Molecular determination of the phylogenetic position of a species in the genus Colpodella (Alveolata)

12 Pages·2001·1.5 MB·English

by  Siddall, Mark Edward

AMERICAN MUSEUM
Novtitates
PUBLISHED BY THE AMERICAN MUSEUM OF NATURAL HISTORY
CENTRAL PARK WEST AT 79TH STREET, NEW YORK, NY 10024
Number 3314, 10 pp., 3 figures January 30, 2001
Molecular Determination of the Phylogenetic
Position of a Species in the Genus Colpodella
(Alveolata)
MARK E. SIDDALL,'’ KIMBERLY S. REECE,” TOM A. NERAD,? AND
EUGENE M. BURRESON*
ABSTRACT
The phylogenetic position of a species of the free-living protist genus Colpodella is assessed
using its SSU rDNA gene sequence in the context of a wide array of other alveolate taxa.
Phylogenetic analyses indicates that this species is not related to species of Bodo as has
previously been suggested. However, SSU rDNA data alone are insufficient to provide a whol-
ly stable hypothesis of relationships for Colpodella sp. among the various alveolate phyla.
Much of this instability can be attributed to alignment procedure sensitivity. Analyses of SSU
rDNA and actin gene sequences in combination provide strong support for a species of Col-
podella as sister group to the ciliates, irrespective of alignment procedure. Moreover, use of
SSU rDNA data alone strongly support a recent common ancestry of the genera Perkinsus
and Parvilucifera with the dinoflagellates, and not with the apicomplexans. These findings,
and the nearly identical ultrastructural characteristics of Colpodella, Perkinsus, and Parvilu-
cifera species, suggest that this morphology is the ancestral condition for all of Alveolata.
INTRODUCTION *“‘super-phylum”’ Alveolata (Gajadhar et al.,
It is now well understood that protistan 1991). Each of these three phyla share only
phyla Apicomplexa, Ciliophora, and Dinozoa a few common features such as a second in-
share a recent common ancestor and form the ner membrane system (alveoli) and micro-
' Assistant Curator, Division of Invertebrate Zoology, American Museum of Natural History.
? Virginia Institute of Marine Science, Gloucester Point, Virginia.
3 American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia.
4 Virginia Institute of Marine Science, Gloucester Point, Virginia.
2 AMERICAN MUSEUM NOVITATES NO. 3314
pores (Siddall et al., 1997) but otherwise and each of these taxa have elongate extru-
they have unique characteristics. For exam- somes that are hypothesized to be homolo-
ple, dinoflagellates once thought to be ances- gous with apicomplexan rhoptries. Previous-
tral to all eukaryotes (Loeblich, 1976), have ly, however, molecular data have not been
uniquely condensed chromosomes. Ciliates available for any species in the genus Col-
stand apart not only with uniquely arranged podella.
cortical ciliature but also by having two nu-
clei. The apicomplexans, so named for their MATERIALS AND METHODS
distinctive apical organelles (Levine, 1988),
also exhibit gliding motility unlike any other An undescribed species of Colpodella
group of organisms. In light of this unique- found feeding on Bodo caudatus was isolated
ness, the ancestral conditions for the alveo- from brown woodland soil in Gambrill State
lates are not obvious. Park, Maryland, in 1993. This species was
Perkinsus marinus was thought to be re- maintained in vitro at the American Type
lated to apicomplexans when it was discov- Culture Collection [ATCC 50594] with its
ered to possess structures reminiscent of a naturally occurring bodonid prey. Frozen 500
conoid and micronemes (Perkins, 1976; Lev- wl samples were thawed, and 50 pl 2M NaCl
ine, 1978). Levine (1988), though he held in 1500 pl cold ethanol was added to precip-
that this oyster parasite may be ancestral to itate any DNA released from ruptured cells
the group, added that this was ‘“‘conjectural prior to DNA isolation. After centrifugation
and never will be proven. There are no fos- and washing with 70% ethanol, the pellet
sils.””, However, several molecular phyloge- was resuspended in lysis buffer and digested
netic analyses clearly have placed Perkinsus with proteinase-K at 55°C. DNA isolation
species with the dinoflagellates (Siddall et then followed standard phenol-chloroform
al., 1995, 1997; Reece et al., 1997). The re- extraction procedures.
