The Journal of Volume 38
Protozoology
January-February 1991
Number 1
J. ProfozooL, 38(1), 1991, pp. 1-6 0 199 I by the Society of Protozoologists
Phylogenetic Relationships of Blpharisma americanum and Colpoda inflata Within the Phylum Ciliophora Inferred from Complete Small Subunit rRNA Gene Sequences SPENCER J. GREENWOOD,' MARTIN SCHLEGEL,*MITCHELL L. SOGIN) and DENIS H. LYNN' 'Department of Zoology, University of Guelph, Guelph, Ontario NlG 2 W I , Canada, Universitat Tubingen, Zoologisches Institut Abteilung Zellbiologie, Auf der Morgenstelle 28, 14 Tubingen. Federal Republic of Germany, and Tenter for Molecular Evolution, Marine Biology Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT. The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 17 19 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters. Key words. Ciliates, colpodid, heterotrich, molecular evolution, taxonomy.
P
The present study further explores the phylogenetic relationships within the phylum Ciliophora by sequencing the complete SSrRNA genes from the heterotrich Blepharisma americanum and the colpodid Colpoda inflata. The relationship of the heterotrichs and colpodids to other ciliate groups will be discussed in relation to their placement in three recent classification schemes [S, 26, 31, 321.
HYLOGENETIC relationships among the ciliates have been based primarily on the structural features of the cortex, the somatic and oral kinetids, and secondarily on information derived from features of the oral apparatus, extrusomes, morphogenesis, and stomatogenesis [ 151. The probable conservative nature of ultrastructural features of the somatic and oral kinetid makes them pivotal characters for assessing phylogeny [ 141. The establishment of several systematic schemes of the Phylum Ciliophora incorporating these ultrastructural features demonstrates their acceptance as good phylogenetic markers [5, 26,27, 31, 321. Small & Lynn [3 1, 321 divided the Phylum Ciliophora into eight monophyletic classes within three subphyla. The relationships between the subphyla are based either on ultrastructural aspects of oral structures (i.e. Subphyla Rhabdophora, Cyrtophora) or somatic kinetids (i.e. Subphylum Postciliodesmatophora) [31, 321. Oral structures, which show great plasticity even within a class (e.g. the colpodids [8]), must be taken as frail characters [ 141, and especially for the separation of subphyla. Since there are few morphological characters to unite the classes, relationships between classes and subphyla are very speculative ~151. Phylogenetic relationships may be investigated further by using molecular sequence information, in particular the determination of small subunit rRNA (SSrRNA) sequences. The number of SSrRNA sequences for ciliate taxa has increased rapidly within the last few years [7, 16, 33, 34, 36, 371. The information from these partial and complete SSrRNA sequences has corroborated some taxonomic relationships based on ultrastructural and other morphological characters [7]. It also has brought other relationships into question. For example, the SSrRNA gene sequence of the hypotrich Euplotes aediculatus [36] did not cluster with the nassophorean Paramecium tetraurelia as predicted by Small & Lynn [31, 321. Instead Euplotes grouped closer to the classical taxonomic position of the hypotrichs with the stichotrichs as predicted by Corliss [5] and de Puytorac et al. [26].
MATERIALS AND METHODS The culturing of the ciliate C. inflata (American Type Culture Collection #30917) and extraction and purification of bulk nucleic acids were performed using the protocols described by Lynn 8~ Sogin [ 161. DNA of B. americanum was isolated following the modified Kavenoff and Zimm isolation procedure [ 11 and purified using cesium chloride centrifugation [ 171. Polymerase chain reaction (PCR) gene amplification of purified ciliate DNA was accomplished with a Perkin-Elmer/Cetus DNA Thermal cycler using a modification of the methods of Medlin et al. [18]. Briefly, the DNA was first denatured at 94" C for 5 min to separate the two complementary strands. Then, during a gradual cooling period of 2 min to 37" C the oligonucleotide primers complementary to the 5' and 3' conserved proximal termini preferentially annealed to the rRNA gene. Primer extension proceeded at 72" C for a period of 6 min in the presence of Thermus aquaticus (Taq) DNA polymerase (New England Biolabs) and deoxynucleotide triphosphates (Pharmacia). The rDNA sequence between the conserved oligonucleotide primers was exponentially amplified through 30 cycles of denaturation, primer annealing, and primer extension. Amplification products were checked for the proper size by electrophoresing a sample on a 2% agarose gel against a 1 kb BRL marker (Bethesda Research Labs) and a sample of Tetrahymena tropicalis PCR amplified rDNA (size 1.8 kb). Samples of the correct size were then extracted with phenol and concentrated by ethanol precipitation at -20" C [ 171. PCR amplified rDNA samples were cloned into M 13 [ 191 and single-stranded templates were prepared for directing DNA syn1
2
J. PROTOZOOL., VOL. 38, NO. 1, JANUARY-FEBRUARY 1991
B.americanun
aacctggttg atcctgccag tAGTCATATG CTTGTCTCAA AGATTMCCC ATCCATCTCT M G T A T M G C M T T A T A C C C CCAGAC-TGC GAATCGCTCA
99
C.inf lata
aacctggttg atcctgccag tAGTCATATG CTTGTCTTAA AGACTAACCC ATCCATGTCT M C T A T M C T A-TTATACAG CGAAACATGC GAATCCCTCA
99
T.thermophila
AACCTGGTTG ATCCTGCCAG TTA-CATATG CTTGTCTTAA ATATTMCCC ATCCATGTGC CAGT-TCAGT A-TTGAACAG CGAAAC-TGC GAATGGCTCA 96
B.americanun
TTAAAACAGT TATACTTTAT TTGCTAGACC T - - - - T T A T A
C.inf lata
TTAAAACAGT TATAGTTTAT TCGATTATTT TCTCCTT-CA TGGATAACCG TACTAATTCT AGAGCTAATA CANCCTGTC- AAACCTCACT TTTCTCCAAC
188 197
T.thermophi1a
TTAAAACACT TATAGTTTAT TTGATAATTA AAGA-TTACA TCGATMCCG ACCTAATTCT TCCCCTMTA CATGCTTAAA ATTCCCT-GT CCTCCGACCC
194
------ GTCG ACGAATCATA
TCGATAACCG T A C T M T T C T AGAGCTAATA CATCCTCGTT ACGCCTGTGC C T G - - - - - - -
B.americanun
----GTATTT
-GAAGCATTC
270
C. inf 1a t a
CCTTCTATTT ATTAGATATA AAACCAATAC CCAGCAATCC CTT-TTCTGA TGAT-TCATA ATMCTGAAC CGACCGCCGC CCTCCTGCCG CGAATCCTTC
295
T.thermophiLa
GAACGTATTT A T T A W T A T T AGACCAATCG CAGCAATCTC ATTGAGAT--
289
ATTAGATAAA ACCCAACGGG GGCGACCCT-
A T M C T T A C C GAACTCGACC T A C T - - - - - -
-GAA-TCAAA GTMCTCATC GGATCGAGGT TTACCTCGAT AAATCA-TCT
B.amer icanun
M C T T T C T G C CCTATCACCT TTCGATGGTA GTCTATTGGA CTACCATCCC GATGACCCCT GACCGAGAAT TACCCTTCGA TTCCGGACAG CGACCCTGAC 370
C. inf l a t a
AACTTTCTCC CCTATCACCT TTCGATGGTA GTGTATTCGA CTACCATGGC GATCACGCGT MCGCGGAAT TACGGTTCGA TTCCGGAGAC CGAGCCTGAC 395
T.thermophi1a
AACTTTCTGC CCTATCACCT CTCCATCGTA CTGTATTCGA CTACCATCCC ACTCACCCCT AACCGAGAAT TACCCTTCGA TTCCGCAGAA CGAGCCTGAC 389
B. a m e r i c a n u n
AAATCCCTAC CACATCTAAG CM-GGCACC AGGCGCCCAA A T T A C C U A T CCTAACTCAG CGAGGTAGTG ACAACAAATA ACAACGCCCC CCTTTCTCTT
469
C.inflata
AAATGGCTAC CACATCTAAC GAA-CCCACC AGCCCCCTAA ATTACCCAAT CCTMTTCAG CGACCTACTC ACMCAAATA ACAACTCCGA CCTCACTCGA
494
1-thermophil a
AAACCGCTAC TACAACTACG CTTCCCCACC ACGGAAGAAA ATTGGCCMT CCTAATTCAG CGAGCCAGTG ACAAGAAATA GCAACCTCGC AAACTTACCT
489
__ - GA TTCGMTGAG T T M G T G T M AACCCT--TC
B.americanun
CC---
CCACCACCCG CGG-TAAT-C CACCTCCACT
552
C. inf t a t a
GGTNACGAGA TTGNMTGAG M C M T T T M ACCTCTTATC GAGTMCAAT TGGACGGCM CCTC-TGCTG CCACCAGCCG CCGCTAATTC CAGCTCCAAT
593
T. thermophi l a
TTCTACGCCA TTGAMTGAG M C A G T C T M ATCTCTTAGC G A G G M U A T TGGACGCCAA G-TCATCGTC CCACCAGCCC CGC-TAATTC CAGCTCCAAT
587
B .emericam
AGCGTATATT M A G - T T G T T - C C - A G T T M AAAGCTCGTA GTGMGC-TC TGCGGG-GCG GGTCGCTCCG CCTCGGTCTC GTGACCTTCC TCGCAT-CCA
646
C-inflata
AGCGTATATT MAGCTTGTT A C G U C T T M AAAGCTCGTA GTTGMTATC TGGCCGGTGC TGTTCTTGGC TCCCTGAGTC CGCTCAGCGC CTCGTCATCC 693
T.themophi1a
AGCGTATATT M A G - T T G T T - - G C A G T T M MAGCTCGTA GTTGMCTTC TGT-1-CAGG TTCATTTCGA TTCGTCCT-G T G A M - - C T G GACATACGTT
B . e r i cawn C. inf l a t a T. t h e m i la
TC-TGAGCM CGGC--TCCG G C - - A T T M C TTG-TCGCTG TCGTG-ATCA GGTAC--TTT ACCTTGAGCA MTGAGAGTG TTCCAGGCAG GCTTGGGCCT 737 GTATGGGMA CTAGCTCGAC C----TTCAC
GAGGACCACT - - G A C G - U A GC---TG-TG
TGGTCGGCTA 6 - T G G A - T U
--__-M M T CGGCCTTCAC TGGTTCGACT
--TACACTTT A C T T T G A M A M T T A G A G T G TTTCAGGCAC CCAATNGCTT
679
785
A C T G T G M M MTTAGAGTG TTCCAGGCAG GTTTTAGCCC
769
B americanun
GATACGTCCA CCATCGAATA ATAGAAGAGC ACTGGCCTCC ATTTATTGCT GTTATGGCCC T T A - G T M T G ATTAATAGGG ATAGTTGCCC GCATTTGTAT
836
C.inf lata
CGATACTGTA GCATCGMTA ATCGAATACC ACTTTGACCT ATTTCTTGGT NTCTCGAGGT U A A G T M T G ATTMTAGCC ACAGTTGGGC GCATTCCTAT
885
T-thermophila
GAATACATTA GCATGGAATA ATGGAATAGG ACTAAGTCCA TTTTATTCCT TCTTCGATTT CC---TAATG
ATTAATAGGC ACAGTTGGCG GCATTACTAT
866
B.americanun
TTAATTCTCA GACCTGAAAT TCTATGATTT ATTAAACACA AACTTATCCC AAACCATTTC CCAAGGATCT TTTCATTAAT CAAGAACCAA ACTTAACCGA
936
C. inf Lata
TTAATNGTCA GACCTGAAAT TCTTCGATTT TTTAAAGACG AACTTATGCG AMCCATTTG CCAACCATCT TTTCATTAAT CAAGAACCAA AGTTACCCCA
985
T. thermophi l a
T T M T A C T C A GACGTGAAAT TCTTGGATTT ATTAAGCACT MCTAATGCC AAACCATTTC CCAAACATCT TTTCATTAAT CAAGAACCAA AGTTAGGGGA 966
.
