complete sequence of the mitochondrial dna of the red alga … · physiology throughout the phylum....

The Plant Cell, Vol. 11, 1675–1694, September 1999, www.plantcell.org © 1999 American Society of Plant Physiologists

Complete Sequence of the Mitochondrial DNA of the Red Alga

Porphyra purpurea

: Cyanobacterial Introns and Shared Ancestry of Red and Green Algae

Gertraud Burger,

a,b,1

Diane Saint-Louis,

b

Michael W. Gray,

a,c

and B. Franz Lang

a,b

a

Program in Evolutionary Biology, Canadian Institute for Advanced Research, 180 Dundas Street West, Toronto, Ontario M5G 1Z8, Canada

b

Département de Biochimie, Université de Montréal, 2900 Boulevarde Edouard-Montpetit, Montréal, Québec H3T 1J4, Canada

c

Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

The mitochondrial DNA (mtDNA) of

Porphyra purpurea

, a circular-mapping genome of 36,753 bp, has been completelysequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animalsand fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins andsubunits of succinate:ubiquinone oxidoreductase. The mitochondrial large subunit rRNA gene contains two group II in-trons that are extraordinarily similar to those found in the cyanobacterium

Calothrix

sp, suggesting a recent lateral in-tron transfer between a bacterial and a mitochondrial genome. Notable features of

P. purpurea

mtDNA include thepresence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rear-rangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis ofred algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs areunusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share acommon ancestry with green algae and plants.

INTRODUCTION

Historically considered to be “red plants,” red algae (rhodo-phytes) were grouped together with protists only in the mid-dle of this century; however, debate continues as to whenthis taxonomic group first appeared in the evolutionary his-tory of mitochondria-containing eukaryotes. Because thered algal cell lacks flagellar basal bodies and centrioles,some workers have claimed that rhodophytes are the mostearly diverging and primitive group among photosyntheticeukaryotes (e.g., Lee, 1989). This view also has been sug-gested by certain molecular phylogenies (e.g., Lipscomb,1989; Stiller and Hall, 1997). Some authors even advocatethat red algae, in particular

Cyanidioschyzon

spp and

Cya-nidium

spp, represent the evolutionary bridge between cy-anobacteria and eukaryotes (Seckbach, 1994). An oppositeinterpretation contends that the red algae represent a de-rived group that has secondarily lost many of the distinctivefeatures of the protistan cytoskeleton (Pueschel, 1990; Scottand Broadwater, 1990). Finally, it has been proposed thatred algae may have given rise to the fungi (Demoulin, 1985)

or that red algae are affiliated with the plant kingdom viacommon ancestry with green algae (see, e.g., Bhattacharyaet al., 1993; Cavalier-Smith, 1993; McFadden et al., 1994;Schlegel, 1994; Paquin et al., 1997; Lang et al., 1998). Thisbroad spectrum of coexisting, mutually exclusive hypothe-ses highlights our limited knowledge about the phylogeneticrelationship between rhodophytes and other eukaryoticphyla.

Rhodophytes form a morphologically heterogeneous phy-lum that has more species (

z

2500 to 6000 in at least 12 or-ders; Woelkerling, 1990) than all other seaweeds combined.Multicellular as well as unicellular rhodophyte genera exist,and there is also considerable variety in morphology andphysiology throughout the phylum. On the basis of the ob-served divergence in nuclear small subunit (SSU) rRNA se-quences, Ragan et al. (1994) concluded that “rhodophytesare more divergent among themselves than are fungi orgreen algae and green plants together.”

In 1995, the first complete mitochondrial DNA (mtDNA)sequence from a red alga,

Chondrus crispus

(Gigartinales),was published (Leblanc et al., 1995a). In molecular phyloge-nies using single nuclear genes, members of the Gigartinalesform one of the late-diverging rhodophyte orders (Ragan etal., 1994; Saunders and Kraft, 1997). In many respects, the

1

To whom correspondence should be addressed. E-mail [email protected]; fax 514-343-2210.

1676 The Plant Cell

genome structure and gene repertoire of

C. crispus

mtDNAresemble those of plants and protists, but the

C. crispus

mi-tochondrial genome displays neither exceptionally primitivecharacters nor especially derived traits. No “new” mitochon-drial genes are present, as occurs in the mtDNA of the het-erotrophic flagellate

Reclinomonas americana

(Lang et al.,1997), nor are otherwise ubiquitous mitochondrial genesmissing, as occurs, for example, in

Chlamydomonas

spp(see Nedelcu, 1998) or animals (see Wolstenholme, 1992).However, the extent to which these unexpectedly “normal”features of the

C. crispus

mtDNA are characteristic of thered algal assemblage as a whole remains to be explored.

We chose to sequence the mtDNA of

Porphyra purpurea

,a member of the Bangiales, because this order is consid-ered to have diverged before the Gigartinales and mostother orders (all except Compsopogonales and some mem-bers of the Porphyridales; Ragan et al., 1994; Saunders andKraft, 1997). In addition, we have sequenced several mito-chondrial genes of

Gracilariopsis lemaneiformis

(Gracilariales;GenBank accession number AF11819). In the meantime, a10-kb portion of the mtDNA from

Cyanidium caldarium

, amember of the Porphyridales, was published (Viehmann,1995), and at a late stage in the preparation of this manu-script, the complete mtDNA sequence of

Cyanidioschyzonmerolae

was released (Ohta et al., 1998). With these newdata, we are now in a position to undertake comparativegene and genome analysis encompassing several early andlate diverging red algal orders. Such phylogenetically broadinformation should not only provide us with a better under-standing of mitochondrial genome diversity within this lin-eage but also allow us to reinvestigate questions aboutwhen red algae emerged in the evolutionary history of eu-karyotes and how rapidly this group has been evolving.

RESULTS

Physical Properties, Gene Content, and Overall Organization of

P. purpurea

mtDNA

The mtDNA of

P. purpurea

maps as a circular molecule of36,753 bp. The overall AT content of the mitochondrial ge-nome is 66.5%, with intergenic spacers being significantlyricher in A

1

T (72.0%) than are coding regions (66.0%). Fig-ure 1 shows that genetic information is densely packed inthe mtDNA, with 91% of the sequence specifying genes andopen reading frames (ORFs) and only 9% without detectablecoding content. Genes are encoded on both strands, andtheir orientation suggests that the genome is transcribed intwo units, starting from a bidirectional promoter located be-tween

trnQ

and

rtl

(Figure 1, double-headed arrow). In fact,this intergenic region contains a stem–loop structure thatmight be involved in transcription initiation, as suggested forthe chicken mitochondrial promoter (L’Abbé et al., 1991). In

Chondrus crispus

mtDNA as well, the bipolar transcription

initiation site coincides with a palindromic repeat (Richard etal., 1998). However, we were unable to detect in the

trnQ

to

rtl

intergenic region of

P. purpurea

a sequence element resem-bling the promoter motif proposed for

C. crispus

mtDNA.Table 1 lists the genes in

P. purpurea

mtDNA that code for26 structural RNAs (i.e., the SSU and large subunit [LSU] rR-NAs and 24 transfer RNAs) and 21 proteins, including 17components of the respiratory chain and ATP synthase andfour ribosomal proteins. In addition, two conserved ORFsare found in

P. purpurea

mtDNA, one of which (

ymf16

) hasbeen identified recently (Table 1). Open reading frames con-tained within the two

rnl

introns are of the reverse tran-scriptase type (Michel and Lang, 1985; Xiong and Eickbush,1990). Also present in

P. purpurea

mtDNA are four uniqueORFs lacking significant similarity to any entry in the publicdomain sequence databanks, as well as fragmented DNApolymerase (

dpo

) and reverse transcriptase (

rtl

) genes.Three classes of genes detected in the

P. purpurea

mtDNA have counterparts in only a few other protist taxa. (1)Among the four ribosomal protein genes (

rps3

,

rps11

,

rps12

,and

rpl16

),

rps11

has been found only in a dozen species,including members of early diverging plants, green algae, ja-kobids, and other protists (Gray et al., 1998; G. Burger, B.F.Lang, and M.W. Gray, unpublished results). The remainingthree ribosomal protein genes are invariably present in pro-tist mtDNAs when these genomes contain ribosomal proteingenes. (2) To date, only four non-rhodophyte species areknown in which subunits of the succinate:ubiquinone oxi-doreductase (respiratory complex II) are mitochondrially en-coded: the liverwort

Marchantia polymorpha

(Oda et al.,1992; Daignan-Fornier et al., 1994; Burger et al., 1996); thejakobid flagellates

R. americana

(Burger et al., 1996; Lang etal., 1997) and

Jakoba libera

(G. Burger, B.F. Lang, C.O’Kelly, and M.W. Gray, unpublished results); and the cryp-tophyte alga

Rhodomonas salina

(Gray et al., 1998). (3) Mito-chondrially encoded DNA polymerase (

dpo

) genes are alsopresent in only a few eukaryotic species, with the majoritybeing fungi and plants. These sequences are generally lo-cated on mitochondrial plasmids (reviewed in Kempken etal., 1992; Weber et al., 1995), whereas the

P. purpurea dpo

is contained within the mtDNA itself. All known mitochon-drial

dpo

genes, whether plasmid encoded or not, belong tothe B-type family, related to DNA polymerase II of

Esche-richia coli

(classification according to Braithwaite and Ito,1993). (4) Finally, mitochondrial reverse transcriptase (or re-verse transcriptase–like) genes (

rtl

) that are free-standing,that is, not in introns, have been detected in only two otherspecies,

M. polymorpha

(Oda et al., 1992) and

Chlamydo-monas reinhardtii

(Boer and Gray, 1988).

