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MOLECULAR PHYLOGENETICS AND EVOLUTION

Molecular Phylogenetics and Evolution 32 (2004) 246263 www.elsevier.com/locate/ympev

Distribution of introns in the mitochondrial gene nad1 in land plants: phylogenetic and molecular evolutionary implications
Olena Dombrovska and Yin-Long Qiu*
Department of Ecology and Evolutionary Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109-1048, USA Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, 8008 Zurich, Switzerland Received 22 July 2003; revised 17 December 2003 Available online 27 February 2004

Abstract Forty-six species of diverse land plants were investigated by sequencing for their intron content in the mitochondrial gene nad1. A total of seven introns, all belonging to group II, were found, and two were newly discovered in this study. All 13 liverworts examined contain no intron, the same condition as in green algae. Mosses and hornworts, however, share one intron by themselves and another one with vascular plants. These intron distribution patterns are consistent with the hypothesis that liverworts represent the basal-most land plants and that the two introns were gained in the common ancestor of mosseshornwortsvascular plants after liverworts had diverged. Hornworts also possess a unique intron of their own. A fourth intron was found only in Equisetum L., Marattiaceae, Ophioglossum L., Osmunda L., Asplenium L., and Adiantum L., and was likely acquired in their common ancestor, which supports the monophyly of moniliformopses. Three introns that were previously characterized in angiosperms and a few pteridophytes are now all extended to lycopods, and were likely gained in the common ancestor of vascular plants. Phylogenetic analyses of the intron sequences recovered topologies mirroring those of the plants, suggesting that the introns have all been vertically inherited. All seven nad1 group II introns show broad phylogenetic distribution patterns, with the narrowest being in moniliformopses and hornworts, lineages that date back to at least the Devonian (345 million years ago) and Silurian (435 million years ago), respectively. Hence, these introns must have invaded the genes via ancient transpositional events during the early stage of land plant evolution. Potentially heavy RNA editing was observed in nad1 of Haplomitrium Dedecek, Takakia Hatt. & Inoue, hornworts, Isoetes L., Ophioglossum, and Asplenium. A new nomenclature is proposed for group II introns. 2004 Elsevier Inc. All rights reserved.
Keywords: Genomic structural characters; Group II introns; Intron nomenclature; Land plant phylogeny; Mitochondrial DNA; nad1; RNA editing

1. Introduction Evolution of land plants (embryophytes) fundamentally altered the terrestrial ecosystem and set the stage for evolution of other land-dwelling life (Gensel and Edwards, 2001; Graham, 1993; Kenrick and Crane, 1997a). A large number of paleobotanical, morphological, and molecular studies have been devoted to understanding this important event in the history of life on earth. However, our knowledge on radiation of early land plants remains incomplete, and in particular several key questions regarding basal land plant phylogeny are still controversial. These include: what represents the
* Corresponding author. Fax: 1-734-764-0544. E-mail address: ylqiu@umich.edu (Y.-L. Qiu).

rst lineage of land plants, which bryophyte group is sister to vascular plants, and how are several extant pteridophyte lineages related to each other (Edwards et al., 1995; Hedderson et al., 1998; Kenrick and Crane, 1997b; Lewis et al., 1997; Mishler et al., 1994; Nickrent et al., 2000; Nishiyama and Kato, 1999; Pryer et al., 2001; Qiu et al., 1998; Renzaglia et al., 2000; Samigullin et al., 2002; Taylor, 1995; Wellman et al., 2003)? Clearly, more data are needed to resolve these issues. The mitochondrial genome in land plants is a rich source of information to investigate plant phylogeny (Beckert et al., 1999, 2001; Qiu et al., 1998, 1999; Vangerow et al., 1999). Two sets of introns have been found in angiosperms (Kubo et al., 2000; Notsu et al., 2002; Unseld et al., 1997) and the liverwort Marchantia polymorpha (Oda et al., 1992), respectively. Most of the angiosperm

1055-7903/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2003.12.013

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introns are now known to extend their presence to gymnosperms, pteridophytes, and even hornworts, mosses, or liverworts (Beckert et al., 1999, 2001; Gugerli et al., 2001; Hashimoto and Sato, 2001; Malek and Knoop, 1998; Malek et al., 1997; Pruchner et al., 2001, 2002; Qiu et al., 1998). The intron distribution patterns can be used to infer phylogenetic relationships among basal lineages of land plants, given that conservative genomic structural changes have proven to be informative markers in resolving dicult phylogenetic issues (Manhart and Palmer, 1990; Raubeson and Jansen, 1992). The plant mitochondrial gene nad1 contains four group II introns in angiosperms (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Kubo et al., 2000; Notsu et al., 2002; Unseld et al., 1997; Wissinger et al., 1991), but none in Marchantia (Oda et al., 1992) nor in several green algae that have been sequenced for their mitochondrial genomes: Prototheca wickerhamii (Wol et al., 1994), Chlorogonium elongatum (Kroymann and Zetsche, 1998), Nephroselmis olivacea and Pedinomonas ck minor (Turmel et al., 1999), Scenedesmus obliquus (Ku et al., 2000; Nedelcu et al., 2000), Mesostigma viride (Turmel et al., 2002a), Chaetosphaeridium globosum (Turmel et al., 2002b), Chara vulgaris (Turmel et al., 2003), and Chlamydomonas reinhardtii [M.W. Gray, unpublished data (GenBank Accession No. U03843); one group I intron has been reported in Chlamydomonas eugametos nad1 (Denovan-Wright et al., 1998)]. The rst three angiosperm introns have been shown to be present in a few basal pteridophytes (Gugerli et al., 2001; Malek and Knoop, 1998), and the last one is present throughout land plants except liverworts (Qiu et al., 1998). An additional group II intron, located upstream of the rst angiosperm intron, has been found in the moss Ceratodon purpureus (Malek and Knoop, 1998). However, the information on the full intron content in nad1 across land plants, especially in bryophytes, is still lacking. It would be desirable to obtain this information to trace origins of these introns, to test whether they follow vertical inheritance or undergo horizontal transfer, and to examine their frequency of secondary losses. Understanding evolution of these introns will not only ensure their proper use as phylogenetic markers, but also will help us to gain insight into their role in shaping evolution of the mitochondrial genome in land plants. In this study, we carry out a broad survey of intron distribution in nad1 in land plants by sequencing the nearly entire length of the gene, with particularly dense taxon sampling in liverworts, mosses, and hornworts. We aim to uncover phylogenetically informative intron distribution patterns to resolve relationships among basal land plant lineages. We also seek to understand the tempo and mode of intron evolution in mitochondrial genomes of early land plants, as a relatively large body of data is now available from both mitochondrial genome sequencing studies and evolutionary surveys.