.
TCWCT--
TAGGGAGTM A---CATTTT
B m r icanun
TCAAAGACGA TCAGATACCG TCCTACTCTT AACCATAAAC TATCCCGACT AGAGATTCGA CGTGCCATTA AAAGTACTCC TTCACCATCT TCCGAGAAAT
C.inflata
TCAAAGACGA TCACATACCC TCCTACTCTT AACCATAAAC TATACCGACT ACGGATTGCT GACCTCTTTT
T.thermophila
TCAAAGACGA TCAGATACCC TCCTAGTCTT AACTATAAAC TATACCCACT CCGGATCCGC TCCAATAAA- ----TCTCCA
- - -MCCCTC
1036
ATCACCACCT TATCAGAAAT
1082
GTCGCCACCC TATCAGAAAT
1061
B. a m e r i c a n u n
CAAACTCTTT CGCTTCTCCC CCCAGTATGC T-CGCAACAC -TGAAACTTA MCGAATTGA CGCAACGCCA CCACCACGAC TGGCA--TGC CCCCTTAATT
1132
C. inf Lata
CAAACTCTTT CGCTTCTCGG CGGACTATCG 1-CCCAAGCC CTGAAACTTA AACGAATTGA CGGAAGCGCA CCACCACGAC TCCACCCT-C CGCCTTAATT
118C
T.thermophila
CAAACTCTTT GGGTTCTGGC GGAAGTATGG TACCCAACTC -TGAAACTTA AAGCAATTGA CGGAACACCA -CACCAGAAC TGGAACCT-C CGGCTTAATT
11%
.
B a m e r icanun
TGACTCAACA CGGGCAAACT TACCAGCTCC AGACATGCCA ACGATTGACA GATTGATAGC TCTTTCTTGA TTCTATCCGT CCTGCTCCAT CCCCGTTCTT
1232
C. i n f Lata
TGACTCAACA CCCCGAAACT TACCACCTCC AGACATACTT CGGATTGACA GATTGAGACC TCTTTCTTGA TTCTATGCCT CGTGGTCCAT GCCCCTTCTT
128C
T.thermophi1a
TGACTCAACA CCCCCAAACT CACGAGCCCA ACACACAGAA CCCATTCACA GATTGAGACC TCTTTCTTCA TTCTTTCCGT GCTGCTCCAT CCCCGTTCTT
125E
Fig. 1. Small subunit rRNA gene sequences of the ciliates Blepharisma americanum ( 1 7 19 nucleotides) and Colpodu inflata ( I 786 nucleotides) aligned with the sequence from Tetruhymenu thermophilu (1 753 nucleotides) [37]. Nucleotides indicated in lowercase correspond to those regions complementary to the oligonucleotide primers used for PCR amplification. Numbers at end of lines indicate the number of nucleotides. The differences in sequence introducing alignment gaps (-) in the sequences. Ambiguities in nucleotide positions for B. umericanum and for C. inj7utu are indicated in the figure by the letter ‘N.’
GREENWOOD ET AL-BLEPHARISMA AMERICANUM A N D COLPODA INFLA TA SMALL SUBUNIT R R N A GENE
- - -TCTTGCC
3
B. a m e r icanun
AGTTCCTCGA CTGATTTCTC TGGTTMTTC CGATMCGAA CGAGACCTTA ACCTCCTMC TAGTCCTC--
MAGACTCGC ACTTCTTAGA
1327
C.inflata
ACTTCGTCGA CTGATTTGTC TGGTTMTTC C C T T M C G M CGAGACCTTA ACCTGCTMC TACTCAC- - C TTTATCTCM TACCCGTCTG ACTTCTTAGA
T.thermophi1a
ACTTGCTGGA CTGATTTGTC TGGTTMTTC CCTTAACGAA CGAGACCTTA ACCTGCTMC TACT----CT
1378 1354
B. amer icanun
CCCACTTTCC CCCCTA- - C T C C M C G M G T T T M C C C M TAACACCTCT GTGANCCCCT TAGANCTCCT GCGCCCCACC CCCGCTACAC TGATCCACCC
C.inflata
GGGACTTTAT G T C C M - - C CATMGGMG TTTGACGCM TMCAGGTCT CTGATGCCCT TAGATGTCCT GCCCCGCA-G CCCCCTACAC TGACACATGC
1424 1474
T.themphi1a
GCGACTATT- C T G C M T M C C C M T C G M C T T T M G C C M TAACAGGTCT CTGATCCCCC TAGACCTCCT CCCCCCCACC CCCCTTACM TGACTCCCCC
1453
GCTTGTAMT MCACGTTCT ACTTCTTAGA
-
B.americanun
ACTAACCCC- ----TCCGCC
C. inf l a t a
M C M C T T T A TTTTTCCTGA CTCCGMCGG TTCCCCTAAT CTTCTATMT CTCTCTCCTG CT-AGGCATA GA-TCTTTGG MTTATAGAT C T T G M C M C
1. t h e r m o p h i l a
A A A M C - - T A TTT--CCTCT CCTCGGMGC TACCCCTMT C T T A T - T M T ACCAGTCGTG TTACCGATAG 1--1CTTTGG MTTGTCGAT CTTGMCGAC
1513 1572 1546
B. amer icanun
GAATTCCTAG T A M C G C M C TCATCMCTT CCATTGACTA CCTCCCTGCC CTTTCTACAC ACCCCCCGTC GCTCCTACCG ATTTCGACTG ATGACCTGM
1613
C. inf l a t a
GMTTCCTAG T M C C A T M C TCATCACCTT CTCCTGATTA CCTCCCTCCC CTTTGTACAC ACCCCCCCTC GCTCCTACCC ATTTTGACTC ATCCCCTGM
1. t h e m p h i l a
GAATTTCTAG T M C T G C M C TCATCAGCTT GCGTTGATTA TGTCCCTGCC CTTTGTACAC ACCGCCCGTC GCTTGTAGTA AC---GMTG
1672 1643
B.americanun
TCCTCCCGAC TGCACACCTC
C. i n f l a t a
CCTTCTGGAC TCTGGTCACG CTTGACCTGA TTCTCCGMG 1-TAAGTAAA CCTTATCACT TAGAGGMGG ACMGTCCTA ACMGGTTTC C g t a g g t g a a
T.thermophila
CCTTCTGGAC TCCGA-CACC M T C - 1 1 - C C
B.americaMm
cctgcagaag gatca
1719
C.inf l a t a
cctgcagaag gatca
1.thermophila
CCTGCAGATG GATCATTA
1786 1753
CU-GAMNC CCCCGCTAAC CTTCAMC-T GCATCCTGAT ---CGCGATT
-------- GT
GACTCTT-CC MTTATTCCT CATCMCGAC
GTCTGGTGM
1704 1771 G G M A AATAACTAM CCCTACCATT T G G M U A C A AGMGTCGTA ACAAGGTATC TGTAGCTGM 1735
CTGTCGGAAG T - T G A C T A M CCTTATUCT TAGAGGMGG AGMCTCGTA AUAGCTATC T g t a g g t g a a
-----
thesis in the Sanger dideoxy-chain termination sequencing protocol [3, 301. Sequences were aligned using a procedure that considers the conservation of both the primary and secondary structure for phylogenetic reconstruction [7]. The sequence information was reduced to approximately 1,600 unambiguously aligned positions for comparisons of the Phylum Ciliophora in relation to other eukaryotes. Sequence data used in the alignments were from the following: corn, Zea mays [20]; chlorophyte, Chlamydomonas reinhardtii [9]; yeast, Saccharomyces cerevisiae [29]; bread mould, Neurospora crassa [35]; oomycete, Achlya bisexualis [9]; chrysophyte, Ochromonas danica [9]; dinoflagellate, Prorocentrum micans [ lo]; and the ciliates Euplotes aediculatus [36], Oxytricha nova [7], Oxytricha granulifera [Schlegel & Sogin, unpubl.], Onychodromus quadricornutus [Schlegel & Sogin, unpubl.], Stylonychia pustulata [7], Paramecium tetraurelia [33], Tetrahymena hegewischi [34], Tetrahymena thermophila [35], and Tetrahymena pyriformis [34]. Aligned sequences were analysed using a Distance Matrix method in which calculations of structural (sequence) similarity and evolutionary distance incorporate the Jukes & Cantor [ 1 11 correction to estimate unobserved nucleotide substitutions [7, 23, 241. Phylogenetic trees were constructed by an additive tree method which determines the tree geometry and the branch lengths that best fit the evolutionary distance data [23, 241. RESULTS The complete small subunit rRNA sequences obtained for B. americanum and C. inflata were 1719 nucleotides and 1786 nucleotides respectively (Fig. 1). The phylogenetic tree generated from the SSrRNA sequence data used a weighting mask that included all regions of the eukaryotic SSrRNAs that can be unambiguously aligned for this specific set of sequences (Fig. 2; Table 1). The almost simultaneous divergence ofthe plants, fungi, and ciliates has been noted earlier [9]. This tree clearly shows that the ciliates diverged as
a monophyletic group [6], and among the taxa chosen for the analysis the dinoflagellates, represented by Prorocentrum micans, are the sister group to the ciliates (Fig. 2). The depth of branching between some of the major ciliate groups is deeper
0.05
Fig. 2. Ciliate phylogeny inerred from complete small subunit rRNA sequence similarities using a distance-matrix method. The analysis was limited to approximately 1,600 positions that could be unambiguously aligned in all of the rRNA sequences. The evolutionary distance (average number of nucleotide substitutions per sequence position) is represented by the horizontal component separating species in the figure. The scale bar equals 0.05 nucleotide substitutions per sequence position. Data are given in Table 1 .
