Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.

Phycobilisomes linker family in cyanobacterial genomes: divergence and evolution

Cyanobacteria are the oldest life form making important contributions to global CO2 fixation on the Earth. Phycobilisomes (PBSs) are the major light harvesting systems of most cyanobacteria species. Recent availability of the whole genome database of cyanobacteria provides us a global and further view on the complex structural PBSs. A PBSs linker family is crucial in structure and function of major light-harvesting PBSs complexes. Linker polypeptides are considered to have the same ancestor with other phycobiliproteins (PBPs), and might have been diverged and evolved under particularly selective forces together. In this paper, a total of 192 putative linkers including 167 putative PBSs-associated linker genes and 25 Ferredoxin-NADP oxidoreductase (FNR) genes were detected through whole genome analysis of all 25 cyanobacterial genomes (20 finished and 5 in draft state). We compared the PBSs linker family of cyanobacteria in terms of gene structure, chromosome location, conservation domain, and polymorphic variants, and discussed the features and functions of the PBSs linker family. Most of PBSs-associated linkers in PBSs linker family are assembled into gene clusters with PBPs. A phylogenetic analysis based on protein data demonstrates a possibility of six classes of the linker family in cyanobacteria. Emergence, divergence, and disappearance of PBSs linkers among cyanobacterial species were due to speciation, gene duplication, gene transfer, or gene loss, and acclimation to various environmental selective pressures especially light.

Distribution and mobility of metals in agricultural soils near a copper smelter in South China

The distribution and mobility of heavy metals in the paddy soils surrounding a copper smelting plant in south China was investigated. We assessed the degree of metal contamination using an index of geoaccumulation. The metals were divided into two groups: (1) Cu, Zn, Pb and Cd, whose concentrations were heavily affected by anthropogenic inputs, and (2) Ni, Co and Cr, which were mainly of geochemical origin. Concentrations of Cu, Cd, Zn, and Pb in the polluted soils were higher than the Chinese soil quality criteria. The chemical partitioning patterns of Pb, Zn and Cu indicated that Pb was largely associated with the residual and NH2OH HCl extractable fractions. In contrast, Cd was predominantly associated with the MgCl2 extractable fraction. A large proportion of Cu was bound to the acidic H2O2 extractable fractions, while Zn was predominantly found in the residual phase. The fraction of mobile species, which potentially is the most harmful to the environment, was found to be elevated compared to unpolluted soils in which heavy metals are more strongly bound to the matrix. The mobility of the metals was studied by water extraction using a modification of Tessier's procedure, and the order of mobility was Zn > Cd > Cu > Ce > Pb.

