Orbilia querci sp. nov. and its knob-forming nematophagous anamorph

Orbilia querci, a new nematode-trapping fungus, was found on rotten wood of Quercus sp. in Huai-rou County, Beijing, China. It is characterized by having a tear-shaped spore body in the cylindrical ascospore. Pure culture was obtained from the ascospores. Conidiophores were simple or occasionally branched, bearing a single conidium on the tip. Conidia were spindle-shaped, mostly with 3-septa. Nematodes were captured by means of adhesive stalked knobs. The adhesive knobs were produced frequently on nutritional agar plates even in the absence of challenging nematodes. Its anamorph is placed in Dactylellina and named as D. querci. The sequence divergence of the ITS1 region between the fungus and the other knob-forming species tested was 23.8-33.4%, supporting O. querci as a distinct species. This is the first report of the connection between a knob-forming nematophagous hyphomycete and an Orbilia teleomorph.     

Phylogeny and taxonomy of the North American clade of the Ceratocystis fimbriata complex

Ceratocystis fimbriata is a widely distributed, plant pathogenic fungus that causes wilts and cankers on many woody hosts. Earlier phylogenetic analyses of DNA sequences revealed three geographic clades within the C. fimbriata complex that are centered respectively in North America, Latin America and Asia. This study looked for cryptic species within the North American clade. The internal transcribed spacer regions (ITS) of the rDNA were sequenced, and phylogenetic analysis indicated that most isolates from the North American clade group into four host-associated lineages, referred to as the aspen, hickory, oak and cherry lineages, which were isolated primarily from wounds or diseased trees of Populus, Carya, Quercus and Prunus, respectively. A single isolate collected from P. serotina in Wisconsin had a unique ITS sequence. Allozyme electromorphs also were highly polymorphic within the North American clade, and the inferred phylogenies from these data were congruent with the ITS-rDNA analyses. In pairing experiments isolates from the aspen, hickory, oak and cherry lineages were interfertile only with other isolates from their respective lineages. Inoculation experiments with isolates of the four host-associated groupings showed strong host specialization by isolates from the aspen and hickory lineages on Populus tremuloides and Carya illinoensis, respectively, but isolates from the oak and cherry lineages did not consistently reveal host specialization. Morphological features distinguish isolates in the North American clade from those of the Latin American clade (including C. fimbriata sensu stricto). Based on the phylogenetic evidence, interfertility, host specialization and morphology, the oak and cherry lineages are recognized as the earlier described C. variospora, the poplar lineage as C. populicola sp. nov., and the hickory lineage as C. caryae sp. nov. A new species associated with the bark beetle Scolytus quadrispinosus on Carya is closely related to C. caryae and is described as C. smalleyi.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon

Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and β-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The β-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS subclade Ib included the P. indigoferae ex-type sequence, this group was considered to be P. vexans since South African isolates in this clade produced globose sporangia. Group C contained four apple isolates that were related to, but distinct from P. cucurbitacearum. Although P. vexans groups A-C might be distinct species, they are not described here as such due to (i) these groups only representing some of the known diversity in P. vexans, (ii) conflicting gene tree phylogenies preventing phylogenetic species identification, and (iii) sexually sterile isolates preventing the broad application of biometrical data.     

Intraspecies Analysis: Comparison of ITS Sequence Data and Gene Intron Sequence Data with Breeding Data for a Worldwide Collection of Gonium pectorale

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis. Received: 9 March 1998 / Accepted: 1 June 1998     

Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

Among many isolates that resulted from four independent surveys of yeasts associated with plants in Brazil, the USA, Portugal and Taiwan, we have characterized eighteen basidiomycetous strains, two of which were conspecific with the type strain of Rhodotorula acheniorum, whereas the remaining sixteen isolates appeared not to correspond to any previously described species. Microsatellite-PCR fingerprinting with primers M13 and (GTG)5 confirmed that the latter strains formed three genetically distinct groups. Each group was considered to represent a distinct species based on nucleotide sequences of the D1/D2 domains of the 26S rRNA gene and the internal transcribed spacer (ITS) region. Phylogenetic analyses of sequence data placed the putative novel species in a clade with R. acheniorum and the dimorphic smut fungus Farysia chardoniana. A novel anamorphic genus, Farysizyma, is created to accommodate the three undescribed species, which were named Farysizyma itapuensis, Farysizyma setubalensis and Farysizyma taiwaniana. A new combination, Farysizyma acheniorum, is proposed for R. acheniorum, which may represent the yeast-phase anamorph of Farysia thuemenii.     

