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Mycology and Plant Pathology |
Laboratory of Plant Taxonomy and Evolution, Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Research Institute of Humanity and Nature, National Institutes for the Humanities, Inter-university Research Institute Corp., Kyoto 603-8047, Japan; Makino Herbarium, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan
Received for publication December 14, 2006. Accepted for publication August 9, 2007.
ABSTRACT
Taxonomical classification of higher fungi remains an important challenge and can benefit from the application of molecular analysis. We propose that the ectomycorrhizal (EM) fungal taxa might include a number of cryptic species because there are few morphological characteristics useful for distinguishing among these fungi. Previously, host specificity in most EM fungi was thought to be low, but we suspect that confusion of cryptic species has led to an underestimate of fungal host specificity. We analyzed both nuclear and mitochondrial DNA sequences from Strobilomyces fungi and obtained evidence that what were previously described as four species can be grouped into 14 distinct lineages, suggesting that these lineages might be distinct biological species. Moreover, we identified host plants for Strobilomyces via nucleotide sequencing of both fungal and plant DNA from EM samples. Most lineages of Strobilomyces tested in this study were associated only with Fagaceae trees, even though Strobilomyces species were previously thought to be generalists with regard to hosts. Thus, we present an approach useful for identifying cryptic species and detecting the true host range of a set of EM fungi in natural conditions.
Key Words: atp6 • cytonuclear system • host range • mutualism • RPB1
The current state of taxonomical classification of higher fungi lags behind our understanding of the number of species. Hawksworth (1991)
estimated the total number of fungal species as ca. 1.5 million, of which only ca. 69 000 have been described (Kirk et al., 2001
). One challenge to classification of higher fungi is that they often have an insufficient number of distinguishing morphological characteristics useful for taxonomy. The simplicity of fungal morphology suggests that higher fungi may contain many cryptic species. In addition, many species of higher fungi have a wide geographical distribution, such as those found throughout the northern hemisphere or old world tropics (Corner, 1972
; Singer, 1986
). The unusually wide distribution of a single morphological species also suggests existence of reproductively isolated cryptic species with similar morphological characteristics. Indeed, the existence of cryptic species among higher fungi is known to be the case for some well-known fungal species. For example, using a sexual intercompatibility test, Aanen and Kuyper (1999)
detected at least 20 reproductively isolated groups among fungi classified as Hebeloma crustuliniforme, even though these fungi could not be distinguished morphologically.
Among mycorrhizas, ectomycorrhizas (EM) are characterized by the formation of a mantle and a Hartig net of intercellular hyphae on roots of predominantly tree species (Smith and Read, 1997
). EM fungi comprise species from multiple families in the phyla Basidiomycota and Ascomycota (higher fungi). EM host plants are typically large woody species such as pine, oak, birch, eucalyptus, and other temperate, and to a lesser extent, tropical tree species. Through mutualistic relationships between EM fungi and plant roots, the plant provides fixed carbon to the fungus, and in return, the fungus provides mineral nutrients, water, and protection from pathogens (Smith and Read, 1997
). Researchers have reported that EM fungi display different degrees of host specificity. The results of direct observation of plant–sporocarp associations, a routine method for estimating fungal host range, suggest that the majority of EM fungi have a broad host range (Molina et al., 1992
; Smith and Read, 1997
; Cairney and Chambers, 1999
). Restriction fragment length polymorphism analysis of PCR-amplified DNA fragments (PCR-RFLP) has also suggested a broad host range for EM fungi (Karen et al., 1997
; Bruns et al., 1998
; Horton and Bruns, 1998
; Cullings et al., 2000
; Kennedy et al., 2003
). Low host specificity of EM fungi may promote resource sharing in plant communities (Read, 1997
; Simard et al., 1997
).
However, the approaches that have been used in the past to estimate EM host specificity were problematic. The results of these studies may have been confused by the existence of cryptic species with indistinguishable morphologies but differentiated host specificities. PCR–RFLP analysis may not be adequate for delimiting closely related fungal species whose genetic differences are small. Instead, cytonuclear approaches, which resolve genetic differences more effectively and provide helpful information about intrinsic reproductive isolation, should be used to accurately discriminate among closely related species of fungi. The method for identifying host plant species used in the past is also deficient. Careful observation of plant–sporocarp associations is the simplest and the prevailing method for testing host specificity of EM fungi (Molina et al., 1992
). But especially in mixed forests, determining the host plant for a given fungal species is difficult because the connection between EM fungi and host plants is difficult to observe in natural settings and has seldom been achieved (Trappe, 1962
; Bills et al., 1986
). Instead, observation of EM root tips is crucial for identifying host plant species. Therefore, information reported to date about host plant specificity in many EM fungal species is not sufficiently reliable.
