Molecular phylogenetics of the clover genus (Trifolium--Leguminosae)

Trifolium, the clover genus, is one of the largest genera of the legume family. We conducted parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences obtained from 218 of the ca. 255 species of Trifolium, representatives from 11 genera of the vicioid clade, and an outgroup Lotus. We confirm the monophyly of Trifolium, and propose a new infrageneric classification of the genus based on the phylogenetic results. Incongruence between the nrDNA and cpDNA results suggests five to six cases of apparent hybrid speciation, and identifies the putative progenitors of the allopolyploids T. dubium, a widespread weed, and T. repens, the most commonly cultivated clover species. Character state reconstructions confirm 2n=16 as the ancestral chromosome number in Trifolium, and infer a minimum of 19 instances of aneuploidy and 22 of polyploidy in the genus. The ancestral life history is hypothesized to be annual in subgenus Chronosemium and equivocal in subgenus Trifolium. Transitions between the annual and perennial habit are common. Our results are consistent with a Mediterranean origin of the genus, probably in the Early Miocene. A single origin of all North and South American species is hypothesized, while the species of sub-Saharan Africa may originate from three separate dispersal events.     

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 ? resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.     

Molecular phylogenetics of Caenogastropoda (Gastropoda: Mollusca)

Caenogastropoda is the dominant group of marine gastropods in terms of species numbers, diversity of habit and habitat and ecological importance. This paper reports the first comprehensive multi-gene phylogenetic study of the group. Data were collected from up to six genes comprising parts of 18S rRNA, 28S rRNA (five segments), 12S rRNA, cytochrome c oxidase subunit I, histone H3 and elongation factor 1alpha. The alignment has a combined length of 3995 base positions for 36 taxa, comprising 29 Caenogastropoda representing all of its major lineages and seven outgroups. Maximum parsimony, maximum likelihood and Bayesian analyses were conducted. The results generally support monophyly of Caenogastropoda and Hypsogastropoda (Caenogastropoda excepting Architaenioglossa, Cerithioidea and Campanilioidea). Within Hypsogastropoda, maximum likelihood and Bayesian analyses identified a near basal clade of nine or 10 families lacking an anterior inhalant siphon, and Cerithiopsidae s.l. (representing Triphoroidea), where the siphon is probably derived independently from other Hypsogastropoda. The asiphonate family Eatoniellidae was usually included in the clade but was removed in one Bayesian analysis. Of the two other studied families lacking a siphon, the limpet-shaped Calyptraeidae was associated with this group in some analyses, but the tent-shaped Xenophoridae was generally associated with the siphonate Strombidae. The other studied hypsogastropods with an anterior inhalant siphon include nine families, six of which are Neogastropoda, the only traditional caenogastropod group above the superfamily-level with strong morphological support. The hypotheses that Neogastropoda are monophyletic and that the group occupies a derived position within Hypsogastropoda are both contradicted, but weakly, by the molecular analyses. Despite the addition of large amounts of new molecular data, many caenogastropod lineages remain poorly resolved or unresolved in the present analyses, possibly due to a rapid radiation of the Hypsogastropoda following the Permian-Triassic extinction during the early Mesozoic.     

Molecular phylogenetics and delimitation of species in Cortinarius section Calochroi (Basidiomycota, Agaricales) in Europe

Cortinarius is the most species rich genus of mushroom forming fungi with an estimated 2000 spp. worldwide. However, species delimitation within the genus is often controversial. This is particularly true in the section Calochroi (incl. section Fulvi), where the number of accepted taxa in Europe ranges between c.60 and c.170 according to different taxonomic schools. Here, we evaluated species delimitation within this taxonomically difficult group of species and estimated their phylogenetic relationships. Species were delimited by phylogenetic inference and by comparison of ITS sequence data in combination with morphological characters. A total of 421 ITS sequences were analyzed, including data from 53 type specimens. The phylogenetic relationships of the identified species were estimated by analyzing ITS data in combination with sequence data from the two largest subunits of RNA polymerase II (RPB1 and RPB2). Seventy-nine species were identified, which are believed to constitute the bulk of the diversity of this group in Europe. The delimitation of species based on ITS sequences is more consistent with a conservative morphological species concept for most groups. ITS sequence data from 30 of the 53 types were identical to other taxa, and most of these can be readily treated as synonyms. This emphasizes the importance of critical analysis of collections before describing new taxa. The phylogenetic separation of species was, in general, unambiguous and there is considerable potential for using ITS sequence data as a barcode for the group. A high level of homoplasy and phenotypic plasticity was observed for morphological and ecological characters. Whereas most species and several minor lineages can be recognized by morphological and ecological character states, these same states are poor indicators at higher levels.     