Mitochondrial Gene Content and Order in

P. purpurea

and Other Red Algae

The gene content of

P. purpurea

and

Chondrus crispus

mtDNAs (Leblanc et al., 1995a) is identical, except that

rrn5

Porphyra purpurea

Mitochondrial DNA 1677

(specifying 5S rRNA) and

rpl20

are missing from

P. purpurea

mtDNA, whereas

trnS(gcu)

,

dpo

, and

rtl

are absent from

C.crispus

mtDNA (identification of

C. crispus

rrn5

is describedbelow). Compared with the mtDNA of the former two rhodo-phytes,

Cyanidioschyzon merolae

mtDNA (Ohta et al., 1998)encodes six or seven more ribosomal proteins and fourcomponents involved in

c

-type cytochrome biogenesis(

yejU

,

yejV

,

yejW

, and

yejR

[

ccl1

]), which are otherwise

known from plant, ciliate, and jakobid mtDNAs (Gray et al.,1998). A 5S rRNA gene is also present in

Cyanidioschyzonmerolae

mtDNA, located downstream of

rpl6

, at the sameposition at which we have identified a homolog in the

Cya-nidium caldarium

mtDNA sequence (see below).Comparison of gene order in the three completely se-

quenced rhodophyte mtDNAs reveals that the highest re-semblance is between the

P. purpurea

and

Chondrus

Figure 1. Physical and Gene Map of P. purpurea mtDNA.

Genes, exons, and nonintronic ORFs are depicted as black blocks and intronic ORFs as green blocks, with gene abbreviations listed in Table 1.Gene blocks outside and inside the circle are transcribed clockwise and counterclockwise, respectively. Regions within which genes overlap areshown in yellow. Transfer RNA genes are shown as thin black bars, with the corresponding letters indicating their amino acid specificities (seeTable 2) and numbers denoting different genes specific for the same amino acid. The anticodons of the numbered tRNA genes are as follows: L1,(UAA); L2, (UAG); S1, (GCU); S2, (UAG); G1, (UCC); G2, (GCC); R1 (ACG); and R2, (UCU). Me and Mf are elongator and initiator trnM(cau), respec-tively. Color coding of names identifies mitochondrial genes/ORFs that are typically present in fungal and animal mtDNAs (black), that have beenfound in protists and plants (blue), or that are unique to P. purpurea (green). A size scale is indicated, and the EcoRI restriction map is shown on theinnermost circles. Two restriction sites, at positions 5198 and 14334 (asterisks), coincide with polymorphic sites. Magenta arrows and blocks de-note the two copies of the inverted repeat element, and the double-headed arrow indicates the position of the proposed bidirectional promoter.

1678 The Plant Cell

crispus

maps. The

P. purpurea

mtDNA map can be brokeninto five pieces, which, when rearranged in order and orien-tation, yields a gene arrangement that differs from the

Chon-drus crispus

map by only a few single-gene transpositions,deletions, and insertions, as well as the presence or ab-sence of unique ORFs. Three gene clusters are conservedamong

P. purpurea

,

C. crispus

, and

Cyanidioschyzon mero-lae

mtDNAs, the longest comprising

atp6

-

atp8

-

nad5

-

nad4

-

nad2

-

nad1

-

nad3

. In

P. purpurea

and

C. crispus

mtDNAs,

sdh4

is inserted between

nad4

and

nad2

, whereas

sdh4

islocated elsewhere in the

Cyanidioschyzon merolae mito-chondrial genome. Furthermore, rns-nad4L-rnl occurs in allthree genomes, except that yejU is inserted between nad4Land rnl in C. merolae mtDNA. Finally, the linkage groupcox2-cox3-ymf39 is found in all three mtDNAs.

Vestiges of bacterial operon structure are seen in theserhodophyte mtDNAs. The ribosomal protein clusters rps3-rpl16-rps11 in P. purpurea, rps3-rpl16 in Chondrus crispus,and rps3-rpl16-rpl14-rpl5-rps14-rps8-rps11 in Cyanidio-schyzon merolae mtDNA maintain the same relative gene or-der as in the adjacent str, S10, spc, and a operons of E. coli.Apparently, four adjacent genes in the cluster of Cyanid-ioschyzon merolae mtDNA, that is, rpl14-rpl5-rps14-rps8,have been lost in P. purpurea and C. crispus mtDNAs, prob-ably in a single event, during the evolution of Bangiales andGigartinales.

In Cyanidium caldarium, a 10-kb stretch of the 30-kb long,circular mtDNA has been sequenced (Viehmann, 1995). Ex-cept for the absence of two tRNA genes in Cyanidium cal-darium mtDNA that appear in the corresponding region ofCyanidioschyzon merolae mtDNA, the order of the 20 genescontained in this fragment is identical in these two species,suggesting that mitochondrial gene order and content arevirtually the same in these two acidothermophilic rhodo-phytes.

rRNA Genes Encoded in P. purpurea mtDNA

The mitochondrial genome of P. purpurea encodes conven-tional, eubacteria-like LSU and SSU rRNAs. Their predictedsizes are 2546 and 1407 nucleotides, respectively, shorterthan those of their E. coli counterparts (2904 and 1542 nu-cleotides, respectively). This length difference reflects thesomewhat truncated variable regions found in the P. pur-purea mitochondrial rRNAs compared with the E. coli ones;otherwise, the secondary structures of the mitochondrialand eubacterial homologs are essentially superimposable.(Secondary structures are available from the WWW rRNAdatabase maintained by R.R. Gutell at the University ofTexas; http://pundit.icmb.utexas.edu/RNA/. Direct links tothese structures are also provided through GOBASE, theOrganelle Genome Database [Korab-Laskowska et al.,1998; http://megasun.bch.umontreal.ca/gobase].)

In the region between positions z240 and 265, the mito-chondrial LSU rRNA of P. purpurea lacks a recognizablesecondary structure element analogous to that found be-tween positions z300 and 340 in the E. coli LSU rRNA andin a separate small 3S rRNA in some naturally fragmentedchloroplast LSU rRNAs (Turmel et al., 1991). In addition, theP. purpurea mitochondrial LSU rRNA sequence contains adeletion in the region corresponding to E. coli 23S rRNA po-sitions z485 and 510. Both of these atypical features char-acterize animal and most fungal mitochondrial LSU rRNAs.Finally, a tertiary base pair interaction corresponding toG570:C866 in E. coli SSU rRNA is an unusual G•G pair inthe case of P. purpurea.

The P. purpurea mitochondrial rRNA secondary structuresare very similar to those encoded by the mtDNA of Chon-drus crispus (Leblanc et al., 1995b), with the same variableregions closely approximating one another in size and most

Table 1. Genes Identified in P. purpurea mtDNAa

rRNAs (2)Small subunit (1): rnsLarge subunit (1): rnl

Transfer RNAs (24) (see Figure 1 and Table 2)Ribosomal proteins (4)

Small subunit (3): rps3, rps11, rps12Large subunit (1): rpl16

Electron transport and oxidative phosphorylation (17)Respiratory chain (14)

NADH dehydrogenase (7): nad1, nad2, nad3, nad4, nad4L,nad5, nad6

Succinate:ubiquinone oxidoreductase (3): sdh2, sdh3, sdh4Ubiquinol:cytochrome c oxidoreductase (1): cobCytochrome c oxidase (3): cox1, cox2, cox3

ATP synthase (3): atp6, atp8, atp9Group II intronic ORFs (2)

orf544 (rnl intron 1)orf546 (rnl intron 2)

Other proteins (3)rtl (reverse transcriptase–like protein)dpo (DNA polymerase)ymf16b (homolog of E. coli mttB [tatC])

Conserved ORFs of unknown function (1)ymf39b

ORFs unique to P. purpurea mtDNA (4)orf132orf176orf238orf284

a Genes are classified according to their function. Numbers withinparentheses indicate the number of genes in a particular class.b The ymf designations for ORFs follow the recommendations of theCommission on Plant Gene Nomenclature (1993). ymf39 has beenshown to specify a membrane component of unknown function inangiosperms (Stamper et al., 1987; Prioli et al., 1993). Note thatymf19 (orfB) homologs have recently been identified as atp8 (Gray etal., 1998) and that ymf16 has been recognized as a homolog of E.coli tatC (or mttB), which codes for a protein involved in a Sec-inde-pendent protein translocation pathway (Bogsch et al., 1998; Weineret al., 1998).