Aurapex penicillata gen. sp. nov. from native Miconia theaezans and Tibouchina spp. in Colombia

Conidiomata of a fungus resembling Chrysoporthe cubensis, a serious canker pathogen of Eucalyptus spp. (Myrtaceae, Myrtales) in tropical and subtropical parts of the world, was found on Eucalyptus grandis in Colombia. Fruiting structures of the fungus could be distinguished from those of C. cubensis by their distinctly orange conidiomatal necks. This fungus also was found on several plant species native to Colombia including Tibouchina urvilleana, T. lepidota and Miconia theaezans (Melastomataceae, Myrtales). Morphological comparisons, as well as those based on sequences of the ITS1/ITS2 region of the ribosomal DNA repeat and the beta-tubulin gene, were used to characterize this fungus. Its pathogenicity was assessed on various plants from which it has been collected, either in field or greenhouse trials. Phylogenetic analyses showed that isolates reside in a clade distinct from the four clades accommodating Chrysoporthe, Cryphonectria, Endothia and Rostraureum. Members of this clade are distinguished by the presence of orange conidiomatal necks with black bases and a unique internal stromatal structure. No teleomorph has been found for this fungus, for which we have provided the name Aurapex penicillata gen. sp. nov. A. penicillata produced only small lesions after inoculation on young T. urvilleana, M. theaezans and E. grandis trees and appears not to be a serious pathogen.     

Molecular and morphological characterization of Leveillula (Ascomycota: Erysiphales) on monocotyledonous plants

Leveillula on monocotyledonous plants have been recorded as L. taurica by several authors, whereas the fungus on Allium has been described as an independent species, namely L. allii, by some authors. We sequenced ca 600 bp of the rDNA ITS region for two Leveillula specimens from Allium and Polianthes (both from monocotyledons) and compared them with several already published sequences from Leveillula isolates from dicotyledons. Pair-wise percentages of sequence divergences were calculated for all Leveillula isolates. The ITS sequence of the Polianthes isolate was identical to L. taurica on Helianthus and Vicia. The sequence of the Allium isolate was 99.5 % identical to L. taurica on Euphorbia, Haplophylum, Peganum, etc. These results suggest close relationships between monocot and dicot pathogenic Leveillula species. The identity between two monocot isolates was 98.4 %. Phylogenetic analysis revealed that the two monocot isolates do not group into a clade together. This result suggests that Leveillula acquired parasitism to monocots at least twice independently.     

Putative natural hybrid between Puccinia lagenophorae and an unknown rust fungus on Senecio madagascariensis in KwaZulu-Natal,South Africa

Several specimens of an aecial rust fungus were collected on Senecio madagascariensis during a field survey carried out in KwaZulu-Natal, South Africa. As telia were not present in the specimens collected, DNA sequence analyses were undertaken to determine the identity of the rust species. ITS and β-tub1 sequencing confirmed that one of the isolates recovered is Puccinia lagenophorae sensu lato. On the other hand, sequencing and RFLP analysis revealed the presence of two divergent copies of ITS and β-tub1 in all the other six isolates investigated. In both phylogenetic trees, one copy of the gene region grouped within a well supported clade with sequences of P. lagenophorae accessions from different geographical origins and hosts, and the Australian rusts Puccinia saccardoi and Puccinia stylidii. The other copy of these gene regions grouped within a separate clade comprising European accessions of Puccinia dioicae (ITS) and Uromyces sommerfeltii (β-tub1) that occur on Asteraceae hosts. Multiple copies of these gene regions were not observed in Australian isolates of P. lagenophorae. Our study provides some evidence that an interspecific hybrid rust fungus, with P. lagenophorae as one of its parents, may occur on S. madagascariensis in South Africa. The identity of the other parent remains unknown.     

