Nuclear ITS Region Research Articles

Phylogenetic Affinities of South American Anemone (Ranunculaceae), including the Endemic Segregate Genera, Barneoudia and Oreithales

This study tests the phylogenetic affinities of 11 South American species of Anemone s.l., including the closely related endemic segregate genera Barneoudia and Oreithales. We analyzed combined sequence data (chloroplast atpB‐rbcL spacer and nuclear ITS regions) for 51 species of Anemone s.l., using both likelihood and cladistic methods. The segregate genera, Oreithales and Barneoudia, nest within Anemone and are included in a clade (subgenus Anemone, sect. Pulsatilloides) consisting largely of South American taxa (Anemone sellowii, Anemone helleborifolia, and Anemone rigida) and other Southern Hemisphere species (e.g., Anemone caffra, Knowltonia vesicatoria, and Anemone crassifolia). As reported previously, Anemone antucensis (Chile and Argentina) is in a separate clade (subgenus and section Anemonidium) and is sister to Anemone tenuicaulis (New Zealand). Anemone multifida, Anemone triternata, and Anemone decapetala are embedded in a clade (subgenus and section Anemone) consisting largely of North American taxa. The tetraploid, Anemone multifida, was found in two separate, highly supported clades in the nuclear and chloroplast trees, suggesting a hybrid origin. Both sections Pulsatilloides and Anemonidium suggest that anemones originated in the Northern Hemisphere and subsequently spread to the Southern Hemisphere, a pattern found in common with other members of Ranunculaceae. Preliminary suggestions are offered for the reclassification of Anemone.

A multi‐locus phylogeny of Euryops (Asteraceae, Senecioneae) augments support for the "Cape to Cairo" hypothesis of floral migrations in Africa

AbstractWith about 100 species, Euryops (Cass.) Cass. ranks among the most speciose genera of the tribe Senecioneae (Asteraceae). The genus has its greatest diversity in South Africa, and displays an interesting disjunct distribution with most of the taxa found in southern Africa and a group of eight endemic species confined to the mountains of tropical East Africa and northeastern Africa. Molecular phylogenetic analyses of DNA sequence data from three chloroplast fragments and the nuclear ITS region were used to reconstruct the evolutionary history of 41 Euryops species in order to unravel species relationships and to determine the origin of the disjunct Afromontane taxa. Our results show a lack of support and resolution in the internal structure of the trees, but also reveal strong incongruence between the ITS and cpDNA datasets as assessed by Bayes Factors. We hypothesise that this is a consequence of the isolation and divergence of many populations over a short time period at some point in the history of the genus. Molecular dating based on our phylogenetic tree suggests that the genus diversified in South Africa around four million years ago. The origin of the East African species, dated at 1.9 Ma, well after the uplift of the East African mountains, is consistent with a scenario of a single dispersal event from South Africa northwards into the tropical East African mountains where diversification occurred, creating a monophyletic group of regional Afromontane endemics.

Molecular phylogenetic studies reveal an undescribed species within the North American concept of Melanelixia glabra (Parmeliaceae)

We employ a molecular phylogenetic approach using nuclear ITS and mitochondrial SSU rDNA gene regions to test the general efficacy of species boundaries in the morphological species Melanelixia glabra. 35 new sequences are generated for this study. Our results provide evidence that M. glabra is polyphyletic, indicating that using only morphological criteria to define species boundaries in this group of lichenised fungi underestimates actual species-level diversity. These analyses also demonstrate that the geographically distant population of the M. glabra complex in North America is the sister group of two Indian species (M. glabroides and M. villosella) and exhibits considerable molecular divergence from the European and Turkish specimens (M. glabra s. str). Additionally, some minor morphological differences support the isolation of the American clade. Such results strongly suggest that this population of M. glabra is a new phylogenetic (morphological+phylogenetic) taxon that is described here as a new species (Melanelixia californica). Our approach using two independent genes appears to be a rigorous method to critically examine species boundaries originally based on traditional morphological approaches in this group of lichenised fungi. Our study shows that the use of morphology, molecular data and geography provide a robust approach to delimitation of phylogenetic species.