Porphyra purpurea Mitochondrial DNA 1679

of the proven or predicted tertiary interactions found in thehomologous red algal rRNA sequences. Overall, however,the structures of the P. purpurea mitochondrial rRNAs ap-pear more derived than those of C. crispus in that atypicalfeatures noted above (absence of several secondary struc-ture elements and of tertiary base pair interactions) are notseen in the C. crispus rRNAs. Nevertheless, in phylogeneticanalyses of mitochondrial SSU rRNA sequences, P. pur-purea and C. crispus form a tight clade without significantdifferences in branch lengths. In such analyses, the relation-ship of the monophyletic red algal clade with other phyla isuncertain (D.F. Spencer and M.W. Gray, unpublished re-sults).

Two group II introns are present in the P. purpurea mito-chondrial rnl gene; both are inserted within the highly con-served peptidyl transferase center in the 39 half of the LSUrRNA between nucleotides corresponding to 2508 to 2509and 2610 to 2611 of the E. coli sequence. Intron 1 is locatedat exactly the same position as intron 4 in the mitochondrialrnl of the brown alga Pylaiella littoralis (Fontaine et al., 1995).The potential RNA structures of these introns and charac-teristics of the ORFs they contain are described in a latersection.

Mitochondrial 5S rRNA Genes in Red Algae

The distribution of the mitochondrial 5S rRNA gene (rrn5) isquite sporadic within various eukaryotic phyla. Originally,this gene was described only in the mtDNA of plants, somegreen algae, and jakobid flagellates. Subsequently, we de-tected rrn5 in the mtDNA of Chondrus crispus, situated be-tween nad3 and rps11 (Gray et al., 1998), after the originalclaim—that a 5S rRNA gene is located between cox2 andcox3 (Leblanc et al., 1995a)—had been discounted (Lang etal., 1996). Moreover, we have determined that Cyanidiumcaldarium contains a mitochondrially encoded rrn5 in the in-tergenic region between rpl6 and trnM(cau); at this same ge-nomic location, Ohta et al. (1998) identified a counterpartrrn5 in Cyanidioschyzon merolae mtDNA. However, despitean exhaustive search, we have failed to unearth such a genein P. purpurea mtDNA.

Figures 2A to 2C depict the mitochondrial 5S rRNAs fromChondrus crispus, Cyanidium caldarium, and Cyanidioschy-zon merolae, which share evident sequence identities andsecondary structure similarities with 5S rRNA sequences ofeubacteria and plant and protist mitochondria (Lang et al.,1996). However, there are two deviations from otherwiseuniversally conserved features of the mitochondrial consen-sus structure. (1) The Cyanidium caldarium 5S rRNA has aU32:A58 pairing instead of the otherwise conserved A:Upair at the corresponding positions in helical domain III,and CCU (positions 40 to 42) instead of CCA in loop C (thelatter feature shared with the Cyanidioschyzon merolaehomolog). (2) Compared with their counterparts in other eu-karyotes, including Chondrus crispus, helices II and V appear

Figure 2. Potential Secondary Structures of Red Algal Mitochon-drial 5S rRNAs.

(A) Chondrus crispus.(B) Cyanidium caldarium.(C) Cyanidioschyzon merolae.The nucleotide sequences were taken from GenBank accessionnumbers Z47547 (Leblanc et al., 1995a), Z48930 (Viehmann, 1995),and D89861 (Ohta et al., 1998), respectively. Structures were mod-eled after that of wheat mitochondrial 5S rRNA (Spencer et al., 1981;De Wachter et al., 1982). Invariant residues in mitochondrial 5S rRNAsare circled, and broken lines enclose variable regions. Nonstandardpairings are highlighted by small filled (purine–pyrimidine), smallopen (purine–purine), and large filled (pyrimidine–pyrimidine) circles.Helices (I to V) and loops (A to E) are denoted as in Moore (1995).

1680 The Plant Cell

thermodynamically much less stable in the mitochondrial 5SrRNA secondary structures of Cyanidium caldarium and Cy-anidoschyzon merolae.

Mitochondrial Transfer RNA Genes and Codon Usage in P. purpurea mtDNA

P. purpurea mtDNA encodes 24 tRNA genes that are scat-tered over the entire genome, either singly or in groups oftwo or three. All of these tRNA sequences can assume stan-dard cloverleaf secondary structures, with very few depar-tures from the conventional structure. Most notable is an extraU between positions 12 and 13 in the D stem of tRNAAsn(GUU).Additional atypical features (using the standard tRNA num-bering system) include the following: G48 (last nucleotide ofthe variable loop) versus Y in tRNACys(GCA); C9 (betweenacceptor and D stems) versus R in tRNAGlu(UUC); G8 (alsobetween acceptor and D stems) versus Y, and G14 (in Dloop) versus A in tRNAHis(GUG); and A32 (first nucleotide ofanticodon loop) versus Y in elongator tRNAMet(CAU). The lat-ter feature is found frequently in the mitochondrial elongatortRNAMet(CAU) of protists (M.W. Gray, unpublished obser-vation), including that of Chondrus crispus and Cyanidio-schyzon merolae. The single polymorphic site (see below)

affecting a tRNA gene falls in the anticodon arm of tR-NAAla(UGC). This polymorphism (C↔T) would have a mini-mal effect on the overall secondary structure stabilitybecause both C42 and U42 would pair with G28. All of theunusual features of the mitochondrial tRNAs in P. purpureaare also found in the counterpart genes of C. crispus, withthe exception of the extra U nucleotide in the D stem of tR-NAAsn(GUU), which occurs in an otherwise strongly con-served stretch of sequence.

Table 2 shows that the 24 P. purpurea mitochondrial tR-NAs are sufficient to decode 57 of 62 sense codons that oc-cur in mtDNA-encoded protein genes of this organism (TAA/TAG being used as stop codons). tRNA genes not found inthis genome are those recognizing isoleucine (ATA) andthreonine (ACN) codons; in these cases, tRNA import fromthe cytosol presumably makes up the deficit. Interestingly, atrnT gene is also absent from the mtDNAs of golden algae,stramenopiles, a jakobid flagellate, and land plants (seeGray et al., 1998), suggesting a number of independentlosses of this particular gene from mtDNA in the course ofevolution.

P. purpurea mtDNA encodes two glycine tRNAs, tRNAGly

(GCC) and tRNAGly(UCC), which would be functionally re-dundant if the latter species were able to recognize all fourglycine codons. However, in E. coli, a C to U substitution at

Table 2. Codon Frequencies and tRNA Recognition Pattern in P. purpurea Mitochondria

AAa Codon Anticodonb %c AAa Codon Anticodonb %c AAa Codon Anticodonb %c AAa Codon Anticodonb %c

F TTT gaa 74 S TCT uga 33 Y TAT gua 63 C TGT gca 59F TTC gaa 26 S TCC uga 11 Y TAC gua 37 C TGC gca 41L TTA uaa 46 S TCA uga 18 * TAA 96 W TGA ucad 74L TTG uaa 12 S TCG uga 4 * TAG 4 W TGG ucad 26L CTT uag 13 P CCT ugg 50 H CAT gug 63 R CGT acge 22L CTC uag 4 P CCC ugg 11 H CAC gug 37 R CGC acge 11L CTA uag 21 P CCA ugg 29 Q CAA uug 77 R CGA acge 9L CTG uag 4 P CCG ugg 10 Q CAG uug 23 R CGG acge 3I ATT gau 9 T ACT — 38 N AAT guu 62 S AGT gcu 21I ATC gau 14 T ACC — 15 N AAC guu 38 S AGC gcu 13I ATA — 47 T ACA — 37 K AAA uuu 84 R AGA ucu 46M ATG cauf 100 T ACG — 11 K AAG uuu 16 R AGG ucu 9V GTT uac 32 A GCT ugc 43 D GAT guc 66 G GGT gcc 42V GTC uac 11 A GCC ugc 13 D GAC guc 34 G GGC gcc 14V GTA uac 44 A GCA ugc 32 E GAA uuc 75 G GGA ucc 32V GTG uac 14 A GCG ugc 12 E GAG uuc 25 G GGG ucc 12

a Amino acid (one-letter code). Asterisks denote termination codons.b Anticodons are shown in lowercase letters. Dashes indicate tRNAs whose genes are not present in P. purpurea mtDNA. tRNAs with U in thewobble position of the anticodon are assumed to be able to decode all four members of four-codon families (i.e., leucine, CTN; valine, GTN;serine, TCN; proline, CCN; alanine, GCN).c Percentage of each amino acid specified by a given codon in P. purpurea mtDNA.d tRNATrp(UCA) is able to recognize TGA as well as TGG, consistent with the presence of numerous TGA codons in P. purpurea mitochondrialprotein-coding genes.e The A is assumed to be converted to I (inosine) post-transcriptionally, with the resulting tRNAArg(ICG) able to decode all four CGN argininecodons (Pfitzinger et al, 1990).f Separate elongator (e) and initiator (f, formyl) tRNA Met are present.