Phylogenetic approaches based on concordance of multiple nuclear gene genealogy can be performed for various fungal taxa and provide helpful information about species boundaries. Those approaches, however, may not reveal reproductive isolation between phylogenetic groups because inheritance of nuclear genes may not be independent owing to linkage disequilibria. Thus, we considered the idea that cytonuclear systems, which are often applied to animal or plant taxa, could be used to detect cryptic species in EM fungi. Nuclear DNA and cytoplasmic DNA are independently inherited (biparental and uniparental, respectively), and thus cytonuclear disequilibria in the hybrid zone can provide information about gene flow or intrinsic reproductive barriers between genotypes (Arnold, 1993
; Avise, 2004
). In fungi, as in animals, nuclear and mitochondrial DNA sequences are available and might facilitate detection of cytonuclear disequilibria. Such molecular methods should be more practical than mating tests, which are difficult to perform for many EM fungal taxa (Martin et al., 1994
). Furthermore, applying molecular methods to EM samples may enable accurate identification of both fungi and plant species, thereby allowing to assess host specificity even for samples from forests containing many tree species.
Several key features suggest that fungi in the genus Strobilomyces may be one of the best models for understanding cryptic species and host specificities of EM fungi. Strobilomyces (Strobilomycetaceae, Boletales) is a genus of EM fungi (Singer, 1986
; Matsuda and Hijii, 1999
). Among Strobilomyces, the following four species have been reported to exist in Japan: S. strobilaceus (Scop.:Fr.) Berk., S. confusus Sing., S. seminudus Hongo, and S. mirandus Corner (Nagasawa, 1987
; Sato et al., 2005
). Potential host plant species for these fungi often coexist at collection sites. For example, in northern Japan (Nagano Prefecture), deciduous oaks (Quercus) and coniferous trees (Abies and Pinus) often grow together; in mid-Japan (Kyoto, Shiga, and Osaka prefectures), evergreen oaks (Castanopsis and Quercus), deciduous oaks (Quercus), and coniferous trees (Pinus) often grow together; and in Taiwan and southern Japan, diverse evergreen oaks (Castanopsis, Quercus, and Lithocarpus) grow together. High host specificity has not been reported for species in this genus; instead, researchers have thought that all species in the genus are generalists with regard to host plant association and can form EM with evergreen or deciduous oaks as well as with coniferous trees (Nagasawa, 1987
; Molina et al., 1992
).
In this study, we analyzed nucleotide sequences of both nuclear and mitochondrial DNA from Strobilomyces fungi in an exhaustive search for cryptic species. In addition, we identified host plants for Strobilomyces via analysis of fungal and plant DNA sequences detected in fungus-colonized roots. The aim of this study was to test the idea that Strobilomyces fungi include several cryptic species and furthermore, that some of those cryptic species possess high host specificity, which would suggest that host specificity of EM fungi in general has been underestimated because some recognized taxa actually comprise multiple species differing in host range.
MATERIALS AND METHODS
Fungal materials and mycorrhizal sampling
We collected 60 fruit bodies of the genus Strobilomyces from mixed forests of Pinus and deciduous Quercus; mixed forests of Pinus, deciduous Quercus, evergreen Quercus and Castanopsis; or mixed forests of evergreen Quercus and Castanopsis in Japan. Collection locations and their forest types were as follows: Kyoto-MZ and Shiga-TN from mixed forests of Pinus and Quercus; Shiga-OM, Miyazaki-AY, Miyazaki-MK, and Miyazaki-TK from mixed forests of evergreen Quercus and Castanopsis; and Kyoto-YS, Kyoto-AT, Kyoto-KY, Kyoto-TK, Kyoto-MT, Kyoto-SM, Shiga-MD, and Osaka-MN from mixed forests of Pinus, deciduous Quercus, evergreen Quercus and Castanopsis. A few samples were collected from mixed forests of evergreen Quercus and Castanopsis in Taiwan. Information about the samples used in this study is shown in Appendix 1. Potential host plant species at each collection site are shown in Table 1. For collecting samples, small sections of fruit bodies were removed and stored in 99.5% ethanol for molecular analysis, and the remaining sections were dried and preserved as voucher specimens. All specimens were deposited in the Makino Herbarium of Tokyo Metropolitan University (MAK).
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DNA extraction, PCR amplification, and nucleotide sequencing
Total DNA was extracted from the fruit body in 99.5% ethanol as follows. CTAB buffer (500 µL, containing 100 mM Tris, 1.4 M NaCl, 20 mM EDTA, 2% CTAB) was added to 10–50 mg of fungal material in 1.5-mL microtubes. The fungal tissue was crushed using a pestle and incubated for 30 min at 65°C. After incubation, the cloudy suspension was extracted using an equal volume of chloroform and was centrifuged for 15 min at 16 000 g. The clear supernatant was then transferred to a new microtube. An equal volume of isopropanol and one-tenth volume of sodium acetate were added, and the sample was centrifuged for 5 min at 16 000 x g. After the precipitated DNA was washed with 70% ethanol, it was dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).
