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GROUP I INTRON VERSUS ITS SEQUENCES IN PHYLOGENY OF CETRARIOID LICHENS
Lichenologist 31(5): 441–449 (1999) Article No. lich.1999.0231 Available online at http://www.idealibrary.com on
GROUP I INTRON VERSUS ITS SEQUENCES IN PHYLOGENY OF CETRARIOID LICHENS Arne THELL*
Abstract: Phylogenetic trees based on group I intron sequences and on internal transcribed spacer (ITS) sequences of mycobiont ribosomal genes were calculated and compared. Eight cetrarioid and four non-cetrarioid species of the Parmeliaceae were compared. The phylogeny based on group I intron sequences is partly congruent with the ITS sequence phylogeny. Group I intron sequences are presumably less informative for infragenic studies. The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. All introns analysed are located between the positions 1516 and 1517 of the fungal 18S ribosomal RNA gene. Cetrarioid lichens form a non-homogeneous group within the Parmeliaceae according to both group I intron and ITS sequences. 1999 The British Lichen Society
Introduction Sequences from the internal transcribed spacers (ITS) of the nuclear rDNA have recently been used in the phylogeny of cetrarioid lichens (Mattsson & Wedin 1998; Thell 1998; Thell & Miao 1998; Thell et al. 1998). The ITS sequences give a good picture of the evolution of closely related species and genera. However, the status of those phylogenetic trees must be questioned until further taxa and other regions of the DNA are investigated to assess the robustness of phylogenetic hypotheses based on ITS data. Phylogenetic analyses of sequence data from more than one gene help to support systematic positions of taxa (Lutzoni 1997). In the present study, phylogenetic trees obtained from ITS and group I introns are compared. Group I introns were detected in lichens by DePriest & Been (1992) for the first time. Such insertions were observed in about one third of all sequenced lichens in the Parmeliaceae within this and a previous study (Thell & Miao 1998). The insertion sequence of a Cetraria ericetorum subsp. ericetorum sample was used for comparison in a public database, and was recognized as a group I intron. This sequence could be aligned with and folded to closely resemble a group I intron from Cladonia grayi (Altschul et al. 1997; DePriest & Been 1992) Only group I introns located between the positions 1516 and 1517 of the 18S gene of the nuclear rDNA with reference to Escherichia coli are considered in this study (Gargas et al. 1995; Gutell et al. 1994). Introns at this site are *Department of Systematic Botany, Lund University, Ö. Vallgatan 18–20, 5–223 61 Lund, Sweden. 0024–2829/99/050441+09 r30.00/0
1999 The British Lichen Society
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Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Parmelia saxatilis 74 Tuckermannopsis subalpina 109 Cetraria arenaria 173 Cetraria islandica 41 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica 93 Tuckermannopsis americana 148 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Vulpicida pinastri 02 Vulpicida pinastri 391 Tuckermannopsis platyphylla 43 Dactylina arctica 160 Melanelia hepatizon 77 Melanelia hepatizon 183 and 224 F. 1. Group I intron sequence phylogeny. Strict consensus tree based on four most parsimonious trees using the heuristic search option and the tree bisection-reconnection method in PAUP 4·0. Tree length=299, RI=0·690 and CI=0·642. The DNA ID-numbers are indicated.
clearly more frequent in some genera, for example, in Cetraria Ach. and Tuckermannopsis Gyelnik, but have so far not or very rarely been found in the genera Cetrariella Kärnefelt & A. Thell (unpublished data), Flavocetraria Kärnefelt & A. Thell and Platismatia W. L. Culb. & C. F. Culb. (Thell et al. 1998; Thell & Miao 1998). However, introns may reside elsewhere in the genome and be transposed to a previously intronless position. There may be informative patterns useful for systematics if the analysis is restricted to one certain position of the genome. The aim of this study is to compare phylogeny based on group I intron and ITS sequences (Figs 1–2). A combined group I intron and ITS data set is also evaluated and compared with the two separate trees (Fig. 3). Among the taxa analysed, Cavernularia lophyrea, Dactylina arctica subsp. beringica, Hypogymnia physodes and Parmelia saxatilis are non-cetrarioid entities. However, the two former species were supposed to have relationships with different cetrarioid taxa (Mattsson et al. unpublished data; Randlane
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Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Parmelia saxatilis 74 Melanelia hepatizon 183 Melanelia hepatizon 77 Melanelia hepatizon 224 Dactylina arctica 160 Tuckermannopsis platyphylla 43 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Tuckermannopsis americana 148 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Vulpicida pinastri 391 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria arenaria 173 Cetraria islandica 41 Cetraria islandica 93 F. 2. ITS sequence phylogeny. Strict consensus tree based on six most parsimonious trees using the heuristic search option and the tree bisection-reconnection method in PAUP 4·0. Tree length=304, RI=0·722 and CI=0·681. The DNA ID-numbers are indicated.
et al. 1997). Cavernularia Degel. has an ascus of Tuckermannopsis form (Mattsson et al. unpublished data). A relationship between the genera Dactylina Nyl. and Allocetraria Kurok. & M. J. Lai was proposed by Kärnefelt & Thell (1996). Considering previous studies, ITS sequences appear as good species fingerprints. However, very closely related species, such as Cetraria ericetorum and C. islandica as one example and Platismatia herrei and P. stenophylla as a second example, were weakly separated or not separated as distinct species (Thell & Miao 1998; Thell et al. 1998). The phylogenetic hypothesis based on ITS sequences suggests that ascus characters cannot be used for distinguishing genera within cetrarioid lichens. The phylogeny based on ITS data of the taxa presented here have been discussed in earlier papers but ITS sequences from some of the populations are presented for the first time in this study.
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Parmelia saxatilis 74 Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Dactylina arctica 160 Melanelia hepatizon 183 Melanelia hepatizon 77 Melanelia hepatizon 224 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Vulpicida pinastri 391 Cetraria arenaria 173 Cetraria islandica 41 Cetraria islandica 93 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 F. 3. Strict consensus tree calculated from three most parsimonious trees, based on a combined matrix of both group I intron and ITS sequences. The heuristic search option and the tree bisection-reconnection method in PAUP 4·0 were used. Tree length=399, RI=0·666 and CI=0·625. The DNA ID-numbers are indicated.
