Phylogeny of Ptychostomum （Bryaceae,Musci） inferred from sequences of nuclear ribosomal DNA internal transcribed spacer （ITS） and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.     

Phylogeny and Biogeography of Thuja L. （Cupressaceae）, an Eastern Asian and North American Disjunct Genus

In order to develop better insights into biogeographic patterns of eastern Asian and North American disjunct plant genera, sequences of nuclear ribosomal DNA internal transcribed spacer (nr DNAITS) region were used to estimate interspecific relationships of Thuja L. (Cupressaceae) and infer its biogeography based on the phylogeny. According to the phylogenetic analysis, two clades were recognized. The first clade included Thuja plicata D. Don (western North America) and T. koraiensis Nakai (northeastern Asia), and the second one contained T. occidentalis (Gord.) Cart. (Japan). The ancestral area of Thuja was inferred to be eastern Asia, and two dispersal events were responsible for the modern distribution of Thuja in North America. Both the North Atlantic land bridge and Bering land bridge were possible routes for the migration of ancestral populations to North America.     

Evolutionary relationships of trichostrongyloid nematodes (Strongylida) inferred from ribosomal DNA sequence data

The evolutionary relationships of 21 species of trichostrongyloid nematodes were determined by use of sequence data of the second internal transcribed spacer of the ribosomal DNA aligned according to secondary structure information. Irrespective of the method of analysis used, the topologies of the phylogenetic trees derived from the molecular data differed with respect to all four hypotheses proposed previously for the evolutionary relationships of the different subfamilies within the Trichostrongylidae based on morphological data. Thus, the molecular data set did not resolve the conflict between the four previous proposals for the subfamilial relationships. Nonetheless, all trees derived from the molecular data showed strong support for the exclusion of the genera Filarinema and Amidostomum from the clade containing the species within the family Trichostrongylidae. This represents a major difference from the most recent proposal of the systematics of the Trichostrongyloidea in which these two genera were included within the Trichostrongylidae. Therefore, the molecular data support an earlier systematic framework in which Filarinema and Amidostomum were considered to be sister groups of the Trichostrongyloidea.     

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.     

ITS sequence data support a single origin for North American Astereae (Asteraceae) and reflect deep geographic divisions in Aster s.l

The Astereae is the largest tribe of Asteraceae in North America. Morphological diversity suggests that the North American assemblage is polyphyletic as 12 endemic genera, as well as lineages of the genus Erigeron and Conyza (Conyzinae), have been hypothesized to represent at least five separate invasions of North America from Africa, Australia, Eurasia, and South America. This hypothesis was tested with a phylogenetic analysis of nucleotide sequence data from the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Sequences for 62 taxa represent seven outgroup taxa and all major Northern and Southern Hemisphere groups of Astereae, including broad taxonomic and geographic sampling of Conyzinae and Aster s.l. (sensu lato). Parsimony analyses indicate that all North American Astereae are members of a strongly supported clade, and that a diverse group of predominantly woody taxa from Africa, Australia, and South America, are basal Astereae. Furthermore, Aster s.l. is deeply polyphyletic as Eurasian taxa, including Aster s.s. (sensu stricto), appear more closely related to Southern Hemisphere taxa than to North American Aster segregates. There is only low to moderate agreement between proposed higher level Astereae relationships based on ITS and those based either on morphology or chloroplast restriction site data.     

