PCR产物的RFLP分析在黄芪亚族（豆科）系统学研究中的应用初探

用聚合酶链式反应（ＰＣＲ）将豆科黄芪亚族７属９种及甘草亚族１属１种植物叶绿体基因组中ｎｄｈＦ和ｐｓｂＡ基因中一段约３．１ｋｂ的ＤＮＡ扩增出来,并摸索出一最佳的ＰＣＲ条件,使得此条带得以特异性扩增,可直接用于限制性内切酶水解。通过对此扩增片段的限制性片段长度多态性（ＲＦＬＰ）的初步分析,认为利用ＰＣＲ扩增出的ＤＮＡ进行ＲＦＬＰ分析来探讨植物的系统进化问题在中国现行条件下大有潜力,其方法简单易行,结果     

EVIDENCE FOR A CLOSE RELATIONSHIP BETWEEN IOSTEPHANE AND VIGUIERA (ASTERACEAE: HELIANTHEAE)

The phylogenetic relationship of Iostephane is assessed using data from morphology, flavonoid chemistry, and chloroplast DNA and nuclear ribosomal DNA restriction fragment analysis. Morphological evidence supports placement of Iostephane in subtribe Helianthinae, but fails to clarify the placement of the genus within this assemblage. Further evidence for the placement of Iostephane in subtribe Helianthinae is provided by the presence in all species of the genus of floral flavonoids of the chalcone/aurone type, which provides a distinctive trait for the subtribe within the tribe Heliantheae. Analysis of chloroplast DNA from two species of Iostephane, I. heterophylla and I. madrensis, in comparison to Viguiera and related genera indicates that the restriction site patterns with 16 enzymes for the Iostephane species are virtually identical to one another as well as to those of Viguiera sect. Maculatae. Data from restriction fragment patterns of nuclear rDNA are concordant with the results from chloroplast DNA in suggesting a direct relationship between the two groups. The close phylogenetic relationship between Iostephane and Viguiera sect. Maculatae suggested by the DNA restriction fragment data was not suggested by any other set of data.     

Phylogeny and molecular evolution of the tribe Harpalini (Coleoptera, Carabidae) inferred from mitochondrial cytochrome-oxidase I

The tribe Harpalini is a group of ground beetles with a world-wide distribution that comprises approximately 2000 species and about 238 genera and subgenera. Hypotheses about the phylogenetic relationships of the subtribes of Harpalini are implicit within the systematic criteria put forward by different authors. A 759 bp fragment of the mitochondrial COI was sequenced in 119 specimens (107 species) of 52 genera and subgenera that represent the main lineages of Harpalines, and 3 species of other tribes used as outgroups. A hierarchical study of sequence divergence (under uncorrected and corrected models) and ts:tv ratio pattern analyses were carried out at different taxonomic levels. A low saturation rate was detected at first and second codon positions, whereas A+T richness causes a low transitions:transversions ratio, which suggests--a priori--a high rate of saturation at the third codon position. A progressive accumulation of sequence divergence and a decreasing ts:tv ratio were found from lower to higher taxonomic levels. MP strict consensus, ML, and minimum evolution distance (under ts+tv and tv only schemes) trees showed similar major clades within the tribe. The subtribe Ditomina is a monophyletic lineage with close affinities to the subtribe Harpalina. Harpalina is a polyphyletic lineage as the genus Daptus is always related to members of the subtribe Stenolophina, and the Selenophorines resulted a polyphyletic group related to the subtribe Anisodactylina. Main lineages proposed by Noonan [Quaest. Entomol. 9 (1973) 266] within the subtribe Anisodactylina have been corroborated in this study. The Australian genus Phorticosomus is not related to Ditomina but to the Australian Notiobioids lineage. Most taxa of the subtribe Stenolophina are always included in the same clade, together with taxa of the subtribe Pelmatellina, which might be considered as a lineage of Stenolophina related to Bradycellus and Dicheirotrichus. The subtribe Amblystomina lacks a well-supported relationship to the other subtribes of Harpalini and could not be consistently related to any of them.     

