Phylogenetic analysis of eastern Asian and eastern North American disjunct Lespedeza (Fabaceae) inferred from nuclear ribosomal ITS and plastid region sequences

Lespedeza (tribe Desmodieae, Fabaceae) follows a disjunct distribution in eastern Asia and eastern North America. Phylogenetic relationships among its species and related taxa were inferred from nuclear ribosomal internal transcribed spacer (ITS) and plastid sequences (trnH‐psbA, psbK‐psbI, trnK‐matK and rpoC1). We examined 35 species of Lespedeza, two of Kummerowia and one of Campylotropis, the sole constituents of the Lespedeza group. An analysis of these data revealed that the genus Campylotropis is sister to the other two genera. However, we were unable to resolve the relationships between Kummerowia and Lespedeza in the strict consensus trees of parsimony analyses based on plastid and combined DNA data. In the genus Lespedeza, the Old World subgenus Macrolespedeza is monophyletic, whereas the transcontinental subgenus Lespedeza is paraphyletic. Monophyly of eastern Asian species and of North American species is strongly supported. Although inconsistent with the traditional classification, this phylogenetic finding is consistent with seedling morphology. Three subgroups recognized in subgenus Macrolespedeza were unresolved in our phylogenetic trees. An incongruence length difference (ILD) test indicated that the two partitions (nuclear ITS and plastid sequences) were significantly incongruent, perhaps because of hybridization between species in Lespedeza. Most of the primary clades of tribe Desmodieae are Asian, implying that the relatively few New World ones, such as those in Lespedeza, are more recently derived from Asia. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164 , 221–235.     

Phylogenetic relationships ofPythium species based on ITS and 5.8S sequences of the ribosomal DNA

The sequences of ITS regions in 30 species and two groups of the genusPythium were resolved. In the phylogenetic trees, the species were generally divided into two clusters, referred to here as the F and S groups. The species in the two groups correspond in terms of their sporangial morphology, with the F group being filamentous/lobulate and the S group being spherical. Genetic divergence within the F group was lower than that within the S group. Other morphological characteristics such as oogonial structure and sexual nature appeared to be unrelated to the groupings in these trees. An alignment analysis revealed common sequences to all the species and arrangements specific to each F or S group. It was found that the ITS region was a good target in designing species-specific primers for the identification and detection ofPythium species. In the tree based on 5.8S rDNA sequences, oomycetes are distantly related to other fungi but separated from algae in Chromista.     

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.     

Phylogenetic analysis of the genus Hexachlamys (Myrtaceae) based on plastid and nuclear DNA sequences and their taxonomic implications

Myrtaceae are one of the most species‐rich families of flowering plants in the Neotropics. They include several complex genera and species; Hexachlamys is one of the complex genera. It has not been recognized as a distinct genus and has been included in Eugenia, based on morphological grounds. Therefore, molecular systematic studies may be useful to understand and to help to solve these relationships. Here, we performed a molecular phylogenetic analysis using plastid and nuclear data in order to check the inclusion of Hexachlamys in Eugenia. Plastid (accD, rpoB, rpoC1, trnH‐psbA) and nuclear (ITS2) sequence data were analysed using Bayesian and maximum parsimony methods. The trees constructed using ITS2 and trnH‐psbA were the best able to resolve the relationships between species and genera, revealing the non‐monophyly of Hexachlamys. The molecular phylogenetic analyses were in agreement with previous morphological revisions that have included Hexachlamys in Eugenia. These results reinforce the importance of uniting knowledge and strategies to understand better issues of delimitation of genera and species in groups of plants with taxonomic problems. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 532–543.     