cent discovery and characterization of a Per- Amplification of the small subunit ribosom-
kinsus-like protist, Parvilucifera infectans, al RNA gene used combinations of primers 5’-
have complicated this picture (Norén et al., AACCTGGTTGATCCTGCCAGT-3’ and 5’-
1999; Erard-Le Denn et al., 2000). Like Per- TGATCCTTCCGCAGGTTCACCT-3’ (prim-
kinsus species, P. infectans is a parasite, but ers “A” and “B” respectively) and internal
one of toxic dinoflagellates not of metazoans. primers (primer “L’: 5’-CCAACTACGAGC-
Its ultrastructural characteristics are marked- TTTTTAACTG-3’, primer “‘C’’: 5’-CGG-
ly similar to those seen in species of Perkin- TAATTCCAGCTCCAATAG-3’, primer ““Y”’:
sus. A phylogenetic analysis of SSU rDNA 5'-CAGACAAATCGCTCCACCAAC:-3’,
strongly supports the monophyly of these primer “O”’’: 5'-AAGGGCACCACCAGG-
two genera, but as the sister group to Api- AGTGGAG-3’'). Reaction mixtures were heat-
complexa, not to Dinoflagellata (Norén et al., ed to 94°C for four minutes and then cycled
1999). 35 times at 94°C (20 s), 47°C (20 s), 68°C (105
Less well studied are species of the genus s) with a final extension at 70°C (seven min-
Colpodella, which except for their free-living utes). “Universal” actin gene primers “‘480”’:
habits, also are nearly indistinguishable from 5'-AA(TC)GGIGA(AG)AA(AG)ATGACICA-
Perkinsus species. Species of Colpodella (AG)AT(TCA)ATGTT-3' and ‘483’: 5’-
(prev. Spiromonas) are predators of other CCAIACI(CG)(AT)(AG)TA(CT)TTIC-
free-living protists (Brugerolle and Mignot, (GT)(CT)TCIGGIGG-3' designed by G. Warr
1979; Myl’nikov, 1991; Simpson and Patter- (Medical University of South Carolina) and M.
son, 1996). With a reinforced rostrum, they Wilson (Mississippi State Medical Center) for
penetrate through the cell membrane of their amplification of vertebrate actin genes were
protistan prey and consume the cytoplasmic used to amplify a central coding region of the
contents or ingest whole cells (Brugerolle actin genes corresponding to residues 127
and Mignot, 1979). The rostrum of Colpo- through 333 in vertebrate actin protein se-
della species is identical in structure and quences. Five to 10 ng of genomic DNA was
function to the so-called “‘conoid” of zoo- amplified in 50 pl reactions using the BRL
spores of Perkinsus and Parvilucifera species PCR Reagent System (Life Technologies, Gai-
2001 SIDDALL ET AL.: PHYLOGENY OF COLPODELLA 3
thersburg, Maryland). Reagent concentrations AF006470, AFO09244, AF060975, AFO80611,
were as follows: 20 mM Tris-HCl (pH 8.4), AF080612, AF111183, LO2366, L19078,
50 mM KCI, 1.5 mM MgCl, 50 pmoles of L19080, L24383, NSPRG18S, U40264,
each primer, 0.2 mM each of dATP, dGTP, U67117, U94787], 18 ciliates [X56171,
dCTP, dTTP, 20% (v/v) BSA, and 1.25 units AF164136, U97111, U17354, U57769,
of Taq DNA polymerase. Following an initial U57770, U5S7771, X03948, X03772, SPU97112,
denaturation of four minutes at 95°C, reac- ECU57765, EMU57766, ECU5776, FVU971 10,
tions were subjected to 40 cycles of one mi- OCU17355, AHU51554, CHU97109], and
nute at 95°C, one minute at 45°C and three Parvilucifera [AF133909] as well as seven
minutes at 65°C. A final extension of five outgroup taxa [X89518, M87327, M32705,
minutes at 65°C was added to each amplifi- J01353, U83128, M97959, M32704]. Bodo
cation reaction. Amplification products were caudatus [X53910] was included in prelim-
electrophoresed in 1.5% low-melting-point inary alignments to assess the identity of
agarose and removed with the QiaQuick Gel- clones. Analyses employing combined SSU
Extraction protocol (QiaGen), or were cloned rDNA and actin gene sequences were con-
into the plasmid vector pCR2.1 (TA Cloning ducted with the same taxonomic composition
kit, InVitrogen) following the manufacturer’s as above and coding actin as missing for
protocol. The ligated plasmids were trans- those taxa for which it is not available. Com-