2. Materials and methods 2.1. DNA extraction, gene amplication and sequencing, intron secondary structure modeling Materials for 46 representatives of all major nonowering land plant lineages, including 12 liverworts, 19 mosses, four hornworts, two lycopods, eight moniliformopses (sensu Kenrick and Crane, 1997b, including Equisetum, Psilotaceae, and ferns), and one gymnosperm were collected in the eld or greenhouses, carefully identied, and properly vouchered (Table 1). All bryophytes were cleaned and checked under a dissecting scope to avoid contamination. The voucher information and GenBank accession numbers are given in Table 1. The green algae (see Figs. 1 and 2) were used as outgroups for comparing intron content and timing the evolutionary origins of the introns in nad1. Total cellular DNAs were extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987), with 2% polyvinyl pyrrolidone, 0.1% diethyldithiocarbamic acid (sodium salt), and 0.1% ascorbic acid added, or alternatively using the Plant DNAeasy kit (Qiagen) according to the manufacturers protocol. Multiple sets of primers were tested to circumvent any potential problems caused by direct and reverse RNAediting at priming sites, and they are listed in Table 2. Where possible, a single amplicon was recovered for the gene. In some species, where the size of nad1 (including both exons and introns) exceeded the capacity of conventional PCR methods, the gene was amplied and sequenced in several overlapping fragments and the nad1 gene contig was assembled subsequently. PCR amplication reactions contained approximately 10 ng template DNA, 10 mM Tris/HCl (pH 8.85), 25 mM KCl, 5 mM (NH4 )2 SO4 , 2 mM MgSO4 , 200 lM each dNTP, 0.20 lg each primer, and 2.0 U Taq DNA polymerase (Qiagen) in a nal volume of 50 ll. The reactions were carried out using 30 cycles, each consisting of 30 s denaturation at 94 C, 30 s annealing at 52 C, and 14 min extension at 72 C. The rst cycle was preceded by an initial denaturation step (4 min, 94 C), and the last one was followed by a 12 min extension step at 72 C. Alternatively, for amplifying fragments larger than 3.5 kb, the Expand Long Template PCR-System (BoehringerMannheim) was used, following the protocol supplied by the manufacturer. PCR fragments, depending on their size, were cloned with TA-TOPO or XL-TOPO cloning kits (Invitrogen). Positive clones were sequenced in both directions using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kits on an ABI 377 sequencer (both from Applied Biosystems). M13 universal primers and those designed to match internal sequences of cloned fragments (Table 2) were used during sequencing. Sequence processing and contig assembly were done using the Sequencher program (Gene Codes Corporation).

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Table 1 Plant material used in this study, intron presence (+) or absence ()) in the mitochondrial gene nad1, and GenBank accession number of the sequences Species Vouchera Accession No. nad1, one accession # Gymnosperms Cycas revoluta Thunb. Moniliformopses Asplenium nidus L. Adiantum sp. Osmunda regalis L. Ophioglossum lusitanicum L. Marattia attenuata Lab. Angiopteris evecta (G.Forst.) Hom. Equisetum arvense L. Psilotum nudum (L.) P.Beauv. Lycopods Isoetes sp. Huperzia lucidula (Michx.) Trevis. Hornworts Notothylas breutelii (Gottsche) Gottsche Phaeoceros carolinianus (Michx.) Prosk. Megaceros tosanus Steph. Anthoceros agrestis Paton Qiu94051 Qiu94081 AY354954 Qiu95118 AY354953 Qiu96171 Qiu98097 Qiu96169 Qiu96170 Qiu94003 AY354940 Qiu94002 + + ) ) ) AY354943 ) AY354948 ) AY354947 ) ) ) ) AY354943 ) AY354948 ) AY354947 ) + + nad1i258 nad1i287 nad1i 348 nad1i394 nad1i477 nad1i669 nad1i728

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+ AY354955 ) ) + AY354950 + AY354943 AY354944 + AY354948 + AY354947 ) ) ) + AY354950 AY354951 + + + AY354951 AY354952 + AY354945 + AY354949 + AY354946 + + AY354941 AY354942

+AY354943 +AY354948 +AY354947 +

) AY354943 +AY354948 +AY354947 +

+ + AY354941

Qiu96272 AY354939 Qiu94173 AY354938 Qiu97078 Qiu97002 Qiu97001 Qiu99112 AY354981 AY354980 AY354979 AY354978

) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

) ) + + + + + + + + + + + + + + + + +

) ) + + + + ) ) ) ) ) ) ) ) ) ) ) ) ) )

+ + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

) + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

) + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

+ + + + + + + + + + + + + + + + + + +

Mosses Hypnum cupressiforme Hedw. Qiu99079 AY354976 Brachythecium rutabulum (Hedw.) BSG. Qiu94071 AY354975 Thuidium recognitum (Hedw.) Lindb. Qiu94174 AY354974 Anomodon viticulosus (Hedw.) Hook. & Tayl. Qiu99066 AY354973 Hedwigia ciliata (Hedw.) P.Beauv. Qiu99056 AY354972 Rhizogonium paramattense (Mull. Hal.) Qiu98069 AY354971 Reichardt Bartramia halleriana Hedw. Leucobryum albidum (Brid.) Lindb. Dicranum scoparium Hedw. Mnium sp. Gymnostomum rucurvirostrum Hedw. Fissidens dubius P.Beauv. Tetraphis pellucida Hedw. Polytrichum juniperum Hedw. Qiu99062 Qiu94075 Qiu94073 Qiu94097 Qiu94072 Qiu99069 Qiu00001 Qiu94176 AY354970 AY354969 AY354968 AY354935 AY354936 AY354967 AY354956 AY354966

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a The vouchers with numbers between Qiu94001 and 97999 are deposited at Indiana University Herbarium (IND, Bloomington, IN, USA), and those with numbers between Qiu98001 and 00999 at University of Zurich Herbarium (Z, Zurich, Switzerland).

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Sequence alignment was performed using ClustalX program (Thompson et al., 1997). Secondary structure models of group II introns were inferred according to the consensus model of Michel et al. (1989) on Foldalign server (Mathews et al., 1999; Zuker et al., 1999). Manual adjustments were made after computer-assisted folding analysis. 2.2. Group II intron nomenclature Because there is a great deal of confusion on intron names in literature due to lack of a standard nomenclature, we develop a system here for group II introns that is similar to what has been proposed for group I introns in ribosomal DNA (Johansen and Haugen, 2001), which has been adopted for archael introns (Nakayama et al., 2003). The intron name in our system will be composed of: (1) gene name, (2) the letter i for intron, and (3) insertion site in the orthologous gene of M. polymorpha, and it will always be italicized. For example, nad1i728 refers to an intron located in the gene nad1 of a given species at the M. polymorpha nucleotide position 728 (an alignment of the exon sequence of the species and that of M. polymorpha will be necessary to identify the intron insertion site). Compared to the group I intron nomenclature, our system has omitted use of the species name acronym and the genome (chloroplast, mitochondrial, or nuclear) designation letter as part of the formal intron name. Instead, we recommend that all that information as well as other information such as trans-splicing (Malek and Knoop, 1998), presence of ORF (Zimmerly et al., 2001), status within a twintron (Hallick et al., 1993), and intron phase (Sharp, 1981) be amended to the formal intron name in particular studies when the need of incorporating such information arises. Thus, this system has advantages of being exible and convenient in communication. Marchantia polymorpha is chosen as a reference species because its mitochondrial and chloroplast genomes have been sequenced (Oda et al., 1992; Ohyama et al., 1986) and they contain most of the genes that have been found so far in organellar genomes of land plants and other photosynthetic eukaryotes. In cases where M. polymorpha lacks an ortholog, or orthologous part due to deletion, of the gene in which an intron has been found in one or several species, the exon sequence of the species in which the intron was rst discovered will be used as the reference. The insertion site in this species will be used with a superscript asterisk followed by the species name in a parenthesis. For example, ycf66i106 (Chaetosphaeridium globosum) refers to an intron present at position 106 in the chloroplast gene ycf66 (which is absent in M. polymorpha) and it was rst discovered in C. globosum, which has been used as the reference species. For introns located in the 50 and 30 untranslated regions, a ) and + sign will be used

+ + + + + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) + + + + + ) ) ) ) ) AY354934 AY354933 AY354932 AY354937 AY354977 Qiu94074 Qiu99083 Qiu94175 Qiu97125 Qiu97126 Atrichum angustatum (Brid.) BSG. Diphyscium foliosum (Hedw.) Mohr Sphagnum recurvum P.Beauv. Takakia ceratophylla (Mitt.) Grolle Takakia lepidozioides Hatt. & Inoue

Liverworts Frullania dilatata (L.) Dumort. Radula complanata (L.) Dum. Plagiochila asplenioides (L.) Dum. Lophocolea heterophylla (Schrad.) Dum. Marsupella emarginata (Ehrh.) Dumort. Pellia sp. Metzgeria temperata Kuwahara Metzgeria conjugata Lindb. Conocephalum sp. Ricciocarpos natans (L.) Corda Blasia pusilla (Micheli) L. Haplomitrium mnioides (Lindb.) Schust.

Qiu99058 Qiu99075 Qiu99064 Qiu98007 Qiu99077 Qiu95001 Qiu98008 Qiu99080 Qiu94096 Qiu97153 Qiu99108 Qiu97127

AY354962 AY354961 AY354960 AY354959 AY354958 AY354964 AY354931 AY354965 AY354957 AY354929 AY354963 AY354930

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

) ) ) ) ) ) ) ) ) ) ) )

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Fig. 1. The exon phylogeny of the mitochondrial nad1 from 64 species of land plants and green algae (Nephroselmis olivacea (AF110138) was used as the outgroup). Shown here is a maximum likelihood tree ()Ln likelihood 14993.51076). The branch length dierence is contributed by both the divergence level and length of the sequences analyzed. Asterisks mark the nodes collapsed in the strict consensus tree of 25,941 most parsimonious trees (length: 3106 steps, CI (consistency index): 0.4858, and RI (retention index): 0.6863). The only dierence between the likelihood and parsimonious trees concerns placement of Ophioglossum, Psilotum, and Osmunda. Bootstrap values higher than 50% are shown above the branches. The taxa with sequences from the GenBank are underlined: Prototheca (NC_001613), Mesostigma (AF353999), Pedinomonas (NC_000892), Scenedesmus (AF204057), Chlorogonium (Y13644), Chlamydomonas (U03843), Chaetosphaeridium (NC_004118), Marchantia (NC_001660), Ginkgo (AF227466, AF227470), Gnetum (AF227467, AF227471), Pinus (AF160261), Arabidopsis (Y08501, Y08502), Petunia (X60400-X60403), Oenothera (AH003143), Beta (NC_002511), Oryza (AB076665), and Triticum (X57965-X57968).

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Fig. 2. Intron distribution in the mitochondrial nad1 of land plants and green algae. The species are arranged into phylogenetic groups. The coding sequences (gray boxes) are drawn to an approximate scale, with all intron positions marked. Rectangular boxes denote complete coding sequences; triangle-edged boxes indicate partial coding sequences. Trans-spliced introns are represented by shifted exon boxes. Species with data from literature are underlined. The numbers in parentheses after intron names indicate intron phases. *For nad1i728, (1) all mosses except Takakia (both species) and Sphagnum have lost the matR, (2) splicing status in Psilotum and Ophioglossum is unknown due to the partial sequences obtained, (3) presence of the cis-spliced intron in the four gymnosperms (except in Pinus where splicing status is unknown) has been demonstrated by Southern hybridizations (Qiu and Palmer, in press) and matR sequencing (Qiu et al., 1999), and (4) trans-splicing in Beta, Petunia, Triticum, and Oryza evolved independently (Qiu and Palmer, in press).

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Table 2 Primers used for mitochondrial nad1 gene amplication and sequencing Forward primers Location Mp nad1-1 Mp nad1-82 Mp nad1-153 Mp nad1-191 Ol nad1i258-763 Ol nad1i258-1213 Mp nad1-264 Aa nad1i287-334 Mp nad1-292 Hl nad1i394-369 Hl nad1i394-705 Hl nad1i394-1060 Hl nad1i394-1341 Mp nad1-625 Hl nad1i669-1185 Hl nad1i669-2391 Hl nad1i669-2628 Mp nad1-671 Mp nad1-703 Mp nad1-636 Hl nad1i669-538 Hl nad1i669-984 Hl nad1i669-1435 Hl nad1i669-1818 At nad1i728-2229 At nad1i728-2490 At nad1i728-2517 Mp nad1-762 At At At At At At At At At At At nad1i728-432 nad1i728-745 nad1i728-766 nad1i728-1028 nad1i728-1249 nad1i728-1852 nad1i728-1852 nad1i728-1859 nad1i728-1870 nad1i728-2890 nad1i728-3128
*

Reverse primers Sequence 5 -ATGAGAATTTATCTAATTGG-3 CTAGCWGAACGWAAAGTCATGGC GTTACAACCTTTDGCWGAYGG AAGARCCTATTTTAYYRAGTAGTGCT CARGTGCGCAAGGACCTKWMAC TTCGGAGAGGTCCTATCG TTTGGTTGCTTGGGCGGTTATC ACTATGCGGACATCTTAC GATTATGGTATGGTATTGTC TGTAYGCTGTCTTGCTTCCT CTCRAATCYCACGAATCC MGRTGGAAACAAGCAGCG CTGGGTACTGAGCCCTTTGC GCGACTRATTCMGCTTCTGCTTCTGGTA TTCTGCYTCDGCYTCTGG ATCTAGGTATATGTTGGTAA GTGTGAAGGCAGGACCMS CAGCCGCAATAACAGCAC AGAGCAAAYCCCATDGAVG CTCATTAAGATCATATTAGCATACTC TACCAGAAGCAGAAGCKGAATYAGTCGC AGCTGAATTMGTHGCDGG RAGTATRATGACCTGGTCG CACRATCMATATACACACC0 TACGCCGTTACAATACGCAGA GGAGCTGGTCTCAGATGATG GCTATGCRAGATYTCTCTTGATCTTTGC TCGTCTGCGTCCTCTTCGTTC GTRAATATAGCRGACCAGCGRAT GARGATTTGTGCTTGTKGGC TAGGAATATCTAGGATGGGCAGC TGAGGTGCGGAGCYTTGCATCYGACATT GTAAACTCATTRRACAGG CGGTACYCGAATCYATTTACGA AKCCCGAGTTTCYAGACACAT CAGTGTACTACTATCACCCCTAC GCYTACACACCAGGCTACCCYTC TAGAGAAGCGTCGGAGAGTA TRGARAAGCGTCGGAGAGTAAAGCACC GCGTCGGAGAGTAAAGCACCGTATC TAAAGCACCGTATCCATCTCACAG RGCCTCATAGTGCCTCCTGT CAAAAGACTGCTAGTGGCGTCC
0 0

