A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances

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1 Mycologia, 98(1), 2006, pp # 2006 by The Mycological Society of America, Lawrence, KS A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances Qi-Ming Zhou Shou-Yu Guo 1 Man-Rong Huang Jiang-Chun Wei Systematic Mycology & Lichenology Laboratory, Institute of Microbiology, Academia Sinica, Bei-yi-tiao No. 13, Zhong-guan-cun, Beijing, , P.R. China Abstract: Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rdna, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi. Key words: chemotype, group I intron, insertion distribution pattern, ITS, speciation, SSU rdna INTRODUCTION Lichens are well known for their secondary metabolic substances that rarely occur elsewhere in nature (Culberson 1969, Culberson et al 1988). For more than 100 y lichenologists have used such unique organic compounds as an aid in lichen identification Accepted for publication 7 Dec Corresponding author. shouyug@yahoo.com and classification (e.g. Narui et al 1996). Recently there has been conclusive evidence that the major secondary metabolism observed in lichens is due to mycobionts (Culberson and Armaleo 1992) and is inherited as a genetic trait (Culberson et al 1988). Some studies also have suggested that the recognition of chemically defined species could be supported by their distinct geographical distributions and ecological preferences (Hawksworth 1976). One of the classical examples was provided by Cladonia chlorophaea complex (DePriest 1992, 1994). However chemical similarity or difference is not an infallible indicator of systematic relationships because chemistry is known to be variable in some species (Culberson 1986, Culberson et al 1988) and sporadically shared by divergent groups. This phenomenon has been reported in Rhizoplaca with several chemotypes detected for R. chrysoleuca, R. melanophalma, R. peltata, and R. subdiscrepans (Leuckert et al 1977, Wei 1984, Ryan and Nash 1997, Arup and Grube 2000). Molecular evidence, particularly variations in nucleotide sequences, offers a large number of potentially informative characters. The ITS regions were used generally in the comparisons of closely related species and genera, and their sequences information has been one of the primary criteria in ascertaining relationships at the specific level (e.g. Groner and LaGreca 1997, Goward and Goffinet 2000). Recently the ITS region also was used in population analysis (e.g. Presa et al 2002, Koch et al 2003). Other genes also have been applied in fungal systematics including chitin synthase genes (e.g. Mehmann et al 1994, Kroken and Taylor 2001), the b-tubulin gene (e.g. Koufopanou et al 2001, Myllys et al 2001, Cruse et al 2002), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (e.g. Myllys et al 2002) and the microsatellite markers (e.g. Carter et al 2001, Walser et al 2005). These genes were used mostly in studying population structure or cryptic species. Studies on population differentiation and isolation in lichens have been carried out recently, especially for the corticolous and epiphytic lichens (e.g. Graphis scripta [Murtagh et al 2000], Letharia vulpina [Högberg et al 2002] and Lobaria pulmonaria [Walser et al 2005]). Rhizoplaca is a small genus of saxicolous lichens with nine species usually containing usnic acid (Ryan and Nash 1997, Arup and Grube 2000, Kirk et al 2001). Species of Rhizoplaca are more or less umbilicate and fixed to the substrate or more rarely 57

2 58 MYCOLOGIA are fruticose or unattached, vagrant lichens. This genus formerly was recognized by Choisy (1929), who described it under the synonym Omphalodina M. Choisy with R. chrysoleuca (Smith) Zopf (5 Omphalodina chrysoleuca) as the type species. R. chrysoleuca is famous for its nearly worldwide distribution and its ability to produce ice nucleation active proteins (Kieft 1988, Kieft and Ruscetti 1990). Sometimes populations of R. chrysoleuca that differ in morphology and secondary products can be found growing intermixed with typical material. For example on Mount Wuling (Hebei Province, north China, ca. 110 km east of Beijing), populations comprising various morphotypes and several distinct chemotypes are found. The presence of a particular nrdna sequence pattern being unique