Chloroplast-Expressed Glutamine Synthetase (ncpGS): Potential Utility for Phylogenetic Studies with an Example from Oxalis (Oxalidaceae)

Chloroplast-expressed glutamine synthetase (ncpGS), a nuclear-encoded gene containing several introns, is introduced as a tool for phylogenetic studies at lower taxonomic levels. This gene is a member of a multigene family, but it diverged long ago from the cytosolic-expressed members of the family and appears to be single copy in the majority of taxa examined to date. The conservation of both coding sequence and position of introns has allowed the design of primers for use in a broad range of dicot taxa to amplify and sequence a region of ncpGS that contains four introns. The utility of this region in phylogenetic studies of congeneric species is illustrated by an example using eight Oxalis species. The four introns in these taxa are typical in size (76 to 136 bp), base composition (high T content), and structure (e.g., sequence of splice sites and putative branch points) for plant internal introns. Levels of variation among these ncpGS sequences compare favorably with those of the internal transcribed spacer of nuclear ribosomal DNA (ITS) from the same taxa, and results of phylogenetic analysis of ncpGS data are generally congruent with previous results using ITS.     

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.     

Molecular phylogeny of the genus Stereocaulon (Stereocaulaceae, lichenized ascomycetes)

In the present study phylogenetic relationships of the genus Stereocaulon (lichenized ascomycetes) were examined using DNA sequences from the ITS1–5.8 S–ITS2 rDNA gene cluster and from the protein-coding β-tubulin gene. In addition to the fruticose species traditionally classified in Stereocaulon, representatives of the crustose species that have recently been transferred to the genus were included. Muhria, a monotypic genus that is morphologically similar to Stereocaulon, differing only in apothecia ontogeny, was also incorporated. The analyses included 101 specimens from the ingroup representing 49 taxa. Sequences from both DNA regions were analysed simultaneously using direct optimization under the parsimony optimality criterion. The results support the inclusion of the crustose species and Muhria in Stereocaulon, while the current infrageneric classification is not supported. As Muhria is securely nested within Stereocaulon the new combination Stereocaulon urceolatum comb. nov. (syn. Muhria urceolata) is made. Further, species concepts need to be re-examined, as some species do not appear as monophyletic entities in the phylogeny.     

南美蟛蜞菊霜霉病病原鉴定及系统发育分析

[目的]为从天敌病原生物方面探索外来入侵植物南美蟛蜞菊的生物防治,对新发现的南美蟛蜞菊霜霉病进行病原鉴定和系统发育分析。[方法]在广东省广州市对南美蟛蜞菊霜霉病的发生及危害情况进行调查,并通过病害症状识别、病原显微形态记录与比较、病原菌及其近似种ITS序列结构比较、LSU序列和ITS序列系统发育分析,对南美蟛蜞菊霜霉病病原进行鉴定和系统发育分析。[结果]南美蟛蜞菊霜霉病在广州零星发生,但该病害在华南农业大学校园内发生较严重,发病率达50%~70%,病情指数为30~35。经鉴定,其病原菌为南美蟛蜞菊单轴霉,是国内一新记录种。基于病原菌LSU序列和ITS序列的系统发育分析显示,侵染菊科植物的单轴霉属菌种聚在一个分枝,亲缘关系密切,与侵染其他不同科寄主植物的单轴霉亲缘关系较远。ITS序列结构比较显示,寄生于菊科向日葵族植物的单轴霉属菌种的ITS2区包含多个重复序列,不同菌种间的ITS2区重复序列相似度不同,说明侵染菊科向日葵簇植物的单轴霉属菌物可细分成多个种,而不是只有向日葵单轴霉。[结论]广州发生的南美蟛蜞菊霜霉病是该寄主上首次正式报道的病害,鉴定的病原菌也是国内一新记录种;寄生在菊科植物上的单轴霉属种类不尽相同,但亲缘关系紧密。     

Chloroplast-expressed glutamine synthetase (ncpGS): potential utility for phylogenetic studies with an example from Oxalis (Oxalidaceae).

