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Keywords:

  • lichens;
  • Nostoc;
  • cyanobacteria;
  • maritime Antarctica;
  • symbiont specificity;
  • tRNALeu intron

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The cyanobionts of lichens and free-living Nostoc strains from Livingston Island (maritime Antarctica) were examined to determine both the cyanobiont specificity of lichens and the spatial distribution of Nostoc strains under extreme environmental conditions.
  • • 
    We collected five different lichen species with cyanobacteria as primary or secondary photobiont (Massalongia carnosa, Leptogium puberulum, Psoroma cinnamomeum, Placopsis parellina and Placopsis contortuplicata) and free-living cyanobacteria from different sample sites and analysed them using the tRNALeu (UAA) intron as a genetic marker to identify the cyanobacterial strains.
  • • 
    Our results showed that the same Nostoc strain was shared by all five lichen species and that an additional strain was present in two of the lichens. Both Nostoc strains associated with lichen fungi also occurred free-living in their surrounding. Bi- and tri-partite lichens were not different in their cyanobiont selectivity.
  • • 
    Contrary to studies on different lichen species in temperate regions, the Antarctic lichen species here did not use species-specific cyanobionts; this could be because of a selection pressure in this extreme environment. Limiting factors under these ecological conditions favor more versatile mycobionts. This results in selection against photobiont specificity, a selection pressure that may be more important for lichen distribution than the effect of cold temperatures on metabolism.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Most lichens are a bipartite symbiosis between a fungal partner, the mycobiont, and a photosynthetic partner that can be either a green alga (phycobiont) or a cyanobacterium (cyanobiont). Tripartite lichens consist of a mycobiont associated with a photosynthetic green alga and a cyanobacterium, which occurs in special structures termed cephalodia. Fixation of N2 in tripartite lichens is restricted to the cephalodia. The ability of cyanolichens to fix atmospheric N2 means that they can make a considerable contribution to the nitrogen budget in nutrient-poor habitats (Potts, 2000; Vincent, 2000). In maritime Antarctica, nitrogen availability is a major factor influencing lichen diversity and the proportion of lichen taxa containing N2-fixing Nostoc species is higher in ice-free inland communities (Smith, 1995) than in coastal communities. Other environmental factors such as cold temperatures and low light during winter are more extreme than in temperate zones and there is a distinct decline in lichen biodiversity with higher latitude (Green et al., 1999; Øvstedal & Lewis Smith, 2001; Pintado et al., 2001).

In temperate regions, mycobionts appear to be strongly selective in their choice of a Nostoc strain as symbiotic partner. The species identity of the lichen seems to determine the cyanobacterial strain rather than the geographical origin of the samples (Paulsrud et al., 1998; Paulsrud et al., 2000; Paulsrud et al., 2001). It is also known that, in contrast to mycobionts, cyanobionts perform poorly under the subzero conditions that are common in Antarctica (Schroeter et al., 1997). Hence, it is possible that the extreme conditions in the Antarctic would cause a decrease in the number of cyanobacterial strains and produce a situation where lichen fungi would be forced to be less selective in their choice of cyanobionts. The result would be less constancy in mycobiont–cyanobiont associations.

It is possible to use molecular techniques to determine whether the mycobiont of each lichen species always selects the same algal or cyanobacterial partner. A group I intron in the tRNALeu gene has been used to distinguish strains of Nostoc symbionts of various organisms (Paulsrud & Lindblad, 1998; Paulsrud et al., 1998, 2000; Costa et al., 1999, 2001) and to investigate horizontal gene transfer (Paquin et al., 1997; Rudi et al., 2002). Variable regions within this highly conserved intron have proved to be very useful in studies of cyanobiont specificity. Cyanobacterial strains have been successfully differentiated in different groups of lichenized ascomycetes (Paulsrud et al., 1998; Summerfield et al., 2002; Linke et al., 2003) and between free-living strains and lichen cyanobionts (Oksanen et al., 2002; Rikkinen et al., 2002). Consequently, we have used this molecular marker to study the cyanobiont specificity of Antarctic lichens and the free-living cyanobacteria in their surroundings.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We determined the genetic identity of the cyanobionts of five lichen species and of free-living Nostoc colonies from eight localities at South Bay and Byers Peninsula (Fig. 1), two ice-free inland habitats on Livingston Island (South Shetland Islands). Two lichens (Leptogium puberulum and Massalongia carnosa; Fig. 2a,b) were bipartite with a Nostoc as the primary cyanobiont and the other three lichens were tripartite species consisting of a mycobiont, a photosynthetic green alga and the cyanobiont Nostoc in cephalodia (Placopsis contortuplicata, Placopsis parellina and Psoroma cinnamomeum; Fig. 2c–e).

image

Figure 1. Map of Livingston Island (South Shetlands, Antarctica). Sample sites are marked by Roman numerals (I–VIII).

