Phylogeny and comparative seed morphology of epiphytic and terrestrial species of <Emphasis Type="Italic">Liparis</Emphasis> (Orchidaceae) in Japan

To elucidate the evolution of epiphytes in Liparis section Liparis, we examined the phylogenetic relationships of 16 species by using internal transcribed spacer regions of 18S–26S nuclear ribosomal DNA (ITS) and three chloroplast DNA regions (trnS-trnG spacer, trnL with trnL-trnF spacer, and partial matK). Results showed that the epiphytic L. fujisanensis is sister to the terrestrial L. koreana and L. kumokiri, while another epiphyte, L. truncata, is sister to the terrestrial L. krameri. Therefore, the two epiphytic species evolved from terrestrial species independently in section Liparis. Comparative seed morphology revealed that the epiphytes have larger embryos than their closely related terrestrial counterparts. A similar trend toward the increase of embryo size in the two epiphytic species belonging to closely related, but distinct clades suggests that the large embryo may have an advantage in the epiphytic lifestyle. The two epiphytic species share another character state, smaller air spaces in the seed than that of closely related terrestrial species, suggesting possible low dispersibility of the epiphytes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

中国兰科羊耳蒜属新记录种——岩生羊耳蒜及其独特的珠芽繁殖方式

报道了中国兰科(Orchidaceae)羊耳蒜属(Liparis Rich.)植物一新记录种——岩生羊耳蒜(Liparis petraea),并提供了其形态描述及彩色图片,凭证标本保存于广西植物研究所标本馆(IBK)。岩生羊耳蒜与见血青(L.nervosa)在形态上相近,但前者叶银灰绿色,叶面平展,唇瓣基部胼胝体为短圆柱状突起,植株通常具珠芽而易与后者区分。岩生羊耳蒜居群内个体呈聚集式分布,并与其具有的珠芽繁殖方式相关。     

Three new species of Liparis s.l. (Orchidaceae: Malaxideae) from Southwest China based on morphological characters and phylogenetic evidence

Liparis aureolabella and L. mengziensis, two new species from the karst region of southwestern China, and L. bingzhongluoensis, a new species from montane region in Yunnan, are described and illustrated. L. aureolabella is easily distinguished from its relatives by having abaxially purple leave with purple reticulate veins prominent adaxially, a lip auriculate at base, and falcate-lanceolate pollinia. Liparis mengziensis is closely related to L. petiolata and L. auriculata, but differs from them by having an ovate to broadly ovate leaf, purple lip and apex connate along the margins. Liparis bingzhongluoensis is similar to Liparis nanlingensis, but the new species is characterized by having a lip with two transparent ridges on its disc, longitudinally concave basal callus and triangular column wings. Phylogenetic analyses based on nuclear ribosomal ITS and plastid matK sequences showed that L. aureolabella and L. mengziensis are nested with L. petiolata or L. auriculata in a monophyletic clade. L. bingzhongluoensis is sister to a clade formed by L. nanlingensis, L. tsii, L. sasakii and L. krameri. Moreover, morphological comparisons strongly support that the three species as separated species newly to science.     

Phylogenetic relationships among subgenera,species, and varieties of Japanese <Emphasis Type="Italic">Salvia</Emphasis> L. (Lamiaceae)

To determine evolutionary relationships among all Japanese members of the genus Salvia (Lamiaceae), we conducted molecular phylogenetic analyses of two chloroplast DNA (cpDNA) regions (rbcL and the intergenic spacer region of trnL−trnF:trnL−trnF) and one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS). In cpDNA, nrDNA, and cpDNA+nrDNA trees, we found evidence that all Japanese and two Taiwanese Salvia species are included in a clade with other Asian Salvia, and Japanese Salvia species were distributed among three subclades: (1) S. plebeia (subgenus Sclarea), (2) species belonging to subg. Salvia, and (3) species belonging to subg. Allagospadonopsis. At the specific level our findings suggest: a close relationship between S. nipponica and S. glabrescens, no support for monophyly of S. lutescens and its varieties in cpDNA, nrDNA and cpDNA+nrDNA trees, and that S. pygmaea var. simplicior may be more closely related to S. japonica than to other varieties of S. pygmaea.     

