1. BotanicalJournal ofthe Linnean Society (1995), 178: 81-105. With 5 figures
Are red algae plants?
MARK A. RAGAN
Canadian Imtitute for Advanced Research Program in Evolutionary Biology, and NRC
Institute for Marine Biosciences, 1417 Oxford Street, Hal+?, Nova Scotia,
Canada B3H 32 7
AND
ROBIN R. GUTELL
Department of Molecular, Cell and Developmental Biology, and Department of
Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-02 15,
U.S.A.
Received September 7994, acceptedfor@blication June 1995
For 200 years prior to the 1938 publication of H. F. Copeland, all authorities (with one
exception) classified red algae (Rhodophyta) within Kingdom Plantae or its equivalent.
Copeland’s reclassification of red algae within Kingdom Protista or Protoctista drew from an
alternative tradition, dating to Cohn in 1867, in which red algae were viewed as the earliest
or simplest eukaryotes. Analyses of ribosomal RNA (rRNA) sequence data initially favoured
Copeland’s reclassification. Many more rRNA gene (rDNA) sequences are now available from
the eukaryote lineages most closely related to red algae, and based on these data, the
hypothesis that red algae and green plants are sister groups cannot be rejected. An increasing
body of sequence, intron-location and functional data from nuclear- and mitochondrially
encoded proteins likewise supports a sister-group relationship between red algae and green
plants. Submerging Kingdoms Plantae, Animalia and Fungi into Eukarya would provide a
more natural framework for the eventual resolution of whether red algae are plants or
protists.
0 199.5 The Linnean Socicly of London
ADDITIONAL KEY WORDS:-Eukarya - Kishino-Hasegawa test L molecular-sequence
phylogeny - Plantae - Protista - Rhodophyta - 5s rRNA gene - 18s rRNA gene -
Templeton-Felsenstein test.
CONTENTS
Rhodophytaas amonophyletic group .
Rhodophyta as plants us Rhodophyta as pro&
. . . . . 82
. . . 87
A second tradition: red algae as ‘the deepest eukaryotes’ . . 88
Resolution: both traditions appear incorrect . . . . . 89
Are red algae plants after all? A new generation of rDNA trees . . 89
The eukatyote 5S rRNA tree revisited . . . . . 94
Evidence from nuclear genes . . . . . . . . 95
Evidence from mitochondrial and plastid genes . . . . 97
0024-4074/95/060081+25 $08.00/O
81
Q 1995 The Linnean Society of London

2. 82 M. A. RAGAN AND R. R. GUTELL
Evidence from nonsequence data .............. 97
Indirect evidence from studies of plastid origin ........... 98
Acknowledgements ................. 99
References .................... 99
RHODOPHYTA AS A MONOPHYLETIC GROUP
The Rhodophyta encompasses 2500-6000 species (Woelkerling, 1990) of
eukaryotes sharing the character states listed in Table 1. None of these
character states is a synapomorphy unique to red algae. Zygomycetes,
ascomycetes and basidiomycetes also lack flagella and store a-1,4 glucans in
their cytosol; glaucophytes and green plants have flattened mitochondrial
cristae and two plastid-envelope membranes; cyanobacteria lack accessory
chlorophylls and typically possess unstacked thylakoids with superficial
phycobilisomes. Red algal pit connections may not be homologous with the
superficially similar structures of some vascular plants and cyanobacteria, and
pit phigs (Pueschel, 1990) are uniquely red algal, but neither pit connections
nor pit plugs occur in all red algae. Red algae are, however, unique in
exhibiting the combination of character states listed in Table 1, and in
practice there is little difficulty in distinguishing what is or is not a red alga.
