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Department of Botany, University of Tennessee, Knoxville, Tennessee 37996
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Artomyces pyxidatus (Auriscalpiaceae) is a lignicolous, coralloid basidiomycete found throughout temperate regions of the Northern Hemisphere. Previous studies established that populations from the eastern United States, Sweden, and China were conspecific based on mating compatibility and enzyme profiles. In this study, mating compatibility was extended to include collections from Russia, Costa Rica, Mexico, and Utah. The molecular diversity of A. pyxidatus was examined by DNA sequence and restriction site analyses of the nuclear ribosomal internally transcribed spacer region (ITS1–5.8S-ITS2). A phylogenetic analysis of twelve isolates based on ITS sequences revealed a broad geographical pattern in which Eurasian isolates comprise a sister clade to North American isolates. North American isolates appear to be further subdivided into northeastern and southwestern clades. A survey of 255 A. pyxidatus isolates using restriction enzymes revealed variable RFLP patterns that follow similar geographical patterns.
Key words: Artomyces pyxidatus, biogeography, Clavicorona pyxidata, RFLP, ribosomal DNA
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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erected subgenus Ramosa to include the branched, lignicolous, amyloid-spored species. Jülich (1981)
elevated this group to genus rank indicating that morphological differences between subgenera Ramosa and Clavicorona were significant enough to warrant genus level status. He established the name Artomyces, and chose A. pyxidatus as the type species. Recent sequence data from large subunit (27S) and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA support this genus rank treatment (Lickey 2001). Morphological and cultural characterizations of A. pyxidatus were elucidated by Dodd (1972)
, James and McLaughlin (1988)
, Wu (1991)
, and Wu et al (1995)
. Additionally, Wu (1991)
and Wu et al (1995)
determined that isolates of A. pyxidatus from China, Sweden, and the eastern United States were all intercompatible and suggested that they represented a single biological species. A different laccase enzyme banding pattern, however, was observed for isolates from China (Wu 1991
, Wu et al 1995
).
There are many examples of Homobasidiomycete species that have very broad geographic distributions and whose populations remain sexually intercompatible in vitro, even though they may be continents apart. These include Collybia dryophila (Vilgalys and Johnson 1987
, Vilgalys 1991
), Flammulina velutipes (Petersen et al 1999
), Panellus stypticus (Petersen and Bermudes 1992a, b
, Jin 2000
), Pholiota spumosa (McCleneghan 1996
), Pleurotopsis longinqua (Petersen 1992
, Petersen and McCleneghan 1995, 1997
), Pleurotus ostreatus and P. pulmonarius (Petersen and Hughes 1993
, Petersen 1995a, b
), Schizophyllum commune (Raper et al 1958
), and Xeromphalina campanella (Johnson 1997
). These taxa have been the subject of recent genetic studies to determine the amount of variation that exists among widely disparate populations (Vilgalys and Johnson 1987
, Vilgalys 1991
, Vilgalys and Sun 1994
, McCleneghan 1996
, Johnson 1997
, Hughes et al 1998, 1999
, James et al 1999
, Methven et al 2000
, Jin et al 2001
). While biological barriers to reproduction are apparently lacking among populations of these taxa, geographic barriers to gene flow seem to exist as evidenced by sequence and allozymic divergence.
The purpose of the present study was to elucidate biogeographic patterns among collections of Artomyces pyxidatus. We report mating compatibility patterns of collections from Costa Rica, Mexico, Russia, and Utah with previous collections and examine biogeographical patterns using DNA sequence and restriction fragment length polymorphism (RFLP) frequencies of the nuclear ribosomal ITS area.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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.