Molecular phylogenetics of the Espeletia complex (Asteraceae): evidence from nrDNA ITS sequences on the closest relatives of an Andean adaptive radiation

The subtribe Espeletiinae (Asteraceae, Heliantheae) comprises morphologically and ecologically diverse plants endemic to the tropical montane paramos of the Andes of Venezuela, Colombia, and Ecuador. Though the ecophysiology and ecology of this adaptive radiation have been well studied, relationships among taxa in the subtribe and between the subtribe and other taxa in the Heliantheae are poorly known. In this study, sequences from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA are used to test previous hypotheses about the phylogenetic position of the Espeletiinae within the Heliantheae and to determine which taxa are the subtribe's closest relatives. Gene phylogenies based on maximum parsimony analyses reveal that the Espeletiinae clade is nested well within the subtribe Melampodiinae and thus should be considered a monophyletic complex of species, not a separate subtribe. The most parsimonious gene trees suggest that the genus Ichthyothere may be the sister taxon to the Espeletia complex and that the genus Smallanthus and a species of Rumfordia are likely among the complex's other closest living relatives. These data offer preliminary insights into the origins of this adaptive radiation and the broader phylogenetic context in which it occurred.     

Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): insights from plastid matK and nuclear ribosomal ITS sequences

We conducted phylogenetic analyses using two DNA sequence data sets derived from matK, the maturase-coding gene located in an intron of the plastid gene trnK, and the internal transcribed spacer region of 18S–26S nuclear ribosomal DNA to examine relationships in subtribe Aeridinae (Orchidaceae). Specifically, we investigated (1) phylogenetic relationships among genera in the subtribe, (2) the congruence between previous classifications of the subtribe and the phylogenetic relationships inferred from the molecular data, and (3) evolutionary trends of taxonomically important characters of the subtribe, such as pollinia, a spurred lip, and a column foot. In all, 75 species representing 62 genera in subtribe Aeridinae were examined. Our analyses provided the following insights: (1) monophyly of subtribe Aeridinae was tentatively supported in which 14 subclades reflecting phylogenetic relationships can be recognized, (2) results are inconsistent with previous classifications of the subtribe, and (3) repeated evolution of previously emphasized characters such as pollinia number and apertures, length of spur, and column foot was confirmed. It was found that the inconsistencies are mainly caused by homoplasy of these characters. At the genus level, Phalaenopsis, Cleisostoma, and Sarcochilus are shown to be non-monophyletic.     

A revised classification of subtribe Helianthinae (Asteraceae: Heliantheae) II. Derived lineages

Sequence data for internal transcribed spacer (ITS) and partial external transcribed spacer (ETS) regions were combined in a phylogenetic analysis with previously obtained plastid DNA restriction site data to provide a comprehensive molecular phylogenetic hypothesis for derived members of subtribe Helianthinae. Analyses of the two molecular datasets provided conflicting evidence on relationships among some groups, supporting the hypothesis that hybridization has played a significant role in the divergence of the subtribe. A revised generic‐level classification is presented that divides the approximately 350 species of the subtribe among 21 genera. The paraphyletic Viguiera is narrowed to embrace only the type species, V. dentata. Four newly described genera, Dendroviguiera, Gonzalezia, Heiseria and Sidneya, are composed of species formerly included in Viguiera. Aldama is expanded to include 118 species extending from southwestern North America and Mexico to South America. This requires 116 new combinations, including 58 that were recently transferred into Rhysolepis, which is a synonym of Aldama, based on molecular phylogenetic results. One species of Viguiera is transferred to Tithonia, and two combinations in Hymenostephium are validated. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 167 , 311–331.     

Variation of DNA amount in 47 populations of the subtribe Artemisiinae and related taxa (Asteraceae, Anthemideae): karyological, ecological, and systematic implications.

Genome size has been estimated by flow cytometry in 47 populations of 40 species of the tribe Anthemideae (Asteraceae), mainly from Artemisia and other genera of the subtribe Artemisiinae and related taxa. A range of 2C values from 3.54 to 21.22 pg was found. DNA amount per basic chromosome set ranged from 1.77 to 7.70 pg. First genome size estimates are provided for one subtribe, 10 genera, 32 species, and two subspecies. Nuclear DNA amount correlated well with some karyological, physiological and environmental characters, and has been demonstrated as a useful tool in the interpretation of evolutionary relationships within Artemisia and its close relatives.     

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.     