Linnaeus (1753) did not establish a separate taxon for the red algae then
known, but instead distributed them among at least three genera within the
Cryptogamia Algae subdivision of Class XXIV (Cryptogamia). Thus red algae
now assigned to the genera Chondrus and Furcellaria were placed in Linnaean
genus Fucus (thalloid cryptogams); Batrachospermum and Lemanea were included
in the Linnaean Conferua (filamentous cryptogams); and Porphyra was considered
within the Linnaean UZua (membranous cryptogams). In the early decades of
the nineteenth century these artificial genera were broken apart, and new
taxa were established based on the colour of the thallus and spores. Only
then did the morphologically more-complex red algae begin to be grouped
together as FlorideCs (Lamouroux,
Rhodospermeae (Harvey, 1836),
1813), Purpureae (C. A. Agardh, 1824),
or Rhodophyceae (Ruprecht, 1851). At the
same time, coralline red algae were distinguished from zoocorals (Schweigger,
1819; Gray, 1821; Philippi, 1837). Thuret (1855) was the first to group both
bangiophycidean and florideophycidean red algae into a single taxon
(Rhodophyceae), although only after the careful work of Berthold (1882) did
this concept gain widespread acceptance.
Since early in the present century (von Wettstein, 1901) red algae have
been afforded their own Division Rhodophyta, containing class I&odophyceae
TARL~ 1. Distinguishing features of red algae
Eukaryotic
No centrioles, flagellar basal bodies, flagella, or other 9+2 structures
Flattened mitochondrial cristae
Plastid envelope composed of two membranes (i.e. no external plastid endoplasmic
reticulum-derived membrane)
Chlorophyll u as only chlorophyll
Unstacked thylakoids
Phycqbiliproteins in stalked phycobilisomes on the thylakoid surface
Storage of a-1,4 glucans (starches) in the cytosol

3. ARE BED ALGAE PLANTS? 83
T~ar.e 2. Characters differentiating members of subclasses Bangiophycidae and
Florideophycidae
Bangiophycidae Florideophycidae
Nuclear condition
Plastid
Plastid location
Cell division
Thallus complexity
Pit connections
Sexual reproduction
Filamentous gonimoblast
Tetrasporangia
uninucleate
usually single stellale
typically axile
intercalary
uni- or multicellula
usually absent
absent, rare, or
controversial, except
in genus Porphyra
absent
absent
usually multinucleate
usually multiple discoidal
usually peripheral
mostly apical
multicellular
present
widespread
usually present
usually present
and usually two subclasses, Bangiophycidae and Florideophycidae. Characters
distinguishing bangiophytes from florideophytes are presented in Table 2.
Again the paucity of clear-cut, positive defining synapomorphies is notable;
but again, in practice there is little difficulty in assigning a red alga to one
or the other subclass.
The modern classification of red algae follows Schmitz (1892) and Schmitz
& Hauptfleisch (1897), who established one bangiophycidean and four
florideophycidean orders (Figure 1). Schmitz based his delineation of
florideophyte orders on characters of female reproductive morphology and
post-fertilization development: structures of the carpogonium and the
carpogonial branch, presence and fate of auxiliary cell(s), orientation of the
planes of cytokinesis, and pattern of zygote amplification. As the ultrastructure,
SANGIALES
CDMPSOPDGON4LES
RHODDCHAETALES
PDRPHYR/D/ALES
GONlOTRKX4LES
CRYPTDNEMIALES
I ‘-
NEMALMLES
SATR4CHDSPERhbUES
ACRDCMUlALES
SONNEMAlSONlALES
GELIDIALES
GlGARTlNALES
AHNFELTIALES
GR4CURMES
CRYPTCNEMMLES
HILDENSRANDIALES
RHODYMENIALES
PALMAFTlALES
1 I I I I I I I I I I I I I I I I I I I I I I
1Eso 1w 1910 1920 1530 1s‘l4 1960 lsso 1970 ls80 1980 2m
t
0 GONlOTRCiALES llSS9l. ABANDONED
SCHMllZ
, CklAETANGlAL!3 llS63~. NOT ACCEPTED
1992
* NEMASTOMALES WZSI, ABANDONED
. SPHAEROCOCCALES l19261, ABANDONED
Figure 1. History or recognition of orders within Bhodophyta, from Schmitz (1892) to the
present. The newly proposed Bhodogorgonales (1994) contains recently discovered genera
(Renouxia, Rhodogorgon) which had not previously been formally placed in any other order.