Reassignment of Species of Paraphyletic Junellia s. l. to the New Genus Mulguraea (Verbenaceae) and New Circumscription of Genus Junellia: Molecular and Morphological Congruence

Fil: O'Leary, Nataly Cristina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Botanica Darwinion. Academia Nacional de Ciencias Exactas, Fisicas y Naturales. Instituto de Botanica Darwinion; Argentina

Molecular phylogeny and biogeography of Holcoglossum (Orchidaceae: Aeridinae) based on nuclear ITS, and chloroplast trnL-F and matK

AbstractThe phylogenetic relationships and biogeography of the Asiatic orchid genus Holcoglossum (Vandeae, Aeridinae) were inferred from sequences of chloroplast matK and trnL‐F genes and nuclear ITS regions of 25 taxa (12 Holcoglossum, 11 Aeridinae, 2 outgroups). Analysis of combined datasets with parsimony and Bayesian methods revealed that Holcoglossum is highly supported as monophyletic, including two debatable species, H. amesianum and H. subulifolium, and consists of three well‐supported major subclades which agree well with biodiversity centers of Holcoglossum: one tropical subclade comprising four species, another forming a polytomy comprising six species restricted to the temperate alpine region of the Hengduan Mountains, and the third with two species somewhat distributed between the first two regions. However, these three major subclades are only partly in accordance with previous infrageneric classifications based on morphological characters. The lip lamella, long and conical spur and tapering stipe seem to have evolved at least twice in Holcoglossum, similarly the pollination system shift has occurred at least twice. Vicariance analyses and molecular phylogeny suggest that Holcoglossum dispersed from tropical regions to the Hengduan Mountains and then radiated there as a result of the uplift of Qinghai‐Tibetan Plateau.

Phylogenetic relationships of the monotypic genera Halacsya and Paramoltkia and the origins of serpentine adaptation in circum‐mediterranean Lithospermeae (Boraginaceae): insights from ITS and matK DNA sequences

AbstractHalacsya and Paramoltkia are monotypic and partially sympatric genera in the Balkans, with no clear relationships among extant Lithospermeae due to striking morphological autapomorphies and scarcity of phylogenetic analyses in this group. The two species H. sendtneri and P. doerfleri show a strict selectivity for serpentine soils, posing the question whether this edaphic specialization reflects a common ancestry or a parallel process of adaptive evolution in unrelated lineages. DNA sequences from the nuclear ITS and chloroplast matK regions were generated from multiple accessions of Halacsya and Paramoltkia, and from representatives of 16 other genera of Mediterranean Lithospermeae. SEM analyses of pollen morphology were also conducted to test relationships indicated by molecular phylogenies. Parsimony analyses retrieved a clade of morphologically well differentiated monotypic Lithospermeae including Halacsya and Paramoltkia. ITS provided a better resolution of relationships and showed the two genera to be sistergroups close to Mairetis and Moltkiopsis, and no affinity to Moltkia as supposed by past authors. Pollen characters corroborated the phylogenetic link between the two Balkan genera. Five further monophyletic clades were recognised: Onosma‐Echium, Moltkia, Lithospermum s.l., Arnebia‐Macrotomia, and Alkanna‐Podonosma. Mapping the edaphic preferences of Lithospermeae onto molecular cladograms showed that serpentinophytism as an obligate condition originated separately in the clade of monotypic genera and in that of Onosma‐Echium. In Halacsya and Paramoltkia it represents an early ecological synapomorphy which probably originated in situ from non‐serpentine ancestors related to Moltkiopsis and Mairetis.

Phylogenetic relationships in the genus Primula L. (Primulaceae) inferred from the ITS region sequences of nuclear rDNA

The nucleotide sequences of the nuclear rDNA ITS regions were determined for 34 species of the genus Primula L. and one species of the genus Cortusa L., family Primulaceae Vent., and used to infer the phylogenetic relationships among these species. This analysis species of the Russian flora and the flora of adjacent territories were studied for the first time. The results clarified the taxomic structure of the genus Primula and confirmed the entity of some of its sections; but not the subgenera sensu J. Richards. Our data do not support an independent status of the genus Cortusa, placing it as one of the terminal lineages of the section Cortusoides Balf. f. in the genus Primula.