Description of two new species of Glypthelmins Stafford, 1905 (Digenea: Macroderoididae) in Rana spp. from Mexico, based on morphology and mtDNA and rDNA sequences

Glypthelmins Stafford, 1905 includes 29 putative species commonly found in the intestine and liver of anurans from all over the world but mainly in the Americas. Partial sequences of the cytochrome c oxidase subunit 1 ( cox 1), ribosomal internal transcribed spacer region 2 (ITS2) and the large subunit 28S rDNA gene were obtained and analysed using pairwise distance matrices and parsimony methods in order to characterise the interrelationships between 14 isolates of four nominal species of Glypthelmins recognised on morphological grounds. The highest intra-specific sequence divergence occurred in the cox 1 (18.53%) sequence, followed by that of the ITS2 (5.44%) and 28S (4.63%). Genetic variability was detected between the three isolates originally identified as G. facioi Brenes et al., 1959 from two localities in Mexico and one locality in Costa Rica. Sequence divergence exhibited among these isolates ranged from 10.70 to 11.22%, from 0.48 to 0.97% and from 1.33 to 1.88% for cox 1, ITS2 and 28S, respectively. Phylogenetic analysis combining all three data-sets generated a single most parsimonious tree. The three isolates of G. facioi form a clade, with an isolate collected from frogs in Veracruz State as the sister group to an isolate from Tabasco State + G. facioi from Costa Rica. The information derived from pairwise distance of independent data-sets plus the phylogenetic information indicate that each of the two isolates from Mexico, identified a priori as G. facioi, represent separate species. A re-examination of specimens was carried out and a re-evaluation made of the morphological characters to find reliable differences that had been overlooked. As a consequence, G. brownorumae n. sp. from Tabasco and G. tuxtlasensis n. sp. from Veracruz are described based on molecular and morphological differences.     

Additional data for a new Theileria sp. from China based on the sequences of ribosomal RNA internal transcribed spacers

Theileria sinensis was recently isolated and named as an independent Theileria species that infects cattle in China. To date, this parasite has been described based on its morphology, transmission and molecular studies, indicating that it should be classified as a distinct species. To test the validity of this taxon, the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were cloned and sequenced from three T. sinensis isolates. The complete ITS sequences were compared with those of other Theileria sp. available in GenBank. Phylogenetic analyses based on sequence data for the complete ITS sequences indicate that T. sinensis lies in a distinct clade that is separate from that of T. buffeli/orientalis and T. annulata. Sequence comparisons indicate that different T. sinensis isolates possess unique sizes of ITS1 and ITS2 as well as species-specific nucleotide sequences. This analysis provides new molecular data to support the classification of T. sinensis as a distinct species from other known Theileria spp. based on ITS sequences.     

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region of Pythium mercuriale sp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.     

基于rDNAITS序列分析Alternaria与相似属的关系及A.leucanthemi的分类地位

对Alternaria,Stemphylium,Ulocladium,Nimbya,Embellisia等五个形态相似属的代表性种进行了5.8S/ITS区段序列测定,连同从GenBank下载的相关有性型Pleospora,Lewia的同项资料,由Neighbor-joining方法构建系统发育树。序列对比结果显示,所有参试的菌株/种的5.8SrDNA序列保守程度较高,ITS序列变化较大,ITS1比ITS2变化更大。由5.8S/ITS构建的系统发育树可以足够将供试菌区分到属;系统发育树显示Alternaria,Ulocladium,Nimbya和Embellisia系统学关系较近,又存在一定的分化;Stemphylium聚在相对较远的分支上,与上述四属关系较远,相对独立进化;有性型的Pleospora与无性型的Stemphylium被聚到一个分支,这种不同阶段菌株在遗传基础上的一致,充分证明所用方法及所选测序的rDNA区段对本类群真菌的分类很有价值。Alternarialeucanthemi位于系统发育树的外侧,较为独立进化,与Alternaria的其它种亲缘关系较远,却与Stemphyliumspp.和Bipolarisspp.关系较近,对其目前的分类地位提出质疑。     

Studies on Morinia: recognition of Morinia longiappendiculata sp. nov. as a new endophytic fungus, and a new circumscription of Morinia pestalozzioides

A new coelomycete, Morinia longiappendiculata sp. nov., isolated from living stems of four plant species in central Spain, is described. The distinctive morphological characteristics of this fungus are the production of conidia with long basal and apical appendages on filiform conidiogenous cells that contrasts with the short-appendaged conidia and cylindrical conidiogenic cells of the type species, M. pestalozzioides. Comparative sequence analysis of the ITS rDNA region and fragments of the translation elongation factor 1alpha, actin and chitin synthase 1 genes and the study of the HPLC profiles of the M. longiappendiculata and M. pestalozzioides isolates supported the recognition of the new species. Comparison of the ITS rDNA sequences of the Morinia isolates with GenBank sequences indicated that the genus belongs to the Amphisphaeriaceae with the highest similarity to Bartalinia and Truncatella. Bresadola's original definition of M. pestalozzioides is updated by adding information on conidiogenesis and molecular data. A lectotype and epitype are designated for the species. A study of bioactive metabolites revealed that M. pestalozzioides cultures produced moriniafungin, a novel sordarin analog with potent antifungal activity.     