Mating studies –
In addition to previously available cultures, single basidiospore isolates (SBI) were established from spore prints of recent collections from Russia, Mexico, Costa Rica, and Utah. Intercompatibility studies were performed as described by Gordon and Petersen (1991)
with the following modifications. Four monokaryons from each collection were paired with four monokaryons from another collection resulting in a set of four pairings between the two collections (i.e., monokaryons of 9932 and 8396 were paired as 1 x 3, 7 x 5, 9 x 7, and 11 x 9, respectively). Five collections from Costa Rica, eight from Mexico, one from Russia, and one from Utah were included in this test of intercompatibility. Where possible, A. pyxidatus monokaryons from collections used by Wu (1991)
were included in this study. Two collections (8896 and 8950 from Russia), were paired only with 8723 (Mexico) and 8927 (Russia). Collection 1541 (China) was represented by only one monokaryon, and collections 1607 (North Carolina) and 8927 (Russia) were represented by only three monokaryons. All isolates included in the mating study are indicated in Table I
. Compatible matings were determined by the presence of well-formed clamp connections in and near the contact zone, viewed directly on the agar surface under 250x magnification.
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. The nuclear ribosomal ITS 1-5.8S-ITS 2 (ITS) region was amplified using forward primer ITS 5 and reverse primer ITS 4 (White et al 1990
). The PCR protocols were: initial melting cycle of 94 C for 4 min 35 repetitions of a three-step amplification cycle of 94 C for 1 min 52 C for 1 min and 72 C for 1 min ending with an extension cycle of 72 C for 1 min.
Twelve isolates of A. pyxidatus were chosen for sequencing in order to represent populations from a wide geographic range (Table I
). Additionally, two isolates A. microspora were chosen for outgroup comparison because of its close relationship with A. pyxidatus (Lickey unpubl). PCR products were directly purified using a Promega Wizard PCR Purification Kit. The purified product was sequenced using primers ITS 5 and ITS 4 on an automated ABI 373 DNA sequencer (ABI Prism Dye Terminator cycle sequencing, Perkin-Elmer, Inc.). Primers ITS 2 and ITS 3 were used in some cases to clarify ambiguous sequences resulting from insertions and/or deletions (indels). Rarely, a smaller secondary, interfering fragment of non-ITS origin would amplify. In this case, the PCR products were first electrophoresed and then the ITS fragment was excised from a 1 x TAE 1.5% low-melting temperature agarose gel (NuSieve GTG agarose, FMC Bioproducts) following manufacturer's directions. Sequences were manually corrected and aligned using the LINEUP and SEQLAB programs in the Genetics Computer Group package (GCG 2001
), and deposited in GenBank. Accession numbers for A. pyxidatus isolates are: 1541 = AF336140, 7263 = AF336139, 8927 = AF336141, 8903 = AF336142, 8723 = AF336143, 5484 = AF336146, h7524 = AF336148, 9709 = AF336149, 9932 = AF336150, 4938 = AF336147, 1513 = AF336144, and 56667 = AF336145. Accession numbers for A. microspora isolates are: ky5352 = AF336137 and 2349 = AF336138.
Variable regions within the ITS1–5.8S-ITS2 region were identified by comparing aligned sequences. Restriction sites were mapped for ITS sequences using the MAP program of GCG (GCG 2001)
and restriction enzymes were selected which produced restriction fragment length polymorphisms (RFLP) among A. pyxidatus collections. ITS PCR products of all collections in the study were surveyed using restriction enzymes BsaJ I, Bsr I, Ear I, Tse I, Xho I (New England BioLabs), and Cfo I (Promega) according to manufacturer's directions. Restriction fragments were separated on 1 x TBE 3% agarose gels stained with ethidium bromide, and were visualized under ultraviolet light. Fragment sizes were estimated and compared with predicted fragment sizes based on the mapped ITS sequences. Isolates were then scored for presence or absence of restriction sites.
