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Sugadaira Montane Research Center, University of Tsukuba, Sugadaira, Sanada, Nagano 386-2201, Japan
Yoshihisa Suyama
Graduate School of Agricultural Science, Tohoku University, Kawatabi, Naruko, Miyagi 989-6711, Japan
Makoto Kakishima
Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We investigated intraspecific diversity and genetic structures of a saprotrophic fungus—Thysanophora penicillioides—based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the ß-tubulin and combined ITS and ß-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.
Key words: Biogeography, ß-tubulin, Cryptic species, Fungal population, Gene flow, rDNA ITS
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Rogers and Rogers 1999, Taylor et al. 2000). Recently, studies of intraspecific diversity of many economically important fungal taxa have been undertaken energetically to examine mushrooms (e.g. Xu et al 1997, Hughes et al 1998, Coetzee et al 2000, James and Vilgalys 2001), plant pathogenic fungi (e.g. Boeger et al 1993, Trigiano et al 1995, Milgroom et al 1996, Koenig et al 1997, Carbone and Kohn 2001), human pathogenic fungi (e.g. Gräser et al 1996, Xu et al 2000), and entomopathogenic fungi (e.g. Boucias et al 2000, Jensen and Eilenberg 2001, Fargues et al 2002). Less information exists regarding saprotrophic fungi that are not related directly to economic activity. Notwithstanding, recent environmental changes derived from human activity have required such information related to saprotrophic fungi because their distributions are not influenced greatly by artificial effects.
Some obstacles remain for biogeography and population studies of fungi. One is that collecting large samples of fungi from a wide area is difficult. In particular, habitats and distribution patterns of most saprotrophic fungi have not been observed clearly. Therefore, efficient sample collection is important for the study of population genetic structures (James and Vilgalys 2001). Another obstacle is the difficulty in defining fungal populations because direct confirmation of their individuals is lacking in the field (Rogers and Rogers 1999). Still, the distribution of a given fungus is probably discontinuous because the host or suitable substrate for the fungus is distributed discontinuously in most cases. Therefore, it is reasonable that a litter fungal species inhabiting leaf litter under a given host community is defined as a local population of the fungal species.
We selected a saprotrophic microfungus, Thysanophora penicillioides (Roum.) W. B. Kendr. as a target fungal species for fungal population study. Advantages of using the fungus include the following. 1) It is possible to collect many isolates because ecology of the fungus is well known. It appears in a specific habitat—decaying fir needles. 2) Its population is definable. One host, Abies mariesii Mast. (o-shirabiso fir) is dominant in discontinuous subalpine forests throughout Japan. For that reason, we can readily recognize species assemblages in respective forests. 3) It is a morphologically well-defined species. Therefore, we can isolate the fungus from substrates without confusion with other species in the same niche.
We conducted a study to reveal intraspecific diversity and population genetic structures of Thysanophora penicillioides in Abies mariesii forests in the upland mountains interspersed with central to eastern parts of Japan. This study revealed that the fungus had unexpectedly great genetic variation and probably contains plural cryptic species. This study elucidates the population genetic structures of A. mariesii forests and the genetic diversity of the fungus through analyses of the ITS sequences and the ß-tubulin gene sequences based on Japanese isolates and several European and North American isolates from public culture collections.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Sieber-canavesi and Sieber 1993, van Maanen and Gourbière 1997). Its natural distribution roughly overlaps with that of Pinaceae in the northern hemisphere. We infer that the fungus is commonly distributed over wide areas of Japan because investigators have reported that the fungus was isolated from fallen conifer needles and soils in several regions of Japan (Kominami et al 1952, Aoki et al 1990, Tokumasu 1996, Iwamoto and Tokumasu 2001), and fir species that appear to be main hosts of the fungus, distribute all over Japan except in subtropical regions such as Okinawa’s islands.
Fungal isolation.— –
Forests of Abies mariesii were selected as sampling sites for this study. The fir is distributed only in subalpine zones in high mountains located in central and eastern Honshu (Hayashi 1960). The distributions in the forests are discontinuous (FIG. 1).
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and FIG. 1
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). Distances between the sites within populations were at least 20 m. Samples for each site were collected by cutting a 10 x 10 cm section of litter from the forest floor. The samples were then brought back to the laboratory in a sterile paper bag.