formed into E. coli DH5a cells and recom- bined SSU rDNA and actin analyses also
binants were selected for sequence analysis. were conducted with a different taxonomic
Some sequencing reactions employed Big- composition using only those taxa for which
Dye (Applied Biosystems) according to the both genes were available. The latter analy-
manufacturer’s instructions and were electro- ses include SSU rDNA and actin for two
phoresed in 4.5% polyacrylamide on an ABI slime molds [X00134, X13160; X03282,
377 automated sequencer. Chromatograms M21500], four fungi [X04971, Z75578,
from fragments sequenced in both directions Le S307 XSOSLSe IGS... NOIZSS,
were reconciled in Sequence Navigator. For U17498, U78026], a chlorophyte [M32703;
the cloned amplification products, both D50839], Acanthamoeba [U07413; VOOOO2],
strands of the plasmid inserts were se- a rhodophyte [Z14140; U03677], four stra-
quenced in a single tube using the menopiles [AB011423, M32705, X53229,
ThermoSequenase kit (Amersham) and flu- X54265; U11697, %59937, X59936,
orescently labeled (IRD700 and IRD800, LI M59715], a haptophyte [M87327; S64188],
COR) M13 forward and reverse primers in two dinoflagellates, [M14649, L13719;
simultaneous bidirectional sequencing U84290, U84289], four ciliates [X03948,
(SBS). The labeled DNA fragments were AF164136, X56165, X03772; X05195,
separated on 4% Long Ranger acrylamide J01163, AF043608, J04533], two apicom-
(Baker) gels and detected by infrared lasers plexans [U12138, AF108864; U10429,
in a LI COR automated sequencer (model M86241], and Perkinsus [L0O7375; U84287].
4200L-2). Alignment was explored using phenetic
Because of the contaminating prey item CLUSTAL alignments, and parsimonious se-
(Bodo caudatus) DNA, multiple clonal iso- quence alignment with MALIGN (Wheeler
lates of amplification products were se- and Gladstein, 1996a) including and exclud-
quenced to ensure that Colpodella sp. se- ing regions of highly variable alignment; in
quences were obtained. An appropriately addition parameter sensitivity was assessed
structured suite of taxa was obtained for phy- with optimization alignment using POY
logenetic analyses of SSU rDNA alone in- (Wheeler and Gladstein, 1996b). Alignment
cluding 18 dinoflagellates [AF0O22153, parameters varied for 18S rDNA sequences
AF022154, AFO22156, AFO22191, AFO22194, was the cost-ratio of insertion-deletion events
AF022197, AFO22199, AFO22200, AFO22201, to base substitutions (1, 2, 3, 4 and 5). The
AFO022202, AFO52190, AFO77055, AFO80096, most parsimonious solution, or that found to
AF080097, L13717, L13719, M14649, be optimal under likelihood with the no com-
SNU52357], two species of Perkinsus [LO7375, mon mechanism model (Tuffley and Steel,
U07701], 15 apicomplexans [M97703, 1997), was determined with Nona (Goloboff,
+ AMERICAN MUSEUM NOVITATES NO. 3314
1998) using the Ratchet option with 10 rep- support for each of the three recognized al-
licates of 50 iterations. Levels of support for veolate phyla and for monophyly of the gen-
clades examined included Bremer support era Parvilucifera and Perkinsus. Exclusion
values (Bremer, 1988) obtained with Nona of highly variable alignment regions (fig. 1B)
(Goloboff, 1998), parsimony jackknife (Far- yielded two equally parsimonious trees of
ris, et al., 1996) values using 100 replicates length 4022. Again Colpodella grouped with
with branch breaking, and 10 random addi- the Apicomplexa; however, this clade could
tion sequences in Xac (Farris, 1998). be refuted with only two additional steps. Al-
though monophyly of Perkinsus and Parvil-
RESULTS ucifera species with the Dinozoa was indi-
cated with these truncated data, neither this
Analysis of SSU rDNA aligned with nor the affiliation of Colpodella were found
CLUSTAL or MALIGN for the preliminary with parsimony jackknifing.