Molecular phylogenetics of Australo-Papuan possums and gliders (family Petauridae)

Phylogenetic relationships within the possums of the family Petauridae, including their affinities with the family Pseudocheiridae, were inferred from DNA sequences obtained for the mitochondrial ND2 gene (1040 bp) combined with previously published partial 12S rDNA sequences. Short, deep internodes characterize some of the divergences obtained. The robustness of these nodes was assessed by several methods such as exclusion of taxa and partitioning of characters. In all analyses a monophyletic Pseudocheiridae was evident, whereas a monophyletic Petauridae was not as well supported. Within the Petauridae, Gymnobelideus was more closely related to Dactylopsila-Dactylonax than to Petaurus. This supports the results obtained from microcomplement fixation of albumin and DNA-DNA hybridization studies but conflicts with morphological data.     

Molecular phylogenetics and historical biogeography of Hawaiian Dryopteris (Dryopteridaceae)

The fern genus Dryopteris (Dryopteridaceae) is represented in the Hawaiian Islands by 18 endemic taxa and one non-endemic, native species. The goals of this study were to determine whether Dryopteris in Hawai'i is monophyletic and to infer the biogeographical origins of Hawaiian Dryopteris by determining the geographical distributions of their closest living relatives. We sequenced two chloroplast DNA fragments, rbcL and the trnL-F intergenic spacer (IGS), for 18 Hawaiian taxa, 45 non-Hawaiian taxa, and two outgroup species. For individual fragments, we estimated phylogenetic relationships using Bayesian inference and maximum parsimony. We performed a combined analysis of both cpDNA fragments employing Bayesian inference, maximum parsimony, and maximum likelihood. These analyses indicate that Hawaiian Dryopteris is not monophyletic, and that there were at least five separate colonizations of the Hawaiian Islands by different species of dryopteroid ferns, with most of the five groups having closest relatives in SE Asia. The results suggest that one colonizing ancestor, perhaps from SE Asia, gave rise to eight endemic taxa (the glabra group). Another colonizing ancestor, also possibly from SE Asia, gave rise to a group of five endemic taxa (the exindusiate group). Dryopteris fusco-atra and its two varieties, which are endemic to Hawai'i, most likely diversified from a SE Asian ancestor. The Hawaiian endemic Nothoperanema rubiginosum has its closest relatives in SE Asia, and while the remaining two species, D. wallichiana and D. subbipinnata, are sister species, their biogeographical origins could not be determined from these analyses due to the widespread distributions of D. wallichiana and its closest non-Hawaiian relative.     

Molecular phylogenetics and character evolution of the "sacaca" clade: novel relationships of Croton section Cleodora (Euphorbiaceae)

Phylogenetic relationships of Croton section Cleodora (Klotzsch) Baill. were evaluated using the nuclear ribosomal ITS and the chloroplast trnL-F and trnH-psbA regions. Our results show a strongly supported clade containing most previously recognized section Cleodora species, plus some other species morphologically similar to them. Two morphological synapomorphies that support section Cleodora as a clade include pistillate flowers in which the sepals overlap to some degree, and styles that are connate at the base to varying degrees. The evolution of vegetative and floral characters that have previously been relied on for taxonomic decisions within this group are evaluated in light of the phylogenetic hypotheses. Within section Cleodora there are two well-supported clades, which are proposed here as subsections (subsection Sphaerogyni and subsection Spruceani). The resulting phylogenetic hypothesis identifies the closest relatives of the medicinally important and essential oil-rich Croton cajucara Benth. as candidates for future screening in phytochemical and pharmacological studies.     

Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness

Members of tribe Vandeae (Orchidaceae) form a large, pantropical clade of horticulturally important epiphytes. Monopodial leafless members of Vandeae have undergone extreme reduction in habit and represent a novel adaptation to the canopy environment in tropical Africa, Asia, and America. To study the evolution of monopodial leaflessness, molecular and structural evidence was used to generate phylogenetic hypotheses for Vandeae. Molecular analyses used sequence data from ITS nrDNA, trnL-F plastid DNA, and matK plastid DNA. Maximum parsimony analyses of these three DNA regions each supported two subtribes within monopodial Vandeae: Aeridinae and a combined Angraecinae + Aerangidinae. Adding structural characters to sequence data resulted in trees with more homoplasy, but gave fewer trees each with more well-supported clades than either data set alone. Two techniques for examining character evolution were compared: (1) mapping vegetative characters onto a molecular topology and (2) tracing vegetative characters onto a combined structural and molecular topology. In both cases, structural synapomorphies supporting monopodial Vandeae were nearly identical. A change in leaf morphology (usually reduced to a nonphotosynthetic scale), monopodial growth habit, and aeration complexes for gas exchange in photosynthetic roots seem to be the most important characters in making the evolutionary transition to leaflessness.     