4
J. PROTOZOOL., VOL. 38, NO. I , JANUARY-FEBRUARY 1991
Table 1. Structural similarity and evolutionary distance data for eukaryote small subunit rRNA gene sequences." Structural similaritv/evolutionarv distance to: Organisms
Z.m.
Z. mays C. reinhardtii
S. cerevisiae
0.107 0.156 0.175 0.163 0.165 0.164 0.250 0.202 0.187 0.198 0.183 0.183 0.195 0.204 0.256 0.250 0.252
C.r.
S.C.
N.c.
A.b.
0.d.
P.m.
E.a.
0.900
0.859 0.860
0.844 0.853 0.907
0.854 0.855 0.852 0.834
0.852 0.859 0.853 0.847 0.899
0.853 0.853 0.852 0.845 0.861 0.864
0.787 0.786 0.801 0.786 0.81 1 0.813 0.828
0.155
N. crassa 0.163 0.099 A. bisexualis 0.161 0.165 0.188 0. danica 0.167 0.164 0.171 0.109 P. micans 0.163 0.165 0.174 0.154 0.150 E. aediculatus 0.25 1 0.232 0.253 0.218 0.2 15 0.196 B. americanum 0.197 0.195 0.208 0.209 0.179 0.164 0.213 0. nova 0.188 0.165 0.184 0.171 0.155 0.130 0.145 0. granulifera 0.201 0.182 0.197 0.184 0.169 0.152 0.159 0. quadricornutus 0.188 0.168 0.184 0.170 0.161 0.129 0.149 S. pustulata 0.192 0.171 0.189 0.173 0.161 0.135 0.147 C. inflata 0.20 1 0.179 0.192 0.168 0.154 0.153 0.190 P. tetraurelia 0.198 0.185 0.197 0.186 0.183 0.162 0.195 T. hegewischi 0.256 0.215 0.238 0.232 0.226 0.216 0.242 T. thermophila 0.25 1 0.216 0.238 0.229 0.221 0.218 0.242 T. pyriformis 0.253 0.2 13 0.236 0.230 0.223 0.2 17 0.242 Note: The upper half of the table gives the structural similarity (S) values for all pairs of aligned small subunit rRNA sequences. The lower half of the table gives the evolutionary distance values (average number of nucleotide substitutions per sequence position) determined by the Jukes & Cantor [ 1I ] formula for conversion of structural similarity. a Sources of sequence data for the aligned eukaryote small subunit rRNA sequences are listed in Materials and Methods. than the separation between plants and fungi. For example, the colpodid C. inflata and the hypotrich E. aediculatus are separated by an evolutionary distance of 0.190 while corn 2. mays and yeast S. cerevisiae are separated by an evolutionary distance of 0.156 (Table 1). This demonstrates that ciliate groups diverged early after the split of the ciliate ancestor from the main eukaryote line. The Class Oligohymenophorea, sensu Small & Lynn [3 1, 321, represented by T. hegewischi, T.pyriformis, and T. thermophila, remains a monophyletic group with the Class Nassophorea (P. tetraurelia) as its sister group. The Subclass Hypotrichia (E. aediculatus) and the Subclass Stichotrichia (0.quadricornutus, 0. granulifera, 0. nova, and S. pustulata) remain sister groups within the Class Spirotrichea, sensu Corliss [ 5 ] and de Puytorac et al. [26]. However, the evolutionary distance separating the hypotrich E. aediculatus from the stichotrichs ( ~ 0 . 1 5 0 is ) almost as great as the distance separating corn Zea and yeast Saccharomyces (0.156) (Table 1). The stichotrich 0. nova is more closely related by sequence similarity (0.993) to S. pustulata than to 0.granulifera (0.989) (Table 1). The Class Colpodea sensu Small & Lynn [3 1,321, represented by C. inflata, is more closely allied with the oligohymenophoreans and nassophoreans than the spirotrichs while the heterotnch B. americanum is placed as the earliest branching ciliate (Fig. 2). DISCUSSION Molecular comparisons. The significance of the phylogenetic placement of B. americanum and C. infata within the Phylum Ciliophora, inferred from complete SSrRNA sequences, may be determined by congruency checks with the phylogenies constructed from other molecules and morphological characters [23, 241. Baroin et al. [2] constructed a global eukaryotic phylogeny inferred from the approximately 400 nucleotides of Domain 1 and its flanking region of the large subunit rRNA (LSrRNA). The Baroin et al. [2] tree placed the ciliates as a monophyletic
group with the dinoflagellates (Prorocentrum micans) as their sister group. The phylogeny was limited to five ciliates (Blepharisma, Stentor, Tetrahymena, and two species of Paramecium) and 10 other eukaryotes and one eubacterium. The tree topology indicated two main branches: the first depicted the two Paramecium species as closely related to Tetrahymena while the heterotrichs Stentor and Blepharisma were placed on a separate branch [2]. The placement of Tetrahymena and Paramecium and the location of the heterotrichs Blepharisma and Stentor on a separate branch within the ciliate lineage is consistent with the phylogeny of the present study (Fig. 2). However, since the Baroin et al. [2] tree did not contain representatives of the colpodids or hypotrichs not all the intra-phylum comparisons can be made. Nanney's group has been concentrating on Domain 2 (D2) of the LSrRNA, which corresponds to a region of 180 nucleotides located just downstream of Domain 1 [21, 22, 251. Their most extensive phylogeny included 14 ciliates (Colpodium striatum, Colpoda maupasi, Colpoda steinii, Glaucoma chattoni, Didinium, Paramecium, and eight tetrahymenas) and one dinoflagellate (Crypthecodinium cohnii) [25]. The Preparata et al. [25] tree placed the ciliates as a monophyletic group with the dinoflagellate C. cohnii as their sister group. Crypthecodinium is a different genus from the dinoflagellate used in the present study, further reinforcing the notion of these protists as the sister group of the ciliates. Their tree placed the ciliates C. steini and C. maupasi with Didinium and Paramecium outside the hymenostome (Colpidium, Glaucoma, and Tetrahymena) cluster [25]. Although the exact branching order of the genera Colpoda, Didinium, and Paramecium was not resolved on the Preparata et al. [25] tree, this may indicate a close association ofthe colpodids with the gymnostomes as predicted by Corliss [5] (see below). Conversely the branching order may be biased by the low number of sites used in the analysis or by the lack of other ciliate taxa (e.g. heterotrichs, hypotrichs, and stichotrichs.) Lynn & Sogin [ 161 assessed the phylogenetic relationships among the ciliates by using partial SSrRNA sequences derived from reverse transcripts. Both the Lynn & Sogin tree [ 161 and
5
GREENWOOD ET AL. -BLEPHARISMA AMERICANUM AND COLPODA INFLATA SMALL SUBUNIT RRNA GENE
Table 1. Continued. Structural similaritv/evolutionarv distance to: B.a.
0.n.
0.g.
0.q.
S.P
C.i.
P.t.
T.h.
T.t.
T.p.
