• Cyanobacteria were isolated from bipartite cyanolichen species of Pseudocyphellaria and the identity of the major photobionts established. The specificity of the cyanobacterial–fungal association was also examined.
• Comparison of 16S rRNA gene sequences distinguished cyanobacterial and green algal isolates, and both 16S rRNA gene and tRNALeu (UAA) intron sequences of isolates and lichen thalli identified candidate photobionts. In addition, the genetic diversity of both the cyanobiont and mycobiont was investigated using the comparison of tRNALeu (UAA) intron sequences and ITS sequences, respectively.
• The 16S rRNA gene sequences identified two species-specific photobionts with similar sequences; however, the tRNALeu (UAA) intron sequences unambiguously discriminated between the two symbiotic cyanobacterial strains. Moreover, the fungal ITS sequences of the two corresponding lichens, Pseudocyphellaria crocata and Pseudocyphellaria neglecta, showed little variation.
• The cyanobacterial–fungal associations of P. crocata and P. neglecta were specific for all samples. However, the similarity of the ITS sequences raised the possibility that they represent the same species and that their different morphology is influenced by the cyanobacterial symbiont.
A lichen symbiotic association may be either bipartite, where the fungus (mycobiont) is associated with a green alga or a cyanobacterium (photobiont), or it may be tripartite with all three organisms present (Rai et al., 2000). Lichenized cyanobionts may exhibit both atypical morphological characteristics and an altered life cycle whereas, once isolated, the morphology of the cyanobacteria can be used to assist in their identification (Hale, 1983; Friedl & Büdel, 1996). However, the isolation of cyanobionts from the fungal component of the lichen has been found to be problematic, with minor symbionts or epiphytes being cultured (Kardish et al., 1990; Miao et al., 1997). Therefore, the use of strain-specific markers to confirm the isolation of the major cyanobiont is essential.
Little is known about the formation or specificity of the lichen symbiosis and classification is tied to the fungal component, with the photobiont having no influence on taxonomic assignment. Investigation of cyanobacterial diversity in lichen species, by comparison of the tRNALeu (UAA) intron sequences of lichen thalli, suggested that the specificity of the cyanobacteria was influenced more by fungal identity than geographical distribution, and that the degree of specificity of the cyanobacterial associations varied among lichen species (Paulsrud & Lindblad, 1998; Paulsrud et al., 1998, 2000). These studies focused on lichens belonging to the families Peltigeraceae and Nephromataceae collected from Sweden, Finland and North America.
The taxonomic assignment of lichenized fungi has traditionally been based on chemistry, morphology, reproductive mode, and habitat preference; however, more recently, molecular data have also been used (Grube & Kroken, 2000). In particular, the nuclear-encoded genes of the ribosomal repeat unit have been used to obtain fungal phylogenetic information over a wide range of taxonomic levels (White et al., 1990). However, previous studies on photobiont diversity have relied on the traditional taxonomic assignment of species in the lichens investigated.
The objective of the present study was to isolate cyanobacteria from bipartite cyanolichens belonging to the genus Pseudocyphellaria Vain. and establish if these lichens share a common cyanobiont or exhibit specificity at the level of the photobiont. To conduct these experiments we used part of the 16S rRNA gene and the tRNALeu (UAA) intron sequence of isolated cyanobacteria to obtain the identity of the primary photobionts. The examination of cyanobiont specificity has also been undertaken to extend previous studies, both taxonomically and geographically, by assessing three species of lichens belonging to the Lobariaceae from New Zealand. To achieve this, tRNALeu (UAA) intron sequences were used to investigate the specificity of the cyanobionts in three species of Pseudocyphellaria, where lichen samples of the same species from different sites were compared. In addition, fungal-specific primers were used to amplify the internal transcribed spacer (ITS) of the nuclear-encoded ribosomal repeat unit in order to examine the variation in the fungal component of the lichen samples between and within species. In this paper, we report the isolation of two cyanobionts from lichen thalli and establish the specificity of their association with different fungal species.