Location Ol nad1i258-1327 Ol nad1i258-2118 Mp nad1-292 Hl nad1i394-1007 Hl nad1i394-1476 Hl nad1i394-1486 Mp nad1-395 Mp nad1-407 At-nad1i477-12 At-nad1i477-21 At nad1i477-479 At nad1i477-720 Mp nad1-623 Hl nad1i394-1350 Mp nad1-404 Mp nad1-408 At nad1i477-379 At nad1i477-481 At nad1i477-586 Mp nad1-623 At nad1i728-168 At nad1i728-126 At nad1i728-450 At nad1i728-1712 At nad1i728-2070 At nad1i728-2087 Hl nad1i669-2290 Mp nad1-670 Mp nad1-709 Mp nad1-714 Mp nad1-725 Mp nad1-789 Mp nad1-832 Mp nad1-858 Mp nad1-966

Sequence 50 -TAGGCCACTCTGAATTCC-30 CTTTCAGTCGAGTGTCTGGAA GACAATACCATACCATAATC GTCGGTTATTCCATCTCC AGCRARGTGCAGARAACRCG CGATAGAGGAAAGCGAGGTGC TCCTGAAAAAGCATATTTGGRAT ATCGTAATGCTCCYARAAADG ACCTTCGCGGATCCAAACGCGCTC TGCTGCGGTTCCCTTCGGTCCGTTA CTTCCAACGTCAACCTGCG GTRTTGAGTAGGCAGCGCC GGAACCWTTGTAMCYTAGAKAC ATGCTTTTYTRGGAGCATTACGAT TTTTTYRGGAGSWTTACGDTCTG CCTGATCTGGATAACGATAG CMAATGGACTCTACRAGGACC GGGCTGTAGGTGATAAYGC CTTAGATARGTCGTCCGATGGGT GARGGGTAGCCTGGTGTGTARGC TCTCCCAGRACCARAATGATTATC GGTTTAGACTKGATTYGCTCATA TCTTTACTAAGTGTCCCTGGCGGT CATTMTTGMTTGGCGAAG TCYTCWATGGGRTTTGCTYTKTT GCYAATATGATCTTAATGAG TATGATCTTAATGAGGTGCGGAG CGAGCCCGGAATCACCTGG CCCATATATATACMAACAAAAGGGA MGATATCGWGGAAATGCTGCRC TTAAGRAAGCCATTCAAAGGCT

Numbers correspond to the 50 and 30 positions of the forward and reverse primers in Marchantia polymorpha (Mp) mitochondrial nad1 gene (NC_001660, Oda et al., 1992), Arabidopsis thaliana (At) introns nad1i477** and nad1i728 (NC_001284, Unseld et al., 1997), and Anthoceros agrestis (Aa) nad1i287, Huperzia lucidula (Hl) nad1i394 and nad1i669, and Ophioglossum lucitanicum (Ol) nad1i258 (all from this study). Amplication primers are underlined. Two 50 -end nucleotides (TT) from Arabidopsis thaliana nad1i477 (NC_001284) actually belong to the upstream exon. Here we follow the corrected exonintron boundary, starting nucleotide counting in the intron from GTGCG. . .

before the insertion site, respectively. Clearly, future discoveries will present situations for which amendment to this nomenclature will be necessary. 2.3. Phylogenetic analyses Because bryophytes often grow as a mixture of several species, the chance of cross-contamination could still exist even though we made an eort to check tissue purity

under a dissecting scope. To conrm that the sequences obtained were indeed from the species we targeted and not from biological contaminants, we performed phylogenetic analyses on the exon sequences of nad1, using both the parsimony and maximum likelihood methods as implemented in PAUP* 4.0b10 (Swoord, 2003). In the parsimony analysis, we weighted all characters and mutations equally. For the maximum likelihood analysis, we used the HYK85 model with the following settings: two

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substitution types (HYK85 variant), a transition/transversion ratio of 2 (j 4.2901951), empirical base frequencies (A 0:23129; C 0:17128; G 0:21140; T 0:38603), no invariable sites, equal rates for all sites, number of distinct data patterns under this model 932, molecular clock not enforced, starting branch lengths obtained using RogersSwoord approximation method, branch-length optimization one-dimensional Newton Raphson with pass limit 20, delta 1e ) 06, and )ln L (unconstrained) unavailable due to missing data and/or ambiguities. A heuristic search was conducted to nd the trees of the shortest length or the highest likelihood using simple (for the parsimony analysis) or as is (for the maximum likelihood analysis) taxon-addition replicates, one tree held at each step during stepwise addition, TBR branch swapping, steepest descent option in eect, MulTrees option in eect and no upper limit of MaxTrees. A bootstrap analysis was conducted using the parsimony method to test robustness of the topology, using 100 resampling replicates and the same tree search procedure as described above except with SPR branch swapping. We also carried out phylogenetic analyses on intron sequences, to evaluate how intron phylogenies reect the plant phylogeny and to determine whether an intron originated via a single acquisition event followed by vertical inheritance or through multiple independent gains caused by horizontal transfers. Because the parsimony and maximum likelihood analyses produced virtually identical results on the exon data (see Fig. 1 legend), we conducted only parsimony analyses for the intron sequences. For two introns (nad1i287 and nad1i728) with data from a relatively large number of species, the same heuristic tree search method and bootstrap analysis procedure as the exon sequence analyses were used, except with the TBR branch swapping option. For other ve introns that had data from only a small number of species, we were able to use the branch-and-bound or exhaustive tree search methods to nd the most parsimonious tree(s). The bootstrap analysis procedure was the same as in the exon analysis.

