Relationships among pansies (Viola section Melanium) investigated using ITS and ISSR markers

Sequences of the nuclear region ITS and the variable molecular markers ISSR were used to estimate the phylogeny of the section Melanium of the genus Viola. We confirm that the so-called pansies form a derived and monophyletic group. Two floral characters, the upturned side petals and the large size of pollen grains appear to be synapomorphies in Melanium. The Melanium species are very closely related, as shown by the reduced genetic variation compared to the other sections of Viola. Our analyses suggest x=5 or x=7 as the possible base chromosome number of the section Melanium. Polyploidy and hybridization would have played an important role in the evolutionary history of this clade resulting in a wide range of chromosome number. The low genetic differentiation and the complex cytological evolution suggest that diversification in Melanium is the result of a reticulate evolution and rapid radiation in Europe and Northern Africa.     

Hamamelose and its derivatives as chemotaxonomic markers in the genus Primula

About 550 species of vascular plants as well as a few mosses were checked for the occurrence of hamamelose, the corresponding alcohol hamamelitol and a galactoside of the latter, clusianose. Hamamelose was found in most of the species investigated. It provides therefore no useful character for chemotaxonomic purposes. Hamamelitol, however, could only be detected in the genus Primula and was therefore more useful. Small amounts of hamamelitol were found in the leaves of species from the subgenera Auganthus, Aleuritia and Auricula but not from Primula and Sphondylia. Clusianose and large amounts of hamamelitol are produced by species of 4 subsections of the section Auricula, but not by species of the subsections Auricula and Erythrodrosum. A taxonomic separation of the two latter from the 4 former subsections is suggested. The taxonomic position of Primula allionii should be reconsidered, since neither clusianose nor hamamelitol could be demonstrated in this species, whereas the other species of the subsection Rhopsidium do contain these carbohydrates.     

A first approach to the molecular phylogeny of the genusEchinops (Asteraceae): Sectional delimitation and relationships with the genusAcantholepis

A first attempt to establish the phylogeny of the generaEchinops andAcantholepis has been carried out using the analysis of the internal transcribed spacers (ITS) sequences of the nuclear ribosomal DNA including 30Echinops species and the only species of the monotypicAcantholepis. The results of this analysis are discussed in the light of morphological and cytogenetic characters. The genusAcantholepis is placed in a robust clade withEchinops nanus, and together they appear in a basal position to other members ofEchinops. The ITS phylogeny and several other characters, such as chromosome number and nuclear DNA amount, do not agree with the sections currently recognized withinEchinops. Some groups are defined in the present approach, but further studies are necessary to reach a complete, stable and natural infrageneric classification of this genus.     

Chromosome and breeding system evolution of the genus Mercurialis (Euphorbiaceae): implications of ITS molecular phylogeny

 The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were amplified and sequenced from 19 samples representing all species of the genus Mercurialis and two outgroup species, Ricinus communis and Acalypha hispida. The length of ITS1 in the ingroups ranged from 223 to 246 bp and ITS2 from 210 to 218 bp. Sequence divergence between pairs of species ranged from 1.15% to 25.88% among the ingroup species in the combined data of ITS1 and ITS2. Heuristic phylogenetic analyses using Fitch parsimony on the combined data of ITS1 and ITS2 with gaps treated as missing generated 45 equally parsimonious trees. The strict consensus tree was principally concordant with morphological classification. Within the genus, the ITS sequences recognised two main infrageneric clades: the M. perennis complex including three Eurasian stoloniferous species (M.␣leiocarpa, M. ovata and M. perennis) and the western Mediterranean group including eight both annual and perennial species. Of the western Mediterranean clade, the annual and perennial species grouped respectively into two different groups, and the annual life form is revealed as a synapomorphic character derived from perennial, whereas in the Eurasian clade ITS phylogeny suggested M. leiocarpa as basal clade sister to M.␣perennis and M. ovata. ITS phylogeny failed to resolve the relationships among the different cytotypes of M. ovata and M. perennis. ITS phylogeny also suggested rapid karyotypic evolution for the genus. The karyotypic divergence among the perennial species of western Mediterranean region did not corroborate the nucleotide sequence divergence among the species. Optimisation of chromosome numbers onto the ITS phylogeny suggested x=8 to be the ancestral basic chromosome number of the genus. ITS phylogeny confirmed that the androdioecy of M. ambigua is derived from dioecy. The nucleotide heterozygosity and additivity in ITS sequences clearly confirm the interspecific hybridisation in the genus Mercurialis. Received December 22, 2001; accepted May 21, 2002?Published online: November 14, 2002 Address of the authors: Martin Kr?henbühl, Yong-Ming Yuan (correspondence) and Philippe Küpfer, Institut de Botanique, Laboratoire de botanique évolutive, Université de Neuchatel, Emile-Argand 11, CH-2007 Neuchatel, Suisse. (e-mail: yong-ming.yuan@unine.ch)     

