Differential patterns of ophiostomatoid fungal communities associated with three sympatric Tomicus species infesting pines in south-western China, with a description of four new species

Bark beetles and their associated fungi, which cause forest decline and sometimes high mortality in large areas around the world, are of increasing concern in terms of forest health. Three Tomicus spp. (T.brevipilosus, T.minor and T.yunnanensis) infect branches and trunks of Pinusyunnanensis and P.kesiya in Yunnan Province, in south-western China. Tomicus spp. are well known as vectors of ophiostomatoid fungi and their co-occurrence could result in serious ecological and economic impact on local forest ecosystems. Nonetheless, knowledge about their diversity, ecology, including pathogenicity and potential economic importance is still quite rudimentary. Therefore, an extensive survey of ophiostomatoid fungi associated with these Tomicus species infesting P.yunnanensis and P.kesiya was carried out in Yunnan. Seven hundred and seventy-two strains of ophiostomatoid fungi were isolated from the adult beetles and their galleries. The strains were identified based on comparisons of multiple DNA sequences, including the nuclear ribosomal large subunit (LSU) region, the internal transcribed spacer regions 1 and 2, together with the intervening 5.8S gene (ITS) and the partial genes of β-tubulin (TUB2), elongation factor 1α (TEF1-α) and calmodulin (CAL). Phylogenetic analyses were performed using maximum parsimony (MP) as well as maximum likelihood (ML). Combinations of culture features, morphological characters and temperature-dependent growth rates were also employed for species identification. Eleven species belonging to five genera were identified. These included six known species, Esteyavermicola, Leptographiumyunnanense, Ophiostomabrevipilosi, O.canum, O.minus and O.tingens and four novel taxa, described as Graphilbumanningense, O.aggregatum, Sporothrixpseudoabietina and S.macroconidia. A residual strain was left unidentified as Ophiostoma sp. 1. The overall ophiostomatoid community was by far dominated by three species, representing 87.3% of the total isolates; in decreasing order, these were O.canum, O.brevipilosi and O.minus. Furthermore, the ophiostomatoid community of each beetle, although harbouring a diversity of ophiostomatoid species, was differentially dominated by a single fungal species; Ophiostomacanum was preferentially associated with and dominated the ophiostomatoid community of T.minor, whereas O.brevipilosi and O.minus were exclusively associated with and dominated the ophiostomatoid communities of T.brevipilosus and T.yunnanensis, respectively. Eight additional species, representing the remaining 12.7% of the total isolates, were marginal or sporadic. These results suggested that sympatric Tomicus populations are dominated by distinct species showing some level of specificity or even exclusivity.

Related papers:

title: Fungi associated with Tomicus piniperda in Poland and assessment of their virulence using Scots pine seedlings
abstract: The species composition and virulence of fungi associated with Tomicus piniperda were studied at eight locations in Poland. The fungi were isolated from phloem and sapwood samples taken from insect galleries and then identified. The virulence of the most common ophiostomatoid species was evaluated through inoculations using two-year-old Scots pine seedlings. A great diversity of fungi were found associated with T. piniperda, including 4 837 cultures and 67 species. The most important groups of fungi were the ophiostomatoids and moulds, including mainly Penicillium, Trichoderma and Mucor species. Among ophiostomatoid fungi, Ophiostoma minus and O. piceae dominated, with a frequency of occurrence of 32.4 and 11.5% of inspected galleries, respectively. Occasionally isolated species included Leptographium lundbergii, L. procerum, L. wingfieldii, Graphium pycnocephalum and Graphium sp. 'W'. In general, the frequency of the ophiostomatoid species was highly variable among locations. Leptographium wingfieldii and O. minus, were the only species capable of killing whole plants and penetrated deeper into the sapwood than other species (87-100% mortality during the 11 week incubation period). Other fungi, including O. piliferum, O. piceae and L. procerum, were considerably less virulent. associated fungi / Leptographium / Ophiostoma / Pinus sylvestris / Tomicus piniperda / virulence

title: Novel associations between ophiostomatoid fungi, insects and tree hosts: current status—future prospects
abstract: Associations between fungal tree pathogens and insects have been recognized for at least 100 years. An important group of these fungi, termed ‘ophiostomatoid fungi’ on account of their morphological similarity, are represented by genera in the families Ceratocystidaceae and Ophiostomataceae. Associations between these fungi, tree-colonizing insects, and host trees have been actively researched since their first discovery. Human activities have led to the global movement of fungi from both families, resulting in the establishment of new and sometimes damaging associations between these fungi, insects and trees. Recent ‘black swan’ events have resulted in an unprecedented increase of ambrosia and bark beetle-associated diseases of forest and fruit trees. We revisit some of the most important emergent diseases caused by the ophiostomatoid fungi, outline the reasons behind the emergence of these diseases, and consider long-term prospects regarding the threats that they pose to forestry and agriculture.

title: Ophiostomatoid fungi associated with the invasive pine-infesting bark beetle, Dendroctonus valens, in China
abstract: State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology (IOZ), Chinese Academy of Sciences (CAS), Beijing, China Tree Protection Co-operation Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa China Eucalypt Research Center (CERC), Chinese Academy of Forestry (CAF), Zhanjiang, Guangdong, China

title: Description of the Yunnan shoot borer, Tomicus yunnanensis Kirkendall & Fac- coli sp. n. (Curculionidae, Scolytinae), an unusually aggressive pine shoot beetle from southern China, with a key to the species of Tomicus
abstract: We describe a new and highly aggressive species of pine shoot beetle, Tomicus yunnanensis Kirkendall & Faccoli, which has been decimating Pinus yunnanensis forests in southwest China for almost three decades. This species was confused with T. piniperda until recent molecular studies showed the SW China populations to be quite divergent from T. piniperda of northeast China and Europe. The clearest morphological differences between these two species lie in the surface sculpture of the elytra: the new species has more widely spaced interstrial granules on the elytral disc, the punctures of interstria 2 on the declivity arranged irregularly and those of striae 1 and 3 smaller. The new species also has dense small hairs on the tip of the antennal club, while T. piniperda has only scattered small hairs on that segment.  T. piniperda specimens are uniformly black, while those of T. yunnanensis have the bulk of the elytra lighter than the base of the elytra and the pronotum. The new species is actually more similar to the Mediterranean species T. destruens, which differs in geographical distribution and in having the punctures of interstria 2 dense on the declivity and light-colored antennae. Species of Tomicus are of general concern to foresters because of their impact on conifer growth, but good illustrations for many species are lacking. We here provide a detailed key to all seven species of the genus (T. minor, T. piniperda, T. destruens, T. brevipilosus, T. yunnanensis and the virtually unknown T. puellus and T. pilifer) as well as diagnostic photographs and drawings. We summarize the biological differences between the new species and T. piniperda and recommend improved communication between taxonomists and forest entomologists, as avoidable taxonomic confusion such as that of T. yunnanensis and T. destruens with T. piniperda hinders the combatting of outbreaks of forest insects.