0.823 0.827 0.828 0.818 0.817 0.84 1 0.852 0.814
0.835 0.834 0.582 0.837 0.847 0.860 0.880 0.868 0.868
0.826 0.823 0.839 0.826 0.837 0.849 0.862 0.857 0.858 0.972
0.838 0.834 0.849 0.837 0.848 0.855 0.88 1 0.865 0.865 0.989 0.968
0.838 0.83 1 0.847 0.833 0.845 0.855 0.877 0.866 0.863 0.993 0.969 0.986
0.828 0.823 0.841 0.830 0.849 0.860 0.861 0.832 0.859 0.895 0.884 0.894 0.892
0.822 0.826 0.836 0.827 0.836 0.837 0.855 0.829 0.855 0.879 0.865 0.880 0.878 0.881
0.783 0.783 0.8 13 0.796 0.800 0.805 0.812 0.793 0.810 0.839 0.829 0.839 0.837 0.843 0.85 1
0.787 0.787 0.8 12 0.796 0.803 0.808 0.810 0.793 0.809 0.836 0.826 0.837 0.833 0.843 0.854 0.986
0.786 0.785 0.814 0.797 0.802 0.807 0.812 0.793 0.813 0.838 0.827 0.839 0.835 0.842 0.855 0.99 1 0.994
0.145 0.157 0.149 0.151 0.156 0.161 0.219 0.220 0.215
0.029 0.01 1 0.007 0.113 0.132 0.181 0.184 0.182
0.032 0.032 0.126 0.149 0.194 0.198 0.197
0.0 13 0.115 0.130 0.180 0.184 0.181
0.112 0.133 0.184 0.189 0.187
the present analysis placed C. inflata on the lineage leading to the nassophoreans and oligohymenophoreans. No representative of the heterotrichs was sequenced in the Lynn & Sogin study [ 161. The partial SSrRNA sequence data appears to have produced a phylogeny consistent with that based on complete sequences. It has been shown previously that phylogenetic trees constructed by using partial SSrRNA sequences from organisms of different kingdoms had topologies identical to that obtained by using complete sequences [ 131. The resolving ability of the reverse transcripts technique has not been fully tested. In both the Kumazaki et al. [ 121 and Walker [38] 5s rRNA analyses, the ciliates (Blepharisma, Bresslaua, Euplotes, Paramecium, and Tetrahymena) were delineated as a monophyletic group and the dinoflagellates, again represented by Crypthecodinium, as their sister group. Both 5s rRNA trees are in agreement with the present analysis with respect to the branching order of Tetrahymena and Paramecium. The position of the colpodid Bresslaua varies between the two 5s rRNA trees: Walker [38] placed Bresslaua on a separate branch from Tetrahymena and Paramecium, while Kumazaki et al. [ 121, in agreement with the present study, placed the colpodids as more closely related to Paramecium and Tetrahymena than to the other ciliates (Fig. 2). The distance between Blepharisma and Euplotes based on the 5s rRNA trees is comparable to that depicted in the present study (Fig. 2), suggesting based on molecular evidence from 5s rRNA and SSrRNA, the heterotrichs should probably be elevated to a higher taxonomic rank as suggested by de Puytorac et al. [26] (see below). Morphological comparisons -taxonomic implications. In the tree derived from the SSrRNA sequences C. inJata was placed on the lineage leading to the nassophoreans and oligohymenophoreans (Fig. 2). Corliss [5] considered the colpodids as an order within the Class Kinetofragminophora together with the trichostome, gymnostome, and hypostome ciliates, and far removed from the nassophoreans and oligohymenophoreans.Small & Lynn [31, 321 and de Puytorac et al. [26] both recognized the colpodids as a distinct group, based on the overlapping posteriorly-directed transverse microtubular ribbons of their somatic dikinetids, and have elevated them to class rank. Small & Lynn [3 1, 321 placed the Class Colpodea along with the Classes Nassophorea, Oligohymenophorea, and Phyllopharyngea (none se-
0.129 0.177 0.177 0.177
0.166 0.162 0.161
0.014 0.009
0.006
quenced) in the Subphylum Cyrtophora since the oral dikinetids possess postciliary microtubular ribbons that support the cytopharynx. On the other hand, de Puytorac et al. [26] placed the colpodids in the Subphylum Prostomatea with the trichostomes and prostomes, based on the polar formation of the cytostome. The deep branching of C. inflata on the SSrRNA trees tends to support the class status given by de Puytorac et al. [26] and Small & Lynn [3 1 , 321. No SSrRNA sequence information is available from representatives of the trichostomes, gymnostomes or prostomes. However, Preparata et al. [25] demonstrated a close association of the gymnostome Didinium with the genus Colpoda. Since their tree lacked representatives of the heterotrichs, hypotrichs, and stichotrichs, no definitive conclusions can be made with respect to the relationships suggested by Corliss [5] and therefore must await further sequence information. Blepharisma americanum was placed as the earliest diverging ciliate after the phylum Ciliophora separated from the main eukaryote line in the phylogeny inferred from complete SSrRNA sequences (Fig. 2). All three classification schemes recognise the heterotrichs as a distinct group. However, Corliss [ 5 ] separated the heterotnchs and hypotrichs at the ordinal level based on differences in their adoral zone of membranelles while Small & Lynn [31, 321 separated the heterotrichs and stichotrichs into different subclasses within the Class Spirotrichea. De Puytorac et al. [26] stated that differences in the adoral zone of membranelles make the heterotrichs distinct from the spirotrichs and have separated the heterotrichs from the spirotrichs, elevating the heterotrichs to a class within the Subphylum Polyhymenophorea. The deep branching of B. americanum in the phylogeny based on complete SSrRNA sequences supports the elevation to class status given the heterotrichs by de Puytorac et al. [26]. Based on Raikov’s hypothesis [28] for explaining nuclear dualism, Corliss [4] initially proposed that the karyorelictid ciliates were the closest living ciliates to the ancestral stock for the phylum. Small & Lynn’s classification [3 1, 321 suggested a close relationship of the heterotrichs to the karyorelictids based on similarities in their somatic dikinetid by placing them together in the Subphylum Postciliodesmatophora [3 1, 321. On the other hand de Puytorac et al. [26] separated the karyorelictids and heterotrichs into the subphyla Karyorelicta and Polyhymeno-
6
J. PROTOZOOL., VOL. 38, NO. 1 , JANUARY-FEBRUARY 1991
phora respectively. In order to fully test the association of the heterotrichs with the karyorelictids, and the deep branching o f the heterotrichs, SSrRNA sequence information from representatives of the karyorelictids and other heterotrichs will be required. ACKNOWLEDGMENTS T h e authors wish t o thank Hille Elwood for technical assistance. This work was completed as part of a University of Guelph M.Sc. thesis by S. J. Greenwood. T h e work was funded by NSERC operating grant #A6544 awarded to D. H. Lynn and by grants of the Universitatsbund of the University o f Tiibingen and the Deutsche Forschungsgemeinschaft t o M. Schlegel a n d by NIH grant GM 32964 to M . L. Sogin. LITERATURE CITED 1. Ammermann, D., Steinbruck, G., Berger, von, L. & Hennig, W.
1974. The development of the macronucleus in the ciliated protozoa, Styonychia mytilus. Chromosoma, 45:401429. 2. Baroin, A,, Perasso, R., Qu, L. H., Brugerolle, G., Bacherllerie, J. P. & Adoutte, A. 1988. Partial phylogeny of the unicellular eukaryotes based on rapid sequencing of a portion of 28s ribosomal RNA. Proc. Natl. Acad. Sci. USA, 85:3474-3478. 3. Biggin, M. D., Gibson, T. J, & Hong, G. F. 1983. Buffer gradient gels and '3label as an aid to rapid sequence determination. Proc. Natl. Acad. Sci. USA, 80:3963-3965. 4. Corliss, J. 0. 1974. Remarks on the composition of the large ciliate class Kmetofragminophora de Puytorac et al., 1974, and recognition of several new taxa therein, with emphasis on the primitive order Protociliatida n. ord. J. Protozool., 21:207-220. 5. Corliss, J. 0. 1979. The ciliated protozoa: characterization, classification, and guide to the literature, 2nd ed. Pergamon Press, New York, New York. 6. Corliss, J. 0. 1984. The kingdom protista and its 45 phyla. BioSystems, 17:87-126. 7. Elwood, H. J., Olsen, G. J. & Sogin, M. L. 1985. The small subunit ribosomal RNA gene sequences from the hypotrichous ciliates Oxytricha nova and Stylonychia pustulata. Mol. Biol. Evol.. 2:399-4 10. 8. Foissner, W. 1985. Klassifikation und phylogenie der Colpodea (Protozoa: Ciliophora). Arch. Protistenk., 129:239-290. 9. Gunderson, J. H., Elwood, H. J., Ingold, A., Kindle, K. & Sogin, M. L. 1987. Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes. Proc. Natl. Acad. Sci. USA, 84:5823-5827. 