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Local population subdivision in the lichen Cladonia subcervicornis as revealed by mitochondrial cytochrome oxidase subunit 1 intron sequences

     Universitetet i Bergen, Botanisk Institutt, Allégaten 41, N-5007 Bergen, Norway

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

A 753–771 bp long intronic sequence from the mitochondrial cox 1 gene of Cladonia subcervicornis (Cladoniaceae, Lecanorales, Ascomycota) was amplified with newly designed PCR primers. The cox 1 intron sequence, which apparently has not been used for phylogenetic or population genetic research in fungi, displays high infraspecific variation. Sequences were obtained from 124 specimens from four neighboring localities in coastal Hordaland, western Norway. An exact test of population differentiation and population pairwise fixation indices F_ST show significantly reduced gene flow between the northernmost locality and the other three populations. Although Cladonia subcervicornis frequently produces apothecia, we conclude that dispersal by ascospores over long distances is rather ineffective in this species.

Key words: Cladonia, dispersal, lichenized Ascomycota, maturase, PCR primers

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The study of lichen population biology has been lagging behind that of other groups of organisms, mostly due to technical problems rather than a lack of interest. Basic questions about lichen dispersal, biogeography, speciation and evolution are still largely unanswered because of this lack of population studies. The simple organization and high morphological plasticity of most lichens made the whole field more or less inaccessible before the development of molecular methods. For a relatively short period, protein-based methods were used for lichen population studies (Fahselt and Hageman 1983, Hageman and Fahselt 1984, 1990, Brown et al 1989, Mattsson 1994). However, poor protein yield and denaturation of enzymes by secondary lichen products made this approach technically difficult, and it did not become widely used, before DNA-based methods more or less replaced allozyme studies.

The development of PCR-based methods is beginning to change this picture because it finally grants lichenologists access to molecular markers, but other problems still impede population genetic studies. Lichens are symbiotic organisms, and DNA extractions of all but the largest thalli almost inevitably contain both fungal and algal or cyanobacterial DNA. Typical datasets in population genetics often comprise hundreds of individuals, but cost-effective methods, such as RAPD-PCR or ISSR-PCR, cannot discriminate among DNA of the symbionts. Unless algal-free mycobiont cultures are used, a costly and laborious approach, the results potentially are flawed. To our knowledge, fungal specific microsatellite primers have not been published for lichens. As a result, nucleotide sequences obtained by fungal-specific PCR-primers are the only reasonable alternative, in most cases. The high cost associated with this approach has not promoted lichen-population genetics, and the number of available marker sequences consequently is low (Zoller et al 1999, Printzen 2002, Printzen and Ekman 2002). Other studies addressing questions of infraspecific variation in lichens with the help of nucleotide sequences include DePriest (1993, 1994) and Beard and DePriest (1996), who employed PCR-RFLP to demonstrate variability of number, size and position of Group 1 introns in the nuclear SSU rDNA of Cladonia chlorophaea and C. subtenuis within populations. However, most nonribosomal gene loci used in lichen phylogeny and population genetics do not contain introns, which makes it unlikely that sufficient variability can be uncovered by PCR-RFLP.

The first objective of our study was to extend the array of molecular markers for population studies in lichens. Fungal mitochondrial genomes are evolutionarily derived and are characterized by accelerated sequence divergence (Gray et al 1999). Considerable length variation has been observed among different species, largely due to the frequent occurrence of Group 1 and Group 2 introns. Both types of introns are self-splicing, mobile elements. Many of them contain ORFs encoding for homing endonucleases, maturases that assist in the splicing of introns, or proteins that function as both (Gimble 2000). An estimated 30% of Group 1 introns contain such ORFs, while they seem to be less common in Group 2 introns (Chevalier and Stoddard 2001). Group 1 introns have been shown to spread rapidly within populations and among species. This mobility causes coding as well as noncoding parts of the intron sequence to be relatively unaffected by selection against deleterious mutations. Instead, they undergo regular cycles of transmission, fixation, degeneration and loss (Goddard and Burt 1999). Mitochondrial sequences thus can be assumed to contain the amount of variability necessary for studies at the population level. The few available studies indeed showed appreciable amounts of infraspecific variation (Zoller et al 1999, Printzen 2002).