海洋环境中有机锡的形态分析及其代谢降解研究

有机锡化合物被广泛用作塑料制品中的稳定剂、船舶油漆的防污剂、工业催化剂、农林业杀虫杀菌剂以及用于木材的防腐保存等，已经引起严重的环境污染。世界上许多国家纷纷制定相应的法规对其使用加以禁止或限制。我国目前还没有明确的限制有机锡使用的法律法规，缺少有机锡污染的第一手资料，更没有长期的控制、监测与研究计划。由于有机锡的种类繁多，理化性质存在差别，所以在提取、分离和测定中均存在较大的困难。从我国这方面己有的工作来看，缺乏各种高选择性的分离方法和高灵敏度的检测方法是制约这项研究广泛开展的原因之一。有机锡的痕量与超痕量分析技术是当今环境和食品安全分析领域的前沿技术。 本论文利用高效液相色谱和电感耦合等离子体质谱联用技术建立了海洋环境中多种有机化合物的同时快速检测方法；发展了多种海洋环境样品中有机锡的前处理技术；研究了有机锡在海洋生物中的分布、代谢及降解过程中化学形态的变化；同时发展了海洋环境中多种痕量元素的快速检测方法。所建立的高效液相色谱和电感耦合等离子体质谱联用技术可同时、快速分析5种有机锡的形态(三甲基锡TMT、二苯基锡DPhT、二丁基锡DBT、三丁基锡TBT和三苯基锡TPhT)，其检出限均低于0.3μg/L。 用所建立方法对南海海洋生物样品中的有机锡污染进行了研究，利用SPSS软件对检测结果进行了探讨，发现在所研究海洋生物样品的97.2%中可检出丁基锡和苯基锡化合物，其浓度分布处于该化合物检出限～1487.8ng/g范围内。其中，贝类样品中总有机锡的平均浓度为416.9ng/g，远远高于鱼类样品中总有机锡的平均浓度（211.9ng/g）。海洋生物中存在高浓度的有机锡说明本海域有机锡污染严重，已经对生态环境造成了严重影响，危害到人类生活。其主要的污染源是防污涂料的应用，目前紧迫的问题是采取必要的措施来控制有机锡的使用。 本工作建立了海水样品和沉积物样品中五种有机锡的简单快速萃取方法。采用加入2%的环庚三烯酚（tropolone）的二氯甲烷CH2Cl2对海水中的有机锡进行萃取，大大提高了有机锡的萃取率，减少了萃取的时间，二苯基锡（DPhT）、二丁基锡（DBT）、三丁基锡（TBT）和三苯基锡（TPhT）的萃取率均在80%以上，仅三甲基锡（TMT）的萃取率较低（在50%左右），究其原因，可能是因为在萃取的过程中三甲基锡（TMT）产生了降解。采用流动相和0.2%环庚三烯酚酮（tropolone）对沉积物国际标准物质PACS-2进行超声萃取及高速离心后，用所建方法进行了分析。结果表明，测定值与标准值吻合。研究表明，所建立的方法可用于实际环境沉积物中有机锡的形态分析。 本文建立了流动注射与电感耦合等离子体质谱联用技术直接同时测定海水中多种痕量元素的方法。该方法采用痕量进样技术，能够有效地减少海水中Na，Mg, Ca和Cl等大量基体元素对待测痕量元素测定的干扰，减少这些元素在电感耦合等离子体采样锥上的盐沉积，可以同时测量海水中的V、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Mo、Cd、Pb，Hg和U等痕量级元素。用所建的方法测定南海海域海水中的重金属元素，发现Cd，Cr，As等有毒有害元素的污染很轻，均符合Ⅰ级海水的限量。 在海洋沉积物样品处理研究中，本工作改进了不需要赶走HF酸就可以对沉积物消解完全的密闭容器消解法，由于减少了赶走HF酸的步骤，使消解的时间由原来的二十个小时降低为十个小时，大大降低了消解的时间。采用该样品消解方法，并用ICP-MS测定了南黄海海域沉积物中锡及其他重金属元素的含量。建立了微波消解-ICP-MS测定海洋生物中锡、砷、镉、汞及铅等有害重金属元素的分析方法，并用于南黄海7个及南海海域29个海产品中的测定。测定结果表明海洋生物中上述有毒有害元素有不同程度的超标问题；不同种类，不同产地的海洋生物中重金属元素的含量有一定的差别，这些研究结果为海产品安全质量控制提供了有价值的科学信息。 在上述各章工作的基础上，本文研究了有机锡在海洋生物中的分布、代谢及降解过程，并初步建立了高效液相-电喷雾-飞行时间质谱（LC-APCI-TOF-MS）测定有机锡的方法，可对未知的有机锡化合物进行结构表征。有机锡在贝类中不同的组织显示，其内脏中有机锡的含量高于肌肉中有机锡含量。常规的煮、炸、蒸及微波的烹饪方式并不能降解海产品中的有机锡化合物。

Determination of Total Arsenic and Arsenic Metabolites in Human Liver Hepatocytes

The effects of direct sampling and three digestion methods were investigated on the determination of arsenic in Chang liver hepatocytes after ultrasonic disintegration were investigated. The results showed that the efficiency of microwave digestion and obturator digestion was better than cold digestion and direct sampling. The day precision (present as RSD) of microwave digestion and obturator digestion were 2.1% and 1.2% the inter-day precision were 1.2% and 2.0%, respectively. The spike recovery for the total As in the sample is 95.7% - 108.1%. The As detection limits with these four sample treatment methods (including direct sampling) were 0.74 - 0.93 mu g/L. In addition, arsenic speciation in Chang liver hepatocytes was also analyzed using the hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma-mass spectrometry. The experimental results indicated that dimethylarsinic acid (DMA) and an intermediate metabolite of DMA were found lit Chang liver hepatocytes besides inorganic arsenic (As(III) and As(V)).