10. Herzog, M. & Maroteaux, L. 1986. Dinoflagellate 17s rRNA sequence inferred from the gene sequence: evolutionary implications. Proc. Natl. Acad. Sci. USA, 83:8644-8648. 1 I . Jukes, T. H. & Cantor, C. R. 1969. Evolution of protein molecules. In: Munro, H. N. (ed.), Mammalian Protein Metabolism. Academic Press, New York, New York, 3:22-126. 12. Kumazaki, T., Hori, H. & Osawa, S. 1983. Phylogeny of protozoa deduced from 5s rRNA sequences. J. Mol. Evol., 19:411-419. 13. Lane, D. J., Pace, B., Olsen, G. J., Stahl, D. A., Sogin, M. L. & Pace, N. R. 1985. Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA, 82:69556959. 14. Lynn, D. H. I98 1. The organization and evolution of microtubular organelles in ciliated protozoa. Biol. Rev., 56:243-292. 15. Lynn, D. H. & Small, E. B. 1988. An update on the systematics of the phylum Ciliophora Doflein, 1901: the implications of kinetid diversity. BioSystems, 21:3 17-322. 16. Lynn, D. H. & Sogin, M. L. 1988. Assessment of the phylogenetic relationships among ciliated protists using partial ribosomal RNA sequences derived from reverse transcripts. BioSystems, 21 :249254. 17. Maniatis, T., Fritsch, E. F. & Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 18. Medlin, L., Elwood, H. J., Stickel, S. & Sogin, M. L. 1988. The
characterization of enzymatically amplified eukaryotic 16s-like rRNA coding regions. Gene, 71:491499. 19. Messing, J. 1983. New MI3 vectors for cloning. In: Wu, R., Grossman, L. G. & Moldave, K. (ed.), Methods in Enzymology: Recombinant DNA. Academic Press, New York, New York, 101:20-78. 20. Messing, J., Carlson, J., Hagen, G., Rubenstein, I. & Oleson, A. 1984. Cloning and sequencing of the ribosomal RNA genes in maize: the 17s region. DNA, 3:3140. 21. Nanney, D. L., Meyer, E. B., Simon, E. M. & Preparata, R. M. 1989. Comparison of ribosomal and isozymic phylogenies of tetrahymenine ciliates. J. Protozool., 36: 1-8. 22. Nanney, D. L., Preparata, R. M., Preparata, F. P., Meyer, E. B. & Simon, E. M. 1989. Shifting ditypic site analysis: heuristics for extending the phylogenetic range of nucleotide sequences in Sankoff analyses. J. Mol. Evol., 28:451459. 23. Olsen, G. J. 1987. Earliest phylogenetic branchings: comparing rRNA-based evolutionary trees inferred with various methods. In: Cold Spring Harbor Symposia of Quantitative Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 52:825-837. 24. Olsen, G. J. 1988. Phylogenetic analysis using ribosomal RNA. In: Noller, H. F. Jr. & Moldave, K. (ed.), Methods Enzymology: Ribosomes. Academic Press, New York, New York, 164:793-812. 25. Preparata, R. M., Meyer, E. B., Preparata, F. P., Simon, E. M., Vossbrinck, C. R. & Nanney, D. L. 1989. Ciliate evolution: the ribosomal phylogenies of the tetrahymenine ciliates. J. Mol. Evol., 28: 427-44 1. 26. Puytorac, P. de, Grain, J. & Mignot, J. P. 1987. PrBcis de protistologie. SocittB Nouvelle des Editions BoubBe, Paris. 27. Puytorac, P. de, Grain, J., Legendre, P. & Devaux, J. 1984. Essai d'application de I'analyse phtnetique a la classification du phylum des Ciliophora. J. Protozool., 31:496-507. 28. Raikov, I. B. 1969. The macronucleus of ciliates. In: Chen, T. T. (ed.), Research in Protozoology. Pergamon Press, New York, New York, 3:l-128. 29. Rubtsov, P. M., Musakhanov, M. M., Zakharyev, V. M., Krayev, A. S., Skryabin, K. G. & Bayev, A. A. 1980. The structure ofthe yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18s ribosomal RNA gene from Saccharomyces cerevisiae. Nucleic Acids Res., 8:5779-5794. 30. Sanger, F., Nicklen, S. & Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74:54635467. 3 1. Small, E. B., Lynn, D. H. I98 1. A new macrosystem for the phylum Ciliophora Doflein, 1901. BioSystems, 14:38740 1. 32. Small, E. B. & Lynn, D. H. 1985. Phylum Ciliophora. In: Lee, J. J., Hutner, S. H. & Bovee, E. C. (ed.). An Illustrated Guide to the Protozoa. Soc. Protozool., Lawrence, Kansas, pp. 393-575. 33. Sogin, M. L. & Elwood, H. J. 1986. Primary structure of the Paramecium tetraurelia small-subunit rRNA coding region: phylogenetic relationships within the Ciliophora. J. Mol. Evol., 23:53-60. 34. Sogin, M. L., Ingold, A., Karlok, M., Nielsen, H. & Enberg, J. 1986. Phylogenetic evidence for the acquisition of ribosomal RNA introns subsequent to the divergence of some of the major Tetrahymena groups. EMBO J., 93625-3630. 35. Sogin, M. L., Miotti, K. & Miller, L. 1986. Primary structure of the Neurospora crassa small subunit ribosomal RNA coding region. Nucleic Acids Res.. 14:9540. 36. Sogin, M. L., Swanton, M. T., Gunderson, J. H. & Elwood, H. J. 1986. Sequence for the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus. J. Protozool., 33:26-29. 37. Spangler, E. A. & Blackburn, E. H. 1985. The nucleotide sequence of the 17s ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin. J. Biol. Chem., 260:63346340. 38. Walker, W. F. 1985. 5s and 5.8s ribosomal RNA sequences and protist phylogenetics. BioSystems. 18:269-278.
Received 5-14-90; accepted 10-15-90
Protozoology
January-February 1991
Number 1
J. ProfozooL, 38(1), 1991, pp. 1-6 0 199 I by the Society of Protozoologists
Phylogenetic Relationships of Blpharisma americanum and Colpoda inflata Within the Phylum Ciliophora Inferred from Complete Small Subunit rRNA Gene Sequences SPENCER J. GREENWOOD,' MARTIN SCHLEGEL,*MITCHELL L. SOGIN) and DENIS H. LYNN' 'Department of Zoology, University of Guelph, Guelph, Ontario NlG 2 W I , Canada, Universitat Tubingen, Zoologisches Institut Abteilung Zellbiologie, Auf der Morgenstelle 28, 14 Tubingen. Federal Republic of Germany, and Tenter for Molecular Evolution, Marine Biology Laboratory, Woods Hole, Massachusetts 02543 ABSTRACT. The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 17 19 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters. Key words. Ciliates, colpodid, heterotrich, molecular evolution, taxonomy.
P
The present study further explores the phylogenetic relationships within the phylum Ciliophora by sequencing the complete SSrRNA genes from the heterotrich Blepharisma americanum and the colpodid Colpoda inflata. The relationship of the heterotrichs and colpodids to other ciliate groups will be discussed in relation to their placement in three recent classification schemes [S, 26, 31, 321.
HYLOGENETIC relationships among the ciliates have been based primarily on the structural features of the cortex, the somatic and oral kinetids, and secondarily on information derived from features of the oral apparatus, extrusomes, morphogenesis, and stomatogenesis [ 151. The probable conservative nature of ultrastructural features of the somatic and oral kinetid makes them pivotal characters for assessing phylogeny [ 141. The establishment of several systematic schemes of the Phylum Ciliophora incorporating these ultrastructural features demonstrates their acceptance as good phylogenetic markers [5, 26,27, 31, 321. Small & Lynn [3 1, 321 divided the Phylum Ciliophora into eight monophyletic classes within three subphyla. The relationships between the subphyla are based either on ultrastructural aspects of oral structures (i.e. Subphyla Rhabdophora, Cyrtophora) or somatic kinetids (i.e. Subphylum Postciliodesmatophora) [31, 321. Oral structures, which show great plasticity even within a class (e.g. the colpodids [8]), must be taken as frail characters [ 141, and especially for the separation of subphyla. Since there are few morphological characters to unite the classes, relationships between classes and subphyla are very speculative ~151. Phylogenetic relationships may be investigated further by using molecular sequence information, in particular the determination of small subunit rRNA (SSrRNA) sequences. The number of SSrRNA sequences for ciliate taxa has increased rapidly within the last few years [7, 16, 33, 34, 36, 371. The information from these partial and complete SSrRNA sequences has corroborated some taxonomic relationships based on ultrastructural and other morphological characters [7]. It also has brought other relationships into question. For example, the SSrRNA gene sequence of the hypotrich Euplotes aediculatus [36] did not cluster with the nassophorean Paramecium tetraurelia as predicted by Small & Lynn [31, 321. Instead Euplotes grouped closer to the classical taxonomic position of the hypotrichs with the stichotrichs as predicted by Corliss [5] and de Puytorac et al. [26].