Phylogenetic Relationships of Gunnera based on Nuclear Ribosomal DNA ITS Region, rbcL and rps16 Intron Sequences

Abstract A previous analysis of two chloroplast gene regions, rbcL and the rps16 intron, showed a clear phylogenetic pattern in Gunnera. However, these regions were not informative enough to completely resolve the phylogeny. In this study the nuclear ITS region was sequenced for 24 specimens representing 22 species of Gunnera. 223 characters out of 819 were informative and supported the same monophyletic groups as the chloroplast gene regions. Because of its greater information content, the ITS region identified additional well-supported clades. In an analysis based on the three gene regions together, 272 characters out of 3154 were informative. The results show that the South American annual, G. herteri is sister to all other species. The African G. perpensa is well-supported as sister to the remaining species, which form two well-defined clades, one with the Malayan G. macrophylla as sister to subgenus Milligania from New Zealand and Tasmania. In the other clade, the South American subgenus Misandra is ...

An unusual new species of Oxalis (Oxalidaceae) from the Knersvlakte, South Africa

Oxalis ericifolia differs from all other southern African Oxalis in having massive papillate protrusions on the adaxial leaflet epidermis, broadly swollen peduncular articulations, a wine-red androecium and an extremely short petal claw, less than one fifth the length of the total petal. Other unusual features include the red colour of the claw and the substrate-induced flattening of the bulbs. It was discovered in a unique locality from which two other endemic Oxalis species have been described. A phylogenetic analysis of the nuclear ITS region places the new species in the informally recognized Oxalis flava clade.

Phylogenetics and Evolution of the Macaronesian Clade of Crassulaceae Inferred from Nuclear and Chloroplast Sequence Data

Abstract The Macaronesian clade of Crassulaceae comprises four genera (Aichryson, Aeonium, Greenovia, and Monanthes) that are largely endemic to Macaronesia, a region encompassing the Azores, Madeira, and the Cape Verde, Canary, and Salvage Islands. The monophyly of this clade has been supported by recent family-level phylogenetic analyses; however, the relationships within the clade remain uncertain. To resolve relationships within the Macaronesian clade, we sequenced several chloroplast DNA regions (matK and the trnL-trnF and psbA-trnH spacer regions) and the nuclear rDNA ITS region. Parsimony analyses of separate ITS and cpDNA data sets recover three major clades, corresponding to Aichryson, the perennial species of Monanthes, and Aeonium (including Greenovia). Congruence tests revealed significant heterogeneity between the ITS and cpDNA data sets. Comparison of the topologies resulting from analyses of these separate data sets indicated five instances of incongruence between the ITS and cpDNA that may...

Phylogenetics of the Antillean Goetzeoideae (Solanaceae) and Their Relationships within the Solanaceae based on Chloroplast and ITS DNA Sequence Data

Coeloneurum, Espadaea, Henoonia, and Goetzea are shrubs and trees that are endemic to the islands of Cuba, Hispaniola, and Puerto Rico in the Greater Antilles. A phylogenetic analysis was conducted to elucidate the evolutionary relationships among them and with other major lineages of the Solanaceae. DNA sequences of the chloroplast genes ndhF, rbcL, and trnL-trnF intron and intergenic spacer were obtained for twenty two taxa and sequences of the nuclear rDNA ITS region were obtained for eight taxa comprising a data set of over 5,000 bp. The inferred phylogeny groups the Antillean genera together with the South American Metternichia and Duckeodendron in a clade within the Solanaceae, pointing to a broader circumscription of the Goetzeoideae. Both chloroplast and nuclear datasets find the following relationships among the Antillean taxa: (Coeloneurum (Henoonia (Espadaea, Goetzea))). The South American genera Metternichia and Duckeodendron are the first and second sister groups, respectively, to the Antillean genera. The close relationship of Metternichia to the Antillean genera also is supported by pollen morphology. Phylogenetic inference suggests that the Antillean taxa first occuppied xeric environments and evolved into more mesic habitats. Floral characteristics indicate evolution of pollination systems from nocturnal, insect-pollination in Duckeodendron and Metternichia to diurnal, bird-pollination in the Antillean genera. Duckeodendron and the Antillean genera produce drupes, but their contrasting morphology and anatomy suggest that these fruit types originated from separate evolutionary events.