Materials and Methods Selected material The lichen samples selected for this study are listed in Table 1 and archived in the herbaria LD (The Botanical Museum, Lund University), TDI (TerraGen Diversity Inc.), and UBC. The extracted DNA belongs to two different series. DNA#AT is kept at LD, while DNA#VM is kept at TDI. Aligned sequences of the group I introns are presented in Table 2. Melanelia hepatizon DNA#AT183, DNA#AT224 and Vulpicida pinastri DNA#VM391 are excluded from Table 1, because almost no infraspecific variation exists. These three sequences were recently published (Thell & Miao 1998). ITS sequences from the same samples used for the group I intron sequences were compared in both a separate and a combined analysis. The alignments used in these analyses are not presented here but available from the author. A small number of earlier published sequences were added to complement the present study (Mattsson et al. unpublished data; Thell 1998; Thell & Miao 1998). All sequences used here are available in a public database at The National Center for Biotechnology Information, NCBI, homepage: http:// www.ncbi.nlm.nih.gov.
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T 1. Samples selected for this study with group I intron or ITS sequences not published earlier Taxon Ahtiana sphaerosporella A. sphaerosporella Cavernularia lophyrea C. lophyrea Cetraria arenaria Dactylina arctica subsp. beringica Hypogymnia physodes H. physodes Melanelia hepatizon Parmelia saxatilis Tuckermannopsis americana T. americana T. platyphylla T. subalpina
Country, Province
DNA#
GenBank‡
BC-9672 (LD) TDI#211 TDI#216 TDI#219 TDI#220
AT73 AT83 AT155 AT164 AT173
AF141859 AF072224 AF141367 AF115757 AF115758
Canada, Alberta Canada, British Columbia Sweden, Scania Canada, British Columbia Canada, British Columbia
TDI#300 BC-96357 (LD) SK-9605 (LD) BC-9677 (LD) 1996, Miao (TDI)
AT160 AT357 AT16 AT77 VM74
AF115760 AF115761 AF141368 AF141369 AF141370
Canada, Canada, Canada, Canada,
TDI#210 TG-961350 (UBC) BC-9643 (LD) BC-9606 (LD)
AT82 AT148 AT43 AT109
AF072232 AF072233 AF072235 AF072237
Canada, Canada, Canada, Canada, Canada,
British Columbia British Columbia British Columbia British Columbia Ontario
British British British British
Columbia Columbia Columbia Columbia
Sample-ID*
* LD =The Botanical Museum, Lund University, TDI =TerraGen Diversity Inc., UBC =University of British Columbia. ‡ homepage, GenBank Accession Numbers: http://www.ncbi.nlm.nih.gov.
Extraction, amplification and sequencing DNA from the AT# series was extracted from 15–25 g dry weight of the lichen, using a hexadecyl trimethylammonium bromide, CTAB, detergent buffer followed by chloroform separation and isopropanol precipitation (Thell et al. 1998). DNA of the VM# series was obtained from a standard method used for filamentous fungi including lysis in a sodium lauroyl sarcosine solution and phenol/chloroform extraction (Miao et al. 1991). The ITS region was amplified from genomic DNA by the polymerase chain reaction (PCR) using the primers ITS5 and ITS4 and standard PCR chemicals from Perkin Elmer. PCR products were electrophoretically purified through agarose twice, and sequenced with the primers ITS5, ITS4, ITS3 and ITS2 (White et al. 1990) using an automatic sequencer, ABI Prism 377 from Perkin Elmer. The group I intron sequences were obtained combining ITS5, ITS2 and partly ITS4. A denaturation temperature of 94C for 1 min, an annealing temperature of 48C for 1 min, and an extension temperature of 72C for 45 s was used in a 30-cycle PCR. A higher annealing temperature, 53C, was used for the reamplification of the ITS, using a 25-cycle PCR programme (Thell et al. 1998). Finally, a 25-cycle sequencing PCR with a denaturation temperature of 96C for 10 s, an annealing temperature of 50C for 5 s, and an extension time of 60C for 4 min was performed to amplify the DNA fragments prior to the sequencing procedure. This DNA was cleaned through CENTRI-SEP columns from Princeton Separations, Inc. and sequenced with an ABI Prism 377 DNA automatic sequencer (Perkin Elmer).
Alignment and phylogenetic analysis A clustal alignment of selected sequences was made in Sequence Navigator with the following preferences: gap penalty 10, gap extend penalty 10, match penalty 10, and help-match penalty 10. The sequences were slightly adjusted by hand and transferred to PAUP 4·0 using the heuristic search option (Swofford 1993). Exclusively minimal trees were saved, received by stepwise addition, 100 random replicates, using the tree bisection-reconnection (TBR) method of swapping branches. Strict concensus trees from the group I intron matrix, the ITS matrix, and a combined group I intron-ITS analysis were calculated (Figs 1–3).
T 2. Group I intron sequences from 12 species of the Parmeliaceae* 5
10
20
Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82
GAACG----...G.ACGGA ...T...... ...T...... ...T...... ...T...... ...T...... ..-....... -----..GGC .......... -.G.-....A ..........
--GTTTGCCT AG...C.... .......... .G........ .G........ .G........ .G........ .....-.... AA........ .......... A......... ..........
Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212
.......... .......... .......... .......... 70 TCGCCC---...T..CAAA .......... ..........
Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41
.......... ..........
.......... .......... .......... .......... 80 CTACGGGGCT TA.TT....C TG.AC..... TG.AC..... * TG.AC....C TG.AC.....
Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82
.......... .......... ..TA..CCTT .......... ...-...TGA ..........
Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02
.......... .......... .......... .......... 130 GGG-TCGACC ...GA..... ...G..A... ...G..A... * ...G...... ...G..A...
Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02
* Infraspecific variation is indicated by stars.
