Evolution in Aeschynanthus (Gesneriaceae) inferred from ITS sequences

 Aeschynanthus Jack, an epiphytic genus with c.160 species, is widespread in SE Asia. We selected 50 species for ITS nrDNA sequencing, to include all biogeographic areas and all infrageneric groupings, which are currently based on seed morphology. Some species were sequenced directly from PCR product; others cloned because of ITS length polymorphisms. The clone sequences were analysed individually and combined in an elision matrix. Results extend earlier findings that Aeschynanthus is divided into two clades, one occurring primarily in mainland SE Asia and the other in Malesia. This pattern is interpreted as indicating an ancient vicariance event followed by dispersal and plate fusion. Clade I has straight or clockwise spiral orientation of the testa cells and clade II anticlockwise spiral orientation. In clade I some species of section Microtrichium form a basal group with other sections being polyphyletic or paraphyletic. In clade II the monophyletic section Aeschynanthus is nested within the paraphyletic basal Microtrichium. Received February 8, 2001 Accepted June 8, 2001     

Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences

Phylogenetic relationships of eight species of Saintpaulia H. Wendl., 19 species of Streptocarpus Lindl. (representing all major growth forms within the genus), and two outgroups (Haberlea rhodopensis Friv., Chirita spadiciformis W. T. Wang) were examined using comparative nucleotide sequences from the two internal transcribed spacers (ITS) of nuclear ribosomal DNA. The length of the ITS 1 region ranged from 228 to 249 base pairs (bp) and the ITS 2 region from 196 to 245 bp. Pairwise sequence divergence across both spacers for ingroup and outgroup species ranged from 0 to 29%. Streptocarpus is not monophyletic, and Saintpaulia is nested within Streptocarpus subgenus Streptocarpella. Streptocarpus subgenus Streptocarpus is monophyletic. The ITS sequence data demonstrate that the unifoliate Streptocarpus species form a clade, and are also characterized by a unique 47-bp deletion in ITS 2. The results strongly support the monophyly of (1) Saintpaulia, and (2) Saintpaulia plus the African members of the subgenus Streptocarpella of Streptocarpus. The data suggest the evolution of Saintpaulia from Streptocarpus subgenus Streptocarpella. The differences in flower and vegetative characters are probably due to ecological adaptation leading to a relatively rapid radiation of Saintpaulia.     

Evolution of Mexican Bursera (Burseraceae) inferred from ITS,ETS, and 5S nuclear ribosomal DNA sequences

I reconstructed a phylogeny of 66 species and varieties of Bursera and 9 outgroup species using sequences of the internal transcribed spacer region (ITS), the 5S non-transcribed region (5S-NTS), and the external transcribed region (ETS) of nuclear ribosomal DNA. This study extends a previously proposed parsimony-based phylogenetic study that used the ITS sequences of 57 Bursera species and five outgroups. Parsimony and maximum likelihood methods were used to infer the phylogeny in this new study. Analyses of the combined data sets largely confirmed the phylogenetic relationships proposed by the previous molecular study but generated a considerably more robust topology. The new phylogenies corroborate the monophyly of the genus, and its division into the two monophyletic subgenera or sections, Bursera and Bullockia. The current analyses also identify four main groups of species in section Bursera, and two in section Bullockia, confirming some of the previously proposed groups based on fruit, flower, and leaf morphology. One previously problematic species B. sarcopoda, which has sometimes been placed in Commiphora, is shown to belong in Bursera. Another controversial species, Commiphora leptophloeos, which was thought to belong to Bursera, falls within Commiphora.     

Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

The genus Alyssum consists of about 195 species native to Europe, Asia and northern Africa. All species were assigned to six sections. Previous molecular phylogeny studies indicate that Alyssum is polyphyletic. However, the divergence time and dispersal of the genus are not well studied. In this study, the phylogenetic relationships within the genus Alyssum were studied with nrDNA ITS sequences obtained from five sections. The divergence time was estimated by fossil calibration and the biogeography was examined by spread analysis. The phylogeny indicated two main lineages: lineage 1 includes the section of Alyssum, Gamosepalum and Psilonema; lineage 2 includes the section of Odontarrhena, Meniocus and Clypeola. The phylogenetic relationship was not congruent with the previous sectional classifications. The age of Alyssum was dated to the upper Miocene. Molecular data suggested the diversification of Alyssum in Mediterranean areas and wide-ranging distribution such as North Africa, eastward into Central Asia and immigration into North America. Climatic aridification and arid/semiarid areas established in the Pliocene/Pleistocene could have provided favourable conditions for the migration and diversification of Alyssum.     

Phylogenetic relationships in the Lejeuneaceae (Hepaticae) inferred using ITS sequences of nuclear ribosomal DNA

Sequences of the ITS1–5.8S–ITS2 region of nuclear ribosomal DNA were generated for 12 species from 9 genera of Lejeuneaceae and a single species of Jubulaceae (outgroup). The taxon sampling of Lejeuneaceae included representatives of the two widely recognized subfamilies, Lejeuneoideae and Ptychanthoideae. The molecular dataset was analysed independently and in combination with a morphological dataset. The nrITS dataset and the combined dataset resulted in identical topologies. The genus Bryopteris, sometimes treated as a separate family Bryopteridaceae, is nested within the Lejeuneaceae subfamily Ptychanthoideae. Lejeuneaceae subfamily Lejeuneoideae proved to be paraphyletic with the tribe Lejeuneeae sister to Ptychanthoideae, albeit without significant bootstrap support. The tribes Brachiolejeuneeae and Cheilolejeuneeae of Lejeuneoideae, established recently based on morphological evidence, are well supported in bootstrap analyses both of the ITS and the combined molecular–morphological datasets. The results support classifications of Lejeuneaceae based on morphological data and demonstrate the usefulness of the ITS region for phylogenetic studies within or among closely related genera of Lejeuneaceae.     

A phylogenetic analysis of Prunus and the Amygdaloideae (Rosaceae) using ITS sequences of nuclear ribosomal DNA

The economically important plum or cherry genus (PRUNUS:) and the subfamily Amygdaloideae of the Rosaceae have a controversial taxonomic history due to the lack of a phylogenetic framework. Phylogenetic analysis using the ITS sequences of nuclear ribosomal DNA (nrDNA) was conducted to construct the evolutionary history and evaluate the historical classifications of PRUNUS: and the Amygdaloideae. The analyses suggest two major groups within the Amygdaloideae: (1) PRUNUS: s.l. (sensu lato) and MADDENIA:, and (2) EXOCHORDA:, Oemleria, and PRINSEPIA: The ITS phylogeny supports the recent treatment of including EXOCHORDA: (formerly in the Spiraeoideae) in the Amygdaloideae. MADDENIA: is found to be nested within PRUNUS: s.l. in the parsimony and distance analyses, but basal to PRUNUS: s.l. in the maximum likelihood analysis. Within PRUNUS:, two major groups are recognizable: (1) the AMYGDALUS:-PRUNUS: group, and (2) the CERASUS:-LAUROCERASUS:-PADUS: group. The clades in the ITS phylogeny are not congruent with most subgeneric groups in the widely used classification of PRUNUS: by Rehder. A broadly defined PRUNUS: is supported.     

Testa and seed appendage morphology in Aeschynanthus (Gesneriaceae): phytogeographical patterns and taxonomic implications