Although lichens produce an astonishingly varied array of diaspores, very little is known about their dispersal. Sexual reproduction in lichens occurs via ascospores or basidiospores, while asexual dispersal can occur either through fungal propagules, such as conidia or structures containing both fungal and algal cells. Büdel and Scheidegger (1996) give a comprehensive overview of the modes of reproduction in lichens. Because fungal ascospores are orders of magnitude smaller than seeds of vascular plants, their dispersal is unlikely to be limited physically. This indeed was supported by findings of lichen propagules in pollen traps on the Atlantic Ocean (Harmata and Olech 1991). These findings, however, did not prove effective dispersal, i.e., dispersal of propagules and establishment in a new environment (Cain et al 2000). Indirect evidence for effective spore dispersal was provided by Tibell (1994) and Wedin (1995), who found statistically significant correlations between small spores and wide geographical distribution in species of the Caliciales and Sphaerophoraceae. On the other hand, investigations of isozyme patterns of Umbilicaria mammulata (Hageman and Fahselt 1992) showed more limited genetic exchange among subpopulations than within, even within a rather limited geographical area. But because Umbilicaria mammulata is very rarely fertile and did not produce vegetative propagules in the study area, the results shed little light on the ability of diaspore dispersal in lichens. In any case, the authors challenged the widespread opinion that lichens disperse easily and effectively and, for the first time, demonstrated that lichen dispersal could be assessed indirectly by measuring genetic differentiation between populations.

To contribute data on a lichen species that frequently produces ascomata and to test the suitability of our new marker for population studies, we compared populations of the macrolichen Cladonia subcervicornis in a windswept, open environment on a local scale. Lichens of the genus Cladonia belong to the most conspicuous and most frequently collected lichens. Cladonia subcervicornis occurs in oceanic parts of northern and western Europe from the British Isles to northern Norway (Purvis and James 1992, Santesson 1993). In Norway it occurs predominantly in coastal heaths, where it forms compact cushions over humus in rock crevices. Cladonia subcervicornis is common in these habitats and may dominate the vegetation of exposed rocks. Strong winds are recorded throughout the year near the coast of southwestern Norway. Calm conditions represent only 6% of all observations at Flesland weather station, and predominant winds change from southeasterly in autumn and winter to northwesterly in summer (Bjørbæk 1993). Ascospores thus should disperse widely in open coastal habitats.

	MATERIALS AND METHODS

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Sampling – Lichen thalli were sampled in four localities near Hammersland (60°15'44'' N, 5°04'00'' E; n = 30) and Ågotnes (60°24'18'' N, 4°59'46'' E; n = 31) on the island of Sotra, the island of Blomøyni (60°31'27'' N, 4°53'44'' E; n = 31) and the island of Alvøyni (60°37'16'' N, 4°48'28'' E; n = 32) in Hordaland off the Norwegian west coast (Fig. 1). Thalli were collected at irregular intervals on areas of about 1000 m² at each locality. No adjacent thalli of less than 0.5 m distance were collected. Almost all thalli had a distance of at least 1 m from the nearest sampled thallus.

View larger version (61K): [in this window] [in a new window]   FIG. 1. Sampling sites are on the islands of Sotra, Blomøyni and Alvøyni, in Hordaland, Norway.

Fungal specific primers were designed following the method proposed by Carbone and Kohn (1999). Primers were checked for possible hairpin structures or dimer formation with Primer Premier MINI version 4.04 (Premier Biosoft, URL: http://www.PremierBiosoft.com). Out of eight forward and six backward primers originally designed, a combination of these two primers amplified part of the cytochrome oxidase subunit 1 sequence in various Cladonia species: (5959F–5') 5'-TCT TAA CGT TGC TGT ATG CTG-3'; (6711R–3') 5'-GAA CCG AAA CTA GTA GAA CCA TA-3'. Primer numbers refer to positions in the cox 1 sequence of Neurospora crassa (Genbank accession X14669).