Chloroplast-expressed glutamine synthetase (ncpGS), a nuclear-encoded gene containing several introns, is introduced as a tool for phylogenetic studies at lower taxonomic levels. This gene is a member of a multigene family, but it diverged long ago from the cytosolic-expressed members of the family and appears to be single copy in the majority of taxa examined to date. The conservation of both coding sequence and position of introns has allowed the design of primers for use in a broad range of dicot taxa to amplify and sequence a region of ncpGS that contains four introns. The utility of this region in phylogenetic studies of congeneric species is illustrated by an example using eight Oxalis species. The four introns in these taxa are typical in size (76 to 136 bp), base composition (high T content), and structure (e.g., sequence of splice sites and putative branch points) for plant internal introns. Levels of variation among these ncpGS sequences compare favorably with those of the internal transcribed spacer of nuclear ribosomal DNA (ITS) from the same taxa, and results of phylogenetic analysis of ncpGS data are generally congruent with previous results using ITS.     

Evolution of rDNA ITS1 and ITS2 sequences and RNA secondary structures within members of the fungal genera Grosmannia and Leptographium

The two internal transcribed spacers (ITS) of the nuclear ribosomal (r) DNA tandem repeat were examined in ophiostomatoid fungi belonging to the genera Grosmannia and Leptographium and closely-related taxa. Although the DNA sequence of the ITS region evolves rapidly, core features of the RNA secondary structure of the ITS1 and ITS2 segments are conserved. The results demonstrate that structural conservation of GC-rich helical regions is facilitated primarily through compensatory base changes (CBCs), hemi-CBCs, and compensating insertions/deletions (indels), although slippage of the RNA strand is potentially an additional mechanism for maintaining basepairing interactions. The major conclusion of the structural analysis of both ITS segments is that two factors appear to be involved in limiting the type of changes observed: a high GC bias for both ITS1 and ITS2 and structural constraints at the RNA level.     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification

The closely related species Rhipicephalus appendiculatus and R. zambeziensis co-occur in a wide area in Zambia. In this area, specimens of both species have been collected on the same individual host at the same time. In addition, specimens that are morphologically intermediate between R. appendiculatus and R. zambeziensis have been found in this area. These observations cast some doubt on the species status of R. appendiculatus and R. zambeziensis. Because the two taxa have varied influences on the epidemiology of East Coast fever (ECF), a cattle disease for which they act as vectors, it is essential that their taxonomic status is clarified and their identification is accurate. Therefore a phylogenetic analysis of nucleotide sequences of the ITS2 and a fragment of the mitochondrial 12S rDNA was performed to reassess the specific status of both taxa. This revealed two well supported clades coinciding with R. appendiculatus and R. zambeziensis as two separate species. In order to facilitate species identification a PCR-RFLP diagnostic assay was developed based on BauI digestion of the ITS2 gene. This assay produced clear diagnostic banding patterns for the two species and is applicable over a wide range of both species’ distribution. Nucleotide sequence data reported in this paper are available in the GenBank™ under the accession numbers DQ849203–DQ849274.     

Evolution of nuclear rDNA its sequences in the Cladophora albida/sericea clade (Chlorophyta)

Ribosomal DNA ITS sequences were compared among 13 different species and biogeographic isolates from the monophyletic albida/sericea clade in the green algal genus Cladophora. Six distinct ITS sequence types were found, characterized by multiple insertions and deletions and high levels of nucleotide substitution. Conserved domains within the ITS regions indicate the presence of ITS secondary structure. Low transition/transversion ratios among the six types and nearly symmetrical tree-length frequency distributions indicate some saturation, and low phylogenetic signal. Although branching order among five of the six ITS sequence types could not be resolved, estimates of ITS sequence divergence as compared with 18S divergence in a subset of the taxa suggests that the origin of the different ITS types is probably in the mid-Miocene (12 Ma ago) but that biogeographic isolates within a single ITS type (including both Pacific and Atlantic representatives) have probably dispersed on a time scale of thousands rather than millions of years.Correspondence to: J.J. Olsen     

