NEW DNA MARKERS FOR DISCRIMINATION BETWEEN CLOSELY ...

CELLULAR & MOLECULAR BIOLOGY LETTERS

Volume 7, (2002) pp 403 – 416 http://www.cmbl.org.pl Received 1 April 2002 Accepted 10 April 2002

NEW DNA MARKERS FOR DISCRIMINATION BETWEEN CLOSELYRELATED SPECIES AND FOR THE RECONSTRUCTION OF HISTORICAL EVENTS; AN EXAMPLE USING LIVERWORTS ZOFIA SZWEYKOWSKA-KULIŃSKA*, ANDRZEJ PACAK, and KAMILA JANKOWIAK Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Międzychodzka 5, 60-371 Poznań, Poland

Abstract: A survey of fully-sequenced chloroplast genomes revealed that in land plants there are six tRNA genes that have introns. Moreover, the length of a particular tRNA gene intron remains relatively stable across species. However, in algae, the presence of chloroplast tRNA genes containing introns is exceptional. A survey of mitochondrial plant genomes revealed introncontaining tRNA genes are rather rare features, with the exception of tRNASerGCU genes in liverworts and peat-mosses. We isolated and sequenced one mitochondrial and three chloroplast intron-containing tRNA genes and a fragment of the mitochondrial coxIII gene containing the first intron from the following liverwort species: Pellia borealis, Pellia epiphylla-species N, Pellia epiphylla-species S and Porella baueri, Porella cordaeana, Porella platyphylla. We showed that, as in the case of higher plants, the rate of nucleotide substitution is lower in the mitochondrial genome than in the chloroplast genome. Moreover, the comparison of intron nucleotide sequences enabled us to show that in the case of one allopolyploid species, Pellia borealis, organelles were transmitted from one parent species, Pellia epiphylla-species N. In the case of another allopolyploid species, Porella baueri, organelles were also inherited from one parent species, Porella cordaeana. Therefore, organellar inheritance in liverworts seems to be uniparental. It remains clear that analysis of carefully chosen chloroplast and mitochondrial DNA sequences allowed us to reconstruct historical events. Key Words: Liverworts, Allopolyploid, Chloroplast Genome, Mitochondrial Genome, Nucleotide Substitutions * Corresponding author, E-mail: [email protected], Fax: (+4861)8292730

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INTRODUCTION Genetic information in plant cells is present in three different compartments: the nucleus, chloroplast and mitochondrion. The majority of the genes responsible for the development and functioning of the whole cell are found in the nucleus. Compared to their ancestors, chloroplasts and mitochondria, have lost most of their genes and mainly retained those responsible for translation: organellar rRNA and tRNA genes, and some protein genes involved in photosynthesis and respiration [1]. Plant chloroplasts have only a few hundred kilobases of DNA in circular genomes present in multiple copies with about 100 genes. Complete loss of redundant and extraneous genes has been reported on [2]. Plant mitochondria have hundreds to thousands of bp of DNA and relatively few genes (e.g. Arabidopsis thaliana has 367kb but only 57 genes). Their genome is arranged in a circular molecule, the so-called master chromosome, containing various numbers of large repeated sequences. Repeated sequences are recombinationally active, generating a number of circular subgenomic molecules [3]. Current knowledge of the rates and mechanisms of the molecular evolution of plant nuclear, chloroplast and mitochondrial genomes has been obtained from comparative studies of their genes and proteins. It has been shown that in higher plants, mtDNA and cpDNA evolve more slowly in nucleotide sequence than in the nuclear genome (5 times and 2 times, respectively) [4]. Additionally, it has been shown that the mt genome evolves rapidly in structure, undergoing frequent rearrangements [5]. Despite growing evidence concerning the structure, function and evolution of higher plant organellar genomes, we still have relatively little knowledge of these areas as regards the lower plants. Some information can be deduced from the mt and cp genomes of the liverwort Marchantia polymorpha (for which full nucleotide sequences are available) [6, 7]. In this paper, we present data showing that the rate of nucleotide substitutions in the mitochondrial and chloroplast genomes of liverworts is similar to that for higher plants. Furthermore, we show that intron sequences of organellar tRNA genes can be used as markers for discrimination between closely-related species, and that the comparison of intron nucleotide sequences from allopolyploid and parental liverwort species can reveal the mode of organellar inheritance during fertilization. MATERIALS AND METHODS Liverwort in vitro cultures The apical parts of thalli were sterilized in a 0.5% sodium hypochlorite solution for 15 minutes. Sterilized sections of the thalli were put on a special modified medium containing per liter: NH4NO3 – 0.12g; KH2PO4 – 0.7g; MgSO4 x 7H2O – 0.246g; CaCl2 – 0.02g; FeCl3 – 0.03g; sucrose – 10g; agar – 10g. The agar medium was adjusted to pH 5.0 [8]. We also used 1/2 x Murashige and Skoog medium, pH 5.6 [9, 10], for liverwort in vitro culture.


