Mark T. Banik

Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus sequencing, which effectively conceals most intragenomic variation, but cloned sequences containing intragenomic variation are becoming prevalent in DNA databases. To understand effects of using cloned rDNA sequences in phylogenetic analyses we amplified and cloned the ITS region from pure cultures of six Laetiporus species and one Wolfiporia species (Basidiomycota, Polyporales). An average of 66 clones were selected randomly and sequenced from 21 cultures, producing a total of 1399 interpretable sequences. Significant variation (≥ 5% variation in sequence similarity) was observed among ITS copies within six cultures from three species clades (L. cincinnatus, L. sp. clade J, and Wolfiporia dilatohypha) and phylogenetic analyses with the cloned sequences produced different trees relative to analyses with consensus sequences. Cloned sequences from L. cincinnatus fell into more than one species clade and numerous cloned L. cincinnatus sequences fell into entirely new clades, which if analyzed on their own most likely would be recognized as “undescribed” or “novel” taxa. The use of a 95% cut off for defining operational taxonomic units (OTUs) produced seven Laetiporus OTUs with consensus ITS sequences and 20 OTUs with cloned ITS sequences. The use of cloned rDNA sequences might be problematic in fungal phylogenetic analyses, as well as in fungal bar-coding initiatives and efforts to detect fungal pathogens in environmental samples.

DNA-based detection of the fungal pathogen Geomyces destructans in soils from bat hibernacula

White-nose syndrome (WNS) is an emerging disease causing unprecedented morbidity and mortality among bats in eastern North America. The disease is characterized by cutaneous infection of hibernating bats by the psychrophilic fungus Geomyces destructans. Detection of G. destructans in environments occupied by bats will be critical for WNS surveillance, management and characterization of the fungal lifecycle. We initiated an rRNA gene region-based molecular survey to characterize the distribution of G. destructans in soil samples collected from bat hibernacula in the eastern United States with an existing PCR test. Although this test did not specifically detect G. destructans in soil samples based on a presence/absence metric, it did favor amplification of DNA from putative Geomyces species. Cloning and sequencing of PCR products amplified from 24 soil samples revealed 74 unique sequence variants representing 12 clades. Clones with exact sequence matches to G. destructans were identified in three of 19 soil samples from hibernacula in states where WNS is known to occur. Geomyces destructans was not identified in an additional five samples collected outside the region where WNS has been documented. This study highlights the diversity of putative Geomyces spp. in soil from bat hibernacula and indicates that further research is needed to better define the taxonomy of this genus and to develop enhanced diagnostic tests for rapid and specific detection of G. destructans in environmental samples.

Molecular phylogeny of Laetiporus and other brown rot polypore genera in North America

Phylogenetic relationships were investigated among North American species of Laetiporus, Leptoporus, Phaeolus, Pycnoporellus and Wolfiporia using ITS, nuclear large subunit and mitochondrial small subunit rDNA sequences. Members of these genera have poroid hymenophores, simple septate hyphae and cause brown rots in a variety of substrates. Analyses indicate that Laetiporus and Wolfiporia are not monophyletic. All North American Laetiporus species formed a well supported monophyletic group (the “core Laetiporus clade” or Laetiporus s.s.) with the exception of L. persicinus, which showed little affinity for any genus for which sequence data are available. Based on data from GenBank, the southern hemisphere species L. portentosus also fell well outside the core Laetiporus clade. Wolfiporia dilatohypha was found to represent a sister group to the core Laetiporus clade. Isolates of Phaeolus, Pycnoporellus and members of the core Laetiporus clade all fell within the Antrodia clade of polypores, while Leptoporus mollis and Laetiporus portentosus fell within the phlebioid clade of polypores. Wolfiporia cocos isolates also fell in the Antrodia clade, in contrast to previous studies that placed W. cocos in the core polyporoid clade. ITS analyses resolved eight clades within Laetiporus s.s., three of which might represent undescribed species. A combined analysis using the three DNA regions resolved five major clades within Laetiporus s.s.: a clade containing conifer-inhabiting species (“Conifericola clade”), a clade containing L. cincinnatus (“Cincinnatus clade”), a clade containing L. sulphureus s.s. isolates with yellow pores (“Sulphureus clade I”), a clade containing L. sulphureus s.s. isolates with white pores (“Sulphureus clade II”) and a clade containing L. gilbertsonii and unidentified isolates from the Caribbean (“Gilbertsonii clade”). Although there is strong support for groups within the core Laetiporus clade, relationships among these groups remain poorly resolved.