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image

Figure 2. (a–e) Lichen species examined: (a) Leptogium puberulum, (b) Massalongia carnosa, (c) Psoroma cinnamomeum, (d) Placopsis parellina, (e) P. contortuplicata. Bars, 1 mm. (f) Free-living Nostoc sp. Bar, 20 µm.

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Large free-living Nostoc colonies were collected from the soil and stored in sealed paper envelopes separated from the collected lichens. The free-living colonies were washed and examined using a microscope to ensure that no fungal hyphae were present.

Neither the cyanobionts of the lichen thalli nor the free-living cyanobacteria were cultured. We isolated DNA directly from washed pieces of thalli from bipartite lichens and pieces of cephalodia from tripartite lichens. Silica sand, liquid nitrogen and pestles were used to corrode the mucilage cover of the cyanobacteria before the extraction. Additional washing steps during the isolation with a NucleoSpin Plant kit (Macherey-Nagel, Düren, Germany) were performed to prevent high concentrations of polysaccharides hampering the polymerase chain reactions (PCR).

To obtain information about the diversity and spatial distribution of the Nostoc symbionts we determined nucleotide sequences of the partly variable cyanobacterial tRNALeu (UAA) intron (Paulsrud & Lindblad, 1998). This DNA region contains elements whose nucleotide sequence is highly conserved, a property that is strongly enforced by their involvement in the secondary and tertiary structure of the intron. However, two highly variable regions, described as P6b and P9 by Costa et al. (2002), are also present in this intron

The undiluted, isolated DNA was used for PCR amplifications of the cyanobacterial tRNALeu (UAA) intron. Primers for amplification were: 5′-GGGGRTRTGGYGRAAT-3′ as forward, and 5′-GGGGRYRGRGGGACTT-3′ as reverse primer. Amplifications were performed in 25 µl volumes containing 12.5 µl Taq PCR Master Mix (Qiagen, Hilden, Germany), 2.5 µl DNA, 2.5 µl of each primer (10 µm) and 5 µl H2O. Stratagene Robocycler (La Jolla, CA, USA) was used with the following program: initial denaturation at 95°C for 15 min, 30 cycles of 94°C for 1.3 min, 48°C for 1.5 min, 72°C for 2 min, one cycle of 72°C for 10 min, and a 4°C soak. Fragments were cleaned using the NucleoSpin Extract kit (Macherey-Nagel) and sequenced using the Ampli-Taq DNA Polymerase FS Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Boston, MA, USA). The sequences were obtained with the sequencing primers: 5′-GGTAGACGCWRCGGACTT-3′ and 5′-TWTACARTCRACGGATTTT-3′. Cycle sequencing was executed with the following program: 25 cycles of 95°C for 30 s, 48°C for 15 s and 60°C for 4 min Sequencing products were precipitated and dried before they were loaded on an ABI 377 (Perkin-Elmer) automatic sequencer. Sequence fragments obtained were assembled with SeqMan 4.03 (DNAStar Inc., Madison, WI, USA) and manually adjusted. Clustal W (Thompson et al., 1994) provided an alignment that was manually adjusted in the variable stem-loop regions. No interrupting sequences were present.

We used the paup*4.0 software package (Swofford, 1998) to construct a phylogenetic tree using a distance-based approach. The main goal of the present study was the identification and comparison of strains not the assessment of evolutionary relationships or the resolution of the phylogeny of the taxa. Nevertheless, in order to achieve this in an easily visible manner, we present a tree to illustrate our results. Distance matrices were obtained with Kimura's two-parameter model (Kimura, 1980) and calculated using the neighbor-joining method (Saitou & Nei, 1987). We included four sequences of the cyanobiont of P. parellina from Argentina as an outgroup because it was the only taxon from which we could obtain fresh material for DNA extraction from a nonAntarctic environment. The tree was drawn using treeview (Page, 1996).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In total, 64 specimens were examined, including four samples of P. parellina collected in Argentina (Table 1). Sequences of cyanobionts are denoted according to the lichen species from which they were amplified. This name is followed by a Roman figure indicating the sample site, as shown in Fig. 1, plus a number for the individual specimen at each site. We obtained sequences of tRNALeu (UAA) introns varying in length between 302 bp in the cyanobiont of P. contortuplicata I-3 and 354 bp in L. puberulum VI-2. See Table 1 for Roman figures and GenBank accession numbers of all nucleotide sequences. Sequences were aligned to produce a matrix of 269 nucleotide-position characters used for the phylogenetic analysis. The alignment started within the P3 element identified by Costa et al. (2002) and ended with the last base pair of the P9 element. We cut approximately 40 bp at the beginning and the end of the alignment so that the remaining length was equal to the shortest sequences obtained. These parts of the alignment removed contained mostly constant sites. The most variable element (P6b, from bp 59 to bp 147) had sufficient numbers of informative characters to allow us differentiate among distinct strains.