position 32 (first position in anticodon loop) restricts the de-coding capacity of a tRNAGly(UCC) to the codons GGA andGGG (Lustig et al., 1993). To recognize the GGY codons, thesecond tRNAGly with a GCC anticodon is required. In fact, P.purpurea mitochondrial tRNAGly(UCC) does have a U ratherthan a C nucleotide at position 32. The two trnG genes showsignificant sequence similarity (64% identical residues), sug-gesting that they arose via a recent gene duplication anddivergence from a common, ancestral trnG gene. Also,tRNASer(GCU) and tRNASer(UGA) have presumably arisen bygene duplication because they are 71% identical in se-quence.

The tRNA gene set encoded in the P. purpurea and Chon-drus crispus mtDNAs is identical, except that the latter lackstrnS(gcu). P. purpurea and Cyanidioschyzon merolae alsohave the same mitochondrial tRNA gene set, except that thelatter encodes an additional trnL(caa) and its trnW has aCCA rather than a UCA anticodon.

Table 2 also lists the codon distribution in P. purpureaprotein-coding genes, which, as in most mtDNAs, is biasedtoward codons ending in an A or T. No significant differencein codon usage was observed in identified protein-codinggenes, conserved ORFs, unique ORFs, and intronic ORFs.Moreover, there is a GTG codon in the P. purpurea cox1gene at exactly the same position as an ATG initiator codonin the corresponding Chondrus crispus gene, strongly sug-gesting that GTG serves as a start codon in mitochondrialtranslation in P. purpurea; use of GTG initiation codons hasbeen inferred previously in several plant and protist mito-chondrial as well as bacterial systems (e.g., Netzker et al.,1982; Kozak, 1983; Lang et al., 1997). Finally, TGA is trans-lated in P. purpurea mitochondria as tryptophan (inferredfrom the presence of a tRNA with anticodon UCA and multi-ple protein sequence alignments). This is the most commondeviation from the standard translation code in mitochondriaand is also found in C. crispus mtDNA; in contrast, mito-chondria of Cyanidium caldarium and Cyanidioschyzonmerolae use the universal genetic code.

Unassigned Reading Frames and Pseudogenes inP. purpurea mtDNA

Four unique ORFs (orf132, orf176, orf238, and orf284) arepresent in the mtDNA of P. purpurea. Whereas the deducedOrf132 and Orf176 proteins exhibit an intermediate degreeof polarity, the amino acid composition of Orf238 is charac-teristic of hydrophobic membrane components (hydropho-bicity 59.5; Kyte and Doolittle, 1982). Orf284, in contrast,contains a high proportion of charged and polar residues(hydrophobicity 210.6), comparable with that of ribosomalproteins.

As mentioned earlier, the coding regions of rtl and dpo arediscontinuous, that is, broken up into two and four adjacentgene fragments, respectively, whose order and orientationare maintained. Fragmentation of both genes is due to in-

frame TAA-termination codons or frameshifts (apparentlygenerated by single-nucleotide deletions). As discussed be-low, rtl seems to be a vestige of a group II intron ORF, andtherefore we assume that it is not expressed, although itscodon usage does not differ significantly from that of con-served mitochondrial genes.

A partial dpo sequence has been obtained from another,uncharacterized Porphyra sp (GenBank accession numberX65264). Figure 3 shows that the sequence translates, incontiguous fashion, into that portion of the dpo gene thatcorresponds to P. purpurea fragments 1 and 2, plus the 59-terminal part of fragment 3. Unexpectedly, the Dpo proteinsof the two Porphyra spp share only 39.5% identity. Such ahigh fluidity in gene sequence and structure within the samegenus indicates that dpo is evolving extremely rapidly and isprobably a pseudogene. Split and scrambled dpo se-quences also occur in the liverwort M. polymorpha, whosemitochondrial genome contains three ORFs that display sig-nificant similarity to the N-terminal, middle, and C-terminalportion of P. purpurea dpo (Figure 3). In contrast, a continu-ous dpo is found in maize mitochondria, where it is encodedby a plasmid (Paillard et al., 1985), and in the mitochondriaof the golden-brown alga Ochromonas danica (G. Burger, A.Coleman, and B.F. Lang, unpublished results), where it iscontained in the mtDNA itself. It has been suggested thatdpo was not originally contained in the proto-mitochondrialgenome but was introduced via mobile plasmids at an ad-vanced point in the evolutionary history of mitochondria(Weber et al., 1995).

Introns and Free-Standing Intron Fragments inP. purpurea mtDNA

Two group II introns, 2483 and 2478 nucleotides long, inter-rupt the LSU rRNA coding region in P. purpurea mtDNA. Ac-cording to their RNA secondary structures (Figure 4), theseintrons belong to subgroup B1 (Michel et al., 1989). Both in-trons contain ORFs, inserted within domain IV, that code forproteins 544 and 546 residues long. These ORFs possess allof the conserved motifs characteristic of reverse tran-scriptases of non-LTR retrotransposons (Michel and Lang,1985; Xiong and Eickbush, 1990) as well as the particularprotein domains (reverse transcriptase, Z, zinc finger, andthe C-terminal HNH motif) that are believed to be involved inendonucleolytic cleavage and intron homing (Mohr et al.,1993; Shub et al., 1994).

Figures 4A and 4B show that introns 1 and 2 in P. pur-purea mtDNA resemble one another to a high degree. Theintron RNA secondary structures are virtually identical, thededuced protein sequences of the two intron ORFs (Orf544and Orf546) share 70% identical residues and 20% con-served changes, and the nucleotide sequence outside thereading frames can be aligned readily (68% identity). Such apronounced resemblance suggests that intron transpositionhas occurred in cis into the same gene (Ferat et al., 1994).

1682 The Plant Cell

Indeed, the sequences surrounding the two intron insertionsites in rnl share nearly 50% identical residues (nine out of20 nucleotides).

Database searches with the Orf544 and Orf546 proteinsreveal striking similarity to intronic reverse transcriptaseORFs of the cyanobacterium Calothrix sp (Ferat and Michel,1993; BLAST score 344, probability 3.0 3 102171). The sec-ond highest score was found with mitochondrial intron ORFproteins from the brown alga Pylaiella littoralis; these algalORFs had earlier been reported to share common featureswith cyanobacterial counterparts (Fontaine et al., 1995).Overall protein similarity is considerably lower between thePylaiella littoralis and Calothrix sp (32%) or Pylaiella littoralisand P. purpurea (23%) ORFs than between the P. purpureaand Calothrix sp homologs (44%).

In addition, the introns from P. purpurea mtDNA and Calo-thrix sp in which these ORFs reside are extraordinarily alike.Figure 4B shows that the few obvious differences in theirpotential RNA secondary structures are three additionalstem–loop elements inserted in domain I of the P. purpureaintrons; otherwise, there are only minor variations in helixlength and loop size in the six domains (for domain assign-ments, see Michel et al., 1989). Finally, the P. purpurea andCalothrix sp introns share significant similarities at the nucle-otide sequence level, primarily in domain V and in the basalregions of domains I and III, which are generally poorly con-served. Such a striking degree of structural resemblance isnot seen between the rnl introns from P. purpurea and Py-laiella littoralis or between the Calothrix sp and Pylaiella lit-toralis introns.

In addition to the two rnl introns described above, we de-tected relics of a group II intron in P. purpurea mtDNA. A typi-cal domain V consensus structure is located downstream ofand partially overlaps with the C-terminal region of rtl; how-ever, a complete intron core structure could not be identified.Presumably, the rtl gene was initially contained in a group IIintron, and fragmentation of its reading frame occurred con-comitantly with the loss of its conserved RNA secondarystructure. Because the intron no longer resided in a vital gene,there would have been no selective pressure to conserve theRNA secondary structure that is required for proper splicing.