Phylogenetic reconstruction –
Phylogenetic relationships were estimated using PAUP 4.0 (Swofford 2000). Two apomorphic insertions were detected among A. pyxidatus sequences and were excluded from the analysis. Gaps in A. pyxidatus sequences that resulted from the alignment with A. microspora (the designated outgroup) were treated as missing data. A branch-and-bound search was used to find the most parsimonious tree. Two bootstrap analyses of 100 replicates were conducted with both a branch-and-bound search using maximum parsimony and a neighbor-joining search (Saitou and Nei 1987
) using Jukes-Cantor distance measures (Jukes 1969
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Phylogenetic reconstruction – A parsimony analysis of the ITS sequences using a branch-and-bound search yielded two most parsimonious trees of 65 steps (Fig. 2 ; TreeBase accession # S661, matrix # M1037). Bootstrap analyses showed strong support for three major branches with little resolution at the tips. The neighbor-joining tree had the same overall topology as the tree produced by the branch-and-bound searches. Two major branches were resolved within A. pyxidatus; a Eurasian clade and a New World clade. Within the New World clade, there appears to be a well-supported separation between eastern isolates and southern isolates with overlap in Georgia. A similar but less well-supported separation occurs in the Eurasian clade where there was an apparent separation between European isolates and the single Asian isolate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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), Pleurotus pulmonarius European and North American collections showed a 0.5% sequence difference (Vilgalys and Sun 1994
) and Panellus stypticus collections differed by 0.7% (unpubl). Continent-specific RFLP patterns based on ITS sequence differences confirmed that European and North American populations were genetically distinct.
Additional evidence for reproductive separation between continents is found in analysis of an 18S Group I intron in A. pyxidatus previously discovered by Hibbett (1996)
. All collections of A. pyxidatus contained a Group I intron in the 18S ribosomal RNA gene (Lickey 2001). However, New World isolates, including the three with geographically inconsistent RFLP patterns, contained an unknown insertional element within the 18S Group I intron. This unknown insertional element was absent from all Eurasian isolates examined (Lickey 2001). Presence of the "unknown" insertional element in collections that were exceptions to biogeographical RFLP patterns suggests that these anomalies may be due to mutations resulting in loss of a restriction site rather than to European introductions into North America.
Presence of widespread A. pyxidatus sequence and RFLP patterns unique to either North America or Europe, suggests that populations on these two landmasses are effectively geographically isolated. ITS sequence differences would further suggest that these geographical domains have been separated for a long time. In contrast, an allozyme study of Schizophyllum commune, which has an almost global distribution, indicated that this species maintains high levels of genetic variation and no fixed allelic differences were observed among populations (James et al 1999
). It was hypothesized that some low levels of gene flow may have occurred through long-distance spore dispersal, and/or that effective population sizes were large enough to overcome the effects of genetic drift.
Biogeographical history –
The apparent reproductive isolation of Europe and North America raises questions as to when these populations were last connected. Several researchers have concluded that effective reproductive isolation may have occurred millions of years ago [Vilgalys and Sun 1994
(Pleurotus), Wu and Mueller 1997
, Redhead 1988
, Hughes et al 1999
(Flammulina), Methven et al 2000
(Flammulina), Coetzee et al 2000
(Armillaria mellea), and Jin et al 2001
(Panellus stypticus)]. All of these have been hypothesized to be very old species with Laurasian distributions. Their populations may have been separated with the disappearance of the last North Atlantic land bridge in the Eocene (
40 Mya), or more recently with the disappearance of any of the Bering land bridges in the Pleistocene (1.6 Mya). During periods when land bridges existed, forests, and probably their fungal counterparts, were much more widely distributed across the connected continents of the Northern Hemisphere (Graham 1999
).