The current sectional classification of the genus Aeschynanthus Jack, essentially based on seed morphology, presents some problems of species placement. A comparative SEM survey of seed and seed appendages was undertaken in order to assess the value of this classification. Seeds of 99 taxa (that is about two‐thirds of the estimated total) were examined and found to fall into two types, A and B. Type A has spiral testa cell orientation, papillae formed from a single cell and short smooth appendages. Type B is recognized by the straight orientation of the testa cells, combined with the presence of papillae formed from the raised ends of two adjacent cells on the long hair‐like appendages and usually on the testa. Only six of the investigated species did not fall into either category. Three have straight testa cell orientation combined with single‐cell papillae and short smooth appendages; the papillae and appendage characters place them in type A. Three have spiral testa cell orientation and short smooth appendages but the testa cells have slightly raised ends; these are also placed in Type A. The three subtypes in Type A are equivalent to the sections Haplotrichium s.s., Microtrichium and Aeschynanthus, but the divisions are less clear than those within Type B. However, other morphological characters support sectional separation. Type B subdivides into three: two subtypes equivalent to sections Polytrichium and Diplotrichium, and a third encompassing section Xanthanthos together with part of the current sect. Haplotrichium, and here referred to as sect. X. There is sufficient morphological correlation with seed type to make the sectional position of many species clear without recourse to seed, particularly in sects Polytrichium, Diplotrichium, Haplotrichium S.S. and Aeschynanthus. There is strong correlation between seed type and geographical distribution. Sects. Microtrichium and Aeschynanthus, with Type A seed, are essentially Malesian. Groups with Type B seed are largely confined to mainland south and south‐east Asia, except for sect. Polytrichium which is more widespread, possibly due to the greater effectiveness of a coma of hairs in wind dispersal. It is suggested that Type A seed, probably sect. Microtrichium, is the least determined and Type B sect. Polytrichium the most derived seed type. Based on these findings a revised key to the sections is provided.     

Delimitation of Sauropus (Phyllanthaceae) based on plastid matK and nuclear ribosomal ITS DNA Sequence data

Background and Aims

A recent molecular phylogenetic study showed that Sauropus is deeply embedded within Phyllanthus together with its allies, Breynia and Glochidion. As relationships within Sauropus are still problematic and the relationship with Breynia has long been doubted, more molecular data are needed to test/corroborate such a broad definition of Phyllanthus. This study aims to clarify the status and delimitation of Sauropus and establish its position within Phyllanthaceae.

Methods

Plastid matK and nuclear ribosomal ITS DNA sequence data for Sauropus and its allies were used to construct phylogenetic trees using maximum parsimony and Bayesian methods.

Key Results

Within Phyllanthus, Sauropus can be split into the mainly south-east Asian Sauropus sensu stricto (s.s.) plus Breynia and the mainly Australian Sauropus (formerly Synostemon). Sauropus s.s. plus Breynia comprise two distinct clades; one is the combination of Sauropus sections Glochidioidei, Sauropus and Schizanthi and the other is the combination of Sauropus sections Cryptogynium and Hemisauropus and the monophyletic genus Breynia.

Conclusions

Molecular data indicate that Synostemon should be reinstated at the same level as Sauropus s.s. and that Sauropus s.s. should be united with Breynia under the latter, older name. The molecular data corroborate only two of the five infrageneric groups of Sauropus recognized on the basis of morphological data.Key words: Breynia, DNA sequence data, Euphorbiaceae, ITS, matK, molecular phylogenetics, Phyllanthaceae, Sauropus, Synostemon     

应用ITS序列分析探讨偏花报春的系统位置

对于偏花报春Primula secundiflora Franch. 的系统位置,主要有两种意见,一种认为偏花报春具有典型的钟状花冠,应置于钟花报春组Sect．Sikkimensis;而另一种意见则依据其他特征将其置于灯台报春组sect．Proliferae。通过对偏花报春、灯台报春组4种植物和钟花报春组3种植物核糖体DNA中的内转录间隔区(ITS)的序列测定及分析,并结合形态学及染色体特征的比较论证,认为偏花报春应置于灯台报春组。     

Molecular phylogeny ofCheirolophus (Asteraceae:Cardueae-Centaureinae) based on ITS sequences of nuclear ribosomal DNA

The genusCheirolophus has an interesting western Mediterranean and Macaronesian distribution. Here we investigate the delimitation of the genus and its exclusion from the large genusCentaurea, the systematic position of the related genusPaleocyanus, the delimitation of some species and the phylogeny of the group. We have carried out a phylogenetic analysis of the PCR-generated sequences of the internal transcribed spacers (ITS-1 and ITS-2) of the nuclear ribosomal DNA. The results suggest that the genus, includingPaleocyanus crassifolius is monophyletic; thus, a new combination of this species underCheirolophus is proposed. The Macaronesian group of species is also monophyletic, indicating a single colonization of the archipelago. The poor resolution of microspecies in the Macaronesian group reinforces the hypothesis of a very recent differentiation of the group.     