Phylogenetic Analysis of Group II Intron ORFs

To investigate the evolutionary distances of the P. purpureaintron ORFs relative to other reverse transcriptase proteins,

440; Porph. purp., Porphyra purpurea DNA polymerase fragments ato d (this report; the start sites of fragments b to d are shown in low-ercase letters at positions 93, 158, and 432); Ochr. dani., Ochromo-nas danica (G. Burger, B.F. Lang, and A. Coleman, unpublished data);and Zea mays (S07183 [protein]; Paillard et al., 1985). Residues 191to 790 of the maize sequence are included in the alignment.

Figure 3. Alignment of Mitochondrially Encoded DNA PolymeraseProtein Sequences from Algae and Plants.

Protein sequences were inferred from the corresponding DNA se-quences. Residues identical in >75% of taxa are boxed. Stretchesof >4 consecutive residues that are identical or that represent con-servative exchanges in all compared taxa are highlighted by whiteletters on a black background; if this feature applies to the two Por-phyra spp only, then the corresponding residues are shown in boldface.Dashes denote alignment gaps, asterisks identify the C terminus ofthe proteins, and dots indicate that the polypeptides continue be-yond the region for which the sequence is shown. Porph. sp, Por-phyra sp (X65264), the partial sequence of which ends at residue


Figure 4. Similarities between Group II Introns from P. purpurea Mitochondria and Calothrix sp.

(A) Intron 1 in rnl of P. purpurea mtDNA. Nucleotide (nt) positions identical with those in intron 2 of the same genome are shown in blue.Residues identical in the two P. purpurea introns and in the group II intron of the cyanobacterium Calothrix sp (Ferat and Michel, 1993;GenBank accession number X71404) are shown in red.(B) Intron 2 in rnl of P. purpurea mtDNA. Secondary structure elements not present in the Calothrix sp intron are marked by green shading.Tertiary base pairings are indicated by EBS1:IBS1, EBS2:IBS2, and a:a9 to h:h9; the involved nucleotides are boxed or marked by arrows (Costaet al., 1997).

1684 The Plant Cell

phylogenetic analyses were performed on a collection ofreadily alignable reverse transcriptase proteins of mito-chondrial, chloroplast, and bacterial origin. Bootstrap sup-port (Felsenstein, 1985) and likelihood estimations(Kishino et al., 1990) were high with all tree constructionmethods applied. Figure 5 depicts the resulting phyloge-netic reconstruction, which reveals two large clusters ofreverse transcriptase proteins, one including the classicmitochondrial intron ORFs of plants and fungi that are allinserted in group IIA introns, the other cluster includingmembers from protist mitochondria, chloroplasts, andbacteria, which reside in group IIB introns. A similar bipar-tite tree structure of group IIA and IIB intron ORFs hasbeen reported previously (Fontaine et al., 1995). Themost closely related reverse transcriptase proteins arethose from P. purpurea mitochondria and Calothrix sp, re-

inforcing the view of a very recent lateral intron transferbetween cyanobacteria and P. purpurea mitochondria.

Inverted Repeats in P. purpurea mtDNA

In P. purpurea mtDNA, a sequence stretch of 291 bp oc-curs twice, with the two copies located roughly oppositeone another on the circular mtDNA map and in invertedorientation (Figure 1, magenta arrows). These repeated el-ements, designated INV1 and INV2, extend into the 5 9

portion of orf238 and orf132, whose protein sequencesare consequently identical between amino acid positions1 and 94. In angiosperm mitochondria, inverted (and alsodirect) repeats appear to promote major genome rear-rangements (Hanson and Folkerts, 1992). Hence, we in-

Figure 5. Phylogenetic Relationships among Group IIB Intron ORF Proteins from Mitochondria and Bacteria.

A combination of PROTDIST and FITCH with a variation coefficient of 0.3 was used, as described in Methods. The same tree topology was ob-tained when maximum likelihood approaches (PROTML and PUZZLE) were used. Bootstrap support, calculated from 1000 replicates, is shownat each branch (in percentages; rounded numbers). Ovals highlight short and poorly supported branches (bootstrap support ,80%). mt, cp, andbc indicate mitochondrial, chloroplast, and bacterial sequences, respectively. Designations (GenBank accession numbers within parentheses)are as follows: Calothrix, orf584 bc, “Orf2,” contained in an intron of an unknown gene of Calothrix sp, see legend to Figure 4; Clostridium,orf609 bc, intronic ORF of C. difficile transposon (X98606; Mullant et al., 1996); E. coli, orf416 bc, intronic ORF in E. coli DNA (S50828 [protein];Ferat et al., 1994); Marchantia, cob-3 mt, ORF in cob intron 3; Marchantia, cox1-2 mt, ORF in cox1 intron 2 of M. polymorpha mtDNA (M68929;Oda et al., 1992); Porphyra, rnl-1 mt, Orf544 in rnl intron 1; Porphyra, rnl-2 mt, Orf546 in rnl intron 2 of P. purpurea mtDNA (this work); Pylaiella,rnl-1 mt, ORF in rnl intron 1; Pylaiella, rnl-2 mt, ORF in rnl intron 2 of P. littoralis mtDNA (S58503, S58504 [protein]; Fontaine et al., 1995); Saccha-romyces, cox1-1 mt, ORF in cox1 intron 1; Saccharomyces, cox1-2 mt, ORF in cox1 intron 2 of S. cerevisiae mtDNA (V00694; Bonitz et al.,1980); Scenedesmus, petD-1 cp, ORF in petD intron 1 of S. obliquus chloroplast DNA (P19593 [protein]; Kück, 1989); Schizosaccharomyces,cob-1 mt, ORF in cob intron 1 of S. pombe mtDNA (X02819; Lang et al., 1985); and Streptococcus, orf425 bc, maturase-related protein in S.pneumoniae DNA (AF030367; Coffey et al., 1998).


vestigated whether this is also the case in P. purpureamtDNA. Polymerase chain reaction (PCR) amplification wasperformed with primers that anneal in the single-copy re-gions, 90 bp upstream of INV1 and 95 bp downstream ofINV2, respectively (see Figure 1, genome map). If, in P. pur-purea mitochondria, molecules are present that had under-gone intramolecular recombination between the invertedrepeats, a PCR product of 476 bp should be found, whereasthe experimental conditions would not amplify the 14,653-bp fragment that corresponds to the configuration (shown inFigure 1) inferred from sequencing the random clone library.In fact, we obtained a PCR product of z480 bp and con-firmed its identity by DNA sequencing. Although potentialPCR artifacts cannot be excluded readily, we infer that the476-bp fragment reflects the presence of a low proportion ofmtDNA molecules in an alternative genomic conformationthat results from intramolecular recombination involving theinverted repeats.

Sequence Polymorphisms in P. purpurea mtDNA

In P. purpurea mtDNA, we observed 64 polymorphisms ofthree different types (Table 3). (1) Single-nucleotide substitu-tions in coding as well as intergenic regions are the mostabundant type. These substitutions involve only two of thefour possible nucleotides and are predominantly transitions.(2) Deletions/insertions (indels) of from 1 to 43 bp are lo-cated exclusively in intergenic regions. (3) A macrosubstitu-tion of 53 or 494 bp is found after nucleotide 11,409 (Figure1). The longer substitution, representing the version of thesequence deposited in GenBank, extends the 39 part of dpoby 36 codons and the 59 portion of rtl by 50 codons, com-pared with the shorter version.

Intergenic regions contain approximately twice as manypolymorphic sites per kilobase than coding regions. Amongcoding regions, rtl, dpo, and ORFs (except orf284, see be-low) harbor approximately sixfold more polymorphic sitesper kilobase and exhibit an approximately fourfold higher ra-tio of nonsilent to silent single-nucleotide substitutions com-pared with conserved genes. ORFs and fragmented genesin P. purpurea mtDNA are apparently subject to minimal se-lective pressure and therefore can evolve at a faster pacethan do genes involved in vital functions such as electrontransfer, ATP synthesis, and translation. Notably, orf284contains significantly fewer polymorphic sites (1.1 per kilo-base) than do other unique ORFs (Table 3). Considering thatthe encoded protein has a high number of charged and po-lar residues, orf284 might code for a poorly conserved, andtherefore unrecognizable, ribosomal protein.

We further investigated whether the states of adjacentpolymorphic sites correlate at the level of individual clones.In most adjacent pairs examined (43 of 49), we detected co-variance, suggesting that the mtDNA used to construct theclone library contained two distinct types of molecules. Re-markably, most noncovariant adjacent pairs of sites are lo-

calized to the inverted repeat copies. This finding confirmsthe conclusion of the previous section that these repeats areinvolved in intramolecular recombination.