title: Chapter 10 – The Genus Tomicus
abstract: The genus Tomicus includes eight species distributed in Europe and/or Asia, with one of them (T. piniperda) having been introduced to North America. Tomicus are monogamous and univoltine species that develop on pines, except for one species that feeds on Picea. Their life cycle alternates between reproductive attacks on stem and sexual maturation in shoots. A species identification key, the phylogenetic tree, and the host ranges are presented. Only the biology of three European species and one Asian species is known sufficiently in detail. They are associated with a large diversity of organisms under the bark of the host tree and whenever possible their role is discussed. They are generally unable to overcome the resistance of healthy trees and thus develop on very weak or recently killed trees, but they can cause considerable damage to shoots, thus sometimes becoming a weakening factor. Factors affecting population abundances and host susceptibility are discussed. Finally, economic and ecological impacts are presented, followed by population management aspects.

title: Interspecific Competition between Tomicus yunnanensis and T.minor(Col.Scolytidae)During Shoot-feeding Period in Yunnan of China
abstract: The possible interspecific competition between Tomicus yunnanensis and T.minor during shoot feeding was studied from May 2000 to May 2001 in Yunnan,China.The vertical distribution of both species in crowns of Yunnan pine,Pinus yunnanensis was studied in the field.The results indicated that both T.minor and T.yunnanensis had own seasonal feeding activity to vertical distribution in crowns of Yunnan pine trees.The average diameters of shoot-attacks by T.minor were less than that of T.yunnanensis,and other shoot feeding characteristics(age of shoot-attack,location and length of burrowed tunnels),were described for two species. indicates that T.yunnanensis is more aggressive to Yunnan pine than T.minor during shoot feeding period.

title: Host Preference and Competion in barkbeetles, Tomicus yunnanensis and T.minor in Breeding Period
abstract: The interspecfic competition of barkbeetle Tocmicus yunnanensis and T.minor during breeding period was studied in room conditions. A breeding experiment with logs of Yunnan pine (Pinus yunnanensis) in cages with presence or absence of the competitor was performed. It was shown that T.minor primarily preferred lower part of stems covered with thick bark for its breeding, while T.yunnanensis preferred upper part of stems with thinner bark. The brood development period of T.yunnanensis was shorter than that of T.minor, which was 86-89 d vs 120-125 d with presence and absence of competitor. T.minor presented as a more secondary occupant species than T.yunnanensis, and less competitive to T.yunanensis in occupying new host on Yunnan pine in Yunnan of China.

title: The shoot-feeding ecology of three Tomicus species in Yunnan Province, southwestern China
abstract: Three Tomicus pine shoot beetles, T. yunnanensis (Kirkendall and Faccoli) (Coleoptera: Scolytidae), T. minor (Hartig), and T. brevipilosus (Wood and Bright), have been causing serious damage to Yunnan pine ( Pinus yunnanensis (Franchet) (Pinales: Pinaceae)) stands in Yunnan, southwestern China. However, their ability to coexist in the crowns of the same trees during the shoot-feeding phase has not been elucidated. In our study, we investigated and compared the shoot-feeding ecology of the three species of pine shoot beetle in P. yunnanensis in Anning County, Yunnan Province. Shoot-feeding by T. yunnanensis, T. minor, and T. brevipilosus initiated in April, May, and June, and ended in February, April, and May, respectively. Individual T. yunnanensis and T. minor adults fed in shoots for about seven months, and T. brevipilosus for nine months, before initiating reproduction. All three Tomicus species fed in the current-year shoots close to the apical bud. No specific overwintering behavior was observed prior to reproduction. The entrance hole of T. yunnanensis was furthest away from the apical bud, and T. minor was the closest to the apical bud. Differences in the spatial distribution of these shoot-feeding sites might reduce competition among the three beetle species. The long-lasting and overlapping shoot-feeding by the three Tomicus species may reduce the resistance of P. yunnanensis and facilitate the reproduction of these beetles in the trunks of living trees, and thus help explain the severe damage by Tomicus in P. yunnanensis.

title: Impacts of Tomicus minor on distribution and reproduction of Tomicus piniperda (Col., Scolytidae) on the trunk of the living Pinus yunnanensis trees
abstract: This study investigated the impacts of Tomicus minor on Tomicus piniperda when the two Tomicus species coexist in the trunks of living Yunnan pine (Pinus yunnanensis L.) trees growing in the Kunming region, in south‐western China. Tomicus piniperda mostly locates in the mid‐ and upper trunks of Yunnan pine tree; whereas T. minor mainly attacks the mid‐ and lower trunks. In the mid‐trunk area from 1.0 to 5.0 m above ground, there are overlapping attack zones for the two Tomicus species, which accounts for an average of 80% of the entire zone occupied by T. piniperda and an average of 70% of the zone occupied by T. minor. In correspondence with their attack distributions, the average attack densities of the two species varied with height along the trunk, with 165.3 egg galleries per m2 at a height of 6 m for T. piniperda, and 138.2 egg galleries per m2 at the 1 m height for T. minor. It is suggested that T. minor adjusts its attack pattern with respect to T. piniperda, and thereby minimizes interspecific competition. No remarkable difference of average T. piniperda egg gallery length was found between the zone in which only T. piniperda occurred and the zone in which T. piniperda and T. minor coexisted; this is suggested to be due to low host quality in the upper trunk region where only T. piniperda was present. The number of T. piniperda larval galleries was highest when only T. piniperda was present, and decreased as T. piniperda and T. minor coexisted, particularly in the case when the density of T. piniperda was less than that of T. minor. Average larval density was 1649 larval galleries per m2 where only T. piniperda occurred. However, when T. piniperda coexisted with T. minor, T. piniperda larval density averaged 1010 per m2 when T. piniperda density was higher than T. minor, and averaged 442 per m2 when T. piniperda density was less than T. minor, which led to the conclusion that T. minor makes a negative impact on T. piniperda reproduction when the two Tomicus species jointly colonize the same trunk of Yunnan pine tree.