our own unpublished analysis of six genes from over 180 land plants. However, there were several anomalies in the exon phylogeny, e.g., Haplomitrium falling into the clade of leafy and simple thalloid liverworts, paraphyly of mosses, and Takakia being sister to hornworts. Several factors may have contributed to these results: short sequences used for such a broad range of taxa, uneven RNA editing patterns (see below), and insucient taxon sampling. Nonetheless, we think that the exon phylogeny does suggest that nad1 is a useful phylogenetic marker and that all of the sequences obtained are indeed from the species we targeted, i.e., not from biological contaminants. A total of seven introns were found, and all of them belong to group II (Table 1, Fig. 2). Five of them have been reported before (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Kubo et al., 2000; Malek and Knoop, 1998; Notsu et al., 2002; Wissinger et al., 1991), and two are newly discovered in this study. They all have characteristic group II intron primary and secondary structural features: the consensus sequence of GBGYG at the 50 -end (the second nucleotide consensus needs to be expanded; the consensus sequence of AY at the 30 -end is not being followed by many group II introns, including three of the seven introns reported here: nad1i348, nad1i669, and nad1i728); six double helical domains radiating from a central wheel; the bulging A residue at the 7th8th position from the 30 -end in domain VI, which is required for lariat formation and intron excision; and the conserved domain V with a GNRA terminal tetraloop (for six out of seven introns) (Lambowitz and Belfort, 1993; Qin and Pyle, 1998). For 12 liverworts, we obtained the full length coding region of nad1 using primers that covered start and stop codons (Metzgeria conjugata and M. temperata missed two codons at the 30 -end), and found no introns at all (Table 1, Fig. 2). This is the same condition as reported in Marchantia (Oda et al., 1992). In other land plants, the introns showed lineage-specic distribution patterns as described below. 3.1. nad1i258

3. Results Among the 46 species of diverse land plants we examined (Table 1), amplication of almost entire nad1 coding region encompassing all known intron positions was possible for a majority of the species. However, only partial coding regions of varying lengths covering some of the intron positions were obtained from Isoetes, Psilotum Sw., Angiopteris Hom., Marattia Sw., Ophioglossum, Osmunda, and Cycas L.. Our analyses of the exon sequences produced a phylogeny (Fig. 1) that is in general agreement with the relationships of these plants as reported in published studies (Hedderson et al., 1998; Lewis et al., 1997; Newton et al., 2000; Pryer et al., 2001) and in

This is a novel intron we discovered in the present study; its secondary structure is shown in Fig. 3A. It is present in all the moniliformopses for which we could get a sequence of this region of the gene: Equisetum, Ophioglossum, Angiopteris, Marattia, Adiantum, and Asplenium, but is absent from green algae, bryophytes and other vascular plants (Table 1, Fig. 2). Its typical length is approximately 900 bp, with an exception in Ophioglossum, where the intron contains a remnant ORF, resulting in a length of 2598 bp. Our phylogenetic analyses show that the evolutionary history of the intron parallels that of the plants (Fig. 4A; Pryer et al., 2001), suggesting that the intron was acquired in the common ancestor of moniliformopses and has been inherited vertically.

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Fig. 3. Inferred secondary structure of two newly discovered group II introns in the mitochondrial nad1. (A) nad1i258 from Marattia attenuata and (B) nad1i348 from Phaeoceros carolinianus.

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Fig. 4. Phylogenies of the seven introns found in the mitochondrial nad1 of land plants. The numbers above the branches are bootstrap values. The taxa with sequences from the GenBank are underlined: Ceratodon (Y17810), Isoetes lac. (Y17812), Pinus (AF160261), Osmunda 1 (Y17815), Equisetum tel. (Y17811), and Notothylas (AF068932). (A) nad1i258, the single shortest tree found in an exhaustive tree search (length: 1033 steps, CI: 0.9555, and RI: 0.8357). (B) nad1i287, the strict consensus tree of 10 shortest trees found in a heuristic search (length: 694 steps, CI: 0.7277, and RI: 0.7970). (C) nad1i348, the single shortest tree found in an exhaustive tree search (length: 73 steps, CI: 0.9863, and RI: 0.8000). (D) nad1i394, the single shortest tree found in an exhaustive tree search (length: 1057 steps, CI: 0.9574, and RI: 0.9240). (E) nad1i477, the single shortest tree found in an exhaustive tree search (length: 2223 steps, CI: 0.9353, and RI: 0.6949). (F) nad1i669, the single shortest tree found in an exhaustive tree search (length: 1708 steps, CI: 0.9895, and RI: 0.9669). (G) nad1i728, the strict consensus tree of 12 shortest trees found in a heuristic search (length: 7556 steps, CI: 0.6546, and RI: 0.5223).

3.2. nad1i287 This intron, being orthologous to the one discovered in Ceratodon purpureus by Malek and Knoop (1998), was found in all 19 mosses and three hornworts we investigated (no PCR product was obtained from

Megaceros D.H. Campbell), and has an average length of 900 bp. It is absent in green algae and other lineages of land plants (Table 1, Fig. 2). The intron phylogeny (Fig. 4B) is in agreement with that of the plants (Newton et al., 2000), indicating vertical transmission of the intron in the mosses and hornworts.

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3.3. nad1i348 This is another new intron we discovered, and its secondary structure is shown in Fig. 3B. It is present in all four hornworts we examined: Anthoceros [Micheli] L., Megaceros, Phaeoceros Proskauer and Notothylas Sull., but absent in green algae and all other land plants (Table 1, Fig. 2). The intron has a typical length of 600 bp. Our phylogenetic analyses indicate that it has been transmitted vertically following its acquisition in the common ancestor of hornworts (Fig. 4C). 3.4. nad1i394 This intron is orthologous to the cis-spliced nad1Ti1 in Isoetes lacustris reported by Malek and Knoop (1998) and the trans-spliced rst intron of nad1 in angiosperms (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Wissinger et al., 1991). According to our survey, it is also present, in a cis-spliced form as in Isoetes lacustris, in Huperzia Bernh., Isoetes sp., Equisetum arvense, Angiopteris, Marattia, Adiantum, and Asplenium, but absent entirely in bryophytes and green algae (Table 1, Fig. 2). Among the species investigated, the intron size ranges from 1400 to 2000 bp in most species, but in Isoetes it drops dramatically to 520 bp. This sharp decrease of intron size in Isoetes has been observed earlier by Malek and Knoop (1998) for this intron as well as for two other introns in nad2 and nad5. It appears now that the decrease of intron size is restricted to only Isoetes; the other lycopod we examined, Huperzia, has a normal-sized intron. The absence of this intron in Ophioglossum (Fig. 2) and Equisetum telmateia (Malek and Knoop, 1998) can be explained by secondary losses. The intron phylogeny (Fig. 4D) essentially agrees with the plant phylogeny (Pryer et al., 2001), with the exception of Equisetum embedded in Marattiaceae. This topological anomaly may be caused by insucient taxon sampling, rather than serving as evidence of intron horizontal transfer. Distribution of the intron in land plants (Fig. 2) is mostly consistent with the hypothesis of a single origin of the intron in the beginning of vascular plant evolution followed by independent losses. Our failure to amplify the portion of the gene covering the intron insertion site in Psilotum, Osmunda, and Cycas, if not due to technical reasons, could be an indication that this intron is present in a trans-spliced state in these plants, as multiple independent evolutions of transsplicing have been detected in another nad1 intron, nad1i728 (Conklin et al., 1991; Kubo et al., 2000; Notsu et al., 2002; Qiu and Palmer, in press; Wissinger et al., 1991). Further investigation in these plants would be desirable to nd out the exact status of the intron.