Ten needles per site were used for fungal detection. Brown or partly darkened brown needles were selected from each litter sample. A modified Tokumasu’s washing method (Tokumasu 1980) was used for washing soil particles and other microorganisms out of the needles: ten needles placed in each test tube were washed with detergent water (0.005% aerosol-OT) and sterilized water for 3 min using an automatic mixer (S-100; Taitec Co.). After washing, the needles were dried for 12 h on sterilized filter paper on a clean bench. Subsequently, each of the needles was incubated at 20 C on a corn meal agar (Nissui Pharmaceutical Co. Ltd.) plate. After incubation, they were observed photomicroscopically for a month. One isolate per plate was reserved as the representative site when T. penicillioides appeared on plural needles in a plate. Isolates were incubated at 20 C on corn meal agar plates (Nissui Pharmaceutical Co. Ltd. or Sigma-Aldrich Corp.) for DNA techniques.
European and North American isolates.— –
Seven European and four North American strains were obtained from public culture collections from Centraalbureau voor Schimmelcultures (CBS), Institute for fermentation, Osaka (IFO), and the University of Alberta Microfungus Collection & Herbarium (UAMH). Their strains were isolated in eight countries over two continents and from various substrates (TABLE II).
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, Iwamoto et al 2002) under a GeneAmp PCR System 2400 (Applied Biosystems). Two-step PCR method was used with these conditions: initial denaturation at 95 C for 15 min, 40 cycles of denaturation at 94 C for 20 s, and annealing at 60 C for 1 min, a final extension period at 72 C for 10 min, and a 4 C soak. A QiAquick PCR Purification Kit (Qiagen Inc.) purified the PCR products.
The ß-tubulin gene partial fragments including three non-coding regions (about 360 bp) were amplified with benA1 and benA2 designed for Aspergillus species. (Geiser et al 1998). The amplification method was the same as that for ITS except that the annealing temperature was 58 C.
Sequencing.— – Sequencing reactions were performed with a BigDyeTM Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) according to the manufacturer’s instructions under a GeneAmp PCR System 2400 (Applied Biosystems). The primer ITP1 was used for reading upper sequences of ITS in all isolates. Representative isolates for phylogenetic analyses and isolates with minor changes were determined in both directions using the ITP2 primer and the mutation sites were confirmed. For isolates used in phylogenetic analyses, the ß-tubulin sequence was determined in both directions using benA1 and benA2 primers. Sequencing reaction products were purified with a DyeEx Spin Kit (Qiagen Inc.). Direct sequencing was performed using a sequencing system (ABI PRISM 377 DNA sequencing system; Applied Biosystems).
Data analyses.— –
Each distinctive sequence (allele) was defined as each ITS type. Among ITS types, sequences with variation of less than two sites were classified into the same group (ITS group). Nei’s gene diversity formulae (Hep) were used to evaluate the genetic diversity of populations (Nei 1973). The uncorrected distance (p-distance) between sequences of ITS or ß-tubulin were calculated using PAUP version 4.0b10 (Swofford 2002).
Phylogenetic analysis.— –
Three data sets—ITS, ß-tubulin, and ITS and ß-tubulin—were prepared of combined data including all ITS types of T. penicillioides (TABLE II, III). Alignments of data sets were determined using Clustal X version 1.81 (Thompson et al 1997) and were refined visually.
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). Consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were measured using PAUP. A partition homogeneity test was performed using PAUP to investigate the statistical congruence between the ITS and ß-tubulin data sets.
Sequences used for phylogenetic analyses were deposited in DDBJ. Accession numbers for ITS are AB175229
[GenBank]
–AB175252
[GenBank]
, AB213264
[GenBank]
–AB213271
[GenBank]
and numbers for ß-tubulin are AB175253
[GenBank]
–AB175276
[GenBank]
, AB213272
[GenBank]
–AB213279
[GenBank]
(TABLE II, III).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), thereby confirming that it abundantly inhabited decaying A. mariesii needles in Japan. Ultimately, 254 isolates were collected from 324 examined sites of 15 local populations (TABLE I).