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Total genomic DNA isolation Total DNA was isolated from 100mg of plant tissue from an in vitro culture. Genomic DNA was extracted using the procedure given in the DNeasy Plant Mini Kit (QIAGEN). 1.5 - 3µg of DNA was usually obtained. DNA was also extracted from dried herbarium material of Pellia species using a modified version of the DNeasy Plant Mini Kit (QIAGEN) procedure. The major modification was connected to the method of initial dried material preparation: 20mg of liverwort thalli was ground in a pre-heated mortar (65ºC) until a fine powder was obtained. Further steps of DNA isolation were as recommended in the QIAGEN protocol. The yield of isolation varied around 0.5µg. PCR reactions The PCR mixture contained the following components for 10µl reactions: 7.5ng of total DNA; 0.5µM of primer A; 0.5µM of primer B; 1µl of 10x PCR buffer supplied by QIAGEN; 1mM of spermidine [11]; 200µM of each dNTP; 0.25 unit of Taq DNA polymerase (QIAGEN). PCR was initiated by denaturation at 95ºC for 5 min, followed by 30 cycles: denaturation at 94ºC for 1 min, annealing at 56ºC for 1 min, elongation at 72ºC for 1 min. The reactions ended with elongation at 72ºC for 5 min (MJ Research PTC-200 Peltier Thermal Cycler). Primers used for the PCR reactions were based on the complete mitochondrial genome (accession no. M68929 in the GenBank Database) and the chloroplast genome sequence (accession nos. X04465 and Y00686 in the GenBank Database) from the liverwort M. polymorpha and represent exon fragments of the studied genes. The primers for PCR amplification were as follows: for the intron sequence of the chloroplast tRNALeu UAA gene A primer 5’ GGG GGT ATG GCG AAA TTG G 3’ B primer 5’ TGG GGG TAG AGG GAC TTG 3’ for the intron sequence of the chloroplast tRNAGly UCC gene A primer 5’ CGG GTA CGG GAA TCG AAC 3’ B primer 5’ GCG GGT ATA GTT TAG TGG 3’ for the intron sequence of the chloroplast tRNALys UUU gene A primer 5’ AAC TCA ATG GTA GAG TAC TC 3’ B primer 5’ GGC TCG AAC CCG GAA CTC 3’ for the intron sequence of the mitochondrial tRNASer GCU gene A primer 5’GGA GGT ATG GCT GAG TGG 3’ B primer 5’GAG GAA ATG GGA TTT GAA CC 3’ for the first intron sequence of the mitochondrial coxIII gene A primer 5’GGA GGC GGA ACA CTT CTT TG 3’ B primer 5’GGA CCA CAA ATG TAT GAT GTC 3’ The products of PCR were separated on a 0.8% agarose gel in 1xTBE and purified using a QIAquick Gel Extraction Kit (QIAGEN).