Table 1.  The tRNALeu (UAA) intron sequences of free-living cyanobacteria and cyanobacteria associated with lichens
SourceSample siteGenBank number
  1. Sample sites on Livingston Island (South Shetland Islands, Antarctica) are indicated with I–VIII and the individual specimens with numbers; A, Argentine samples.

Leptogium puberulum I-1Byers Peninsula, Lago Escondido (I)AY304240
Leptogium puberulum I-2Byers Peninsula, Lago Escondido (I)AY304235
Leptogium puberulum I-3Byers Peninsula, Lago Escondido (I)AY304241
Leptogium puberulum II-1Byers Peninsula, Usnea Plug (II)AY304233
Leptogium puberulum II-2Byers Peninsula, Usnea Plug (II)AY304239
Leptogium puberulum II-3Byers Peninsula, Usnea Plug (II)AY304295
Leptogium puberulum II-4Byers Peninsula, Usnea Plug (II)AY304238
Leptogium puberulum III-1Byers Peninsula, Chester Cone (III)AY304237
Leptogium puberulum III-2Byers Peninsula, Chester Cone (III)AY304242
Leptogium puberulum VI-1Byers Peninsula, north of Cerro Negro (VI)AY304236
Leptogium puberulum VI-2Byers Peninsula, north of Cerro Negro (VI)AY304243
Leptogium puberulum VI-3Byers Peninsula, north of Cerro Negro (VI)AY304244
Leptogium puberulum VII-1South Bay, Punta Polaca (VII)AY304245
Massalongia carnosa I-1Byers Peninsula, Lago Escondido (I)AY304250
Massalongia carnosa I-2Byers Peninsula, Lago Escondido (I)AY304246
Massalongia carnosa I-3Byers Peninsula, Lago Escondido (I)AY304247
Massalongia carnosa III-1Byers Peninsula, Chester Cone (III)AY304249
Massalongia carnosa V-1Byers Peninsula, South Beaches (V)AY304251
Massalongia carnosa VII-1South Bay, Punta Polaca (VII)AY304248
Massalongia carnosa VIII-1South Bay, Reina Sofia (VIII)AY304252
Placopsis contortuplicata I-1Byers Peninsula, Lago Escondido (I)AY304256
Placopsis contortuplicata I-2Byers Peninsula, Lago Escondido (I)AY304257
Placopsis contortuplicata I-3Byers Peninsula, Lago Escondido (I)AY304258
Placopsis contortuplicata I-4Byers Peninsula, Lago Escondido (I)AY304259
Placopsis contortuplicata I-5Byers Peninsula, Lago Escondido (I)AY304261
Placopsis contortuplicata II-1Byers Peninsula, Usnea Plug (II)AY304262
Placopsis contortuplicata VII-1South Bay, Punta Polaca (VII)AY304263
Placopsis contortuplicata VIII-1South Bay, Reina Sofia (VIII)AY304260
Placopsis parellina I-1Byers Peninsula, Lago Escondido (I)AY304271
Placopsis parellina I-2Byers Peninsula, Lago Escondido (I)AY304264
Placopsis parellina I-3Byers Peninsula, Lago Escondido (I)AY304272
Placopsis parellina II-1Byers Peninsula, Usnea Plug (II)AY304266