Genetic variation in the popular lab worm Lumbriculus variegatus (Annelida: Clitellata: Lumbriculidae) reveals cryptic speciation

Genetic variation in the freshwater oligochaete Lumbriculus variegatus from Europe, North America and Japan was studied by sequencing and analysing the mitochondrial 16S and COI genes, and the nuclear ITS region. What hitherto has been regarded as L. variegatus was found to consist of at least two distinct clades (I and II), both of which occur in Europe as well as North America (clade I also in Japan). Specimens from a single locality in Sierra Nevada, California, also morphologically identified as L. variegatus, represent a third clade, which appears to be more closely related to clade II than to clade I, based on 16S data only. Average COI genetic distances were 17.7% between clades I and II, 0.6% within clade I, and 1.3% within clade II. Further, for these two clades, the mitochondrial (16S and COI) gene trees, which consider only the maternal lineages, are congruent with the ITS gene tree, which is the result of recombinations of paternal as well as maternal genomes. Finally, chromosome counts revealed clade I specimens to be highly polyploid, and clade II specimens to be diploid. We therefore conclude that clades I–II are separately evolving lineages, and that they should be regarded as separate species. This will have to be taken into account in the continued use of L. variegatus as a model organism in biological sciences.

Neotropical roots of a Polynesian spice: the hybrid origin of Tahitian vanilla, Vanilla tahitensis (Orchidaceae)

Absent in the wild, Tahitian vanilla (V. tahitensis) is a gourmet spice restricted in distribution to cultivated and feral stands in French Polynesia and Papua New Guinea. Its origins have been elusive. Our objective was to test the purported hybrid derivation and parentage of V. tahitensis from aromatic, neotropical progenitors. Nucleotide sequences from V. tahitensis and neotropical Vanilla were assayed for phylogenetic relatedness in two independently inherited genomic regions, the nuclear ITS region, and the trnH-psbA noncoding region of chloroplast DNA. As predicted to occur for early generation hybrids, placement of V. tahitensis was nonconcordant. All V. tahitensis clustered with V. planifolia from analysis of cpDNA sequences, suggesting V. planifolia as the maternal genome contributor. Phylogenetic reconstruction of ITS sequences showed that most V. tahitensis nested incongruently with V. odorata, but others remained sister to V. planifolia. Recovery of ITS clones in V. tahitensis related to both V. planifolia and V. odorata also supports its biphyletic origin from these two taxa. We interpret the high percentage (95%) of additive polymorphic sites in V. tahitensis relative to its parents as indication of a recent, and probably human-mediated, evolutionary origin.

Taxonomic characterization of Shiraia-like fungi isolated from bamboos in Japan

The molecular phylogeny of nuclear LSU rDNA sequences (D1/D2 domain), ITS regions, and beta-tubulin gene (tub2) showed that the seven strains of Shiraia-like fungi obtained from fresh bamboo tissues as endophytes were closely related to Shiraia bambusicola and had three distinctive lineages (groups A-C). The closest group (group A) to S. bambusicola produced distinctive prawn-shaped conidioma-like structures that differed from conidiomata in the anamorph of S. bambusicola. Currently, none of the morphological structures and molecular database records were compatible with our Shiraia-like fungi. These results reveal that Shiraia-like fungi group A is supposed to be a new species that should be assigned into a novel genus/species related to S. bambusicola.

Molecular phylogenetics of Alchemilla, Aphanes and Lachemilla (Rosaceae) inferred from plastid and nuclear intron and spacer DNA sequences, with comments on generic classification

Alchemilla (the lady’s mantles) is a well known but inconspicuous group in the Rosaceae, notable for its ornamental leaves and pharmaceutical properties. The systematics of Alchemilla has remained poorly understood, most likely due to confusion resulting from apomixis, polyploidisation and hybridisation, which are frequently observed in the group, and which have led to the description of a large number of (micro-) species. A molecular phylogeny of the genus, including all sections of Alchemilla and Lachemilla as well as five representatives of Aphanes, based on the analysis of the chloroplast trnL–trnF and the nuclear ITS regions is presented here. Gene phylogenies reconstructed from the nuclear and chloroplast sequence data were largely congruent. Limited conflict between the data partitions was observed with respect to a small number of taxa. This is likely to be the result of hybridisation/introgression or incomplete lineage sorting. Four distinct clades were resolved, corresponding to major geographical division and life forms: Eurasian Alchemilla, annual Aphanes, South American Lachemilla and African Alchemilla. We argue for a wider circumscription of the genus Alchemilla, including Lachemilla and Aphanes, based on the morphology and the phylogenetic relationships between the different clades.