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DNA sequencing PCR products were cloned using the pGEM-T Easy Vector System (Promega). The DNA sequencing of the direct PCR products and PCR products cloned in plasmid was performed using the fmol DNA Cycle Sequencing System (Promega) and the CEQ DTCS-Quick Start Kit and CEQ 2000 XL Sequencer from Beckman Coulter. All sequences were carried out using direct PCR product sequencing and PCR products cloned in the pGEM-T Easy. Two populations of liverworts were always used for sequencing, and an additional 14 populations were analysed using restriction enzymes. Accession numbers cp tRNALeu(UAA) P.borealis-AF 244086 P.epiphylla-species S and P.epiphylla-species N have an identical intron sequence to P.borealis. cp tRNAGly(UCC) P.borealis-AF 240473, P.epiphylla-species S-AF 240161, P.epiphylla-species NAF 217210. cp tRNALys(UUU) P.borealis-AF 238498, P.epiphylla-species S-AF 238497, P.epiphylla-species N-AF 238496. mt tRNASer(GCU) P.borealis-AF 244576, P.epiphylla-species S-AF 242357, P.epiphylla-species N-AF 242358. mt coxIII, intron no 1- P.borealis-AF 441788, P.epiphylla-species S-AF 443198, P.epiphylla-species N-AF 443199. Restriction analysis The PCR products of chosen intron sequences were digested using the following enzymes: HaeIII for a fragment of the mitochondrial coxIII gene; and HgaI for the chloroplast tRNAGlyUCC gene. The digestion reactions were performed according to the protocol recommended by the producer (Boehringer MannheimHaeIII, New England BioLabs-HgaI). The digested products were separated electrophoretically on an 8% polyacrylamide gel or 0.8% agarose gel, stained with ethidium bromide and analyzed under UV light. Software Clustal X (version 1.64b and 1.81) [12] was used for nucleotide sequence alignment. RESULTS The survey of the Marchantia polymorpha chloroplast genome revealed the presence of six tRNA genes which have introns (tRNALeuUAA, tRNALysUUU, tRNAGlyUCC, tRNAIleGAU – two copies, tRNAAlaUGC – two copies and tRNAValUAC). We did an additional inspection of the chloroplast genomes in the


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Tab. 1. Survey of fully sequenced chloroplast genomes for the introns in tRNA genes. The numbers represent the length of introns in tRNA genes in basepairs; ps – pseudogene.

Organism

Astasia longa Euglena gracilis Nephroselmis olivacea Chlorella vulgaris Guillardia theta Cyanidium caldarium Porphyra purpurea Odontella sinensis Mesostigma viride Toxoplasma gondii Marchantia polymorpha Psilotum nudum

Accession number in tRNALeu tRNALys tRNAGly tRNAIle tRNAAla tRNAVal GenBank (UAA) (UUU) (UCC) (GAU) (UGC) (UAC) Database AJ294725 X70810 AF137379 AB001684 AF041468 AF022186

218 -

-

-

-

-

-

U38804

-

-

-

-

-

-

Z67753 AF166114

-

-

-

-

-

-

U87145

188

-

-

-

-

-

X04465

315

2111

593

886

768

533

AP004638

384

2410

719

862

769

541

D17510

488

2501

741

986

774

543

Oryza X15901 sativa Triticum aestivum AB042240 Zea mays X86563

540

2504

678

947

812

597

588 458

2404 2489

676 698

806 949

805 806

598 603

Arabidopsis AP000423 thaliana Epifagus M81884 virginiana Spinacia oleracea AJ400848

512

2559

714

729

801

599

-

-

-

ps. 887

ps. 474

-

304

2496

708

733

819

667

Lotus japonicus

AP002983

570

2627

709

721

806

599

Z00044

503

2526

691

707

709

571

AC093544

353

2487

683

691

816

581

Pinus thunbergii

Nicotiana tabacum Medicago truncatula

GenBank database [13]. The search showed that among algae and in Apicomplexa phylum, plastid genomes do not contain introns in tRNA genes,