Descriptions of Protospathidium serpens (Kahl, 1930) and P-fraterculum n. sp (Ciliophora, Haptoria), two species based on different resting cyst morphology

Protospathidium serpens (Kahl, 1930) is frequent in semiterrestrial and terrestrial habitats worldwide. Conventionally, all populations are considered as conspecific because they have very similar overall morphologies and morphometrics. We studied in detail not only the morphology of the vegetative cells but also the resting cysts using live observation, protargol impregnation, and scanning electron microscopy. These revealed a cryptic diversity and biogeographic pattern in details of the dorsal brush and cyst wall morphology. The cyst wall is spiny in the Austrian specimens, while smooth in the South African and Antarctic populations. Accordingly, P. serpens consists of at least two species: P. serpens (with spiny cyst wall) and P. fraterculum n. sp. (with smooth cyst wall); the latter is probably composed of two distinct taxa differing by the absence (South African)/presence (Antarctic) of a monokinetidal bristle tail in brush row 3, the number of dikinetids comprising brush row 1 (seven versus three), and the total number of brush dikinetids (29 versus 17). Protospathidium serpens is neotypitied with the new population from Austria. The significance of resting cyst morphology is discussed with respect to alpha-taxonomy and overall ciliate diversity.

Simultaneous Determination of Multi-Organotin Compounds in Seawater by Liquid-Liquid Extraction-High Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry

The hyphenated technique of high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) was applied to the simultaneous determination of five organotin compounds (trimethyltin, dibutyltin, tributyltin, diphenyltin and triphenyltin) in seawater samples. Agilent TC-C18 column was used for the separation, the mobile phase of HPLC was CH3CN : H2O: CH3COOH=65 : 23 : 12 (phi), 0.05% TEA, and pH value was adjusted to 3.0 by diluent ammonia. The flow rate was 0.6 mL . min(-1). Five mixed organotin compounds in a mix standard solution from 100 to 0.5 mu g . L-1 were applied for the method assessment. The experimental results indicate that the correlation coefficient of calibration curves (R-2) for each organotin compound was over 0.998 and the detection limits of the five organotin compounds were lower than 3 ng . L-1. Different mixed organic solvents including dichloromethane or toluene were used for extraction of organotin and the extraction condition of organotin from seawater was optimized. The 100 mL seawater acidized by hydrochloric acid was extracted by 10 mL carbon dichloride (CH2Cl2) with 2% tropolone for 10 min twice. Extracted organic solvents were mixed And blown to one drop by nitrogen with the rate of 1.7 mL . min(-1), then 1 mL acetonitrile was added to the drop for redissolving the organotin compounds. Finally, the mixed redissolution was filtered by 0.22 mu m organic filter membrane before analysis. it was found that the only organotin compound in seawater was triphenyltin (TPHT) and the content was 53.2 ng . L-1. The recoveries test from the standard addition for diphenyltin (DPHT), dibutyltin (DBT), tributyltin (TBT) and triphenyltin (TPHT) were over 80%. However, the recovery for trimethyltin (TMT) was relatively low and the value was 50%. The reason might be attributed to the decomposition or adsorption of those compounds during the extraction procedure. Further study on this subject is in progress.

Copper complexation by fulvic acid affects copper toxicity to the larvae of the polychaete Hydroides elegans

Copper toxicity is influenced by a variety of environmental factors including dissolved organic matter (DOM). We examined the complexation of copper by fulvic acid (FA), one of the major components of DOM, by measuring the decline in labile copper by anodic stripping voltammetrically (ASV). The data were described using a one-site ligand binding model, with a ligand concentration of 0.19 mu mol site mg(-1) C, and a logK' of 6.2. The model was used to predict labile copper concentration in a bioassay designed to quantify the extent to which Cu-FA complexation affected copper toxicity to the larvae of marine polychaete Hydroides elegans. The toxicity data, when expressed as labile copper concentration causing abnormal development, were independent of FA concentration and could be modeled as a logistic function, with a 48-h EC50 of 58.9 mu g 1(-1). However, when the data were expressed as a function of total copper concentration, the toxicity was dependent on FA concentration, with a 48-h EC50 ranging from 55.6 mu g 1(-1) in the no-FA control to 137.4 mu g 1(-1) in the 20 mg 1(-1) FA treatment. Thus, FA was protective against copper toxicity to the larvae, and such an effect was caused by the reduction in labile copper due to Cu-FA complexation. Our results demonstrate the potential of ASV as a useful tool for predicting metal toxicity to the larvae in coastal environment where DOM plays an important role in complexing metal ions. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular evidence for double maternal origins of the diploid hybrid Hippophae goniocarpa (Elaeagnaceae)

