Phylogeny of the ectomycorrhizal mushroom genus Alnicola (Basidiomycota, Cortinariaceae) based on rDNA sequences with special emphasis on host specificity and morphological characters

Alnicola (=Naucoria, pro parte) is a mushroom genus of strictly temperate, obligately ectomycorrhizal species, traditionally included in the family Cortinariaceae. Most Alnicola spp. are primarily host specific on Alnus, although a few are mycobionts of Salix or other hosts. The different species of Alnicola exhibit unique morphological (cystidia, pileipellis) and cytological (dikaryotic or monokaryotic hyphae) characters. This makes the genus Alnicola of particular interest for studying the evolution of host specificity and morphological characters in ectomycorrhizal basidiomycetes. We used a combination of classical morphological and phylogenetic methods (rDNA ITS and LSU sequences) to address the following questions: (i) Is Alnicola monophyletic? And (ii) Are characters like host specificity or microscopical structures synapomorphic for certain clades? The study included nearly all currently known European Alnicola sp. Our results demonstrated that, on one hand, the genus Alnicola is polyphyletic, with sistergroup relationships to Hebeloma, Anamika or the clades /Hymenogaster I and /Hymenogaster II. On the other hand, Alnicola splits into three well-supported clades corresponding to the sections Alnicola, Submelinoides, and Salicicolae. The strict host-specificity to Alnus is a derived character and has occurred at least twice. The following morphological characters are synapomorphic for defined clades: the spindle-shaped hymenial cystidia for sect. Alnicola, the hymeniform pileipellis for sect. Submelinoides, and monocaryotic/clampless hyphae for sect. Salicicolae and its sistergroup /Hymenogaster II. As a taxonomical consequence, polyphyly of Alnicola implies that the sects. Submelinoides and Salicicolae need to be segregated from Alnicola.     

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacer of the nuclear ribosomal DNA

The internal transcribed spacer (ITS1, 5.8S rDNA, and ITS2) region of nuclear ribosomal DNA (nrDNA) was sequenced from 53 species, which represent most of the living species diversity in the genus Phalaenopsis (Orchidaceae). A phylogeny was developed for the genus based on the neighbor-joining and maximum parsimony analyses of molecular data. Results of these analyses provided support for the monophyly of the genus Phalaenopsis and concurred in that the genera Doritis and Kingidium should be treated as being parts of the genus Phalaenopsis as suggested by Christenson (2001). Within the genus Phalaenopsis, neither subgenera Aphyllae nor Parishianae were monophyletic, and they were highly clustered with subgenus Proboscidioides plus sections Esmeralda and Deliciosae of subgenus Phalaenopsis based on ITS data. Those species also have the same characters of morphology of four pollinia and similar biogeographies. Furthermore, neither subgenus Phalaenopsis nor Polychilos was monophyletic. Within the subgenus Phalaenopsis, only section Phalaenopsis was highly supported as being monophyletic. As for the subgenus Polychilos, only section Polychilos was moderately supported as being monophyletic. In conclusion, the present molecular data obtained from the ITS sequence of nrDNA of the genus Phalaenopsis provide valuable information for elucidating the phylogeny of this genus.     

The phylogenetic relationships of Cynoglottis (Boraginaceae- Boragineae) inferred from ITS, 5.8S and trnL sequences

A molecular phylogenetic analysis of Cynoglottis was performed to evaluate previous hypotheses based on non-molecular evidence concerning the position of this genus within Boraginaceae tribe Boragineae. ITS-5.8S and trnL_UAA sequences from the nuclear and chloroplast non-coding genomes were obtained for four Cynoglottis taxa and selected members of the related genera Anchusa, Anchusella, Gastrocotyle, Brunnera and Pentaglottis. Cynoglottis is monophyletic, but neither trnL nor ITS support a close relationship with Brunnera, unlike previously supposed on morphological grounds. Brunnera is, instead, related to the southwestern European monotypic genus Pentaglottis, with which it forms a basal clade. ITS-5.8S sequences show that Anchusa thessala, a southeastern European annual species of Anchusa subg. Buglossellum, is sister to Cynoglottis and that the two taxa form a clade which also includes the Balkan endemic Gastrocotyle macedonica. Species of Anchusa subg. Anchusa form a separate lineage with high bootstrap support, suggesting that this heterogeneous genus is paraphyletic with respect to Cynoglottis. ITS sequences also discriminate between the Balkan-Apenninic diploid C. barrelieri and the Anatolian tetraploid C. chetikiana, albeit with low support. The molecular results are discussed in the light of karyological, morphological and chorological aspects.This work has been supported by M.I.U.R. 40% 2003 and the University of Firenze.     