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with the exception of Chlorella vulgaris and Toxoplasma gondii, which contain introns in their plastid tRNALeuUAA genes. Starting from Bryophyta subdivision introns were present in all the plant chloroplast genomes in the six tRNA genes mentioned above. According to the available data, the only exception was Epifagus virginiana – a parasitic plant that does not contain introns in its plastid tRNA genes. In this case, a reduction of the whole plastid genome is visible (its length represents a half of the length of the average plastid genome – 70028bp). It still possesses two pseudogenes for tRNAIleGAU and tRNAAlaUGC containing introns (887bp and 474bp respectively). Table 1 summarizes these data. Generally it can be stated that intron content in chloroplast tRNA genes is a rather stable feature of land plant chloroplasts. The survey of mitochondrial plant genomes revealed that tRNA genes containing introns are rather rare. However, one has to remember that only a limited number of complete plant mitochondrial genome sequences are available. Table 2 presents the list of plants with mitochondrial tRNA genes containing introns, while Table 3 presents the list of plants having all mitochondrial tRNA genes without introns. An interesting case is the presence of an intron in mitochondrial tRNASerGCU genes in several species of liverworts (from the genera Marchantia, Pellia and Porella) and at least two species of peat-moss (Sphagnum rubellum, S.subsecundum – unpublished data). However, it is not present in the sequenced mitochondrial genomes of algae and higher plants. It would be interesting to elucidate the history of intron acquisition in tRNASer genes by liverworts and peat-mosses. Tab. 2. List of plants with mitochondrial tRNA genes containing introns. The numbers represent the length of introns in tRNA genes in basepairs.

Organism Chondrus crispus Marchantia polymorpha

Accession number in GenBank Database Z47547 M68929

tRNAIle tRNASer (GAU) (GCU) 474 991

Using PCR and specific primers, we amplified one mitochondrial and several chloroplast tRNA genes containing introns from the following liverwort species: Pellia borealis, Pellia-epiphylla-species S, Pellia epiphylla-species N, Porella baueri, Porella platyphylla and Porella cordaeana. Pellia borealis and P. baueri are allopolyploid species that originated after the hybridization and chromosome set duplication of Pellia epiphylla-species S and Pellia epiphylla-species N [14, 15] and Porella platyphylla and Porella cordaeana [16], respectively. These two triplets of liverwort species represent closely related species – one in the genus Pellia and the other in the genus Porella. We used the following chloroplast tRNA genes: tRNALeu, tRNAGly, tRNALys and mitochondrial tRNASer gene. Since the tRNASer gene is, as mentioned above, the only mitochondrial tRNA gene containing an intron, we also used the first intron in the mitochondrial coxIII


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gene amplified in PCR. Figure 1 represents the electrophoretic separation of PCR products for the liverwort species studied. As shown in Figure 1, the majority of PCR fragments obtained exhibit very similar, if not identical, mobility during electrophoresis. The only exception is seen in the chloroplast tRNALeu gene from Porella platyphylla, Porella cordaeaena and Porella baueri: the DNA fragment amplified from the Porella platyphylla total DNA migrates more slowly, suggesting that its intron sequence is longer than that of the other two species (Figure. 1A). Since Porella platyphylla is morphologically very similar to Porella baueri and proper species recognition is difficult, this sequence can be used as a marker for the discrimination between these two species. Tab. 3. Plants with a complete mitochondrial genome sequence and no intron in the mitochondrial tRNA genes.

Organism

Accession number in GenBank Database

Prototheca wickerhamii Chlamydomonas reinhardtii Rhodomonas salina Arabidopsis thaliana Beta vulgaris

U02970 U03843 AF288090 NC 001284 NC 002511

All PCR products were sequenced, both directly and in plasmids. Sequencing was always done on two different populations of each species. Table 4 presents the intron length in chloroplast tRNA genes for leucine, glycine, lysine and in the mitochondrial tRNASer gene and coxIII gene. Figure 2 shows the scheme of introns studied where nucleotide substitutions, deletions and insertions are depicted. Table 5 shows the comparison of the number of substitutions, deletions and insertions in two compartments: chloroplast and mitochondrion. There is one feature of intron sequences that allows us to reconstruct some details of hybridization events in Pellia borealis and Porella baueri. Intron sequences derived from both genomes – mitochondrial and chloroplast – were identical in Pellia borealis and Pellia epiphylla-species N and different in the case of Pellia epiphylla-species S. It suggested that during hybridization and chromosome duplication between two Pellia epiphylla species, Pellia epiphyllaspecies N was the donor of both types of organelles. We tested fourteen different populations of Pellia borealis using restriction enzymes that specifically digest the PCR products derived from the chloroplast tRNAGly gene and mitochondrial coxIII gene. In all the populations, the chloroplast and mitochondrial restriction patterns were identical to those of Pellia borealis and Pellia epiphylla-species N, suggesting that in all the populations tested Pellia epiphylla-species N