assessment including Bodo species yielded Because these analyses of protists neces-
one optimal tree. This tree placed Bodo cau- sarily entail the use of highly divergent out-
datus with the other sequence available for group taxa (specifically a combination of
this species, Perkinsus species with the di- fungal and algal taxa), the preceding analyses
noflagellates, and Colpodella sp. as sister to were conducted again in the absence of the
the Ciliophora which together were hypoth- outgroup taxa. Analyses both with and with-
esized to be the sister clade to the apicom- out variable aligned regions did not group
plexans. More than 20 extra steps were re- Colpodella with the apicomplexans but rath-
quired to lose the Bodo clade. Monophyly of er suggested a closer relationship to the cil-
Perkinsus spp. and dinoflagellates had a Bre- iates (fig. 1 C,D). Nonetheless, the most par-
mer support of 17 but nonmonophyly of Col- simonious solution grouping Colpodella with
podella and Ciliophora could be found in a Ciliophora using all aligned sites (5873
tree only two steps longer. Analysis of these steps) or using only more conservative sites
same data, aligned with CLUSTAL in the ab- (3338 steps) each could be refuted with only
sence of the distantly related Bodo species, three additional steps.
resulted in two optimal trees disagreeing Combined analyses using 63 eukaryotic
only on relationships within the dinoflagel- taxa and including actin for 17 taxa for
lates. These trees indicated monophyly of the which this gene is available, yielded three
genus Perkinsus and the dinoflagellates but equally parsimonious trees of length 8385 for
placed Colpodella sp. as sister to the apicom- the analysis using all 18S rDNA sites (fig.
plexans. Analyses of parameter sensitivity 2A). An additional 12 steps were required to
with optimization alignment when substitu- group Colpodella with the Apicomplexa and
tions and indels had equal costs indicated an 37 steps were required to group Colpodella
apicomplexan relationship for Colpodella sp. with its look-alikes, species of Perkinsus and
but failed to find monophyly of any alveolate Parvilucifera. Using only conservative 18S
phyla. Optimization alignment with double rDNA sites and actin combined (fig. 2B)
or higher costs for indels all supported Col- minimum length trees (5287 steps) were 13
podella as sister to the Ciliophora. steps shorter than those grouping Colpodella
Realignment with MALIGN (using a 2:1 with the Apicomplexa and 30 steps shorter
indel:substitution cost) yielded six trees of than those grouping Colpodella with species
length 7136 with Colpodella as sister to the of Perkinsus and Parvilucifera.
