Patterns of photobiont diversity were examined in some Nostoc-containing lichens using the nucleotide sequence of the cyanobacterial tRNA Leu (UAA) intron. Lichen specimens collected in northwestern USA were analysed and the sequence data were compared with tRNA Leu (UAA) intron sequences previously obtained from lichens in northern Europe. Generally, it is the species identity of a lichen rather than the geographical origin of the specimen that determines the identity of the cyanobiont. Identical intron sequences were found in Peltigera membranacea specimens collected in Oregon (USA) and in Sweden, and very similar sequences were also found in Nephroma resupinatum thalli collected in Oregon and Finland. Furthermore, in mixed assemblages where two Peltigera species grew in physical contact with each other, the different lichen species housed different photobiont strains. There is however not a one-to-one relation between mycobiont and photobiont as some intron sequences were found in more than one lichen species, and different intron sequences were found in different samples of some lichen taxa. Peltigera venosa exhibited a higher level of photobiont diversity than any other lichen species studied, and several intron sequences could for the first time be obtained from a single thallus. It is not clear whether this is evidence of lower cyanobiont specificity, or reflects an ability to exhibit different degrees of lichenization with different Nostoc strains. In one specimen of P. venosa, which contained bipartite cyanosymbiodemes and tripartite, cephalodiate thalli, both thallus types contained the same intron sequence.

The cyanobacterial symbionts in some Nostoc-containing lichens were investigated using the nucleotide sequence of the highly variable cyanobacterial tRNA Leu (UAA) intron. When comparing different Nostoc-containing lichens, identical intron sequences were found in different samples of the same lichen species collected from two remote areas. This was true for all species where this comparison was made (Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and Nephroma arcticum (L.) Torss.). With one exception, a specific intron sequence was never found in more than one lichen species. However, for two of the species, Peltigera aphthosa and Nephroma arcticum, two different cyanobionts were found in different samples. By examining a P. aphthosa photosymbiodeme it could be shown that the same Nostoc is present in both bipartite and tripartite lobes of this lichen. It is thus possible for one cyanobiont\Nostoc to form the physiologically different symbioses that are found in bipartite and tripartite lichens. The connection between photobiont identity and secondary chemistry is discussed, as a correlation between differences in secondary chemistry and different cyanobionts\Nostocs in the species Peltigera neopolydactyla (Gyeln.) Gyeln. was observed. It is concluded that more knowledge concerning the photobiont will give us valuable information on many aspects of lichen biology.

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNALeu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNALeu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNALeu (UAA) intron can be of great value when examining cyanobacterial diversity.

The diversity and host specificity of the cyanobionts of several cycad species (Cycas circinalis L., C. rumphii Miq., Encephalartos lebomboensis I. Verd., E. villosus Lem., and Zamia pumila L.) collected in a botanical garden were examined using the tRNALeu(UAA) intron sequence as a genetic marker. Nested PCR was used to specifically amplify the tRNALeu(UAA) intron directly from the freshly isolated symbiotic cyanobionts. By direct amplification of the biological material the laborious isolation of the cyanobionts can be avoided. A single DNA fragment, ranging in size from 287 bp to 329 bp, was consistently amplified from the different biological samples. The intron sequences obtained from the cycad cyanobionts show high similarities to the corresponding sequences in the free‐living strains Nostoc sp. strain PCC 73102 and N. muscorum as well as in several lichen cyanobionts. Although different Nostoc strains were found in the present study, no sequence variation was observed when analyzing a single coralloid root. However, different coralloid roots from a single cycad individual may harbor different cyanobacteria. Moreover, cyanobionts in coralloid roots of both Encephalartos lebomboensis and E. villosus were found to possess the same intron sequence.

Summary• Possibilities of introducing new cyanobacterial strains into established lichen symbioses were examined by manipulating thalli of Peltigera aphthosa and by using the trnL (UAA) intron to identify specific cyanobacterial strains.• P. aphthosa has Nostoc symbionts in cephalodia on the upper surface of the thallus. In three different P. aphthosa populations, each consisting of seven thalli, all cephalodia were experimentally removed. The manipulated lichens were then inoculated with known strains of cultured cyanobacteria and left to develop new cephalodia. After a summer in the field the lichens were harvested and the strain identities of cyanobacterial symbionts in 80 newly formed cephalodia were determined. All epiphytic colonies of free-living cyanobacteria found were also analysed.• Foreign cyanobacteria were not readily incorporated into established P. aphthosa thalli. All newly formed cephalodia contained the same intron sequence, which was identical to that found in the removed cephalodia. At least two inoculated Nostoc strains were able to survive as epiphytic colonies on experimental thalli. Both strains had originally been isolated from bipartite Peltigera species.• Results indicate that associations between cyanobacteria and lichen-forming fungi can be very specific and stable, which contrasts with the general view that cyanobacterial symbioses are rather unspecific.