Secondary chemistry and ribosomal DNA data congruencies in Arnica (Asteraceae)

To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa ( Layia hieracioides and Madia sativa ), using phylogenetic inference and principal component analysis (PCA). Several groups supporting multiple accessions of the same species (of A. montana , A. longifolia , A. gracilis , and A. chamissonis ) are congruent between the phylogenetic trees based on nrDNA and STL data. Sesquiterpene lactone profiles were found to be highly consistent within multiple samples of A. montana and A. longifolia respectively . Moreover, sesquiterpene lactone data support subspecies classifications of A. chamissonis and A. parryi , with additional support from DNA sequence data and cytometric data. Morphology, STL data (PCA), cytometric data and DNA sequence data suggest a hybrid origin of one accession ( A. gracilis  ×  longifolia ). In A. gracilis , A. latifolia , and Layia hieracioides , previously not investigated for STLs, we found large amounts of xanthalongin derivatives. This is the first time STLs have been reported from subtribe Madiinae.
© The Willi Hennig Society 2009.     

FINS (forensically informative nucleotide sequencing): a procedure for identifying the animal origin of biological specimens.

The keys to identifying different species normally rely heavily on morphological characteristics. However, when an animal has been killed for food or sport, these markers are often destroyed or intentionally removed from the animal. This presents a problem for government agencies who are involved in determining the species origin of an animal or products derived from it in order to enforce conservation and/or health-related regulations. The problem is compounded if the meat of the animal has been processed in any way. We have developed a procedure called FINS (Forensically Informative Nucleotide Sequencing) that overcomes these problems. FINS has four components. First, methods have been developed that can isolate DNA from a wide range of biological samples including processed foods (e.g., canned, partially cooked, pickled, salted or smoked). Second, a specific segment of DNA is amplified using PCR. Third, the nucleotide sequence of the amplified segment of DNA is determined. Fourth, this nucleotide sequence is subjected to a phylogenetic analysis using a database, and the most closely related species is identified. FINS is a rapid, reliable and reproducible procedure that is based on established techniques. This procedure fills the need for an accurate method of determining the species identity of a specimen when this is not possible by conventional means.     

Kauriphanes n. gen., a new genus of braconid parasitoid wasp (Hymenoptera: Braconidae: Doryctinae) from New Zealand

A new genus belonging to the braconid wasp subfamily Doryctinae, Kauriphanes n. gen. (type species K. khalaimi n. sp.), is described from New Zealand. This genus is placed within the doryctine subtribe Caenophanina. The extent of this subtribe is discussed and the phylogenetic relationships of three of its genera were investigated using one mitochondrial and one nuclear DNA sequence markers. Similar to previous studies, the Bayesian analyses performed significantly support a clade with the included members of Caenophanina as a sister group of a clade with the examined species of Spathiini sensu stricto. The placement of the Caenophanini within Doryctini, however, is left pendant to further exhaustive phylogenetic studies. A key to genera and subgenera belonging to Caenophanina is given.     

Phylogeny of the Spiny African Daisies (Compositae, tribe Arctotideae, subtribe Gorteriinae) based on trnL-F, ndhF, and ITS sequence data

The tribe Arctotideae (African Daisies), of the flowering plant family Compositae (Asteraceae), is a diverse and interesting group with a primarily southern African distribution (ca. 13 genera, 215 species) and many species in the Cape Floristic Region. It is divided into two subtribes: Arctotidinae (ca. 5 genera, 85 species) and Gorteriinae (ca. 8 genera, 130 species). The monophyly of the genera within the subtribe Gorteriinae and their relationship to one another was investigated using 71 samples/212 sequences including 64/141 of which are newly reported from three phylogenetic markers, two from chloroplast DNA (trnL-F and ndhF) and one from the nuclear genome (ITS). The outgroup was composed of seven members from the sister subtribe. Results show the subtribe Gorteriinae to be divided into three monophyletic groups, the Gazania-Hirpicium-Gorteria group, the Didelta group, and the Berkheya-Cullumia group. Within these three groups are 13 sub-groups, one of which has sub-clades. The genus Berkheya Ehrh. is paraphyletic, falling into five different sub-groups. The two monotypic genera, Cuspidia and Heterorhachis are not nested within any of the Berkheya clades. Hirpicium and Cullumia each have most of their taxa in a monophyletic group, but they also have one or two taxa associated with other clades. Four of the five sub-groups of Berkheya have morphologically recognizable shared characters, such as habit and spines that have been recognized by past studies. However, the grouping of one species with Didelta is difficult to explain. Support for the major clades and most of the sub-groups is strong but the relationships among some of the terminal taxa are variable.     