Reticulate evolution in the natural range of the invasive wetland tree species Melaleuca quinquenervia

The Melaleuca leucadendra complex (broad-leaf paperbarks; Myrtaceae) is a dominant component of the tropical and sub-tropical biota of Australia, particularly in wetlands of high conservation significance. In Florida and other parts of the Americas, however, one member of the group (Melaleuca quinquenervia) is a serious ecological and economic weed. Understanding the relationships and evolution of the group is integral to both conservation and biocontrol efforts. Although the complex is currently considered to include up to 14 species, there has been some concern over taxonomic boundaries within the complex because most species are circumscribed only by combinations of characters, each of which also occurs in other species. Here, DNA sequence data derived from the chloroplast and two nuclear regions are used to explore the relationships of M. quinquenervia. We find little evidence for clear species boundaries within the M. leucadendra complex in general, with regional sharing of chloroplast haplotypes across morphologically defined taxa, indicating asymmetrical introgression or retention of ancestral haplotypes (lineage sorting). Phylogenies were further confounded by the recovery of multiple copies of both nuclear regions sequenced (ITS and rpb2) from many individuals. There was no clear evidence of polyploidy or pseudogenes, but multiple duplications of rpb2 could not be ruled out. Parsimony networks of the nuclear ITS region show some clustering of haplotypes by morphospecies but there is also evidence of both hybridisation and recombination. Signals of introgression were also evident in rpb2, supporting an hypothesis of recent or ongoing gene flow between M. quinquenervia and other members of the M. leucadendra complex. Both relaxed and fixed molecular-clock dating estimate the introgression to have occurred sometime within the past seven million years (95% CI: 0.7–18). The New Caledonian population of M. quinquenervia appears to have been established by dispersal from Australia during this period. M. quinquenervia is found to have alleles closely related to multiple different morphotaxa within the M. leucadendra complex, suggesting considerable past introgression into this taxon from some other members of the M. leucadendra complex, and this has implications for biocontrol efforts. The M. leucadendra complex appears to reflect early to intermediate stages of speciation, possibly driven by different ecologies.

A phylogenetic classification of tribe Poranthereae (Phyllanthaceae, Euphorbiaceae sensu lato)

Summary. A phylogenetic classification of tribe Poranthereae Gruning (Phyllanthaceae Martynov) is presented, based on molecular studies using DNA sequence data of nuclear ITS and plastid matK regions in conjunction with morphological characters. The tribe contains eight genera, four subgenera and 111 species. Circumscription of the genera differs considerably from previous systems. Only Actephila Blume remains unchanged. Andrachne sensu lato is divided into five genera: Andrachne L., Leptopus Decne., Notoleptopus Voronts. & Petra Hoffm., Phyllanthopsis (Scheele) Voronts. & Petra Hoffm., Pseudophyllanthus (Mull. Arg.) Voronts. & Petra Hoffm.; two further species are transferred to Meineckia Baill. subgenus Petaliferae Voronts. & Petra Hoffm. Archileptopus P. T. Li is included in Leptopus; Oreoporanthera (Gruning) Hutch. is included in Poranthera Rudge; Zimmermannia Pax and Zimmermanniopsis Radcl.-Sm. are included in Meineckia subgenus Meineckia. The genus Notoleptopus and Meineckia subgenus Petaliferae are newly described; Phyllanthopsis and Pseudophyllanthus are raised to generic level; Andrachne subgen. Phyllanthidea (Didr.) Voronts. & Petra Hoffm. is raised to subgeneric level. Six new combinations and a nomen novum are made at species level: Leptopus fangdingianus (P. T. Li) Voronts. & Petra Hoffm., Phyllanthopsis phyllanthoides Voronts. & Petra Hoffm., P. arida (Warnock & M. C. Johnst.) Voronts. & Petra Hoffm., Pseudophyllanthus ovalis (E. Mey. ex Sond.) Voronts. & Petra Hoffm., Notoleptopus decaisnei (Benth.) Voronts. & Petra Hoffm., Meineckia cerebroides (Petra Hoffm.) Voronts. & Petra Hoffm. and M. gracilipes (Petra Hoffm.) Voronts. & Petra Hoffm. Morphology and geographical distribution is discussed with special reference to petals, disc morphology and seed.