title: Shoot damage by Tomicus sp. (Coleoptera: Scolytidae) and effect on Pinus yunnanensis resistance to subsequent reproductive attacks in the stem
abstract: 1 In South‐western China, Yunnan pines Pinus yunnanensis, suffer considerable damage from an undescribed Tomicus sp. previously thought to be T. piniperda.

title: A new Leptographium species associated with Tomicus piniperda infesting pine logs in Korea.
abstract: During the course of a survey of sapstaining fungi in Korea, a Leptographium was isolated from Pinus densiflora and P. koraiensis logs, infested with the bark beetle Tomicus piniperda. The fungus grew optimally at 25 C on 2% malt extract agar and showed a high level of tolerance to cycloheximide. The Leptographium has unusually short conidiophores and is morphologically similar to L. pini-densiflorae, L. lundbergii, L. yunnanense, and the Leptographiumn anamorph of Ophiostoma crassivaginatum. Comparisons of DNA sequence data for three gene regions, as well as morphological characteristics, confirmed that this fungus represents an undescribed taxon. We consequently provide the name Leptographium koreanum sp. nov. for it here.

title: Fungi associated with Tomicus minor on Pinus sylvestris in Poland and their succession into the sapwood of beetle-infested windblown trees
abstract: The study identified and measured frequency of fungal species associated with Tomicus minor (Hart.) on Pinus sylvestris L. (Scots pine) in Poland. Additionally, fungal succession in P. sylvestris sapwood was investigated during a 12 week period following an attack by this insect. Fungi were isolated from five populations of overwintered adult beetles and their galleries with 59 species of fungi being represented among the 2880 cultures obtained. The most frequent species, Ophiostoma canum (Munch) Syd. & P. Syd., Hormonema dematioides Lagerb. & Melin, and Ambrosiella tingens (Lagerb. & Melin) L.R. Batra, appeared to be specifically associated with T. minor. The succesional changes in species composition during a 12 week period following an attack by T. minor were observed. The pattern of fungal succession in P. sylvestris sapwood essentially agreed with a general scheme of fungal succession in tree sapwood infested by bark beetles. Ambrosiella tingens was the first invader of sapwood and occurred most freq...

title: Fungal flora associated with Tomicus piniperda L. in an area close to a timber yard in southern Poland
abstract: The association between Tomicus piniperda L.  and fungi was studied in a Pinus sylvestris L. forest in Mielec‐Mościska. Fungi were isolated from overwintered adult beetles taken from two stands situated in different distance from timber yard. Two media were used for isolation. The results showed great diversity of fungi associated with T. piniperda: 1895 cultures, representing 64 species, were isolated. Penicillia and Hormonema dematioides were the dominant species, found in 20.2% and 17.8% of all beetles, respectively. A frequently isolated ophiostomatoid fungi was Ophiostoma minus. Qualitative and quantitative differences in the mycobiota composition of this insect between two stands were detected. The highest richness and diversity of fungal species appeared in the samples taken from the location where the trees were heavily damaged by shoot‐feeding of T. piniperda. Differences were most clear for the pathogenic O. minus, which was a common fungal associate of the insects in this stand.

title: The virulence of phytopathogenic fungi associated with the bark beetles Tomicus piniperda and Orthotomicus erosus in Tunisia
abstract: Phytopathogenic fungi associated with the bark beetles Tomicus piniperda and Orthotomicus erosus were isolated in various pine forests of Tunisia. Tomicus piniperda and its galleries yielded Leptographium wingfieldii, Ophiostoma minus, and Ophiostoma ips. Ophiostoma minus was the most frequent species associated with T. piniperda, in both the attacking and the emerging beetles. It was collected from most investigated forests, whereas O. ips and L. wingfieldii were obtained only from forests located in Central and Northern Tunisia. Frequencies of association with T. piniperda were always low, reaching 11.1% only once, for O. ips. Ophiostoma ips was the only blue stain fungus associated with O. erosus and its galleries. It was found in all the localities, but at a low and variable frequency, exceeding 15% very rarely. The virulence of 16 fungal isolates was tested by single inoculations into Pinus halepensis (Aleppo pine) at two localities. Differences were detected among species; L. wingfieldii was the most virulent and O. minus the least virulent species in terms of phloem reaction zone formation and fungal growth in the phloem. In a separate experiment, mass inoculations (400 and 800 inoculations per m2 of bole on 1-m high belts) were performed with two isolates of L. wingfieldii on Aleppo pine and on Pinus brutia (Brutia pine). Three months later, measurements of sapwood status (% of conductive transversal section) and of its specific hydraulic conductivity, as well as of the phloem reaction zone lengths, did not show any isolate or density effect. At these experimental sites, Brutia pines appeared significantly more susceptible than Aleppo pines.

title: FIRST REPORT OF BLUE-STAIN IN PINUS YUNNANENSIS CAUSED BY OPHIOSTOMA TINGENS ASSOCIATED WITH TOMICUS MINOR IN CHINA
abstract: Yunnan pine (Pinus yunnanensis Franch.) is a crucial economic and ecological tree species in Yunnan Province, China. In February 2015, small pieces of stained phloem were collected from the galleries of Tomicus minor infesting P. yunnanensis and transferred to 2% malt extract agar (MEA) at 20°C. The resulting brown to black colonies had light brown and irregular margins. The globose conidia (5.6–) 6x10 (–11) µm in size exhibited non-phialidic conidiogenesis. The morphological characteristics suggested an identification of Ophiostoma tingens (Lagerb. & Melin) Z.W. de Beer & M.J. Wingf., previously described as Ambrosiella tingens (Lagerb. & Melin) L.R. Batra (Batra, 1967). PCRs were conducted using primers NS1/NS4, ITS3/LR3 and BT2E/BT12 for nSSU, nLSU and β-tubulin, respectively (Alamouti et al., 2009). The obtained sequences of strain YMF 1.04939 (1023 bp, 931 bp and 730 bp) were submitted to GenBank with the accession Nos. MF043741, MF043742 and MF043743. BLASTn analysis showed 99% identity with the corresponding sequences of A. tingens isolate CBS 366.53, now O. tingens (EU170277, AF282871 and EU977468). A pathogenicity test was conducted by placing mycelia and sterile agar with 1 cm iron borers in healthy wood of P. yunnanensis; each treatment had ten repeats. After 10 days at 20°C, the staining in the phloem of wood inoculated with O. tingens reached 3.64 cm in average length, while that for control was 2.18 cm. The fungus was re-isolated only from inoculated wood. Although it has been reported in some European countries (Harrington et al., 2010), this represents the first report that O. tingens causes blue-stain in P. yunnanensis in China.