Apicomplexa but in which Perkinsus species Using only 24 taxa for which both 18S
and Parvilucifera infectans did not group ei- rDNA and actin sequences are currently
ther with dinoflagellates or with apicomplex- available, phenetically aligned data (length =
ans. The relationship between Colpodella 7243) placed Colpodella and Perkinsus spe-
and Apicomplexa could be refuted with two cies as sister taxa to Ciliophora, and Dino-
extra steps. Parsimony jackknife values of flagellata, respectively. Identical results (fig
whole SSU rDNA sequences are illustrated 2C) were found with the phylogenetically
for higher taxonomic groups in the consensus aligned data dength = 7176). The latter re-
of these trees in figure 1A. There was strong sult was strongly supported by parsimony
2001 SIDDALL ET AL.: PHYLOGENY OF COLPODELLA 5
Candida Candida
Saccharomyces Chlamydomonas Saccharomyces Emniliania
Emiliania OchroCmFoAuoscctnhauiarsysi a a CAhcclh iaymaycCF odhusorctoumasmro ionana ass
MOextSyottprroiucmshb ai diumE uplotes stoneidsi niunf Dsaastryitcrhiac ha
100 PFaDrirodnaitmnoeinEcEuiuDiipmaEasd i uonsidmtfypir tolOdirdocpiiihdTuhcanimer hin tyauiro muagm hi eynmae na CGiltephora 100 ProOxryoCStdotrPolFrianeroco prmhnsabat mi odenEiciuEuaidpm iui pdmEini uotdpodildiuoinmtni eiusum m
99 CoBlapboedseitaA lnao phAyTr'CCorCoiolr dioeecdpshost n h yoptherius 95 ColpedeifaA nAopThCyCr oides rftephhp trehytyaorgpait ehnemare inuas
71 30 FrfeNsTneakoosexsplSoTpCoiahporyaerla rticSa taaoaes ucbrrmeyxiclasazolt ico iyossLn ta inhsko emsittnaeniresel ltai a 100 FrefNnsTekBoooeasxlsbpoipeoSTapCsorhla lyiaresrta aiaoclas Sso beumaecprlrxyoailcsrz aio ta oci oysnrs o ibhiiosnmi i tnainse lla
83 95, PNeoPrcektriiknfiusncusasu so ClamslErPeasCiaynorymriEosicev ipsnlrim puolieoosusarrcr piaiaoa ff r aeranrome abla ilxnaii ma Apicomplexa 81 87 PNeorPcketirinklsiuucnass uECsiyo mCclEeaslmireroaminysreiaopir Psoniarptaura aosvn r iemallaLuf xaca iinfkmeears at eralla
a2 PLCAePerSmnhpyGcGyetirpySysladitrrytmoppooooempnhodrpdrsbaodioeiiiirnidcraneoisei nilidunodiinuimatodAumi n rnim muuionp mmpuih imuis md inium Binozea 73 ScPrCriProphyApriespmosiicyteoeielpnrcieitaoar rdC iuiCedmenro rainatuoAtomplici euosxmr a ynsd riuPmyr ocAymspthiis dinGiounmy aulax
PeridiniAuml exandriPyurmo cystis PentaphSayrmsboidoidPnieinruiimud mi nium
Cerafocorys Lepidediniumn
Ceratium Gonyaulax GGyyrmondoidniiunmi um
60 Colpodelia ATCMeCi cpus Calpedetia ATCC
100 OxyStirrDPiFoicarmdhrobianain tmdioeiunumciEE maiDufp EuasdEinomsiudtty ppirotlldidrocoiiihdtuncaiemhi ns aui mu m 53 100 PrMoOerxtoyodStpotrDunrii csodhm iabn iiduimEu uEmdfp DsiiaopdstiirynEodit‘f udciropihuidalnmci oi hntuaiem us m
Anophyroriodr; 5elo sadi pnorhtnerh tyyoaegphlt ebnMeaOr iNuGs ACnoolPpFeharpyrosran atmioedncieias:u m 3 jgiehymena
Babesia Coleps Babesia icphMtyhoyogpitehnerai u|s
TChyetilaeurxiza oon TChyetialuenxaz oon
87 FreSnakrecloicay stis hominis 100 Frenkelia Sarcocystis} homin; is
fsospora bSelalric ocystis tanella fsospora beSlta rcocysts tanetia
60 70 9826 PLeeCPSnrpNecGtyGTiPNerrhyAaSpodyoieoekrrmpytpxocsmrsiooyhpomttdo.knr pndseplabiipoioosirieCrolicnlniedrunrasoueogliafssaiiEiro cndldussuCi u dnytaadioimysmmmo rio insoEn ce a nusunlpiPirli mopsommimeuiou:peroaeur ams imnarrim rp s i aid io t a iPrrna noanaLubierm asAiluvCnan lmmiekixaAp l re ilhusamecittaxiedoa frcieneonClrdreilaryrua isam u tP miy- urmo cysis. Gonyaulax r8e3: ] PeLCnreNSPtypocPeGapiGcrNrry—pttdfiyPeTorkeAihpoomeoosrloipm—padrinsxusodSneyirekopcoicisyrr nlsiaeodpieniumioo lnarlinifCEdsubodt saisatuynam iadi-u mo rmncsi oCnisesoEa ulmudainrp— fi CpmoaCnriuiiosmei s eymn maurPeersproa iean rma Aoasr iriu ttlrti paimaoreaid onon cx uiebrPuamtamniaarsiini n~r ayai |udv sxmriLi lFi an—u—u necmk i efsetPreyaArr meoplciyhasi idi—is n iGuom nyaulax