The polymorphic sites within the inverted repeats of P.purpurea mtDNA account for a 1.4% sequence variationwithin these repeats. This high variability stands in markedcontrast to the situation in the chloroplast genome ofmany green algae and land plants, in which the two in-verted repeat copies, which include the z10-kb rRNAgene cluster, are identical over their entire length. A copycorrection mechanism similar to gene conversion in bac-teria is thought to maintain the sequence identity of thetwo repeat regions in these chloroplasts (Lemieux andLee, 1987). If recombination processes do indeed takeplace in P. purpurea mitochondria, as indicated by the twodifferent molecular conformations detected by PCR experi-ments, it is puzzling why the recombination machinerywould support homologous recombination but not geneconversion. It should be noted that the chloroplast DNAof P. purpurea also contains two repeats encompassingrRNA genes; however, unlike in green algal and plantchloroplast DNAs, these repeats are arranged in directorientation, differ from one another in sequence, and donot appear to support intramolecular recombination (Reithand Munholland, 1993b).

To our knowledge, P. purpurea is the only protist in whichconspicuous mtDNA sequence polymorphisms have beendocumented to date, with two major molecular classes de-tected in the present study. Moreover, there are indica -tions of two additional classes generated by inversion of

Table 3. Sequence Polymorphisms in P. purpurea mtDNA

Gene/Regiona

Substitution

Single ntb

Indel Macrod

Total Numberd

Numberper kbTotal

Non-silentc

Intergenic regions (55) 7 — 4 0 11 3.3

Intron cores (2) 5 — 0 0 5 3.0RNAs (26) 5 — 0 0 5 0.9Conserved

proteins (23) 10 2 0 0 10 0.6Rare proteins (2) 8 7 0 1* 9* 3.6Intronic ORFs (2) 14 10 0 0 14 4.2Unique ORFs (4) 10 6 0 0 10 4.0Total mtDNA 59 — 4 1 64 1.7

a Classes of genes and genome regions in P. purpurea mtDNA inwhich polymorphic sites occur. Numbers within parentheses denotethe total number of genes or regions within a class.b nt, nucleotide.c A dash means not applicable.d Asterisks indicate that a single macro substitution affects both dpoand rtl.

1686 The Plant Cell

approximately half of the mitochondrial chromosome rela-tive to the other half. These findings raise questions aboutthe homogeneity of the DNA material that was used for con-structing the clone library. For mtDNA extraction, gameto-phytes (folious thalli) of P. purpurea were collected in thewild, offshore Nova Scotia, Canada, and propagated in thelaboratory (Reith and Munholland, 1993a). The observedvariance in the sequenced DNA material suggests a consid-erable heterogeneity of the mitochondrial genome withincross-fertilizing populations of P. purpurea.

Global Phylogenetic Analysis Using Mitochondrial Protein-Coding Genes

We have analyzed the phylogenetic relationship within rhodo-phytes and between rhodophytes and chlorophytes by in-cluding new mitochondrial sequence data from the redalgae P. purpurea (reported here), Cyanidioschyzon merolae(Ohta et al., 1998), and Gracilariopsis lemaneiformis (Langand Goff, 1999) as well as of the green alga Tetraselmismaculata (this report). For the analysis, we used the concat-enated, deduced protein sequences of four genes, cox1,cox2, cox3, and cob, from 33 different taxa. Both distanceand likelihood algorithms were applied, with the analysesgenerating identical topologies and similar bootstrap sup-port and likelihood estimations.

Figure 6 shows within the red algal clade a deep diver-gence between Cyanidium caldarium and Cyanidioschzonmerolae, on the one hand, and Porphyridales, Bangiales,and Gracilariales on the other. P. purpurea (representingBangiales) emerges after the divergence of the “Cyanidiumcomplex” but before Porphyridales and Gracilariales. Nota-bly, the Cyanidium caldarium and Cyanidioschyzon merolaebranch lengths are approximately two times as long asthose of the other three rhodophyte taxa, reflecting an ac-celerated rate of evolution at the sequence level in these ac-idothermophilic species.

The tree as a whole is characterized by coherent clades ofrhodophytes, chlorophytes/plants, animals, and fungi; theoverall branching pattern implies that green and red algaeare sister taxa that evolved from a common ancestor to theexclusion of animals and fungi. The distance between thebacterial and mitochondrial clades is conspicuously large,whereas within the mitochondrial clade, the intervals be-tween the major nodes are small, implying a nearly simulta-neous, “explosive” radiation of all eukaryotic taxa.

To resolve further the branching order of green and red al-gae relative to that of the common ancestor of animals andfungi, we included in the analysis additional protists, such asR. americana, Malawimonas jakobiformis, and Jakoha libera(jakobid flagellates; Lang et al., 1997; G. Burger, B.F. Lang,C. O’Kelly, and M.W. Gray, unpublished results), Acan-thamoeba castellanii (rhizopod amoeba; Burger et al., 1995),Phytophthora infestans (oomycete; B.F. Lang and L. Forget,unpublished results), and O. danica (chrysophyte alga; G.

Burger, B.F. Lang, and A. Coleman, unpublished results)(Figure 6). Stramenopiles (O. danica and P. infestans) on theone hand and jakobids on the other form strongly supportedclades that, together with A. castellanii, do not specificallygroup with either the green or red algae, or with animals andfungi. However, stramenopiles, jakobids, and rhizopodscannot be positioned on the tree at a significant confidencelevel. We attribute this topological instability particularly tothe absence of a suitable outgroup (reflected by the largedistance between bacterial and mitochondrial proteins), aswell as to a lack of data from early diverging eukaryotic taxa.

DISCUSSION

Very Recent Intron Transfer between the Genomes ofP. purpurea Mitochondria and a Cyanobacterium

ORFs in group II introns fall into two distinct subclasses ofreverse transcriptases. Those contained in group IIB intronsare small and share distinctive protein signatures that setthem apart from those residing in group IIA introns (Ferat etal., 1994; Fontaine et al., 1995). A large majority of the100 or so group II introns known to date belong to sub-group IIA and are found in mitochondrial and chloroplastgenomes. The two mitochondrial introns from P. purpureabelong to subgroup B, as do the only three currently knownbacterial group II introns.

Our phylogenetic analysis of reverse transcriptase ORFs(Figure 4) as well as primary sequence and RNA secondarystructure comparisons show that the P. purpurea mitochon-drial introns are very closely related to counterparts from thecyanobacterium Calothrix sp, a finding of relevance to thelong-standing question about intron origins and propaga-tion. Several ORF-containing group I and II introns havebeen shown to be mobile elements in the sense that in ge-netic crosses, they invade intronless copies of the genes inwhich they are inserted (Dujon et al., 1986; Skelly et al.,1991). This has led to the inference in a number of casesthat introns are also able to spread laterally between unre-lated species, and between genomes from different cellularcompartments (e.g., Lang et al., 1985; Lonergan and Gray,1994; Turmel et al., 1995; Vaughn et al., 1995; Cho et al.,1998). In the case of P. purpurea, cyanobacterial intronsmight have invaded mitochondria via chloroplasts that in-herited their introns directly from a cyanobacterial ancestor.However, the chloroplast DNA of P. purpurea, which hasbeen sequenced completely (Reith and Munholland, 1995),does not contain any introns or reverse transcriptase ORFs,making the hypothesis of intron transfer via chloroplast DNAless likely. A direct, recent intron transmission between a cy-anobacterium and a red algal mitochondrion remains themost plausible scenario to account for the above observa-tions.


Figure 6. Phylogeny of Red and Green Algal Mitochondrial Genomes.

