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Phylogeny of the Hypochnicium punctulatum complex as inferred from ITS sequence data

     Botanical Institute, Göteborg University, Carl Skottsbergs Gata 22b, S-405 30 Göteborg, Sweden

	ABSTRACT

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Parsimony analysis based on ITS sequence data was carried out to investigate the Hypochnicium punctulatum complex (Basidiomycota). The study gives full support to earlier, crossing test-based species delimitations. Altogether, 18 specimens were sequenced and their spore sizes plotted together with measurements from the corresponding type specimens. Spore sizes were found to cluster readily into four groups, all of which were supported by the phylogenetic analysis. However, in the case of H. punctulatum and H. albostramineum, the morphological delimitation is unsatisfactory and a zone of potential spore size overlap is shown to exist. The new combination Hypochnicium cremicolor is proposed for a species previously known as a small-spored taxon in the H. punctulatum complex, and H. caucasicum is shown to be a younger synonym to H. wakefieldiae. A key to the species is provided.

Key words: Corticiaceae, Hypochnicium, ITS, ribosomal DNA, parsimony, phylogeny, species complex

	INTRODUCTION

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Many genera among corticioid fungi (Basidiomycota) contain species complexes, where specimens with seemingly identical or very similar basidiomata are genetically isolated from each other, hence forming distinct biological species in the concept's strict definition (Boidin 1977, Hallenberg 1985). Within the genus Hypochnicium J. Erikss., such species complexes are found, one of which is in focus for the present study.

The genus Hypochnicium was formed by Eriksson in 1958 to include species formerly placed in the genera Corticium Pers., Gloeocystidium Karst., Peniophora Cke., and Thelephora Ehrh. ex Willd. Key characters of the new genus were rather big, thick-walled spores (the walls were later found to stain with Cotton Blue) and to some extent, distinct and richly ramified hyphae. In 1976, Eriksson and Ryvarden depicted Hypochnicium as an easily circumscribed genus that was comparatively homogeneous and relatively well separated from other genera. Several species have been added to the genus since Eriksson's publication (1958). Boidin (2000) presented a key to all 29 species described so far.

Six morphological subgroups were recognized among the North European species, one of which was referred to as the H. punctulatum-group (Eriksson and Ryvarden 1976). In this group, a new combination, H. eichleri (Bres.) Erikss. and Ryv., was introduced. According to the flora "Corticiaceae of North Europe" (Eriksson and Ryvarden 1976), H. punctulatum J. Erikss. and H. eichleri could be separated by means of spore size; H. punctulatum was said to have spores 5.5–6.6 x 4.5–5 µm in size, whereas the spores of H. eichleri were somewhat bigger, 8–10 x 6–7 µm. Nevertheless, several herbarium specimens of the H. punctulatum group seemed intermediate in terms of spore size. Even resorting to ecological and distributional criteria, no unequivocal line could be drawn with respect to exact species delimitation. Using incompatibility tests and cultural studies, Hallenberg (1985) surprisingly found three subgroups inside the H. punctulatum group. The first subgroup was heterothallic, tetrapolar, and had small spores (5–6.5 x 4.5–5.5 µm), hence corresponding well to H. punctulatum sensu Eriksson and Ryvarden (1976); the second subgroup was heterothallic, tetrapolar, had bigger spores (7–8 x 5.5–7 µm), and was designated H. eichleri s. str.; the third subgroup was homothallic, had even bigger spores (8–10 x 6–7 µm), and represented a distinct species, which was named H. albostramineum (Bres.) Hallenb. Spores in the last mentioned taxon were uninucleate, and single-spore mycelia were found to be regularly dikaryotic.

Hallenberg (1985) concluded that it seemed impossible to separate H. eichleri from H. punctulatum and H. albostramineum based on distribution, biotope, and substrate preference; however, these criteria could be used to separate H. punctulatum from H. albostramineum. Thus, H. eichleri seemed to account for the specimens with the intermediate spore size of the H. punctulatum group sensu Eriksson and Ryvarden (1976).

Several species have been described within this complex, and uncertainty of species delimitation has made taxonomy troublesome. It is the purpose of the present study to compare the result from a phylogenetic analysis with morphology and crossing test data and re-establish taxonomy and nomenclature on a firm basis. For the benefit of the reader, proposed taxon names are used from this point. An overview of past and present names is found in Table II.