Phylogeny of <Emphasis Type="Italic">Litsea</Emphasis> and related genera (Laureae-Lauraceae) based on analysis of <Emphasis Type="Italic">rpb2</Emphasis> gene sequences

The relationship between Litsea and related genera is currently unclear. Previous molecular studies on these taxa using cpDNA and nrITS were unable to produce well-resolved phylogenetic trees. In this study, we explored the potential of the rpb2 gene as a source of molecular information to better resolve the phylogenetic analysis. Although rpb2 was believed to be a single-copy gene, our cloning results showed that most species examined possessed several copies of these sequences. However, the genetic distance among copies from any one species was low, and these copies always formed monophyletic groups in our molecular trees. Our phylogenetic analyses of rpb2 data resulted in better resolved tree topologies compared to those based on cpDNA or nrITS data. Our results show that monophyly of the genus Litsea is supported only for section Litsea. As a genus, Litsea was shown to be polyphyletic. The genera Actinodaphne and Neolitsea were resolved as monophyletic groups in all analyses. They were also shown to be sisters and closer to the genus Lindera than to the genus Litsea. Our results also revealed that the genus Lindera is not a monophyletic group.     

Phylogenetic investigations of Sordariaceae based on multiple gene sequences and morphology

The family Sordariaceae incorporates a number of fungi that are excellent model organisms for various biological, biochemical, ecological, genetic and evolutionary studies. To determine the evolutionary relationships within this group and their respective phylogenetic placements, multiple-gene sequences (partial nuclear 28S ribosomal DNA, nuclear ITS ribosomal DNA and partial nuclear β-tubulin) were analysed using maximum parsimony and Bayesian analyses. Analyses of different gene datasets were performed individually and then combined to generate phylogenies. We report that Sordariaceae, with the exclusion Apodus and Diplogelasinospora, is a monophyletic group. Apodus and Diplogelasinospora are related to Lasiosphaeriaceae. Multiple gene analyses suggest that the spore sheath is not a phylogenetically significant character to segregate Asordaria from Sordaria. Smooth-spored Sordaria species (including so-called Asordaria species) constitute a natural group. Asordaria is therefore congeneric with Sordaria. Anixiella species nested among Gelasinospora species, providing further evidence that non-ostiolate ascomata have evolved from ostiolate ascomata on several independent occasions. This study agrees with previous studies that show heterothallic Neurospora species to be monophyletic, but that homothallic ones may have a multiple origins. Although Gelasinospora and Neurospora are closely related and not resolved as monophyletic groups, there is insufficient evidence to place currently accepted Gelasinospora and Neurospora species into the same genus.     

Molecular characterisation of Hanseniaspora species

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6–7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.     

Genetic diversity and phylogenetic position of the subclass Astomatia (Ciliophora) based on a sampling of six genera from West African oligochaetes (Glossoscolecidae, Megascolecidae), including description of the new genus Paraclausilocola n. gen

To more confidently assess phylogenetic relationships among astome ciliates, we obtained small subunit (SSU) rRNA sequences from nine species distributed in six genera and three families: Almophrya bivacuolata, Eudrilophrya complanata, Metaracoelophrya sp. 1, Metaracoelophrya sp. 2, Metaracoelophrya intermedia, Metaradiophrya sp., Njinella prolifera, Paraclausilocola constricta n. gen., n. sp., and Paraclausilocola elongata n. sp. The two new species in the proposed new clausilocolid genus Paraclausilocola n. gen. are astomes with no attachment apparatus, two files of contractile vacuoles, and an arc-like anterior suture that has differentiations of thigmotactic ciliature on the anterior ends of the left kineties of the upper surface. Phylogenetic analyses were undertaken using neighbor-joining, Bayesian inference, maximum likelihood, and maximum parsimony. The nine species of astomes formed a strongly supported clade, showing the subclass Astomatia to be monophyletic and a weakly supported sister clade to the scuticociliates. There were two strongly supported clades within the astomes. However, genera assigned to the same family were found in different clades, and genera assigned to the same order were found in both clades. Thus, astome taxa appear to be paraphyletic when morphology is used to assign species to genera.     