MATERIALS AND METHODS The culturing of the ciliate C. inflata (American Type Culture Collection #30917) and extraction and purification of bulk nucleic acids were performed using the protocols described by Lynn 8~ Sogin [ 161. DNA of B. americanum was isolated following the modified Kavenoff and Zimm isolation procedure [ 11 and purified using cesium chloride centrifugation [ 171. Polymerase chain reaction (PCR) gene amplification of purified ciliate DNA was accomplished with a Perkin-Elmer/Cetus DNA Thermal cycler using a modification of the methods of Medlin et al. [18]. Briefly, the DNA was first denatured at 94" C for 5 min to separate the two complementary strands. Then, during a gradual cooling period of 2 min to 37" C the oligonucleotide primers complementary to the 5' and 3' conserved proximal termini preferentially annealed to the rRNA gene. Primer extension proceeded at 72" C for a period of 6 min in the presence of Thermus aquaticus (Taq) DNA polymerase (New England Biolabs) and deoxynucleotide triphosphates (Pharmacia). The rDNA sequence between the conserved oligonucleotide primers was exponentially amplified through 30 cycles of denaturation, primer annealing, and primer extension. Amplification products were checked for the proper size by electrophoresing a sample on a 2% agarose gel against a 1 kb BRL marker (Bethesda Research Labs) and a sample of Tetrahymena tropicalis PCR amplified rDNA (size 1.8 kb). Samples of the correct size were then extracted with phenol and concentrated by ethanol precipitation at -20" C [ 171. PCR amplified rDNA samples were cloned into M 13 [ 191 and single-stranded templates were prepared for directing DNA syn1
2
J. PROTOZOOL., VOL. 38, NO. 1, JANUARY-FEBRUARY 1991
B.americanun
aacctggttg atcctgccag tAGTCATATG CTTGTCTCAA AGATTMCCC ATCCATCTCT M G T A T M G C M T T A T A C C C CCAGAC-TGC GAATCGCTCA
99
C.inf lata
aacctggttg atcctgccag tAGTCATATG CTTGTCTTAA AGACTAACCC ATCCATGTCT M C T A T M C T A-TTATACAG CGAAACATGC GAATCCCTCA
99
T.thermophila
AACCTGGTTG ATCCTGCCAG TTA-CATATG CTTGTCTTAA ATATTMCCC ATCCATGTGC CAGT-TCAGT A-TTGAACAG CGAAAC-TGC GAATGGCTCA 96
B.americanun
TTAAAACAGT TATACTTTAT TTGCTAGACC T - - - - T T A T A
C.inf lata
TTAAAACAGT TATAGTTTAT TCGATTATTT TCTCCTT-CA TGGATAACCG TACTAATTCT AGAGCTAATA CANCCTGTC- AAACCTCACT TTTCTCCAAC
188 197
T.thermophi1a
TTAAAACACT TATAGTTTAT TTGATAATTA AAGA-TTACA TCGATMCCG ACCTAATTCT TCCCCTMTA CATGCTTAAA ATTCCCT-GT CCTCCGACCC
194
------ GTCG ACGAATCATA
TCGATAACCG T A C T M T T C T AGAGCTAATA CATCCTCGTT ACGCCTGTGC C T G - - - - - - -
B.americanun
----GTATTT
-GAAGCATTC
270
C. inf 1a t a
CCTTCTATTT ATTAGATATA AAACCAATAC CCAGCAATCC CTT-TTCTGA TGAT-TCATA ATMCTGAAC CGACCGCCGC CCTCCTGCCG CGAATCCTTC
295
T.thermophiLa
GAACGTATTT A T T A W T A T T AGACCAATCG CAGCAATCTC ATTGAGAT--
289
ATTAGATAAA ACCCAACGGG GGCGACCCT-
A T M C T T A C C GAACTCGACC T A C T - - - - - -
-GAA-TCAAA GTMCTCATC GGATCGAGGT TTACCTCGAT AAATCA-TCT
B.amer icanun
M C T T T C T G C CCTATCACCT TTCGATGGTA GTCTATTGGA CTACCATCCC GATGACCCCT GACCGAGAAT TACCCTTCGA TTCCGGACAG CGACCCTGAC 370
C. inf l a t a
AACTTTCTCC CCTATCACCT TTCGATGGTA GTGTATTCGA CTACCATGGC GATCACGCGT MCGCGGAAT TACGGTTCGA TTCCGGAGAC CGAGCCTGAC 395
T.thermophi1a
AACTTTCTGC CCTATCACCT CTCCATCGTA CTGTATTCGA CTACCATCCC ACTCACCCCT AACCGAGAAT TACCCTTCGA TTCCGCAGAA CGAGCCTGAC 389
B. a m e r i c a n u n
AAATCCCTAC CACATCTAAG CM-GGCACC AGGCGCCCAA A T T A C C U A T CCTAACTCAG CGAGGTAGTG ACAACAAATA ACAACGCCCC CCTTTCTCTT
469
C.inflata
AAATGGCTAC CACATCTAAC GAA-CCCACC AGCCCCCTAA ATTACCCAAT CCTMTTCAG CGACCTACTC ACMCAAATA ACAACTCCGA CCTCACTCGA
494
1-thermophil a
AAACCGCTAC TACAACTACG CTTCCCCACC ACGGAAGAAA ATTGGCCMT CCTAATTCAG CGAGCCAGTG ACAAGAAATA GCAACCTCGC AAACTTACCT
489
__ - GA TTCGMTGAG T T M G T G T M AACCCT--TC
B.americanun
CC---
CCACCACCCG CGG-TAAT-C CACCTCCACT
552
C. inf t a t a
GGTNACGAGA TTGNMTGAG M C M T T T M ACCTCTTATC GAGTMCAAT TGGACGGCM CCTC-TGCTG CCACCAGCCG CCGCTAATTC CAGCTCCAAT
593
T. thermophi l a
TTCTACGCCA TTGAMTGAG M C A G T C T M ATCTCTTAGC G A G G M U A T TGGACGCCAA G-TCATCGTC CCACCAGCCC CGC-TAATTC CAGCTCCAAT
587
B .emericam
AGCGTATATT M A G - T T G T T - C C - A G T T M AAAGCTCGTA GTGMGC-TC TGCGGG-GCG GGTCGCTCCG CCTCGGTCTC GTGACCTTCC TCGCAT-CCA
646
C-inflata
AGCGTATATT MAGCTTGTT A C G U C T T M AAAGCTCGTA GTTGMTATC TGGCCGGTGC TGTTCTTGGC TCCCTGAGTC CGCTCAGCGC CTCGTCATCC 693
T.themophi1a
AGCGTATATT M A G - T T G T T - - G C A G T T M MAGCTCGTA GTTGMCTTC TGT-1-CAGG TTCATTTCGA TTCGTCCT-G T G A M - - C T G GACATACGTT
B . e r i cawn C. inf l a t a T. t h e m i la
TC-TGAGCM CGGC--TCCG G C - - A T T M C TTG-TCGCTG TCGTG-ATCA GGTAC--TTT ACCTTGAGCA MTGAGAGTG TTCCAGGCAG GCTTGGGCCT 737 GTATGGGMA CTAGCTCGAC C----TTCAC
GAGGACCACT - - G A C G - U A GC---TG-TG
TGGTCGGCTA 6 - T G G A - T U
--__-M M T CGGCCTTCAC TGGTTCGACT
--TACACTTT A C T T T G A M A M T T A G A G T G TTTCAGGCAC CCAATNGCTT
679
785
A C T G T G M M MTTAGAGTG TTCCAGGCAG GTTTTAGCCC
769
B americanun
GATACGTCCA CCATCGAATA ATAGAAGAGC ACTGGCCTCC ATTTATTGCT GTTATGGCCC T T A - G T M T G ATTAATAGGG ATAGTTGCCC GCATTTGTAT
836
C.inf lata
CGATACTGTA GCATCGMTA ATCGAATACC ACTTTGACCT ATTTCTTGGT NTCTCGAGGT U A A G T M T G ATTMTAGCC ACAGTTGGGC GCATTCCTAT
885
T-thermophila
GAATACATTA GCATGGAATA ATGGAATAGG ACTAAGTCCA TTTTATTCCT TCTTCGATTT CC---TAATG
ATTAATAGGC ACAGTTGGCG GCATTACTAT
866
B.americanun
TTAATTCTCA GACCTGAAAT TCTATGATTT ATTAAACACA AACTTATCCC AAACCATTTC CCAAGGATCT TTTCATTAAT CAAGAACCAA ACTTAACCGA
936
C. inf Lata
TTAATNGTCA GACCTGAAAT TCTTCGATTT TTTAAAGACG AACTTATGCG AMCCATTTG CCAACCATCT TTTCATTAAT CAAGAACCAA AGTTACCCCA
985
T. thermophi l a
T T M T A C T C A GACGTGAAAT TCTTGGATTT ATTAAGCACT MCTAATGCC AAACCATTTC CCAAACATCT TTTCATTAAT CAAGAACCAA AGTTAGGGGA 966
.
.
TCWCT--
TAGGGAGTM A---CATTTT
B m r icanun
TCAAAGACGA TCAGATACCG TCCTACTCTT AACCATAAAC TATCCCGACT AGAGATTCGA CGTGCCATTA AAAGTACTCC TTCACCATCT TCCGAGAAAT
C.inflata
TCAAAGACGA TCACATACCC TCCTACTCTT AACCATAAAC TATACCGACT ACGGATTGCT GACCTCTTTT
T.thermophila
TCAAAGACGA TCAGATACCC TCCTAGTCTT AACTATAAAC TATACCCACT CCGGATCCGC TCCAATAAA- ----TCTCCA
- - -MCCCTC
1036
ATCACCACCT TATCAGAAAT
1082
GTCGCCACCC TATCAGAAAT
1061
B. a m e r i c a n u n
CAAACTCTTT CGCTTCTCCC CCCAGTATGC T-CGCAACAC -TGAAACTTA MCGAATTGA CGCAACGCCA CCACCACGAC TGGCA--TGC CCCCTTAATT
1132
C. inf Lata
CAAACTCTTT CGCTTCTCGG CGGACTATCG 1-CCCAAGCC CTGAAACTTA AACGAATTGA CGGAAGCGCA CCACCACGAC TCCACCCT-C CGCCTTAATT
118C
T.thermophila
CAAACTCTTT GGGTTCTGGC GGAAGTATGG TACCCAACTC -TGAAACTTA AAGCAATTGA CGGAACACCA -CACCAGAAC TGGAACCT-C CGGCTTAATT
11%
.
B a m e r icanun
TGACTCAACA CGGGCAAACT TACCAGCTCC AGACATGCCA ACGATTGACA GATTGATAGC TCTTTCTTGA TTCTATCCGT CCTGCTCCAT CCCCGTTCTT
1232
C. i n f Lata
TGACTCAACA CCCCGAAACT TACCACCTCC AGACATACTT CGGATTGACA GATTGAGACC TCTTTCTTGA TTCTATGCCT CGTGGTCCAT GCCCCTTCTT
128C
T.thermophi1a
TGACTCAACA CCCCCAAACT CACGAGCCCA ACACACAGAA CCCATTCACA GATTGAGACC TCTTTCTTCA TTCTTTCCGT GCTGCTCCAT CCCCGTTCTT
125E
Fig. 1. Small subunit rRNA gene sequences of the ciliates Blepharisma americanum ( 1 7 19 nucleotides) and Colpodu inflata ( I 786 nucleotides) aligned with the sequence from Tetruhymenu thermophilu (1 753 nucleotides) [37]. Nucleotides indicated in lowercase correspond to those regions complementary to the oligonucleotide primers used for PCR amplification. Numbers at end of lines indicate the number of nucleotides. The differences in sequence introducing alignment gaps (-) in the sequences. Ambiguities in nucleotide positions for B. umericanum and for C. inj7utu are indicated in the figure by the letter ‘N.’