We thank Carolyn J. Bird and Bob Richards for assistance with this figure.

4. 84 M. A. RAGAN AND R. R. GUTELL
NEIWLIALES
::
2
_______..__ _.---.-. ___.--. -..
=
L
ACRtKHAETIALES
2
I
g.
lcystes monospermesl
.f-
----.--.------.I------- --
_--_.--__ ---
_-___- - _- - -
:
I
BANGIALE:
I 1
; __-_-- _- --_---.-_-
lcystes plurispermes!
---.
I
[EUR~~~DOP~~YCIDAEI -1
(cystes d production
__ -.-. _-_-.
-r
RtlOC’X-l4ETALES
----JL
I
ERYTHROPELTIDALES
cmPsoPOGONA’-=
PORPHYRIDIALES
(pas de vrais cystesl
Figure 2. Phylogeny of red algae according to Magne (1989), based on morphology and
mode of fertile cell formation within gametocysts and sporocysts. Reproduced with permission.
reproduction and life histories of red algae became better understood during
the twentieth century, additional characters were introduced, and existing
ones reinterpreted @raft, 1981; Garbary & Gabrielson, 1990: 479-481); as a
consequence, additional florideophycidean orders were erected (Fig. 1). Most
authorities now recognize 11-13 orders of florideophytes (two new orders,
Rhodogorgonales (Fredericq & Morris, 1994) and Plocamiales (Saunders &
Kraft, 1994), were recently proposed); at least two of these orders,
Acrochaetiales and Gigartinales, are widely suspected not to be monophyletic.
Delineation of bangiophyte orders has received somewhat less attention.
Skuja (1939) recognized four or five orders based on macromorphological
characters. As the ultrastructure and reproductive morphology of these
organisms became better known, orders were merged, a new order erected,
and families reassigned (Feldmann, 1967; Chapman, 1974; Garbary, Hansen
& Scagel, 1980). Most authorities now recognize four orders of bangiophycidean
red algae, at least one of which (Porphyridiales) may not be monophyletic.
Classification of red algae, as indeed of most algae, has historically been
carried out without reference to phylogeny. Few if any delineations of new
orders mention character synapomorphy, and various subjective, aesthetic,
and didactic factors have played important roles in algal taxonomy (Silva,
1984). Garbary and Gabrielson were the first to apply phylogenetic principles
in classifying red algae (reviewed by Garbary & Gabrielson, 1990). Mag-rre
(1989; Fig. 2) proposed that two lineages of multicellular red algae exist, one

5. ARE RED ALGAE PLANTS? a5
composed of multicellular bangiophytes other than order Bangiales, the other
of Bangiales plus a monophyletic Florideophycidae.
The first red algal phylogenies based on molecular sequences were put
forward by Lim et aZ. (1986) and Hori & Osawa (1986, 1987). These
authors sequenced nuclear-encoded 5s ribosomal RNAs from six genera of
florideophytes and one bangiophyte, and inferred trees by cluster analysis
(UPGMA). Their trees show red algal 5s rRNAs to form a single cluster,
and the 5s r-RNA of Por@yra to diverge most deeply within this cluster. As
5s rRNAs are small, highly constrained molecules with few informative sites
(Halanych, 1991; Steele et aZ., 1991), the value of these trees in revealing
red algal phylogeny has been doubted.