Genetic diversity and genetic structures based on ITS in Abies mariesii forests in Japan.— –
ITS of all Japanese isolates were determined and 21 variable sites were recognized in the aligned sequences of ITS regions (FIG. 2). Finally, 12 distinctive sequences (ITS types 1a–5a) were detected. These ITS types were classified into five groups in which groups 1 and 2 were included more than two ITS types (FIG. 2). Frequencies of respective ITS types and diversity indexes (Hep) within local populations are shown in TABLE IV. Overall, type 1a was dominant (59.8%), followed by type 2a (19.3%), whereas other ITS types had low frequencies (less than 6.7%). Among ITS groups, group 1 was dominant (65.4%), followed by group 2 (25.2%).
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Type 1a was the only ITS type that was detected in all local populations; its allele frequency exceeded 0.500 in ten local populations. Type 2a occurred at relatively high frequency in three northern and three southern local populations. Remarkably, the three northern populations (HD, HT and HY) had minor haplotypes belonging to group 2 (2c–2e); they were found only in this region. Minor ITS types of group 1 (1b–1d) were restricted to western A. mariesii forests (MY, NB, TY, NH, YG, AS and SJ). Type 3a was detected from two adjacent local populations (KK and ZO) and type 5a in two geographically separate northern populations (HY and HA). A member of minor ITS group Type 4a occurred in a wide area.
Genetic diversity in European and North American isolates based on ITS.— –
When European and North American isolates were added, 28 variable sites were found in ITS of the fungus (FIG. 2). Four ITS types and two ITS groups were detected from 11 isolates collected in Europe and North America. It is noteworthy that none had been detected in Japan; new ITS types 6a, 6b, 6c and 7a were classified into new ITS groups 6 and 7 (TABLE II). Type 6a was dominant in the examined isolates and detected from both Europe (four countries) and North America (two countries). Types 6b and 6c were detected respectively in one European isolate, whereas type 7a was found in three North American isolates (TABLE II).
Genetic distances and Phylogenetic relationships.— –
TABLE V shows genetic distances between ITS types. The distances within each ITS group (for groups 1, 2 and 6) are 0.002–0.003. On the other hand, the genetic distance between ITS groups was an average of 0.013 for values of 0.003–0.029. The distance of group 3 vs. group 7 was not very distant (0.003) because the variation between groups was mainly contributed by some indels (FIG. 2).
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, III). The analyzed ß-tubulin region was more variable than ITS and many alleles were detected (TABLES II, III). The genetic distance matrix between each ß-tubulin allele is shown in TABLE VI. Considered based on the classification of its groups, grouping for groups 1–5 of ß-tubulin well corresponded to that of ITS; the distances within their groups and among groups were 0–0.028 and 0.031–0.082 respectively (TABLE VI). As for groups 6 and 7, however, the grouping of ITS was inconsistent to that of ß-tubulin: the ß-tubulin distances among isolates indicating ITS types 6a or 6b vs. 6c (CBS 345.33, 348.64, 553.68, 576.68. UAMH 3391, 4691, 9768 vs. CBS 292.60) were 0.036–0.042 and 0.041 between two groups of three isolates, indicating ITS type 7a (CBS 314.56 vs. UAMH 9248, NBRC 9011). These values corresponded with that between group vs. group for groups 1–5 (TABLES VI). In other words, these isolates accumulated genetic variations in the ß-tubulin locus but few or no genetic changes in ITS. Therefore, the strain CBS 292.60 indicating ITS type 6c is treated as a member of ß-tubulin group 8 (TABLE II). Similarly, UAMH 9248 and NBRC 9011, indicating ITS type 7a, are treated as ß-tubulin group 9 (TABLE II). Consequently, the genetic distance within a group ranged 0.003–0.028 and that between groups was 0.031–0.094 overall. This grouping was supported by phylogenetic analyses.
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). On the other hand, 18 MP trees were constructed using the ITS data set without the gap sites (603 sites including 15 parsimony-informative sites, length = 26 steps, CI = 0.808, RI = 0.957, RC = 0.773, HI = 0.192). Although the bootstrap values of groups 1–3 and 5 were not different from the data set including gaps, that of group 4 and group 6 clades were very low in comparison to it (FIG. 3).