Internal transcribed spacer 2 (ITS2) regions were amplified from nuclear ribosomal DNA using the ITS3 and ITS4 primers as described by White et al. (1990)
. To amplify the largest subunit (RPB1) of RNA polymerase II region, we designed RPB1A-st forward and RPB1C-st reverse primers based on sequence comparison among Strobilomyces species (Table 2, Fig. 1a). After a first round of PCR amplification using ATP6–1 and ATP6–2 primers, we performed nested PCR to amplify the ATPase subunit 6 (atp6) region using the ATP6–3 and ATP6–4 primers described by Kretzer and Bruns (1999)
. Sequence information for all PCR primers used in this study is provided in Table 2. PCR amplification was done using 1 µL total DNA in a 20-µL reaction mixture containing 1x PCR buffer, 2 nmol dNTPs, 10 pmol of both forward and reverse primers, and 0.5 units Ex-Taq polymerase (Takara Bio, Otsu City, Shiga, Japan). Cycling parameters for PCR were as follows: denaturation at 95°C for 3 min; followed by 35 cycles of 30 s at 95°C, 30 s at 50–60°C ,and 72°C for 30 s increasing to 90 s during cycling; followed by a final extension of 7 min at 72°C.
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The purified PCR products were sequenced using the same primers used for amplification. Nucleotide sequencing was performed using an ABI 3100 automated sequencer (Applied Biosystems, Foster City, California, USA) with Big Dye-Terminator ver. 3.1 (Applied Biosystems), following the manufacturer's protocols.
Phylogenetic analyses using maximum parsimony and Bayesian analysis
Sequence data were aligned using the CLUSTAL W program (Thompson et al., 1994
). Alignment of ITS2 sequences and intron sequence regions of RPB1 were refined manually using BioEdit (Hall, 1999
). Maximum parsimony (MP) analyses were conducted using PAUP* version 4.0b10 (Swofford, 2002
). In all analyses, gaps were treated as missing data. The MP trees were obtained using the heuristic search option, simple sequence additions, and tree-bisection-reconnection (TBR) branch swapping, holding one tree at each step during stepwise addition, and with the MulTrees option on. MaxTrees was set to 50 000 trees. Characteristics were treated as unordered and unweighted. We first selected Boletus edulis Bull.Fr. as a sister group to Strobilomyces to compare atp6 sequences among Boletales fungi. Then, we designated the following four fungal taxa as outgroups: Austroboletus gracilis (Peck) Wolfe, Boletus edulis (DNA databank accession AF002141), Russula rosacea (Pers.) S.F. Gray (AF002148), and Suillus luteus (L.Fr.) S.F. Gray. Similarly, for RPB1 comparison, B. edulis (DQ067991), Lactarius deceptivus Peck (AY864883), and Suillus pictus (Peck) A.H. Smith & Thiers (AY858965) were designated as outgroups. For ITS2 analyses, B. edulis (AY278764), Gomphidius subroseus Kauffman (DQ384576), and S. pictus (AY854069) were designated as outgroups. For nonparametric bootstrapping (Felsenstein, 1985
), 10 000 bootstrap replicates were performed for each maximum parsimony analysis.
For the Bayesian analysis, the RPB1 data sets were divided into four partitions. The coding region was divided in three partitions (first, second, and third codon positions). The noncoding region consisted of two introns that were treated as one partition. The ITS2 data set was also treated as one partition. The computer program MrModeltest ver. 2.1 (Nylander, 2004
) was used to select an appropriate substitution model, using the Akaike information criterion (AIC) for partitioned data from the RPB1 and ITS2 data sets. Modelselect script (Tanabe, 2006
), a Perl script for selecting a nucleotide substitution model from among 70 models with an arbitrary site-specific rate heterogeneity model, was used to select best-fit models via AIC for the atp6 data sets. Using the model of substitution indicated by AIC, we then ran Bayesian inference analyses using MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck, 2003
). Separate and combined analyses consisted of four simultaneous runs each with four simultaneous Markov chain Monte Carlo (MCMC) chains initially run for 4 000 000 generations, saving the current tree to a file every 100 generations. Default cold and heated chain parameters were used. At the end of each run, we considered the sampling of the posterior distribution to be adequate if the average standard deviation of split frequencies was <0.01. The MCMC runs were summarized and further investigated for convergence of all parameters, using the "sump" and "sumt" commands in MrBayes. Trees prior to log likelihood stabilization and convergence (burn in = 10 000) were discarded before a majority rule consensus tree was generated.
To trace the history of host associations, we used a parsimony-based reconstruction method as implemented in the program Mesquite ver. 1.12 (Maddison and Maddison, 2006
) on the Bayesian 50% majority-role consensus tree as inferred from nucleotide sequences of the RPB1 region.
Determination of host plant species using molecular analyses of EM samples
In addition to morphological observations, we performed molecular analyses to identify combinations of fungal taxa and their host plants. The DNA of both fungi and host plants were extracted from EM root tips as described previously for fungal fruit bodies. We carefully washed the EM root tips in water and removed soil and extra hyphae to avoid detecting nonmycorrhizal fungi. For nucleotide sequencing of fungal DNA from EM, we designed four fungal primers based on a comparison of mitochondrial atp6 sequences of Boletales fungi (Table 2, Fig. 1b) instead of using PCR primers of the nuclear ITS region (Gardes and Bruns, 1993
). These primers were designed to be somewhat specific to Boletaceae and to avoid amplifying products from nonmycorrhizal fungi such as endophytes, saprophytic fungi, or soil fungi. Four universal primers were also newly designed to amplify the rbcL gene in the chloroplast genome of host plants (Table 2, Fig. 1c). We amplified and sequenced the atp6 region and compared the sequences with those from the fruit bodies of Strobilomyces fungi. We considered the sampled EM fungi Strobilomyces when the sequences were identical. To identify host plant species, we amplified and sequenced rbcL genes from the EM samples and compared them to previously identified sequences in the NCBI GenBank database using the BLAST search tool.