Sequences of nuclear-encoded small-subunit rRNA genes (SSU rDNAs)
have proven useful in investigating phylogenetic relationships within or among
many problematic taxa (e.g. Woese, 1987; Cedergren et al, 1988; Sogin,
Edman & Elwood, 1989; Ariztia, Andersen & Sogin, 1991; Berbee & Taylor,
1992; Bird et al, 1992; Saunders & Druehl, 1992; van Keulen et al, 1992;
Andersen et aZ., 1993; Gagnon et aC, 1993; Goggin & Barker, 1993; Hinkle
& Sogin, 1993). SSU rDNA s are relatively large and information-rich, and
the presence of highly conserved regions as their immediate 5’ and 3’ ends
facilitates their amplification via the polymerase chain reaction (Sogin, 1990).
The first detailed trees of red algal SSU rDNAs showed Rhodophyceae and
Florideophycidae, but not Bangiophycidae, to be monophyletic (Figure 3 and
others from Ragan et aZ., 1994 and unpublished). In these trees, bangiophyte
SSU rDNAs constitute the three or four most basal branches within
Rhodophyceae. All 71 tested alternative topologies consistent with a
monophyletic (holophyletic) Bangiophycidae could be rejected using the
Kishino-Hasegawa test under maximum likelihood, and 68 of these could
also be rejected using the Templeton-Felsenstein test under parsimony (Ragan
et al, 1994); thus the SSU rDNA sequences strongly suggest that
Bangiophycidae is paraphyletic. Further SSU rDNA sequences are available
for the monophyletic orders (Gracilariales (Bird et al, 1992) and Bangiales
(Oliveira et al, 1995), for the large and possibly heterogeneous order
Gigartinales (Saunders & Kraft, 1994; Lluisma & Ragan, in press), and for
the monophyletic Nemaliales-Acrochaetiales-Palmariales complex (Saunders et
al, 1995).
Remarkably (given the difficulties inherent in phylogenetic inference based
on 5s rRNAs, cited above), the 5s rRNA UPGMA tree of Hori C Osawa
(1987) is congruent with the SSU rDNA tree not only in showing the
Bangiales to branch basally within Rhodophyceae, but also in showing
Palmariales to branch more basally within Florideophycidae than members
of Gigartinales, Gelidiales and Gracilariales.
The monophyly of rhodophytan SSU rDNAs apparent from Figure 3 is
not an artifact of outgroup selection. Replacement of the cryptomonad nuclear
rDNA sequences with small numbers of other SSU rDNAs (unpublished
data) or with a wide selection of eukaryote rDNAs (below) likewise yields a
monophyletic Rhodophyta. There is, however, one complication. SSU rDNAs
from the nucleomorph genomes of the cryptomonads Cryptomonas sp. phi
and Qrenomonas salina S&tore group among red algal rDNAs (Douglas et
al, 1991; Maier et al, 1991), specifically with those of genera Erythrocludia,

6. 86 M. A. RAGAN AND R R. GUTELL
I 4
.05 units
IC
Palmariales
Acrochaedales
Nemaliales
GigarQnales
Gigarttnales
Gigartinales
Ceramiales
Gelidia es
IRhodymenta es
Grgartmales
Bonnemarsoniales
Ceramiales
Ceramiales
Ceramiales
Ceramiales
Gracilariales
Gracilariales
- Gracilaria cornea Gracilariales
1 ’ Gracilaria verrucosa Gracilariales
4 IO
- Gracilariopsis lemaneifotis Gracilariales
-7 Curdiea
Gracilariales
Melanthalia . Gracilariales
L Gracilariopsts sp. Gracilariales
-Ahnfehia Ahnfeltiales
Corallina
Hildenbrandia
Corallinales
Hi@p$!;~
Porphyridiales
Bangiales
Palmariales
Palmariales
Compsopogonales
Porphyridiales
Figure 3. Bootstrapped maximum-likelihood tree (DNAML) of red algal SSU rDNA sequences,
based on most-conservative sequence regions (1566 nucleotide positions). With permission
from Ragan et al (1994). Authorities for taxa in Figs 3-5 are as follows:
Anabaena variabilis Kiitz. Nicotiana tabacum L.
Aspergillus nidulans (Eidam) Winter Physcomitrella patens (Hedw.) B.S.G.