GREENWOOD ET AL-BLEPHARISMA AMERICANUM A N D COLPODA INFLA TA SMALL SUBUNIT R R N A GENE
- - -TCTTGCC
3
B. a m e r icanun
AGTTCCTCGA CTGATTTCTC TGGTTMTTC CGATMCGAA CGAGACCTTA ACCTCCTMC TAGTCCTC--
MAGACTCGC ACTTCTTAGA
1327
C.inflata
ACTTCGTCGA CTGATTTGTC TGGTTMTTC C C T T M C G M CGAGACCTTA ACCTGCTMC TACTCAC- - C TTTATCTCM TACCCGTCTG ACTTCTTAGA
T.thermophi1a
ACTTGCTGGA CTGATTTGTC TGGTTMTTC CCTTAACGAA CGAGACCTTA ACCTGCTMC TACT----CT
1378 1354
B. amer icanun
CCCACTTTCC CCCCTA- - C T C C M C G M G T T T M C C C M TAACACCTCT GTGANCCCCT TAGANCTCCT GCGCCCCACC CCCGCTACAC TGATCCACCC
C.inflata
GGGACTTTAT G T C C M - - C CATMGGMG TTTGACGCM TMCAGGTCT CTGATGCCCT TAGATGTCCT GCCCCGCA-G CCCCCTACAC TGACACATGC
1424 1474
T.themphi1a
GCGACTATT- C T G C M T M C C C M T C G M C T T T M G C C M TAACAGGTCT CTGATCCCCC TAGACCTCCT CCCCCCCACC CCCCTTACM TGACTCCCCC
1453
GCTTGTAMT MCACGTTCT ACTTCTTAGA
-
B.americanun
ACTAACCCC- ----TCCGCC
C. inf l a t a
M C M C T T T A TTTTTCCTGA CTCCGMCGG TTCCCCTAAT CTTCTATMT CTCTCTCCTG CT-AGGCATA GA-TCTTTGG MTTATAGAT C T T G M C M C
1. t h e r m o p h i l a
A A A M C - - T A TTT--CCTCT CCTCGGMGC TACCCCTMT C T T A T - T M T ACCAGTCGTG TTACCGATAG 1--1CTTTGG MTTGTCGAT CTTGMCGAC
1513 1572 1546
B. amer icanun
GAATTCCTAG T A M C G C M C TCATCMCTT CCATTGACTA CCTCCCTGCC CTTTCTACAC ACCCCCCGTC GCTCCTACCG ATTTCGACTG ATGACCTGM
1613
C. inf l a t a
GMTTCCTAG T M C C A T M C TCATCACCTT CTCCTGATTA CCTCCCTCCC CTTTGTACAC ACCCCCCCTC GCTCCTACCC ATTTTGACTC ATCCCCTGM
1. t h e m p h i l a
GAATTTCTAG T M C T G C M C TCATCAGCTT GCGTTGATTA TGTCCCTGCC CTTTGTACAC ACCGCCCGTC GCTTGTAGTA AC---GMTG
1672 1643
B.americanun
TCCTCCCGAC TGCACACCTC
C. i n f l a t a
CCTTCTGGAC TCTGGTCACG CTTGACCTGA TTCTCCGMG 1-TAAGTAAA CCTTATCACT TAGAGGMGG ACMGTCCTA ACMGGTTTC C g t a g g t g a a
T.thermophila
CCTTCTGGAC TCCGA-CACC M T C - 1 1 - C C
B.americaMm
cctgcagaag gatca
1719
C.inf l a t a
cctgcagaag gatca
1.thermophila
CCTGCAGATG GATCATTA
1786 1753
CU-GAMNC CCCCGCTAAC CTTCAMC-T GCATCCTGAT ---CGCGATT
-------- GT
GACTCTT-CC MTTATTCCT CATCMCGAC
GTCTGGTGM
1704 1771 G G M A AATAACTAM CCCTACCATT T G G M U A C A AGMGTCGTA ACAAGGTATC TGTAGCTGM 1735
CTGTCGGAAG T - T G A C T A M CCTTATUCT TAGAGGMGG AGMCTCGTA AUAGCTATC T g t a g g t g a a
-----
thesis in the Sanger dideoxy-chain termination sequencing protocol [3, 301. Sequences were aligned using a procedure that considers the conservation of both the primary and secondary structure for phylogenetic reconstruction [7]. The sequence information was reduced to approximately 1,600 unambiguously aligned positions for comparisons of the Phylum Ciliophora in relation to other eukaryotes. Sequence data used in the alignments were from the following: corn, Zea mays [20]; chlorophyte, Chlamydomonas reinhardtii [9]; yeast, Saccharomyces cerevisiae [29]; bread mould, Neurospora crassa [35]; oomycete, Achlya bisexualis [9]; chrysophyte, Ochromonas danica [9]; dinoflagellate, Prorocentrum micans [ lo]; and the ciliates Euplotes aediculatus [36], Oxytricha nova [7], Oxytricha granulifera [Schlegel & Sogin, unpubl.], Onychodromus quadricornutus [Schlegel & Sogin, unpubl.], Stylonychia pustulata [7], Paramecium tetraurelia [33], Tetrahymena hegewischi [34], Tetrahymena thermophila [35], and Tetrahymena pyriformis [34]. Aligned sequences were analysed using a Distance Matrix method in which calculations of structural (sequence) similarity and evolutionary distance incorporate the Jukes & Cantor [ 1 11 correction to estimate unobserved nucleotide substitutions [7, 23, 241. Phylogenetic trees were constructed by an additive tree method which determines the tree geometry and the branch lengths that best fit the evolutionary distance data [23, 241. RESULTS The complete small subunit rRNA sequences obtained for B. americanum and C. inflata were 1719 nucleotides and 1786 nucleotides respectively (Fig. 1). The phylogenetic tree generated from the SSrRNA sequence data used a weighting mask that included all regions of the eukaryotic SSrRNAs that can be unambiguously aligned for this specific set of sequences (Fig. 2; Table 1). The almost simultaneous divergence ofthe plants, fungi, and ciliates has been noted earlier [9]. This tree clearly shows that the ciliates diverged as
a monophyletic group [6], and among the taxa chosen for the analysis the dinoflagellates, represented by Prorocentrum micans, are the sister group to the ciliates (Fig. 2). The depth of branching between some of the major ciliate groups is deeper
0.05
Fig. 2. Ciliate phylogeny inerred from complete small subunit rRNA sequence similarities using a distance-matrix method. The analysis was limited to approximately 1,600 positions that could be unambiguously aligned in all of the rRNA sequences. The evolutionary distance (average number of nucleotide substitutions per sequence position) is represented by the horizontal component separating species in the figure. The scale bar equals 0.05 nucleotide substitutions per sequence position. Data are given in Table 1 .
4
J. PROTOZOOL., VOL. 38, NO. I , JANUARY-FEBRUARY 1991
Table 1. Structural similarity and evolutionary distance data for eukaryote small subunit rRNA gene sequences." Structural similaritv/evolutionarv distance to: Organisms
Z.m.
Z. mays C. reinhardtii
S. cerevisiae
0.107 0.156 0.175 0.163 0.165 0.164 0.250 0.202 0.187 0.198 0.183 0.183 0.195 0.204 0.256 0.250 0.252
C.r.
S.C.
N.c.
A.b.
0.d.
P.m.
E.a.
