North American Biological Species Research Articles

Armillaria altimontana, a new species from the western interior of North America

Armillaria altimontana, previously considered North American biological species (NABS) X, is described as new. To date, it appears that A. altimontana prefers higher-elevation, mesic sites within the dry, conifer forest zone of western interior North America. This species has been found on hardwoods and conifers and is associated most commonly with Abies-dominated forest types in southern British Columbia, Washington, Oregon, Idaho and northern California. Partial elongation factor 1-alpha (tef1) sequences were generated from six isolates of A. altimontana originating from three locations in northern Idaho. Phylogenetic analyses of all 10 North American Armillaria species were carried out with maximum parsimony and maximum likelihood. Results indicate that isolates of A. altimontana formed a monophyletic group and clustered with A. calvescens, A. cepistipes, A. gallica and A. nabsnona, which is in agreement with recent phylogenetic studies of Armillaria.

Advances toward DNA-based identification and phylogeny of North American Armillaria species using elongation factor-1 alpha gene

The translation elongation factor-1 alpha (EF-1α) gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The phylogenetic relationships revealed clear separation of all ten North American Armillaria species, with the exception of one A. gallica isolate that perhaps represents an unnamed cryptic species. These results indicate that the EF-1α gene could potentially serve as a diagnostic tool for distinguishing among currently recognized North American biological species of Armillaria.

Advances toward DNA-based identification and phylogeny of North American <i>Armillaria</i> species using elongation factor-1 alpha gene

The translation elongation factor-1 alpha (EF-1α) gene was used to examine the phylogenetic relationships among 30 previously characterized isolates representing ten North American Armillaria species: A. solidipes (=A. ostoyae), A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, North American biological species X, A. cepistipes, and A. tabescens. The phylogenetic relationships revealed clear separation of all ten North American Armillaria species, with the exception of one A. gallica isolate that perhaps represents an unnamed cryptic species. These results indicate that the EF-1α gene could potentially serve as a diagnostic tool for distinguishing among currently recognized North American biological species of Armillaria.

A new species of Epichloë symbiotic with Chinese grasses

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of β-tubulin gene (tub2) introns and translation elongation factor 1-α gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

A new species of Epichloe symbiotic with Chinese grasses

Epichloë species are fungal symbionts (endophytes) of grasses, six European and four North American biological species in genus Epichloë have been described in previous researches. In this study we describe a new Epichloë species, Epichloë yangzii Li et Wang, found in natural symbioses with Roegneria kamoji native to China. We investigated the host specificity, morphology, interfertility tests and molecular phylogenetic evidences of this new species. The results indicated that E. yangzii is host specific and seedborne. Most morphological characteristics of this new species are typical in the genus. However differences are evident in several features including size of perithecia, asci and ascospores. In mating tests E. yangzii was not interfertile with E. elymi isolates from related hosts in genera Elymus. Phylogenetic relationships based on sequences of beta-tubulin gene (tub2) introns and translation elongation factor 1-alpha gene (tef1) introns showed that members of the new species grouped into exclusive clades with high bootstrap value.

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

SummaryPhylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.

Interfertility between Armillaria cepistipes and A. sinapina

European strains of Armillaria cepistipes were reported to be interfertile with strains from three American Armillaria species known as North American Biological Species (NABS) V (A sinapina), NABS X and NABS XI. Such interfertility between species raises some doubts about using different Latin binomials for species capable of mating. This interfertility was reinvestigated by mating 24 haploid isolates of European A cepistipes with 23 isolates of A sinapinafrom North America and Asia. Individual pairings were independently performed at least once at Universite Laval, Canada and at INRA Clermont-Ferrand, France. From the 420 interspecific pairings performed at Laval, two were positive and seven were ambiguous for a total of 2.1% of all the pairings. From the 506 pairings made at Clermont-Ferrand, 10 were positive and 24 were ambiguous for a total of 6.7%. The differences in the pairing results may be explained by incubation temperatures, and the different types and concentrations of malt extract used at each laboratory. The low levels of interfertility found between A. cepistipes and A. sinapina may result from the absence of genetic barriers that are usually present between sympatric species. This low level of interfertility reflects differences in morphology, distribution, and habitat for these two species of Armillaria and this supports the retention of different species denominations.