Bacillus megaterium de Bary Pisum sativum L.
Bacillus stearothermophilus Donk Porphyra acanthophora Oliveira & Coil
Caenorhabditis elegans (Maupas) Dough. Porphyra leucosticfa Tltur. in Le Jol.
Chlamydomonas reinhardtii Dang. Porphyra miniata (C. Ag.) C. Ag.
Chondrus crispus Stackh. Porphyra purpurea (Roth) C. Ag.
Cochliobob heteroskophus Drech. Porphyra spiralis Oliveira & Coil
Drosophila melanogaster Meig. Porphyra umbilicalis (L.) J. Ag.
Escherichia coli (Migula) Cast. & Chalm. Saccharomyces cerevisiae Hansen
Gallus gallus L. Schistosoma mansoni Sambon
Gracilaria chilensis Bird et al Sinapis alba L.
Gracilan’a cornea J. Ag. Spinacia oleracea L.
Gracihrria Uvahiae McLachlan
Gracilaria venucosa (Huds.) Papenf.
Tiypanosoma brucei Plimm. & Bradf.
U&ago maydb (DeCand.) Corda
Gracilariopti lemaneiformis (Bory) Dawson Zea mays L.
et al Zygosaccharomyces rouxii (Boutr.) Yarrow
Homo sapiens L. Zymomonas mobilis (Lindner) Kluyver &
Magnolia lilh$ora Desr. in Lam. van Neil
Erythrotrichirz, and Stylonema (Fig. 3 and below). As cryptomonad nucleomorphs
have never been considered to be red algae, Rhodophyceae might technically
be paraphyletic: similar problems arise with viruses, which may have arisen
on multiple occasions from various taxa. Various solutions are possible, the

7. ARE RED ALGAE PLANTS? 87
easiest being simply to acknowledge that the cryptomonad nucleomorph has
apparently arisen from a red alga, and formally or informally cross-reference
this compartment and its genes to Rhodophyta.
A large sequence database is also available for the plastid-encoded gene
encoding the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase
(LS-RuBisCO) (Ch ase et al, 1993). Freshwater et al. (1994) have recently
sequenced 81 red algal LS-RuBisCO genes and inferred a parsimony tree.
As their tree is not rooted outside the red algae, no conclusions can be
drawn about monophyly of Rhodophyta or of its two subclasses; but if the
LS-RuBisCO gene tree were rooted as in Figure 3 (placing Compsopogonales
but not Bangiales on the deepest branch), Florideophycidae would again be
monophyletic and Bangiophycidae again not monophyletic. Although some
topological details differ, both the LS-RuBisCO and SSU rDNA trees indicate
that Hildenbrandiales, Corallinales, Acrochaetiales, Palmariales, Nemaliales
and Ahnfeltiales are the earliest-diverging lineages within Florideophycidae.
RHODOPHYTA AS PLANTS KS RHODOPHYTA AS PROTISTS
All available molecular data support the decision of the early phycologists
to recognize a monophyletic grouping for red algae. At which point did this
lineage diverge from the other eukaryotes. 3 What are the closest relatives of
red algae? From West & Fritsch (1927: 416) and Drew (1950: 190) to
Garbary & Gabrielson (1990: 493), Woelkerling (1990: 2) and Corliss (1994:
25), answers to these questions have been ‘unknown’ and ‘problematical’.
Historically, red algae were considered to be plants. From the days of
Pliny the Elder if not earlier, all organisms had been considered to be either
plant or animal; not surprisingly, de Jussieu (1789), Velley (1795), Lamouroux
(1805, 1813), T umer (1808), de Candolle (1813), C. A. Agardh (1824),
Bartling (1830), Harvey (1836, 1841), Endlicher (1836-1840), J. G. Agardh
(1842-1901), Endlicher & Unger (1843), Ktitzing (1843), Nageli (1847), Lindley
(1853) and Berkeley (1857) followed Linnaeus in treating red algae as plants.