Phylogenetic relationships in the genus <Emphasis Type="Italic">Primula</Emphasis> L. (Primulaceae) inferred from the ITS region sequences of nuclear rDNA

The nucleotide sequences of the nuclear rDNA ITS regions were determined for 34 species of the genus Primula L. and one species of the genus Cortusa L., family Primulaceae Vent., and used to infer the phylogenetic relationships among these species. In this analysis species of the Russian flora and the flora of adjacent territories were studied for the first time. The results clarified the taxomic structure of the genus Primula and confirmed the entity of some of its sections; but not the subgenera sensu J.Richards. Our data do not support an independent status of the genus Cortusa, placing it as one of the terminal lineages of the section Cortusoides Balf. f. in the genus Primula.     

Phylogenetic relationships in genus <Emphasis Type="Italic">Arachis</Emphasis> based on ITS and 5.8S rDNA sequences

Background  

The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.     

PHYLETIC TRENDS IN SECTIONS EUBLEPHARIS AND CALLIOPSIS OF THE GENUS COREOPSIS (COMPOSITAE)

Artificial hybridization, fertility of interspecific and intersectional hybrids, chromosome numbers, and trends in habit and morphology in taxa of sections Eublepharis and Calliopsis of the genus Coreopsis are used to consider the sectional relationships and to derive a presumed phylogeny for the two sections. The two sections are closely related and show a low level of interfertility, but this level is as high as in some interspecific crosses within sections. The sections differ in base chromosome number and achene winging. The problematic C. rosea evidently should remain in section Eublepharis. Both sections probably arose from x = 13 stock resembling C. integrifolia or C. pubescens. Descending aneuploidy was involved in the derivation of section Calliopsis, while chromosome evolution in section Eublepharis has involved polyploidy.     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

A phylogenetic analysis of Primulaceae s.l. based on internal transcribed spacer (ITS) DNA sequence data

 Phylogenetic relationships in Primulaceae were investigated by analysis of nuclear rDNA ITS sequences. Thirty-four species of Primulaceae, two of Myrsinaceae and four outgroup taxa were analyzed. In accordance to the results of recently published papers on the phylogeny of Primulaceae we found the family to be paraphyletic and resolved the positions of some genera. Our results show (a) the rather basal position of Centunculus within Lysimachieae, the genus thus being rather distantly related to Anagallis, (b) the close relationship between Lysimachia sect. Lerouxia, Anagallis, Asterolinon, and Pelletiera, (c) the well-supported monophyly of a group consisting of the four genera Hottonia, Omphalogramma, Bryocarpum, and Soldanella, and (d) the affinity of Stimpsonia to the Myrsinaceae-Lysimachieae-Ardisiandra clade. The ITS sequence data do not provide sufficient information to resolve basal relationships within the Primulaceae s.l. There is evidence against the monophyly of the large genera Primula, Androsace, and Lysimachia. In contrast to the phylogenetic reconstructions based on plastid gene sequences, Cyclamen does not appear as a member of the Myrsinaceae-Lysimachieae clade, but its position remains unclear. Revised July 10, 2002; accepted November 21, 2002 Published online: March 20, 2003     