GC balance in the internal transcribed spacers ITS 1 and ITS 2 of nuclear ribosomal RNA genes

Summary The internal transcribed spacer (ITS) 1 and 2, the 5.8S rRNA gene, and adjacent 18S rRNA and 25S rRNA coding regions of two Cucurbitaceae (Cucurbita pepo, zucchini, ITS 1: 187 bp, and ITS 2: 252 bp in length, andCucumis sativus, cucumber, ITS 1: 229 bp, and ITS 2: 245 bp in length) have been sequenced. The evolutionary pattern shown by the ITSs of these plants is different from that found in vertebrates. Deletions, insertions, and base substitutions have occurred in both spacers; however, it is obvious that some selection pressure is responsible for the preservation of stem-loop structures. The dissimilarity of the 5 region of ITS 2 found in higher plants has consequences for proposed models on U3 snRNA-ITS 2 interaction in higher eukaryotes.The two investigated Cucurbitaceae species show a G+C content of ITS 1 that nearly equals that of ITS 2. An analysis of the ITS sequences reveals that in 19 out of 20 organisms published, the G+C content of ITS 1 nearly equals that of ITS 2, although it ranges from 20% to 90% in different organisms (GC balance). Moreover, the balanced G+C content of the ITSs in a given species seems to be similar to that of so-called expansion segments (ESs) in the 25/28S rRNA coding region. Thus, ITSs show a phenomenon called molecular coevolution with respect to each other and to the ESs. In the ITSs of Cucurbitaceae the balanced G+C composition is at least partly achieved by C to T transitions, via deamination of 5-methylcytosine. Other mutational events must be taken into account. The appearance of this phenomenon is discussed in terms of functional constraints linked to the structures of these spacers.     

Phylogenetic analysis of Stylosanthes (Fabaceae) based on the internal transcribed spacer region (ITS) of nuclear ribosomal DNA

 Phylogenetic relationships in Stylosanthes are inferred by DNA sequence analysis of the ITS region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA in 119 specimens, representing 36 species of Stylosanthes and 7 species of the outgroup genera Arachis and Chapmannia. In all examined specimens of any particular diploid and (allo)polyploid species, only a single ITS sequence type was observed. This allowed us to identify a parental genome donor for some of the polyploids. In several diploid and polyploid species, different specimens contained a different ITS sequence. Some of these sequence types were present in more than one species. Parsimony analysis yielded several well-supported clades that agree largely with analyses of the chloroplast trnL intron and partially with the current sectional classification. Discordances between the nuclear and cpDNA analyses are explained by a process of allopolyploidization with inheritance of the cpDNA of one parent and fixation of the ITS sequences of the other. S. viscosa has been an important genome donor in this process of speciation by allopolyploidy. Received August 14, 2001; accepted March 4, 2002 Published online: November 14, 2002 Addresses of the authors: Jacqueline Vander Stappen, Steven Van Campenhout and Guido Volckaert (E-mail: guido.volckaert@agr.kuleuven.ac.be), Katholieke Universiteit Leuven, Laboratory of Gene Technology, Kasteelpark Arenberg 21, B-3001 Leuven, Belgium. Jan De Laet, American Museum of Natural History, Division of Invertebrate Zoology, Central Park West at 79th Street, New York 10024–5192, USA. Susana Gama-López, Universidad Nacional Autónoma de México, Unidad de Biología, Tecnología y Protipos (UBIPRO), FES-Iztacala, Laboratorio de Recursos Naturales, Av. de Los Barrios S/N, Colonia Los Reyes Iztacala, Municipio Tlalnepantla, Estado de México, C.P. 54090, México. Present address: Apartado Postal 154, Cto. Parque No. 3, C.P. 53102, México.     

