Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

Liang Bua Homo floresiensis mandibles and mandibular teeth: a contribution to the comparative morphology of a new hominin species

In 2004, a new hominin species, Homo floresiensis, was described from Late Pleistocene cave deposits at Liang Bua, Flores. H. floresiensis was remarkable for its small body-size, endocranial volume in the chimpanzee range, limb proportions and skeletal robusticity similar to Pliocene Australopithecus, and a skeletal morphology with a distinctive combination of symplesiomorphic, derived, and unique traits. Critics of H. floresiensis as a novel species have argued that the Pleistocene skeletons from Liang Bua either fall within the range of living Australomelanesians, exhibit the attributes of growth disorders found in modern humans, or a combination of both. Here we describe the morphology of the LB1, LB2, and LB6 mandibles and mandibular teeth from Liang Bua. Morphological and metrical comparisons of the mandibles demonstrate that they share a distinctive suite of traits that place them outside both the H. sapiens and H. erectus ranges of variation. While having the derived molar size of later Homo, the symphyseal, corpus, ramus, and premolar morphologies share similarities with both Australopithecus and early Homo. When the mandibles are considered with the existing evidence for cranial and postcranial anatomy, limb proportions, and the functional anatomy of the wrist and shoulder, they are in many respects closer to African early Homo or Australopithecus than to later Homo. Taken together, this evidence suggests that the ancestors of H. floresiensis left Africa before the evolution of H. erectus, as defined by the Dmanisi and East African evidence.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Molecular phylogeny of Phoma and allied anamorph genera: Towards a reclassification of the Phoma complex

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.     

RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

Chemical composition and antimicrobial activity of Cymbopogon citratus and Cymbopogon giganteus essential oils alone and in combination

As part of ongoing research on the chemical composition and the antimicrobial properties of Burkinabe plants essential oils alone and in combination, essential oils (EOs) from leaves of Cymbopogon citratus and Cymbopogon giganteus from Burkina Faso were analyzed by GC-FID and GC-MS. Five constituents, which accounted for 96.3% of the oil, were identified in the EO of C. citratus. Geranial (48.1%), neral (34.6%) and myrcene (11.0%) were the major constituents. For C. giganteus a total of eight compounds were identified which represented 86.0% of the oils extracted. The dominant compounds were limonene (42%) and a set of monoterpene alcohols: trans-p-mentha-1(7),8-dien-2-ol (14.2%), cis-p-mentha-1(7),8-dien-2-ol (12%), trans-p-mentha-2,8-dien-1-ol (5.6%) and cis-p-mentha-2,8-dien-1-ol (5.2%). The EOs were tested against nine bacteria by using disc diffusion and microdilution methods. C. giganteus EO showed antimicrobial effects against all microorganisms tested whereas C. citratus EO failed to inhibit Pseudomonas aeruginosa. The antimicrobial activity of combinations of the two EOs was quantified by the checkerboard method. Combinations of the two EOs exerted synergistic, additive and indifferent antimicrobial effects. Results of the present investigation provide evidence that the combinations of plant EOs could be assessed for synergistic activity in order to reduce their minimum effective dose.     

Essential mycorrhizal partners of the endemic Chilean orchids Chloraea collicensis and C. gavilu

Orchids are obligate mycoheterotrophic plants, relying on fungal nutrient resources to grow for their entire life or until they develop into photosynthetic seedlings. In Chile, orchids are represented by 7 genera and 63 species, 27 of which are endemic. Some Chilean species are considered endangered or rare, but many are insufficiently known. This study aims to isolate, culture, and identify fungal species found in symbiosis with the endemic Chilean orchids Chloraea collicensis Kraenzl. and Chloraea gavilu Lindl. for their potential to be used in future conservation programs. Roots of both species of orchids were collected in the field and those presenting pelotons were firstly cultured in agar-water and thereafter sub-cultured in potato dextrose agar media. Fungal colony growth was measured under the dissecting microscope. Fungal isolates from C. gavilu showed a higher growth rate than isolates from C. collicensis and could be used as inoculum for seed germination in further studies. Isolated colonies showed morphological characteristics of the form genus Rhizoctonia and presented two nuclei per cell. The ITS-nrDNA sequences confirmed their morphological identification as species of Tulasnella.     

