ryukyu Islands and mainland. And we also sought the possible selection pressures involved in the evolution and maintenance of leaf trichome polymorphism. Results showed leaf trichome density in wild radish was highly variable across the Japanese archipelago, with plants from southern insular populations tending to exhibit glabrous leaves, while those of northern mainland Japan were completely covered by dense trichomes. This trichome variation in Japanese populations of wild radish was probably attributing to external stress involving in protecting against frost damage and resistance to herbivores. This study will be helpful to understand the morphological variation under divergent selections in wild radish and contribute to the areas of breeding and evolutionary studies in radish.ハマダイコンは日本においては西表島から北海道までの幅広い緯度の海岸に生育する。著者らの従来の観察では，琉球列島では葉や茎が無毛である傾向があり，反対に本州北部では粗長毛が密に生える傾向が見られた。そこで本研究では曖昧な印象であった毛の密度について調べるために，日本列島内の緯度に沿った11集団に由来する個体の，葉の向軸側の毛の密度を実体顕微鏡で計測した。その結果，琉球列島だけにおいて無毛の個体が存在するとともに，少数ながら粗長毛が密に生える個体も共存した。その一方で，北緯33度以上の本州の集団ではすべての個体で葉に粗長毛が密に生えていた。近年のアブラナ科植物での研究では，毛が草食性昆虫などの食害を防ぐなどの役割があると報告されており，今後には，なぜ南西諸島の集団で無毛の個体が共存できるのかという検討が必要である

海浜植物ハマヒルガオ(ヒルガオ科) における葉の厚さの地理的分化: 海岸と内陸淡水湖の集団間の比較

琵琶湖に内陸隔離されている海浜植物ハマヒルガオCalystegia soldanella（L.）Roem. et Schult. が，海岸の個体との間で葉の厚さに分化を起こしているかを検証した。比較をしたのは，琵琶湖の自生地6集団と海岸の自生地9 集団である。葉の厚さを測定して多重比較検定にかけたところ，5 グループに分類された。そのうちの1 グループに全ての琵琶湖の集団が含まれ，海岸の集団は残りの4 グループに含まれた。琵琶湖のハマヒルガオの葉は，海岸のものに比べて，有意に厚さが薄くなっていることが示唆された。琵琶湖は淡水湖であるために，塩ストレスへの適応が不要であることが，その要因の一つとして考えられる

琉球列島におけるソテツ(ソテツ科) の小羽片形態の地理的変異

Sixteen populations of Cycas revoluta（Cycadaceae）, covering almost all distribution areas on the Ryukyu Islands and Kyushu, Japan, were examined to determine geographical differences in leaflet morphology. We assessed four morphological characteristics of leaflets and applied one-way analysis of variance（ANOVA）to test thedifference in each morphological character. Cluster analysis based on four morphological properties resulted in two clear major clusters comprising northerly and southerly populations that were demarcated north of Yorontou and south of Tokunoshima Islands, respectively. Northern cycads tended to have leaflet margins with shallow recurving and narrower leaflet lamina width, while the southern populations had deeply recurved leaflet margins and wider leaflet lamina width. The morphological differentiation may imply intraspecific differentiation.琉球列島から九州にかけて分布するソテツ（Cycas revoluta Thunb.）の自生地16 集団を対象にして，小羽片の形態の地理的な分化を検証した。小羽片の横断面における小羽片の幅と厚さ，中肋の厚さ，小羽片の縁の巻き込みの高さの4 形質を測定してクラスター解析にかけたところ，北方型と南方型の2 つのグループに分かれた。北方型には主に与論島以北の集団が含まれ，南方型には主に徳之島以南の集団が含まれる。ただし，この境界にある徳之島は南方型に，沖永良部島は北方型に含まれる。これには，小羽片の幅と縁の巻き込みの高さに南北間で違いがあることが寄与しており，とくに縁の巻き込みの高さは，南側で大きく巻き込む傾向が見られた

Application of a Simplified Method of Chloroplast Enrichment to Small Amounts of Tissue for Chloroplast Genome Sequencing

Premise of the study: High-throughput sequencing of genomic DNA can recover complete chloroplast genome sequences, but the sequence data are usually dominated by sequences from nuclear/mitochondrial genomes. To overcome this deficiency, a simple enrichment method for chloroplast DNA from small amounts of plant tissue was tested for eight plant species including a gymnosperm and various angiosperms. Methods: Chloroplasts were enriched using a high-salt isolation buffer without any step gradient procedures, and enriched chloroplast DNA was sequenced by multiplexed high-throughput sequencing. Results: Using this simple method, significant enrichment of chloroplast DNA-derived reads was attained, allowing deep sequencing of chloroplast genomes. As an example, the chloroplast genome of the conifer Callitris sulcata was assembled, from which polymorphic microsatellite loci were isolated successfully. Discussion: This chloroplast enrichment method from small amounts of plant tissue will be particularly useful for studies that use sequencers with relatively small throughput and that cannot use large amounts of tissue (e.g., for endangered species)

Application of the isolation with migration model demonstrates the pleistocene origin of geographic differentiation in cardamine nipponica (brassicaceae), an endemic japanese alpine plant

The Pleistocene was characterized by a cyclic pattern of cold and warm climatic periods, or climatic oscillations, which caused fluctuations in the distributions of organisms. This resulted in drastic changes in demography, thereby accelerating the genetic divergence of populations. Phylogeographic studies have elucidated the history of populations during the Pleistocene. However, given the lack of model-based analysis of population histories, previous phylogeographic studies could not adequately evaluate the effect of these Pleistocene climatic oscillations on the genetic divergence and migration events between populations. Populations of Japanese alpine plants in central and northern Japan are highly differentiated, and a history of isolation between regions during the Pleistocene was inferred. Using sequences of 10 nuclear genes (ca. approximately 7, 000 bp in total) from Cardamine nipponica (Brassicaceae), we applied an isolation with migration (IM) model to test the significance of the isolation history between central and northern Japan and to assess whether range shifts during the Pleistocene climatic oscillations were involved in the genetic differentiation between regions. The estimated divergence time indicates that the two regions were separated about 100, 000-110, 000 years ago. The exclusive occurrence of closely related haplotypes within each region (parsimony network) and the high level of genetic differentiation between the regions (mean FST = 0.417) indicate that genetic divergence occurred following the isolation of the two regions. Therefore, the genetic differentiation between regions was shaped during the Pleistocene, especially during the last glacial and inter and postglacial periods. In addition, our multilocus analysis showed that populations in central and northern Japan were completely isolated after they split. Geographic separation and subsequent restricted migration events among mountains could explain this isolation history between regions. Furthermore, genetic drift in the reduced populations would remove evidence of occasional migration, emphasizing the isolation history. Therefore, our application of a demographic model demonstrated the Pleistocene origin of geographic differentiation statistically and provided a plausible migration history for C. nipponica

Development of microsatellite markers for Vitex rotundifolia (Verbenaceae), an endangered coastal plant in Lake Biwa, Japan

[Premise of the study]: Microsatellite loci were developed forVitex rotundifolia, an endangered species isolated to Lake Biwa, to investigate its genetic diversity and population structure. [Methods and Results]: Ten primer sets were identified in Japanese populations of V. rotundifolia. The number of alleles per locus ranged from one to six and gene diversity per locus ranged from 0.040 to 0.697 between two populations. In addition, all loci could be successfully amplified in V. trifolia. [Conclusions]: These markers will be useful for studies of genetic diversity and population structure of endangered species isolated to Lake Biwa of V. rotundifolia, to aid in the development of conservation strategies