The PCR conditions used for EM samples were as follows. The mixture contained 1x Ampdirect buffer, 1x Ampdirect addition-3 buffer (Shimadzu, Kyoto City, Kyoto, Japan), 1 nmol dNTPs, 5 pmol each forward and reverse primers, and 0.25 units Ex-Taq polymerase. An initial round of PCR was followed by amplification with nested-PCR primers. Cycling parameters included an initial denaturation at 95°C for 3 min; followed by 35 cycles of 30 s at 95°C, 30 s at 48–52°C, and 72°C for 30 s; and a final extension of 7 min at 72°C.
Morphological observation of fungi and EM samples
The pileus and stipe of fungi were examined without magnification or with the aid of a stereoscopic microscope. Basidiospores were examined using a light microscope or a scanning electron microscope (SEM, XL-Series; Philips Electronics, Minato Region, Tokyo, Japan) at 8000x.
We identified the EM of Strobilomyces fungi with a stereoscopic microscope. We distinguished the EM of Strobilomyces genus members from other fungi based on the morphology of S. confusus EM, which was described by Matsuda and Hijii (1999)
. Stereoscopic micrographs of Strobilomyces EM are shown in Fig. 2. Strobilomyces EM samples were then stored, prepared, and used for molecular analyses as described earlier.
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Nucleotide sequence variation among Strobilomyces samples and inferred phylogenetic relationships
Genetic distances were calculated from RPB1 and ITS2 sequences obtained from our specimens. The data indicated distinct segmentation among Strobilomyces species, because these fungi could be grouped into a total of 14 distinct lineages. Fungi typically categorized as Strobilomyces confusus and S. seminudus could be grouped into four lineages (lineages 1–4), Strobilomyces strobilaceus could be grouped into seven lineages (lineages 6–12), and S. mirandus comprised only one lineage (lineage 13). For the RPB1 data, the average similarity within each lineage was 99.95% (SD = 0.085), whereas that among lineages was 92.14% (SD = 4.68). For the ITS2 data, the average similarity within each lineage was of 99.51% (SD = 0.51), whereas similarity among lineages was 85.31% (SD = 6.92).
The RPB1 data set comprised 27 taxa and 1149 total characters, of which 318 were parsimony informative. We found few indels and no significantly variable sites in the nuclear RPB1 sequence data. For this reason, we included all character data in the analysis. Using MrModeltest, we selected a general time reversible (GTR) model that used gamma distribution (Rodriguez et al., 1990
) as the best-fit model for the RPB1 first position. A version of the HKY model that includes a proportion of invariable sites (Hasegawa et al., 1985
) was the best-fit model for the RPB1 second position. A version of the HKY model that includes a gamma distribution parameter was selected for the RPB1 third position and intron partition. The Bayesian inference topology is shown in Fig. 3. The topology of the MP trees (>50 000 MP trees; TL = 1049, CI = 0.724, RI = 0.903) was the same as that for the Bayesian tree and thus, only the Bayesian trees are shown in Fig. 3. Analysis used to generate these trees included posterior probabilities and BP values based on MP analysis.
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The ITS2 data set comprised 30 taxa, 542 total characters, and 192 parsimony informative characters. Several samples were removed prior to data analysis because they failed at the PCR amplification step. Ambiguously aligned regions (e.g., regions around the ITS4 primer) were also removed prior to analysis of the data. Based on the result of MrModeltest, a general time reversible (GTR) model that incorporates a proportion of the invariable sites and a gamma distribution parameter was selected for ITS2. The Bayesian inference topology is shown in Fig. 4. MP trees (>50 000 MP trees, TL = 557, CI = 0.682, RI = 0.891) had nearly identical topologies. Monophyly of Strobilomyces was supported by high posterior probabilities (1.00) and BP (94.8%). The existence of two large clades (A and B) was also supported by high posterior probabilities (0.98 and 0.95, respectively), but these also had lower BPs (81.4 and 73.8%). In addition, 14 distinct lineages were well supported by posterior probabilities in the range 0.95–1.00 and BPs in the range 98.4–100%. Little information about phylogenetic relationships among lineages could be inferred. Moreover, neither the clade formed by lineages 3 and 4 nor that formed by lineages 8 and 9 were supported by the ITS2 trees.