Effects of cloning and root-tip size on observations of fungal ITS sequences from Picea glauca roots

To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was investigated by selecting 20 small root tips (SRT, 1.0–2.0 mm long) and 20 large root tips (LRT, 5.0–6.0 mm long). DNA was isolated from each tip and PCR-amplified with fungal-specific primers. PCR reactions were divided into two portions, one of which was sequenced directly and one of which was cloned first followed by sequencing of 12 random clones. With direct sequencing all 20 SRT produced an identifiable sequence, while only 13 of 20 LRT (65%) yielded an identifiable sequence. With cloning and sequencing all 40 tips produced identifiable fungal ITS sequences regardless of size. Failure of direct sequencing in LRT was associated with the presence of multispecies assemblages. Cloning identified 18 taxa overall while direct sequencing identified four. Cloning was not affected by tip size and identified more taxa relative to direct sequencing, although cost and probability of observing lab-based contaminants (e.g. airborne or reagent-based) were higher. We suggest that standardized controls be run whenever clones are sequenced from environmental samples, including positive controls derived from pure cultures and negative controls that cover the entire extraction, amplification and cloning process. Additional studies on larger root segments and bulked samples are needed to determine whether cloning can detect fungi accurately and cost-effectively in complex environmental samples.

Armillaria species of the Olympic Peninsula of Washington state, including confirmation of North American biological species XI.

Forty-eight collections of Armillaria from the Olympic Peninsula of Washington State were identified to species using the conventional pairing protocol and/or a polymerase chain reaction-based (PCR) technique utilizing restriction fragment poly? morphisms (RFLPs) of the intergenic spacer region (IGS) of the ribosomal DNA. The PCR-based tech? nique yielded results comparable to those obtained by the conventional pairings. Several restriction pat? terns not previously reported in North American iso? lates were found using the enzyme Alu I. Collections from eight different host species were identified as A. ostoyae, A. sinapina, A. nabsnona, A. gallica, or North American biological species (NABS) XI. Ar? millaria ostoyae, A. sinapina, and A. nabsnona were the most often collected, with the first two occurring predominantly on gymnosperms and the latter most? ly on angiosperms. Armillaria gallica and NABS XI were collected rarely and only from angiosperms. This is only the second report of the occurrence of NABS XI.

Assessment of compatibility among Armillaria cepistipes, A. sinapina, and North American biological species X and XI, using culture morphology and molecular biology

Ten single-spore isolates each of Armillar- ia sinapina, A. cepistipes, and North American bio- logical species (NABS)X and XI were paired in all combinations. A second set of ten single-spore iso- lates of each species was likewise paired. Each pairing was duplicated for a total of 3280 pairs. Using the standard morphological criteria (e.g., fluffy, crustose) to assess the pairings, A. sinapina exhibited the fol- lowing compatibility levels: 68% with itself, 5.5% with A. cepistipes, 3.5% with NABS X, and 5% with NABS XI, respectively. Armillaria cepistipes was rated 53%, 6.5%, and 57% compatible with itself and NABS X and XI, respectively. NABS X was 70% selfcompatible and 4.0% compatible with NABS XI. NABS XI was 55% selfcompatible. The intergenic spacer region of the rDNA of each isolate was amplified using poly- merase chain reaction and digested with Alu I. Seven distinct restriction fragment length polymorphic (RFLP) patterns were observed: two in A. cepistipes, two in A. sinapina, one in NABS X, one in NABS XI, and one that was common to NABS XI and A. cep- istipes. By using the RFLP patterns as markers to as- sess the outcome of the pairings, it was determined that, in general, the morphological ratings were an accurate reflection of nuclear combination. Combin- ing the RFLP and pairing results confirms that NABS XI is compatible with A. cepistipes and should be con- sidered conspecific. A. sinapina, NABS X, and A. cep- istipes are compatible to a very limited degree in cul- ture, but not enough evidence exists to warrant re- ducing any to synonomy. Therefore, we believe NABS X should be formally described as a new species of

The genus Laetiporus (Basidiomycota, Polyporales) in East Asia.