title: Action mechanisms of phytotoxin produced by Leptographium yunnanense associated with Tomicus piniperda
abstract: Leptographium yunnanense was the main pathogenic fungus associated with Tomicus piniperda attacking Pinus yunnanensis ,which could assist the beetles in overcoming the resistance of the host tree.The phytotoxin of L.yunnanense could restrain the activities of peroxidase(PO),polyphenol oxidase(PPO)and phenylalanine ammonia lyase(PAL) in the tree and thus reduce the resistance of the host tree and benefit the fungus to intrude into the tree successfully.The enzyme activities of Pinus yunnanensis was lower than that of P.armandi ,and restrained stronger by the phytotoxin,this indicated that the resistance of P.armandi was higher than that of P.yunnanensis .And this was an important cause of serious damage of Tomicus piniperda on P.yunnanensis ,while little damage on P.armandi in Yunnan Province.

title: Resistance of conifers to bark beetle attack: Searching for general relationships
abstract: Bark beetles are among the few native insects that can kill large numbers of trees in a single year. The present paper reviews recent work on the relationship between conifer resistance to bark beetle attack and tree vigor, e.g. in terms of wood production per unit of foliage. Experimental studies in the Pacific Northwest and the southeast U.S.A., and in Norway, are drawn upon to show that tree resistance to attack may be closely related to the amount of current and stored photosynthate that is available for defense. An experimental approach is advocated to critically test the relationship between host-tree resistance and the limitations on the transfer of critical resources to the site of attack.

title: Fungal Associates of European Bark Beetles With Special Emphasis on the Ophiostomatoid Fungi
abstract: Fungi are common and well-known associates of bark beetles (Coleoptera: Scolytidae). The relationship between fungi and scolytids was recognized relatively long ago. Schmidberger (1836) described an “ambrosia” in the galleries of the wood-inhabiting bark beetle Xyleborus dispar, and Hartig (1844) discovered the fungal nature of this “ambrosia” lining the tunnels of the insects. Likewise, Hartig (1878) first recognized the interrelationships between insect damage, discoloration of wood and fungi, and during his studies on blue-stain in the sapwood of conifers, Munch (1907, 1908) observed that blue-stain in living trees and lumber is associated with attack by bark beetles. Since these early discoveries a large number of investigations on various aspects of the association of fungi with bark beetles have been carried out. Scolytids are among the most economically important pests of the world s forests, especially conifer forests in the boreal and temperate regions of the Northern hemisphere (Postner 1974; Schwerdtfeger 1981; Wood 1982; Wood and Bright 1992). A considerable number of fungal associates of bark beetles are known as forest pathogens in their own right, causing vascular wilt or vascular stain diseases (Webber and Gibbs 1989; Harrington 1993a, 1993b; Wingfield et al. 1993). Many other species give rise to discoloration in the sapwood of conifers and cause enormous losses to forestry and wood industry worldwide (Whitney 1982; Seifert 1993; Butin 1996). Although the association between scolytids and fungi has been recognised for more than one century, many fundamental aspects of this relationship are still poorly

title: The role of phytopathogenicity in bark beetle-fungus symbioses: a challenge to the classic paradigm.
abstract: The idea that phytopathogenic fungi associated with tree-killing bark beetles are critical for overwhelming tree defenses and incurring host tree mortality, herein called the classic paradigm (CP), has driven research on bark beetle-fungus symbiosis for decades. It has also strongly influenced our views of bark beetle ecology. We discuss fundamental flaws in the CP, including the lack of consistency of virulent fungal associates with tree-killing bark beetles, the lack of correspondence between fungal growth in the host tree and the development of symptoms associated with a successful attack, and the ubiquity of similar associations of fungi with bark beetles that do not kill trees. We suggest that, rather than playing a supporting role for the host beetle (tree killing), phytopathogenicity performs an important role for the fungi. In particular, phytopathogenicity may mediate competitive interactions among fungi and support survival and efficient resource capture in living, defensive trees.

title: Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species
abstract: Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.

title: Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics
abstract: Comparative studies of the nucleotide sequences of ribosomal RNA (rRNA) genes provide a means for analyzing phylogenetic relation­ ships over a wide range of taxonomic levels (Woese and Olsen 1986; Zimmer et al 1988; Medlin et al 1988; ). The nuclear small-subunit rDNA sequences (16S-like) evolve rela­ tively slowly and are useful for studying distantly related organisms, whereas the mitochondrial rRNA genes evolve more rapidly and can be useful at the ordinal or family level. The internal transcribed spacer region and intergenic spacer of the nuclear rRNA repeat units evolve fastest and may vary among species within a genus or among populations. Numerous sequences of rRNA genes have been obtained primarily by isolating and sequencing individual cloned genes (Medlin et al 1988). Direct rRNA sequencing (Lane et al 1985) has also been used to rapidly obtain sequence data. However, this method requires rela­ tively large amounts of RNA and is prone to errors since only one strand is sequenced. The polymerase chain reaction (PCR) and direct sequencing offer several advantages over cloning and direct rRNA sequencing: (1) the method utilizes relatively crude preparations of total DNA such as

title: Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes
abstract: We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.

title: Leptographium wingfieldii introduced into North America and found associated with exotic Tomicus piniperda and native bark beetles.
abstract: Leptographium wingfieldii is a well-known fungal associate of the pine shoot beetle, Tomicus piniperda, in Europe. This fungus is pathogenic to pines and is an important cause of blue-stain in the sapwood of infested trees. Tomicus piniperda was first found in a Christmas tree plantation in Ohio, USA, 1992, but isolation of the fungi associated with these intercepted insects was not attempted. Fungal strains resembling L. wingfieldii were recently isolated from pines attacked by T. piniperda, Dendroctonus valens and Ips pini in the northeastern United States. These strains were morphologically similar to the ex-type and other reference strains of L. wingfieldii. Strains were also compared based on sequences of the partial ITS ribosomal DNA operon, beta-tubulin and elongation factor 1-alpha (EF-1alpha) genes. Based on these DNA sequence comparisons, reference strains of European L. wingfieldii were conspecific with North American strains from pines attacked by T. piniperda, D. valens and I. pini. A single strain from Canada, collected in 1993 near the Ontario border with the USA, shortly after the discovery of T. piniperda in that area and tentatively identified as L. wingfieldii, was also included in this study. Its identification was confirmed, suggesting that L. wingfieldii has been present in this region and probably over the whole range of the insect's distribution for at least a decade. This represents the first record of L. wingfieldii associated with the introduced and damaging pine shoot beetle T. piniperda in North America. It shows that the fungus is well established and can become associated with other native bark beetles that attack stressed and/or dying trees. The occurrence and spread of this highly pathogenic fungus associated with North American bark beetles should be monitored.