基于 rDNA ITS 1序列探讨臂尾轮属轮虫的系统发生关系

本文通过对剪形臂尾轮虫、矩形臂尾轮虫、十指臂尾轮虫、红臂尾轮虫、角突臂尾轮虫、双棘臂尾轮虫、裂足臂尾轮虫和萼花臂尾轮虫等八种臂尾轮虫rDNAITS 1序列分析,并以西氏晶囊轮虫为外群,使用PAUP和贝叶斯软件分别构建臂尾轮属轮虫系统发生树( MP树、NJ树、ML树和贝叶斯树) ,以探讨臂尾轮属的系统发生关系,并解决其中的一些分类问题。结果表明:本研究所涉及的轮虫rDNAITS 1平均序列差异百分比较高,为29 %;海水和淡水臂尾轮虫被明显分为不同的进化枝;除双棘臂尾轮虫外,在淡水臂尾轮虫中具有三对前棘刺且营附着生活的种类与前棘刺少于三对且营浮游生活的种类聚在不同支系中,这与以形态特征为主所进行的系统发生研究结果基本一致;所有的系统树均支持将十指臂尾轮虫作为一个独立的支系分离出来,裂足臂尾轮虫应归入臂尾轮属。     

Phylogenetic relationships of coffee-tree species (Coffea L.) as inferred from ITS sequences of nuclear ribosomal DNA

 Phylogenetic relationships of Coffea species were estimated from the sequences of the internal transcribed spacer (ITS 2) region of nuclear ribosomal DNA. The ITS 2 region of 37 accessions belonging to 26 Coffea taxa and to three Psilanthus species was directly sequenced from polymerase chain reaction (PCR)-amplified DNA fragments. The level of variation was high enough to make the ITS 2 a useful tool for phylogenetic reconstruction. However, an unusual level of intraspecific variation was observed leading to some difficulty in interpreting rDNA sequence divergences. Sequences were analysed using Wagner parsimony as well as the neighbour-joining distance method. Coffea taxa were divided into several major groups which present a strong geographical correspondence (i.e. Madagascar, East Africa, Central Africa and West Africa). This organisation is well supported by cytogenetic evidence. On the other hand, the results were in contradiction with the present classification of coffee-tree taxa into two genera, namely Coffea and Psilanthus. Furthermore, additivity of parental rDNA types was not observed in the allotetraploid species C. arabica. Received: 25 July 1996 / Accepted: 18 October 1996     

Phylogeny of the subgenus Culicoides and related species in Italy, inferred from internal transcribed spacer 2 ribosomal DNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) include vectors for the economically important animal diseases, bluetongue (BT) and African horse sickness (AHS). In the Mediterranean Basin, these diseases are transmitted by four species of Culicoides: the first three belong in the subgenus Avaritia Fox and are Culicoides imicola Kieffer, Culicoides obsoletus (Meigen) and Culicoides scoticus Downes and Kettle; the fourth is Culicoides pulicaris (Linnaeus) in the subgenus Culicoides Latreille. In the Palaearctic Region, this subgenus (usually referred to as the C. pulicaris group) now includes a loose miscellany of some 50 taxa. The lack of clarity surrounding its taxonomy stimulated the present morphological and molecular study of 11 species collected in Italy. Phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer 2 (ITS2) sequence variation demonstrated a high degree of divergence. These results, combined with those from a parallel morphological study, disclosed: (1) that some previously described taxa should be resurrected from synonymy; (2) that there are new species to be described; (3) that the subgenus Culicoides (as currently employed) is a polyphyletic assemblage of four lineages - the subgenus Culicoides sensu stricto, the subgenus Silvicola Mirzaeva and Isaev, the subgenus Hoffmania Fox and the hitherto unrecognized Fagineus species complex. Each is discussed briefly (but not defined) and its constituent Palaearctic taxa listed. Strong congruence between morphological and molecular data holds promise for resolving many of the difficult taxonomic issues plaguing the accurate identification of vector Culicoides around the world.     