...G..A... ...TC.A..T A..GC.A... ...TC.A..T A......... ...GC.A.T. ** ...T..A.T. ...T..A..T ...G..A... ...GC.A... 190 CTTCG-GTGG ....TC.... .......... .......... * ....C..... .......... * ....C..... .A.-.C.... .AAT-..... .A...C.... ...TTC.... .......... * *** * -.CG.C.... ..A-.A.... ...-.T.... .G.T..AC..
TG.AC..... TAG.T..... ACCA.....G TAG.T...T. AATAC....A TC.T...... ** * T.GTA..... TC..C...... T..TT...... ...T....CA 140 AGCAGCTCCG ........-.........A .........A
30
40
50
60
GCTGGATGCC A...A.GA C.G.A.C... C.G.A.C... C.G.A.C... C.G.A.C... C.G.A.C... A..A..A..T ATC.A.A... A..A..A..T A.C.-.-... ........... * A......... AT.A..A... C.G...A... .......... 90 GGCAACGCCA ........T. .........G .........G
TCCGCAGCGA A.T....... C.A....... C.A....... C.A....... C.A....... C.A....... CT........ CT........ CT........ .AG....... ........... * C......... .T.......... C.-....... .TG....... 100 TCAGTCTGCG .......... ...C.G.... ...C.G....
CTCTAAAAAA .......G.. .......C.. .......C... .......C.. .......C.. .......C.. .......G.. .......T.. .......G.. .C.....T.. .......... * .......G.. .......G.. .......G.. .......... 110 CCGGGA-GAC .T.......C. ....A.A.T. ....-.A.T.
GTGCATCAAG C...TATT.. C.....T-.. C.....T-.. C.....T-.. C.....T-.. C.....T-.. A.......... C...-CCG.. A.....C-.. C...-CTT.. ......C-.. * T.....C-.. C...G.C-.. C..TG.T-.. -.....C-.. 120 CCC----TTA T.TCGTACCC ...CGAAC.G ...TGAAC.C
..........G ..........G
...C.G.... ...C.G....
.........G .......... ........T. .......... ........T. .......... .......... .......... .......... ........T. 150 -GA---GTCC .--TTCA-T. ..CTTT-.TT .CTTT-.TT
...C.G.... ...A...... .......... ...A...... .T.A...... .......... * ........T. ...A..... .......... .T........ 160 CGGAGTCCA TA....T... -.....T... -.....T...
....A-.T. ....A.A.T. * ....-.A.T. .T....A... .T..A.GCTC .T....A... .T..-.A... .T....G.T. * * .T..-.A.T. .T....-.TA ....-.A.T. .T....A--C 170 AGATCAAACG ..........
...TGAAC.C ...CGAAC.C ** ...T-AAC.C ..-.GAA... .T.TCGTCAG ..-.GAA... .TTT..TCAT ..-.GAA..C * * * ....GAA-.. ....GAA..G ...CGAAC.G ...CGAACA. 180 ATAGCGGCCA .CG.......
..........
..........
.........A G........A * * ........TA G........A .......T.A G........A ..A....T.A G......T.G * * G........A G........A .........A G......... 200 TT-GAGATAT .C.T...... .......... ..........
..CTTT-.TT ..CTT.-.TT
-.....T... -.....T...
.......... ..........
.......... ..........
..CTT.-.TT A-.GTC..TT G.GAGC-C.. A-.GTC..TT ...GA.CCT. C..GCTACT. * **** A..ATC.CT. -..ATC...T ---G.C..T. CGAGT.ACT. 210 GATCGGCCTT ..C.....CC ..A.....CC ..A.....CC
-.....T... -.....T... T......... T.....T... T.....T... .......... * * T..... T... T.....T... T.....T... C.....T... 220 GGGCGATGAC AAC..C.A.G T.CT.T.ACT T.CT.T.AC.
.......... .......... .......... .......... .......... .......... * ..G....... .......... .......... .......... 230 CCGCCCAAGA .G..TGGC.. A-..TGG... A-..TGG...
.......... .......... .......... .......... ..G....... .......... * .........G .......... .......... ....T....G 3′ A---TCCGCG .ATT.TT... C.....TT... C....TT...
.......... ..........
..A.....CC ..A.....CC
T.CT.T.AC. T.CT.T.AC.
A-..TGG... A-..TGG...
C....TT... C....TT...
.......... ..C....C.. ...T...... ..C....C.. ...A...... ..........
..A.....CC .........C ..C.....CC .........C .........C ........C * ........CC .........C .........C ........C.
T.CT.T.AC. ..CT.CCATT AAC..C.A.G ..CT.CCATT T.C-.C..T. ..--.CGACT * ** ***** T.CT.T..T. ..CT-T.C.T ..CTA-.... ..CT.T..T.
A-..TGG... A-...-G...G.ATGG... A-...-G....-.TAGCC. AT-..GCCCC ** ****** A-..TTGT.G TA...-.... A.....GG.. A-..TAGG.G
C....TT... CATT.G.... AATT.TT... CA...TT... GAGATTA--. .AGAATT... **** .....TT... ....----.. -..T.TT... -....TT...
.......... ..C....C.. .......... ..........
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The three parmelioid taxa, Cavernularia lophyrea, H. physodes and Parmelia saxatilis, were selected as outgroup in the cladistic analyses. These species were in an earlier study and in a recent preliminary extended analysis exposed as rather closely related and, furthermore, not very closely related to the cetrarioid group (Mattson & Wedin 1998).