曲唇羊耳蒜在中国东北分布的真实性研究

曲唇羊耳蒜分布于日本、朝鲜和俄罗斯远东地区,中国东北的黑龙江、吉林和辽宁也有报道,但是《中国植物志》认为,该种在中国东北是否存在尚有很大的疑问。本研究证明曲唇羊耳蒜的确在中国东北有分布。     

Nanocnide zhejiangensis sp. nov. (Urticaceae: Urticeae) from Zhejiang Province,East China

Nanocnide zhejiangensis X.F. Jin & Y.F. Lu, a new species of Urticaceae from Zhejiang, east China, is described with illustrations. The new species is morphologically similar to Nanocnide japonica in having staminate inflorescence longer than leaves, but differs by having glabrous stems, petioles, peduncles and abaxial leaf surfaces, glabrous perianth lobes of staminate flowers, dorsally glabrous perianth lobes of pistillate flowers, and acuminate or solitary spinose–setaceous at the apex. Analysis based on ITS, atpF–H, atpB–rbcL and trnL–F sequences also demonstrate that Nanocnide japonica is the closest extant relative to the new species.     

Zingiber pseudosquarrosum sp. nov. (Zingiberaceae) from Andaman and Nicobar Islands,India

Zingiber pseudosquarrosum L. J. Singh & P. Singh sp. nov. (Zingiberaceae) from Andaman and Nicobar Islands, India is described and illustrated and its conservation status is evaluated. This new species seems related to Z. squarrosum Roxb. but can be distinguished by the presence of a small pseudostem, long ligule and petiole, broadly lanceolate leaves with cuneate base, caudate apex and paxillate venation, urceolate or prolate inflorescences, very long underground peduncle, long calyx, very long corolla tube, labellum deep purple red (claret) at centre, orange red (vermillion) at lateral sides with ivory (off white) spots, tip truncate and slightly notched, margin folded back, stamen with short filament, yellow orange anther, light brown ovary, blood red (cinnabar) seeds covered by a white aromatic aril. A key to the species of Zingiber Mill. from the Andaman and Nicobar Islands is also provided.     

Hybridization between the threatened plant, <Emphasis Type="Italic">Lespedeza leptostachya</Emphasis> Englem. and its co-occurring congener <Emphasis Type="Italic">Lespedeza capitata</Emphasis> Michx.: morphological and molecular evidence

Natural hybridization is common in the genus Lespedeza. No hybrids between Lespedeza leptostachya Englem. and Lespedeza capitata Michx. are formally recognized in any of the current floras, however observations in the field suggest that hybridization might occur in many of their shared habitats. Putative hybrids were compared to L. leptostachya and L. capitata using morphological measurements and screened for the presence of species-specific trnL-F gene region (cpDNA) and the ITS gene region (nrDNA). A discriminate analysis of 10 morphological measurements identified the hybrids as intermediate to both parents with two PCA axes explaining 99% of the variation between taxa. The presence of hybrids was confirmed by genetic markers with individuals morphologically identified as hybrids having cpDNA trnL-F genotypes identical to L. leptostachya and the ITS (nrDNA) phenotypes in most cases contain the ITS genotype of both parents, however, some putative hybrid individuals contained the ITS genotype of only one parents. Those individuals with L. leptostachya ITS and trnL-F could be a case of misclassification, but the presence of both L. capitata ITS genotypes and L. leptostachya trnL-F genotypes suggest segregation has occurred, which may result from either selfing or backcrossing.     