3.5. nad1i477 This intron is orthologous to the second intron in nad1 of angiosperms (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Wissinger et al., 1991). It has been reported to be present in Huperzia lucidula and a large number of seed plants by Gugerli et al. (2001) and Won and Renner (2003) using either sequencing or Southern hybridization. In this survey, we found that this intron is present in Huperzia lucidula (the same DNA was used for both Gugerli et al., 2001, and this study), Angiopteris, Marattia, Ophioglossum, Osmunda, and Cycas, but absent in green algae, bryophytes, and four vascular plants that were sequenced through the intron insertion site, Isoetes, Equisetum, Adiantum, and Asplenium (Table 1, Fig. 2). Clearly, absence of the intron in the four vascular plants represents secondary losses, as has been observed earlier in some seed plants (Gugerli et al., 2001). The size of this intron shows a full range of variation from 896 bp in Arabidopsis to 2844 bp in Ophioglossum, unlike nad1i394 which has a bimodal size distribution. The intron phylogeny (Fig. 4E) agrees well with the plant phylogeny. Judging from the data shown here and in Gugerli et al. (2001), the intron was most likely acquired in the common ancestor of vascular plants and has been vertically inherited ever since, with secondary losses in a small number of plant lineages. 3.6. nad1i669 This is another trans-spliced intron in angiosperms (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Wissinger et al., 1991), and its cis-spliced ortholog has been isolated from Equisetum telmateia and Osmunda regalis (Malek and Knoop, 1998). In this study, we obtained sequences of cis-spliced introns from Huperzia and Psilotum as well as from a dierent species of Equisetum (E. arvense) and a dierent accession of Osmunda regalis than those used by Malek and Knoop (1998) (Table 1, Fig. 2). The size of the intron varies from 1561 bp in E. arvense to 3090 bp in Huperzia, with size in Psilotum and Osmunda being intermediate. The intron phylogeny (Fig. 4F) shows no sign of horizontal transfer. Besides its entire absence in green algae and bryophytes, the intron is also absent in Isoetes, Adiantum, and Asplenium, which can be attributed to secondary losses (Fig. 2). Its distribution pattern is consistent with the idea that the intron was gained in the ancestor of vascular plants. Failure of obtaining PCR product in Angiopteris, Marattia, Ophioglossum, and Cycas again could indicate presence of transspliced introns in these plants. 3.7. nad1i728 This intron is orthologous to the nad1.i4 that has previously been isolated from Sphagnum L. and

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Notothylas (Qiu et al., 1998) as well as several angiosperms (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Kubo et al., 2000; Notsu et al., 2002; Thomson et al., 1994; Unseld et al., 1997; Wahleithner et al., 1990; Wissinger et al., 1991). It has also been shown to be present in a large number of land plants except liverworts according to the Southern hybridizations (Qiu et al., 1998; Qiu and Palmer, in press) and matR (an ORF encoded within the intron) sequencing (Anderberg et al., 2002; Qiu et al., 1999). Here, we report sequences of this intron from 19 mosses, 3 hornworts (the Notothylas intron sequence was taken from Qiu et al. (1998) and hence was not included in this count), and 9 pteridophytes (Table 1, Fig. 2). The intron in hornworts and pteridophytes shows a range of size variation from 2221 bp in Osmunda to 4037 bp in Marattia. However, in mosses, while two Takakia species still have a full-sized intron (slightly over 2800 bp), Sphagnum and other mosses have lost most or all of their matR (with the intron size being 1452 bp in Sphagnum and around 850 bp in all other mosses). The portion of matR still present in Sphagnum is the X domain, which is implicated in intron splicing and is usually retained in any group II intron that contains an ORF (Mohr et al., 1993). The phylogeny of the intron (Fig. 4G) mirrors that of the plants rather well, indicating that the intron has been inherited vertically since its acquisition in the common ancestor of non-liverwort land plants as suggested by Qiu et al. (1998). For both Psilotum and Ophioglossum, we obtained only partial sequences of the intron using primers in the adjacent exons and in 50 - or 30 -end of the intron, and thus cannot conrm splicing status of the intron. Isoetes was reported to lack the intron in a Southern hybridization survey (Qiu et al., 1998), and our attempt to use PCR to check for the presence of the intron failed this time.

4. Discussion 4.1. Phylogenetic implications of intron distribution in nad1 of land plants A total of seven introns, all belonging to group II, have been discovered in the mitochondrial gene nad1 through surveys of a large number of land plants by this study and many others (Chapdelaine and Bonen, 1991; Conklin et al., 1991; Gugerli et al., 2001; Kubo et al., 2000; Malek and Knoop, 1998; Notsu et al., 2002; Qiu et al., 1998; Qiu and Palmer, in press; Thomson et al., 1994; Unseld et al., 1997; Wahleithner et al., 1990; Wissinger et al., 1991). Yet, no intron has been found in the 12 liverworts sequenced in this study nor in Marchantia by Oda et al. (1992), which span most of the liverwort diversity: Calobryales, Marchantiales, Metzgeriales, and Jungermanniales (Schuster, 1966, 2000).

This complete absence of introns in liverworts is the same condition as in the green algal outgroups (Kroyck et al., 2000; Nedelcu mann and Zetsche, 1998; Ku et al., 2000; Turmel et al., 1999, 2002a,b, 2003; Wol et al., 1994; M.W. Gray, unpublished data). The sole group I intron in nad1 of Chlamydomonas eugametos (Denovan-Wright et al., 1998), but not in C. reinhardtii (M.W. Gray, unpublished data (GenBank Accession No. U03843)), is irrelevant to the discussion here, as its distribution clearly suggests that it was gained via horizontal transfer. Earlier, Qiu et al. (1998) used the hypothesized gains of nad1i728 (nad1.i4 in their nomenclature) and two other introns in the mitochondrial gene cox2, cox2i373 and cox2i691, to argue for the basal-most position of liverworts among land plants. Nickrent et al. (2000) suggested that absence of these introns in the liverworts could be caused by secondary losses, as seen in some plants reported in Qiu et al. (1998). Further, Nishiyama and Kato (1999), Nickrent et al. (2000), and Renzaglia et al. (2000) conducted phylogenetic analyses of multigene data sets (with limited taxon sampling) and spermatogenesis characters and concluded that hornworts occupy the basal-most position in land plants. In light of the data presented here, we believe that the most parsimonious explanation for intron distribution in nad1 is still that liverworts, not hornworts, represent the oldest land plants, and that both nad1i287 and nad1i728 were gained in the common ancestor of mosseshornworts vascular plants after liverworts had diverged. While the former was lost in the ancestor of vascular plants, the latter had been retained. The idea of a single gain of nad1i287 in a common ancestor of mosses and hornworts can only be entertained if the two groups form a clade, but both published studies (Kenrick and Crane, 1997b; Mishler et al., 1994; Nickrent et al., 2000; Nishiyama and Kato, 1999; Renzaglia et al., 2000; see also Qiu and Lee, 2000) and our unpublished multigene analysis of land plant phylogeny have failed to recover this topology. If we follow the hornwort basal hypothesis, both introns would have to be gained in the common ancestor of land plants and lost in liverworts, with an additional loss of nad1i287 in vascular plants since no study so far has suggested a sister relationship of liverworts and vascular plants (Qiu and Lee, 2000). This scenario is signicantly less parsimonious than the explanation oered by the liverwort basal hypothesis. The hornwort basal hypothesis is also contradicted by the distribution of two other mitochondrial group II introns, one in nad2 (nad2i1282, Pruchner et al., 2002) and the other in nad5 (nad5i477, Malek and Knoop, 1998), both of which are present in vascular plants and hornworts but not in liverworts and mosses. Meanwhile, two introns in nad7, nad7i40, and nad7i209, have now been found in mosses, hornworts, and vascular plants but not in liverworts, thus adding more evidence to the liverwort