Results and Discussion Infraspecific variation The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. The infraspecific variation of group I introns is generally very small, with one striking example – Tuckermannopsis americana. Introns were observed in both of the two samples investigated. They show dissimilarities to an unexpectedly large extent from each other, being different at 51 positions (Table 2). A comparison between the ITS sequences from the same samples unveils differences at eight positions only (Fig. 2). Cetraria ericetorum, C. islandica, M. hepatizon and V. pinastri are taxa with a slight infraspecific variation. The group I intron sequences of the Cetraria species was recently discussed by Thell & Miao (1998). New material from British Columbia of M. hepatizon was sequenced as a complement to two earlier, identical, sequences, originating from different continents (Thell & Miao 1998). The new one (DNA#AT77) differs from those only at one position, resulting in a length of 219 nucleotides (Table 1). For V. pinastri only one intron, 217 base-pairs long, is included here. This one, from southern Sweden, was compared with a second sample, collected in British Columbia, published by Thell & Miao (1998). These sequences were different at one position as well. Hypogymnia physodes, Cavernularia lophyrea and Ahtiana sphaerosporella show no infraspecific variation of the intron sequences (Table 2). Noteworthy, this was observed for the geographically well-separated specimens of H. physodes as well (southern Sweden and British Columbia). Only one of the studied populations of T. platyphylla contained an intron, and for Cetraria arenaria, T. subalpina and Dactylina arctica subsp. beringica only one sample was available for each taxon. Concerning D. arctica subsp. beringica, four additional introns have been found in the 18S gene (Miao pers. comm.). Monophyletic groups Three strict consensus trees are presented here; one from the group I intron matrix (Fig. 1), one originating from the ITS data set (Fig. 2) and, finally, a third one based on a combined matrix with both group I intron and ITS sequences (Fig. 3). The three species representing Cetraria s. str., C. arenaria, C. ericetorum and C. islandica, is the only monophyletic group that is supported both by group I intron and ITS sequences (Figs 1 & 2). Vulpicida pinastri and T. subalpina join Cetraria s.str. to form a monophyletic clade in the ITS tree. Those positions are supported by the tree based on the combined matrix (Figs 2 & 3). Further monophyletic groups are recognized when comparing at least two different consensus trees. Except for Cetraria s. str, a group composed of
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D. arctica, M. hepatizon and T. platyphylla is observed when comparing the group I intron tree with the combined tree (Figs 1 & 3). Tuckermannopsis americana forms a clade together with Ahtiana sphaerosporella in the ITS analysis, which is supported by the phylogeny obtained from the combined matrix (Figs 2 & 3). The heterogeneous nature of the genus Tuckermannopsis was observed in a separate study (Thell 1998). Both trees, calculated from the ITS and the combined matrices, gather H. physodes and Cavernularia lophyrea on a separate branch (Fig. 2). This is supported by morphology, anatomy and secondary chemistry, although Cavernularia differs from Hypogymnia in producing cavernulae in the lower cortex and in having smaller, more spherical ascospores (Goward 1986). The asci of the genera Cavernularia and Hypogymnia, however, belong to different groups (Mattsson et al. unpublished data; Thell et al. 1995). In previous phylogenetic studies based on ITS sequences, the genus Vulpicida is close to Cetraria s. str., a position that is presumably more likely, supported by six samples from two additional species, V. canadensis and V. juniperinus (Mattsson & Wedin 1998; Thell & Miao 1998). However, the ‘odd’ position of V. pinastri in the intron tree is supported by two almost identical sequences with European and North American origins. Thus, the most striking incongruities between group I intron and ITS phylogeny in this study concern the positions of M. hepatizon and V. pinastri and the separation of the T. americana populations in the intron tree (Figs 1 & 2). Transpositions of introns Only group I introns situated between positions 1516 and 1517 at the small subunit of the rDNA were considered here. The differences between phylogenies of ITS and group I introns, first of all the different positions of V. pinastri, represented by two populations from different continents, and the split of the T. americana samples, both from British Columbia, in the intron analysis, arouse suspicion that introns might have been transposed to position 1516 but originated elsewhere in the rDNA. The different taxonomic positions of V. pinastri, comparing the group I intron and ITS phylogenies could be explained if such transpositions occurred early in the evolution of cetrarioid lichens. Studying further intron locations in the small subunit may answer if such transpositions really occur. Group I introns should, according to this very limited and preliminary study, be less reliable than ITS at the infrageneric level. I would like to express my sincere thanks to Drs Ingvar Kärnefelt and Vivian Miao for comments on and improvements to the manuscript. Dr Ulf Swensson is thanked for generous help with the phylogenetic analyses. The project was financed by European Commission, Training and Mobility of Researchers (TMR) ‘Large Scale Facility’ Contract numbers ERBCHGECT940065 and ERBFMGECT980118 between the EU and the University of Helsinki, Department of Ecology and Systematics. My sincere thanks to Prof. Timo Koponen and Dr Johannes Enroth who administered the grant and to M. Sc. Inkeri Ahonen for practical help during the laboratory work. Travel expenses in connection with the first meeting devoted to the progress of molecular lichenology, Graz, Austria, 11–15 August 1998, was covered by Bokelunds Foundation, administered by Lund University.