Molecular phylogenetics of Thysanoptera

Analyses of morphological data on the higher-level phylogenetics of Thysanoptera have suggested two alternative hypotheses: (1) sister-taxon status of the monophyletic suborders Terebrantia and Tubulifera. and (2) paraphyly of Terebrantia with respect to Tubulifera. Analyses of nucleotide sequence data from the mitochondrial gene cytochrome oxidase I and the nuclear gene I8S rONA using maximum parsimony. neighbour joining. and maximum likelihood provide strong support for the hypothesis of a sister-taxon relationship between the Terebrantia and Tubulifera. These data resolve this long-standing controversy in thysanopteran phylogenetics and provide a framework for further studies of higher-level relationships in this order.     

Molecular phylogenetics of tribe Poranthereae (Phyllanthaceae; Euphorbiaceae sensu lato)

Novel insights into the evolutionary history of a taxonomically complex tropical plant group were gained in this study using DNA sequence data. A molecular phylogenetic analysis of the newly circumscribed and expanded tribe Poranthereae (Phyllanthaceae) is presented. Sampling included 97 accessions for 63 of c. 120 species. Largely congruent results have been obtained from nuclear ribosomal ITS and plastid matK sequences. These analyses support the recognition of Andrachne and Leptopus as distinct genera. The deceptively similar Andrachne section Phyllanthopsis, Andrachne ovalis, and Leptopus decaisnei are separate lineages to be segregated. Zimmermannia and Zimmermanniopsis are embedded in Meineckia; Oreoporanthera is embedded in Poranthera; and Archileptopus is embedded in Leptopus. Andrachne section Pseudophyllanthus is polyphyletic, the two Madagascan endemics emerging as a sister clade to Meineckia. The noncontiguous distributions of Andrachne sensu lato and Leptopus sensu lato were found to be the result of separate evolutionary histories of morphologically similar clades, whereas Andrachne sensu stricto and Meineckia remain geographically disjunct. Actephila and Leptopus are sisters with a sympatric distribution in humid Asia. Presence of petals appears to be plesiomorphic for the tribe. Petals are reduced in Actephila and Oreoporanthera and lost in the Meineckia clade.     

Boletus roseoflavus,a new species of Boletus in section Appendiculati from China

Boletus roseoflavus is described as a new species based on morphological and molecular studies of the type collection and additional materials. Boletus roseoflavus is morphologically distinct from other related species (B. regius, B. appendiculatus, and B. speciosus) by its light pink, pink, or rosy pileus, lemon yellow or golden yellow context, and lemon yellow or light yellow, enlarged download stipe with yellow reticulation. Phylogenetic analyses based on ribosomal DNA internal transcribed spacer regions and nuclear large subunit ribosomal RNA indicate that B. roseoflavus belongs to Boletus section Appendiculati.     

Molecular phylogenetics of Candida albicans

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.     

Molecular phylogenetics reveals Leontodon (Asteraceae, Lactuceae) to be diphyletic

The plastid matK gene, trnL/F spacer, and nuclear rDNA ITS were sequenced for 36 species of Leontodon and 29 taxa of related genera of tribe Lactuceae. Phylogenetic relationships inferred from the independent and combined data are largely congruent and reveal that Leontodon sensu lato (s.l.) as presently defined is diphyletic: L. subgenus Leontodon forms a clade with Helminthotheca, Picris and Hypochaeris as sister genera, whereas L. subgenus Oporinia appears as a separate clade with strong bootstrap support and is thus better treated as a separate genus. Previous sectional classifications of Leontodon s.l. are considered in the light of DNA and additional morphological and karyological data. Support is presented for a core group of Hypochaeridinae sensu stricto (s.s.) with the two clades of Leontodon s.l., Helminthotheca, Picris, and Hypochaeris, whereas Urospermum, Hyoseris, Aposeris, and Rhagadiolus appear to be positioned more distantly.     