	MATERIALS AND METHODS

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PCR amplification and DNA sequencing – The polymerase chain reactions were carried out using Ready-To-Go^TM PCR Beads kit (Amersham Pharmacia Biotech) and PCR primers ITS1F and ITS4B (Gardes and Bruns 1993) in a Biometra TRIO-Thermoblock (Biometra, Germany). PCR conditions followed Gardes and Bruns (1993). The PCR product was purified using QIAquick^TM Spin procedure (QIAGEN) in accordance with the manufacturer's recommendations. For quantification, a GeneQuant II (Pharmacia Biotech) spectrophotometer was used. Sequence reactions were conducted using the RPN 2438/RPN 2538 Thermo Sequenase Fluorescent Labelled Primer Cycle Sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech), following the manufacturer's recommendation. For each reaction, 50 ng of template DNA and 2.5 pmol 5'-cy-labeled primer (ITS1F_cy or ITS4B_cy) were used. The sequences were obtained using an ALFexpress^TM (Pharmacia Biotech) automated sequencer. GenBank numbers are given in Table I.

Data analysis – The obtained sequences (ITS1, 5.8S, and ITS2) were edited in the ALF manager® (Pharmacia Biotech) and Sequencher® (GeneCodes Inc.) software, and were aligned manually in PAUP* 4.0b8 (Swofford 2000). A 10 bp long insert was recoded as two characters. All characters were unordered and gaps were treated as "missing data" or as "fifth characters".

Parsimony analysis was performed in PAUP* using the branch and bound search method and outgroup rooting when gaps were treated as "fifth characters". When gaps were treated as "missing data" a heuristic search was performed with 2000 replications of stepwise random addition with three trees held at each step, TBR swapping, MULPARS, and MaxTrees set to 10 000. Hypochnicium geogenium (Bres.) J. Erikss. together with H. subrigescens Boidin were selected as outgroup because of similarities in basidiome morphology with species in the H. punctulatum complex. The computing scheme used for the analyses was Addition sequence furthest and MulTrees.

Clade support was estimated through bootstrap analysis as implemented in PAUP*, using 1000 bootstrap replications, 1000 MaxTrees, TBR swapping, MULPARS, and 1000 replications of stepwise random addition with 3 trees held at each step. TreeBASE Study accession number = S726, Matrix accession number = M1157.

Morphological studies – Micro- and macromorphological studies were done with a dissecting microscope (x12) and light microscope (x1000). Spore measurements were undertaken from spore prints where available. For each specimen, spore length and width were measured for 30 spores; mean and SD values were calculated. Several type specimens were studied in order to compare spore sizes and other morphological details with those of the specimens used for sequencing and crossing tests.

Crossing tests – Since the incompatibility between H. punctulatum, H. albostramineum, and H. cremicolor has been previously shown (Hallenberg 1985), complementary crossing tests were performed for all specimens assigned to H. wakefieldiae. Moreover, as a verification of earlier results, interspecific crossings were performed between representatives of all species studied here [an exception was FCUG 269 (H. albostramineum), for which homothallism has been indicated].

Single-spore mycelia from different specimens were placed in pairs on malt-extract agar (1.25% malt extract) and left at room temperature for five weeks. From each specimen, 2–4 single spore mycelia were used. Paired cultures were then checked for clamp formation in three different regions: at the immediate contact zone and on opposite sides of the inocula, some 20 mm from respective inoculum. Plates with negative results were rechecked after an additional three weeks.

	RESULTS

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Molecular analysis – Nucleotide sequence data for the internal transcribed spacers ITS1 and ITS2, and 5.8S rDNA of the 20 studied Hypochnicium specimens (Table I) were obtained and processed as described above. It was found that the whole of ITS1, 5.8S, and ITS2 could be used for the analysis; an additional 13 bp of the first part of nuclear large-subunit rDNA allowed for alignment and was included in the data matrix. After the recoding of a 10 bp-long insert as two characters, the data matrix consisted of 630 characters, of which 382 were constant and 47 were uninformative with respect to parsimony, leaving 201 informative characters (gaps treated as "fifth characters").

Parsimony analysis yielded 27 most-parsimonious trees of 393 steps with a CI (ensemble consistency index) of 0.8524 and an RI (ensemble retention index) of 0.9255. One of the most parsimonious trees is shown together with branch support values as estimated through bootstrapping of the consensus tree in Fig. 1. The variation between the most parsimonious trees was small and confined to the internal arrangement of taxa within respective clade. When gaps were treated as "missing data" a heuristic search had to be performed. Tree topology was the same as above, while tree length was 283 and number of informative characters reduced to 142. Four clades can be distinguished in the consensus tree: (1) the H. punctulatum clade, (2) the H. wakefieldiae clade, (3) the H. albostramineum clade, and (4) the H. cremicolor clade, all of which are strongly and in some cases very strongly supported.