Fig,. 1; Consenses of optimal trees found from parsimony analysis of SSU rDNA aligned with
MALIGN using all aligned sites (A), only conservative sites (B), and excluding outgroup taxa for all
sites (C) and conservative sites only (D). Branches are drawn proportional to amount of change. Values
at internodes for groups of interest are parsimony jackknife support indices (asterisk indicates not sup-
ported with this method). Thickened branches indicate implied retention of the Colpodella/Perkinsus
morphology.
Candidi a Candida
Saccharomyces Saccharomyces Emiliania
Chiamydornaras Chlamydomonas
Emiliania “Achiva
Oakromngs Ochromonas
Fucus. UEUS.
ag aoe colpodetia ATCC
Colpodella ATCC £ lefopus
Metopus Didinium
—— Euplotes fsotricha
69 Sirombidium 72 Dasytricha
Oxytricha Eudiplodinium
sill t| s anaht odinium
4100 Dasytrnitcohdai ni| um 100 Strombidaiumi e
Eudiplodinium Oxytricha
Epiaidium Proradon
Paramecium Coleps
Frontonia * Paramecium
Tetrahymena Frontonia
Ophryogiena Anophyroides
_ichthyaptherius Tetrahymena
Anophyroides Ophryogilena
rorodon ichthyoptherius
Babesia Coleus is Babesia
Theileria Theileria
Cytauxzoon Cytauxzoon
36 98 Frénkelia Frenkelia
Sarcocystis hominis Sarcocystis hominis
Sarcocystis fanella 100 Sarcocystis tanella
fsospora belli fsospora belli
Neospora Neospora
Toxoplasma Toxoplasma
Lankesterella sospora robini
Caryospora Caryospora
fsospora robini Lankesterella
Cyclospora Cyclospora
Eimeria maxima Eimeria maxima
93 EimeriaP atravniellulca ifera Eimeria taneifa
Perkinsus olseni 86 Parvilucifera
Perkinsus marinus Perkinsus olseni
Noctiluca Perkinsus marinus
Amyloodinium Noctituca
74 Symbiodinium Ceratccorys
Peridinium ws 80 Pyrocystis
Amphidinium Alexandrium
90 Cryploperidonopis e2 Ceratium
Pflesteria Gonyaulax
Lepidodinium Amphidinium
Gymnodinium Amyloodinium
Gyrodinium Cryptoperidanopis
Prorocentrum Piiesteria
Scrippsiella Proracentrum
Pentapharsodini : Scrippsiella
Pyrocystis Pentapharsodini
Alexandrium B Symbiodinium
A Ceratocorys Peridinium
Gonyaulax Lepidodinium
Ceratium Gymnodinium
Gyrodinium
Dictyostelium
Physarum
Chondrus
>20) Candida
an >20 | >20 Saccharomyces
>5 30 >20 |9 Hias toplasma
1 Neurospora
5 6 Chiamydomonas
A : 3 Acanthamoeba
; Emiliania
>20 Fucus
8 >20 ©| >20 Costaria
. >20 | >20 Achlya
Phytophthora
g Colpodella ATCC
. >20 Tetrahymena
>20 19 Paramecium
z >20 >20 Euplotes
> >20 Oxytri- cha
> ard
16 Cryptosporidium
10 Toxoplasma
Cc 12 Perkinsus
9 111 Prorocentrum
44 Amphidinium
Fig. 2. Consenses of optimal trees found from parsimony analysis of combined 18S rDNA and actin
nucleotide sequences (SSU rDNA data were only available for 24 taxa) for all available sites (A) and
for conservative SSU rDNA sites (B); values at internodes are Bremer support indices. Optimal tree
(C) found for combined analyses using only those taxa for which both genes are available; values at
internodes are Bremer support indices for the 18S rDNA data (above nodes) and for the actin data
(below nodes). Thickened branches indicate implied retention of the Colpodella/Perkinsus morphology.