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). In the group 1 clade, three subclades were generated along with two subclades in the group 2 clade. Of the isolates indicating the ITS group 6, four isolates of ITS types 6a or 6b were clustered to one clade comprising two subclades. However, one isolate (CBS 292.60) of ITS type 6c was not clustered with it; its lineage was treated as 8th group on the ß-tubulin tree (FIG. 4). Isolates of ITS group 7 were divisible into two lineages that were genetically distant from each other. These two lineages were not supported with a significant bootstrap value and one lineage was treated as 9th group (FIG. 4). Relationships among these four lineages were unknown by analyses of the ß-tubulin data set.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) was found to have uncommon genetic variations within a species. Respective genetic variations of regions among alleles were 8.57%, 5.56% and 6.29% in ITS1, ITS2, and total. These values apparently differ from those of related taxa belonging to Penicillium subgenus Penicillium, a close relativity of the genus Thysanophora (Iwamoto et al 2002), for which very low variations at ITS regions were indicated (Skouboe et al 1999, Scott et al 2004, Peterson 2004). The phylogenetic analyses showed that multiple lineages were recognizable in the fungus. Especially, the combined data of ITS and ß-tubulin strongly support nine lineages with high bootstrap values (93–100%, FIG. 5). According to phylogenetic species recognition by the concordance of genealogies (GCPSR, Taylor et al 2000), each lineage corresponds to a phylogenetic species. However, we should treat the fungus as a species complex that includes several cryptic species because we have found no clear phenotypic differentiations among lineages. Examples for which morphological characteristics are consistent with species boundaries by GCPSR have been reported for many fungi. In rare cases, combinations of GCPSR and other phenotypic characteristics or geographical information have been used to discern morphologically indistinguishable species complexes (Fisher et al 2002, O’Donnell et al 2004). Whether this species complex deserves to be separated depends on the result of further morphological, physiological and phylogenetic studies.
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, 1999), Colletotrichum species (Lu et al 2004), Lophodermium pinastrii (Müller et al 2001), and Phialocephara fortinii (Grünig et al 2004). Grünig et al. (2004) reported that different cryptic species in the P. fortinii species complex occurred in the same root segments of Norway spruce. The authors isolated two ITS types from a single decaying needle several times (data not shown). Each lineage of the species complex has undergone gene dispersion without geographic limitation among local populations because the distribution of each lineage shows a wide expansion through the studied areas in A. mariesii forests. However, ITS type 1a was detected frequently in all local populations: it was dominant in most local populations even though multiple ITS types coexisted in all populations. The fact that group 1, including ITS type 1a, is overwhelmingly dominant and other groups are minor in A. mariesii forests of Japan is interesting, but its reason remains unclear. Perhaps members of group 1 acquired more advantageous characteristics for fitness than other groups in the ecosystem.
In marked contrast, the minor ITS types within groups 1 and 2 were recorded only in restricted areas. It is assumed that types 1b, 1c, 1d were derived from type 1a, and types 2c, 2d, 2e from type 2a based on results of parsimony analysis (FIGS. 3–5). Probably, they were generated by a recent point mutation at ITS of types 1a or 2a. Therefore, their distributional areas might remain small even though they began to disperse from their places of origin.
We initially surmised that any clonal lineage might be distributed around the world and that various lineages found in Japanese population were also detected in overseas samples. Thereby, the fungus might be capable of dispersing its propagules throughout the world for a relatively short period because of abundant production of small dry conidia; it might be colonizable on fallen needles of various pinaceous trees that are distributed over a wide area. For sequencing the strains out of Japan, we obtained strains of Europe and North America via public culture collections. Results showed that ITS types of European and North American isolates differed from Japanese isolates and that they formed separate clades for Japanese isolates in phylogenetic trees. Against our expectations, this fact suggests that genetic diversification occurred independently in two separate regions and that numerous differentiated populations of the fungus might exist on a global scale. Although genetic structures among three separate regions might be different, we cannot draw a conclusion about the relationships between genetic structures of the three regions because the isolates obtained from European and North American populations are too few.
This study detected numerous genetic variations using ITS sequence polymorphism in an anamorphic fungus inhabiting leaf litter of Pinaceaeous forests. Results revealed genetically diverged groups, suggesting a species complex including multiple cryptic species. These facts foreshadow that more genetic variations for the species might be detectable in other areas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author, Present address: BioFrontier Laboratories, Kyowa Hakko Kogyo Co. Ltd., Asahi-machi, Machida, Tokyo 194-8533, Japan, E-mail: susumu.iwamoto{at}kyowa.co.jp
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