to particular chemotypes would support chemotypic divergence. In contrast the random association of nrdna sequence patterns and chemotypes would support chemotypic polymorphism within the species (DePriest, 1994). The differences in nrdna sequences combined with the chemical variability were used to identify genetically distinct individuals and genetic diversity in the present study. We investigated the morphotypic and chemotypic polymorphisms of R. chrysoleuca from a molecular-genetic view and studied the population structure of R. chrysoleuca on Mount Wuling. MATERIALS AND METHODS Samples. Lichen samples selected for this study are listed (TABLE I). Forty-three Rhizoplaca specimens were selected from five localities in Hebei Province, Jilin Province, Xinjiang Uygur Autonomous Region and Nei Mongol Autonomous Region, China (FIG. 1A). Among them 26 R. chrysoleuca samples were taken from nine lichen mats (each ca cm diam) in two sites at different elevation (1700 m and 2100 m, respectively) on Mount Wuling, Hebei Province, north China (FIG. 1B, C). All samples were carefully brushed to remove possible epiphytic contaminants. These samples are deposited in the Lichen Section of the Mycological Herbarium, Academia Sinica (HMAS-L), as voucher specimens for the ITS and SSU rdna sequences. Morphology. Morphology of the thalli of R. chrysoleuca was examined first. Observations of external features were made under a dissecting microscope (Zeiss SV11). Two main morphotypes (TABLE II) were established based on the morphological data that were obtained from ca. 200 specimens (including the samples used in the molecular study) collected in different regions (most of them from China and 17 specimens from Europe and North America). Chemistry. Individual thalli of 36 R. chrysoleuca (TA- BLE I) were examined chemically (chemotypes given in TABLE III) with standard thin-layer chromatographic (TLC) methods (Culberson 1972). The compounds in each thallus were extracted with acetone, and the extracts were spotted on cm silica gel plates (supported by glass, Qingdao Ocean Chemical Factory) and developed in solvent system C. Spots were visualized under UV light at 254 nm before spraying with 10% sulfuric acid and heating at 110 C for 10 min. Lethariella cladonioides which contains norstictic acid and atranorin was selected as a control sample. PCR amplification and sequencing. Total DNA was extracted from all 43 individual Rhizoplaca thalli (TABLE I) using the modified CTAB method (Rogers and Bendich 1988). DNA extracts were used for PCR amplification of the ITS regions including the 5.8S gene of the nuclear rdna with ITS5 and ITS4 (White et al 1990) as primers. PCR reactions were performed in a DNA Thermal Cycler (Biometra) as follows: initial denaturation at 95 C for 3 min, followed by 35 cycles of 30 s denaturation at 94 C, 45 s annealing at 58 C, 1 min extension at 72 C and completed with a final 8 min extension at 72 C. Products were purified with Gel Extraction Mini Kit (SABC). Sequencing reactions were carried out by Shanghai Genecore Corp. with an ABI 3700 Sequencer. Both complementary strands of each sample were sequenced. Partial SSU rdna sequences were obtained from all R. chrysoleuca samples after amplifying with primer pair NS21UCB and NS24UCB (Gargas and Taylor 1992) except for 5A and 17A, but the length of PCR products differed significantly among individuals (TABLE I). The fragments obtained from samples 111, 211, 221, 223, G8, 231, 234, 7A, 8A, 9A, 10A and 15A were chosen to represent the different length products and sequenced by Shanghai Bioasian Corp. using an ABI 377 Sequencer. However only the 59 and 39 flank sequences of the PCR product for 15A were obtained because it failed in walking reactions. The near complete SSU rdna sequence of R. chrysoleuca 144 had been obtained in a previous study (AY530888). Data analysis. ITS sequences (including ITS1, 5.8S rdna and ITS2) of Rhizoplaca acquired in this study and those from GenBank (Parmelia sulcata, AF410840; R. cerebriformis U312, AF159942; R. chrysoleuca U192, AF159924; R. chrysoleuca U302, AF159940; R. cylindrica U305, AF159941; R. haydenii U285, AF159937; R. idahoensis U313, AF159943; R. melanophthalma U219, AF159929; R. melanophthalma U278, AF159934; R. melanophthalma U281, AF159935; R. peltata U198, AF159925; R. peltata U282, AF159936; R. subdiscrepans U387, AF163113; R. subidahoensis U314, AF159944) were aligned with DNAMAN4.0 (Lynnon Biosoft) without the flanking regions of the small subunit and large subunit rdna. Phylogenetic analysis was executed with software MEGA2 (Kumar et al 2001) and Parmelia sulcata was selected as the outgroup taxon. We used the Kimura 2- parameter model and retained gaps initially but excluded them in the pairwise distance estimation. The neighbor joining (NJ) method was used in constructing the phylogenetic tree, and the reliability