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Fig. 1. 0.8% agarose gel electrophoresis of PCR products for: A) the chloroplast tRNALeuUAA gene; B) the chloroplast tRNAGlyUCC gene; C) the chloroplast tRNALysUUU gene; D) the mitochondrial tRNASerGCU gene; E) the mitochondrial coxIII gene with intron number 1. Analyzed species: 1. – Pellia epiphylla-species S, 2. – Pellia borealis, 3. – Pellia epiphylla-species N; 4. – Porella baueri, 5. – Porella cordaeana, 6. – Porella platyphylla; Used markers: M1 – GeneRuler 100bp DNA Ladder, MBI Fermentas; M2 – GeneRuler 1kb DNA Ladder, MBI Fermentas; M3 – MassRuler DNA Ladder Mix, MBI Fermentas.


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Tab. 4. The length (in bp) of introns in chloroplast tRNA genes for leucine, glycine, lysine and in the mitochondrial tRNA gene for serine and coxIII gene.

The length of intervening sequences (IVS) in nucleotides Organism

Pellia borealis Pellia epiphylla N Pellia epiphylla S Porella baueri Porella cordaeana Porella platyphylla

cp tRNALeuUAA

cp tRNAGlyUCC

cp tRNALysUUU

mt tRNASerGCU

mt no 1 intron in coxIII gene

310

738

2156

1316

1035

310

738

2156

1316

1035

310

741

2152

1315

1036

342

688

~ 2000

919

886

342

688

~ 2000

919

883

369

688

~ 2000

907

883

Tab. 5. The comparison of the number of substitutions, deletions and insertions in two compartments: chloroplast and mitochondrion Pellia epiphylla-species S and Porella platyphylla.

Pellia epiphylla-species S relative to Pellia borealis Intron cp tRNAGlyUCC cp tRNALysUUU mt tRNASerGCU mt no1intron in coxIII gene

Substitution Deletion 6 9 0 1

Insertion

1 1 3 5

2 0 3 6

Porella platyphylla relative to Porella baueri Intron

Substitution

Deletion

Insertion

cp tRNALeuUAA

2

1

2

cp tRNAGlyUCC mt tRNASerGCU

16 0 0

0 2 1

0 0 1

mt no 1intron in coxIII gene

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Fig. 2. Introns of chloroplast and mitochondrial tRNA and mitochondrial coxIII genes studied with marked differences between the following species: A – Pellia borealis/Pellia epiphylla-species N versus Pellia epiphylla-species S and B – Porella baueri/Porella cordaeana versus Porella platyphylla.

transmitted its organelles to Pellia borealis. Figure 3 shows the electrophoretical separation of restriction fragments from PCR products of the chloroplast tRNAGly gene and the mitochondrial coxIII gene. In the allopolyploid species Porella baueri, the intron sequences were identical with those derived from Porella cordaeana and different from those derived from Porella platyphylla. It suggests that Porella cordaeana was the donor of organelles during the hybridization event that gave rise to Porella baueri. DISCUSSION As mentioned earlier, the intron content in chloroplast tRNA genes is a rather stable feature of land plant chloroplasts (Table 1). Our results confirm this


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statement with respect to liverworts, which until now were only represented by data from the sequencing of one liverwort species – M. polymorpha.

Fig. 3A. 0.8% agarose gel electrophoresis of chloroplast tRNAGlyUCC genes digested with HgaI. S – P. epiphylla – species S populations, N – P. epiphylla- species N populations, M.- GeneRuler 100bp DNA Ladder, MBI Fermentas. 3B. 8% polyacrylamide gel electrophoresis of mitochondrial coxIII genes (with intron number 1) digested with HaeIII. N – P. epiphylla- species N populations, S – P. epiphylla – species S populations, M. – GeneRuler 100bp DNA Ladder, MBI Fermentas.