Breakdown of distyly in a tetraploid variety of <Emphasis Type="Italic">Ophiorrhiza</Emphasis><Emphasis Type="Italic"> japonica</Emphasis> (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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A new species of Govenia (Orchidaceae) from Chiapas, Mexico

Govenia rubellilabia, a new terrestrial orchid from Sierra Madre de Chiapas, Mexico, is described and illustrated. Diagnostic characters of this new species are compared with other species ofGovenia. Govenia rubellilabia is similar in appearance toG. matudae, but it can be distinguished from this species by a shorter inflorescence with fewer flowers, green-yellowish tepals, a reddish lip lacking spots in the margin of the apex, and by a shorter anther beak.     

Taxonomic Status and Genetic Variation of Korean Endemic Plants, <Emphasis Type="Italic">Eranthis byunsanensis</Emphasis> and <Emphasis Type="Italic">Eranthis pungdoensis</Emphasis> (Ranunculaceae) based on nrDNA ITS and cpDNA Sequences

To reappraise the taxonomic status of two Korean endemic plants, Eranthis byunsanensis and Eranthis pungdoensis, we analyzed five taxa of that genus plus three out-group genera (Cimicifuga, Anemonopsis, and Anemone). In all, 52 representative accessions were examined for nrDNA (ITS) and 43 accessions for cpDNA (trnH–psbA, rps16, trnL, and trnLF). In the ITS region, all of the aligned sequences from E. byunsanensis had the same ribotype as from E. pungdoensis. For the ITS phylogeny, E. pungdoensis could not be distinguished from E. byunsanensis. However, the clade of E. byunsanensis, including E. pungdoensis, was separated from Eranthis pinnatifida of Japan, which has a distinct ribotype and forms a sister group. In our cpDNA analysis, E. byunsanensis showed paraphyly, and the clade of E. pungdoensis was nested within the E. byunsanensis clade. Haplotypes of each population of E. byunsanensis were highly variable, and TCS analysis of that species implied that the Jeju population is ancestral. Moreover, the cp-group of E. pungdoensis was separated from the other cp-groups by five substitutions and five indels. Therefore, based on these DNA data and TCS analysis, we advise that the taxonomic status of E. pungdoensis be treated as E. byunsanensis B. Sun var. pungdoensis (B.U. Oh) S.H. Yeau, C.S. Lee & N.S. Lee, stat. nov.     

Species delimitation tests of endemic <Emphasis Type="Italic">Lepidium papilliferum</Emphasis> and identification of other possible evolutionarily significant units in the <Emphasis Type="Italic">Lepidium montanum</Emphasis> complex (Brassicaceae) of western North America

Lepidium papilliferum of southwest Idaho was previously treated as an infraspecific variety of Lepidium montanum. Chloroplast (cpDNA) sequences, nuclear ribosomal internal transcribed spacer (ITS) sequences, and AFLPs were used to test species delimitations and other possible evolutionarily significant units (ESU) based on genetic differentiation, isolation by distance (IBD), and genetic admixture among 32 L. montanum and 21 L. papilliferum collections from the western US. The L. papilliferum AFLP genotypes formed a monophyletic clade. However, the AFLP genotypes of L. montanum samples from eight western sites were more similar to L. papilliferum, which together comprise a regionally significant West clade showing significant differentiation from eastern L. montanum collections (East clade). Bayesian analysis of AFLP genotypes detected possible admixture between L. papilliferum and related western L. montanum collections. Neither taxa nor regionally significant AFLP clades displayed reciprocally monophyletic cpDNA or ITS sequences, but the AFLP clades showed stronger cpDNA differentiation and unique ITS alleles. The East and West clades fit models of speciation with relatively strong IBD within groups and weak IBD between groups, based on correlations between the average number of AFLP differences and geographic distances among collection sites, but comparisons between taxa did not fit this model. Conversely, relatively strong partial correlations between AFLP and taxonomic differences, controlling for geography, support taxonomic delimitations. Results suggest that L. papilliferum is a distinct subgroup of L. montanum influenced by speciation. However, gene flow or common ancestry between L. papilliferum and western forms of L. montanum provide a basis for other possible ESUs.     