Molecular phylogeny of Old WorldTrifolium (Fabaceae), based on plastid and nuclear markers

Phylogenies of Old WorldTrifolium species were constructed using nucleotide sequence data of the internal transcribed spacers (ITS) of nuclear ribosomal DNA and chloroplast DNA restriction site data from PCR-amplified genes and genic regions (rbcL,trnK, andrpoC1–C2). Biogeography, morphological evolution, and the existing classification forTrifolium were examined. The genusTrifolium is strongly supported as monophyletic, however, only one small section (Chronosemium) is monophyletic, although the data are in conflict regarding its placement. The two largest sections of the genus, Sects.Lotoidea andTrifolium, are not supported as monophyletic, as currently circumscribed. Many members of Sect.Lotoidea are basal within the genus, supporting previously-proposed hypotheses concerning plesiomorphic morphological characters and a Mediterranean-Mideast biogeographic origin of the genus.     

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

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

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Hebeloma species associated with Cistus

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.     

Systematic implications of sesquiterpene lactones in Lactuca species

A chemosystematic study of eleven species from the genus Lactuca (Asteraceae) was performed, based on the distributional data for eight sesquiterpene lactones as diagnostic characters. The lactones were identified in leaf and root extracts by comparison of their HPLC retention times and on-line UV spectra with those of reference compounds. Our results support the status of the section Lactuca, subsection Lactuca as a recognizable group within the genus, although Lactuca aculeata is a distinct species. Moreover, sesquiterpene lactone patterns of Lactuca perennis, Lactuca tatarica, Lactuca indica and Lactuca capensis are also given. It is worth noting that the sesquiterpene lactones are absent from Lactuca tenerrima. The chemosystematic impact of lactucin-type guaianolides and the germacranolide lactuside A is discussed briefly.     

The phylogenetic positions of the conifer genera Amentotaxus,Phyllocladus, and Nageia inferred from 18s rRNA sequences

To determine the evolutionary positions of the conifer genera Amentotaxus, Phyllocladus, and Nageia, we obtained 18S rRNA sequences from 11 new taxa representing the major living orders and families of gymnosperms. With the published Chlamydomonas as an outgroup, phylogenetic analyses of our new data and available sequences indicate that (1) the Gnetales form a monophyletic group, which is an outgroup to the conifers, (2) the conifers are monophyletic, (3) Taxaceae, Cephalotaxaceae, Cupressaceae, and Taxodiaceae form a monophyletic group, (4) Amentotaxus is closer to Torreya than to Cephalotaxus, suggesting that Amentotaxus is better to be classified as a member of Taxaceae, (5) Phyllocladus, Dacrycarpus, Podocarpus, and Nageia form a monophyletic group, and (6) Pinaceae is an outgroup to the other families of conifers. Our finding that Phyllocladus is a sister group of the Podocarpaceae disagrees with the suggestion that the phylloclade of the genus is an ancient structure and that the genus is a terminal taxon within the Podocarpaceae. The genus Nageia is more closely related to Podocarpus than to Dacrycarpus and was derived from within the Podocarpaceae. In conclusion, our data indicate that in conifers, the uniovulate cone occurred independently in Taxacaeae and Cephalotaxaceae, and in Podocarpaceae after the three families separated from Pinaceae, and support the hypothesis that the uniovulate cone is derived from reduction of a multiovulate cone.Correspondence to: S.-M. Chaw     

Phylogenetic analysis of the subfamily Alpinioideae (Zingiberaceae), particularly Etlingera Giseke, based on nuclear and plastid DNA