We showed that in the case of other liverworts from the genera Pellia and Porella, which represent quite different groups of liverworts than M.polymorpha, introns are also present in the case of the tRNALeu, tRNALys and tRNAGly genes. It still remains to be elucidated whether introns are present in the chloroplast tRNAIle, tRNAAla, and tRNAVal genes. It has been shown that the rate of nucleotide substitutions in plant chloroplast and mitochondrial genomes is much lower than in the case of nuclear genomes [4]. Our results seem to confirm the lower rate of substitutions when the mitochondrial sequences are compared to chloroplast sequences in liverworts (in the case of Pellia complex there are 15 substitutions in chloroplast genes versus 1 in mitochondrial genes and in the case of Porella complex there are 18 substitutions in chloroplast genes versus 0 in the case of mitochondrial genes) (Table 5). It is important to note that the length of DNA fragments compared was approximately equal. A lot of nucleotide sequence differences were represented by insertions and deletions (Table 5). Probably, the mechanisms leading to nucleotide substitutions and

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insertions/deletions are quite different and can be a subject of independent studies. A frequently-used chloroplast sequence in plant systematic studies is the tRNALeu gene (anticodon UAA) containing a group I intron. This intron, several hundred nucleotides in length, exhibits great variability in its nucleotide sequence when cyanobacteria strains or closely-related plant species are compared [17, 18]. Our results show that in the case of three closely-related species of liverworts in the genus Pellia: P.borealis, P.epiphylla-species N and P.epiphylla-species S, the tRNALeu gene intron sequences were identical and could not be used as molecular markers for the discrimination between these closely related species. However, the comparison of intron nucleotide sequences for all liverwort species from the genus Pellia showed that it is possible to construct a phenetic dendrogram for the genus, based on nucleotide sequence differences in other Pellia species [19]. On the other site, the comparison of the chloroplast tRNALeu gene sequence in three closely related species in the genus Porella allowed us to discriminate between P.baueri/P.cordaeana and P.platyphylla at the level of the PCR product length. This is because of two insertions (20 bp and 8 bp respectively) in the tRNALeu gene intron of P.platyphylla. Sequence analysis of other mitochondrial and chloroplast tRNA gene introns studied showed that in all cases we were able to discriminate between two closely-related species that gave rise to allopolyploid species. Thus we postulate that these intron sequences are even better molecular markers than those derived from the chloroplast tRNALeu intron – frequently used in systematic studies. We strongly recommend the use of these intron sequences for studying taxonomic relationships between closely related species of land plants. To our knowledge, organellar intron-containing tRNA genes, with the exception of the chloroplast tRNALeu gene, have not been used in these types of studies. In the case of allopolyploid species like Pellia borealis and Porella baueri, analysis of chloroplast and mitochondrial tRNA gene intron sequences allowed us to point to the parental species that was the organelle donor during hybridization events. Since both organelles were given by one parent, their transmission was uniparental. It suggests the main avenue of organellar inheritance in liverworts, but more examples should be studied before making such a generalization. It remains clear that analysis of carefully chosen chloroplast and mitochondrial DNA sequences allowed us to reconstruct historical events. Acknowledgements. This work was supported by a Research Grant of the State Committee of Scientific Research (No. 1128/P04/2000/19) and by a research Grant of the Faculty of Biology at the Adam Mickiewicz University, Poznań, Poland (No. PBWB 304/2001).


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16. Boisselier-Dubayle, M.C., Lambourdiere, J. and Bishler, H. The leafy liverwort Porella baueri (Porellaceae) is an allopolyploid. Pl. Syst. Evol. 210 (1998) 175-197. 17. Meissner, K., Frahm, J-P., Stech, M. and Frey, W. Molecular divergence and infrageneric relationship of Monoclea (Monocleales, Hepaticae). Nova Hedwigia 67 (1998) 289-302. 18. Paulsrud, P. and Lindblad, P. Sequence variation of the tRNALeu intron as a marker for genetic diversity and specificity of symbiotic cyanobacteria in some lichens. Appl. Environm. Microbiol. 64 (1998) 310-315. 19. Pacak, A. and Szweykowska-Kulińska, Z. Molecular data concerning alloploid character and the origin of chloroplast and mitochondrial genomes in the liverwort species Pellia borealis. J. Plant Biotechnol. 2 (2000) 101108.