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R Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389–3402. DePriest, P. T. & Been, M. D. (1992) Numerous group I introns with variable distribution in the ribosomal DNA of a lichen fungus. Journal of Molecular Biology 228: 315–321. Gargas, A., DePriest, P. T. & Taylor, J. W. (1995) Positions of multiple insertions in SSU rDNA of lichen-forming fungi. Molecular Biology and Evolution 12: 208–218. Goward, T. (1986) Brodoa, a new lichen genus in the Parmeliaceae. Bryologist 89: 219–233. Gutell, R. R., Larsen, N. & Woese, C. R. (1994) Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiology Reviews 58: 10–26. Kärnefelt, I. & Thell, A. (1996) A new classification for the Dactylina/Dufourea complex. Nova Hedwigia 91: 595–605. Lutzoni, F. (1997) Phylogeny of lichen- and non-lichen forming comphalinoid mushrooms and the utility of testing for compatibility among multiple data sets. Systematic Biology 46: 373–406. Mattsson, J.-E. & Wedin, M. (1998) Phylogeny of the Parmeliaceae – DNA data vs. morphological data. Lichenologist 30: 463–472. Mattsson, J.-E., Thell, A. & Wedin, M. (1999) The importance of ascus structures when identifying monophyletic groups within the Parmeliaceae – an evaluation based on DNA data (in prep.). Miao, V., Matthews, D. E. & VanEtten, H. D. (1991) Identification and chromosomal location of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca. Molecular and General Genetics 226: 24–223. Randlane, T., Saag, A. & Thell, A. (1997) A second updated world list of cetrarioid lichens. Bryologist 100: 109–122. Swofford, D. L. (1993) PAUP: Phylogenetic Analysis Using Parsimony, 3·1·1. Champaign: Illinois Natural History Survey. Thell, A. (1995) A new position of the Cetraria commixta group in Melanelia (Ascomycotina, Parmeliaceae). Nova Hedwigia 60: 407–422. Thell, A. (1998) Phylogenetic relationships of some cetrarioid species in British Columbia with notes on Tuckermannopsis. Folia Cryptogamica Estonica 32: 113–122. Thell, A. & Miao, V. (1998) Phylogenetic analysis of ITS and group I intron sequences from European and North American samples of cetrarioid lichens. Annales Botanici Fennici 35: 275–286. Thell, A., Mattsson, J.-E. & Kärnefelt, I. (1995) Lecanoralean ascus types in the lichenized families Alectoriaceae and Parmeliaceae. Cryptogamic Botany 5: 120–127. Thell, A., Berbee, M. & Miao, V. (1998) Phylogeny of the genus Platismatia based on rDNA ITS sequences (Lichenized Ascomycotina). Cryptogamie, Bryologie Lichénologie 19: 43–54. White, T. J., Burns, T., Lee, S. & Taylor, J. (1990) Amplification and direct sequencing of fungal ribosomal DNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White, eds): 315–322. San Diego: Academic Press. Accepted for publication 9 May 1999
GROUP I INTRON VERSUS ITS SEQUENCES IN PHYLOGENY OF CETRARIOID LICHENS Arne THELL*
Abstract: Phylogenetic trees based on group I intron sequences and on internal transcribed spacer (ITS) sequences of mycobiont ribosomal genes were calculated and compared. Eight cetrarioid and four non-cetrarioid species of the Parmeliaceae were compared. The phylogeny based on group I intron sequences is partly congruent with the ITS sequence phylogeny. Group I intron sequences are presumably less informative for infragenic studies. The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. All introns analysed are located between the positions 1516 and 1517 of the fungal 18S ribosomal RNA gene. Cetrarioid lichens form a non-homogeneous group within the Parmeliaceae according to both group I intron and ITS sequences. 1999 The British Lichen Society
Introduction Sequences from the internal transcribed spacers (ITS) of the nuclear rDNA have recently been used in the phylogeny of cetrarioid lichens (Mattsson & Wedin 1998; Thell 1998; Thell & Miao 1998; Thell et al. 1998). The ITS sequences give a good picture of the evolution of closely related species and genera. However, the status of those phylogenetic trees must be questioned until further taxa and other regions of the DNA are investigated to assess the robustness of phylogenetic hypotheses based on ITS data. Phylogenetic analyses of sequence data from more than one gene help to support systematic positions of taxa (Lutzoni 1997). In the present study, phylogenetic trees obtained from ITS and group I introns are compared. Group I introns were detected in lichens by DePriest & Been (1992) for the first time. Such insertions were observed in about one third of all sequenced lichens in the Parmeliaceae within this and a previous study (Thell & Miao 1998). The insertion sequence of a Cetraria ericetorum subsp. ericetorum sample was used for comparison in a public database, and was recognized as a group I intron. This sequence could be aligned with and folded to closely resemble a group I intron from Cladonia grayi (Altschul et al. 1997; DePriest & Been 1992) Only group I introns located between the positions 1516 and 1517 of the 18S gene of the nuclear rDNA with reference to Escherichia coli are considered in this study (Gargas et al. 1995; Gutell et al. 1994). Introns at this site are *Department of Systematic Botany, Lund University, Ö. Vallgatan 18–20, 5–223 61 Lund, Sweden. 0024–2829/99/050441+09 r30.00/0
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Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Parmelia saxatilis 74 Tuckermannopsis subalpina 109 Cetraria arenaria 173 Cetraria islandica 41 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica 93 Tuckermannopsis americana 148 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Vulpicida pinastri 02 Vulpicida pinastri 391 Tuckermannopsis platyphylla 43 Dactylina arctica 160 Melanelia hepatizon 77 Melanelia hepatizon 183 and 224 F. 1. Group I intron sequence phylogeny. Strict consensus tree based on four most parsimonious trees using the heuristic search option and the tree bisection-reconnection method in PAUP 4·0. Tree length=299, RI=0·690 and CI=0·642. The DNA ID-numbers are indicated.
clearly more frequent in some genera, for example, in Cetraria Ach. and Tuckermannopsis Gyelnik, but have so far not or very rarely been found in the genera Cetrariella Kärnefelt & A. Thell (unpublished data), Flavocetraria Kärnefelt & A. Thell and Platismatia W. L. Culb. & C. F. Culb. (Thell et al. 1998; Thell & Miao 1998). However, introns may reside elsewhere in the genome and be transposed to a previously intronless position. There may be informative patterns useful for systematics if the analysis is restricted to one certain position of the genome. The aim of this study is to compare phylogeny based on group I intron and ITS sequences (Figs 1–2). A combined group I intron and ITS data set is also evaluated and compared with the two separate trees (Fig. 3). Among the taxa analysed, Cavernularia lophyrea, Dactylina arctica subsp. beringica, Hypogymnia physodes and Parmelia saxatilis are non-cetrarioid entities. However, the two former species were supposed to have relationships with different cetrarioid taxa (Mattsson et al. unpublished data; Randlane
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Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Parmelia saxatilis 74 Melanelia hepatizon 183 Melanelia hepatizon 77 Melanelia hepatizon 224 Dactylina arctica 160 Tuckermannopsis platyphylla 43 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Tuckermannopsis americana 148 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Vulpicida pinastri 391 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria arenaria 173 Cetraria islandica 41 Cetraria islandica 93 F. 2. ITS sequence phylogeny. Strict consensus tree based on six most parsimonious trees using the heuristic search option and the tree bisection-reconnection method in PAUP 4·0. Tree length=304, RI=0·722 and CI=0·681. The DNA ID-numbers are indicated.
et al. 1997). Cavernularia Degel. has an ascus of Tuckermannopsis form (Mattsson et al. unpublished data). A relationship between the genera Dactylina Nyl. and Allocetraria Kurok. & M. J. Lai was proposed by Kärnefelt & Thell (1996). Considering previous studies, ITS sequences appear as good species fingerprints. However, very closely related species, such as Cetraria ericetorum and C. islandica as one example and Platismatia herrei and P. stenophylla as a second example, were weakly separated or not separated as distinct species (Thell & Miao 1998; Thell et al. 1998). The phylogenetic hypothesis based on ITS sequences suggests that ascus characters cannot be used for distinguishing genera within cetrarioid lichens. The phylogeny based on ITS data of the taxa presented here have been discussed in earlier papers but ITS sequences from some of the populations are presented for the first time in this study.