title: Phylogeography and evolutionary patterns in Sporothrix spanning more than 14 000 human and animal case reports
abstract: Pathology to vertebrate hosts has emerged repeatedly in the order Ophiostomatales. Occasional infections have been observed in Sporothrix mexicana at a low level of virulence, while the main pathogenic species cluster in a derived clade around S. schenckii s.str. In this paper, phylogeny and epidemiology of the members of this clade were investigated for 99 clinical and 36 environmental strains using four genetic loci, viz. rDNA ITS and partial CAL, TEF1, and TEF3; data are compared with amplified fragment length polymorphism (AFLP) genotyping. The four main species of the pathogenic clade were recognised. The species proved to show high degrees of endemicity, which enabled interpretation of literature data where live material or genetic information is lacking. The clade of four species comprised nine subclusters, which often had limited geographic distribution and were separate from each other in all partitions, suggesting low degrees of interbreeding between populations. In contrast, S. globosa exhibited consistent global distribution of identical AFLP types, suggesting another type of dispersal. Sporothrix brasiliensis is known to be involved in an expanding zoonosis and transmitted by cats, whereas S. globosa infections originated from putrid plant material, causing a sapronosis. Sporothrix schenckii s.str., the most variable species within the clade, also had a plant origin, with ecological similarities to that of S. globosa. A hypothesis was put forward that highly specific conditions in the plant material are required to promote the growth of Sporothrix. Fermented, self-heated plant debris may stimulate the thermodependent yeast-like invasive form of the fungus, which facilitates repeated infection of mammals.

title: MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability
abstract: We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.

title: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods.
abstract: Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.

title: RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models
abstract: RAxML-VI-HPC (randomized axelerated maximum likelihood for high performance computing) is a sequential and parallel program for inference of large phylogenies with maximum likelihood (ML). Low-level technical optimizations, a modification of the search algorithm, and the use of the GTR+CAT approximation as replacement for GTR+Gamma yield a program that is between 2.7 and 52 times faster than the previous version of RAxML. A large-scale performance comparison with GARLI, PHYML, IQPNNI and MrBayes on real data containing 1000 up to 6722 taxa shows that RAxML requires at least 5.6 times less main memory and yields better trees in similar times than the best competing program (GARLI) on datasets up to 2500 taxa. On datasets > or =4000 taxa it also runs 2-3 times faster than GARLI. RAxML has been parallelized with MPI to conduct parallel multiple bootstraps and inferences on distinct starting trees. The program has been used to compute ML trees on two of the largest alignments to date containing 25,057 (1463 bp) and 2182 (51,089 bp) taxa, respectively.


AVAILABILITY
icwww.epfl.ch/~stamatak

title: The divorce of Sporothrix and Ophiostoma: solution to a problematic relationship
abstract: One of the causal agents of human sporotrichosis, Sporothrix schenckii, is the type species of the genus Sporothrix. During the course of the last century the asexual morphs of many Ophiostoma spp. have also been treated in Sporothrix. More recently several DNA-based studies have suggested that species of Sporothrix and Ophiostoma converge in what has become known as Ophiostoma s. lat. Were the one fungus one name principles adopted in the Melbourne Code to be applied to Ophiostoma s. lat., Sporothrix would have priority over Ophiostoma, resulting in more than 100 new combinations. The consequence would be name changes for several economically important tree pathogens including O. novo-ulmi. Alternatively, Ophiostoma could be conserved against Sporothrix, but this would necessitate changing the names of the important human pathogens in the group. In this study, we sought to resolve the phylogenetic relationship between Ophiostoma and Sporothrix. DNA sequences were determined for the ribosomal large subunit and internal transcribed spacer regions, as well as the beta-tubulin and calmodulin genes in 65 isolates. The results revealed Sporothrix as a well-supported monophyletic lineage including 51 taxa, distinct from Ophiostoma s. str. To facilitate future studies exploring species level resolution within Sporothrix, we defined six species complexes in the genus. These include the Pathogenic Clade containing the four human pathogens, together with the S. pallida-, S. candida-, S. inflata-, S. gossypina- and S. stenoceras complexes, which include environmental species mostly from soil, hardwoods and Protea infructescences. The description of Sporothrix is emended to include sexual morphs, and 26 new combinations. Two new names are also provided for species previously treated as Ophiostoma.

title: Multigene phylogenies and morphological characterization of five new Ophiostoma spp. associated with spruce-infesting bark beetles in China.
abstract: Ophiostoma spp. . Some of these are serious tree pathogens, while the majority is blue-stain agents of timber. In recent years, various bark beetle species have been attacking spruce forests in Qinghai province, China, causing significant damage. A preliminary survey was done to explore the diversity of the ophiostomatoid fungal associates of these beetles. The aims of the present study were to identify and characterize new Ophiostoma spp. associated with spruce-infesting bark beetles in Qinghai Province, and to resolve phylogenetic relationships of Ophiostoma spp. related to the Chinese isolates, using multigene phylogenetic analyses. Results obtained from four gene regions (ribosomal internal transcribed spacer regions, β-tubulin, calmodulin, translation elongation factor-1α) revealed five new Ophiostoma spp. from Qinghai. These included O. nitidus sp. nov., O. micans sp. nov., and O. qinghaiense sp. nov. in a newly defined O. piceae complex. The other two new species, O. poligraphi sp. nov. and O. shangrilae sp. nov., grouped in the O. brunneo-ciliatum complex. Based on DNA sequence and morphological comparisons, we also show that O. arduennense and O. torulosum are synonyms of O. distortum, while O. setosum is a synonym of O. cupulatum.