Placopsis parellina II-2Byers Peninsula, Usnea Plug (II)AY304274
Placopsis parellina II-3Byers Peninsula, Usnea Plug (II)AY304280
Placopsis parellina III-1Byers Peninsula, Chester Cone (III)AY304283
Placopsis parellina III-2Byers Peninsula, Chester Cone (III)AY304269
Placopsis parellina III-3Byers Peninsula, Chester Cone (III)AY304278
Placopsis parellina V-1Byers Peninsula, South Beaches (V)AY304275
Placopsis parellina VI-1Byers Peninsula, north of Cerro Negro (VI)AY304273
Placopsis parellina VI-2Byers Peninsula, north of Cerro Negro (VI)AY304265
Placopsis parellina VI-3Byers Peninsula, north of Cerro Negro (VI)AY304296
Placopsis parellina VI-4Byers Peninsula, north of Cerro Negro (VI)AY304281
Placopsis parellina VI-5Byers Peninsula, north of Cerro Negro (VI)AY304282
Placopsis parellina VIII-1South Bay, Reina Sofia (VIII)AY304268
Placopsis parellina VIII-2South Bay, Reina Sofia (VIII)AY304277
Placopsis parellina A1-1Argentina, Provincia Rio Negro, San Carlos de Bariloche, Playa Serena (Lumbsch/Guderley 1997) (A1)AY304267
Placopsis parellina A1-2Argentina, Provincia Rio Negro, San Carlos de Bariloche, Playa Serena (Lumbsch/Guderley 1997) (A1)AY304276
Placopsis parellina A2-1Argentina, Provincia Rio Negro, P.N. Nahuel Huapi, Cerro Tronador, Ventisquero Negro (Mesutti 1990) (A2)AY304270
Placopsis parellina A2-2Argentina, Provincia Rio Negro, P.N. Nahuel Huapi, Cerro Tronador, Ventisquero Negro (Mesutti 1990) (A2)AY304279
Psoroma cinnamomeum I-1Byers Peninsula, Lago Escondido (I)AY304284
Psoroma cinnamomeum III-1Byers Peninsula, Chester Cone (III)AY304285
Psoroma cinnamomeum III-2Byers Peninsula, Chester Cone (III)AY304289
Psoroma cinnamomeum IV-1Byers Peninsula, near Smellie Point (IV)AY304234
Psoroma cinnamomeum IV-2Byers Peninsula, near Smellie Point (IV)AY304288
Psoroma cinnamomeum IV-3Byers Peninsula, near Smellie Point (IV)AY304292
Psoroma cinnamomeum V-1Byers Peninsula, South Beaches (V)AY304286
Psoroma cinnamomeum V-2Byers Peninsula, South Beaches (V)AY304294
Psoroma cinnamomeum V-3Byers Peninsula, South Beaches (V)AY304291
Psoroma cinnamomeum V-4Byers Peninsula, South Beaches (V)AY304287
Psoroma cinnamomeum VI-1Byers Peninsula, north of Cerro Negro (VI)AY304290
Psoroma cinnamomeum VI-2Byers Peninsula, north of Cerro Negro (VI)AY304293
Free-living Nostoc sp. II-1Byers Peninsula, Usnea Plug (II)AY304253
Free-living Nostoc sp. VI-1Byers Peninsula, north of Cerro Negro (VI)AY304255
Free-living Nostoc sp. VI-2Byers Peninsula, north of Cerro Negro (VI)AY304254