Phylogenetic status and matrilineal structure of the biting midge, Culicoides imicola, in Portugal, Rhodes and Israel

The biting midge Culicoides imicola Kieffer (Diptera: Ceratopogonidae) is the most important Old World vector of African horse sickness (AHS) and bluetongue (BT). Recent increases of BT incidence in the Mediterranean basin are attributed to its increased abundance and distribution. The phylogenetic status and genetic structure of C. imicola in this region are unknown, despite the importance of these aspects for BT epidemiology in the North American BT vector. In this study, analyses of partial mitochondrial cytochrome oxidase subunit I gene (COI) sequences were used to infer phylogenetic relationships among 50 C. imicola from Portugal, Rhodes, Israel, and South Africa and four other species of the Imicola Complex from southern Africa, and to estimate levels of matrilineal subdivision in C. imicola between Portugal and Israel. Eleven haplotypes were detected in C. imicola, and these formed one well-supported clade in maximum likelihood and Bayesian trees implying that the C. imicola samples comprise one phylogenetic species. Molecular variance was distributed mainly between Portugal and Israel, with no haplotypes shared between these countries, suggesting that female-mediated gene flow at this scale has been either limited or non-existent. Our results provide phylogenetic evidence that C. imicola in the study areas are potentially competent AHS and BT vectors. The geographical structure of the C. imicola COI haplotypes was concordant with that of BT virus serotypes in recent BT outbreaks in the Mediterranean basin, suggesting that population subdivision in its vector can impose spatial constraints on BT virus transmission.     

Phylogenetic analysis of the mitochondrial cytochrome oxidase subunit I gene of five species of the Culicoides imicola species complex

The phylogenetic status of members of the Culicoides imicola Kieffer (Diptera: Ceratopogonidae) species complex of haematophagous midges is unknown, and simple means to identify the members using all life stages are unavailable. In this study, the status of three confirmed (C. imicola s.s., C. bolitinos Meiswinkel and C. loxodontis Meiswinkel) and two provisional (C. tuttifrutti Meiswinkel and C. kwagga Meiswinkel) members of the complex from South Africa was assessed using phylogenetic analysis of partial DNA and amino acid sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The four or five individuals of each species analysed contained one or two haplotypes each. Interspecific divergence was significant and characterized by strong A <--> T transversion bias. Phylogenetic trees constructed using neighbour-joining, parsimony and maximum likelihood showed each species to be distinct. Combinations of sites for two restriction enzymes in the COI sequences were species-specific and could form the basis of a diagnostic PCR assay.     

Phylogenetic relationships within Korthalsella (Viscaceae) based on nuclear ITS and plastid trnL-F sequence

The nuclear encoded internal transcribed spacer (ITS) region and the plastid encoded trnL-F region were sequenced for 25 populations of Korthalsella, a genus of reduced, monoecious, Old World misletoes. The molecular study confirms the hypothesis that branch shape and cladotaxy (the arrangement of branches with respect to their parent axis) are unreliable indicators of relationship in the genus and demonstrates that many of the taxa previously recognized are not monophyletic. Both gene regions identify three major subgroups within the genus and find lower level relationships within these subgroups highly correlated with geographic distance. An analysis based upon 18S and rbcL sequences identifies Ginalloa as the sister group to Korthalsella, which together with the branching order within the genus, indicates that Korthalsella originated in Papuasia and aids in elucidating evolution of the peculiar inflorescence structure. There are problems associated with species delimitation when evolutionary units are more restricted than morphological lineages, and justification is offered for recognizing only morphologically diagnosable monophyletic lineages as species. Varying substitution rates and differing modes of inheritance in ITS and trnL-F result in complementary utility of the two regions for elucidating infrageneric relationships in Korthalsella.     

Cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, based on the DNA sequences of nuclear ribosomal internal transcribed spacer 1

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otae-related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae-related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1-homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1-homologous group of M. gypseum complex are more diverse. This ITS1-based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time-saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non-culturable state.     

Phylogenetic relationships between European and Chinese truffles based on parsimony and distance analysis of ITS sequences

Phylogenetic relationships among truffle species from Europe and China were investigated through parsimony analysis of the ITS sequences. Three major clades were obtained among the species analysed. The so-called white truffles appeared polyphyletic since Tuber magnatum was grouped with brown truffles and not with the other white species (T. maculatum, T. borchii, T. dryophilum, T. puberulum). The black truffles investigated in this study, T. brumale, T. melanosporum, T. indicum and T. himalayense, were grouped in an independent clade. The Périgord black truffle T. melanosporum and the Chinese black truffles T. indicum and T. himalayense, were very closely related. The delimitation of these species was estimated by a distance analysis on several isolates collected from different geographic areas. In spite of intraspecific variations of the internal transcribed spacers (ITS) sequences, T. melanosporum and the Chinese black truffles can be unambiguously attributed to distinct taxa.     