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basal hypothesis (Hashimoto and Sato, 2001; Pruchner et al., 2001; N. Sato, personal communication). Therefore, the overall intron distribution in the mitochondrial genes (cox2, nad1, nad2, nad5, and nad7) is more consistent with the liverwort basal hypothesis than with the hornwort basal hypothesis. The liverwort basal hypothesis received an indirect support from recently completed studies of chloroplast genome sequencing of Physcomitrella patens (a moss, Sugiura et al., 2003), Anthoceros formosae (Kugita et al., 2003a), Psilotum nudum (Wakasugi et al., unpublished data, GenBank Accession No. AP004638), and Adiantum capillus-veneris (Wolf et al., 2003). In Marchantia (Ohyama et al., 1986), Physcomitrella, and Chaetosphaeridium (Turmel et al., 2002b), the inverted repeat in the chloroplast genome does not include the genes ndhB, rps7, and rps12 (partial). However, Anthoceros cpDNA has these three genes as part of its inverted repeat, a condition otherwise only seen in Psilotum, Adiantum, and other vascular plants such as rice (Hiratsuka et al., 1989) and tobacco (Wakasugi et al., 1998). This expansion of the inverted repeat to encompass these three genes can almost certainly be regarded as a synapomorphy to unite hornworts and vascular plants, a relationship alluded to by the aforementioned phylogenetic distribution of nad2i1282 and nad5i477 as well as some sequence analyses (Lewis et al., 1997; Samigullin et al., 2002; see also Qiu and Lee, 2000) (admittedly, taxon sampling, in particular of vascular plants, in these sequence analyses is rather thin). Lastly, the most recent report of fossil evidence from the Ordovician rocks of Oman on sporangium structure that contain spore tetrads characteristic of early land plants also support the liverwort basal hypothesis (Wellman et al., 2003). A recent study found that certain genomic structural changes, such as ribosomal protein gene loss and transfer from the mitochondrial to nuclear genomes, occur at rather high frequencies (Adams et al., 2002). Another study also showed that a group II intron had likely experienced horizontal transfer from angiosperms to Gnetum (Won and Renner, 2003). Moreover, deep losses of introns have clearly happened, as can be seen from this study and others that investigated mitochondrial introns in basal land plants (Beckert et al., 1999, 2001; Pruchner et al., 2002). These studies raise the issue of utility of genomic structural characters in inferring plant phylogenetic relationships. However, our phylogenetic analyses of intron sequences showed little sign of independent gains and horizontal transfer of the group II introns investigated here (Fig. 4). Hence, we contend that while one certainly cannot use genomic structural characters, nor any other kind of characters, blindly for phylogenetic inference, it is appropriate to use intron distribution patterns, particularly intron gains, if they are demonstrated to occur at very low frequencies, to evaluate competing phylogenetic hypotheses. Intron

distribution patterns as well as other genomic structural characters such as gene order and content in the cpDNA inverted repeat will add a new source of information for plant phylogenetic reconstruction. Another issue that these intron distribution data may help to resolve is the phylogenetic position of the enigmatic bryophyte genus Takakia. Though rst thought to be related to liverworts (Hattori and Inoue, 1958; Schuster, 1966) or as an independent lineage at the same rank as liverworts, mosses, and hornworts (CrandallStotler, 1986), discovery of antheridia and sporophytes has revived an earlier idea that the genus shows anity to mosses (Mizutani, 1967; Smith and Davison, 1993). Recent phylogenetic analyses of morphological and molecular data seem to support this conclusion, but the exact placement of Takakia within mosses is far from being certain (Hedderson et al., 1998; Kenrick and Crane, 1997b; Mishler et al., 1994; Newton et al., 2000; Renzaglia et al., 2000). In this study, we found that the intron content in nad1 of both Takakia species is the same as 17 diverse mosses we investigated, but dierent from that of liverworts and hornworts (Table 1, Fig. 2). Hence, a moss anity for the genus seems most probable. Furthermore, our examination of the ORF (matR) in nad1i728 indicates that the two Takakia species still have an intact matR, whereas Sphagnum has only the X domain of the ORF and the other 16 mosses lack the ORF entirely. Two lines of evidence support the following ordering of the three states in these taxa: Takakia (ancestral), Sphagnum (intermediate), and all other mosses (derived). One is that the X domain has been suggested to be the least dispensable element in group II intron ORFs since it is involved in intron splicing (Mohr et al., 1993). The other is that no matR loss has been observed in any of the large number of other land plants that have been investigated (Anderberg et al., 2002; Qiu et al., 1999; Qiu and Palmer, in press). Consequently, the matR distribution data would support a topology in which Takakia is sister to the clade of Sphagnum plus other mosses, as has been recovered in some recent sequence analyses (Renzaglia et al., 2000; Qiu et al., unpublished data). The data of the present study may also address the monophyly of moniliformopses, a group that comprises traditionally circumscribed ferns, Equisetum, and Psilotaceae. The clade was rst identied by Kenrick and Crane (1997b) after they analyzed both extant and extinct pteridophytes, and was subsequently supported by both multigene sequence analysis and a shared unique insertion in the chloroplast gene rps4 (Pryer et al., 2001). In this study, we found that the ferns (both eusporangiate and leptosporangiate) and Equisetum share the intron nad1i258. Unfortunately, no sequence was obtained from Psilotaceae. Our phylogenetic analyses of the intron sequences indicate that the intron was most likely acquired in a single event in the common ancestor