Infrageneric phylogeny of the genus Gentiana (Gentianaceae) inferred from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacers (ITSs) of nuclear ribosomal DNA have been sequenced for 20 species of Gentiana. By incorporating previously released sequence data of eight species, phylogenelic analyses using Fitch parsimony and character-state weighted parsimony were carried out. The length of ITS 1 in the taxa surveyed ranged from 223 to 238 bp and ITS2 from 216 to 234 bp. Sequence divergence between pairs of species ranged from 5.0% to 48.9% in ITS1, from 1.1% to 45.3% in ITS2, and from 3.2% to 46.1% in combined data of ITS1 and ITS2. The ITS phylogeny was generally congruent with morphological classifications except that G. asclepiadea was revealed to be closely related to section Gentiana instead of section Pneumonanthe and section Stenogyne was shown to be a paraphyletic group of the genus Gentiana that would be better excluded from the genus. A divergence among the three European endemic sections and the remaining sections of the genus other than section Stenogyne was revealed. Thus the European species of the genus together do not form a monophyletic group. A close relationship between the sections Chondrophyllae s. l. (including section Dolichocarpa), Cruciata and Pneumonanthe was suggested. The section Frigidae s. l. (including sections Monopodiae, Isomeria, Microsperma, and Phyllocalyx) contained two well-supported clades: section Frigidae s. str. and all others together. The monophyly of the typically dysploid group section Chondrophyllae s. l. was confirmed. Optimization of chromosome numbers on the ITS phylogeny suggested that 2/1 = 26 is a plesiomorphic state for the clade comprising sections Frigidae s. l., Cruciata, Pneumonanthe, and Chondrophyllae s. l., and probably 2n = 20 is a plesiomorphic state for the dysploid group, section Chondrophyllae s. l.     

DNA Barcoding Evaluation and Its Taxonomic Implications in the Species-Rich Genus Primula L. in China

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Unexpected high divergence in nrDNA ITS and extensive parallelism in floral morphology of Pedicularis (Orobanchaceae)

Internal transcribed spacer (ITS) region sequences of nuclear ribosomal DNA (nrDNA) were used in the phylogenetic reconstruction of Pedicularis, a genus with strong adaptive radiation. Forty-two species representing 12 greges of the genus were all, except P. resupinata, sampled from the Hengduan Mountain region, China. A high level of ITS sequence variation was found among the species distributed in such a small geographical area, which had been rarely reported in other groups. The great ITS divergence in Pedicularis could be explained by a relatively ancient origin and diversification of the genus followed by migration of different floristic components into the Hengduan Mountains, or accelerated rates of molecular evolution in parasitic lineages of Orobanchaceae. In the present ITS phylogeny, almost all the main clades are not consistent with the high hierarchical taxa within Pedicularis, which implies that significant parallel evolution occurred in floral morphology of the genus, and that undue attention has been paid to corolla characters in the intragenus classifications.We thank the two anonymous reviewers for valuable comments and suggestions; Professor Guo You-Hao, Drs. Tan Dun-Yan, Luo Yi-Bo, Bao Ying, Luo Yan, Mr. Mao Jian-Feng and Mr. Qin Wei for their great help in the field collection; Drs. Song Bao-Hua, Wei Xiao-Xin and Miss Sun Ying-Xue for their help in the laboratory work. This study was supported by State Key Basic Research and Development Plan (Grant No. G2000046804) and the National Natural Science Foundation of China (Grant No. 30121003).     