Nonetheless “. . . there are numerous expressions in the works of naturalists
of all times, which show a suspicion that organisms exist which are not to
be regarded properly as either animal or vegetable in their structure and
nature” (Wilson & Cassin, 1863: 120). The latter authors cite Buffon,
Daubenton and Pliny himself to this effect (pp. 119-121), while Bory de
Saint V%rcent (1824: 660 and 1825: 6) reads these sentiments into Linnaeus’s
Zoo~hytu and Lamar&s Animaux u~at/ziques.
Bory de Saint Vincent was apparently the first to propose a third kingdom
of life (Psycho&are, or two-souled organisms) encompassing “les Arthrodiees,
les Spongiaires, [et] la plupart des Polypiers”; his Regne Vegetal remained
compositionally similar to the classical concept of plants “minus some of
their cryptogams” (1824: 659 and 1825: 8). H. F. Copeland (1947: 351) later
denied priority to Bory’s Regne Psychodiare on grounds that it was not
Latinate.
Without citing Bory, Owen (1860: 4) recognized that “. . . there are numerous
beings, mostly of minute size and retaining the form of nucleated cells,
which manifest the common organic characters, but without the distinctive
superadditions of true plants or animals. Such organisms are called ‘Protozoa’,

8. 88 M. A. RAGAN AND R. R. GUTELL
and include the sponges or Amorphozoa, the Foraminifera or Rhizopods,
the Polycystineae, the Diatomaceae, Desmidiae, Gregarinae, and most of the
so-called Polygastria of Ehrenberg, or infusorial animalcules of older authors”.
Owen did not mention red algae, but from his delineation of Protozoa it is
clear that he too considered red algae to be plants. Hogg (1860) accepted
Owen’s grouping but challenged his choice of the term Protozoa for it,
preferring instead Regnum Primigenum. This kingdom comprised Protoctista
(“all the lower creatu&s, or the primary organic beings”) and Amorphoctista
(sponges). Owen later (1861: 6) proposed an alternative name, Acrita.
Wilson & Cassin (1863) erected an alternative third kingdom of life,
Primalia, with five subkingdoms, the first of which was Algae. These authors
did not mentioned red (or brown or green) algae, but made it clear (pp
118-119) that Primalia was to be much more inclusive than Owen’s
Protozoa. As by the 1860s scholarly treatments of algae usually included the
florideophytes, Wilson & Cassin appear to be the first authorities to have
removed red algae from among the plants. The influential Haeckel (1866,
1878, 1904), however, expressly included Florideae among Plantae, not among
his own third kingdom, Protista.
Haeckel’s placement of red algae was not seriously challenged in the
botanical (Eichler, 1886) or protistological (Dobell, 1911) literature until H.
F. Copeland (1938) grouped red algae with Protista-with disclaimers that
rhodophytes are “a highly evolved group of unknown origin” (Copeland,
1938: 402, 409) and a diagram (p. 410) hs owing the red algal lineage closely
appressed to Plantae. Copeland later (1947, 1956) substituted Hogg’s term
Protoctista on the basis of priority. Since Copeland, most authorities have
considered red algae among the Protista or Protoctista, although Whittaker
(1969) and D odson (1971) continued treating them as plants.
A SECOND TRADITION: RED ALGAE AS ‘THE DEEPEST EUKARYOTES’
As early as 1867, Cohn concluded that “. . .in a natural system, the
filamentous cyanobacteria must be separated from the unicellular green algae,
with which they are usually arranged, and moreover placed as the bottommost
step of a different organizational ranking immediately before the red algae”
(1867: 36; translation by M. R.). This idea of red algae constituting the
oldest, deepest-branching, or most ‘primitive’ group of eukaryotes permeates
the subsequent phycological and botanical literature (Tilden, 1933, 1935;
Copeland, 1947, 1956; Cronquist, 1960; Christensen, 1964; Whittaker, 1969
Figure 3; Ragan & Chapman, 1978; Taylor, 1978; Lipscomb, 1989; Bremer,
1989). Dougherty & Allen (1958) formalized
as ‘mesoprotists’.