Cryptic speciation in Herbertus (Herbertaceae, Jungermanniopsida): Range and morphology of Herbertus sendtneri inferred from nrITS sequences

Maximum likelihood and parsimony analyses of nrITS1-5.8S-ITS2 sequences place Austrian Herbertus sendtneri in a well bootstrap supported clade with Herbertus azoricus and Neotropical accessions which were alternatively assigned to Herbertus subdentatus or Herbertus dicranus. Asian and African Herbertus dicranus form a separate lineage which is placed sister to several European Herbertus taxa. Herbertus borealis from the British Isles is placed sister to H. stramineus in a robust sister relationship, indicating that the species does not belong in the synonymy of H. dicranus. As a result of the molecular investigation, the range of Herbertus sendtneri is extended to the Neotropics and H. azoricus is placed into the synonymy of H. sendtneri. Without much doubt, Herbertus dicranus does not occur in tropical America. Morphologically, H. sendtneri and H. dicranus can at most be separated by weak tendencies. Herbertus sendtneri is characterized by the frequent occurrence of coarse appendages at the leaf margins and often completely papillose leaves. H. dicranus often has small appendages and a nearly smooth basal leaf half. Herbertus borealis, which is nonmonophyletic with H. dicranus in the molecular trees, cannot be separated morphologically from forms of H. dicranus with small appendages. Herbertus subdentatus is not identical with H. sendtneri. Morphological homoplasy and cryptic speciation are obviously common in Herbertus.We thank the owners and curators of the herbaria cited in the text for the loan of specimens, P. Schwartz for assistance with SEM techniques, and M. Lindner for help with laboratory work. M. Schmull, S.R. Gradstein, F.A. Hartmann, and R.-L. Zhu helped to collect Herbertus sendtneri. D.S. Rycroft (Glasgow) collected H. borealis. G. Gärtner and D. Miller kindly organized authorizations. Parts of this study were supported by the German Science Foundation (DFG grant HE 3584/1).     

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.     

Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species

Fusarium species causing wilt diseases in different plants were characterised by comparing nonpathogenic and different pathogenic species using rDNA RFLP analysis. The ITS (internal transcribed spacer) region of 12 isolates belonging to the section Elegans, Laseola, Mortiella, Discolor, Gibbosum, Lateritium and Sporotrichiella were amplified by universal ITS primers (ITS-1 and ITS-4) using polymerase chain reaction (PCR). Amplified products, which ranged from 522 to 565 bp were obtained from all 12 Fusarium isolates. The amplified products were digested with seven restriction enzymes, and restriction fragment length polymorphism (RFLP) patterns were analysed. A dendrogram derived from PCR-RFLP analysis of the rDNA region divided the Fusarium isolates into three major groups. Assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships.     

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.     

The Genus Clusia L.: Molecular Evidence for Independent Evolution of Photosynthetic Flexibility

Abstract: Members of the Clusiaceae genus Clusia (tropical trees and shrubs) belong to the small group of dicotyledonous trees which are able to perform crassulacean acid metabolism (CAM). Most of the species are able to switch between C₃ and CAM modes of photosynthesis and only a few are restricted to either C₃ or CAM. In order to discover possible phylogenetic relationships with regard to the mode of photosynthesis, we investigated 17 species of the genus Clusia, and one species each of the Clusiaceae genera Oedematopus and Hypericum on the basis of internal transcribed spacer (ITS) sequences between the 18S and 26S coding regions of nuclear ribosomal DNA. Little length variation was detected in the ITS region of Clusia species. ITS1 sequences ranged from 255 to 260 bp and ITS2 sequences from 208 to 210 bp. Neighbour-joining and parsimony analyses of these sequences resulted in considerable differences in cluster formation when compared to a classification based on morphological characteristics. The molecular data also give no indication of a group-specific evolution of modes of photosynthesis, i.e., C₃ and CAM. We thus conclude that CAM has evolved independently several times within the genus Clusia.     

Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassiniinae) based on sequences from Nuclear Ribosomal DNA internal transcribed spacers (ITS)

The phylogenetic relationships among sexually reproducing species of Antennaria (Asteraceae) are poorly understood. An earlier cladistic analysis based on morphology did not fully resolve the phylogeny of these taxa and therefore a different approach using molecular data was explored. The internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA were sequenced for 30 species of Antennaria and one species from each of the outgroup genera Anaphalis, Ewartia, Leontopodium, and Pseudognaphalium. The ITS-1 sequence in Antennaria ranged from 253 to 260 base pairs (bp) in length, and the proportion of nucleotide differences between pairs of species of Antennaria ranged from 1 to 14%. For ITS-2, the divergence between pairs of species of Antennaria ranged from 0 to 8%. ITS-2 is shorter than ITS-1, ranging from 213 to 219 bp. Phylogenetic analysis indicates that, relative to the outgroups included, Antennaria is a well-supported monophyletic group. Based on the genera surveyed, Leontopodium appears to be the sister genus of Antennaria. The general topology of the molecular trees agrees with that based on previous morphological analyses and indicates that Antennaria is composed of six clades of equal rank, corresponding to the traditionally recognized informal groups, the Geyeriae, Argenteae, Arcuatae, Dimorphae, Pulcherrimae, and Catipes. Sequence and morphological data indicate that the Alpinae and Dioicae are unnatural, polyphyletic units that should be abandoned and redefined as the monophyletic Catipes group. Phylogenetic analysis of ITS sequences also suggests the dissociation of A. stenophylla from the Dimorphae, where it is traditionally placed, and its affiliation with the Argenteae, as well as the placement of A. arcuata in its own group.     

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.     

Infrageneric phylogeny of the genus Gentiana (Gentianaceae) inferred from nucleotide sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacers (ITSs) of nuclear ribosomal DNA have been sequenced for 20 species of Gentiana. By incorporating previously released sequence data of eight species, phylogenelic analyses using Fitch parsimony and character-state weighted parsimony were carried out. The length of ITS 1 in the taxa surveyed ranged from 223 to 238 bp and ITS2 from 216 to 234 bp. Sequence divergence between pairs of species ranged from 5.0% to 48.9% in ITS1, from 1.1% to 45.3% in ITS2, and from 3.2% to 46.1% in combined data of ITS1 and ITS2. The ITS phylogeny was generally congruent with morphological classifications except that G. asclepiadea was revealed to be closely related to section Gentiana instead of section Pneumonanthe and section Stenogyne was shown to be a paraphyletic group of the genus Gentiana that would be better excluded from the genus. A divergence among the three European endemic sections and the remaining sections of the genus other than section Stenogyne was revealed. Thus the European species of the genus together do not form a monophyletic group. A close relationship between the sections Chondrophyllae s. l. (including section Dolichocarpa), Cruciata and Pneumonanthe was suggested. The section Frigidae s. l. (including sections Monopodiae, Isomeria, Microsperma, and Phyllocalyx) contained two well-supported clades: section Frigidae s. str. and all others together. The monophyly of the typically dysploid group section Chondrophyllae s. l. was confirmed. Optimization of chromosome numbers on the ITS phylogeny suggested that 2/1 = 26 is a plesiomorphic state for the clade comprising sections Frigidae s. l., Cruciata, Pneumonanthe, and Chondrophyllae s. l., and probably 2n = 20 is a plesiomorphic state for the dysploid group, section Chondrophyllae s. l.     

Delineation of Chondrus (Gigartinales, Florideophyceae) in China and the origin of C. crispus inferred from molecular data