3 ZHOU ET AL: GENETIC VARIABILITY OF RHIZOPLACA CHRYSOLEUCA 59 TABLE I. Information for 43 samples included in this study GenBank accession numbers Species or specimens ITS SSU rdna (Length) Collection information R. chrysoleuca 111 AY AY (1000 bp) Hebei, Guo & Huang 111 R. chrysoleuca 113 AY (1600 bp) Hebei, Guo & Huang 113 R. chrysoleuca 121 AY (1000 bp) Hebei, Guo & Huang 121 R. chrysoleuca 123 AY (1000 bp) Hebei, Guo & Huang 123 R. chrysoleuca 124 AY (1600 bp) Hebei, Guo & Huang 124 R. chrysoleuca 126 AY (1000 bp) Hebei, Guo & Huang 126 R. chrysoleuca 127 AY (1600 bp) Hebei, Guo & Huang 127 R. chrysoleuca 133 AY (1000 bp) Hebei, Guo & Huang 133 R. chrysoleuca 141 AY (1000 bp) Hebei, Guo & Huang 141 R. chrysoleuca 143 AY (1600 bp) Hebei, Guo & Huang 143 R. chrysoleuca 144 AY AY (1600 bp) Hebei, Guo & Huang 144 R. chrysoleuca 145 AY (1000 bp) Hebei, Guo & Huang 145 R. chrysoleuca 146 AY (1000 bp) Hebei, Guo & Huang 146 R. chrysoleuca 156 AY (1000 bp) Hebei, Guo & Huang 156 R. chrysoleuca 157 AY (1600 bp) Hebei, Guo & Huang 157 R. chrysoleuca 211 AY AY (1000 bp) Hebei, Guo & Huang 211 R. chrysoleuca 216 AY (1000 bp) Hebei, Guo & Huang 216 R. chrysoleuca 221 AY AY (1000 bp) Hebei, Guo & Huang 221 R. chrysoleuca 222 AY (1000 bp) Hebei, Guo & Huang 222 R. chrysoleuca 223 AY AY (1000 bp) Hebei, Guo & Huang 223 R. chrysoleuca 224 AY (1000 bp) Hebei, Guo & Huang 224 R. chrysoleuca 231 AY AY (1600 bp) Hebei, Guo & Huang 231 R. chrysoleuca 234 AY AY (1600 bp) Hebei, Guo & Huang 234 R. chrysoleuca 235 AY (1000 bp) Hebei, Guo & Huang 235 R. chrysoleuca 242 AY (1000 bp) Hebei, Guo & Huang 242 R. chrysoleuca 246 AY (1000 bp) Hebei, Guo & Huang 246 R. chrysoleuca 5A AY Xinjiang, Wei, d R. chrysoleuca 7A AY AY (1450 bp) Xinjiang, Wei, c R. chrysoleuca 8A AY AY (1450 bp) Xinjiang, Wei, b R. chrysoleuca 9A AY AY (1450 bp) Xinjiang, Wei, R. chrysoleuca 10A AY AY (1450 bp) Nei Mongol, Wang et al., a R. chrysoleuca 12A AY (1450 bp) Nei Mongol, Wang et al., R. chrysoleuca 14A AY (1300 bp) Nei Mongol, Wang et al., R. chrysoleuca 15A AY (1300 bp) Nei Mongol, Wang et al., R. chrysoleuca 17A AY Nei Mongol, Wei, R. chrysoleuca G8 AY AY (1000 bp) Xinjiang, Guo, 3515a R. melanophthalma 6A AY Xinjiang, Wei, a R. peltata G10 AY Xinjiang, Guo, 3557 R. peltata G11 AY Xinjiang, Guo, 3516 R. peltata G9 AY Xinjiang, Guo, 3515b R. subdiscrepans 4A AY Nei Mongol, Wang et al., 13-2 R. subdiscrepans m1 AY Jilin, Zhou, R. subdiscrepans m2 AY Jilin, Zhou, of the inferred tree was tested by 1000 bootstrap replications. RESULTS Morphology and chemistry. Two main morphotypes (TABLE II) were identified in the specimens examined. Morphotype I was characterized by the gray thalli and narrow lobes or granules and morphotype II by the yellow-green thalli and wide lobes. However morphotype I was found only in the samples from Mount Wuling (FIG. 2) in the specimens we examined. A total of 12 chemotypes were detected in the present study. Neither morphotype correlated with the two major groups of chemotypes (TABLE III). The first major group of chemotypes (gray thallii and partial yellowgreen thalli, lacks pseudoplacodiolic acid and unidentified substance 2, Group 1 in TABLE III) includes chemotypes I to X. The second major