Comparative Analysis of Phylogenetic Relationships of Grain Amaranths and Their Wild Relatives (Amaranthus; Amaranthaceae) Using Internal Transcribed Spacer, Amplified Fragment Length Polymorphism, and Double-Primer Fluorescent Intersimple Sequence Repeat Markers

The most economically important group of species in the genus Amaranthus is the A. hybridus species complex, including three cultivated grain amaranths, A. cruentus, A. caudatus, and A. hypochondriacus, and their putative wild progenitors, A. hybridus, A. quitensis, and A. powellii. Taxonomic confusion exists among these closely related taxa. Internal transcribed spacer (ITS) of nuclear ribosomal DNA, amplified fragment length polymorphism (AFLP), and double-primer fluorescent intersimple sequence repeat (ISSR) were employed to reexamine the taxonomic status and phylogenetic relationships of grain amaranths and their wild relatives. Low ITS divergence in these taxa resulted in poorly resolved phylogeny. However, extensive polymorphisms exist at AFLP and ISSR loci both within and among species. In phylogenetic trees based on either AFLP or ISSR or the combined data sets, nearly all intraspecific accessions can be placed in their corresponding species clades, indicating that these taxa are well-separated species. The AFLP trees share many features in common with the ISSR trees, both showing a close relationship between A. caudatus and A. quitensis, placing A. hybridus in the same clade as all grain amaranths, and indicating that A. powellii is the most divergent taxon in the A. hybridus species complex. This study has demonstrated that both AFLP and double-primer fluorescent ISSR have a great potential for generating a large number of informative characters for phylogenetic analysis of closely related species, especially when ITS diversity is insufficient.     

Revision of some Thecaphora species (Ustilaginomycotina) on Caryophyllaceae

Specimens of Thecaphora saponariae s. lat. from several caryophyllacean host plants belonging to the genera Cerastium, Dianthus, Petrorhagia, Saponaria, Silene, and Stellaria were studied by means of both LM, SEM, and molecular phylogenetic analyses using ITS and LSU rDNA sequences. The data show that T. saponariae s. lat. is not uniform but represents several taxa. Molecular phylogenetic analyses, correlated with morphology of the sori, spore balls, and spores, permitted the recognition of five species. Two new species, Thecaphora italica and T. cerastii are described, and two new combinations, T. alsinearum and T. melandrii are proposed. The anamorph of Thecaphora saponariae is reported for the first time. A lectotype is designated for Sorosporium silenes-inflatae. Evolutionary aspects are discussed.     

基于低拷贝核基因GBBSI的禺毛茛复合体系统进化研究

禺毛茛多倍体复合体及其近缘种系统进化关系复杂,杂交与多倍化现象同时存在。该复合体内高倍性植物的形成及扩散过程仍需进一步研究。首次克隆了毛茛属植物低拷贝核基因颗粒结合型淀粉合成酶 I (GBBSI )基因,并利用其构建禺毛茛多倍体复合体及其近缘种的系统进化树和网状进化关系,进而证明其适合于研究毛茛属植物种下系统发育研究。结果表明：匍枝毛茛与多倍体复合体关系密切,参与了该多倍体复合体的起源和进化;禺毛茛起源于茴茴蒜和卷喙毛茛,扬子毛茛起源于茴茴蒜和匍枝毛茛;在该类群中茴茴蒜是个关键种,它在多倍体复合体中可能起着枢纽基因组的重要作用。     

Molecular phylogenetic analyses based on the nuclear rRNA genes and the intron–exon structures of the nuSSU rRNA gene in Dictyocatenulata alba (anamorphic Ascomycota)