title: The Ophiostoma clavatum species complex: a newly defined group in the Ophiostomatales including three novel taxa
abstract: Two species of blue-stain fungi with similar morphologies, Ophiostoma brunneo-ciliatum and Ophiostoma clavatum, are associates of bark beetles infesting Pinus spp. in Europe. This has raised questions whether they represent distinct taxa. Absence of herbarium specimens and contaminated or mistakenly identified cultures of O. brunneo-ciliatum and O. clavatum have accentuated the uncertainty regarding their correct identification. The aim of this study was to reconsider the identity of European isolates reported as O. brunneo-ciliatum and O. clavatum by applying DNA-based identification methods, and to provide appropriate type specimens for them. Phylogenetic analyses of the ITS, βT, TEF-1α and CAL gene sequences revealed that the investigated isolates represent a complex of seven cryptic species. The study confirmed that ITS data is insufficient to delineate species in some Ophiostoma species clusters. Lectotypes and epitypes were designated for O. clavatum and O. brunneo-ciliatum, and three new species, Ophiostoma brunneolum, Ophiostoma macroclavatum and Ophiostoma pseudocatenulatum, are described in the newly defined O. clavatum-complex. The other two species included in the complex are Ophiostoma ainoae and Ophiostoma tapionis. The results suggest co-evolution of these fungi in association with specific bark beetles. The results also confirm the identity of the fungus associated with the pine bark beetle Ips acuminatus as O. clavatum, while O. brunneo-ciliatum appears to be mainly associated with another pine bark beetle, Ips sexdentatus.

title: Esteya , a new nematophagous genus from Taiwan, attacking the pinewood nematode ( Bursaphelenchus xylophilus )
abstract: Esteya vermicola gen. et sp. nov. isolated from infected pinewood nematodes, Bursaphelenchus xylophilus, is described and illustrated. The new species is characterized by the production of two types of conidiophores, conidiogenous cells, and conidia. The first type of conidiophore is subhyaline to greyish green, flask-shaped, apex tapering at the apex into a thin neck, sometimes crooked; the conidiogenous cells are integrated, phialidic, rarely percurrent; the conidia are hyaline, one-celled, lunate, concave, containing an endospore-like apparatus, adhesive. The second type of conidiophore is subhyaline to greyish green, cylindrical, subulate, septate, the base somewhat swollen; conidiogenous cells integrated, phialidic; conidia hyaline, one-celled, bacilloid, often forming an aggregate droplet at the apex, non-adhesive. Esteya vermicola exhibits high infectivity towards the pinewood nematode, and the potential for its development as a biocontrol agent against the pinewood nematode is briefly discussed.

title: High infection activities of two Esteya vermicola isolates against pinewood nematode
abstract: Esteya vermicola, as the first recorded endoparasitic fungus of pinewood nematode, exhibits high infection activity and shows potential as a biological control agent to combat the devastating pine wilt disease. However, there is still a paucity of data about this rare hyphomycete. In this article, two E. vermicola isolates, CBS 100821 and CBS 115803, were studied in the morphological characteristics and infection activities against pinewood nematode. Although both isolates parasitized the tested nematode, CBS 115803 showed significantly higher infection effectiveness than that of CBS 100821, to kill all the tested nematodes within 3 - 4 days. As to lunate conidia, a novel germination mode was observed and recorded. 
 
   
 
  Pinewood nematode, Esteya vermicola, Infection activity, germination mode of conidia.

title: Morphological, molecular and biological characterization of Esteya vermicola, a nematophagous fungus isolated from intercepted wood packing materials exported from Brazil.
abstract: The nematophagous fungus Esteya vermicola, strain NKF 13222, was purified from an isolate of Bursaphelenchus rainulfi which was intercepted from wood packaging materials originating in Brazil and arriving at Tianjin port in China. The fungus produced two types of conidiogenous cells and conidia, each with different germination modes. More lunate adhesive conidia than bacilloid conidia were produced on nutrient-poor water agar medium. Morphological comparisons revealed the NKF 13222 strain closely resembled the Taiwan strain E. vermicola (ATCC 74485) previously isolated from the pinewood nematode B. xylophilus. Phylogenetic analysis of the β-tubulin and elongation factor 1-α genes indicated that the NKF 13222 grouped with other strains of E. vermicola including the Taiwan strain. This was the first record of E. vermicola from B. rainulfi in South America. Infection tests demonstrated that NKF 13222 was more infective to aphelenchid than tylenchid nematodes and that only lunate adhesive conidia were infectious. The results suggest that the fungus might be a pathogen of plant parasitic nematodes with a broad distribution and provide new information for the potential biocontrol of plant diseases caused by B. xylophilus, Aphelenchoides spp. and Ditylenchus destructor.

title: Specific and promiscuous ophiostomatalean fungi associated with Platypodinae ambrosia beetles in the southeastern United States
abstract: Ambrosia beetles in the subfamily Platypodinae (Coleoptera: Curculionidae) have been farming fungi for over 50 million y, yet they remain understudied and most of their fungal symbionts are unknown. We identified fungal communities associated with all four platypodine species native to the southeastern United States: Euplatypus compositus, Euplatypus parallelus, Myoplatypus flavicornis, and Oxoplatypus quadridentatus. Forty-eight samples were analyzed by quantitative culturing and DNA sequencing. Phylogenetic analyses of 28S rDNA sequences revealed that the four platypodines were routinely associated with several genera in the Ophiostomatales. E. compositus is associated primarily with Raffaelea campbellii 1 and Raffaelea sp. 6 and, to a lesser extent, Raffaelea sp. 2. M. flavicornis is associated with Raffaelea sp. 5. E. parallelus and O. quadridentatus are less specific; the latter mostly associated with Raffaelea cyclorhipidia. Three of the four beetle species were also associated with Ceratocystiopsis spp. This is the first report of Raffaelea associated with E. parallelus, which is invasive in Asia and Africa.

title: Two new Ophiostoma species lacking conidial states isolated from bark beetles and bark beetle-infested Abies species in Japan
abstract: Two ophiostomatoid fungi without conidiophores and conidia were isolated from bark beetles and bark beetle-infested Abies spp. The teleomorph characters were similar to species of Ophiostoma, but the absence of the conidial stage hampered their exact generic assignment. Detailed morphological observations and molecular analysis clarified their generic affiliation. Consequently, they were placed in the genus Ophiostoma, but their morphology and DNA sequences did not coincide with any other species of Ophiostoma. Here, we propose these two species as Ophiostoma aoshimae sp. nov. and Ophiostoma rectangulosporium sp. nov. The former species is characterized by perithecial necks ornamented with projections and ellipsoid to oblong ascospores, and the latter species is characterized by perithecial necks ornamented with rectangular ascospores and white colonies.

title: Phylogeny and taxonomy of the Ophiostoma piceae complex and the Dutch elm disease fungi
abstract: The Ophiostoma piceae complex forms a monophyletic group of insect-dispersed pyrenomycetes with synnemata (Pesotum) and micronematous (Sporothrix) synanamorphs. Other species of Ophiostoma outside ...