Relationships among East Asian, North American and European Laetiporus sulphureus s. lat., a cosmopolitan brown rot species complex, were assessed with phylogenetic analyses and incompatibility tests. Three East Asian taxa, Laetiporus cremeiporus sp. nov., Laetiporus montanus and Laetiporus versisporus, are described and illustrated as well as compared with related taxa from Southeast Asia, North America and Europe. Phylogenetic analyses showed that L. cremeiporus and L. versisporus are clearly distinct species among Laetiporus taxa. The three conifer inhabiting species, Laetiporus conifericola, Laetiporus huroniensis and L. montanus, are closely related to each other. The European population of L. montanus exhibits two sequence variants of the EF1alpha: one is the same as observed in L. sulphureus in Europe and the other is that observed in East Asian population of L. montanus. A key to the known species of Laetiporus in the northern hemisphere is provided.

Identification of Armillaria species from Wisconsin and adjacent areas.

Single-spore isolates from 218 basidiomata of Armillaria from Wisconsin, the upper peninsula of Michigan, and eastern Minnesota were identified to species by pairing with known haploid testers. Collec? tions were made from 25 different host species and were identified as A. ostoyae, A. gallica, A. calvescens, A. mellea, or A. sinapina. The most frequently collected species were A. ostoyae and A. gallica. Within the sam? pling area, A. ostoyae and A. sinapina were collected only in the northern portions, and A. mellea was col? lected predominantly in the southern portions. Ar? millaria gallica and A. calvescens were collected throughout the area. Armillaria sinapina and A. ostoyae basidiomata were found equally on gymnosperms and angiosperms, whereas the other three species were collected predominantly from angiosperms. In addi? tion to the basidiomata collections, 71 cultures of Ar? millaria were isolated from roots and rhizomorphs in seven 10- to 20-year-old quaking aspen stands in northern Wisconsin. These were subsequently iden? tified by pairing with known haploid testers; A. sina? pina and A. ostoyae were recovered most often, whereas A. gallica was isolated less frequently. In comparison

Incompatibility groups among North American populations of Laetiporus sulphureus sensu lato

Mycelial interactions and allozyme analysis for glucose-6-phosphate isomerase activity were used to evaluate compatibility of pairings of single spore isolates (SSIs) within and between Laetiporus restric- tion groups (LRGs). SSIs from six collections of LRG II were completely compatible. SSIs from four LRG III collections from western North America were completely compatible, but those from a fifth collec- tion of LRG III, from Michigan, were only partially compatible with those from the western LRG III col- lections. LRG II SSIs were incompatible with LRG III SSIs, and SSIs of these LRGs were incompatible with SSIs of L. sulphureus, L. cincinnatus, and LRG VI. SSIs from the lone collection of LRG VI were com- patible with those of L. sulphureus, which also con- tains LRGs I and VII. Thus, L. sulphureus s. 1. is com- posed of four Laetiporus incompatibility groups (LIGs): L. sulphureus s. s., L. cincinnatus, LIG II, and LIG III. The LIGs differ in host, pore layer color, geographical distribution, and LRG. Pairings of tester SSIs from each of the LIGs with SSIs from an addi- tional 42 collections of Laetiporus confirm the mutual exclusiveness of the LIGs and the association of the LIGs with the LRGs as well as other characteristics. Therefore, these four LIGs are worthy of recognition at the species level.

Relationships among North American and Japanese Laetiporus isolates inferred from molecular phylogenetics and single-spore incompatibility reactions

Relationships were investigated among North American and Japanese isolates of Laetiporus using phylogenetic analysis of ITS sequences and single-spore isolate incompatibility. Single-spore isolate pairings revealed no significant compatibility between North American and Japanese isolates. ITS analysis revealed 12 clades within the core Laetiporus clade, seven of which are known to occur in North America (including Hawaii and the Caribbean), three in Japan, two in South America, three in Europe and one in South Africa. The identity of L. sulphureus s.s. has yet to be determined and could be either L. “sulphureus” (clade C), which appears to be restricted to Europe and occurs on angiosperms and gymnospersm, or L. “sulphureus” (clade E), which is found in Europe, North America and South America exclusively on angiosperms. Three clades, one from the Caribbean, one from Hawaii and one from South Africa, have yet to be named formally. Of the three Laetiporus species found in Japan two have been named recently (L. cremeiporus and L. montanus) and one has been epitypified (L. versisporus). The single-spore incompatibility and ITS data support recognition of the three Japanese taxa as distinct biological and evolutionary species.