0.900
0.859 0.860
0.844 0.853 0.907
0.854 0.855 0.852 0.834
0.852 0.859 0.853 0.847 0.899
0.853 0.853 0.852 0.845 0.861 0.864
0.787 0.786 0.801 0.786 0.81 1 0.813 0.828
0.155
N. crassa 0.163 0.099 A. bisexualis 0.161 0.165 0.188 0. danica 0.167 0.164 0.171 0.109 P. micans 0.163 0.165 0.174 0.154 0.150 E. aediculatus 0.25 1 0.232 0.253 0.218 0.2 15 0.196 B. americanum 0.197 0.195 0.208 0.209 0.179 0.164 0.213 0. nova 0.188 0.165 0.184 0.171 0.155 0.130 0.145 0. granulifera 0.201 0.182 0.197 0.184 0.169 0.152 0.159 0. quadricornutus 0.188 0.168 0.184 0.170 0.161 0.129 0.149 S. pustulata 0.192 0.171 0.189 0.173 0.161 0.135 0.147 C. inflata 0.20 1 0.179 0.192 0.168 0.154 0.153 0.190 P. tetraurelia 0.198 0.185 0.197 0.186 0.183 0.162 0.195 T. hegewischi 0.256 0.215 0.238 0.232 0.226 0.216 0.242 T. thermophila 0.25 1 0.216 0.238 0.229 0.221 0.218 0.242 T. pyriformis 0.253 0.2 13 0.236 0.230 0.223 0.2 17 0.242 Note: The upper half of the table gives the structural similarity (S) values for all pairs of aligned small subunit rRNA sequences. The lower half of the table gives the evolutionary distance values (average number of nucleotide substitutions per sequence position) determined by the Jukes & Cantor [ 1I ] formula for conversion of structural similarity. a Sources of sequence data for the aligned eukaryote small subunit rRNA sequences are listed in Materials and Methods. than the separation between plants and fungi. For example, the colpodid C. inflata and the hypotrich E. aediculatus are separated by an evolutionary distance of 0.190 while corn 2. mays and yeast S. cerevisiae are separated by an evolutionary distance of 0.156 (Table 1). This demonstrates that ciliate groups diverged early after the split of the ciliate ancestor from the main eukaryote line. The Class Oligohymenophorea, sensu Small & Lynn [3 1, 321, represented by T. hegewischi, T.pyriformis, and T. thermophila, remains a monophyletic group with the Class Nassophorea (P. tetraurelia) as its sister group. The Subclass Hypotrichia (E. aediculatus) and the Subclass Stichotrichia (0.quadricornutus, 0. granulifera, 0. nova, and S. pustulata) remain sister groups within the Class Spirotrichea, sensu Corliss [ 5 ] and de Puytorac et al. [26]. However, the evolutionary distance separating the hypotrich E. aediculatus from the stichotrichs ( ~ 0 . 1 5 0 is ) almost as great as the distance separating corn Zea and yeast Saccharomyces (0.156) (Table 1). The stichotrich 0. nova is more closely related by sequence similarity (0.993) to S. pustulata than to 0.granulifera (0.989) (Table 1). The Class Colpodea sensu Small & Lynn [3 1,321, represented by C. inflata, is more closely allied with the oligohymenophoreans and nassophoreans than the spirotrichs while the heterotnch B. americanum is placed as the earliest branching ciliate (Fig. 2). DISCUSSION Molecular comparisons. The significance of the phylogenetic placement of B. americanum and C. infata within the Phylum Ciliophora, inferred from complete SSrRNA sequences, may be determined by congruency checks with the phylogenies constructed from other molecules and morphological characters [23, 241. Baroin et al. [2] constructed a global eukaryotic phylogeny inferred from the approximately 400 nucleotides of Domain 1 and its flanking region of the large subunit rRNA (LSrRNA). The Baroin et al. [2] tree placed the ciliates as a monophyletic
group with the dinoflagellates (Prorocentrum micans) as their sister group. The phylogeny was limited to five ciliates (Blepharisma, Stentor, Tetrahymena, and two species of Paramecium) and 10 other eukaryotes and one eubacterium. The tree topology indicated two main branches: the first depicted the two Paramecium species as closely related to Tetrahymena while the heterotrichs Stentor and Blepharisma were placed on a separate branch [2]. The placement of Tetrahymena and Paramecium and the location of the heterotrichs Blepharisma and Stentor on a separate branch within the ciliate lineage is consistent with the phylogeny of the present study (Fig. 2). However, since the Baroin et al. [2] tree did not contain representatives of the colpodids or hypotrichs not all the intra-phylum comparisons can be made. Nanney's group has been concentrating on Domain 2 (D2) of the LSrRNA, which corresponds to a region of 180 nucleotides located just downstream of Domain 1 [21, 22, 251. Their most extensive phylogeny included 14 ciliates (Colpodium striatum, Colpoda maupasi, Colpoda steinii, Glaucoma chattoni, Didinium, Paramecium, and eight tetrahymenas) and one dinoflagellate (Crypthecodinium cohnii) [25]. The Preparata et al. [25] tree placed the ciliates as a monophyletic group with the dinoflagellate C. cohnii as their sister group. Crypthecodinium is a different genus from the dinoflagellate used in the present study, further reinforcing the notion of these protists as the sister group of the ciliates. Their tree placed the ciliates C. steini and C. maupasi with Didinium and Paramecium outside the hymenostome (Colpidium, Glaucoma, and Tetrahymena) cluster [25]. Although the exact branching order of the genera Colpoda, Didinium, and Paramecium was not resolved on the Preparata et al. [25] tree, this may indicate a close association ofthe colpodids with the gymnostomes as predicted by Corliss [5] (see below). Conversely the branching order may be biased by the low number of sites used in the analysis or by the lack of other ciliate taxa (e.g. heterotrichs, hypotrichs, and stichotrichs.) Lynn & Sogin [ 161 assessed the phylogenetic relationships among the ciliates by using partial SSrRNA sequences derived from reverse transcripts. Both the Lynn & Sogin tree [ 161 and
5
GREENWOOD ET AL. -BLEPHARISMA AMERICANUM AND COLPODA INFLATA SMALL SUBUNIT RRNA GENE
Table 1. Continued. Structural similaritv/evolutionarv distance to: B.a.
0.n.
0.g.
0.q.
S.P
C.i.
P.t.
T.h.
T.t.
T.p.
0.823 0.827 0.828 0.818 0.817 0.84 1 0.852 0.814
0.835 0.834 0.582 0.837 0.847 0.860 0.880 0.868 0.868
0.826 0.823 0.839 0.826 0.837 0.849 0.862 0.857 0.858 0.972
0.838 0.834 0.849 0.837 0.848 0.855 0.88 1 0.865 0.865 0.989 0.968
0.838 0.83 1 0.847 0.833 0.845 0.855 0.877 0.866 0.863 0.993 0.969 0.986
0.828 0.823 0.841 0.830 0.849 0.860 0.861 0.832 0.859 0.895 0.884 0.894 0.892
0.822 0.826 0.836 0.827 0.836 0.837 0.855 0.829 0.855 0.879 0.865 0.880 0.878 0.881
0.783 0.783 0.8 13 0.796 0.800 0.805 0.812 0.793 0.810 0.839 0.829 0.839 0.837 0.843 0.85 1
0.787 0.787 0.8 12 0.796 0.803 0.808 0.810 0.793 0.809 0.836 0.826 0.837 0.833 0.843 0.854 0.986
0.786 0.785 0.814 0.797 0.802 0.807 0.812 0.793 0.813 0.838 0.827 0.839 0.835 0.842 0.855 0.99 1 0.994
0.145 0.157 0.149 0.151 0.156 0.161 0.219 0.220 0.215
0.029 0.01 1 0.007 0.113 0.132 0.181 0.184 0.182
0.032 0.032 0.126 0.149 0.194 0.198 0.197
0.0 13 0.115 0.130 0.180 0.184 0.181
0.112 0.133 0.184 0.189 0.187
the present analysis placed C. inflata on the lineage leading to the nassophoreans and oligohymenophoreans. No representative of the heterotrichs was sequenced in the Lynn & Sogin study [ 161. The partial SSrRNA sequence data appears to have produced a phylogeny consistent with that based on complete sequences. It has been shown previously that phylogenetic trees constructed by using partial SSrRNA sequences from organisms of different kingdoms had topologies identical to that obtained by using complete sequences [ 131. The resolving ability of the reverse transcripts technique has not been fully tested. In both the Kumazaki et al. [ 121 and Walker [38] 5s rRNA analyses, the ciliates (Blepharisma, Bresslaua, Euplotes, Paramecium, and Tetrahymena) were delineated as a monophyletic group and the dinoflagellates, again represented by Crypthecodinium, as their sister group. Both 5s rRNA trees are in agreement with the present analysis with respect to the branching order of Tetrahymena and Paramecium. The position of the colpodid Bresslaua varies between the two 5s rRNA trees: Walker [38] placed Bresslaua on a separate branch from Tetrahymena and Paramecium, while Kumazaki et al. [ 121, in agreement with the present study, placed the colpodids as more closely related to Paramecium and Tetrahymena than to the other ciliates (Fig. 2). The distance between Blepharisma and Euplotes based on the 5s rRNA trees is comparable to that depicted in the present study (Fig. 2), suggesting based on molecular evidence from 5s rRNA and SSrRNA, the heterotrichs should probably be elevated to a higher taxonomic rank as suggested by de Puytorac et al. [26] (see below). Morphological comparisons -taxonomic implications. In the tree derived from the SSrRNA sequences C. inJata was placed on the lineage leading to the nassophoreans and oligohymenophoreans (Fig. 2). Corliss [5] considered the colpodids as an order within the Class Kinetofragminophora together with the trichostome, gymnostome, and hypostome ciliates, and far removed from the nassophoreans and oligohymenophoreans.Small & Lynn [31, 321 and de Puytorac et al. [26] both recognized the colpodids as a distinct group, based on the overlapping posteriorly-directed transverse microtubular ribbons of their somatic dikinetids, and have elevated them to class rank. Small & Lynn [3 1, 321 placed the Class Colpodea along with the Classes Nassophorea, Oligohymenophorea, and Phyllopharyngea (none se-
0.129 0.177 0.177 0.177
0.166 0.162 0.161
0.014 0.009
0.006
quenced) in the Subphylum Cyrtophora since the oral dikinetids possess postciliary microtubular ribbons that support the cytopharynx. On the other hand, de Puytorac et al. [26] placed the colpodids in the Subphylum Prostomatea with the trichostomes and prostomes, based on the polar formation of the cytostome. The deep branching of C. inflata on the SSrRNA trees tends to support the class status given by de Puytorac et al. [26] and Small & Lynn [3 1 , 321. No SSrRNA sequence information is available from representatives of the trichostomes, gymnostomes or prostomes. However, Preparata et al. [25] demonstrated a close association of the gymnostome Didinium with the genus Colpoda. Since their tree lacked representatives of the heterotrichs, hypotrichs, and stichotrichs, no definitive conclusions can be made with respect to the relationships suggested by Corliss [5] and therefore must await further sequence information. Blepharisma americanum was placed as the earliest diverging ciliate after the phylum Ciliophora separated from the main eukaryote line in the phylogeny inferred from complete SSrRNA sequences (Fig. 2). All three classification schemes recognise the heterotrichs as a distinct group. However, Corliss [ 5 ] separated the heterotnchs and hypotrichs at the ordinal level based on differences in their adoral zone of membranelles while Small & Lynn [31, 321 separated the heterotrichs and stichotrichs into different subclasses within the Class Spirotrichea. De Puytorac et al. [26] stated that differences in the adoral zone of membranelles make the heterotrichs distinct from the spirotrichs and have separated the heterotrichs from the spirotrichs, elevating the heterotrichs to a class within the Subphylum Polyhymenophorea. The deep branching of B. americanum in the phylogeny based on complete SSrRNA sequences supports the elevation to class status given the heterotrichs by de Puytorac et al. [26]. Based on Raikov’s hypothesis [28] for explaining nuclear dualism, Corliss [4] initially proposed that the karyorelictid ciliates were the closest living ciliates to the ancestral stock for the phylum. Small & Lynn’s classification [3 1, 321 suggested a close relationship of the heterotrichs to the karyorelictids based on similarities in their somatic dikinetid by placing them together in the Subphylum Postciliodesmatophora [3 1, 321. On the other hand de Puytorac et al. [26] separated the karyorelictids and heterotrichs into the subphyla Karyorelicta and Polyhymeno-
6
J. PROTOZOOL., VOL. 38, NO. 1 , JANUARY-FEBRUARY 1991
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Received 5-14-90; accepted 10-15-90