title: Characterisation of synnematous bark beetle-associated fungi from China, including Graphium carbonarium sp. nov.
abstract: Ophiostomatoid fungi on trees are typically bark beetle associates that cause sapstain in timber and some are pathogens. Very little is known regarding the ophiostomatoid fungi associated with bark beetles in China and the aim of this study was to identify a collection of these fungi with synnematous anamorphs. Micromorphology and DNA sequences of the internal transcribed spacer regions (ITS) of the ribosomal DNA and the partial β-tubulin gene were used for identifications. The isolates could be divided in six morphological groups. DNA sequence comparisons with published data confirmed that these groups represented six species, four in the Ophiostomatales (Sordariomycetidae) and two in the Microascales (Hypocreomycetidae). The majority of these were isolated from conifer hosts. Ophiostoma quercus, O. setosum, Pesotum fragrans (Ophiostomatales) and Graphium pseudormiticum (Microascales) were found on Tsuga dumosa infested by a Pissodes sp. In addition, O. quercus and P. fragrans were found associated with Tomicus yunnanensis on Pinus yunnanensis, P. fragrans with a Pissodes sp. on P. armandi, and O. piceae with Ips subelongatus on Larix olgensis. Only two species, O. quercus and a new species in the Graphium penicilliodes complex, described here as Graphium carbonarium sp. nov., were isolated from Pissodes galleries on Salix babylonica. These results include several new fungus-host and fungus-insect associations, and G. pseudormiticum is reported here for the first time from China.

title: The teleomorph of Leptographium yunnanense, discovered in crosses among isolates from Thailand, China, and Japan
abstract: A teleomorph was discovered in crosses among isolates of Leptographium yunnanense isolated from Pinus spp. originating in Thailand, China, and Japan. The ascocarps are black, globose to subglobose, and lacking necks. Ascospores are hyaline, 1-celled, surrounded by hyaline sheaths, and appear cucullate in side view, quadrangular in face view, and triangular in end view. Three species were known to have teleomorphs morphologically similar to the present fungus. However, their anamorphs were distinguishable from L. yunnanense. Thus, this teleomorph is described as Grosmannia yunnanensis.

title: A newLeptographium species associated withTomicus piniperda in south-western China
abstract: Tomicus species (Coleoptera: Scolytidae) are serious pests of pines with a wide distribution in Europe, Asia and America. In Yunnan, south-western China,T. piniperda has destroyed more than 0.5 million ha ofPinus yunnanensis in the past 15 years. A blue stain fungus belonging to the genusLeptographium is associated with both the shoot-feeding and trunk-attacking stages of the beetle's life cycle. The fungus is morphologically similar to the anamorph ofOphiostoma crassivaginatum and toL. pyrinum, which are both characterised by short robust conidiophores and hyphae covered by a granular layer. Both these species have been isolated from conifers and are associated with insects. After comparing the fungus fromT. piniperda with similarLeptographium species, using light and scanning electron microscopy, we concluded that it represents a new taxon, which is described here asL. yunnanense sp. nov.

title: Three new Ophiostoma species with Pesotum anamorphs associated with bark beetles infesting Abies species in Nikko, Japan
abstract: Three species of Ophiostoma possessing Pesotum anamorphs isolated from bark beetles and their galleries infesting Abies species in Nikko, Japan, are described as new species. Ophiostoma nikkoense is characterized by brush-shaped synnemata producing long septate clavate conidia, perithecia with neck, and allantoid ascospores. Ophiostoma microcarpum has smaller perithecia with hyphoid ostiolar hyphae on the neck, and the ascospores are cylindrical or ossiform in side and face views. Ophiostoma abieticola has perithecia without ostiolar hyphae on the neck and produces orange-section-shaped or reniform ascospores.

title: Sporothrix brunneoviolacea and Sporothrix dimorphospora, two new members of the Ophiostoma stenoceras-Sporothrix schenckii complex
abstract: Sporothrix inflata is a saprobic member of the Ophiostoma stenoceras-Sporothrix schenckii species complex, reported mainly from soil. Ophiostoma bragantinum, an ascomycete described from Brazil, has been proposed as its possible teleomorph. Previous studies revealed that Sporothrix inflata is phenotypically and genetically variable, suggesting the existence of cryptic species. During a continued survey on the biodiversity of microfungi from different countries, seven isolates morphologically similar to S. inflata were obtained from soil samples collected in Spain and USA. In this study their phenotypic features and phylogenetic relationships were assessed. DNA sequence data of two nuclear loci revealed that these isolates correspond to two unnamed clades in S. inflata s.l., one of which also included the type strain of Humicola dimorphospora, a species that traditionally has been considered a synonym of S. inflata. These two groups are proposed herein as Sporothrix brunneoviolacea sp. nov. and Sporothrix dimorphospora comb. nov. S. brunneoviolacea is characterized phenotypically by the production of a diffusible violet-brown pigment in culture and mostly globose, pigmented, lateral blastoconidia. On the other hand S. dimorphospora lacks diffusible pigments and shows mostly subglobose to obovoid pigmented lateral blastoconidia. In contrast to the type strain of S. inflata S. brunneoviolacea and S. dimorphospora assimilate raffinose. The phylogenetic analysis suggested that the proposed anamorph-teleomorph connection between S. inflata and O. bragantinum might not be correct.

title: A diverse assemblage of Ophiostoma species, including two new taxa on eucalypt trees in South Africa
abstract: Fungi in the Ophiostomatales include important pathogens of trees as well as agents of wood stain, reducing the economic value of timber. They rely on insects, such as bark beetles, for dispersal and are commonly associated with wounds on trees. Although Ophiostoma spp. have been reported from eucalypt wood chips in South Africa, very little is known about the diversity of the Ophiostomatales, or their insect associates, on plantation-grown Eucalyptus spp. The aim of this study was to consider the diversity and distribution of the Ophiostomatales infecting fresh wounds on Eucalyptus trees in the country. Additionally, knowledge regarding their association with nitidulid beetles, which have previously been shown to carry Ophiostoma spp., was sought. Surveys were conducted in five provinces where Eucalyptus spp. are commonly grown, and the fungi collected were identified using morphological comparisons and multigene sequence phylogenies. Of the 139 isolates collected, five Ophiostoma spp. were identified including O. quercus, O. tsotsi and O. tasmaniense. These were from cut stumps as well as from the nitidulid beetles Brachypeplus depressus and Carpophilus spp. In addition, two new taxa in the O. stenoceras–Sporothrix schenckii complex were identified from Eucalyptus trees infested by Phoracantha semipunctata. The two new taxa are described as O. candidum sp. nov., and O. fumeum sp. nov., respectively. The results of this study clearly show that the diversity and ecology of Ophiostoma spp. on Eucalyptus trees in South Africa is poorly understood and that further studies are required to determine the possible economic relevance of these fungi.