Coarse-scale population structure of pathogenicArmillariaspecies in a mixed-conifer forest in the Blue Mountains of northeast Oregon

The coarse-scale population structure of pathogenic Armillaria (Fr.) Staude species was determined on approximately 16 100 ha of relatively dry, mixed-conifer forest in the Blue Mountains of northeast Oregon. Sampling of recently dead or live, symptomatic conifers produced 112 isolates of Armillaria from six tree species. Armillaria species identifications done by using a polymerase chain reaction based diagnostic and diploid–diploid pairings produced identical results: 108 of the isolates were Armillaria ostoyae (Romagn.) Herink and four were North American Biological Species X (NABS X). Five genets of A. ostoyae and one of NABS X were identified through the use of somatic incompatibility pairings among the putatively diploid isolates. Armillaria ostoyae genet sizes were approximately 20, 95, 195, 260, and 965 ha; cumulative colonization of the study area was at least 9.5%. The maximum distance between isolates from the 965-ha A. ostoyae genet was approximately 3810 m, and use of three estimates of A. ostoyae spread rate in conifer forests resulted in age estimates for the genet ranging from 1900 to 8650 years. Results are discussed in relation to possible mechanisms that influenced the establishment, expansion, and expression of these genets; the genetic structure and stability of Armillaria; and the implications for disease management in this and similar forests.

Distribution of Armillaria species in California

To determine the identity and distribution of Armillaria species in California, we collected 589 isolates from managed areas (urban landscapes, orchards, and vineyards) and 20 natural forest types. From our collection of isolates, we identified A. mellea, A. gallica, A. nabsnona, and North American biological species X (NABS X). State-wide ranges of each species were estimated based on distributions of forest types from which they were collected. Only A. mellea and A. gallica are widely-distributed in California; A. nabsnona and NABS X appear to have restricted ranges. Armillaria mellea is common in most regions of the state dominated by development, such as the Central Valley, the San Francisco Bay area, and Los Angeles. It was the only species, with the exception of a single isolate of NABS X, isolated from symptomatic hosts, including both forest trees and planted hosts. Armillaria mellea does cause mortality in California forests, but large disease centers are rare. The distribution of A. gallica in California includes the greatest variety of forest types of all Armillaria species identified from our collection. Although it shares many forest types in common with A. mellea, A. gallica is extremely rare in developed areas. The range of A. nabsnona in California appears to be restricted to northern redwood forests. NABS X was only identified in high elevation red and white fir forests of the southern Cascades.

Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids, diploids (doubled haploids), or a mixture of haploids and diploids (doubled haploids). Depending on the nuclear status of the basidiospore-derived lines of NABS X, intraspecifically mated cultures can exist as diploids or tetraploids, and possibly triploids or aneuploids under in vitro conditions. Based on previous in vitro mating studies, seven basidiospore isolates were specifically selected to assess rare, interspecific mating among Armillaria cepistipes, A. sinapina, NABS X, and NABS XI. Cultures from basidiospore-derived isolates were paired to produce four interspecifically paired cultures, and matings were assessed using flow cytometry and restriction fragment length polymorphism (RFLP) analyses. Based on flow cytometric analysis, the A. cepistipes isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolates were individually compatible with isolates of NABS X and NABS XI. Mean fluorescence intensities of A. cepistipes‹NABS X, A. sinapina‹NABS X, and A. sinapina‹NABS XI mated cultures revealed a triploid or tetraploid nuclear status compared to the haploid or diploid (doubled haploid) nuclear status of initial basidiospore-derived isolates. Polymerase chain reaction (PCR) and RFLP of the intergenic spacer (IGS) region generated banding patterns for basidiospore-derived isolates and mated cultures. Four species-specific RFLP banding patterns were observed in basidiospore-derived isolates of A. cepistipes, A. sinapina, NABS X, and NABS XI. PCR-RFLP analysis showed combined banding patterns from mated cultures. Flow cytometry and PCR-RFLP analysis are eective tools to assess matings of Armillaria species.

Characterization of North American Armillaria species by nuclear DNA content and RFLP analysis

Twenty-six isolates representing nine North American Armillaria species were investigated with flow cytometry and RFLP (restriction fragment length polymorphism) analyses to determine their nuclear DNA content and RFLP profile. Three putatively diploid isolates of A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. mellea, A. gallica, A. nabsnona, and North American Biological Species (NABS) X were analyzed, and two putatively diploid isolates of NABS XI also were analyzed. Nuclear DNA contents of Armillaria species were 0.11–0.17 pg per nucleus (55–84 X 106 bp/C), depending on species. Among the nine North American Armillaria species tested, A. ostoyae, A. gemina, and A. mellea possessed relatively small nuclear DNA contents (0.11–0.12 pg per nucleus), whereas A. gallica possessed a relatively large nuclear DNA content (0.17 pg per nucleus). A. nabsnona has a slightly larger nuclear DNA content (0.13 pg per nucleus) than A. ostoyae, A. gemina, and A. mellea. Other species (A. calvescens, A. sinapina, NABS X, and NABS XI) possessed moderate nuclear DNA contents (ca 0.15 pg per nucleus). Polymerase chain reaction (PCR) and RFLP of the intergenic spacer region-1 (IGS-1) generated banding patterns for nine Armillaria species. In addition to previously reported banding patterns, new banding patterns are presented for A. gemina, A. calvescens, A. mellea, and A. gallica.