The phylogenetic tree was inferred from an alignment of the concatenated protein sequences of Cob, Cox1, Cox2, and Cox3. A combination ofPROTDIST and FITCH was used, as described in Methods, with a variation coefficient of 0.5. The same tree topology was obtained when maxi-mum likelihood approaches (PROTML and PUZZLE) were used, except for branches with ,80% bootstrap support. Bootstrap support (in per-centages), calculated from 1000 replicates, is shown for all branches. Short and poorly supported branches (,80% bootstrap support) areindicated by thin lines. Animals, fungi, rhizopods, red algae, green algae/plants, jakobids, stramenopiles, and bacteria are indicated and groupedtogether by square brackets. Ciliates, chlamydomonads, kinetoplastids, apicomplexans, and slime molds have been excluded from the analysisbecause these taxa cannot be placed reliably in the tree due to the accelerated evolutionary rate of their mitochondrial genes. Species (withGenBank accession numbers in parentheses) are as follows: Homo, H. sapiens (J01415; Anderson et al., 1981); Mus, M. musculus (J01420; Bibbet al., 1981); Xenopus, X. laevis (M10217; Roe et al., 1985); Strongylocentrotus, S. purpuratus (X12631; Jacobs et al., 1988); Drosophila, D.yakuba (fruitfly; X03240; Clary and Wolstenholme, 1985); Metridium, M. senile (AF000023; Beagley et al., 1998); Rhizopus, R. stolonifer (chytridi-omycete fungus; Laforest et al., 1997; Saccharomyces, S. cerevisiae (ascomycete fungus; X84842, V00694, V00695, J01478; de Zamaroczy andBernardi, 1986); Pichia, P. canadensis (Hansenula wingei) (ascomycete fungus; D31785; Sekito et al., 1995); Aspergillus, A. (Emericella) nidulans(ascomycete fungus; J01387, X15441, X06960; Netzker et al., 1982; Brown, 1993); Neurospora, N. crassa (ascomycete fungus; K01181, A28755[protein], X01850, K00825, V00668; Collins, 1993); Podospora, P. anserina (ascomycete fungus; X55026; Cummings et al., 1990); Allomyces, A.macrogynus (chytridiomycete fungus; U41288; Paquin and Lang, 1996; Paquin et al., 1997); Acanthamoeba, A. castellanii (rhizopod; U12386;Burger et al., 1995); Malawimonas, M. jakobiformis (jakobids; G. Burger, B.F. Lang, C. O’Kelly, and M.W. Gray, unpublished data); Cyanid-ioschyzon, C. merolae; Cyanidium, C. caldarium; Gracilariopsis, G. lemaneiformis, Cox1, Cox2, Cox3 (rhodophyte; AF118119); Chondrus, C.crispus (rhodophytes; see legend to Figure 2); Porphyra, P. purpurea (rhodophyte; this report); Triticum, T. aestivum (P07747 [protein], Y00417,X01108, P15953 [protein]; Bonen et al., 1984, 1987; Boer et al., 1985; Gualberto et al., 1990); Marchantia, M. polymorpha (liverwort; see legendto Figure 5); Prototheca, P. wickerhamii (green alga; U02970; Wolff et al., 1994); Tetraselmis m., T. maculata, Cob (300 amino acids), Cox1 (266amino acids), Cox2 (complete) (green alga; this report); Tetraselmis s., T. (Platymonas) subcordiformis (green alga; Z47797; Kessler and Zetsche,1995); Reclinomonas, R. americana (jakobid; AF007261; Lang et al., 1997); Jakoba, J. libera; Phytophthora, P. infestans (oomycete; B.F. Langand L. Forget, unpublished data); Ochromonas, O. danica (chrysophyte; G. Burger, A. Coleman, and B.F. Lang, unpublished data); Rickettsia, R.prowazekii (a-Proteobacterium; AJ235270 to AJ235273; Andersson et al., 1998); Bradyrhizobium, B. japonicum (a-Proteobacterium; J03176,X68547; Thony-Meyer et al., 1989; Bott et al., 1992); Rhodobacter, R. sphaeroides (a-Proteobacterium; X56157, X62645, M57680, C45164 [pro-tein]; Yun et al., 1990; Cao et al., 1991, 1992; Shapleigh and Gennis, 1992); and Paracoccus, P. denitrificans (a-Proteobacterium; X05829,M17522, X05934, X05828; Kurowski and Ludwig, 1987; Raitio et al., 1987). Unpublished protein sequences are available at http://megasun.bch.umontreal.ca/People/lang/FMGP/proteins.html.

1688 The Plant Cell

Rhodophytes Emerged Simultaneouslywith Chlorophytes

For the first time, mitochondrial sequence information is avail-able from five rhodophyte orders, including Bangiales (P.purpurea, complete sequence; described here), Gigartinales(Chondrus crispus, complete sequence; Leblanc et al.,1995a), Porphyridales (Cyanidium caldarium, 10-kb frag-ment; Viehmann, 1995), Gracilariales (Gracilariales lemanei-formis, 3-kb fragment; Lang and Goff, 1999; GenBankaccession number AF118119), and Cyanidioschyzon (C.merolae, complete sequence; Ohta et al., 1998). In addition,we have information about mtDNA size and shape inGracilariopsis pulvinata (Goff and Coleman, 1995). Taken to-gether, these data should fully encompass mtDNA diversitywithin the red algae. It should be noted that some taxonomichierarchies include the genus Cyanidioschyzon within theCyanidiales, which comprises several small, unicellular, aci-dothermophilic red algae (Ott and Seckbach, 1994). In con-trast, the National Center for Biotechnology Informationtaxon database consortium recognizes Cyanidioschyzon asa separate order comprising a single genus and species.

In nuclear and chloroplast phylogenies, the genus Cyanid-ium forms the deepest branch of the red algal clade (Raganet al., 1994; Saunders and Kraft, 1996; Palmer andDelwiche, 1998), although Porphyridiales as a whole is pos-sibly not a monophyletic group (Gabrielson et al., 1985;Ragan et al., 1994). The order Bangiales diverges basally tomost of the 13 or so other orders, whereas Gigartinales andGracilariales are more recently diverging clades (reviewed inSaunders and Kraft, 1997). The red algal topology in the treethat we infer from mitochondrially encoded proteins (Figure6) is in complete agreement with these nuclear and chloro-plast phylogenies.

Whereas the phylogenetic relationships within the red al-gae are well established, the association of rhodophyteswith other eukaryotic phyla is still highly controversial. In nu-clear SSU rRNA trees, the majority of eukaryotic taxa, in-cluding rhodophytes, cluster tightly together in a nearlysimultaneous radiation (termed the crown group), basal towhich emerge slime molds and a number of other groups(Sogin, 1997). On the other hand, analyses of b-tubulin pro-teins place Chondrus crispus together with slime molds(Liaud et al., 1995). Finally, in a tree based on the large sub-unit of RNA polymerase II (RNAPII; Stiller and Hall, 1997),green algae group together with fungi and animals, to theexclusion of red algae. Although bootstrap support is high inthis study, the green algal/fungal/animal alliance is likely anartifact due to the pronounced divergence of the red algalRNAPII sequences compared with those of green algae;also, insufficient taxonomic sampling (only one red and twogreen algal species) is a concern. As reviewed in detail byPalmer and Delwiche (1998), nuclear phylogenies that in-clude representatives of red algae together with members ofthe major eukaryotic kingdoms are largely unresolved, oftensuffering from long-branch attraction, weak support, or poor

overall sampling (e.g., Zhou and Ragan, 1995; Liu et al.,1996; Keeling and Doolittle, 1997).

In contrast to nuclear trees, several recent phylogeneticanalyses of mitochondrial data point to a sister-group rela-tionship between red and green algae; however, these anal-yses also lack either rigorous statistical or bootstrap support(e.g., Bhattacharya and Schmidt, 1997) or include only a lim-ited number of eukaryotic taxa (e.g., Boyen et al., 1994) or ofrhodophyte taxa in particular (e.g., Paquin et al., 1997; Langet al., 1998). To our knowledge, the mitochondrial phylogenypresented here (Figure 6) is the only analysis to date com-bining high bootstrap support with broad sampling of mi-tochondriate eukaryotes, showing robust coherence ofmembers of the red algae, green algae/plants, animals, andfungi, and providing strong evidence for the divergence ofred and green algae from a shared ancestor. The sister-group relationship of red and green algae is congruent withresults obtained from the analysis of chloroplast genes inwhich green, red, and glaucocystophyte algae cluster to-gether (reviewed in Palmer and Delwiche, 1998). This con-gruence supports the idea that the origin of plastids ismonophyletic and that the primary plastid-containing organ-isms diverged from a single common ancestor.

Rhodophytes Exhibit a Derived Pattern of mtDNA Organization and Complexity

The mtDNAs of P. purpurea, Chondrus crispus, Cyanid-ioschyzon merolae, Cyanidium caldarium, and Gracilariopsisspp are quite similar in size (25 to 36.8 kb), and the fourmtDNAs of the first four species are similar in gene comple-ment and gene order as well. Apart from ribosomal proteingenes, six more of which are present in Cyanidioschyzonmerolae (and probably also Cyanidium caldarium) mtDNAthan in P. purpurea mtDNA, the mitochondrial gene reper-toire in rhodophytes is relatively small. Having a total of z60genes and encoding essentially the animal set of subunits ofrespiratory complex I, III, IV, and V, rhodophyte mtDNAshave apparently lost a substantial number of genes to thenucleus. These features set rhodophyte mtDNAs apart frommitochondrial genomes present in certain green algae, cryp-tophyte algae, and many nonphotosynthetic protists, whichall display a more ancestral pattern of mtDNA organization.