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Parmelia saxatilis 74 Cavernularia lophyrea 155 and 164 Hypogymnia physodes 16 and 357 Ahtiana sphaerosporella 83 Tuckermannopsis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Dactylina arctica 160 Melanelia hepatizon 183 Melanelia hepatizon 77 Melanelia hepatizon 224 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Vulpicida pinastri 391 Cetraria arenaria 173 Cetraria islandica 41 Cetraria islandica 93 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 F. 3. Strict consensus tree calculated from three most parsimonious trees, based on a combined matrix of both group I intron and ITS sequences. The heuristic search option and the tree bisection-reconnection method in PAUP 4·0 were used. Tree length=399, RI=0·666 and CI=0·625. The DNA ID-numbers are indicated.
Materials and Methods Selected material The lichen samples selected for this study are listed in Table 1 and archived in the herbaria LD (The Botanical Museum, Lund University), TDI (TerraGen Diversity Inc.), and UBC. The extracted DNA belongs to two different series. DNA#AT is kept at LD, while DNA#VM is kept at TDI. Aligned sequences of the group I introns are presented in Table 2. Melanelia hepatizon DNA#AT183, DNA#AT224 and Vulpicida pinastri DNA#VM391 are excluded from Table 1, because almost no infraspecific variation exists. These three sequences were recently published (Thell & Miao 1998). ITS sequences from the same samples used for the group I intron sequences were compared in both a separate and a combined analysis. The alignments used in these analyses are not presented here but available from the author. A small number of earlier published sequences were added to complement the present study (Mattsson et al. unpublished data; Thell 1998; Thell & Miao 1998). All sequences used here are available in a public database at The National Center for Biotechnology Information, NCBI, homepage: http:// www.ncbi.nlm.nih.gov.
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T 1. Samples selected for this study with group I intron or ITS sequences not published earlier Taxon Ahtiana sphaerosporella A. sphaerosporella Cavernularia lophyrea C. lophyrea Cetraria arenaria Dactylina arctica subsp. beringica Hypogymnia physodes H. physodes Melanelia hepatizon Parmelia saxatilis Tuckermannopsis americana T. americana T. platyphylla T. subalpina
Country, Province
DNA#
GenBank‡
BC-9672 (LD) TDI#211 TDI#216 TDI#219 TDI#220
AT73 AT83 AT155 AT164 AT173
AF141859 AF072224 AF141367 AF115757 AF115758
Canada, Alberta Canada, British Columbia Sweden, Scania Canada, British Columbia Canada, British Columbia
TDI#300 BC-96357 (LD) SK-9605 (LD) BC-9677 (LD) 1996, Miao (TDI)
AT160 AT357 AT16 AT77 VM74
AF115760 AF115761 AF141368 AF141369 AF141370
Canada, Canada, Canada, Canada,
TDI#210 TG-961350 (UBC) BC-9643 (LD) BC-9606 (LD)
AT82 AT148 AT43 AT109
AF072232 AF072233 AF072235 AF072237
Canada, Canada, Canada, Canada, Canada,
British Columbia British Columbia British Columbia British Columbia Ontario
British British British British
Columbia Columbia Columbia Columbia
Sample-ID*
* LD =The Botanical Museum, Lund University, TDI =TerraGen Diversity Inc., UBC =University of British Columbia. ‡ homepage, GenBank Accession Numbers: http://www.ncbi.nlm.nih.gov.
Extraction, amplification and sequencing DNA from the AT# series was extracted from 15–25 g dry weight of the lichen, using a hexadecyl trimethylammonium bromide, CTAB, detergent buffer followed by chloroform separation and isopropanol precipitation (Thell et al. 1998). DNA of the VM# series was obtained from a standard method used for filamentous fungi including lysis in a sodium lauroyl sarcosine solution and phenol/chloroform extraction (Miao et al. 1991). The ITS region was amplified from genomic DNA by the polymerase chain reaction (PCR) using the primers ITS5 and ITS4 and standard PCR chemicals from Perkin Elmer. PCR products were electrophoretically purified through agarose twice, and sequenced with the primers ITS5, ITS4, ITS3 and ITS2 (White et al. 1990) using an automatic sequencer, ABI Prism 377 from Perkin Elmer. The group I intron sequences were obtained combining ITS5, ITS2 and partly ITS4. A denaturation temperature of 94C for 1 min, an annealing temperature of 48C for 1 min, and an extension temperature of 72C for 45 s was used in a 30-cycle PCR. A higher annealing temperature, 53C, was used for the reamplification of the ITS, using a 25-cycle PCR programme (Thell et al. 1998). Finally, a 25-cycle sequencing PCR with a denaturation temperature of 96C for 10 s, an annealing temperature of 50C for 5 s, and an extension time of 60C for 4 min was performed to amplify the DNA fragments prior to the sequencing procedure. This DNA was cleaned through CENTRI-SEP columns from Princeton Separations, Inc. and sequenced with an ABI Prism 377 DNA automatic sequencer (Perkin Elmer).
Alignment and phylogenetic analysis A clustal alignment of selected sequences was made in Sequence Navigator with the following preferences: gap penalty 10, gap extend penalty 10, match penalty 10, and help-match penalty 10. The sequences were slightly adjusted by hand and transferred to PAUP 4·0 using the heuristic search option (Swofford 1993). Exclusively minimal trees were saved, received by stepwise addition, 100 random replicates, using the tree bisection-reconnection (TBR) method of swapping branches. Strict concensus trees from the group I intron matrix, the ITS matrix, and a combined group I intron-ITS analysis were calculated (Figs 1–3).