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

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 Armillaria sinapina, A. cepistipes, and North American biological species (NABS) X and XI were paired in all combinations. A second set of ten single-spore isolates 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 following 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 polymerase 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. cepistipes. By using the RFLP patterns as markers to assess the outcome of the pairings, it was determined that, in general, the morphological ratings were an accurate reflection of nuclear combination. Combining the RFLP and pairing results confirms that NABS XI is compatible with A. cepistipes and should be considered conspecific. A. sinapina, NABS X, and A. cepistipes are compatible to a very limited degree in culture, but not enough evidence exists to warrant reducing any to synonomy. Therefore, we believe NABS X should be formally described as a new species of Armillaria.

Molecular Phylogeny and Evolutionary Divergence of North American Biological Species ofArmillaria

Armillariais a genus of root infecting basidiomycetes, which includes nine North American biological species. Anonymous nucleotide sequences obtained from four different primer pairs were combined to produce a data set which was analyzed phylogenetically. The data indicated that randomly chosen sequences from the genome were capable of resolving the phylogenetic history of species ofArmillariaand provided strong support for intraspecies clustering. NABS III and VII formed a significant monophyletic clade, with III being derived from the more broadly distributed NABS VII. Sequences of isolates of NABS V showed a high degree of variation. This variation may be an indication of recent sympatric speciation, with NABS IX and X diverging from a genetically diverse NABS V. NABS I formed a monophyletic clade despite the variation in geographic distance among the isolates. The position of NABS II as ancestral to NABS I was discussed. However, literature evidence favored divergence of NABS II from NABS I, while this study illustrated genetic similarity of NABS II with NABS VI. NABS VI was the most divergent of the North American species and represented the outgroup. A molecular clock of NABSArmillariawas proposed.

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.

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.

Armillaria nabsnona, a New Species from Western North America

Armillaria nabsnona is characterized morphologically and described as a new species. It is compatible with tester strains of North American biological species (NABS) IX. Using restriction fragment length polymorphism (RFLP) of the polymerase chain reaction (PCR) products amplified from the intergenic spacer (IGS) region of the ribosomal DNA, we also demonstrate an association of cultures of specimens with tester strains of NABS IX. Armillaria nabsnona appears to be restricted to several species of hardwood trees in the states of Washington, Oregon, California, Idaho and Alaska, and in the Canadian province of British Columbia. Basidiomata have been found in both the fall and spring.

Species delimitation in North American species of Armillaria as measured by DNA reassociation

The relatedness among biological species in Armillaria was explored using DNA reassociation experiments. Forty strains including nine of the ten reproductively isolated NABS (North American biological species) were used in the study. All of the biological species have significant levels of DNA sequence similarity with each other. Four major DNA sequence similarity clusters were found. The first includes A. calvescens, A. gallica, A. sinapina , NABS IX and NABS X; the second includes A. mellea , with 17 isolates; the third includes A. gemina and A. ostoyae ; and the fourth includes A. tabescens , which is the most distant with about 30% similarity with the other taxa. The results are discussed in regard to the relationships estimated by rRNA RFLP data.

Identification of Armillaria Species in California

Mating experiments were conducted with 30 California isolates of Armillaria species to determine their intersterility grouping. Basidiomes were collected from a variety of coniferous and hardwood hosts in urban, agricultural and forested regions in northern and central California. Monosporous cultures were obtained and paired with haploid tester strains representing 10 North American biological species. A majority of the California isolates were compatible with Armillaria mellea sensu stricto (= North American biological species VI). The exceptions were four isolates from a low elevation, coastal forest. Three of these were compatible with North American biological species IX, a still unidentified Armillaria species, and one was compatible with A. gallica (= North American biological species VII). These findings show that Armillaria species diversity exists in California, but that A. mellea predominates on several hosts and in diverse habitats.

Clamp Connections in North American Armillaria Species: Occurrence and Potential Application for Delimiting Species

Clamp connections were observed in basidiospore dilutions from each of four North American biological species of Armillaria (A. gallica, A. sinapina, A. ostoyae, and A. calvescens) following spore germination and hyphal anastomosis. The occurrence of clamp connections in intrabasidioma pairings of single-germinated basidiospores and of excised margin-hyphae of Armillaria ostoyae and A. gallica was that expected of a bifactorial incompatibility system. All intraspecific pairings between basidiospores or excised margin-hyphae from actively growing haploid cultures from two different basidiomata of A. ostoyae and of A. gallica produced clamp connections. The development of the crustose colony morphology was almost fully coincident with the formation of clamp connections in compatible matings with either A. ostoyae or A. gallica. Results indicate that clamp connections occur predictably and provide an additional precise and useful criterion for recognizing biological species of Armillaria. For unexplained reasons, haploid isolates paired more than 3 mm apart failed to produce clamp connections.