title: Reevaluation of the status of the bluestain fungus and bark beetle associate Ophiostoma minus
abstract: Thirty isolates of the morphological spe- cies Ophiostoma minus, obtained from Europe and North America, were compared using several criteria. These included sexual compatibility, linear growth rate, sensitivity to cycloheximide, perithecial dimen- sions and RAPD profiles. Within the species complex, three distinct mating populations were identified: sexually compatible heterothallic isolates originating from both Europe and North America, homothallic isolates occurring only in North America, and anoth- er group of heterothallic isolates corresponding to individuals previously identified as 0. pseudotsugae from North America. Isolates of 0. pseudotsugaewere sexually incompatible with the heterothallic 0. minus isolates. Differences in linear growth rate, perithecial size and RAPD banding patterns confirmed the dif- ferences among the three groups, and the combined evidence suggested these are three distinct species.

title: Phylogenetic analysis of the bluestain fungus Ophiostoma minus based on partial ITS rDNA and beta-tubulin gene sequences.
abstract: In an attempt to clarify the relationship between fungi classified as Ophiostoma minus, but of different geographic origins and mating systems, sequencing of the 5.8S and ITS 2 rDNA, and beta-tubulin gene was carried out. The beta-tubulin gene was highly informative, supporting the sub-division of O. minus into two groups based on geographic origin. Furthermore, isolates previously classified as O. pseudotsugae were confirmed as being clearly distinct from O. minus. However, sequencing did not reveal any polymorphisms between isolates with homothallic as compared to heterothallic mating systems. This was supported by crosses using methylbenzamidazole-2-yl carbamate nuclear markers which showed that hybridisation between isolates of different mating systems was possible. However, we propose that different mating systems may still signal a divergence of isolates of O. minus.

title: Competition between a biological control fungus, Ophiostoma piliferum, and symbionts of the southern pine beetle
abstract: A colorless isolate of 0. piliferum was paired in a series of competitive interactions with three fungal symbionts of Dendroctonus frontalis, the southern pine beetle. Two of these fungi, Ceratocys- tiopsis ranaculosus and Entomocorticium sp. A, are considered to be mutualists of the southern pine beetle. The third fungal symbiont, 0. minus, is con- sidered to be an antagonist. I found strong evidence of differential competition between 0. piliferum and all three symbionts. In primary and secondary re- source capture contests on an artificial medium O. piliferum outcompeted all three fungi. In inocula- tions of natural substrate, 0. piliferum outcompeted the two mutualists but did not outcompete 0. minus. The ability of 0. piliferum to outcompete beetle mu- tualists on both artificial and natural substrates indi-

title: The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from community pyrosequencing
abstract: Symbioses are increasingly seen as dynamic ecosystems with multiple associates and varying fidelity. Symbiont specificity remains elusive in one of the most ecologically successful and economically damaging eukaryotic symbioses: the ambrosia symbiosis of wood-boring beetles and fungi. We used multiplexed pyrosequencing of amplified internal transcribed spacer II (ITS2) ribosomal DNA (rDNA) libraries to document the communities of fungal associates and symbionts inside the mycangia (fungus transfer organ) of three ambrosia beetle species, Xyleborus affinis, Xyleborus ferrugineus and Xylosandrus crassiusculus. We processed 93 beetle samples from 5 locations across Florida, including reference communities. Fungal communities within mycangia included 14–20 fungus species, many more than reported by culture-based studies. We recovered previously known nutritional symbionts as members of the core community. We also detected several other fungal taxa that are equally frequent but whose function is unknown and many other transient species. The composition of fungal assemblages was significantly correlated with beetle species but not with locality. The type of mycangium appears to determine specificity: two Xyleborus with mandibular mycangia had multiple dominant associates with even abundances; Xylosandrus crassiusculus (mesonotal mycangium) communities were dominated by a single symbiont, Ambrosiella sp. Beetle mycangia also carried many fungi from the environment, including plant pathogens and endophytes. The ITS2 marker proved useful for ecological analyses, but the taxonomic resolution was limited to fungal genus or family, particularly in Ophiostomatales, which are under-represented in our amplicons as well as in public databases. This initial analysis of three beetle species suggests that each clade of ambrosia beetles and each mycangium type may support a functionally and taxonomically distinct symbiosis.

title: Phenotypic and DNA sequence data comparisons reveal three discrete species in the Ceratocystis polonica species complex.
abstract: Ceratocystis polonica and C. laricicola are two morphologically similar species that occur on conifers and reside in the Ceratocystis coerulescens species complex. They, however, represent two ecologically distinct entities. C. polonica causes blue stain on Norway spruce (Picea abies) and other spruce species (Picea spp.) in Eurasia and is associated with the bark beetles Ips typographus, I. typographus japonicus, I. amitinus and I. duplicatus. In contrast, C. laricicola lives in a symbiotic relationship with the bark beetles Ips cembrae and I. subelongatus that infest various larch species (Larix spp.). The objective of this study was to consider the phylogenetic relationships of C. polonica and C. laricicola and more specifically to determine the identity of Japanese isolates from both spruce and larch, based on sequences derived from the ITS regions of the rRNA operon, the beta-tubulin gene and the HMG box of the MAT-2 gene. Isolates were also compared based on morphology and cultural characteristics. Comparisons of anamorph and teleomorph structures confirmed that C. polonica and C. laricicola are indistinguishable based on morphology. Both species had an optimal growth temperature of 25 degrees C. However, at temperatures between 31-33 degrees, C. polonica isolates grew slowly or not at all, while C. laricicola isolates grew more actively at these temperatures. Thus, a growth test at 32 degrees can differentiate these species. Phylograms generated using parsimony for the three gene regions were strongly congruent. These showed three distinct clades supported by high bootstrap values. Two of the clades clearly separate C. laricicola from Europe and C. polonica, supporting the view that they represent two discrete taxa. A third clade included isolates obtained from galleries of Ips subelongatus on Larix kaempferi in Japan. This fungus clearly represents a discrete taxon that is closely related to, but distinct from C. laricicola, which is described here as C. fujiensis sp. nov.