Materials and Methods
Lichen specimens of four bipartite species of Pseudocyphellaria were collected from sites in the South Island of New Zealand and were identified according to Galloway (1988). The cyanolichen species examined were: Pseudocyphellaria crocata (L.) Vain., typically found in exposed habitats; Pseudocyphellaria maculata D. J. Galloway, usually from moist, shaded environments; Pseudocyphellaria murrayi D. J. Galloway, usually located in moderately to densely shaded niches, and Pseudocyphellaria neglecta (Müll. Arg.) H. Magn., typically from low humidity locations exposed to high light.
Lichens were collected from three sites in Otago (subcoastal, hill suburb, altitude 365 m, moderate rainfall (site 1); dry, high-alpine grassland, altitude 1310 m, high light and high insolation (site 2); and lowland, coastal forest, altitude 5–10 m, moderate shade and moderate rainfall (site 4)), three sites in Southland (pastureland at edge of rainforest, altitude 600 m, high light and moderate to high rainfall (site 3); subalpine rainforest, altitude 820 m, moderate shade and very high rainfall (site 5); and subalpine rainforest/scrub, altitude 800 m, very high precipitation (site 7)) and one site in Canterbury (parkland, altitude 530 m, high light, moderate rainfall (site 6)). Voucher specimens were deposited in the herbarium of Otago University. Table 1 lists the sites from which the samples were collected and Fig. 1 provides the locations of these sites in the South Island.
Table 1. Description of lichen samples and location of collection sites
The isolation of cyanobionts was only undertaken from the thalli PC2, PMa1, PMi1 and PN1.
pcA, pcB, pcC
Cyanobacteria were isolated from thalli using either the thallus fragmentation method of Ahmadjian (1993b) or the homogenization protocol of Miao et al. (1997). The strain Nostoc PCC 73102 was obtained from the Pasteur Culture Collection. Cyanobacteria were grown on Bold’s Basal Medium zero nitrogen (BBM0N) agar and agarose plates (Deason & Bold, 1960; Ahmadjian, 1993b) at 21°C in continuous light at an intensity of 30 µmol m−2 s−1 measured with a LI-189, Li-Cor (Lincoln, NE, USA) light meter.
The DNA was extracted from isolated cultures using a protocol modified from Arteaga-Nieto et al. (2000). After cell lysis and centrifugation the supernatant was extracted with an equal volume of phenol followed by an equal volume of chloroform; both the phenol and the chloroform treatments were repeated and the DNA was precipitated with ethanol. DNA from lichen thalli was extracted as described in Cubero et al. (1999). A minimum thallus dry weight of 50 mg was used for each DNA extraction.
A region of the 16S rRNA gene, corresponding to Escherichia coli nucleotides 359–805, was amplified from cyanobacterial and lichen DNA using the cyanobacterial and plastid-specific primers CYA359F and CYA781Rb from Nübel et al. (1997). The polymerase chain reaction (PCR) included 1.5 mm MgCl2, 10 ng µl−1 genomic DNA and 0.05 U µl−1 Platinum Taq (Invitrogen, Groningen, The Netherlands). Samples were initially denatured at 95°C for 5 min. This was followed by three cycles of 45 s at 95°C and 90 s at 60°C, three cycles of 45 s at 95°C and 90 s at 57°C, three cycles of 45 s at 95°C and 90 s at 54°C, three cycles of 45 s at 95°C and 90 s at 51°C, and 20 cycles of 45 s at 95°C and 90 s at 48°C. The reaction then concluded with an extension at 72°C for 3 min.
Cyanobacterial colonies were photographed on agar plates using an Olympus zoom stereomicroscope SZ-40 with an Olympus SC35 camera and high-power microscopy was carried out using an Axiophot photomicroscope (Carl Zeiss, Göttingen, Germany).