4 60 MYCOLOGIA FIG. 1. Sketch maps of collecting sites. A, five localities in China (N Mount Wuling, Hebei;. Mount Arxan, Motianling and Mount Jiguan, Inner Mongol; m Mount Tuding, Jilin; X Jimsar, Xinjinag; & Hanas and Guanyuting, Xinjiang). B and C are the loci at 2100 m and 1700 m elevations respectively. Each dot represents a single mat. Samples were taken from every mat. Samples serial numbers indicate their positions. For example, R. chrysoleuca111 was collected from 1-1 mat in B locus (2100 m), and R. chrysoleuca246 was collected from 2 4 mat in C locus (1700 m). group of chemotypes (yellow-green thalli from Mount Wuling, contains pseudoplacodiolic acid and unidentified substance 2, Group 2 in TABLE III) includes chemotypes XI and XII. Chemotypes VII, V and XI correspond respectively to the chemotypes 1, 2 and 4 in Leuckert et al (1977). Whereas chemotypes 3 (usnic + pseudoplacodiolic acids), 5 (usnic + placodiolic + psoromic acids) and 6 (usnic + pseudoplacodiolic + psoromic acids) detected by Leuckert et al (1977) for R. chrysoleuca were not found in our specimens examined. Chemotypes I, II, III, VI and XII were reported for the first time. It is interesting that almost all samples collected from Mount Wuling lack lecanoric acid, which is present in most samples from other areas. ITS phylogenetic analysis. A total of 43 ITS sequences were determined from the mycobiont of Rhizoplaca. The ranges of the length of the ITS region in the samples are bp. The samples of R. chrysoleuca from Mount Wuling formed two distinct well-supported clades (clade I and clade II, marked respectively with # andn in FIG. 2). Little variations in ITS rdna sequences were found within either group. The R. chrysoleuca samples from other areas did not form monophyletic groups according to their collection sites; R. chrysoleuca G8 was an exception because only one collection was obtained from Jimusaer, Xinjiang. R. chrysoleuca samples14a and 15A grouped with U192, and this clade was supported by a 99% bootstrap value. With 97% support other R. chrysoleuca samples appeared as a sister branch to this clade. Although the nucleotides differences in ITS rdna were obvious, all R. chrysoleuca individuals grouped together and this topology has 100% TABLE II. Morphological variations in the Rhizoplaca chrysoleuca Character Color of apothecia Color of thallus (upper surface) Color of thallus (lower surface) Form of thallus Morphotype I Orange-yellow Gray Black Granule Morphotype II Green Yellow-green Brown Lobe

5 ZHOU ET AL: GENETIC VARIABILITY OF RHIZOPLACA CHRYSOLEUCA 61 TABLE III. The chemotypes of the Rhizoplaca chrysoleuca in China a Samples Major secondary products Chemotype atr usn pla UIS1 UIS2 pso psu UIS3 lec Group1 Group2 R. chrysoleuca I R. chrysoleuca XI R. chrysoleuca tra tra III R. chrysoleuca tra IV R. chrysoleuca XI R. chrysoleuca VII R. chrysoleuca XII R. chrysoleuca VII R. chrysoleuca II R. chrysoleuca XII R. chrysoleuca XII R. chrysoleuca tra IV R. chrysoleuca VII R. chrysoleuca VII R. chrysoleuca XI R. chrysoleuca tra + VI R. chrysoleuca tra + VI R. chrysoleuca V R. chrysoleuca V R. chrysoleuca V R. chrysoleuca V R. chrysoleuca XII R. chrysoleuca XII R. chrysoleuca V R. chrysoleuca V R. chrysoleuca V R. chrysoleuca 10A + + VIII R. chrysoleuca 12A + + IX R. chrysoleuca 14A + + VIII R. chrysoleuca 15A + + VIII R. chrysoleuca 17A + + VIII R. chrysoleuca 5A + + VIII R. chrysoleuca 7A + + VIII R. chrysoleuca 8A X R. chrysoleuca 9A + + VIII R. chrysoleuca G8 + + V a atr 5 atranorin, usn 5 usnic acid, pla 5 placodiolic acid, pso 5 psoromic acid, psu 5 pseudoplacodiolic acid, lec 5 lecanoric acid, UIS1 5 unidentified substance in Rf class 6, UIS2 5 unidentified substance in Rf class 5 6, UIS3 5 unidentified substances in Rf class major and constant constituents, tra 5 trace constituents. bootstrap support. R. melanophthalma, R. cerebriformis, R. subidahoensis, R. cylindrical, R. idahoensis and R. haydenii composed the core group of Rhizoplaca (Arup and Grube 2000) with 99% bootstrap support. R. subdiscrepans and R. chrysoleuca appeared as a sister taxon to the core group with 99% support. R. peltata, on the other hand, was on a separate branch. Insertion distribution patterns in SSU rdna. Four types of length polymorphism were observed in the SSU rdna of R. chrysoleuca as revealed by the PCR reaction (TABLE I), and the result of sequencing indicated that it was caused by different numbers of insertions. Twelve PCR products were sequenced in this study. Together with R. chrysoleuca 144 whose SSU rdna sequence had been determined in a previous study, partial SSU rdna sequences were obtained for 13 individuals. Because the sequences of some individuals were identical only one was submitted to GenBank (TABLE I, the samples sharing the same accession number had identical sequences). The result of sequencing showed that the same length fragments were caused by homological insertions in the same positions. Four kinds of insertion patterns were observed corresponding to four