Phylogenetic analyses of Septoria species based on the ITS and LSU-D2 regions of nuclear ribosomal DNA

The phylogenetic relationships of 17 selected Septoria spp. (eight with a known Mycosphaerella teleomorph), Phloeospora ulmi (teleomorph M. ulmi) and 18 additional taxa (10 with a Mycosphaerella teleomorph) were inferred from ITS and D2-LSU nrDNA sequences. In total, 10 anamorph genera associated with Mycosphaerella were represented. Intraspecific variation in ITS was limited in Septoria, with the exception of three strains that all were identified as S. rubi but originated from different Rubus spp. and probably belong to different species of Septoria. The results of D2 region sequencing confirmed Mycosphaerella sense lato (including Davidiella and Eruptio) as monophyletic. The cereal pathogen Septoria tritici, which is closely related to S. passerinii as found in ITS analysis, clusters with Ramularia spp. in the D2 analyses, distant from the other Septoria spp. The pathogens S. apiicola, S. linicola and S. populicola cluster in a major clade containing Phl. ulmi, and other Septoria spp. and Cercospora spp. Short branch lengths in this clade suggest a very recent evolution. Septoria castaneicola and S. pyricola also might represent relatively distant lineages. Both analyses of the regions indicated that Septoria is not monophyletic within Mycosphaerella and that conidiomatal structure (acervulus, pycnidium) has little value for predicting phylogenetic relatedness. As a consequence, the separation between the acervular Phloeospora and pycnidial Septoria is untenable. The loss of the teleomorph most likely has occurred several times in the evolutionary history of Mycosphaerella.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Systematics of the subfamily Haplorchiinae (Trematoda: Heterphyidae), based on nuclear ribosomal DNA genes and ITS2 region

Phylogenetic relationships of 6 species in the trematode subfamily Haplorchiinae were analyzed using small and large subunit of ribosomal DNA genes (18S rDNA and 28S rDNA) and internal transcribed spacer subunit II (ITS2) region as molecular markers. Maximum Likelihood and Bayesian inference analyses of combined rDNAs and ITS2 indicated a close relationship between the genera Haplorchis and Procerovum, while these two genera were distinct from Stellantchasmus falcatus. These phylogenetic relationships were consistent with the number of testes but not with the characters of the modification of the seminal vesicle or of the ventral sucker. Although three Haplorchis spp. were, together with Procerovum, in the same cluster, their mutual topology was incongruent between rDNA and ITS2 trees. Phylogenetic analyses using other molecular markers with more species are necessary to work out solid phylogenetic relationships among the species in this subfamily.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences

The nuclear ITS region of 19 species of Alnus was amplified and sequenced. The inferred molecular phylogeny shows that all species of the genus Alnus form a monophyletic group close to Betula and that the fundamental dichotomy within the genus lies between the subgenera Alnaster and Gymnothyrsus, sensu Murai (1964). The subgenus Alnaster appears to be basal in the genus, based on archaism of morphological features, and branching close to the root of the trees due to low ITS divergence from genus Betula. The monophyly of the section Clethropsis is not supported by the present data: Alnus nepalensis is positioned in the subgenus Gymnothyrsus, away from A. nitida and A. maritima. Surprisingly, A. formosana sect. Japonicae is closely tied to A. maritima sect. Clethropsis, with which it shares few morphological traits, and is separate from A. japonica sect. Japonicae with which it shares many traits. An increase in substitution rate is noted in the group comprising A. formosana, A. maritima and A. nitida relative to the rest of the genus, which appears to have had, on the average, a very slow mutation rate. Alnus glutinosa, the designated type for the genus, appears to be representative of the genus both for morphological characters and evolutionary rate. North-East Asia is comforted in its position of origin of the genus since not only does it have the highest number of species and representatives in all deep branching lineages, there are also fewer transcontinental migrations when a North-East Asian ancestor is postulated than when a North American ancestor is postulated.