2001 SIDDALL ET AL.: PHYLOGENY OF COLPODELLA 7
jackknifing and 38 to 40 extra steps were re- the absence of any unambiguous synapo-
quired to make the genera Colpodella and morphies”’ since they considered micropores
Perkinsus monophyletic. Topological results and the apical complex to be necessary and
for these taxa did not change when gapped sufficient conditions for inclusion of Perkin-
sites were excluded from these data sets. sus or Colpodella species in the phylum Ap-
icomplexa. However, Siddall et al. (1997)
DISCUSSION noted that micropores are exhibited by all al-
veolate taxa and that the anterior structures
On the whole, the molecular evidence in Perkinsus species are no more like an api-
points to this species of Colpodella as sister cal complex than they are like feeding pe-
to the ciliates. Species of Colpodella, previ- duncles in some dinoflagellates. The com-
ously included in the genus Spiromonas, had bined analyses with two genes, and even
been thought to be related to Bodo spp. on some of the results found from the use of
the basis of light microscopy alone. Electron only SSU rDNA, indicate that this species of
microscopy, however, provided ample evi- Colpodella is sister to the ciliates. To the best
dence that these predators are not closely re- of our knowledge, no such arrangement pre-
lated to kinetoplastids (Brugerolle and Mig- viously has been suggested for these free-liv-
not, 1979; Mylnikov, 1991; Simpson and ing flagellates. Simpson and Patterson re-
Patterson, 1996). The ultrastructural findings tained the genus Perkinsus for the parasitic
of Brugerolle and Mignot (1979), for exam- taxa.
ple, led them to speculate on a close affinity That neither Colpodella nor Perkinsus
for Colpodella perforans with the dinofla- Species grouped with apicomplexans, is not
gellates or the apicomplexans. Inclusion of entirely surprising. Lipscomb et al. (1998) at-
the bodonid prey item and another species of tributed the notion of a recent ancestry for
Bodo in preliminary analyses confirms the Perkinsus and the dinoflagellates to Levine
distinct isolation of the two co-occurring se- (1985). However, the hypothesis that Perkin-
quences and clearly separates Colpodella sus species are more closely related to the
from the kinetoplastids. Small subunit ribo- dinoflagellates than to the apicomplexans
somal sequences remain the tool of choice originated with Vivier (1982) who argued
for protozoologists. Although the use of 18S rather forcefully against the prevailing views
rDNA data alone corroborated the expected Stating ‘“‘j’estime que Dermocystidium
alveolate affinity for Colpodella sp., com- (=Perkinsus) et Spiromonas ont des charac-
parison of phenetic and cladistic alignment ters incontestables de Mastigophora et sans
regimes and a variety of additional parame- doubte peuvent-ils étre rapproches des Di-
ters suggests that this gene alone is insuffi- noflagelles.’’ In contrast, Levine (1985)
cient to resolve higher-level relationships in clearly drew Perkinsasida as sister to the eu-
the group. Even with a broad taxonomic apicomplexans and later articulated his belief
composition it is highly sensitive to align- that Perkinsus marinus “is an apicomplex-
ment optimality criterion, to alignment pa- an”’ (Levine, 1988). Vivier’s (1982) position
rameters within an optimality criterion, and repeatedly has been vindicated with respect
to the choice of taxa used to root the ingroup. to Perkinsus species. Morphological data,
It is surprising that Colpodella does not SSU rDNA and actin sequences, separately
group with Perkinsus or Parvilucifera spe- and in combination, consistently have sup-
cies in any of the analyses. Colpodella spe- ported a more recent common ancestry be-
cies are morphologically indistinguishable tween Perkinsus species and the Dinozoa