6 62 MYCOLOGIA The NJ tree inferred from ITS data. The individuals that are marked with same tag were taken from the same area indicate that samples were taken from Mount Wuling, and # refers to samples with gray thallus). The numbers in each node represent bootstrap support values, the absence of a number indicating,50% bootstrap support. (HB 5 Hebei, NM 5 Nei Mongol, XJ 5 Xinjiang, JL 5 Jilin). The morphological types, the chemotypes and the insertion distribution patterns are listed at the right columns. FIG. 2. and these tags are corresponding to those in FIG. 1 (both # and N

7 ZHOU ET AL: GENETIC VARIABILITY OF RHIZOPLACA CHRYSOLEUCA 63 types of length polymorphism (FIG. 3). The insertions in position 1462 (according to Saccharomyces cerevisiae with accession number Z75578) were spliceosomal introns according to their terminal-sequences (Cubero et al 2000); while the rest were group I introns according to their secondary structures (Cech 1988). The group I introns in position 1779 were determined in the ITS region sequencing because primer NS24UCB was ahead of this site. The 59 and 39 flank sequences of the PCR product from R. chrysoleuca 15A indicated that there was one spliceosomal intron in position 1462 and no group I introns in positions 999 or 1779 but there was at least one other insertion according to the length of the PCR product in the region not sequenced (FIG. 3D). The common parts of the SSU rdna for these individuals (not including 15A) were compared with DNAMAN 4.0 (after excluding the introns) and there were only 14 variable positions among about 900 nucleotides, and the number of parsimony-informative characters was just three (data not shown). As a result the data from SSU rdna was not used in constructing phylogenetic trees. DISCUSSION After examining many specimens of R. chrysoleuca from different regions it was obvious to us that this species is quite variable in thallus morphology and chemistry. The morphological differences among R. chrysoleuca individuals were so obvious that we could readily divide them into two main groups merely on the basis of the color of the upper surface and the size of the lobe. Although Arup & Grube (1998, 2000) considered that the growth form of the thallus was highly variable within Rhizoplaca, the distinction between gray and green individuals of R. chrysoleuca is so marked that one would suspect that they are different species. The insertion distribution patterns in R. chrysoleuca populations. Multiple insertions were found in the SSU rdna of R. chrysoleuca. This phenomenon first was reported in lichenized fungi in the Cladonia chlorophaea complex (DePriest and Been 1992, DePriest 1993). Group I introns are considered to be gained by horizontal transfer between different organisms (e.g. Gargas et al 1995, Nishida and Sugiyama 1995, Hibbett 1996, Adams et al 1998, Perotto et al 2000), and spliceosomal introns also are considered to be of recent origin (e.g. Bhattacharya et al 2000). Consequently group I introns and spliceosomal introns generally are excluded in systematic analysis. However some studies revealed that group I introns had been immobile for the most part and were transmitted vertically during diversification at lower taxonomic levels (e.g. Nikoh and Fukatsu 2001, Bhattacharya et al 2002). Hence they have been used in some systematic analysis (e.g. Thell 1999, Myllys et al 2001). Because no insertions occur in the same position in all samples of R. chrysoleuca, we did not apply them in the phylogenetic analysis but simply compared them. The result shows that the insertions in the same position are high homologous. There are five positions of insertion in the partial SSU rdna ( in FIG. 3)of R. chrysoleuca, so there should be type of insertion distribution patterns. However only four patterns were observed. Even if the positions of group I introns, which are generally accepted transposable elements, are only considered (and assuming that samples 5A and 17A had alternative patterns), just 5(3 + 2) of combinations were found. Of interest, the samples from clades I