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of moniliformopses and has been inherited vertically since the acquisition (Fig. 4A). This intron gain would then be a synapomorphy to support the monophyly of moniliformopses. Finally, three introns that were rst characterized in angiosperms, nad1i394, nad1i477, and nad1i669, are now found to be present throughout vascular plants (Fig. 2). Thus, they were most likely gained in the common ancestor of the group. These characters would add to the large body of information already in existence that supports the monophyly of vascular plants (Kenrick and Crane, 1997b). 4.2. Molecular evolutionary implications of intron distribution in nad1 of land plants One conspicuous aspect of distribution of the seven nad1 introns in land plants is that they are all of broad phylogenetic occurrence. The intron that shows the narrowest phylogenetic range is nad1i258, which is still found in Equisetum, and eusporangiate and leptosporangiate ferns (Table 1; Fig. 2). The common ancestor of these plants likely existed between the Silurian and Devonian (435345 million years ago) (Kenrick and Crane, 1997a,b). The other intron that is restricted to only one lineage is nad1i348, which occurs in all four genera (belonging to the only two families) of hornworts that were examined (Table 1; Fig. 2). The hornworts as a lineage must have existed between the mid-Ordovician (about 450 million years ago) and Silurian, as fossils of liverwort anity have been reported from the midOrdovician (Edwards et al., 1995; Taylor, 1995; Wellman et al., 2003) and those of vascular plants from the lower Silurian (Kenrick and Crane, 1997a,b). All other ve introns are found to be shared by at least two major lineages of land plants (Table 1; Fig. 2). Hence, these data suggest that these introns arrived at their current positions at least during the Silurian to Devonian period. There is also evidence suggesting that these and other group II introns in the non-liverwort land plant mitochondrial genome originated via transposition from pre-existing introns in other genes of liverworts [currently based on data mostly from Marchantia (Oda et al., 1992)] and other organisms (Dombrovska and Qiu, in preparation). If these observations hold, it seems that group II intron transposition was quite active in the mitochondrial genome during the early stage of land plant evolution. Meanwhile, among studies that have surveyed introns in a broad range of land plants (Hashimoto and Sato, 2001; Malek and Knoop, 1998; Pruchner et al., 2001; Qiu et al., 1998), only one intron has been shown to be restricted to a single species, the nad5i392 in Huperzia selago (Vangerow et al., 1999). This general lack of group II introns with extremely narrow phylogenetic distribution leads us to conclude that they have not been actively undergoing transposi-

tion in the recent history of land plant evolution. Further, our phylogenetic analyses of all seven nad1 introns uncovered no evidence of horizontal transfer, specically intron homing (Fig. 4). This situation is in stark contrast to what was reported by Cho et al. (1998) of rampant horizontal transfer (homing) by a group I intron in the mitochondrial gene cox1 of angiosperms. The only explanation we can oer is that the cox1 intron carries an endonuclease ORF whereas most of the group II introns in vascular plant mitochondrial genomes have lost their ORFs, which contain domains involved in intron mobility (Mohr et al., 1993; Zimmerly et al., 2001). Despite stable inheritance after ancient gains, some of the introns did experience losses, possibly very deep losses. The distribution pattern of nad1i287 potentially suggests a deep loss in the common ancestor of vascular plants. The likelihood that this intron was gained once in a common ancestor of mosses and hornworts or twice in ancestors of each lineage is much less than that of a single gain in the ancestor of non-liverwort land plants and a subsequent loss in vascular plants, given the intron phylogeny (Fig. 4B) and current results on land plant phylogeny reconstruction (Kenrick and Crane, 1997b; Mishler et al., 1994; Renzaglia et al., 2000; Qiu et al., unpublished data). Three other introns, nad1i394, nad1i477, and nad1i669, also showed a few sporadic losses (Fig. 2; Gugerli et al., 2001), but they are all phylogenetically restricted. Both deep and shallow losses have also been observed for two cox2 introns in angiosperms (Joly et al., 2001; Kudla et al., 2002; Rabbi and Wilson, 1993; Qiu and Palmer, unpublished data). The precise mechanisms for how both deep and shallow intron losses have occurred remain largely unknown at this stage. Among all plants studied, Isoetes is distinct in that it has lost two, if not three (the third one possibly being nad1i728, the exact status of which still cannot be conrmed by sequencing because of our failure to obtain a PCR product), of the four introns that are usually present in nad1 in vascular plants (Fig. 2). Even when introns are present in this and other mitochondrial genes (nad2 and nad5) in Isoetes, they are unusually small (384520 bp, this study; Malek and Knoop, 1998; Pruchner et al., 2002). These two lines of evidence suggest that the mitochondrial genome in Isoetes is probably under strong selection pressure for genome size reduction, as has been seen in some abnormal genomes (Douglas et al., 2001). 4.3. RNA editing in nad1 of land plants RNA editing, post-transcriptional modication of cytidine to uridine and vice versa in RNAs, has been reported to occur commonly in organellar genes and genomes of certain land plants (Giege and Brennicke, 1999; Kugita et al., 2003b; Steinhauser et al., 1999).

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In this study, we noticed that after aligning the exon sequences from all the plants, some lineages seem to have numerous aberrant C ! T(U) and T(U) ! C mutations in the rst or second codon positions. Hence, we checked for the possibility of RNA editing by looking for whether the mutation has altered a conserved amino acid or generated an internal stop codon in some cases. Obviously, this approach is not as conclusive and

accurate as cDNA analysis, but has been employed and proven to be valid by others (Steinhauser et al., 1999). With this approach, we found that the nad1 gene in the liverwort Haplomitrium, two Takakia species, all four hornworts, and several pteridophytes (Isoetes, Ophioglossum, Adiantum, and Asplenium) potentially experience heavy editing, with up to 5.11% of all the nucleotide positions in Anthoceros being edited (Fig. 5).

Fig. 5. Selected codons of the mitochondrial nad1 in land plants showing the extent of RNA editing. Inferred edited nucleotides are underlined, bodyfaced ones having been conrmed by cDNA analysis in the studies that reported editing. Editing frequency (%) equals the number of edited nucleotides divided by the exon length examined. Boxes mark stop codons that require editing for conversion. Dots indicate nucleotides identical to those in Marchantia polymorpha and dashes denote missing data. Marchantia position refers to the position of the rst nucleotide in a codon.

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Previously, high frequencies of RNA editing have been noticed in many of these plants in other mitochondrial genes, cox3, nad2, nad4, and nad5 (Malek and Knoop, 1998; Malek et al., 1996; Pruchner et al., 2001, 2002). However, questions remain as to why there is RNA editing in the rst place and why it occurs in certain lineages but not in others.

Acknowledgments We thank J.D. Palmer for providing initial ideas on starting this project, and V. Knoop for stimulating discussion on plant mitochondrial intron evolution, and both of them for their advice on developing the intron nomenclature. We also thank B. Crandall-Stotler and E. Urmi for help with obtaining plant material. This work was supported by a Swiss NSF research grant (3100053602) and a NSF CAREER Award (DEB 0093012) to Y.Q.

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