空心莲子草响应南方菟丝子寄生的生长-防御权衡

为探讨全寄生植物南方菟丝子(Cuscuta australis)防治入侵植物空心莲子草(Alternanthera philoxeroides)的可行性,以二者野外天然生长的种群为研究对象,分析南方菟丝子寄生对空心莲子草生长及防御的影响,阐明空心莲子草在受到寄生胁迫时如何权衡自身生长与防御的关系,进而发展出一套应对南方菟丝子寄生的生长-防御策略。结果显示:(1)南方菟丝子寄生显著改变空心莲子草茎的形态,茎直径和平均节间长均增加,茎直径变化极显著(P0.01);(2)南方菟丝子寄生显著减少空心莲子草叶片数,但同时显著增加后者茎的分枝数,而茎上的节是潜在的无性繁殖体,故有利于空心莲子草的克隆繁殖;此外,南方菟丝子寄生显著降低了空心莲子草的根、茎、叶生物量和总生物量,抑制空心莲子草的生长;(3)南方菟丝子寄生显著增加空心莲子草茎的单宁、总酚、三萜皂苷含量,增强其防御能力;(4)南方菟丝子寄生的空心莲子草的生物量与茎部木质素、三萜皂苷、单宁和总酚含量均呈现显著负相关性(P0.01),对照组则不存在相关性;且寄生组较对照组相比,生物量的相对百分比显著低于对照组(P0.01),而用于防御的次生代谢产物总含量的相对百分比显著高于对照组(P0.01)。以上结果表明,受到南方菟丝子寄生胁迫后,空心莲子草改变自身的生长-防御策略,减少营养生长投入而将更多的资源投向克隆繁殖,同时增强对"防御"物质的投入,增强其防御能力,以利于后代生存和繁衍。     

Ecosystem engineers: Interactions between eelgrass Zostera capensis and the sandprawn Callianassa kraussi and their indirect effects on the mudprawn Upogebia africana

Increasing recognition is being given to the concept of ‘ecosystem engineers’, which alter the physical nature of the environment and thereby influence other species by means that are neither competitive nor trophic. This paper examines a case study of such effects, in which negative correlations between the abundances of the sandprawn Callianassa kraussi and the eelgrass Zostera capensis imply mutual exclusion because Z. capensis stabilises sediment and inhibits burrowing by C. kraussi, whereas sandprawn bioturbation smothers eelgrass. To test this, we undertook manipulative experiments in which Z. capensis was transplanted into sandflats where C. kraussi was either left undisturbed or eliminated by defaunation. This demonstrated that (1) Z. capensis thrived and expanded in sandflats in the absence of bioturbation but deteriorated and disappeared if C. kraussi was present. (2) In the short term, introduction of Z. capensis reduced densities of C. kraussi, and in established beds of eelgrass, C. kraussi was rare. (3) The mudprawn Upogebia africana was largely restricted to treatments that contained Z. capensis, and its densities were greater in the absence of C. kraussi than in its presence. The presence of eelgrass and the exclusion of C. kraussi also diminished sediment penetrability, suspension of particles and surface burial rates compared to the situation in undisturbed Callianassa-dominated sandflats. These results confirm a mutually negative interaction between C. kraussi and Z. capensis and strengthen the belief that the underlying mechanisms are the antagonistic effects of bioturbation by C. kraussi versus sediment stabilisation by Z. capensis. U. africana seems to benefit directly from Z. capensis but possibly also benefits indirectly from the exclusion of C. kraussi by Z. capensis, since its filter-feeding mode of life requires sufficient sediment stability to maintain semi-permanent U-tubes.     