Our data do not address either the temporal or geographic origin of species A. pyxidatus, however, the European and North American populations must have shared a gene pool at some point in the past. Both the Bering land bridge and the North Atlantic land bridge seem to be equally feasible possibilities for places of last contact. The Bering land bridge was an important corridor for the migration of fauna and flora through the Middle Pliocene (3.5 Mya) and through its intermittent existence during the Quaternary Period (1.6 Mya) (Graham 1999
). Evidence from biogeographical studies with Flammulina velutipes and Panellus stypticus suggest that populations of these species migrated between Asia and western North America (Methven et al 2000
, Jin et al 2001
). It is possible that A. pyxidatus followed that route. However, A. pyxidatus is not known from west of the Rocky Mountains, and the forests that presently occupy that area are largely coniferous. Artomyces pyxidatus seems to occur only on decaying hardwood logs in deciduous forests, and in North America it appears to have a preference for diffuse-porous wood such as Acer, Liriodendron, and Populus (our personal observation). Up to the Late Tertiary (3.4 Mya), climates were warmer and western North America may have been more suitable for deciduous forests favored by A. pyxidatus. During the later Quaternary period, however, the western US and Canada maintained mostly boreal to tundra vegetation types (Delcourt and Delcourt 1993
). If a Bering land bridge was a migration route for A. pyxidatus, it may have been extirpated from the western US and Canada during the Quaternary period.
The North Atlantic land bridge was much more ancient, and the separation between North America and Europe is estimated to have been complete by the Eocene (40 Mya) (Graham 1999
). It was also an important corridor for the exchange of flora and fauna throughout much of the Tertiary, where deciduous forests presumably extended uninterrupted across North America, Europe, and Asia (Graham 1999
). If this was the point of last contact, and the time of separation was approximately 40 Mya, then A. pyxidatus and other fungi with similar distributions are extremely old. Alternatively, after the disappearance of the land bridge, islands such as Greenland and Iceland could have provided "stepping-stones" during warmer periods before and during the Quaternary. Long-distance spore dispersal has not been studied in Clavicorona, but basidiospores of Heterobasidium annosum have been estimated to travel at least 200 miles (322.6 km) (Rishbeth 1959
). Presently, populations of A. pyxidatus are known from as far north as Maine and Sweden, but A. pyxidatus has not been collected from Greenland (Rostrup 1888
, Kobayasi et al 1971
, Watling 1977, 1983
, Knudsen et al 1993
).
A North Atlantic connection is suggested by RFLP frequencies (Fig. 4 ). Even though there is measurable ITS sequence divergence between Eurasian and New World isolates, eastern North American isolates share alleles for BsaJ I and Cfo I "b" with Eurasian isolates. This is consistent with a North Atlantic land bridge connection but might also be explained by rare long-distance spore dispersal. With the prevailing winds moving in an easterly direction, it is conceivable that spores could have been dispersed long distances from eastern North America to Europe. A founder effect may have caused the resulting populations in Europe to be fixed for the shared alleles BsaJ I and Cfo I "b".
Within the New World, isolates can be subdivided into a northeastern and a southwestern group based on ITS sequence data (Fig. 2
). This pattern may reflect the influence of glaciation on populations of A. pyxidatus and its host trees. Throughout the Quaternary, the ranges of forest species across the Northern Hemisphere contracted and expanded with the advance and retreat of glaciers (Delcourt and Delcourt 1993
). Genetic divergence is hypothesized to have occurred when populations were forced southward into isolated refugia during glacial maxima. No fixed restriction site differences were observed in the ITS region among North American isolates, but there are clearly differences in allele frequencies in the two restriction sites Cfo I site "b" and BsaJ I (Fig. 4
). Artomyces pyxidatus was collected in Mississippi (9201), Louisiana (not cultured), and eastern Texas (9828), and there is a potential for gene flow between northeastern populations and southwestern populations through this Gulf Coast corridor. The ITS phylogeny (Fig. 2
) places collections from Georgia in both the northeastern clade (collection 56667) and the southwestern clade (collection 5484). Possibly, the southeastern US represents a hybrid zone where these two populations may introgress.
Geographically consistent crossing barriers in vitro were not observed in this study or in earlier studies (Wu 1991
, Wu et al 1995
) in spite of sequence and RFLP differences. This suggests that mating ability is conserved across diverging allopatric populations and that reproductive isolation is not congruent with sequence divergence.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication October 1, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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