Complete nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences were obtained for 18 Chondrus populations collected at 15 sites from eight countries worldwide. Pairwise comparisons with the multiple alignment revealed that intraspecific divergences of ITS sequences ranged from 0.3 to 1.8% in C. crispus Stackhouse (except for the entity SVLH from France) and from 0.0 to 0.6% in C. ocellatus Holmes, whereas interspecific divergences in Chondrus varied from 1.4 to 5.0%. Three phylogenetic methods (neighbour joining, maximum parsimony and maximum likelihood) confirmed three main lineages: the North Atlantic lineage containing entities of C. crispus from Canada, France, Germany, England, Portugal, Ireland and Wales; a second lineage comprising three species: C. sp. 1, C. armatus (Harvey) Yamada et Mikami, and C. pinnulatus (Harvey) Okamura from the Northern Pacific; and a third lineage containing just one species: C. ocellatus from the Northern Pacific. Chondrus yendoi Yamada et Mikami separated from other Chondrus species singly. nrDNA ITS data indicate that a previous assignment of C. sp. 2 to Mazzaella japonica (Mikami) Hommersand may be incorrect, and additional evidence is needed to resolve the generic placement of this entity. It is inferred from the nrDNA ITS data that three Chondrus species are presently known in China with two, C. ocellatus and C. nipponicus, in Qingdao and two, C. armatus and C. nipponicus, in Dalian. We hypothesize that the ancestor of North Atlantic C. crispus had a Pacific origin, and that the present distribution of C. crispus in the Atlantic Ocean correlates with a trans-Arctic dispersal and vicariance events associated with Pleistocene glaciation maxima.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER REGIONS (ITS1 AND ITS2) DEFINE DISCRETE BIOGEOGRAPHIC GROUPS IN CLADOPHORA ALBIDA (CHLOROPHYTA)1

Nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2), the 5.8S, and short stretches of the adjacent 18S and 26S coding regions were determined in isolates from four disjunct Cladophora albida (Huds.) Kütz. populations (NE-America, W-Europe, Japan, and W-Australia). The two Pacific isolates share nearly identical ITS sequences as do the two Atlantic isolates. In contrast, interoceanic comparisons exhibit a 21% sequence difference. Variation within ITS regions is useful for identification of population groups on a regional or oceanic scale. However, both spacers are characterized by numerous repeat motifs as well as point mutations, which result in alignment problems at the interspecific level within Cladophora.     

Molecular appraisal of Bunium and some related arid and subarid geophilic Apiaceae–Apioideae taxa of the Ancient Mediterranean

Bunium is unusual in Apiaceae in having a variable cotyledon number and broad infrageneric dysploidy. To test the monophyly of the genus, phylogenetic relationships among 39 Bunium species were investigated with DNA sequence data from nuclear (nrITS) and plastid (psbA‐trnH intergenic spacer) regions. Several other taxa with a similar ecology and geography were also included in the analyses. Our results suggest that Bunium is not monophyletic. Bunium spp. in the eastern part of the study area are more closely related to the Central Asian genera Elaeosticta, Galagania, Hyalolaena, Mogoltavia and Oedibasis than to those in the western part. Our study revealed that molecular, morphological (cotyledon number and width of fruit commissure) and karyological data reveal similar patterns in the taxa studied. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 149–170.     

CRYPTIC DIVERSITY AND PHYLOGENETIC RELATIONSHIPS WITHIN THE MASTOCARPUS PAPILLATUS SPECIES COMPLEX (RHODOPHYTA,PHYLLOPHORACEAE)1

Mastocarpus papillatus (C. Agardh) Kütz. is a common intertidal red alga occurring along the west coast of North America from Baja California to Alaska. Sequencing of both the chloroplast‐encoded rbcL gene and the nuclear ribosomal internal transcribed spacer (ITS) regions of ～200 specimens from California to Alaska revealed that M. papillatus is actually a complex of at least five species. All five species have high bootstrap support in phylogenetic analyses of both genetic regions, and in the case of the ITS marker, the species also have distinctive patterns of indels. Three of the species are localized in the mid‐ to upper intertidal, whereas two of the species occur in the low intertidal. The species also have different geographic ranges that overlap in the Vancouver Island area of British Columbia. No distinctive, reliable morphological differences were observed among the species. Although a variety of names are available for species in the complex, it is not yet clear which name goes with which species. As part of the survey, I also sequenced other species of Mastocarpus in the northeast Pacific region, and I provide new distribution records for M. jardinii ( J. Agardh) J. A. West and for a nonpapillate and probably undescribed species of Mastocarpus.