Coding of insertion-deletion events of the chloroplastic intergene atp beta-rbcL for the phylogeny of the Valerianeae tribe (Valerianaceae)

A preliminary analysis of the sequence alignment of the chloroplast intergene atp beta-rbcL in tribe Valerianeae reveals that insertion-deletion evolutionary events ('indels'), combined with nucleotide substitutions, have occurred in large zones in some of the studied taxa. Due to the frequent occurrence and large size of indels within this tribe, intergene length varies from 531 to 788 base pairs within the studied species. This situation poses gap coding problems that we had to tackle before phylogenetic analysis. Four methods of gap coding were used: elimination of gapped sites ('complete omission'), 'missing data', 'fifth base' and Barriel's coding method, which translates indels into new multistate characters in the data matrix. After application of these four methods of data treatment, phylogenetic analyses (maximum parsimony) did not lead to very different results. Three robust clades emerged in each case, corresponding to the Centranthinae subtribe (genus Centranthus), the Fediinae subtribe (genera Fedia and Valerianella), and the American species of Valeriana. The theoretical basis and biological significance of these four methods are discussed in order to apply the best ones in future studies.     

Phylogenetic relationships and generic delimitation in subtribe Arctotidinae (Asteraceae: Arctotideae) inferred by DNA sequence data from ITS and five chloroplast regions

Asteraceae are the largest family in southern Africa. Elucidating its origins and radiation in the region requires well-supported species-level phylogenies of the lineages. This paper presents a phylogenetic framework for subtribe Arctotidinae, which have a southern and eastern African-Australian distribution centered in the winter-rainfall region of South Africa. DNA sequence data from five chloroplast fragments (ndhF, psbA-trnH, rps16, trnS-trnfM, and trnT-trnF) and the nuclear ITS region were analyzed separately and in combination using parsimony and Bayesian methods. The data sets comprised exemplars from 18 ingroup species, representing the five currently accepted genera, and four outgroup species from Gorteriinae. All analyses indicated Arctotis and Haplocarpha are polyphyletic as presently circumscribed. The Australian-endemic Cymbonotus lawsonianus was placed within a strongly supported clade also containing A. arctotoides from South Africa and H. schimperi from eastern Africa. Retention of Dymondia and resurrection of Landtia at generic level are strongly supported. The phylogenetic hypotheses indicate the subtribe might have originated in temperate southern or eastern Africa, or it was ancestrally widespread in southern Africa and has diversified vicariously. The derived placement of C. lawsonianus indicates long-distance dispersal from southern Africa to Australia occurred.     

Phylogenetic analysis of Silphium and subtribe Engelmanniinae (Asteraceae: Heliantheae) based on ITS and ETS sequence data

The phylogenetic relationships of Silphium and subtribe Engelmanniinae were examined using DNA sequence data. The internal transcribed spacer (ITS) region and the external transcribed spacer (ETS) region were sequenced for 39 specimens representing the six genera of subtribe Engelmanniinae (Berlandiera, Chrysogonum, Dugesia, Engelmannia, Lindheimera, and Silphium), plus five additional genera identified as closely related to the Engelmanniinae by chloroplast DNA restriction site analysis, and three outgroups. Phylogenetic analysis supported the monophyly of Silphium with Lindheimera as sister. Silphium can be divided into two sections based upon two well-supported clades that correspond to root type and growth form. These results also supported the expansion of subtribe Engelmanniinae to include Balsamorhiza, Borrichia, Rojasianthe, Vigethia, and Wyethia. We hypothesize that subtribe Engelmanniinae originated in Mesoamerica and later radiated to the United States. We suggest that the cypsela complex, which is present in Berlandiera, Chrysogonum, Engelmannia, and Lindheimera, arose only once and was subsequently lost in Silphium.     