Molecular phylogenetics and evolutionary diversification of labyrinth fishes (Perciformes: Anabantoidei)

Labyrinth fishes (Perciformes: Anabantoidei) are primary freshwater fishes with a disjunct African-Asian distribution that exhibit a wide variety of morphological and behavioral traits. These intrinsic features make them particularly well suited for studying patterns and processes of evolutionary diversification. We reconstructed the first molecular-based phylogenetic hypothesis of anabantoid intrarelationships using both mitochondrial and nuclear nucleotide sequence data to address anabantoid evolution. The mitochondrial data set included the complete cytochrome b, partial 12S rRNA, complete tRNA Val, and partial 16S rRNA genes (3332 bp) of 57 species representing all 19 anabantoid genera. The nuclear data set included the partial RAG1 gene (1494 bp) of 21 representative species. The phylogenetic analyses of a combined (mitochondrial+nuclear) data set recovered almost fully resolved trees at the intrafamily level with different methods of phylogenetic inference. Phylogenetic relationships at this taxonomic level were compared with previous morphology-based hypotheses. In particular, the enigmatic pike-head (Luciocephalus) was confidently placed within the "spiral egg" clade, thus resolving the long-standing controversy on its relative phylogenetic position. The molecular phylogeny was used to study the evolution of the different forms of parental care within the suborder. Our results suggest that the evolution of breeding behavior in anabantoids is highly correlated with phylogeny, and that brood care evolved three times independently from an ancestral free spawning condition without parental care. Ancestral character state reconstructions under maximum parsimony and maximum likelihood further indicated that both bubble nesting and mouthbrooding have evolved recurrently during anabantoid evolution. The new phylogenetic framework was also used to test alternative biogeographic hypotheses that account for the disjunct African-Asian distribution. Molecular divergence time estimates support either a drift vicariance linked to the breakup of Gondwana or Late Mesozoic Early Tertiary dispersal from Africa to Asia or vice versa.     

Molecular phylogenetics and evolution of the endemic Hawaiian genus Adenophorus (Grammitidaceae)

Recent studies of the phylogeny of several groups of native Hawaiian vascular plants have led to significant insights into the origin and evolution of important elements of the Hawaiian flora. No groups of Hawaiian pteridophytes have been subjected previously to rigorous phylogenetic analysis. We conducted a molecular phylogenetic analysis of the endemic Hawaiian fern genus Adenophorus employing DNA sequence variation from three cpDNA fragments: rbcL, atpbeta, and the trnL-trnF intergenic spacer (IGS). In the phylogenetic analyses we employed maximum parsimony and Bayesian inference. Bayesian phylogenetic inference often provided stronger support for hypothetical relationships than did nonparametric bootstrap analyses. Although phylogenetic analyses of individual DNA fragments resulted in different patterns of relationships among species and varying levels of support for various clades, a combined analysis of all three sets of sequences produced one, strongly supported phylogenetic hypothesis. The primary features of that hypothesis are: (1) Adenophorus is monophyletic; (2) subgenus Oligadenus is paraphyletic; (3) the enigmatic endemic Hawaiian species Grammitis tenella is strongly supported as the sister taxon to Adenophorus; (4) highly divided leaf blades are evolutionarily derived in the group and simple leaves are ancestral; and, (5) the biogeographical origin of the common ancestor of the Adenophorus-G. tenella clade remains unresolved, although a neotropical origin seems most likely.     

DNA sequence analyses reveal abundant diversity, endemism and evidence for Asian origin of the porcini mushrooms

The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species) and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions.     

Molecular phylogenetics and diversification of the genus Sporophila (Aves: Passeriformes)

The evolutionary affinities within and among many groups of nine-primaried oscines remain unresolved. One such group is Sporophila, a large genus of New World tanager-finches. Our study focused particularly on clarifying the relationship between this genus and a closely related one, Oryzoborus, and on examining the phylogenetic affinities of the "capuchinos," a group of 11 Sporophila species that share a similar male plumage coloration pattern. Our phylogenetic analyses, based on 498 bp of mitochondrial DNA sequence, indicated that: (1) Oryzoborus is embedded within a well-supported clade containing all Sporophila species, which strongly suggests that both genera should be merged, (2) the species of capuchinos comprise a monophyletic group, implying that the plumage patterns common to all probably arose only once, and (3) the capuchinos clade is comprised of two sub-clades, one including two species that are distributed in northern South America and the other one containing eight species that are present south of the Amazon River. Mean sequence divergence among the southern capuchinos species was extremely low, suggesting a rapid radiation within the last half-million years that may be related to the high level of sexual selection present in the genus and might have been promoted by marine ingressions and egressions that occurred in some southern coastal regions of South America in the Late Pleistocene.