the idea by classifying red algae
It is not hard to understand the roots of this idea, given the character
states defining red algae (Table 1). In pigmentation and ultrastructure, red
algal plastids most closely resemble cyanobacteria; and even if this were
ascribed to a particular course of endosymbiosis, red algae lack flagellar
basal bodies and centrioles. Moreover, the UPGMA tree of Hoi-i & Osawa
(1987) showed red algal 5s rRNAs as the most basally diverging lineage
within the eukaryotes (but see below).

9. ARE RED ALGAE PLANTS?
RESOLUTION: BOTH TRADITIONS APPEAR INCORRECT
89
Primarily through the efforts of Sogin and colleagues, a unified view of
eukaryote phylogeny, based on analysis of SSU rDNA sequences, came into
focus in the late 1980s (Sogin, 1989; Sogin et al, 1989; Schlegel, 1991; Van
de Peer et al, 1993). Two features of the emerging SSU rDNA tree
immediately illuminated the debate on classification of red algae. Red algal
SSU rDNA sequences clearly grouped not at the base of the eukaryote tree,
but just basally to the divergence of morphologically complex taxa including
animals, plants, fungi, and straminipiles (Bhattacharya et al, 1990; Hendriks
et al, 1991). [Organisms with tubular mitochondrial cristae and tripartite
tubular flagellar hairs (bicosoecids, oomycetes, hyphochytrids, opanlinids,
proteromonads, raphidiophytes, eustigmatophytes, chrysophytes, xantophytes,
synurophytes, phaeophytes, bacillariophytes, labyrinthulids, and
thraustochytrids) are straminipiles (see Patterson (1989) for origin of the term
‘stramenopiles’). Haptophytes, cryptomonads, and dinoflagellates, which have
sometimes been included among the Chromophyta, are excluded from
straminipiles.] Moreover, the earliest-diverging rDNA lineages were those of
amitochondriate, not aflagellate, eukaryotes. Thus the cyanobacteria-like
pigmentation of red algae did in fact reflect a primary endosymbiotic event,
and the absence of flagella was clearly reductive. In these initial trees,
however, red algal SSU rDNAs did not appear to be specifically related to
SSUs of green plants; thus neither of the two historical traditions was
confirmed by the early SSU rDNA data.
ARE RED ALGAE PLANTS AFTER ALL? A NEW GENERATION OF rDNA TREES
Although the shape and major features of the eukaryote SSU rDNA tree
were well-established by the end of the 198Os, resolution of the relationships
among red algae, plants, fungi, animals and other closely related lineages
has not become stabilized. Both for analytical reasons (Cavender & Felsenstein,
1987; Lake, 1987; Hendy & Penny, 1989) and from practical experience,
several researchers realized that resolution would require determination of
SSU rDNA sequences of many more early-diverging animals, fungi, red
algae, cryptomonads, apicomplexans, and ciliates. The SSU rDNAs of many
such organisms were subsequently sequenced (Barta, Jenkins & Danforth,
1991; Chapman & Buchheim 1991; Douglas et ah, 1991; Gajadhar et al,
1991; Kelly-Borges, Bergquist & Bergquist, 1991; Wainright et ah, 1993; West
& Powers, 1993; Oliveira & Ragan, 1994; Ragan et al, 1994; Steinkotter et
al, 1994). It also became apparent that the 8rst ‘PO@@ SSU rDNA
(Hendriks et cd, 1991) actually belonged to a member of Pahnariales
(Florideophycidae).