直突摇蚊亚科部分属28S rDNA D1序列的分子系统发育关系(双翅目,摇蚊科)

利用28S rDNA D1部分基因序列对直突摇蚊亚科代表性属级阶元进行了分子系统学研究。测定了12个内群属和2种外群的28SrDNAD1片段,并结合GenBank中3个同亚科种类该基因的同源序列进行了分析。采用2种建树方法(距离邻近法NJ和最大俭约法MP)分析了直突摇蚊亚科内属级分类单元的分子系统发育关系。结果表明,滨海摇蚊属Clunio位于系统发育树的基部,与该属营海洋生活的特殊性一致。心突摇蚊属和真开氏摇蚊属互为姐妹群,流环足摇蚊属和刀突摇蚊属互为姐妹群,此结果与基于形态学的系统发育研究相结果一致。其它属间的系统发育关系因尚无前人研究而有待做进一步研究。本研究同时证明28S rDNA D1基因片段在分析摇蚊科昆虫属级及属内阶元关系上具有一定的指导意义。     

Phylogenetic analysis of Chloraeinae (Orchidaceae) based on plastid and nuclear DNA sequences

The phylogenetic relationships of subtribe Chloraeinae, a group of terrestrial orchids endemic to southern South America, have not been satisfactorily investigated. A previous molecular phylogenetic analysis based on plastid DNA supported the monophyly of Chloraeinae and Gavilea, but showed that Chloraea is non‐monophyletic and that the sole species of Bipinnula analysed is sister to Geoblasta. However, that analysis included only 18 of the 73 species belonging to this subtribe. Here, the phylogenetic relationships of Chloraeinae were assessed by analysing aproximately 7500 bp of nucleotide sequences from nuclear ribosomal internal transcribed spacer (ITS) and plastid DNA (rbcL, matK, trnL‐trnF, rpoB‐trnC) for 42 species representing all four currently accepted genera of Chloraeinae and appropriate outgroups. Nuclear and plastid data were analysed separately and in combination using two different methods, namely parsimony and Bayesian inference. Our analyses support the monophyly of Chloraeinae and their inclusion in an expanded concept of Cranichideae, but none of the genera of Chloraeinae that includes more than one species is monophyletic. Gavilea and Bipinnula are paraphyletic, with Chloraea chica nested in Gavilea and Geoblasta penicillata in Bipinnula. As currently delimited, Chloraea is polyphyletic. The taxonomic changes proposed recently are for the most part not justifiable on phylogenetic grounds, except for recognition of the monotypic genus Correorchis. The lack of resolution for the relationships among species of ‘core’Chloraea suggests a relatively recent diversification of this group. The current generic classification is in need or revision, but additional study is advisable before carrying out further taxonomic changes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 258–277.     

A phylogenetic analysis of the palm subtribe Oncospermatinae (Arecaceae) based on morphological characters

Subtribe Oncospermatinae (Arecaceae: Arecoideae: Areceae) is a diverse group of spiny Old World palms. The subtribe includesOncosperma, a widespread Asian genus of five species, along with seven monotypic genera, all endemic to the Seychelles and Mascarene Islands of the western Indian Ocean. A phylogenetic analysis was conducted in order to test the monophyly of subtribe Oncospermatinae with respect to other Old World genera of tribe Areceae. A matrix of 38 morphological characters was scored for 29 taxa, including 11 species of the Oncospermatinae. A single most parsimonious tree was found, resolving the subtribe as a polyphyletic group of two distinct clades. One clade containingAcanthophoenix, Deckenia, Oncosperma, andTectiphiala was placed as sister to a large group that includes members of subtribes Archontophoenicinae, Arecinae, Iguanurinae, and Ptychospermatinae. The other clade of Oncospermatinae, including the Seychelles endemic generaNephrosperma, Phoenicophorium, Roscheria, andVerschaffeltia, was resolved as sister to the Madagascar endemic subtribe Masoalinae, and may have arisen in the western Indian Ocean region.     

16S to 23S rDNA spacer fragment length polymorphism of Aeromonas sp

We analyzed polymorphism of the PCR-amplified 16S-23S rDNA spacer of Aeromonas species. A total of 69 isolates representing 18 DNA hybridization groups were used in this study. The analysis of PCR products of 16S-23S rDNA spacers revealed patterns consisting of two to eight DNA fragments. The fragment sizes ranged from 730 to 1050 bp. DNA patterns revealed a considerable genetic diversity between species and within a species. When a procedure to eliminate heteroduplex formation was performed, the number of bands was reduced to 2-5. Nevertheless the homoduplex ISR (intergenic spacer region) patterns obtained were not useful for species distinguishing.