Parsimony analyses based on DNA sequence data of the plastid group II intron rps16 and the internal transcribed spacer (ITS) were performed in order to examine the relationship of the pantropical subfamily Alpinioideae in Zingiberaceae (Zingiberales). Special emphasis was given to the large genus Etlingera placed in the tribe Alpinieae. A total of 50 taxa were included in the analysis. The strict consensus tree obtained by combining all data (280 parsimony informative characters of ITS, rps16, and coded indels) is well resolved with strongly supported clades. The subfamily Alpinioideae (excluding Pommereschea and Rhynchanthus) is strongly supported as monophyletic. The basal part of the tree is unresolved but a clade containing the derived genera of Alpinieae (Geocharis, Amomum, Hornstedtia, and Etlingera) is strongly supported. The establishment of Etlingera as the inclusive name for Achasma, Geanthus, and Nicolaia is also strongly supported: Etlingera is monophyletic with Hornstedtia as sister group.     

ITS phylogeny of West Asian Heracleum species and related taxa of Umbelliferae–Tordylieae W.D.J.Koch, with notes on evolution of their psbA-trnH sequences

Summary Heracleum is a large and taxonomically complex genus of the Umbelliferae–Tordylieae. The phylogenetic relationships of West Asian Heracleum species and related taxa were explored using data from sequences of the internal transcribed spacer (ITS1 and ITS2) region of nuclear ribosomal DNA. The data set consists of 56 species, of which 47 were analyzed for the first time; it represents all subdivisions of the genus Heracleum, as well as some representatives of Pastinaca complex. Heracleum was shown to be a polyphyletic genus, as its species fall into two different clades, one of which comprises also Symphyoloma and Mandenovia. Section Pubescentia was confirmed, in contrast to the sections Villosa and Heracleum being polyphyletic. A separate position of the section Wendia was supported. H. marashicum was shown to be a member of a clade comprising Pastinaca and related genera. The sequences of chloroplast psbA-trnH intergenic spacer, the region recently proposed for DNA barcoding in plants, were also analyzed for 33 species, representing all principal clades within Heracleum and its relatives. They have been proven to be very similar and not suitable for DNA barcoding in this group. However, some sequence variation was revealed. This variation could be explained by the combination of such evolutionary events as inversion and duplication. It was shown that these events are rather common in Tordylieae and can occur independently in different lineages. The evolution patterns of psbA-trnH spacer are hypothesized.     

Phylogeny of Rhododendron section Vireya (Ericaceae) based on two non-coding regions of cpDNA

Section Vireya (Blume) Copel.f. is one of the most morphologically diverse groups of the genus Rhododendron. Vireyas have a unique distribution for the genus, being predominantly found throughout the Malesian Archipelago. The alpha taxonomy of section Vireya is relatively well understood and taxa are easily distinguished from other rhododendrons by their general appearance. Defining characteristics of the section the possession of seeds with tailed appendages at both ends, the twisting of capsule valves after opening, placentas that separate as thread-like structures from the central axis as the capsule opens are all subject to exceptions. Phylogenetic analyses of two cpDNA regions, psbA-trnH and trnT-trnL intergenic spacers are reported. The results of each analysis were generally congruent, with clades relating strongly to geographic areas. Section Vireya is monophyletic with the inclusion of at least one species of section Rhododendron. Only two of the seven subsections currently circumscribed are monophyletic: Malayovireya Sleumer and Siphonovireya Sleumer. ``Euvireya', all subsections excluding the paraphyletic subsection Pseudovireya, is monophyletic and includes two major clades, one restricted to eastern Malesia and the other to the western and middle Malesia.     

Phylogenetic Framework for Trema (Celtidaceae)

We used ITS and trnL sequence data, analyzed separately and combined by MP, to explore species relationships and concepts in Trema (Celtidaceae), a pantropical genus of pioneer trees. Whether Trema is monophyletic or includes Parasponia is still unresolved. Three clades within Trema received moderate to high support, one from the New World and two from the Old World, but their relationships were not resolved. In the New World, specimens of T. micrantha formed two groups consistent with endocarp morphology. Group I, with smaller brown endocarps, is a highly supported clade sister to T. lamarckiana. Group II, with larger black endocarps, is poorly resolved with several subclades, including the highly supported T. integerrima clade. Both Old World clades contain Asian and African species, with three or more species in each region. Trema orientalis is not monophyletic: specimens from Africa formed a highly supported clade sister to T. africana, while those from Asia were sister to T. aspera from Australia.     