DNA sequence analysis of herbarium specimens facilitates the revival of Botrytis mali, a postharvest pathogen of apple

The fungus Botrytis cinerea has been widely accepted as the species responsible for causing gray mold decay of apple, although a second species causing apple decay, B. mali, was reported in 1931. Botrytis mali was validly published in 1931, nevertheless it has always been considered a doubtful species. To study the relationship of Botrytis isolates causing gray mold on apple, DNA sequence analysis was employed. Twenty-eight Botrytis isolates consisting of 10 species were sampled, including two B. mali herbarium specimens from apple originally deposited in 1932. The DNA sequence analysis of the beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) genes placed the isolates into groupings with defined species boundaries that generally reflected the morphologically based model for Botrytis classification. The B. cinerea isolates from apple and other host plants were placed in a single clade. The B. mali herbarium specimens however always fell well outside that clade. The DNA sequence analysis reported in this study support the initial work by Ruehle (1931) describing the apple pathogen B. mali as a unique species.     

A new <Emphasis Type="Italic">Dactylella</Emphasis> species from <Emphasis Type="Italic">Orbilia alba</Emphasis>

A new Dactylella species, Dactylella alba was isolated from the ascospores of Orbilia alba collected in Wenshan County, Yunnan Province, China. Conidiophores were either not branched or occasionally branched, bearing divergent sterigmata on the tip with single conidium on each. Conidia were elongated ellipsoids, 1–2 septate, mostly 1 septate. By combining the ITS sequence with morphological characteristics, a new anamorphic species is described and illustrated together with its teleomorph.     

Phylogenetic analyses reveal deeply divergent species lineages in the genus Sphaerobolus (Phallales: Basidiomycota)

Phylogenetic analyses of 27 artillery fungus (Sphaerobolus sp.) isolates were conducted to identify species boundaries in the genus Sphaerobolus. Multiple gene genealogies inferred from maximum likelihood, Bayesian, and maximum-parsimony analyses of sequence data from individual loci (mtSSU, ITS, EF 1-alpha, and LSU) and a combined dataset (mtSSU, ITS, and EF 1-alpha) concordantly indicate the existence of three deeply divergent lineages in the genus Sphaerobolus, each representing a phylogenetic species. These three phylogenetic species correspond to two known species: Sphaerobolus iowensis and Sphaerobolus stellatus, and a newly discovered species. Suprageneric phylogenetic analyses of the mtSSU and LSU datasets containing representatives of related genera of the gomphoid-phalloid clade of Homobasidiomycetes suggested that the undescribed taxon likely is more closely related to S. stellatus than to S. iowensis.     

Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes

The phylogenetic relationships of 51 isolates representing 27 species of Phytophthora were assessed by sequence alignment of 568 bp of the mitochondrially encoded cytochrome oxidase II gene. A total of 1299 bp of the cytochrome oxidase I gene also were examined for a subset of 13 species. The cox II gene trees constructed by a heuristic search, based on maximum parsimony for a bootstrap 50% majority-rule consensus tree, revealed 18 species grouping into seven clades and nine species unaffiliated with a specific clade. The phylogenetic relationships among species observed on cox II gene trees did not exhibit consistent similarities in groupings for morphology, pathogenicity, host range or temperature optima. The topology of cox I gene trees, constructed by a heuristic search based on maximum parsimony for a bootstrap 50% majority-rule consensus tree for 13 species of Phytophthora, revealed 10 species grouping into three clades and three species unaffiliated with a specific clade. The groupings in general agreed with what was observed in the cox II tree. Species relationships observed for the cox II gene tree were in agreement with those based on ITS regions, with several notable exceptions. Some of these differences were noted in species in which the same isolates were used for both ITS and cox II analysis, suggesting either a differential rate of evolutionary divergence for these two regions or incorrect assumptions about alignment of ITS sequences. Analysis of combined data sets of ITS and cox II sequences generated a tree that did not differ substantially from analysis of ITS data alone, however, the results of a partition homogeneity test suggest that combining data sets may not be valid.     

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.     

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.