Preliminary geoherbalism study of Dendrobium officinale food by DNA molecular markers

Dendrobium officinale is commonly used as a traditional valuable Chinese tonic hygienic food. In order to authenticate the quality of hygienic food in the market and geoherbalism in the process of GAP (good agriculture practice), sequences of chloroplast genes (matK and rbcL), the nuclear rDNA ITS region and mitochondria gene nad1 intron2 were used to survey eight populations of D. officinale. No difference was found in sequences of matK, rbcL and nad1 intron2 between populations, except for nine single nucleotide polymorphisms (SNPs) sites in rDNA ITS regions. Two pairs of allele-specific PCR primers based on SNPs were designed to authenticate two genuine populations (GSG and FSC populations). The AS-PCR (allele-specific PCR) fragments of GSG and FSC populations are about 600 and 560 bp, and annealing temperatures are 55–57 and 58–60°C, respectively. Dried “Fengdou” food samples from genuine populations could also be authenticated using two pairs of diagnostic primers. It is demonstrated that methods based on SNPs of rDNA ITS region and AS-PCR are simple, practical and effective for genuine germplasm authentication of D. officinale during the process of GAP and hygienic food quality control.

Taxonomic characterization of <i>Shiraia</i>-like fungi isolated from bamboos in Japan

The molecular phylogeny of nuclear LSU rDNA sequences (D1/D2 domain), ITS regions, and beta-tubulin gene (tub2) showed that the seven strains of Shiraia-like fungi obtained from fresh bamboo tissues as endophytes were closely related to Shiraia bambusicola and had three distinctive lineages (groups A–C). The closest group (group A) to S. bambusicola produced distinctive prawn-shaped conidioma-like structures that differed from conidiomata in the anamorph of S. bambusicola. Currently, none of the morphological structures and molecular database records were compatible with our Shiraia-like fungi. These results reveal that Shiraia-like fungi group A is supposed to be a new species that should be assigned into a novel genus/ species related to S. bambusicola.

The systematic position of <i>Cubanthus</i> and other shrubby endemic species of <i>Euphorbia</i> (Euphorbiaceae) in Cuba

Steinmann, V.W., Van Ee, B., Berry, P.E. & Gutierrez, J. 2007. The systematic position of Cubanthus and other shrubby endemic species of Euphorbia (Euphorbiaceae) in Cuba. Anales Jard. Bot. Madrid 64(2): 123-133. Cubanthus (Euphorbiaceae) has traditionally been recognized as a small genus of three similar species restricted to Cuba and Hispaniola. In this study we used DNA sequence data from the nuclear ITS and chloroplast ndhF gene regions to investigate its systematic position and the position of four other shrubby Euphorbia species endemic to Cuba: E. cubensis, E. helenae, E. munizii, and E. podocarpifolia. The results demonstrate that all of these taxa belong to a well-supported Antillean clade nested within Euphorbia that also includes E. punicea and E. gymnonota. For that reason, we treat Cubanthus as a section of Euphorbia instead of a separate genus. Euphorbia sect. Adenorima is relegated to a synonym of sect. Cubanthus. New names are proposed for Cubanthus brittonii and Cubanthus linearifolius (Euphorbia millspaughii and E. scutiformis, respectively), and a new combination is made for Cubanthus umbelliformis (Euphorbia umbelliformis). Section Cubanthus belongs to clade C of Euphorbia and is part of a New World assemblage that includes members of sections Euphorbiastrum, Pteroneurae, Portulacastrum, Stachydium, and the former genus Pedilanthus. Based on both the molecular results and morphological differences, two subsections are proposed: subsect. Cubanthus and subsect. Moa. The molecular phylogeny supports the hypothesis that section Cubanthus radiated entirely within the Antilles and the nearby Bahamian archipelago.