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

Details of the phylogenetic relationships among tetrahymenine ciliates remain unresolved despite a rich history of investigation with nuclear gene sequences and other characters. We examined all available species of Tetrahymena and three other tetrahymenine ciliates, and inferred their phylogenetic relationships using nearly complete mitochondrial cytochrome c oxidase subunit 1 (cox1) and small subunit (SSU) rRNA gene sequences. The inferred phylogenies showed the genus Tetrahymena to be monophyletic. The three “classical” morphology-and-ecology-based groupings are paraphyletic. The SSUrRNA phylogeny confirmed the previously established australis and borealis groupings, and nine ribosets. However, these nine ribosets were not well supported. Using cox1 gene, the deduced phylogenies based on this gene revealed 12 well supported groupings, called coxisets, which mostly corresponded to the nine ribosets. This study demonstrated the utility of cox1 for resolving the recent phylogeny of Tetrahymena, whereas the SSU rRNA gene provided resolution of deeper phylogenetic relationships within the genus.     

Phylogenetic relationships in Carex, subgenus Vignea (Cyperaceae), based on ITS sequences

To evaluate the sectional classification in Carex, subgenus Vignea, the ITS region of 58 species of 20 sections was analyzed with Neighbor Joining (NJ) and Markov chain Monte Carlo (MCMC) methods. Sections Dioicae, Physodeae and Ovales are found to be monophyletic, with C. bohemica well integrated in the section Ovales. Section Heleonastes turns out to be monophyletic, if C. canescens is treated separately in section Canescentes. Section Elongatae is monophyletic, but C. remota is placed in section Remotae and C. bromoides in section Deweyanae. In both analyses, six representatives of section Arenariae cluster together in a terminal group, whereas C. disticha, C. repens and C. siccata form a basal cluster. C. maritima, as the only member of section Incurvae, shares this basal position. C. chordorrhiza is ascribed to section Chordorrhizeae and not ascribed to the paraphyletic section Divisae. C. vulpina and C. otrubae are assigned to section Vulpinae and separated from the heterogeneous section Stenorhynchae. The other members of sections Divisae, Muehlenbergianae, Multiflorae and Stenorhynchae are scattered throughout the trees. The representatives of section Foetidae are dispersed in both analyses, section Paniculatae appears to be non-monophyletic in the molecular results as well. The subgenus appears subdivided in at least four larger subgroups in all analyses. Whereas these subgroups are strongly supported, the relationships between these subgroups remain only poorly resolved.     

Molecular phylogeny of genus Vigna subgenus Ceratotropis based on rDNA ITS and atpB-rbcL intergenic spacer of cpDNA sequences

The genetic diversity and phylogenetic relationships among species in the genus Vigna subgenus Ceratotropis were investigated using sequence data from the ribosomal DNA ITS and atpB-rbcL intergenic spacer of chloroplast DNA regions. While both sets of sequences were of similar lengths about 700bp the rDNA-ITS was more informative than atpB-rbcL having 170% more polymorphic sites and five times as many parsimony-informative sites. The atpB-rbcL spacer may be appropriate for analysis of taxa above the species level in the genus Vigna. Results of analyzing rDNA-ITS revealed, with low level of statistical bias, separation of the subgenus into three groups that correspond to the three sections Aconitifoliae, Angulares, and Ceratotropis. The ancestral section is Aconitifoliae based on comparison with the outgroup species cowpea, Vigna unguiculata. The V. minima complex, V. minima, V. riukiuensis, and V. nakashimae, has a distinct evolutionary path within section Angulares. Other species in section Angulares are very closely related except V. trinervia. Vigna trinervia has an intermediate position between sections. Sequence data suggests one genome donor to V. reflexo-pilosa came from a lineage within section Angulares close to V. exilis, V. hirtella, and V. umbellata. Data presented supports the view that section Angulares is the most recently diversified section in the subgenus, as inferred by short terminal branch lengths among the species of this section.