Taxonomy and phylogeny of Leptopilina species (Hymenoptera: Cynipoidea: Figitidae) attacking frugivorous drosophilid flies in Japan,with description of three new species

Despite the intensive use of the Leptopilina genus and its drosophilid hosts as model systems in the study of host–parasitoid interactions, the diversity and distribution of the species occurring in the Asian region remain elusive. Here we report the phylogeny of Japanese Leptopilina species attacking frugivorous drosophilid flies, based on COI, ITS1 and ITS2 sequences. Consistent with molecular data, hybridization experiments and morphological examination, five species were recorded in Japan: Leptopilina heterotoma, L. victoriae and three new species, two occurring in the Ryukyu archipelago, L. ryukyuensis and L. pacifica, and another species, L. japonica, distributed in Honshu and Hokkaido. Leptopilina japonica is further divided into two subspecies, L. j. japonica occurring in Japan, and L. j. formosana occurring in Taiwan. According to these results, we discuss the evolution, speciation and colonization history of Japanese Leptopilina species.     

Phylogeny of the Altingiaceae based on cpDNA matK, PY-IGS and nrDNA ITS sequences

 Phylogenetic relationships of the three genera of the family Altingiaceae, i.e., Altingia, Liquidambar and Semiliquidambar, based on matK sequences and the intergenic spacer between the psaA and ycf3 genes (PY-IGS) of cpDNA, and on the internal transcribed spacer (ITS) of nrDNA were studied. Phylogenetic trees based on the three data sets (matK, PY-IGS and ITS) were generated using Hamamelis japonica and Mytilaria laosensis (Hamamelidaceae), Cercidiphyllum japonicum (Cercidiphyllaceae), and Daphniphyllum calycinum (Daphniphyllaceae) as outgroups. The partition-homogeneity tests indicated that the three data sets and the combined data are homogeneous. A combined analysis also generated a strongly supported phylogeny. The phylogenetic trees show that the North American and western Asian species, L. styraciflua and L. orientalis, respectively, form a monophyletic group which is sister to the clade including all Asian species in the family. The genus Liquidambar is paraphyletic with Altingia and Semiliquidambar nested within. Phylogenetic analyses of the molecular data indicate that taxonomic reexamination of the generic delimitation in the Altingiaceae is needed. Received December 20, 2000 Accepted June 25, 2001     

Phylogenetic Use of Noncoding Regions in the GenusGentianaL.: ChloroplasttrnL (UAA) Intron versus Nuclear Ribosomal Internal Transcribed Spacer Sequences

Sequence divergence was estimated within noncoding sequences of both chloroplast DNA (cpDNA)trnL (UAA) intron and nuclear ribosomal DNA (nrDNA) internal transcribed spacer sequences (ITS1 and ITS2) for 10 species of the genusGentianaL. (Gentianaceae). Comparisons of evolutionary rates among these sequences (cpDNA versus nrDNA, ITS1 versus ITS2) were performed. It appears that sequence divergence is on average two to three times higher in ITSs than in thetrnL intron sequences and higher in ITS1 than in ITS2. Both the cpDNA intron and ITSs of nrDNA give concordant phylogenetic trees. However, the ITS-based phylogeny displays higher bootstrap values. At the intrageneric level, at least inGentiana,ITSs (especially ITS2) sequences seem to be more appropriate in the assessment of plant phylogenies. Nevertheless, the cpDNAtrnL intron seems to be preferable at the intergeneric level.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

Phylogenetic reticulation in subtribe Helianthinae

Incongruence between phylogenetic estimates based on nuclear and chloroplast DNA (cpDNA) markers was used to infer that there have been at least two instances of chloroplast transfer, presumably through wide hybridization, in subtribe Helianthinae. One instance involved Simsia dombeyana, which exhibited a cpDNA restriction site phenotype that was markedly divergent from all of the other species of the genus that were surveyed but that matched the restriction site pattern previously reported for South American species of Viguiera. In contrast, analysis of sequence data from the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed Simsia to be entirely monophyletic and placed samples of S. dombeyana as the sister group to the relatively derived S. foetida, a result concordant with morphological information. A sample of a South American species of Viguiera was placed by ITS sequence data as the sister group to a member of V. subg. Amphilepis, which was consistent with cpDNA restriction site data. Samples of Tithonia formed a single monophyletic clade based on ITS sequence data, whereas they were split between two divergent clades based on cpDNA restriction site analysis. The results suggested that cpDNA transfer has occurred between taxa diverged to the level of morphologically distinct genera, and highlight the need for careful and complete assessment of molecular data as a source of phylogenetic information.     