In fact, rhodophyte mitochondrial genomes are reminis-cent of animal (particularly coelenterate) mtDNAs, both withrespect to their conservation of gene order and constancy ofgenome size (reviewed in Wolstenholme and Fauron, 1995).In stark contrast to rhodophytes and animals, the chloro-phyte/plant lineage displays extraordinary variability inmtDNA size, gene content, and overall organization. ThemtDNAs of Prototheca wickerhamii, Nephroselmis olivacea,and M. polymorpha, which are of the ancestral type, containnumerous extra genes compared with animal and fungalmtDNAs. In contrast, derived mitochondrial genomes suchas those of Chlamydomonas spp and Pedinomonas minor


have been subject to considerable gene loss, exceeding eventhat seen in animal mtDNAs, as well as fragmentation andscrambling of rRNA genes (reviewed in Gray et al., 1998).

As inferred from the gene complement in rhodophyte andchlorophyte mtDNAs, the common ancestor of both groupsmust have possessed a mitochondrial gene set that approx-imates that of the heterotrophic flagellate R. americana (94genes; Lang et al., 1997). Approximately half of this set musthave been lost in the red algal lineage soon after its diver-gence from the common progenitor shared with chlorophytes,whereas subsequently, only minor evolutionary changes seemto have occurred.

Are Rhodophytes a Highly Uniform or HighlyDiverse Assemblage?

The remarkable similarities evident among red algal mtDNAs,particularly at the level of gene order and mtDNA size, aswell as the solid coherence and moderate branch lengths ofrhodophyte taxa in the phylogenetic tree shown in Figure 6,exemplify the uniformity of the mitochondrial genome in thisphylum. These findings are in stark contrast to the viewbased on nuclear gene phylogenies, which characterizerhodophytes as an exceptionally diverse phylum (Ragan etal., 1994). Although nuclear and mitochondrial trees are typ-ically congruent in topology, they can differ considerably inphylogenetic distances of particular taxa. The most extremeexamples involve the Chlamydomonas-like green algae.Chlamydomonads are strongly affiliated with other green al-gae and plants in nuclear rRNA phylogenies, whereas in mi-tochondrial rRNA and protein trees, their branches lengthenconsiderably, generating unstable and artifactual topologies(reviewed in Gray and Spencer, 1996). In red algae, the op-posite is true: red algae exhibit exceptionally high diversityin nuclear SSU rRNA phylogenies but are characterized bycompact clustering and moderate distances between oneanother in mitochondrial protein phylogenies. It appears thatthe nuclear SSU rRNA gene in rhodophytes has undergoneaccelerated evolutionary change. Much more nuclear se-quence data will be required, including data from nuclearprotein-coding genes, to evaluate how accurately the nu-clear SSU rRNA sequence reflects the evolutionary pace ofthe red algal nuclear genome.

METHODS

Isolation, Purification, and Cloning of Mitochondrial DNA

Porphyra purpurea was collected near Avonport, Nova Scotia, Can-ada, and then grown in the laboratory in the form of gametophytecultures (Reith and Munholland, 1993b). An AT-rich fraction of P.purpurea DNA, consisting of a mixture of mitochondrial and chloro-plast DNAs, was kindly provided by M. Reith.

Mitochondrial DNA (mtDNA) was isolated by cutting the AT-richDNA fraction with the restriction enzyme SacI and resolving the re-sulting fragments by agarose gel electrophoresis. The largest (35 kb)restriction fragment proved to be the linearized mtDNA, whereas thechloroplast DNA was cut into several fragments of ,15 kb (Reith andMunholland, 1993b). The mtDNA was recovered by electroelutionand physically sheared by nebulization (Okpodu et al., 1994), and afraction of 500 to 3000 bp was recovered after agarose gel electro-phoresis. The DNA was incubated with a mixture of T7 DNA poly-merase and Escherichia coli DNA polymerase I (the Klenowfragment) to generate blunt ends and then cloned into the SmaI siteof the phagemid pBluescript II KS1 (Stratagene). Recombinant plas-mids containing mtDNA inserts were identified by colony hybridiza-tion, using the 35-kb SacI restriction fragment as a probe. Clonescontained in this random library encompassed the entire P. purpureamitochondrial genome. A region of z500 bp bridging the SacI sitewas amplified by the polymerase chain reaction (PCR), cloned, andsequenced, confirming the presence of a single such site in themtDNA.

Tetraselmis maculata CCMP897 was obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (WestBoothbay Harbor, ME) and cultured in seawater. An AT-rich DNAfraction, consisting of chloroplast and mitochondrial DNA, was iso-lated from total cellular DNA by CsCl/Hoechst-33258 dye (Aldrich,Milwaukee, WI) equilibrium centrifugation. Shearing of DNA and sub-sequent steps were conducted as described above.

DNA Sequencing and Data Analysis

DNA sequencing was performed by the dideoxy chain terminationmethod (Sanger et al., 1977), using single-stranded DNA as templateand 35S-dATP as label. Polyacrylamide gels, dried onto the glassplate (Lang and Burger, 1990), were autoradiographed, and se-quences were entered manually into computer files. In addition, au-tomated sequencing was performed on a Li-Cor (Lincoln, NE) 4000Lapparatus, using end-labeled primer and a cycle-sequencing proto-col (Amersham).

Data processing and analysis were performed on SUN worksta-tions (Sun Microsystems, Palo Alto, CA). Sequences were assem-bled using the XBAP package (Dear and Staden, 1991). Multipleprotein alignments were performed with the CLUSTAL W program(Thompson et al., 1994), integrated into the GDE package (GeneticData Environment; Smith et al., 1994). The FASTA program (Pearson,1990) was employed for searches in local databases and the BLASTnetwork service (Altschul et al., 1990) for similarity searches in Gen-Bank at the National Center for Biotechnology Information. Custom-made batch utilities were used for submitting queries and browsingthe output (BBLAST, TBOB, BFASTA, and FOB). A number of addi-tional programs, including multiple sequence file manipulation, pre-processing, and conversion utilities for XBAP, FASTA, and GDE,have been developed by the Organelle Genome MegasequencingProgram (OGMP). These utilities are described in more detail and areavailable through the OGMP website at http://megasun.bch.umontreal.ca/ogmp/ogmpid.html.

The phylogeny programs applied to these sequence data in-clude PROTDIST, FITCH, NEIGHBOUR, PROTML, and PUZZLE (Fitchand Margoliash, 1967; Saitou and Nei, 1987; Felsenstein, 1993;Strimmer and von Haeseler, 1996). The most recent implementationof PROTDIST/FITCH (Phylip, version 3.6; Felsenstein, 1993) wasused, which allows a Jin/Nei correction for unequal rates of change

1690 The Plant Cell

at different amino acid positions. Bootstrap or likelihood estimationswere performed according to Felsenstein (1985) and Kishino et al.(1990), respectively.

The complete sequence of P. purpurea mtDNA has GenBank ac-cession number AF114794. At single-nucleotide substitution sites inthis published sequence record, we have provided the nucleotidethat is the most abundant in the mtDNA population rather than show-ing the IUB ambiguity code. The long version of the macrosubstitu-tion in the rtl/dpo region (see Results) is given in the record, whereasthe short substitution is listed in the “/note” field of the correspond-ing feature.

The DNA sequences of T. maculata cob, cox1, and cox2 haveGenBank accession numbers AF116776, AF116777, and AF116778.

ACKNOWLEDGMENTS

We thank Michael Reith (Institute for Marine Biosciences, NationalResearch Council of Canada, Halifax, Nova Scotia) for providing pu-rified organellar DNA and François Michel (Centre de GénétiqueMoléculaire du CNRS, Gif-sur-Yvette, France) for help in the model-ing of intron RNA secondary structures and in reverse transcriptaseprotein alignments. We thank Charles J. O’Kelly for helpful discus-sions and Isabelle Plante, Yun Zhu, and Isabelle Robert for excellenttechnical assistance with library construction and sequencing. Sup-port of the Organelle Genome Megasequencing Program (OGMP) bythe Medical Research Council (Grant Nos. SP-34/SP-14226) and theCanadian Genome Analysis and Technology Program (Grant No.GO-12323) is also gratefully acknowledged. Generous donations ofan automated sequencer from Li-Cor (Lincoln, NE) and computerequipment from Sun Microsystems (Palo Alto, CA) greatly assistedthis study. G.B. is an Associate and M.W.G. and B.F.L. are Fellows inthe Program in Evolutionary Biology of the Canadian Institute for Ad-vanced Research (CIAR), whom we thank for salary support.

Received January 6, 1999; accepted June 30, 1999.

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DOI 10.1105/tpc.11.9.1675 1999;11;1675-1694Plant Cell

Gertraud Burger, Diane Saint-Louis, Michael W. Gray and B. Franz LangIntrons and Shared Ancestry of Red and Green Algae

: CyanobacterialPorphyra purpureaComplete Sequence of the Mitochondrial DNA of the Red Alga

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