T 2. Group I intron sequences from 12 species of the Parmeliaceae* 5
10
20
Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82
GAACG----...G.ACGGA ...T...... ...T...... ...T...... ...T...... ...T...... ..-....... -----..GGC .......... -.G.-....A ..........
--GTTTGCCT AG...C.... .......... .G........ .G........ .G........ .G........ .....-.... AA........ .......... A......... ..........
Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212
.......... .......... .......... .......... 70 TCGCCC---...T..CAAA .......... ..........
Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41
.......... ..........
.......... .......... .......... .......... 80 CTACGGGGCT TA.TT....C TG.AC..... TG.AC..... * TG.AC....C TG.AC.....
Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82
.......... .......... ..TA..CCTT .......... ...-...TGA ..........
Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02
.......... .......... .......... .......... 130 GGG-TCGACC ...GA..... ...G..A... ...G..A... * ...G...... ...G..A...
Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02 Ahtiana sphaerosporella 73/83 Cavernularia lophyrea 155/164 Cetraria arenaria 173 Cetraria ericetorum subsp. ericetorum 212 Cetraria ericetorum subsp. reticulata 44 Cetraria islandica subsp. islandica 41 Cetraria islandica subsp. islandica 93 Dactylina arctica subsp. beringica 160 Hypogymnia physodes 16/357 Melanelia hepatizon 77 Parmelia saxatilis 74 Tuckermannop´sis americana 82 Tuckermannopsis americana 148 Tuckermannopsis platyphylla 43 Tuckermannopsis subalpina 109 Vulpicida pinastri 02
* Infraspecific variation is indicated by stars.
...G..A... ...TC.A..T A..GC.A... ...TC.A..T A......... ...GC.A.T. ** ...T..A.T. ...T..A..T ...G..A... ...GC.A... 190 CTTCG-GTGG ....TC.... .......... .......... * ....C..... .......... * ....C..... .A.-.C.... .AAT-..... .A...C.... ...TTC.... .......... * *** * -.CG.C.... ..A-.A.... ...-.T.... .G.T..AC..
TG.AC..... TAG.T..... ACCA.....G TAG.T...T. AATAC....A TC.T...... ** * T.GTA..... TC..C...... T..TT...... ...T....CA 140 AGCAGCTCCG ........-.........A .........A
30
40
50
60
GCTGGATGCC A...A.GA C.G.A.C... C.G.A.C... C.G.A.C... C.G.A.C... C.G.A.C... A..A..A..T ATC.A.A... A..A..A..T A.C.-.-... ........... * A......... AT.A..A... C.G...A... .......... 90 GGCAACGCCA ........T. .........G .........G
TCCGCAGCGA A.T....... C.A....... C.A....... C.A....... C.A....... C.A....... CT........ CT........ CT........ .AG....... ........... * C......... .T.......... C.-....... .TG....... 100 TCAGTCTGCG .......... ...C.G.... ...C.G....
CTCTAAAAAA .......G.. .......C.. .......C... .......C.. .......C.. .......C.. .......G.. .......T.. .......G.. .C.....T.. .......... * .......G.. .......G.. .......G.. .......... 110 CCGGGA-GAC .T.......C. ....A.A.T. ....-.A.T.
GTGCATCAAG C...TATT.. C.....T-.. C.....T-.. C.....T-.. C.....T-.. C.....T-.. A.......... C...-CCG.. A.....C-.. C...-CTT.. ......C-.. * T.....C-.. C...G.C-.. C..TG.T-.. -.....C-.. 120 CCC----TTA T.TCGTACCC ...CGAAC.G ...TGAAC.C
..........G ..........G
...C.G.... ...C.G....
.........G .......... ........T. .......... ........T. .......... .......... .......... .......... ........T. 150 -GA---GTCC .--TTCA-T. ..CTTT-.TT .CTTT-.TT
...C.G.... ...A...... .......... ...A...... .T.A...... .......... * ........T. ...A..... .......... .T........ 160 CGGAGTCCA TA....T... -.....T... -.....T...
....A-.T. ....A.A.T. * ....-.A.T. .T....A... .T..A.GCTC .T....A... .T..-.A... .T....G.T. * * .T..-.A.T. .T....-.TA ....-.A.T. .T....A--C 170 AGATCAAACG ..........
...TGAAC.C ...CGAAC.C ** ...T-AAC.C ..-.GAA... .T.TCGTCAG ..-.GAA... .TTT..TCAT ..-.GAA..C * * * ....GAA-.. ....GAA..G ...CGAAC.G ...CGAACA. 180 ATAGCGGCCA .CG.......
..........
..........
.........A G........A * * ........TA G........A .......T.A G........A ..A....T.A G......T.G * * G........A G........A .........A G......... 200 TT-GAGATAT .C.T...... .......... ..........
..CTTT-.TT ..CTT.-.TT
-.....T... -.....T...
.......... ..........
.......... ..........
..CTT.-.TT A-.GTC..TT G.GAGC-C.. A-.GTC..TT ...GA.CCT. C..GCTACT. * **** A..ATC.CT. -..ATC...T ---G.C..T. CGAGT.ACT. 210 GATCGGCCTT ..C.....CC ..A.....CC ..A.....CC
-.....T... -.....T... T......... T.....T... T.....T... .......... * * T..... T... T.....T... T.....T... C.....T... 220 GGGCGATGAC AAC..C.A.G T.CT.T.ACT T.CT.T.AC.
.......... .......... .......... .......... .......... .......... * ..G....... .......... .......... .......... 230 CCGCCCAAGA .G..TGGC.. A-..TGG... A-..TGG...
.......... .......... .......... .......... ..G....... .......... * .........G .......... .......... ....T....G 3′ A---TCCGCG .ATT.TT... C.....TT... C....TT...
.......... ..........
..A.....CC ..A.....CC
T.CT.T.AC. T.CT.T.AC.
A-..TGG... A-..TGG...
C....TT... C....TT...
.......... ..C....C.. ...T...... ..C....C.. ...A...... ..........
..A.....CC .........C ..C.....CC .........C .........C ........C * ........CC .........C .........C ........C.
T.CT.T.AC. ..CT.CCATT AAC..C.A.G ..CT.CCATT T.C-.C..T. ..--.CGACT * ** ***** T.CT.T..T. ..CT-T.C.T ..CTA-.... ..CT.T..T.