Delineation of the European Armillaria species based on the sequences of the internal transcribed spacer (ITS) of ribosomal DNA

Variation within the internal transcribed spacer (ITS) of the ribosomal RNA gene of 15 isolates representing seven European Armillaria species, was examined by sequencing of the PCR-amplified products. The analysis of an 744-bp region showed that the 5.8S gene appeared to be highly conserved in the 15 isolates and in other Basidiomycetes and Ascomycetes, whereas ITS1 and especially ITS2 spacers exhibited polymorphisms due to base substitutions, insertions or deletions of up to eight nucleotides. An initial dendrogram for the full sequence was drawn using cluster analysis (UPGMA), and a tree was constructed using the maximum parsimony method. Both methods indicated that the isolates could be divided into four major groups. One group, consisting of A. ectypa , was distinct from all the other species. Examination of the other groups indicated that A. tabescens and A. mellea were in a separated cluster, with a significant variation between the two isolates of the latter species. A. gallica and A. cepistipes constituted another closely related group distinguishable from A. ostoyae and A. borealis , these latter two species exhibiting the highest similarity. The results are consistent with, and discussed in regard to, the relationships estimated previously by pairing tests, morphological and physiological comparisons, as well as by restriction fragment length polymorphism of the rDNA.     

Spatial distribution of Culicoides species in Portugal in relation to the transmission of African horse sickness and bluetongue viruses

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midge vectors was carried out at 87 sites within a 50 x 50 km grid distributed across Portugal, using light trap collections at the time of peak midge abundance. Culicoides imicola (Kieffer) made up 66% of the 55 937 Culicoides in these summer collections. It was highly abundant in the central eastern portion of Portugal, between 37 degrees 5' N and 41 degrees 5' N, and in a band across to the Lisbon peninsula (at around 38 degrees 5' N). Of all the complexes, its distribution was most consistent with that of previous outbreaks of Culicoides-borne disease, suggesting that it may remain the major vector in Portugal. Its distribution was also broadly consistent with that predicted by a recent climate-driven model validating the use of remote sensing datasets for modelling of Culicoides distribution. Adult C. imicola were found to have overwintered at 12 of 20 sites re-surveyed in winter but it did so in very low numbers. Culicoides obsoletus (Meigen) and Culicoides pulicaris (Linnaeus) complex midges were widespread despite their low summer abundance. The observed coincidence of high abundances of C. imicola and high abundances of C. pulicaris in summer lead us to suggest that C. imicola could bring African horse sickness virus or bluetongue virus into contact with C. pulicaris and the latter complex, together with C. obsoletus, could then transmit these viruses across much wider areas of Europe. The fact that adult C. pulicaris are present in high abundances in winter may provide a mechanism by which these viruses can overwinter in these areas.     

Tribal delimitation and phylogenetic relationships of Loteae and Coronilleae (Faboideae: Fabaceae) with special reference to Lotus: evidence from nuclear ribosomal ITS sequences

The temperate herbaceous tribes Loteae and Coronilleae have traditionally been regarded as taxonomically distinct entities. More recent morphological assessments, however, have challenged this view and suggest combining the two tribes under Loteae. Two key features used to distinguish the Coronilleae from Loteae include jointed fruits and branched root nodules. We evaluate the taxonomic utility of these characters using information derived from phylogenetic analyses of the internal transcribed spacers ITS1 + 2, and the intervening 5.8S region of nuclear ribosomal DNA. Results from this study show that neither the Loteae nor Coronilleae form individual monophyletic groups, and that key fruit and root nodule characters used to distinguish the Coronilleae are homoplastic. Given these data, we support the recognition of a single tribe, Loteae. We also find that Lotus, the largest and most morphologically complex genus in either tribe, is not monophyletic. Rather, it consists of two geographically distinct lineages, Old and New World, each of which are more closely related to other Loteae genera: Old World Lotus are more closely related to Old World Anthyllis, while New World Lotus show closer affinities to Old World Coronilla. These data also have important implications for the biogeography of New World Lotus: equally most parsimonious reconstructions suggest a complex scenario of intercontinental dispersals that involve not only Old World Lotus but Coronilla as well.     