from Perkinsus species and are quite similar (Siddall et al., 1995, 1997; Reece et al.,
developmentally. Simpson and Patterson 1997; Litaker et al., 1999; de la Herran et al.,
(1996) created a new apicomplexan family 2000). That relationship is again corroborat-
Colpodellidae for those predatory, non-en- ed by the analyses conducted here, despite of
doparasitic flagellates that otherwise look recent suggestions that the inclusion of Par-
like Perkinsus species. They argued that vilucifera infectans might alter this arrange-
“any attempt to define the Apicomplexa to ment (Norén et al., 1999). These phyloge-
include only endoparasites ... founders on netic conclusions have received additional
8 AMERICAN MUSEUM NOVITATES NO. 3314
flagellum a
mastigonemes
anterior microtubular basket
micropore Fe,
alveolar membrane
rhoptry-like extrusomes
flagellum
Fig. 3. Illustration of the morphology of Colpodella, Perkinsus, and Parvilucifera which is indicated
to be the plesiomorphic condition for the Alveolata. Thickened branches indicate implied retention of
the Colpodella/Perkinsus morphology.
corroboration. Polyclonal antibodies thought condition for each of the various alveolate
to have binding specificities limited to Per- groups. It long has been suggested that a Per-
kinsus spp. (Dungan and Roberson, 1993) kinsus-like organism gave rise to the apicom-
now are known to share epitopes with free- plexans. That hypothesis would appear to be
living and parasitic dinoflagellates (Chris correct with the important caveat that Per-
Dungan, personal commun.). Vivier’s (1982) kinsus species do not themselves share a re-
belief in a close association of Colpodella cent common ancestor with Apicomplexa.
and Perkinsus species, however, cannot be There are several other species of Colpodella
supported. for which there are sufficient morphological
One necessary implication of these flagel- data to include them in this genus (Simpson
lates not grouping together, in spite of their and Patterson, 1996), however, in light of the
ultrastructural identity, is that this same mor- implication that this morphology is entirely
phology must have been retained from their plesiomorphic, there is no particular reason
common ancestor. That is, for this morphol- to expect all of these to form a monophyletic
ogy to be found in the sister of the ciliates group. If the phylum Perkinsozoa is to be
and in the sister to the dinoflagellates simul- recognized (Norén et al., 1999) for the sister
taneously, it must have been retained from group of the Dinoflagellata, yet another new
the ancestor of all Alveolata. This morphol- phylum would be required for this and any
ogy (fig. 3) then, appears to be the original other species of Colpodella grouping sister to
2001 SIDDALL ET AL.: PHYLOGENY OF COLPODELLA 9
the Ciliophora, but yielding two distinct phy- Gajadhar, A. A., W. C. Marquardt, R. Hall, J.
la for morphologically identical taxa. Alter- Gunderson, E. V. Ariztia-Carmona, and M. L.
natively, Perkinsus and Parvilucifera could Sogin
1991. Ribosomal RNA sequences of Sarco-
be included in Dinoflagellata and Colpodella
cystis muris, Theileria annulata and
left without phylum rank within the Alveo-
Crypthecodinium cohnii reveal evolu-
lata.
tionary relationships among apicom-
plexans, dinoflagellates, and ciliates.
ACKNOWLEDGMENTS Mol. Biochem. Parasitol. 45: 147-154.
Goloboff, P A.
We thank Susan Perkins and Ward Wheel- 1998. Nona. Version 1.9. Program and docu-
er for commenting on earlier drafts. This mentation. INSUE Fundacion e Insti-
tuto Miguel Lillo, Miguel Lillo 205,
work was supported by BIO-DEB 9629487
4000 S. M. de Tucuman, Argentina.
to EMB and MES, as well as BIO-BIR
Software available from http://
9401876 to TAN.
www.cladistics.com/.
Levine, N. D.
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