A-..TGG... A-...-G...G.ATGG... A-...-G....-.TAGCC. AT-..GCCCC ** ****** A-..TTGT.G TA...-.... A.....GG.. A-..TAGG.G
C....TT... CATT.G.... AATT.TT... CA...TT... GAGATTA--. .AGAATT... **** .....TT... ....----.. -..T.TT... -....TT...
.......... ..C....C.. .......... ..........
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The three parmelioid taxa, Cavernularia lophyrea, H. physodes and Parmelia saxatilis, were selected as outgroup in the cladistic analyses. These species were in an earlier study and in a recent preliminary extended analysis exposed as rather closely related and, furthermore, not very closely related to the cetrarioid group (Mattson & Wedin 1998).
Results and Discussion Infraspecific variation The introns have a length of 214–233 nucleotides, and differ at up to 33% of the bases between species. The infraspecific variation of group I introns is generally very small, with one striking example – Tuckermannopsis americana. Introns were observed in both of the two samples investigated. They show dissimilarities to an unexpectedly large extent from each other, being different at 51 positions (Table 2). A comparison between the ITS sequences from the same samples unveils differences at eight positions only (Fig. 2). Cetraria ericetorum, C. islandica, M. hepatizon and V. pinastri are taxa with a slight infraspecific variation. The group I intron sequences of the Cetraria species was recently discussed by Thell & Miao (1998). New material from British Columbia of M. hepatizon was sequenced as a complement to two earlier, identical, sequences, originating from different continents (Thell & Miao 1998). The new one (DNA#AT77) differs from those only at one position, resulting in a length of 219 nucleotides (Table 1). For V. pinastri only one intron, 217 base-pairs long, is included here. This one, from southern Sweden, was compared with a second sample, collected in British Columbia, published by Thell & Miao (1998). These sequences were different at one position as well. Hypogymnia physodes, Cavernularia lophyrea and Ahtiana sphaerosporella show no infraspecific variation of the intron sequences (Table 2). Noteworthy, this was observed for the geographically well-separated specimens of H. physodes as well (southern Sweden and British Columbia). Only one of the studied populations of T. platyphylla contained an intron, and for Cetraria arenaria, T. subalpina and Dactylina arctica subsp. beringica only one sample was available for each taxon. Concerning D. arctica subsp. beringica, four additional introns have been found in the 18S gene (Miao pers. comm.). Monophyletic groups Three strict consensus trees are presented here; one from the group I intron matrix (Fig. 1), one originating from the ITS data set (Fig. 2) and, finally, a third one based on a combined matrix with both group I intron and ITS sequences (Fig. 3). The three species representing Cetraria s. str., C. arenaria, C. ericetorum and C. islandica, is the only monophyletic group that is supported both by group I intron and ITS sequences (Figs 1 & 2). Vulpicida pinastri and T. subalpina join Cetraria s.str. to form a monophyletic clade in the ITS tree. Those positions are supported by the tree based on the combined matrix (Figs 2 & 3). Further monophyletic groups are recognized when comparing at least two different consensus trees. Except for Cetraria s. str, a group composed of
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D. arctica, M. hepatizon and T. platyphylla is observed when comparing the group I intron tree with the combined tree (Figs 1 & 3). Tuckermannopsis americana forms a clade together with Ahtiana sphaerosporella in the ITS analysis, which is supported by the phylogeny obtained from the combined matrix (Figs 2 & 3). The heterogeneous nature of the genus Tuckermannopsis was observed in a separate study (Thell 1998). Both trees, calculated from the ITS and the combined matrices, gather H. physodes and Cavernularia lophyrea on a separate branch (Fig. 2). This is supported by morphology, anatomy and secondary chemistry, although Cavernularia differs from Hypogymnia in producing cavernulae in the lower cortex and in having smaller, more spherical ascospores (Goward 1986). The asci of the genera Cavernularia and Hypogymnia, however, belong to different groups (Mattsson et al. unpublished data; Thell et al. 1995). In previous phylogenetic studies based on ITS sequences, the genus Vulpicida is close to Cetraria s. str., a position that is presumably more likely, supported by six samples from two additional species, V. canadensis and V. juniperinus (Mattsson & Wedin 1998; Thell & Miao 1998). However, the ‘odd’ position of V. pinastri in the intron tree is supported by two almost identical sequences with European and North American origins. Thus, the most striking incongruities between group I intron and ITS phylogeny in this study concern the positions of M. hepatizon and V. pinastri and the separation of the T. americana populations in the intron tree (Figs 1 & 2). Transpositions of introns Only group I introns situated between positions 1516 and 1517 at the small subunit of the rDNA were considered here. The differences between phylogenies of ITS and group I introns, first of all the different positions of V. pinastri, represented by two populations from different continents, and the split of the T. americana samples, both from British Columbia, in the intron analysis, arouse suspicion that introns might have been transposed to position 1516 but originated elsewhere in the rDNA. The different taxonomic positions of V. pinastri, comparing the group I intron and ITS phylogenies could be explained if such transpositions occurred early in the evolution of cetrarioid lichens. Studying further intron locations in the small subunit may answer if such transpositions really occur. Group I introns should, according to this very limited and preliminary study, be less reliable than ITS at the infrageneric level. I would like to express my sincere thanks to Drs Ingvar Kärnefelt and Vivian Miao for comments on and improvements to the manuscript. Dr Ulf Swensson is thanked for generous help with the phylogenetic analyses. The project was financed by European Commission, Training and Mobility of Researchers (TMR) ‘Large Scale Facility’ Contract numbers ERBCHGECT940065 and ERBFMGECT980118 between the EU and the University of Helsinki, Department of Ecology and Systematics. My sincere thanks to Prof. Timo Koponen and Dr Johannes Enroth who administered the grant and to M. Sc. Inkeri Ahonen for practical help during the laboratory work. Travel expenses in connection with the first meeting devoted to the progress of molecular lichenology, Graz, Austria, 11–15 August 1998, was covered by Bokelunds Foundation, administered by Lund University.
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