Molecular phylogenies inferred from the nuclear small subunit rRNA gene (nuSSU), nuclear large subunit rRNA gene D1/D2 region (nuLSU), and ITS-5.8S rRNA gene (ITS) indicated that five cultures of the lichenized hyphomycete Dictyocatenulata alba isolated from Japan form a monophyletic clade with high bootstrap support, and a close relationship to the Ostropomycetidae (Lecanoromycetes, Pezizomycotina, Ascomycota). Insertion sequences were found in the nuSSU of all isolates [e.g., nine insertions in the strain JCM 5358 (Japan Collection of Microorganisms)], some of which were group I introns. Five new insertion positions were found among the D. alba isolates. Using BLAST, none of the insertion sequences of D. alba were closely related to those of fungi or other organisms deposited in public DNA databases. Insertion positions were similar in some isolates, and two positions were common to all isolates. Although all phylogenetic analyses based on nuSSU, nuLSU, and ITS revealed the monophyly of D. alba, the isolates were divided into two (in the nuSSU tree) or three (in the nuLSU and ITS trees) groups. Based on the phylogenetic analyses and the intron–exon structures, the five isolates identified as D. alba belong to three cryptic species and therefore D. alba should be considered a species complex. The very slow-growing, tough agar colonies of the isolates, the occurrence of the species on both slightly lichenized and nonlichenized surfaces of trees, or pebbles (rarely on soil), suggest that the members of the D. alba complex may be lichenized. The photobiont was not clearly identified in this study.     

Exon junction sequences as cryptic splice sites: implications for intron origin

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.     

基于分子证据确认秦岭藤属与驼峰藤属(夹竹桃科)的系统位置

秦岭藤属(Biondia Schltr.)是中国特有属,约13种,驼峰藤属(Merrillanthus Chun&Tsiang)是仅在我国与柬埔寨分布的单种属,目前两个属均被归并到白前属(Vincetoxicum Wolf),由于取样和系统发育分析尚存一些欠缺,因此其系统位置和归属尚需进一步确认。该文使用2个核糖体基因序列片段(ITS、ETS)、5个叶绿体基因序列片段(psbA-trnH、trnG、trnL、trnL-F、trnT-L),以及二者的合并数据,重建娃儿藤亚族(Tylophorinae)(包含属的模式秦岭藤[B.chinensis Schltr.=V.shaanxiense(Schltr.) Meve&Liede]与驼峰藤[M.hainanensis Chun&Tsiang=V.hainanense(Chun&Tsiang) Meve, H. H. Kong&Liede])的系统发育树。结果表明：秦岭藤属与驼峰藤属均包含在白前属中,秦岭藤与青龙藤[B.henryi(Warb.) Tsiang&Li=V.henryi(Warb....     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

Molecular characterization of strawberry pathogen Gnomonia fragariae and its genetic relatedness to other Gnomonia species and members of Diaporthales

Gnomonia fragariae is a poorly studied ascomycete belonging to Diaporthales. Originally G. fragariae was considered a saprophyte occurring on dead tissues of strawberry plants. Recently this fungus was found in Latvia and Sweden, and it was proven to be the cause of severe root rot and petiole blight of strawberry. Thirteen isolates of this pathogen and several other Gnomonia species occurring on rosaceous hosts were characterized by molecular analysis using nucleotide sequences of partial LSU rRNA gene and the total ITS region. The homologous regions from relevant diaporthalean taxa available in the GenBank were also included and compared with the taxa sequenced in this study. Phylogenetic analyses revealed that G. fragariae, G. rubi, and Gnomonia sp. (CBS 850.79) were genetically different from G. gnomon, the type species of the genus, and other members of Gnomoniaceae. The analyses showed that G. fragariae and Hapalocystis were genetically very closely related, forming a phylogenetic clade, which is possibly presenting a new family in the Diaporthales. Morphological comparisons of the Gnomonia species on the basis of commonly used criteria for the taxonomy of Diaporthales, so far did not reveal any evident features for the polyphyletic status of Gnomonia.