A total of 10 µL of DNA extractions were used in 50 µL PCR reactions together with 5 µL Herculase^TM Reaction Buffer (Stratagene 600260–52), 1 mM MgCl₂, 2.5 mM each dNTP, 2.5 U polymerase (Herculase^TM, Stratagene 600264), and 0.8 µM each of the 5'- and 3'- primers. PCR cycling conditions were: 95 C (5'), six cycles of 95 C (45''), 52–46 C (touchdown, 45''), 72 C (1'45''), 34 cycles of 95 C (30''), 46 C (30''), 72 C (1'45''), and a final extension of 72 C (10'). PCR products were purified with the QIAquick^TM PCR Purification Kit (QIAGEN 28106) or the QIAquick^TM Gel Extraction Kit (QIAGEN 28706). Purified DNA was labelled with the BigDye Terminator^TM Kit (Applied Biosystems) and cycle sequenced at 94 C (30''), and 29 cycles at 95 C (15''), 50 C (15''), 60 C (4'). Sequences were determined on an ABI PRISM® 3700 DNA Analyzer (Applied Biosystems), assembled with SeqMan^TM II, version 4.05 (DNASTAR) and manually aligned in BioEdit, version 4.8.8 (Hall 1999). Sequences of each observed haplotype are deposited in GenBank (accession numbers AY148463–AY148469).

Sequence identification – To verify the identity of the nucleotide sequences amplified by us, a BLAST search in GenBank was performed. Cox 1 sequences from lecanoralean ascomycetes have not been published before. To rule out the possibility that we were sequencing populations of parasitic fungi, we also sequenced Cladonia polydactyla (Flörke) Sprengel, collected in different habitats, for comparison. Cladonia sequences were aligned with ClustalW as implemented in Bioedit version 4.8.8 (Hall 1999). One ORF within our sequences was detected and translated with the same program. The protein sequence of the ORF was subsequently subjected to a protein BLAST search to find similar protein sequences.

Data analysis – To compare the variability of cox 1 intron sequences with that of other sequences that have been used for infraspecific studies in lichens, average pairwise differences were calculated for the entire dataset using SITES (Hey and Wakeley 1997) and compared with unpublished datasets of IGS and ITS sequences from Cavernularia hultenii.

The recovered sequences were collapsed into haplotypes and the occurrence of the haplotypes noted for each locality. Genealogy information was not used in the subsequent data analysis because only seven different haplotypes were observed (see below). The dataset was tested for population subdivision by two different approaches using the program Arlequin version 2.001 (Schneider et al 2001). Population pairwise fixation indices F_ST were calculated and their significance tested with a nonparametric permutation approach with 1000 permutations of haplotypes among localities. An exact test of population differentiation (Raymond and Rousset 1995) was performed and significance tested using Markov chain Monte Carlo with 1 000 000 steps, 50 000 of which were dememorization steps.

	RESULTS

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A 753–771 bp long DNA fragment was amplified from DNA extractions of C. subcervicornis. The BLAST search in GenBank showed that the PCR product was most similar to published cox 1 sequences of Neurospora crassa, Schizosaccharomyces pombe, Aspergillus nidulans, A. niger, A. tubingensis, Yarrowia lipolytica and Podospora anserina. Our query sequence showed a high degree of similarity to that of Neurospora crassa, indicating that the gene amplified by us represents fungal cox 1. We concluded that our PCR product represents part of the cox 1 sequence from C. subcervicornis because aligning sequences from both Cladonia species was possible over the total sequence range. The complete amplified fragment corresponds to positions 5980–6710 of the Neurospora sequence. The cox 1 gene of N. crassa is interrupted by four Group 1 introns, and our fragment is homologous to intron 4 (i4) of N. crassa. The sequences of C. subcervicornis contain an ORF of 447 bp encoding an intronic maturase or homing endonuclease of the LAGLIDADG family (Fig. 2). The protein BLAST search revealed a high similarity of this sequence to a mitochondrial maturase of S. pombe, the endonuclease I-SceII of S. cerevisiae and uncharacterized intronic proteins of A. nidulans, P. anserina, Y. lipolytica, and the basidiomycetes Agrocybe aegerita and Flammulina velutipes.

View larger version (7K): [in this window] [in a new window]   FIG. 2. Part of the aligned cox 1 sequences of Cladonia subcervicornis, C. polydactyla and Neurospora crassa. This sequence corresponds to part of an ORF in intron 4 (i4) of N. crassa encoding an intronic maturase with a conserved LAGLIDADG motif

Despite the high variability, only seven different haplotypes were recognized in our samples. The genetic relationships of these haplotypes and their distribution among the four sampled subpopulations is shown in Fig. 3. The results of the test for population differentiation are shown in Table I. Population pairwise fixation indices, as well as the exact test, suggest significant population differentiation between Alvøyni and the other three populations. Neither test indicates significant differentiation between the other three localities.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Frequency of and haplotype network of the seven haplotypes of Cladonia subcervicornis found at four different localities on the western Norwegian coast. Gaps under base number 47 and 56 denote two identical indels TGCGCAGCA. Ovals represent sampled haplotypes, small circles are intermediate haplotypes not sampled by us. Each line in the network corresponds to one nucleotide substitution or an indel. Connections of up to 11 substitutions have a parsimony probability of >95%