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The mitochondrial sequence data contained 25 taxa and 630 total characters, of which 129 were parsimony informative. Few indels were found, and thus all character data were included in Bayesian and MP analyses. The Felsenstein 81 model (Felsenstein, 1981
), incorporating arbitrary site-specific rate heterogeneity, was selected as the best-fit model in Bayesian analysis of atp6 by the Modelselect script. The topology of the MP trees (16 MP trees, TL = 263, CI = 0.821, RI = 0.905) was the same as that of the Bayesian trees. he Bayesian inference topology is shown in Fig. 5. As mentioned in the previous paragraph, phylogenetic relationships were more obscure with the mitochondrial trees than with the nuclear trees, perhaps because the atp6 region is more conserved than the other loci used in this study and is also heavily saturated at the third positions. A posterior probability of 0.98 and a BP of 88.6% support the monophyly of Strobilomyces. Clades designated in the nuclear DNA trees as clades A and B were also detected in the mitochondrial tree but the supporting BPswere lower for the mitochondrial data than for the nuclear data, possibly because the genetic distances among lineages were much lower in the mitochondrial trees than in the nuclear trees.
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). However, some morphological differences among the clades and lineages were also found (Fig. 5). As mentioned previously, for example, most members of clade A had spiny basidiospores whereas most members of clade B had reticulated basidiospores. Additionally, some lineages, including lineage 5 (clade A) and lineage 14 (clade B), had conspicuous morphological traits that helped to distinguish them from the others. More specifically, lineage 5 had a grainy pileus and a thick and long stipe, whereas lineage 14 had a pileus and stipe that were covered with light-brown, woolly scales. Lineage 13 (clade B), one of the types previously classified as S. mirandus, had conspicuous yellowish basidiocarps and could easily be discriminated from the others based on this feature, even though the molecular differences were not substantial.
More subtle morphological differences were detected among lineages 1 through 4 (clade A). For example, fungi of lineage 1 had a pileus with smooth scales and a stipe that is reticulated in its upper section. The lineage 2 had a pileus and stipe with wooly scales, and lineage 3 had a pileus with a rigid spine. The stipe of lineage 4 narrowed toward the top. We could not find morphological traits that distinguished lineages 6 through 12 (clade B).
Host specificity among Strobilomyces species
We identified host plants by amplification and nucleotide sequencing of the rbcL gene from chloroplast DNA in the EM samples. Although the collection sites were mixed forests composed of species in Fagaceae, Pinaceae, and Betulaceae (Table 1), Strobilomyces fungi were associated with only some of the possible host plant species present at a given site. For example, Alnus pendula Matsum., the only species of Betulaceae at our collection sites, was not detected as a host plant of Strobilomyces fungi. Moreover, the Fagaceae hosts we identified only included Castanopsis and Quercus, but the latter did include members of both the deciduous subgenus Quercus and the evergreen subgenus Cyclobalanopsis. Although Lithocarpus often existed at our collection sites (detected at Shiga-OM, Miyazaki-AY, Nantoh-SN, and Taipei-FS sites; Table 1), they were not detected as host plants of Strobilomyces fungi. As for Pinaceae hosts, we can conclude that it was P. densiflora that we identified as a Pinaceae host plant based on rbcL sequences, because we found no other related species at our collection sites. Similarly, the Castanopsis host species we detected is C. cuspidata (Thunb. ex Murray) Schottky.
Several species of subgenus Quercus (including Q. acutissima Carruthers, Q. serrata Thumb., and Q. variabilis Blume) were present at the collection sites (Table 1), and we could distinguish among them based on differences in the rbcL sequence. Consequently, we found that Q. serrata is a host plant for Strobilomyces fungi. We could not identify a particular host species of subgenus Cyclobalanopsis because several members of the subgenus (including Q. glauca Thunb. ex Murray, Q. gilva Blume, Q. hondae Makino, Q. myrsinaefolia Blume, Q. salicina Blume, and Q. sessilifolia Blume) were all growing in Shiga-OM sites (Table 1) and had the same nucleotide sequence of rbcL gene.
Interestingly, fungal lineages 1 through 4 had different host plant associations. Members of lineages 1, 2, and 4 associated only with Fagaceae hosts, whereas lineage 3 fungi associated with both Fagaceae and Pinaceae. Strobilomyces fungi of each of the four lineages and several possible host plants (including C. cuspidata, P. densiflora, Q. serrata, and two evergreen oaks Q. glauca and Q. myrsinaefolia) coexist at the Kyoto-YS and Shiga-MD sites (Table 1).
It is noteworthy that P. densiflora was associated only with Strobilomyces fungi of lineage 3, whereas Fagaceae species were associated with all four lineages at both sites included in this study. The results of reconstructing the ancestral state of the ectomycorrhizal host of Strobilomyces spp. using parsimony suggest that the ability to associate with pine trees is a newly acquired trait for lineage 3. Finally, information was insufficient to infer host associations for clade B, although members of lineage 6 inhabited only the Shiga-OM site and seemed to be associated only with evergreen oaks, even though other possible hosts (such as Castanopsis and Lithocarpus trees) were also growing at the site.