Association of a luminous Vibrio sp., taxonomically related to Vibrio harveyi, with Clytia linearis (Thornely, 1900) (Hydrozoa, Cnidaria)

A previously unknown association between a luminous Vibrio sp., taxonomically related to the species Vibrio harveyi and a common member of the shallow/mid water communities of the Mediterranean Sea, the hydrozoan Clytia linearis is described. All the specimens of C. linearis observed under blue light excitation showed both a natural luminescence appearing as a series of fine dots due to clytin, and a clear fluorescence on the external side of the perisarc around the colonies due to the presence of luminous bacteria. Luminous bacteria were isolated from the surface of C. linearis, their phenotypic characterization as isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a Vibrio sp. taxonomically related to V. harveyi. The association of the V. harveyi-related species with C. linearis, as already suggested for another hydroid, Aglaophenia octodonta, could be explained with the activity of these bacteria of feeding on the chitinous structures present in these hydroids. Moreover, the adhesion of the luminous bacterium (here referred to as Vibrio sp. CL1) on C. linearis may contribute to the survival of this Vibrio species in the marine environment providing a suitable growth habitat.     

板栗和锥栗同域居群的空间遗传结构

采用微卫星技术研究了板栗(Castanea mollissima)和锥栗(C. henryi)同域分布居群的空间遗传结构, 探讨了它们的遗传变异空间分布特征及其形成机制。结果表明,采用的7个微卫星在两物种中共扩增出173个等位基因,两物种都具有较高的遗传多样性且种间遗传分化水平较低(F_ST=0.051), 但空间自相关分析揭示板栗和锥栗在同域居群中具有不同的空间遗传结构,锥栗在100 m内有空间遗传结构,而板栗没有;同时,基于亲缘关系系数F_ij的Sp统计值也显示锥栗具有比板栗更强的空间遗传结构(板栗的Sp=0.002;锥栗的Sp=0.018)。居群遗传变异的空间结构是其传粉和种子散播及生境共同作用的结果,其中种子的近距离散播和居群密度可能是主要的因素。板栗和锥栗居群在小尺度上空间遗传结构的差异可能反映了它们种子的大小及扩散过程的差异。     

Study on Cortinarius subgenus Telamonia section Hydrocybe in Europe, with especial emphasis on Mediterranean taxa

In this paper we have attempted to clarify the taxonomy and nomenclature of thirteen taxa of the genus Cortinarius subgenus Telamonia (sections Hydrocybe, Fraternii) well represented in the southwestern Mediterranean area of Europe (C. atrocoeruleus, C. bombycinus, C casimiri, C. contrarius, C. decipiens, C. fraternus, C. gallurae, C. hoffmannii, C. petroselineus, C. sertipes, C. subturibulosus, C. urdaibaiensis and C. vernus). To this end we have performed a combined study of morphological and molecular data (rDNA ITS sequences). The morphological analysis was carried out on 114 collections and the molecular analysis involved 31 of the 114 collections, including 11 type collections (types for C. casimiri and C. fraternus were not available). In addition, a study of spores under field emission scanning electron microscopy (FESEM) was conducted. The results of the combined analysis allowed us to asign the studied material to five species (C. casimiri s.l., C. decipiens s.l., C. gallurae, C. subturibulosus s.l. and C. vernus s.l.). Thus, all collections from more continental areas, which were originally identified as six different taxa (C. atrocoeruleus, C. contrarius, C. decipiens, C. fraternus, C. sertipes, C. flexipes fo. sertipes) corresponded to C. decipiens sensu lato, a widely distributed, genetically and morphologically variable species. Cortinarius casimiri is also found in such habitats, but it is confirmed as distinct taxon. Collections from Mediterranean sclerophyllous communities correspond to C. gallurae, C. vernus sensu lato and C. subturibulosus sensu lato. Due to close phylogenetic relationships we propose the new combinations C. casimiri var. hoffmannii (=C. decipiens var. hoffmannii non C. hoffmannii) and C. subturibulosus var. bombycinus (=C. bombycinus), and the new variety C. vernus var. nevadavernus (=C. vernus H. Lindstr. & Melot sensu auct.).     