View larger version (16K): [in this window] [in a new window]    FIG. 1. One of 27 most parsimonious trees from the parsimony analysis. Numbers above branches denote branch support from bootstrap analysis and branches in bold are present in the strict consensus tree

Morphological analysis – Macromorphological studies with a dissecting microscope failed to show any significant difference between the clades. In micromorphological studies, the specimens were found to be virtually inseparable if spore characters were not included, the only exception being H. wakefieldiae, the thick-walled basal hyphae of which facilitated identification (Parmasto 1967). This is largely congruent with previous findings (Eriksson and Ryvarden 1976, Hallenberg 1983, 1985). Spore size, however, was found to partially allow for species separation as shown in Fig. 2. By measuring spore size in a number of type specimens, it was possible to assign each of them to one of the four clades revealed in this study.

View larger version (16K): [in this window] [in a new window]    FIG. 2. Spore sizes measured as average values for 30 spores per specimen. Symbols in the diagram refer to species in the H. punctulatum complex, viz. H. albostramineum = •, H. punctulatum = , H. wakefieldiae = , and H. cremicolor = . Symbols without further indications refer to samples used in the phylogenetic analysis while similar symbols with species names refer to type specimens studied

Hypochnicium cremicolor (Bres.) H. Nilsson & Hallenb. comb. nov.

Basionyme: Hypochnus cremicolor Bres., in Ann. Mycol. 1, p 109, 1903.

The type of this species corresponds well with the small-spored taxon in the H. punctulatum complex, which has been referred to by several authors as H. punctulatum s.str. The species is distinguished by its spore size, 6–6.5 x 5–5.5 µm.

H. albostramineum (Bres.) Hallenb. Synonym: H. eichleri (Bres.) J. Erikss. & Ryv.

This is the taxon with the largest spores of the complex, distinguished by a spore size of 8–9.5 x 6.5–7.5 µm. Even though respective type specimens listed here are divergent in their spore sizes, they probably represent the same taxon (Fig. 2). It is here proposed that the name H. albostramineum be used for this large-spored taxon in spite of the fact that H. eichleri is an older name. The reasons are (1) that the type specimen of H. eichleri differs only minutely from H. punctulatum and could possibly belong to that taxon, and (2) for stability of nomenclature as the name H. albostramineum has previously been used for the large-spored taxon.

H. punctulatum (Cooke) J. Erikss. Synonym: H. sphaerosporum (Höhn. & Litsch.) J. Erikss.

The spore size of this taxon, 7.5–8 x 6.5–7 µm, falls between those of the aforementioned species. In particular, big-spored forms in H. punctulatum are virtually impossible to distinguish from small-spored forms of H. albostramineum. As already concluded by Rogers and Jackson (1943), H. sphaerosporum is a synonym to H. punctulatum, even though this name was in common use in Europe for a smooth-spored taxon, now referred to as H. erikssonii Hallenb. & Hjortst. After re-examination of the type specimen (Hallenberg & Hjortstam, 1989), the synonymy was confirmed.

H. wakefieldiae (Bres.) J. Erikss. Synonym: H. caucasicum Parm.

Hypochnicium wakefieldiae was described on material from Britain in 1920 and the name has since then been little used. Hypochnicium caucausicum was, in Parmasto's 1967 description, already clearly distinguished from H. punctulatum by the thick-walled basal hyphae of its basidiomata. Several specimens were mentioned in the description, all of which originated from Georgia. The small spore size of the type specimen made H. caucasicum a possible synonym to the "small-spored H. punctulatum", but no synonymy was ever proposed. From the present study it has become evident that the type specimen represents a form with very short spores compared with other specimens, among them several originating from the Caucasus. On the other hand, variation in spore width appears to be small; the spore size measured in this study is 6.3–8 x 5.5–6 µm. From crossing tests it became evident that the species was distributed in Europe as well, and the type specimen of H. wakefieldiae fits the general spore size of the taxon very well. Moreover, the type specimen contains thick-walled basal hyphae, which are of high diagnostic value.

KEY TO THE SPECIES OF THE H. PUNCTULATUM COMPLEX. Spore measurements are based on means of 30 spores.

1. Spores 6–6.5 x 5–5.5 µm . . . . . H. cremicolor

1. Spores bigger . . . . . 2

     2. Spores 6.5–8 x 5.5–6 µm, thick-walled basal hyphae present . . . . . H. wakefieldiae

     2. Spores 7.5–9.5 x 6.5–7.5 µm, thick-walled basal hyphae absent . . . . . H. albostramineum-punctulatum group (3)

          3. Spores 7.5–8 x 6.5–7 µm . . . . . H. punctulatum

          3. Spores 8–9.5(–12) x 6.5–7.5 µm . . . . . H. albostramineum

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Sequence information from the internal transcribed spacers of nuclear ribosomal DNA has been shown to be very useful in establishing phylogenetic relationships among fungi as well as other organisms, and has been used for that purpose on numerous occasions (e.g., Phillips et al 1994, Hibbett et al 1995, Gernand and Stone 1999). It has also been successfully applied to questions of gene flow between populations and to issues of allopatric speciation as a result of geographic separation (Mayer and Soltis 1999, Hughes et al 1999). In the present study, ITS data was used to investigate the Hypochnicium punctulatum complex, the species of which are difficult to tell from each other on morphological basis.