色季拉山10种报春花属植物花粉形态及其分类学意义

采用扫描电镜法,观察和比较了西藏色季拉山10种报春花的花粉形态特征,同时进行聚类分析,以期为该属植物分类提供孢粉学证据,并进一步为西藏报春花属植物杂交育种及种质资源的利用提供参考。结果表明：(1)供试10种报春花属植物的花粉形状为扁球形、近球形,其中,工布报春(Primula kongboensis)的花粉粒最小,暗紫脆蒴报春(P.calderiana)的花粉粒最大,西藏报春(P.tibetica)为多沟型花粉,其余报春花的花粉一般具3孔沟,大部分孔沟在极区汇合形成复合沟。(2)花粉外壁纹饰大多为穴状或网状,其中,中甸灯台报春(P.chungensis)和西藏报春为网状纹饰中的粗网状类型。(3)虽然基于花粉形态的聚类分析与植物学分类表现出一定的一致性,但粉报春组的西藏报春和工布报春则由于在孢粉学特征上具有明显差异,因此保持了相对较远的亲缘关系。该研究初步认为,色季拉山10种报春花粉形态存在种间差异,研究结果可为植物分类提供一定的参考依据。然而,在进行分类时,仍然需要结合形态学特征、分子生物学等方面综合考虑。     

Primula vallicola sp. nov. (Primulaceae) from Sichuan,China

A new species of Primula, P. vallicola Y. Xu, G. Hao & C.M. Hu, is described and illustrated from Sichuan province, China. Based on general morphology the new species is assigned to Primula sect. Aleuritia subsect. Yunnanensis. It is closely allied to P. kialensis, but can be distinguished by its larger stature, leaf shape, and the well‐differentiated petiole. The leaf shape of the new species is ovate to ovate‐elliptic and usually sub‐rounded at base. The new species also resembles P. homogama, but differs in being glabrous rather than with appressed hairs, and with longer petiole.     

ITS2 secondary structure improves phylogeny estimation in a radiation of blue butterflies of the subgenus Agrodiaetus (Lepidoptera: Lycaenidae: Polyommatus)

Background  

Current molecular phylogenetic studies of Lepidoptera and most other arthropods are predominantly based on mitochondrial genes and a limited number of nuclear genes. The nuclear genes, however, generally do not provide sufficient information for young radiations. ITS2, which has proven to be an excellent nuclear marker for similarly aged radiations in other organisms like fungi and plants, is only rarely used for phylogeny estimation in arthropods, although universal primers exist. This is partly due to difficulties in the alignment of ITS2 sequences in more distant taxa. The present study uses ITS2 secondary structure information to elucidate the phylogeny of a species-rich young radiation of arthropods, the butterfly subgenus Agrodiaetus. One aim is to evaluate the efficiency of ITS2 to resolve the phylogeny of the subgenus in comparison with COI, the most important mitochondrial marker in arthropods. Furthermore, we assess the use of compensatory base changes in ITS2 for the delimitation of species and discuss the prospects of ITS2 as a nuclear marker for barcoding studies.     

Molecular evidence for the phylogenetic position ofHanabusaya asiatica Nakai (Campanulaceae), an endemic species in Korea

The phylogenetic relationship of the Korean endemic genus,Hanabusaya, to other campanulaceous genera has been controversial since it was described by Nakai in 1911. Three genera of Campanuloideae,Symphyandra, Adenophora, andCampanula, have been considered closely related by various taxonomists on the basis of anther shape, gross morphology, and pollen characters, respectively. We have tested these competing taxonomic hypotheses using the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA from 12 taxa representing 7 genera of Campanulaceae. The molecular phylogeny indicates strongly thatHanabusaya is more closely allied toAdenophora than toCampanula orSymphyandra. The phylogenetic affinity ofHanabusaya andAdenophora is supported by a 100% bootstrap value and a high decay index (13). The average sequence divergence value (Kimura’s 2-parameter method) betweenHanabusaya and theAdenophora species is 2.58. The value is significantly (about ten times) lower than the ones observed betweenHanabusaya and the species ofCampanula (average of 23.52) and betweenHanabusaya andSymphyandra (24.95). The ITS sequence phylogeny suggests that some morphological characters, such as fused anthers and corolla shape, are homoplastic in the Campanulaceae genera.     