The cyanobacterial tRNALeu (UAA) intron was amplified from cyanobacterial and lichen DNA using nested primers designed to the tRNALeu (UAA) exons by Paulsrud & Lindblad (1998). The primers annealed at the following nucleotide positions corresponding to the Anabaena azollae (caroliania) tRNA gene: 1–16 and 361–375 for the external primers and 17–34 and 326–344 for the internal primers. These were then used to amplify the 291 bp intron. For PCR with both the external and internal primers, 2 mm MgCl2 and 0.05 U µl−1 Platinum Taq were used. Genomic DNA (10 ng µl−1) was used as the template for the first PCR amplification, using the external primers, and the resultant PCR product was diluted 500-fold for the internal primer PCR. The PCR conditions for the external primers were: 95°C for 5 min, followed by 30 cycles of 95°C for 45 s, 58°C for 1 min and 72°C for 1 min, followed by an extension at 72°C for 5 min. The PCR conditions using the internal primers were: 95°C for 5 min, 30 cycles of 95°C for 45 s, 62°C for 1 min and 72°C for 1 min; this was also followed by an extension at 72°C for 5 min.
Fungal-specific primers were used to amplify the ITS region of nuclear-encoded rRNA genes from DNA isolated from lichen thalli. Nested PCR was carried out using external primers ITS1-f and NL6Amun designed by Gardes & Bruns (1993) and Egger (1995), respectively, and internal primers ITS5 and ITS4 designed by White et al. (1990). In the first round of PCR, using the external primers, each reaction contained 2 mm MgCl2 10 ng µl−1 genomic DNA and 0.05 U µl−1 Platinum Taq. In the second round of PCR, using the internal primers, each reaction contained 2 mm MgCl2, a 500-fold dilution of the first round PCR product and 0.05 U µl−1 Platinum Taq. The conditions for the first round of PCR were: 95°C for 5 min, 30 cycles of 95°C for 45 s, 58°C for 1 min and 72°C for 1 min 30 s, followed by an extension at 72°C for 5 min. For the second round of PCR the conditions were: 95°C for 5 min, 30 cycles of 95°C for 45 s, 62°C for 1 min and 72°C for 1 min, followed by an extension at 72°C for 5 min.
All PCR products were purified using the QIAquick gel extraction kit (Qiagen Inc., Valencia, CA, USA). PCR products were sequenced with the primers used for PCR, using an ABI PRISM 377 DNA Sequencer and BigDye Terminators (Applied Biosystems, Foster City, CA, USA).
Four species of Pseudocyphellaria were obtained from seven different sites in the South Island, New Zealand (Fig. 1). Specimens of P. crocata were obtained from six different sites and these samples are designated PC1 to PC6 in Table 1, and the province where each site is located is also listed. One sample of P. maculata, and one of P. murrayi, were also obtained from site 1 and were designated PMa1 and PMi1, respectively. A further two P. maculata samples (PMa2 and PMa3) were collected from site 2 in Otago. In addition, four samples of P. neglecta were obtained from three sites, one from site 4 in Otago (PN1), one from site 7 in Southland (PN2) and two from site 6 in Canterbury (PN3 and PN4).
Photoautotrophic organisms were isolated from lichen thalli listed in Table 1 by fragmentation or homogenization according to Ahmadjian (1993b) and Miao et al. (1997). These procedures led to the cultivation of organisms that displayed a wide range of colony morphologies (data not shown). Three isolates were obtained from the PC2 specimen of P. crocata: pcA, pcB and pcC. One photoautotroph was cultured from the P. maculata thallus, PMa1, and one from the P. murrayi thallus, PMi1. Two isolates from the PN1 specimen of P. neglecta, pnA and pnB, were also obtained. The different photobionts isolated are summarized in Table 1.
The identity of the isolates was investigated using cyanobacterial- and plastid-specific primers to amplify part of the 16S rRNA gene and comparison of the sequences from the pcC and pnA isolates with sequences available in GenBank indicated that these were green algae. However, the five isolates, pcA, pcB, pmaA, pmiA and pnB, had cyanobacterial sequences consistent with being strains of Nostoc (data not shown).