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Three of the four populations studied by us display little genetic differentiation, with pairwise F_ST values well below 0.05. This result is not surprising in the case of a fertile lichen in an open environment. The northernmost population on Alvøyni, however, is genetically very different from the other three populations (Table I). Such pronounced population differentiation in a fertile lichen species on a small geographic scale is unexpected. The question remains whether this differentiation is really the result of reduced gene flow among populations. Inferring levels of gene flow from F_ST values relies on demographic and population genetic models, the applicability of which is difficult to judge (Ouborg et al 1999). These difficulties are especially pronounced in lichens, where information on reproductive biology, ecology and population dynamics either is lacking or rudimentary. McCauley et al (1995) demonstrated that, in plant metapopulations with high extinction-recolonization dynamics, the genetic differentation between populations indicates much lower dispersal than is actually present.

The few reports on growth rates in Cladonia (e.g., Jahns and Ott 1982) indicate that all specimens collected for this study were several years old. Consequently, it is unlikely that the population at Alvøyni was established recently. The fact that the more-or-less barren rock surfaces on which C. subcervicornis grows are not easily colonized by vascular plants further supports the fact that populations of C. subcervicornis are not subject to high extinction-recolonization dynamics. Regardless of the underlying mathematical model and the fact that we cannot determine Nm—the number of migrants per generation between populations—genetic differentiation hence is likely to result from limited dispersal.

Figure 3 shows that the haplotypes sampled by us cluster into three groups of two closely related alleles. These groups are separated by 6–21 steps. A seventh, single haplotype is separated from the rest of the network by 14 steps. Cryptic speciation recently has been detected in the Letharia vulpina-columbiana species complex (Kroken and Taylor 2001). Thus it is conceivable that the individuals sampled for this study in fact belong to different phylogenetic species. Instead of observing genetic differentiation among populations, in this case we would observe differences in the distribution of four species. Cladonia subcervicornis occurs over a range of 3000 km along the European coast. In the absence of any ecological and morphological variability among localities, the probability of encountering four different cryptic species by chance when sampling over a latitudinal range of just 45 km appears minute, unless the number of cryptic species is very high or the cryptic species are all sympatric. It cannot be concluded from the single example of Letharia that cryptic speciation is common among lichens. In fact, the "cryptic" species detected within that genus by Kroken and Taylor (2001) already had been described as infraspecific taxa (under L. vulpina) based on morphological differentiation (Schade 1955). Without further evidence, we consider it more likely that we are dealing with populations of a single species.

Our results corroborate a study by Hageman and Fahselt (1992), who reported among-stand variation of isozyme patterns on a similar geographical scale. Those authors used Umbilicaria mammulata, a species that rarely is found fertile and that lacks specialized vegetative propagules, as their study object. Our results indicate that ascospores do not help to disperse C. subcervicornis over longer distances, although it frequently is found with apothecia. Two recent studies by Sillett et al (2000) and Hilmo and Såstad (2001) add to our results. In transplantation and growth experiments, these authors showed that some lichens are restricted to old-growth forests, not because of ecological specialization but because of dispersal limitations. Perhaps effective dispersal is much rarer among many lichens than would be predicted by the abundant production of sexual and asexual diaspores. Using molecular markers in studies on the population dynamics of lichens could yield new insights into their dispersal biology. With only few nucleotide markers for infraspecific lichen studies available, cox 1 intron sequences could prove a valuable addition to this small arsenal of tools.

	ACKNOWLEDGMENTS

 
Our thanks are due to Birgit Kanz for technical help and Louise Lindblom and Nora Wirtz for testing primers on Xanthoria and Usnea. Louise Lindblom also is thanked for her constructive criticism of the manuscript. This study was supported by the Research Council of Norway through the Strategic University Programme Applications of Molecular Techniques in Systematic Biology.

	FOOTNOTES

 
¹ Corresponding author. Current address. Abteilung Botanik/ Paläobotanik, Forschungsinstitut Senckenberg, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany. E-mail: christian.printzen{at}senckenberg.de

Accepted for publication November 25, 2002.

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