Functional Significance of Labellum Pattern Variation in a Sexually Deceptive Orchid (Ophrys heldreichii): Evidence of Individual Signature Learning Effects

Mimicking female insects to attract male pollinators is an important strategy in sexually deceptive orchids of the genus Ophrys, and some species possess flowers with conspicuous labellum patterns. The function of the variation of the patterns remains unresolved, with suggestions that these enhance pollinator communication. We investigated the possible function of the labellum pattern in Ophrys heldreichii, an orchid species in which the conspicuous and complex labellum pattern contrasts with a dark background. The orchid is pollinated exclusively by males of the solitary bee, Eucera berlandi. Comparisons of labellum patterns revealed that patterns within inflorescences are more similar than those of other conspecific plants. Field observations showed that the males approach at a great speed and directly land on flowers, but after an unsuccessful copulation attempt, bees hover close and visually scan the labellum pattern for up to a minute. Learning experiments conducted with honeybees as an accessible model of bee vision demonstrated that labellum patterns of different plants can be reliably learnt; in contrast, patterns of flowers from the same inflorescence could not be discriminated. These results support the hypothesis that variable labellum patterns in O. heldreichii are involved in flower-pollinator communication which would likely help these plants to avoid geitonogamy.     

君子峰鸢尾，福建鸢尾科植物一新种

描述了福建省中北部山区鸢尾科(Iridaceae)鸢尾属(Iris)一新种：君子峰鸢尾(I. junzifengensis S. P. Chen, X. Y. Chen&L.Ma),新种与蝴蝶花(I. japonica Thunb.)、台湾鸢尾(I. formosana Ohwi)相近。与蝴蝶花不同之处在于叶片宽大,叶脉明显,表面粗糙;花为顶生稀疏总状聚伞花序,分枝2～6;花大,直径5.0～7.0 cm,花被裂片边缘波状皱褶,全缘,顶端稍有凹缺;蒴果三棱状圆柱形。与台湾鸢尾不同之处在于花小,花被裂片边缘波状皱褶,全缘,顶端稍有凹缺;蒴果三棱状圆柱形。形态特征比较及分子系统发育分析均支持该新种的成立。     

Mitochondrial DNA sequence variation of four <Emphasis Type="Italic">Saccharina</Emphasis> species (Laminariales,Phaeophyceae) growing in Japan

The complete mitochondrial DNA (mtDNA) was sequenced for four Saccharina species including three varieties of Saccharina japonica in Hokkaido: S. japonica; S. japonica var. religiosa; S. japonica var. ochotensis; S. japonica var. diabolica; Saccharina longipedalis; Saccharina angustata; and Saccharina coriacea. Furthermore, the structure and the sequence were compared among them. The total nucleotide length was 37,500–37,657 bp. All mtDNAs were mapped and no differences in the organization of the coding region were found. From the total alignment of S. japonica including the three varieties and S. longipedalis, nucleotide substitutions were detected at 68 sites, and a nucleotide insertion/deletion was detected at one site on rps19 for the whole genome. Variable regions useful for varieties distinction of S. japonica were trnI, trnM, rps19, ORF41, and seven spacers. The pairwise distance between S. japonica, S. angustata, and S. coraiacea was 0.000–0.116. Sequencing comparisons of rps7, rps11, rpl5, and tatC were thought to be useful tools for discrimination and phylogenetic analysis of Saccharina species having similar morphologies.