To examine whether the isolated cyanobacteria represented the major photosynthetic symbiont of the lichen, as opposed to a minor symbiont, epiphyte or commensal, the cyanobacterial- and plastid-specific primers CYA359F and CYA781Rb were used to amplify a region of the 16S rRNA gene from lichen DNA. All cyanobacterial 16S rRNA gene sequences from lichen thalli of P. crocata, P. maculata, P. murrayi and P. neglecta were similar to Nostoc sequences (data not shown). The sequences of two regions of the 16S rDNA from the isolates and lichen thalli corresponding to nucleotides 534–604 and 665–693 of Anabaena PCC 7120 are compared in Fig. 2. The P. crocata cyanobacterial isolate pcA, the P. maculata isolate pmaA and the P. murrayi isolate pmiA have different sequences compared with their respective lichen thalli (Fig. 2b). All the sequences obtained from PCR products of the lichen thalli resulted in unambiguous peaks after electrophoresis, indicating the presence of one genotype and therefore a single photobiont in the thallus from which the DNA had been extracted (Kroken & Taylor, 2000). Therefore, none of the above isolates represented major cyanobionts.
By contrast, the other P. crocata isolate, pcB, and the P. neglecta isolate, pnB, had sequences identical to the lichens from which they were cultured and therefore these isolates were considered potential major photobionts. In addition, the 16S rDNA sequences of the two cyanobacterial isolates pcB and pnB differed by only two bases, and this number of nucleotide changes was seen between sequences of different thalli of P. crocata (data not shown). Interestingly, the sequence similarity between the isolates was not reflected in either the pigmentation of the colonies or their cellular morphologies, which were distinct (compare Fig. 3a,b and c,d). Therefore, these data could not determine whether pcB and pnB were the same strain exhibiting two different morphologies or whether they were two distinct strains.
To ascertain if the pcB and pnB isolates were different cyanobacterial strains, their respective tRNALeu (UAA) intron sequences were obtained. The tRNALeu (UAA) intron sequences of cyanobacteria of the family Nostocaceae have been shown to be highly variable (Xu et al., 1990). A comparison of the tRNALeu (UAA) intron sequences of the cyanobacterial isolates and lichen samples is presented in Fig. 4. These data agree with the 16S rRNA gene sequence data affirming that the majority of the isolates were not the major photobionts of the lichen thalli. However, identical tRNALeu (UAA) intron sequences were found when the pcB isolate was compared with lichen PC2 and when the pnB isolate was compared with lichen PN1. Significantly, while the isolates pcB and pnB had similar 16S rDNA sequences, the tRNALeu (UAA) intron sequences shown in Fig. 4b varied in five places, including a 38 nucleotide segment that was absent in pnB but present in the pcB sequence. Therefore, although the pcB and pnB isolates are strong candidates for the major photobiont found in the PC2 and PN1 thalli, respectively, these two isolates represent distinct Nostoc strains.
To investigate whether the isolated cyanobionts were characteristic of photobionts from the two lichen species, P. crocata and P. neglecta, the cyanobacterial tRNALeu (UAA) intron sequences in lichen specimens from different sites were examined. Highly similar sequences were found in the six P. crocata thalli listed in Table 1; these contained only two nucleotide changes across the entire intron and the sequences of four P. neglecta specimens were identical to each other (data not shown). To further establish the specificity of the association of one cyanobacterial strain with P. crocata and a different strain with P. neglecta, a comparison was made of the tRNALeu (UAA) intron sequence of one P. crocata sample (PC6) and two P. neglecta samples (PN3 and PN4) collected from the same rock. The cyanobiont tRNALeu (UAA) intron sequence of the P. crocata specimen differed from that of the two P. neglecta specimens found growing in close proximity. However, the P. crocata cyanobacterial tRNALeu (UAA) intron sequence was identical to that obtained from other P. crocata samples from different sites and, furthermore, the two P. neglecta samples had cyanobacterial tRNALeu (UAA) intron sequences that were identical to each other and to P. neglecta samples collected from two other locations. The tRNALeu (UAA) intron sequences of three P. maculata thalli from different sites were also compared and these were found to be identical. Furthermore, these P. maculata intron sequences were identical to those obtained for P. crocata.