Molecular phylogeny and biogeography of the narrow endemic Coelonema and affinitive Draba (Brassicaceae) based on two DNA regions

To clarify the relationship between two genera, Draba and the narrow genus Coelonema, endemic to the QilianMountains of the northeastern Qinghai-Tibet Plateau, phylogenetic analyses were conducted using nuclear ribosomal DNA ITS, and the chloroplast DNA trnL, from Coelonema draboides and 30 species of Draba representing eight sections, including 25 species of Chinese Draba, seven of which were endemic to the study region. The results unambiguously support several previously published proposals to unite Coelonema with Draba and accommodate C. draboides in the latter genus on the basis of morphological re-examination. Our molecular data presented here also provide evidence that these two genera should be combined as a monophyletic group with high support. In addition, it is estimated that Draba may have originated about 1.36–2.71 Mya, with C. draboides diverging from Draba about 0.15–0.31 Mya, based on the molecular calibration of ITS datasets. The assumed speciation and rapid expansion of these two genera is likely to have occurred in the eastern edge of the QilianMountains area according to molecular phylogeny and estimated divergence times, which correspond well with the known geological and paleobotanical histories of the Qinghai-Tibet Plateau.     

The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.     

Phylogenetic position of Octosporea muscaedomesticae (Microsporidia) and its relationship to Octosporea bayeri based on small subunit rDNA analysis

Comparative phylogenetic analysis of the small subunit rDNA sequence of Octosporea muscaedomesticae (Flu, 1911) (type species) (Microsporidia) isolated from the blowfly Phormia regina (Diptera:Calliphoridae) is presented. Neighbor Joining bootstrap, Maximum Parsimony and Maximum Likelihood analyses with 38 microsporidian taxa representing five major clades of Microsporidia placed O. muscaedomesticae on a separate branch within a clade containing parasites of freshwater hosts. O. muscaedomesticae differed from Octosporea bayeri, a parasite of the microcrustacean, Daphnia magna (Cladocera:Daphniidae) by 29% demonstrating that the latter microsporidium is not closely related to the type species at the generic level, and should not be placed within the genus Octosporea, a conclusion that is further supported by morphological and developmental differences. Considering the number of disparately related hosts from which Octosporea species have been previously described based mostly on developmental and morphological characters it is likely that many will not fit the current definition of the genus, and it is possible that molecular analysis of these species will show that this genus as defined represents a polyphyletic grouping of unrelated taxa.     

Identification of Mediterranean Diplodus spp. and Dentex dentex (Sparidae) by means of DNA Inter-Simple Sequence Repeat (ISSR) markers

The aim of this paper was to test the effectiveness of Inter-Simple Sequence Repeat (ISSR) technique in detecting specific molecular markers to genetically discriminate some species of teleost fish. As model species, we used four Mediterranean sea bream species (i.e. Diplodus annularis, D. puntazzo, D. sargus sargus, and D. vulgaris), and the common Mediterranean dentex Dentex dentex. Furthermore, the occurrence in our samples of two partially albino sparids (suspected, on a morphological basis, to probably belong to D. sargus sargus and to D. dentex, respectively) allowed ISSRs to be tested on doubtful specimens. Eight ISSR primers produced 95 out of 97 scorable polymorphic loci with a large number (63) of species-private bands. Maximum parsimony analysis, ΔK statistics, and assignment test were consistent in separating the five species and solving the taxonomic position of specimens of dubious attribution. The results obtained evidence the usefulness of ISSR markers in specimen and species identification, particularly when the morphological traits of fish make the classification problematic.     

Molecular and morphological relationships between two closely related species, Turbinaria ornata and T. conoides (Sargassaceae,Phaeophyceae)

We employed four markers (two chloroplastic, one mitochondrial and one nuclear) to investigate whether Turbinaria ornata and Turbinaria conoides, two morphologically similar brown algae, are distinct species. The molecular data revealed reciprocal monophyly, thus confirming the two algae as discrete species. Nevertheless, taxonomical ambiguities remain. T. conoides is more morphologically variable than T. ornata. We identified an atypical T. conoides morphotype whose blade possesses intramarginal teeth, a morphological characteristic of the T. ornata group that is normally absent within T. conoides. The molecular data indicate, nonetheless, that this morphotype belongs genetically to the T. conoides group. Thus, the results cast doubt upon the validity of using intramarginal teeth for species diagnosis.