The Antarctic sequences formed two major, strongly supported clades in the phylogenetic tree (Fig. 3). Clade II contained 51 very similar sequences from the cyanobionts of all five lichen species included in this study collected at all eight sites, along with two free-living Nostoc specimens (Table 1). Cyanobiont sequences from specific lichen species were intermixed and did not form distinct groups within the clade. There was a variability of alignment position of 13% within this group and also one additional and one missing repeat unit (each 7 bp) in some sequences. The sequences of free-living Nostoc colonies comprising this clade were homogeneous and revealed no differences in repeat units. Clade I was composed of sequences of nine cyanobionts collected from five different sites. This clade contained sequences of two of the five lichen species examined (M. carnosa and P. parellina, which are also present in clade II) and a second free-living Nostoc strain. The variability within this group (8%) was lower than in clade II. Again, there was some mixing of the cyanobionts within the clade. In some cases, collections of one lichen species at a single site (e.g. M. carnosa at Byers Peninsula, Lago Escondido (I) or P. parellina at Byers Peninsula, Chester Cone (III) and Byers Peninsula, North of Cerro Negro (VI)) contained different Nostoc strains.

image

Figure 3. Neighbor joining tree based on a clustal alignment of tRNALeu (UAA) intron sequences. Bootstrap supports > 50% are shown. The Roman numerals correspond to the sites indicated in Fig. 1.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results indicate that at least two free-living Nostoc strains occurred on Livingston Island and that they were also present in lichen symbiosis (clade I and II, Fig. 3). The Nostoc strain that was most common and which formed the associations in clade II, seems to be novel. A comparison with cyanobacterial tRNALeu (UAA) intron sequences available in GenBank did not yield any closely related sequence. However, comparison of sequences of clade I with those obtained from named culture collections (Linke et al., 2003) suggested that these strains could belong to Nostoc punctiforme. Nostoc punctiforme is a cosmopolitan species that is reported from a variety of lichen associations (Tschermak-Woess, 1988). There was no indication of geographic patterns of Nostoc strains present between the Antarctic localities sampled. The cyanobionts found in Argentine populations of Placopsis parellina, which were used as an outgroup, differed considerably from the Antarctic cyanobionts but were similar in the four specimens studied. It is uncertain whether these sequences belong to a Nostoc spp. It is known that Placopsis spp. may include additional cyanobiont genera, such as Scytonema or Stigonema (Lamb, 1947). Further studies are needed to examine the cyanobiont diversity of Placopsis parellina in extra-Antarctic environments.

The two distinct clades depicted in Fig. 3 corresponded with different heptanucleotide repeats already known to occur in Nostoc sp. (Costa et al., 2002; Rikkinen et al., 2002). Two distinct forms of tRNALeu (UAA) group I introns that have independent phylogenies have been described in cyanobacteria (Rudi & Jakobsen, 1997; Rudi & Jakobsen, 1999), although this has been disputed by Costa et al. (2002). We compared our sequences with those in GenBank and confirmed that all sequences belonged to one form (i.e. cluster I in Rudi & Jakobsen (1999)), allowing a phylogenetic analysis of these sequences.

Both of the free-living Nostoc strains on Livingston Island were used by mycobionts in lichen associations. This result contrasts with previous studies in temperate regions (Paulsrud et al., 1998, 2000, 2001; Summerfield et al., 2002), which showed that lichens were very restrictive and had species-specific cyanobionts. The Antarctic lichens investigated here did not use species-specific cyanobionts, but associated with free-living cyanobacterial strains found in their immediate surroundings. Utilization of free-living cyanobacteria in symbiotic systems has not yet been reported for lichens or hornworts. In both cases, differences were found between cyanobacterial strains in symbioses and free-living strains (West & Adams, 1997; Oksanen et al., 2002). In our study, the two sequences of free-living Nostoc strains in clade II were identical to those in symbiotic strains. The sequence of free-living Nostoc in clade I differed from the sequences of symbiotic strains in the loss of two repeat units. This finding is consistent with reports from other studies (West & Adams, 1997; Oksanen et al., 2002). Bipartite and tripartite lichens did not show differences in cyanobiont selectivity.

We suggest that the low photobiont specificity of Antarctic lichens may be due to the decreased number of Nostoc strains available, as a result of the extreme Antarctic environmental conditions. Mycobionts would then be able to survive in a lichenized form only by becoming less specific in their choice of cyanobionts. While this clearly introduces variety into the mycobiont–cyanobiont partnerships, it also represents a possible selective pressure on the mycobionts themselves. Only those mycobionts capable of multi-strain cyanobacterial associations will be viable. The decrease in the number of lichen species with increase in latitude might, in part, represent the removal of constitutive single-partner mycobionts.

This may be a general pattern for lichens in Antarctica where the extreme conditions seem to select for flexible mycobionts, a suggestion supported by a study of Romeike et al. (2002) on lichens containing green-algal photobionts. These authors found that Antarctic Umbilicaria spp. were capable of forming symbioses with an astonishing number of different Trebouxia strains.

It is possible that the contrast in results between our study on Antarctic taxa and those made with temperate species may be due to the different species involved rather than to any different behavior of Antarctic and temperate lichens. Such an interpretation, however, appears to be unlikely since the taxa included in our study belong to a wide variety of phylogenetically unrelated families such as Agyriaceae, Collemataceae or Pannariaceae and, furthermore, our results agree well with those obtained by Romeike et al. (2002) on Antarctic lichens associated with green algal photobionts.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Maria-Ines Messuti for kindly providing lichen material from Argentina and Paul Broady for providing Nostoc cultures from Antarctica and New Zealand. Greg Mueller and Imke Schmitt is thanked for helpful comments on the manuscript. We are very grateful to the Ministerio de Ciencia y Tecnología in Madrid for the invitation and stay at the Base Antártica Española (BAE) Juan Carlos I on Livingston Island (ANT 99-0680-C01). This study was supported by a grant from the Deutsche Forschungsgemeinschaft. T. G. A. Green thanks Professor Bryan Gould, Vice-Chancellor of Waikato University for a special grant supporting research in Antarctica and Antarctica New Zealand for assistance with clothing.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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