Lichens are classified according to the fungal component of the symbiosis, with each lichen species containing a unique fungal species. To examine the diversity of the mycobionts of P. crocata and P. neglecta, primers were used that were specific to the fungal rDNA repeat (Egger, 1995). The least conserved region of nuclear-encoded rDNA, the ITS, was amplified from whole-lichen DNA extracts of P. crocata and P. neglecta. Identical ITS sequences were obtained from the PN3 and PN4 samples of P. neglecta collected from the same location, while the P. crocata specimen PC6, also from the same site, varied by four base changes (Fig. 5). Moreover, the ITS was as polymorphic within the samples from the same species as it was between samples of the two species. These results indicated that either the samples represented the same species or a very closely related species that could not be distinguished by the ITS sequence.
Culturing the major photobiont from a lichen thallus may be hampered by the presence of epiphytes and commensals. Accordingly, a comparison of DNA sequences from the cultured isolate, and lichen thallus, is essential to confirm that the isolates represent primary symbionts. The 16S rRNA gene has been widely used for such studies because the primary structure contains both conserved and variable regions (Wilmotte, 1994; Miao et al., 1997). In this report, part of the 16S rDNA was sequenced and this was sufficiently informative to exclude the possibility that the three isolates, pcA, pmaA and pmiA, were major photobionts. By contrast, the 16S rDNA sequences indicated that the pcB and pnB isolates represented the primary photobionts from the P. crocata PC2 and P. neglecta PN1 thalli, respectively, but could not distinguish between these strains. However, this was achieved by comparing the shorter, more variable, tRNALeu (UAA) intron sequences of these isolates, thus establishing that the PC2 and PN1 lichens possessed different primary photobionts.
Previous studies of photobiont specificity revealed highly specific cyanobacterial associations (Paulsrud et al., 1998) and more diverse green algal associations (Helms et al., 2001). Investigation of cyanobiont specificity in three bipartite foliose lichens, Peltigera canina, Peltigera membranacea and Nephroma resupinatum, each collected from two different countries, found the presence of only one cyanobacterial tRNALeu (UAA) intron type per lichen species (Paulsrud & Lindblad, 1998; Paulsrud et al., 1998, 2000). However, Miao et al. (1997) found different cyanobacterial 16S rRNA gene sequences in colourmorphs of the species Peltigera membranacea. Furthermore, Paulsrud et al. (2000) identified the same cyanobacterial strains in the thalli of more than one lichen species. Moreover, multiple intron types were found in species able to form tripartite lichens, with two intron types present in the three species, Peltigera aphthosa, Nephroma arcticum and Peltigera britannica, four present in Peltigera neopolydactyla and five in Peltigera venosa. These authors suggested that the decreased specificity of cyanobionts may be partly accounted for by fungal heterogeneity in P. neopolydactyla, which has highly variable morphology, and that different forms of cephalodia in P. venosa may reflect the presence of cyanobacteria that are not truly lichenized. Paulsrud et al. (2001) have also confirmed the identity of the cultured cyanobiont from the tripartite lichen, P. aphthosa, by sequencing the tRNALeu (UAA) intron.
We found that P. crocata had a cyanobacterial strain that was distinct from that of P. neglecta. These results suggest a highly specific cyanobiont association for these two bipartite species that is consistent with the results of Paulsrud et al. (1998, 2000). This specificity of cyanobacterial–fungal association has now been demonstrated in lichens belonging to three families of the suborder Peltigerineae from both the northern and southern hemispheres. However, because little information is available regarding which is the dominant reproductive mode of lichens, we cannot determine whether the apparent invariability of the association reflects joint dispersal of the two symbionts or the highly specific formation of new lichen thalli. Also in agreement with Paulsrud et al. (2000), we found the same cyanobacterial strain associated with more than one mycobiont, in this case P. crocata and P. maculata. Interestingly, these are species with different habitat preferences (Galloway, 1988). Furthermore, the cyanobacterium from P. crocata, isolate pcB, had a similar intron sequence, differing by only one nucleotide from the cyanobacterial sequence of a Nephroma resupinatum sample described by Paulsrud et al. (1998). The third nucleotide of variable region II is G in pcB (Fig. 4b) and T in N. resupinatum. These data therefore suggest that the pcB cyanobiont is also found lichenized in N. resupinatum in Finland (see Paulsrud et al., 2000).