Homoploid hybrid plant species are rare, and the mechanisms of their speciation are largely unknown, especially for homoploid hybrid tree species. Two contrasting hypotheses have been proposed to explain the origin of Hippophae goniocarpa: (1) it is a diploid hybrid originating from H. rhamnoides ssp. sinensis x H. neurocarpa ssp. neurocarpa, and (2) it originated via marginal differentiation from H. rhamnoides ssp. sinensis. Regardless of which of these hypotheses is true (if either), previous studies have suggested that H. rhamnoides ssp. sinensis is the only maternal donor for this hybrid species. In this study, we aim to elucidate the maternal composition of H. goniocarpa and to test the two hypotheses. For this purpose, we sequenced the maternal chloroplast DNA trnL-F region of 75 individuals representing H. goniocarpa, H. rhamnoides ssp. sinensis, and H. neurocarpa ssp. neurocarpa in two co-occurring sites of the taxa. Seven haplotypes were identified from three taxonomic units, and their phylogenetic relationships were further constructed by means of maximum parsimony, maximum likelihood, and network analyses. These seven haplotypes clustered into two distinct, highly divergent lineages. Two haplotypes from one lineage were found in H. rhamnoides ssp. sinensis, and five (representing the other lineage) in H. neurocarpa ssp. neurocarpa. Hippophae goniocarpa shared four common haplotypes from both lineages, but the haplotypes detected from the two populations differed to some extent, and in each case were identical to local haplotypes of the putative parental species. Thus, both H. rhamnoides ssp. sinensis and H. neurocarpa ssp. neurocarpa appear to have together contributed to the maternal establishment of H. goniocarpa. These results clearly demonstrate that the marginal origin hypothesis should be rejected, and support the hybrid origin hypothesis. Hippophae goniocarpa exhibits a sympatric distribution with its two parent species, without occupying new niches or displaying complete ecological isolation. However, this species has effectively developed reproductive isolation from its sympatric parent species. Our preliminary results suggest that H. goniocarpa may provide a useful model system for studying diploid hybrid speciation in trees. (c) 2008 The Linnean Society of London.

Mitochondrial DNA phylogeny of two morphologically enigmatic fishes in the subfamily Schizothoracinae (Teleostei : Cyprinidae) in the Qinghai-Tibetan Plateau

The complete mitochondrial DNA (mtDNA) cytochrome b gene (1140 bp) was sequenced in Herzenstein macrocephalus and Gymnocypris namensis and in 13 other species and sub-species (n = 22), representing four closely related genera in the subfamily Schizothoracinae. Conflicting taxonomies of H. macrocephalus and G. namensis have been proposed because of the character instability among individuals. Parsimony, maximum likelihood and Bayesian methods produced phylogenetic trees with the same topology and resolved several distinctive clades. Previous taxonomic treatments, which variously placed these two species of separate genera or as sub-species, are inconsistent with the mtDNA phylogeny. Both H. macrocephalus and G. namensis appear in a well-supported clade, which also includes nine species of Schizopygopsis, and hence should be transferred to the genus Schizopygopsis. Morphological changes are further illustrated, and their adaptive evolution in response to the local habitat shifts during the speciation process appears to be responsible for conflicting views on the systematics of these two species and hence the contrasting taxonomic treatments. These species are endemic to the Qinghai-Tibetan Plateau, a region with a history of geological activity and a rich diversity of habitats that may have result in the parallel and reversal evolution of some morphological characters used in their taxonomies. Our results further suggest that speciation and morphological evolution of fishes in this region may be more complex than those previously expected. (c) 2007 The Authors Journal compilation (c) 2007 The Fisheries Society of the British Isles.