and II have distinct insertion distribution patterns. It seems that individuals maintain the same insertion distribution pattern within a recombination population and that it is difficult to form new multiform patterns by intron acquisition and loss. This means that the mechanisms of intron acquisition and intron loss created equilibrium in these populations. Because group I introns without ORF would be eliminated rapidly by intron loss (Dujon 1989), we think that sexual reproduction is the main mechanism for intron gain. Further intronless loci would acquire introns from their intronharboring alleles in zygotes, which suggest that the insertions distribution patterns in a recombination population would be the same. Thus the two different insertion distribution patterns found in gray and green individuals of R. chrysoleuca on Mount Wuling respectively suggests that no gene exchange occurs between the two populations and the isolation mechanism has established. The phylogenetic analysis of R. chrysoleuca. Individuals from Mount Wuling with a gray thallus (morphotype I) assemble in a monophyletic clade (clade I) that is supported by a 97% bootstrap value and those with a green thallus assemble in a second clade (clade II) with 97% support (FIG. 2). Neither clade is well resolved, which is the result of an absence of informative characters within each. So it means that the variation in the ITS sequence is insufficient to discriminate the inner structures of R. chrysoleuca populations on Mount Wuling or on the other hand that reproduction in these small populations is clonal. If

8 64 MYCOLOGIA FIG. 3. Insertions in the different positions of the SSU rdna in R. chrysoleuca. An inverted triangle indicates the presence of an intron in a particular position. The number below the horizontal line gives an indication of the nucleotide s position (according to Saccharomyces cerevisiae with accession number Z75578). The region which was not sequenced is shown with the broken line. it implied exclusive clonal reproduction, one would expect that the genotype of all individuals would correspond to their chemotypes. However our data did not support such a result (FIG. 2). One genotype could share more than one chemotype, which means that the individuals with the same genotype are not necessarily clonal descendants and recombination must occur at some time. In fact the species of Rhizoplaca, including R. chrysoleuca, produce abundant sexual structures (ascomata) and sexual spores seem to provide the only means of dispersal because no symbiotic vegetative propagules are observed. Therefore recombination should occur frequently in Rhizoplaca. The two groups of chemotypes do not cross the boundary between clade I and clade II on Mount Wuling, which demonstrates the evidence of genetic isolation. If only samples collected from Mount Wuling are considered, combining the data from the ITS sequences, chemotype analyses and the insertion distribution patterns indicates that there is no gene flow between clade I and clade II and the recombination occur just within each clade. It is surprising because the individuals were collected in a small geographic area so that the fungi certainly had the opportunity to interbreed (FIG. 1B, C). This indicates that sexual isolating mechanisms have been established between gray and green thalline groups on Mount Wuling, which implies that the specimens of R. chrysoleuca from Mount Wuling should be divided into two phylogenetic species. However, when all the collections of R. chrysoleuca are taken into consideration, the conclusion is quite different. R. chrysoleuca is supported as a monophyletic group by a 100% bootstrap value in the ITS phylogenetic analysis. If we accepted that clade I and clade II represented different phylogenetic species, we would confront much more difficult problems in dealing with individuals outside those two clades. In fact R. chrysoleuca is a taxon with a broad distribution; therefore there are many separate geographical and