Culicoides species as potential vectors of African horse sickness virus in the southern regions of South Africa

African horse sickness (AHS), a disease of equids caused by the AHS virus, is of major concern in South Africa. With mortality reaching up to 95% in susceptible horses and the apparent reoccurrence of cases in regions deemed non‐endemic, most particularly the Eastern Cape, epidemiological research into factors contributing to the increase in the range of this economically important virus became imperative. The vectors, Culicoides (Diptera: Ceratopogonidae), are considered unable to proliferate during the unfavourable climatic conditions experienced in winter in the province, although the annual occurrence of AHS suggests that the virus has become established and that vector activity continues throughout the year. Surveillance of Culicoides within the province is sparse and little was known of the diversity of vector species or the abundance of known vectors, Culicoides imicola and Culicoides bolitinos. Surveillance was performed using light trapping methods at selected sites with varying equid species over two winter and two outbreak seasons, aiming to determine diversity, abundance and vector epidemiology of Culicoides within the province. The research provided an updated checklist of Culicoides species within the Eastern Cape, contributing to an increase in the knowledge of AHS vector epidemiology, as well as prevention and control in southern Africa.     

Phylogeny of Rubus (rosaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences

We used nuclear ribosomal DNA internal transcribed spacer region (ITS 1 - 5.8S - ITS 2; ITS) sequences to generate the first phylogeny of Rubus based on a large, molecular data set. We sampled 57 taxa including 20 species of subgenus Rubus (blackberries), one to seven species from each of the remaining 11 subgenera, and the monotypic and closely related Dalibarda. In Rubus, ITS sequences are most informative among subgenera, and variability is low between closely related species. Parsimony analysis indicates that Rubus plus Dalibarda form a strongly supported clade, and D. repens may nest within Rubus. Of the subgenera with more than one species sampled, only subgenus Orobatus appears monophyletic. Three large clades are strongly supported: one contains all sampled species of nine of the 12 subgenera; another includes extreme Southern Hemisphere species of subgenera Comaropsis, Dalibarda, and Lampobatus; and a third clade consists of subgenus Rubus plus R. alpinus of subgenus Lampobatus. Rubus ursinus appears to be a hybrid between a close relative of R. macraei (subgenus Idaeobatus, raspberries) and an unidentified subgenus Rubus species. ITS sequences are generally consistent with biogeography and ploidy, but traditionally important morphological characters, such as stem armature and leaf type, appear to have limited phylogenetic value in Rubus.     

Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Previously reported virus recovery rates from Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel (Diptera, Ceratopogonidae) orally infected with vaccine strain of African horse sickness virus serotype 7 (AHSV-7) were compared with results obtained from concurrently conducted oral infections with five recent AHSV-7 isolates from naturally infected horses from various localities in South Africa. Culicoides were fed sheep bloods spiked with 10(7.6) TCID(50)/mL of a live-attenuated vaccine strain AHSV-7, and with five field isolates in which virus titre in the bloodmeals ranged from 10(7.1) to 10(8.2) TCID(50)/mL). After an extrinsic incubation of 10 days at 23.5 degrees C, virus recovery rates were significantly higher in C. imicola (13.3%) and C. bolitinos (4.2%) infected with the live-attenuated virus than in midges infected with any of the field isolates. The virus recovery rates for the latter groups ranged from 0% to 9.5% for C. imicola and from 0% to 1.5% for C. bolitinos. The C. imicola population at Onderstepoort was significantly more susceptible to infection with AHSV-7 isolated at Onderstepoort than to the virus strains isolated from other localities. Results of this study suggest that tissue culture attenuation of AHSV-7 does not reduce its ability to orally infect competent Culicoides species and may even lead to enhanced replication in the vector. Furthermore, oral susceptibility in a midge population appears to vary for geographically distinct isolates of AHSV-7.