Origin of the Qinghai-Tibetan Plateau endemic Milula (Liliaceae): further insights from karyological comparisons with Allium

Milula, a monotypic genus endemic to the Qinghai-Tibetan Plateau, was found to be nested deeply within Allium by the molecular phylogeny despite the aberrant morphology. It remains unknown what had contributed to the rapid evolution of morphology and origin of this exceptional species. In contrast to a previous report of its karyotypes with 2n = 16 = 8M+8SM (2SAT), similar to most species of Allium, a rather different karyotype, 2n = 20 = 4M +10SM+6T (2SAT), was found in examined 31 individuals from 6 populations of M. spicata distributed in the central Tibet. Karyotypes of 7 Allium species occurring in the Qinghai-Tibetan Plateau were further reported. The basic number x = 8 was confirmed for all of them and their karyotypes consist mainly of metacentric and submetacentric chromosomes with rare subterminal and terminal chromosomes. The karyotype of M. spicata is distinctly different from that of most Allium species occurring in the plateau through a complete comparison of all available species in this region and adjacent areas. However, the same chromosome number and similar karyotypic structure were found in A. fasciculatum of Sect. Bromatorrhiza, indicating a possible close relationship between them. But this similarity is contradictory to the preliminary molecular phylogenetic analysis that Milula was closely related to A. cyathophorum of Sect. Bromatorrhiza with x=8, but the other species with x=10 and 11 in this section were clearly placed in the other clade. We therefore suggested that the paralleling evolution from x=8 to x=9, 10 and 11 with increasing asymmetry of karyotype possibly due to the chromosomal Robertsonian translocation might occur separately in the two recognized phylogenetic lineages of Allium. In addition to aneuploidy and following change of the chromosomal structures, the habitat isolation due to the recent uplift of the Qinghai-Tibetan Plateau and the Quaternary climatic oscillation, plays a greater role in origin of Milula and other endemic species (genera) with aberrant morphology from their progenitors.

Radiation and diversification within the Ligularia-Cremanthodium-Parasenecio complex (Asteraceae) triggered by uplift of the Qinghai-Tibetan Plateau

The Ligularia-Cremanthodium-Parasenecio (L-C-P) complex of the Tussilagininae (Asteraceae: Senecioneae) contains more than 200 species that are endemic to the Qinghai-Tibetan Plateau in eastern Asia. These species are morphologically distinct; however, their relationships appear complex. A phylogenetic analysis of members of the complex and selected taxa, of the tribe Senecioneae was conducted using chloroplast (ndhF and trnL-F) and nuclear (ITS) sequences. Phylogenetic trees were constructed from individual and combined datasets of the three different sequences. All analyses suggested that Doronicum, a genus that has been included in the Tussilagininae, should be excluded from this subtribe and placed at the base of the tribe Senecioneae. In addition, the Tussilagininae should be broadly circumscribed to include the Tephroseridinae. Within the expanded Tussilagininae containing all 13 genera occurring in eastern Asia, Tussilago and NSPetasites diverged early as a separate lineage, while the remaining I I genera comprise an expanded L-C-P complex clade. We suggest that the L-C-P clade, which is largely unresolved, most likely originated as a consequence of an explosive radiation. The few monophyletic subclades identified in the L-C-P clade with robust support further suggest that some genera of Tussilagininae from eastern Asia require generic re-circumscriptions given the occurrence of subclades containing species of the same genus in different parts of the phylogentic tree due to homoplasy of important morphological characters used to delimit them. Molecular-clock analyses suggest that the explosive radiation of the L-C-P complex occurred mostly within the last 20 million years, which falls well within the period of recent major uplifts of the Qinghai-Tibetan Plateau between the early Miocene to the Pleistocene. It is proposed that significant increases in geological and ecological diversity that accompanied such uplifting, most likely promoted rapid and continuous allopatric speciation in small and isolated populations, and allowed fixation or acquisition of similar morphological characters within unrelated lineages. This phenomenon, possibly combined with interspecific diploid hybridization because of secondary sympatry during relatively stable stages between different uplifts, could be a major cause of high species diversity in the Qinghai-Tibetan Plateau and adjacent areas of eastern Asia. (c) 2005 Elsevier Inc. All rights reserved.