Evidence of hybrid speciation in the North American primroses Primula suffrutescensP. parryiP. rusbyi and P. angustifolia (Primulaceae)

The genetic structure of tetraploid (4x = 44) North American species of Primula subgenus Auriculastrum: P. suffrutescens, P. parryi, P. rusbyi, and P. angustifolia was analyzed at seven enzyme loci and compared with that of the related diploid P. cuneifolia and P. nipponica. The studied tetraploid species showed fixed or almost fixed heterozygosity at various loci, indicating a hybrid origin. About half of their alleles were shared with Cuneifolia taxa sampled, suggesting that they arose from crosses involving a maternal Cuneifolia-like ancestor. A preliminary survey of their possible paternal species, as inferred from their genotypes, was carried out among different Primula groups. Several paternal alleles expected were observed in subgenus Aleuritia, particularly among taxa of sect. Crystallophlomis (e.g., P. chionantha, P. minor, and P. nivalis xanthobasis). However, none of these taxa proved to be a suitable paternal species. Hybridization events that originated P. suffrutescens, P. parryi, P. rusbyi, and P. angustifolia presumably occurred during Pleistocene secondary contacts in Beringia and involved a maternal Cuneifolia-like ancestor and at least two paternal species, for sect. Suffrutescens and Parryi, respectively. Further studies, involving a genetic survey of Aleuritia taxa from Siberia will be needed to detect such paternal species, if not extinct.     

DNA序列在悬钩子属植物分子系统学研究中的应用进展

悬钩子属植物种类繁多,类群复杂,而且多为多倍体和杂种。该文就近年来国内外有关DNA序列在悬钩子属植物分子系统学研究中的应用现状和进展进行了综述,并对中国悬钩子属植物系统发育研究进行了展望。研究认为:叶绿体DNA序列多应用非编码区,且多与ITS序列联合分析;核基因组中ITS序列应用最为广泛,主要用于研究悬钩子属空心莓组与木莓组的进化关系、栽培品种间亲缘关系及部分杂种和多倍体的起源等;在该属植物中发现了ITS个体内多态性,但未进行ITS假基因检测,其系统学应用价值需重新评价;低拷贝核基因只有GBSSI和LEAFY有相关应用。同时认为,悬钩子属植物系统学研究中应用的DNA序列及研究类群均较少,缺乏对整个悬钩子属全面而系统的研究。指出应进一步选择具有代表性的样本、筛选合适的DNA片段,并结合形态学、孢粉学和细胞学等手段对中国悬钩子属植物系统关系进行深入研究。     

Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny

The economically important genus Arachis (Fabaceae) comprises 80 species restricted to South America. One monograph on the genus divided it into nine sections and included an intuitive assessment of evolutionary relationships. There is no comprehensive phylogenetic study of the genus. To test the current systematic treatment of the genus, we reconstructed a phylogeny for Arachis using nuclear ITS and plastid trnT–trnF sequences from 46 species representing all nine sections. ITS cloning of the allotetraploid species of section Arachis indicated the presence of A and B genome alleles and chimeric sequences. Our study revealed that species from section Extranervosae were the first emerging lineage in the genus, followed by sections Triseminatae and Caulorrhizae, and two terminal major lineages, which we refer to as erectoides and arachis. The lineage erectoides comprises members of sections Erectoides, Heteranthae, Procumbentes, Rhizomatosae, and Trierectoides. Species in the arachis lineage form two major clades, arachis I (B and D genomes species and the aneuploids) and arachis II (A genome species). Our results substantiated the sectional treatment of Caulorrhizae, Extranervosae, and Triseminatae, but demonstrated that sections Erectoides, Procumbentes, and Trierectoides are not monophyletic. A detailed study of the genus Arachis with denser taxon sampling, additional genomic regions, plus information from morphology and cytogenetics is needed for comprehensive assessment of its systematics.