DISCUSSION
Cryptic species and systematics in Strobilomyces
Molecular phylogenetic trees based on nucleotide sequences of nuclear RPB1 and ITS2 region indicate that, with the exception of S. mirandus, the previously described "species" we analyzed comprised nonmonophyletic groups and included highly differentiated plural lineages. The data strongly suggest that S. confusus and S. seminudus have been confused and that they should be divided into at least four species. Similarly, the sequence data suggest that S. strobilaceus can be divided into seven or more species. We assert that division of Strobilomyces fungi is valid because S. mirandus, which has a conspicuous appearance and seems to be an independent taxon, did not differ remarkably from neighboring lineages in terms of nuclear DNA sequence. Mitochondrial DNA sequence data indicated only a small degree of genetic difference among the recognized lineages. Nevertheless, molecular data of the mitochondrial atp6 region corresponded well with the nuclear DNA information. The distinct cytonuclear disequilibria observed among the 14 lineages suggests that at least to some extent, a reproductive barrier may exist among these lineages in addition to the genetic differences we observed: if random mating had occurred among different mitochondrial lineages, the lineages would have collapsed in the nuclear phylogenetic trees.
Our results indicate, therefore, that nucleotide sequences of nuclear and mitochondrial DNA provide much more information pertinent to detecting reproductively isolated cryptic species than do the previously used analyses based on PCR-RFLP (Karen et al., 1997
; Bruns et al., 1998
; Horton and Bruns, 1998
; Cullings et al., 2000
; Kennedy et al., 2003
). However, to calculate cytonuclear disequilibria, one must first be able to distinguish homozygote from heterozygote in nuclear genotypes (Arnold, 1993
; Avise, 2004
). In this study, we could not make this distinction, and thus we cannot determine whether gene flow among Strobilomyces lineages is absent or whether sex-based directionality among lineages has occurred. Absence of hybridization among several lineages should be verified by population genetic analyses using codominant genetic markers in sympatric populations of various lineages. Nevertheless, comparison of molecular information of both nuclear and mitochondrial DNA is clearly useful for detecting candidates of cryptic species because it enables an exhaustive search for cryptic species. Finding such species is difficult in population genetics studies, and we propose that this type of analysis would serve as an appropriate first step before population genetic analyses.
The morphological differences detected among some lineages supports the idea that Strobilomyces can be categorized in a larger number of distinct groups than previously thought. Lineages 1 through 4, which include S. confusus and S. seminudus, were distinguishable by pileus or stipe morphology. To further classify these lineages and describe their morphology, researchers will need to compare them with type specimens from several Strobilomyces species that are morphologically similar. Using the methods described here, we cannot currently detect morphological differences among lineages 6 through 12, which were originally described as S. strobilaceus. Analysis of more samples from each lineage will be necessary before we can be certain about their taxonomical status.
In conclusion, relying solely on morphological characteristics to identify distinct species may be problematic for organisms with few distinguishing morphological characteristics. However, comparing the nucleotide sequences of nuclear and cytoplasmic DNA will help researchers detect species boundaries even if the organism includes many cryptic species.
Host specificity in Strobilomyces
Sequencing of the host plant rbcL gene from EM samples clearly revealed that Strobilomyces fungi do not associate with all possible host plant species at the collection sites. Instead, the fungi usually appear to associate only with particular plant species. This fungal genus may prefer Fagaceae species over Pinaceae species as host plants. However, the relative preference for Betulaceae trees remains unclear because these trees were not common at our collection sites. Although more detailed information is needed, the data suggest that Strobilomyces fungi prefer some species of Fagaceae (such as Castanopsis cuspidata, Quercus serrata, and evergreen oak species) over other Fagaceae species.
Fungi of lineage 3 were associated with both Fagaceae and Pinaceae species hosts; in contrast, other lineages were associated only with Fagaceae species. If we assume that each lineage represents an independent biological species, then lineage 3 might be a generalist EM fungus and the others might be specialists. Indeed, additional specialist species of Strobilomyces might be found in future studies. For example, lineage 6 was associated only with evergreen oaks at the Shiga-OM site, but additional analysis of host plants would be needed to confirm this level of specificity. The Shiga-OM location is distinctive because it contained several evergreen oak species, including the rare oak species Quercus gilva and Q. hondae (Table 1). Perhaps lineage 6 might be found in other collection sites with similar potential host flora, such as Kyoto-SM, Miyazaki-AY, Miyazaki-TK, and Miyazaki-MK (Table 1). In this study, however, we were able to obtain only a few samples at each of these sites. A more extensive investigation of EM fungi and hosts at these sites might clarify the host specificity of Strobilomyces among evergreen oak species.
Most EM fungi had been thought to have low host specificity (Molina et al., 1992
; Cairney and Chambers, 1999
), and some studies have reported that generalist fungi can use distantly related plant species as hosts in natural systems (Gardes and Bruns, 1993
; Horton and Bruns, 1998
; Stendell et al., 1999
; Taylor and Bruns, 1999
; Kennedy et al., 2003
). The results obtained in this study, however, suggest that a broad host range for EM fungi might not be as common as formerly thought and that many cryptic fungal species with tighter host specificity may exist.
Generation and analysis of phylogenetic trees can help to clarify our understanding of the evolution of host specificity and/or host switching among EM fungi. As inferred from the tree drawn for clade A based on nuclear RPB1 sequence data, Strobilomyces fungi appear to have acquired a new trait after speciation of lineages 3 and 4, a trait that allows them to associate with Pinaceae in addition to Fagaceae (Fig. 3). This would suggest that pine hosts were a recent acquisition of Strobilomyces fungi. Such evolutionary patterns correlate with a hypothesis presented by den Bakker et al. (2005)
that genetic isolation of allopatric populations during glaciation in the Quaternary accounts for evolution of host specificity or host switching. These researchers suggested that coexistence of several host trees or disappearance of specific host trees at that time might have relaxed host specificity of fungi. However, the available data are insufficient to conclude that EM fungi evolved from specialist into generalist or vice versa. Analysis of molecular data to study the evolutionary patterns of acquisition or loss of host specificity of EM fungi should increase our understanding of the evolutionary relationships among fungi and their host plants.