9 ZHOU ET AL: GENETIC VARIABILITY OF RHIZOPLACA CHRYSOLEUCA 65 ecological populations in the world. No evidence indicates that the potential for gene exchange among these different R. chrysoleuca populations has been lost. Only when populations differ enough to inhibit any gene exchange at all do we commonly view them as separate species. However a series of intermediate forms between clades I clade II can be found in other areas (FIG. 2) and some samples have close genetic relation with these two clades (for example, 17A and clade I, or 5A and clade II) and may be able to mate them. Furthermore the close genetic relationship among individuals of R. chrysoleuca is also reflected by the conservative SSU rdna sequences. Therefore we think that R. chrysoleuca as a monophyletic taxon is still accepted and that the graythalline and green-thalline individuals from Mount Wuling that represented different evolutional lineages could be treated as to represent two varieties. Sexual isolation and speciation. In general there are two main modes of speciation: allopatric and sympatric. And speciation should occur in this sequence: (i) geographical or ecological isolation between populations; (ii) increased genetic divergence; (iii) selection for increased reproductive isolation; (iv) speciation completed. Our result indicates that on Mount Wuling R. chrysoleuca consist of two distinct populations based on morphological, chemical and genetic characters. Although reproductive isolation between these two populations exists we think speciation is still in the third stage and does not complete because a series of intermediate forms (such as 5A, 7A 10A, 12A, 17A, G8 and U302) that have close genetic relation with these two populations can be found in other areas and may be able to mate either of them. Thus it may provide a good example of speciation in lichenized fungi. We speculate that the population with a gray thallus eventually will evolve into a separate species. It has been shown that sexual reproduction predominates in lichen communities growing directly on rocks and at the frontiers of terrestrial life in polar regions. As a pioneer species R. chrysoleuca thus may provide a model for the speciation of saxicolous organisms and for the evolution of breeding systems in extreme environments. The variation in ITS rdna sequence patterns and secondary chemistry among individuals in R. chrysoleuca within a small area would suggest that most species mats do not represent a single genetic clone but multiple genetic clones or that these lichen thalli may have been composed of more than one sexually active fungal genotypes. Our results indicate that the relationships between genotypes and chemotypes are complicated and it should be avoided and that single individuals or few individuals from the same site should be used to represent the population or whole species in systematics study. The interior branches within R. chrysoleuca and R. subdiscrepans are longer than the interspecific branches in the core group and those within R. peltata (FIG. 2). In fact, Arup and Grube had noticed the unusual longer branch of R. chrysoleuca in their study (2000); however, they did not make any interpretation. Such a large variety of R. chrysoleuca is the most likely to be an adoption to the multiple geographical and ecological zones. Another interesting phenomena is that the ITS sequences within R. chrysoleuca from the same site are sometimes more variable than those from different sites. It seems that it was caused by the movement of different R. chrysoleuca populations to the same geographical area. However the further conclusion can not be drawn until an extensive investigation of R. chrysoleuca on a large scale is undertaken. ACKNOWLEDGMENTS We thank Mr Hai-ying Wang, Mr Jun Yang and Miss Xin-Li Wei for help with the field collections. The authors thank Dr J. Elix (Chemistry Department, Australian National University) for reviewing the manuscript. We would like to thank two anonymous reviewers for their helpful comments. This work was supported by grants from National Natural Science Foundation of China (grants , ) and Chinese Academy of Sciences (Grant kscxz-sw-101c). LITERATURE CITED Adams KL, Clements MJ, Vaughn JC The Peperomia mitochondrial coxi group I intron: timing of horizontal transfer and subsequent evolution of the intron. J Mol Evol 46: Arup U, Grube M Molecular systematics of Lecanora subgeneus Placodium. Lichenologist 30: , Is Rhizoplaca (Lecanorales, lichenized Ascomycota) a monophyletic genus? 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