The parsimony analysis yielded four unambiguous clades, all of which receive very strong branch support (Fig. 1). The clades correspond to H. albostramineum, H. cremicolor, H. punctulatum, and H. wakefieldiae. The result is largely congruent with previously accepted species delimitations based on morphology and intercompatibility tests. The clades also find partial support in morphology, where it was shown that spore size, at least to some extent, can be used to separate the species (Fig. 2).

The present study explicitly delimits H. albostramineum and H. punctulatum phylogenetically; the morphological delimitation is, however, not as unambiguous, with an overlapping spore size as the only known criterion that allows for separation. Since the two species can be distinguished in most cases, it is suggested that they remain two separate taxa. With an increasing tendency to delimit species phylogenetically, an alternative would be to group H. albostramineum, H. punctulatum, and H. wakefieldiae together as a separate species, as they share a unique common ancestor (Fig. 1). While pleasing some, this solution would decrease the resolution inside the group and ignore the clearly defined H. wakefieldiae. By keeping H. albostramineum and H. punctulatum and referring specimens of in-between spore size as species of the H. albostramineum-punctulatum group, little precision is lost, and no extensive nomenclatural changes are needed.

The grouping of H. wakefieldiae to H. punctulatum and H. albostramineum within the main clade of the consensus tree (Fig. 1) was an unexpected finding. While H. wakefieldiae in most respects shows considerable similarity to the species of the H. punctulatum complex, it is distinguished from the latter by having distinctly thick-walled basal hyphae, which is probably one of the main reasons that the possibility of a close relationship to the mentioned species was previously overlooked. The present analyses suggest that the hypotheses synonymizing H. wakefieldiae with the small-spored H. cremicolor be disregarded, and show that H. wakefieldiae appears as an independent species in the H. punctulatum complex.

	ACKNOWLEDGMENTS

 
We wish to thank Vivian Aldén for invaluable help in the laboratory. Financial support was given by The Bergwall Foundation and A.N.S. Maskinteknik, and are gratefully acknowledged.

	FOOTNOTES

 
¹ Corresponding author, nils.hallenberg{at}systbot.gu.se

Accepted for publication May 15, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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Eriksson J., 1958 Studies in the Heterobasidiomycetes and Homobasidiomycetes—Aphyllophorales of Muddus National Park in North Sweden. Symb Bot Ups 16:1-172

———, Ryvarden L., 1976 The Corticiaceae of North Europe. Vol. 4. Blindern, Oslo: Fungiflora. p 547–886

Gardes M, Bruns TD., 1993 ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol 2:113-118[Medline]

Gernandt DS, Stone JK., 1999 Phylogenetic analysis of nuclear ribosomal DNA places the nematode parasite, Drechmeria coniospora, in Clavicipitaceae. Mycologia 91:993-1000

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———. 1985 On the Hypochnicium eichleri complex (Basidiomycetes). Mycotaxon 24:431-436

———, Hjortstam K., 1989 Hypochnicium eriksonii sp. nov. instead of H. sphaerosporum—a necessary name alternation. Windahlia 18:43-46

Hibbett DS, Fukumasa-Nakai Y, Tsuneda A, Donoghue MJ., 1995 Phylogenetic diversity in shiitake inferred from nuclear ribosomal DNA sequences. Mycologia 87:618-638

Hughes KW, McGhee LL, Methven AS, Johnson JE, Petersen RH., 1999 Patterns of geographic speciation in the genus Flammulina based on sequences of the ribosomal ITS1–5.8S-ITS2 area. Mycologia 91:978-986

Mayer MS, Soltis PS., 1999 Intraspecific phylogeny analysis using ITS sequences: insights from studies of the Streptanthus glandulosus complex (Cruciferae). Syst Bot 24:47-61

Parmasto E., 1967 Corticiaceae U.R.S.S. IV. Descriptiones taxorum novorum. Combinationes novae. Eesti NSV Tead Akad Toimet Biol 16:377-394

Phillips RB, Manley SA, Daniels TJ., 1994 Systematics of the salmonid genus Salvelinus inferred from ribosomal DNA sequences. Can J Fish Aquat Sci 51: (Suppl 1) 198-204

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