APPENDIX 1.
Taxa and voucher sample information of the fungal and EM samples, and GenBank accession numbers for sequence data obtained in this study. Voucher specimens are deposited in the Makino Herbarium of Tokyo Metropolitan University (MAK).
Taxon—GenBank accessions: RPB1 (basidiocarp), ITS2 (basidiocarp), atp6 (basidiocarp), atp6 (EM fungi), rbcL (EM plant); Source, Voucher specimen.
Austroboletus gracilis (Peck) Wolfe.— —, —, AB275166, —, —; a002, Mt. Kiyomizu, Higashiyama-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-KY), MAK.
Boletellus obscurecoccineus (v. Hohn.) Sing.— —, —, AB275167, —, —; b003, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Siga-MD), MAK.
Strobilomyces confusus Sing.— AB275254, AB275201, AB275173, AB275229, AB275249; s119, Mt. Tanakami, Otsu-shi, Shiga Pref., Japan (Shiga-TN), MAK. Strobilomyces confusus Sing.— AB275258, AB275202, AB275173, AB275232, AB275247; s189, Mt. Tanakami, Otsu-shi, Shiga Pref., Japan (Shiga-TN), MAK. Strobilomyces confusus Sing.— AB275250, AB275192, AB275169, AB275223, AB275247; s249, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces confusus Sing.— AB275250, AB275190, AB275168, AB275220, AB275247; s274, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces confusus Sing.— AB275250, AB275190, AB275169, AB275221, AB275247; s296, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK.
Strobilomyces confusus Sing.— AB275250, AB275194, AB275170, AB275222, AB275244; s323, Mino-shi, Osaka Pref., Japan (Osaka-MN), MAK. Strobilomyces confusus Sing.— AB275258, —, AB275173, AB275233, AB275249; s331, Mt. Atago, ukyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-AT), MAK. Strobilomyces confusus Sing.— AB275254, —, AB275173, AB275234, AB275249; s348, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275258, —, AB275173, AB275234, AB275249; s349, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275191, AB275168, AB275220, AB275244; s362, Takahara-cho, Seishoken-gun, Miyazaki Pref., Japan (Miyazaki-TK), MAK. Strobilomyces confusus Sing.— AB275250, AB275190, AB275168, AB275220, AB275246; s367, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces confusus Sing.— AB275250, AB275195, AB275169, AB275221, AB275247; s372, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275191, AB275169, AB275221, AB275244; s377, Shimogamo, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-SM), MAK. Strobilomyces confusus Sing.— AB275257, AB275199, AB275173, AB275229, AB275245; s384, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275190, AB275169, AB275221, AB275247; s386, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275196, AB275169, AB275221, AB275247; s391, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275254, AB275200, AB275173, AB275229, AB275249; s394, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275254, AB275199, AB275173, AB275229, AB275247; s396, Mt. Kiyomizu, Higashiyama-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-KY), MAK. Strobilomyces confusus Sing.— AB275258, AB275200, AB275173, AB275230, AB275247; s397, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275193, AB275169, AB275221, AB275247; s407, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces confusus Sing.— AB275258, AB275200, AB275173, AB275231, AB275249; s409, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces confusus Sing.— AB275255, AB275199, AB275173, AB275229, AB275247; s410, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces confusus Sing.— AB275250, AB275195, AB275168, AB275220, AB275244; s415, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces confusus Sing.— AB275256, AB275199, AB275173, AB275229, AB275244; s416, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces confusus Sing.— AB275250, AB275191, AB275168, AB275220, AB275245; s426, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275190, AB275168, AB275220, AB275244; s429, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces confusus Sing.— AB275250, AB275195, AB275169, AB275221, AB275245; s434, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces miranudus Corner— AB275274, AB275218, AB275187, —, —; s234, Mt. Mukabaki, Nobeoka-cho, Miyazaki Pref., Japan (Miyazaki-MK), MAK. Strobilomyces miranudus Corner— AB275275, AB275218, AB275188, AB275243, AB275245; s310, Fu-shan Research Station, Taipei, Taiwan (Taipei-FS), MAK. Strobilomyces seminudus Hongo.— AB275251, AB275197, AB275171, AB275224, AB275244; s120, Mt. Kiyomizu, Higashiyama-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-KY), MAK. Strobilomyces seminudus Hongo.— AB275251, AB275197, AB275172, AB275225, AB275244; s170, Mino-shi, Osaka Pref., Japan (Osaka-MN), MAK. Strobilomyces seminudus Hongo.— AB275260, AB275204, AB275174, AB275235, AB275247; s243, Takahara-cho, Seishoken-gun, Miyazaki Pref., Japan (Miyazaki-TK), MAK. Strobilomyces seminudus Hongo.