Molecular systematics and biogeography of Crawfurdia, Metagentiana and Tripterospermum (Gentianaceae) based on nuclear ribosomal and plastid DNA sequences

Background and Aims The systematic position of the genus Metagentiana and its phylogenetic relationships with Crawfurdia, Gentiana and Tripterospermum have not been explicitly addressed. These four genera belong to one of two subtribes (Gentianinae) of Gentianeae. The aim of this paper is to examine the systematic position of Crawfurdia, Metagentiana and Tripterospermum and to clarify their phylogenetic affinities more clearly using ITS and trnL intron sequences.Methods Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and the plastid DNA trnL (UAA) intron were analysed phylogenetically. Ten of fourteen Metagentiana species were sampled, together with 40 species of other genera in the subtribe Gentianinae.Key Results The data support several previously published conclusions relating to the separation of Metagentiana from Gentiana and its closer relationships to Crawfurdia and Tripterospermum based on studies of gross morphology, floral anatomy, chromosomes, palynology, embryology and previous molecular data. The molecular clock hypothesis for the tested sequences in subtribe Gentianinae was not supported by the data (P < 0.05), so the clock-independent non-parametric rate smoothing method was used to estimate divergence time. This indicates that the separation of Crawfurdia, Metagentiana and Tripterospermum from Gentiana occurred about 11.4-21.4 Mya (million years ago), and the current species of these three genera diverged at times ranging from 0.4 to 6.2 Mya.Conclusions The molecular analyses revealed that Crawfurdia, Metagentiana and Tripterospermum do not merit status as three separate genera, because sampled species of Crawfurdia and Tripterospermum are embedded within Metagentiana. The speciation and rapid radiation of these three genera is likely to have occurred in western China as a result of upthrust of the Himalayas during the late Miocene and the Pleistocene.

Molecular phylogeny, recent radiation and evolution of gross morphology of the rhubarb genus Rheum (Polygonaceae) inferred from chloroplast DNA trnL-F sequences

Background and Aims Rheum, a highly diversified genus with about 60 species, is mainly confined to the mountainous and desert regions of the Qinghai-Tibetan plateau and adjacent areas. This genus represents a good example of the extensive diversification of the temperate genera in the Qinghai-Tibetan plateau, in which the forces to drive diversification remain unknown. To date, the infrageneric classification of Rheum has been mainly based on morphological characters. However, it may have been subject to convergent evolution under habitat pressure, and the systematic position of some sections are unclear, especially Sect. Globulosa, which has globular inflorescences, and Sect. Nobilia, which has semi-translucent bracts. Recent palynological research has found substantial contradictions between exine patterns and the current classification of Rheum. Two specific objectives of this research were (1) to evaluate possible relationships of some ambiguous sections with a unique morphology, and (2) to examine possible occurrence of the radiative speciation with low genetic divergence across the total genus and the correlation between the extensive diversification time of Rheum and past geographical events, especially the recent large-scale uplifts of the Qinghai-Tibetan Plateau.Methods The chloroplast DNA trnL-F region of 29 individuals representing 26 species of Rheum, belonging to seven out of eight sections, was sequenced and compared. The phylogenetic relationships were further constructed based on the sequences obtained.Key Results Despite the highly diversified morphology, the genetic variation in this DNA fragment is relatively low. The molecular phylogeny is highly inconsistent with gross morphology, pollen exine patterns and traditional classifications, except for identifying all samples of Sect. Palmata, three species of Sect. Spiciformia and a few species of Sect. Rheum as corresponding monophyletic groups. The monotypic Sect. Globulosa showed a tentative position within the clade comprising five species of Sect. Rheum. All of the analyses revealed the paraphyly of R. nobile and R. alexandrae, the only two species of Sect. Nobilia circumscribed by the possession of large bracts. The crude calibration of lineages based on trnL-F sequence differentiation implied an extensive diversification of Rheum within approx. 7 million years.Conclusions Based on these results, it is suggested that the rich geological and ecological diversity caused by the recent large-scale uplifts of the Qinghai-Tibetan Plateau since the late Tertiary, coupled with the oscillating climate of the Quaternary stage, might have promoted rapid speciation in small and isolated populations, as well as allowing the fixation of unique or rare morphological characters in Rheum. Such a rapid radiation, combined with introgressive hybridization and reticulate evolution, may have caused the transfer of cpDNA haplotypes between morphologically dissimilar species, and might account for the inconsistency between morphological classification and molecular phylogeny reported here.