A number of molecular studies on lichenized fungi used nuclear ribosomal RNA gene sequences to investigate phylogenetic relationships (Crespo & Cubero, 1998; Miadlikowska & Lutzoni, 2000) and genetic variation in lichen populations (Crespo et al., 1999; Zoller et al., 1999). Goffinet & Bayer (1997) used fungal ITS sequence to identify the mycobiont species of photomorph pairs. They reported that ITS nucleotide variation is usually low within a species. However, Bridge & Hawksworth (1998) stated that different levels of ITS sequence variation have been reported in different fungal species. We found the same variation between the fungal ITS sequences of P. crocata and P. neglecta as within samples of each species, suggesting that either the ITS is insufficiently variable to be informative or the fungal component of these lichens represents the same species. LaGreca (1999) used fungal ITS sequences to define species of Ramalina, which contain green algal photobionts and a variation of 1.3–2.6% was reported between samples of the same species and variations of 4.0–4.5% between samples of different species. Moreover, P. crocata ITS sequences in GenBank (Accession Nos. AF401978–AF401981) had a variation of only 0.2% between samples, while Pseudocyphellaria perpetua (Accession Nos. AF401971–AF401977) showed 0.0–1.9% sequence variation. For the sequences shown in Fig. 5 the ITS1 and ITS2 regions for P. crocata showed a variation of 0.0–1.2%, those from P. neglecta showed a variation of 0.0–0.8%, and the variation between P. crocata and P. neglecta was 0.6–1.2%. These data contrast with the 6.2–7.2% ITS sequence variation seen when comparing both the P. crocata and P. neglecta samples with the PMa1 sample, supporting the possibility that the P. crocata and P. neglecta samples represent the same species. Interestingly, the sequence variation between our samples of P. crocata and P. neglecta and the P. crocata sequences from GenBank was 6.6–11.6%. By contrast, the sequences of P. perpetua differed from our P. crocata and P. neglecta by only 1.5–5.6%. Therefore, our southern hemisphere P. crocata samples may be genetically distinct from the northern hemisphere P. crocata samples.
Morphological characteristics have been used to distinguish P. crocata and P. neglecta, and these include the presence of soredia in P. crocata and phyllidia in P. neglecta (Galloway, 1988). It has been deduced that the mycobiont primarily determines the thallus structure (Ahmadjian, 1993a; Büdel & Scheidegger, 1996), and Stocker-Wörgötter (2001a) has shown that the mycobionts of Cladonia imperialis and Cladonia crinita are able to form thallus-like structures under certain culture conditions. However, in photosymbiodemes the same mycobiont exhibits different morphologies in the bipartite and tripartite forms, for example Lobaria fendleri where the same mycobiont may form a lobate green algal thallus or a cyanobacterial fruticose structure (Stocker-Wörgötter, 2001b). Accordingly, if P. crocata and P. neglecta do contain the same lichenized fungal species, a possibility is that their morphological differences may be influenced by the presence of different cyanobacterial strains.
The sequences in this study have been deposited in the EMBL database. The 16S rRNA sequences are AJ437559–AJ437567; the tRNALeu (UAA) intron sequences are AJ421996–AJ422009 and AJ437321–AJ437325, and the ITS sequences are AJ437679–AJ437689.
We thank Dr Michael A. Thomas for helpful discussions and assistance with collecting field samples. This research was supported by a New Zealand Marsden grant (UOO805).