— AB275259, AB275207, AB275175, AB275236, AB275247; s284, Mizorogaike, Kita-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-MZ), MAK. Strobilomyces seminudus Hongo.— AB275261, AB275205, AB275174, AB275235, AB275244; s345, Mt. Kiyomizu, Higashiyama-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-KY), MAK. Strobilomyces seminudus Hongo.— AB275259, AB275203, AB275174, AB275235, AB275247; s346, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces seminudus Hongo.— AB275252, AB275197, AB275171, AB275226, AB275247; s370, Takaragaike, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-TK), MAK. Strobilomyces seminudus Hongo.— AB275251, AB275197, AB275172, AB275225, AB275245; s381, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces seminudus Hongo.— AB275251, AB275197, AB275186, AB275227, AB275247; s399, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces seminudus Hongo.— AB275251, AB275198, AB275172, AB275225, AB275247; s405, Mt. Tanakami, Otsu-shi, Shiga Pref., Japan (Shiga-TN), MAK. Strobilomyces seminudus Hongo.— AB275262, AB275206, AB275174, AB275235, AB275248; s406, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces seminudus Hongo.— AB275260, AB275205, AB275174, AB275235, AB275247; s411, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces seminudus Hongo.— AB275253, AB275198, AB275171, AB275228, AB275244; s421, Miidera-cho, Otsu-shi, Shiga Pref., Japan (Shiga-MD), MAK. Strobilomyces seminudus Hongo.— AB275259, AB275203, AB275174, AB275235, AB275245; s428, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275269, AB275214, AB275181, —, —; s108, Aya-cho, Toshoken-gun, Miyazaki Pref., Japan (Miyazaki-AY), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275270, AB275214, AB275181, —, —; s109, Aya-cho, Toshoken-gun, Miyazaki Pref., Japan (Miyazaki-AY), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275270, —, AB275179, AB275240, AB275244; s110, Aya-cho, Toshoken-gun, Miyazaki Pref., Japan (Miyazaki-AY), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275266, AB275214, AB275182, AB275241, AB275244; s111, Aya-cho, Toshoken-gun, Miyazaki Pref., Japan (Miyazaki-AY), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275265, AB275211, AB275178, —, —; s174, Mino-shi, Osaka Pref., Japan (Osaka-MN), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275268, AB275213, AB275180, —, —; s192, Minenohara-Hill, Susaka-shi, Nagano Pref., Japan (Nagano-MN), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275271, AB275215, AB275183, —, —; s240, Aya-cho, Toshoken-gun, Miyazaki Pref., Japan (Miyazaki-AY), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275264, AB275209, AB275177, AB275238, AB275245; S290, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275264, AB275209, AB275177, AB275238, AB275245; S293, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275273, AB275217, AB275185, —, —; S305, Sanlinchi, Nantoh, Taiwan (Nantoh-SN), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275265, AB275211, AB275178, —, —; S322, Mino-shi, Osaka Pref., Japan (Osaka-MN), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275264, AB275209, AB275177, AB275239, AB275245; S363, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275264, AB275210, AB275177, AB275238, AB275245; s379, Ohmi-Shrine, Jingu-cho, Otsu-shi, Shiga Pref., Japan (Shiga-OM), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275272, AB275216, AB275184, AB275242, AB275247; s380, Mt. Yoshida, Sakyo-ku, Kyoto-shi, Kyoto Pref., Japan (Kyoto-YS), MAK. Strobilomyces strobilaceus (Scop. : Fr.) Berk.— AB275267, AB275212, AB275179, AB275240, AB275247; s404, Mt. Tanakami, Otsu-shi, Shiga Pref., Japan (Shiga-TN), MAK. Strobilomyces sp. 1— AB275263, AB275208, AB275176, AB275237, AB275244; s359, Mito-shrine, Joyo-shi, Kyoto Pref. Japan (Kyoto-MT), MAK. Strobilomyces sp. 2— AB275276, AB275219, AB275189, —, —; s309, Fu-shan Research Station, Taipei, Taiwan (Taipei-FS), MAK.
Suillus luteus (L.:Fr.) S.F.Gray.— —, —, AB275165, —, —; s001, Mt. Tanakami, Otsu-shi, Shiga Pref., Japan (Shiga-TN), MAK.
FOOTNOTES
1 The authors thank K. Yokoyama (Shiga University) and T. Hattori (Forestry and Forest Products Research Institute) for valuable information on fungal systematics. They are grateful to J. Kikuchi (Nara University of Education) and T. Kadokawa (Kyoto University) for help with ectomycorrhizal observation of Strobilomyces and to S. Kurogi (Miyazaki Prefectural Museum of Nature and History), T. W. Hsu, and H. M. Chang (Institute of Endemic Species Research) for their help collecting Strobilomyces fungi. The authors are thankful for the JSPS Research Fellowships for Young Scientists to H.S. and Grant-in-Aid for Scientific Research No. 18370035 to N.M. 
4 Author for correspondence (h-sato{at}sys.bot.kyoto-u.ac.jp
)
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