Our alignment of red algal with other eukaryote SSU rDNAs has proceeded
in stages over more than three years. An initial group of red algal rDNAs
was integrated into the existing database of aligned eukaryote SSU rDNAs,
with alignment based on conservation of secondary- and higher-order structures
as revealed by covariation of nucleotides (Gutell, 1993a,b; Gutell, Larsen &
Woese, 1994); folded structures of these red algal rRNAs were simultaneously
constructed (unpublished). T rees were inferred from the most conservative

10. 90 M. A. RAGAN AND R. R. GUTELL
(i.e. most credibly aligned) regions of this initial alignment matrix, and, based
on the results, sequences were reordered within the database to reflect better
the phylogenetic relationships. More red algal and other sequences were
added as they became available, alignments in the less-structured regions
were refined, trees were again inferred from the (now more extensive)
credibly aligned regions, and the database was again reordered. Five complete
iterations of this pro$ess, and numerous local modifications, have yielded a
structure-based alignment of 62 red algal and approximately 650 other
eukaryote SSU rDNa sequences. From this alignment we have selected 87
aligned SSU rDNAs: two eubacterial and three archaeal rRNAs as outgroups,
and 19 red algal and 63 other eukaryote sequences. The latter were selected
to overrepresent lineages which in our analyses diverge closest to red algae,
and within those lineages to include earlier-diverging representatives (i.e. to
minimize the lengths of internal edges, and thereby to try to avoid topological
artifacts arising from differential apparent rates of acceptance of mutations).
As documented elsewhere (Ragan et al, 1994), we inferred trees from two
variants of the sequence matrix: the ‘essentially complete’ matrix (i.e. after
removing only the primer regions and the most sparsely populated variable
regions: 1815 nucleotide positions), and the securely aligned ‘conservative
core’ (1098 nucleotide positions). Distances were calculated under Felsenstein’s
‘maximum likelihood’ (generalized Kimura two-parameter) model of sequence
change. Bootstrapped (rz = 500 to 1000) neighbour-joining trees were inferred
on a Sun lo/30 workstation using SEQBOOT, DNADIST, NEIGHBOR and
CONSENSE from PHYLIP version 3.53c, and bootstrapped (n = 400 to 763)
parsimony trees on an AVX series 2 computer (64 parallel Intel 860 boards
linked through an Intel 805 transponder) using SEQBOOT, DNAPARS and
CONSENSE from PHYLIP 3.53~ (Felsenstein, 1989). Parsimony trees were
sometimes ‘refit by least squares’ by appending a user tree (the consensus
topology) to the distance matrix for analysis using FITCH and PHYLIP
3.53~; when parsimony and distance trees are topologically similar, this yields
rough estimates of the numbers of steps separating nodes. Consistent with
the experience of Olsen et al. (1994) for matrices of this size, we did not
obtain stable results using fastDNAml.
Not surprisingly, in all our trees, the greatest distances (or numbers of
steps) separated prokaryote from eukaryote SSU rDNAs. The five most
basally branching eukaryote rDNA lineages were respectively those of
Encephalitozoon and Vairimorpha; Hexamita and three Giardia species; Physarum;
Euglena, Bodo, Trypanosoma, Leishmania and Crithidia; and Entamoeba, sometimes
together with Naegleriu. To make the relationships among red algal and
nearby lineages more easily visible, branches more basal than Entumoeba
have been removed from Figure 4A and B.
Results from bootstrapped parsimony and neighbour-joining trees inferred
from the conservative core regions are shown in Figure 4A and B, respectively.
Red algal rDNAs appear monophyletic in both analyses, and the two
cryptomonad nucleomorph rDNAs group among the bangiophycidean lineages.
In agreement with Baldauf & Palmer (1993) and Wainright et al. (1993),
both trees show a sister-group relationship between animal and fungal rDNAs;
nuclear rDNAs of two cryptomonads plus Goniomonas and Glaucocystis likewise
group together (cf. McFadden, Gilson & Hill, 1994). In both analyses (Fig.

11. ARE RED ALGAE PLANTS? 91
4A,B), rDNAs of four groups (red algae; green plants including green algae
sensu ho; cryptomonads, Goniomonas and Gluucocystk and animals plus fungi)
form an exclusive monophyletic group, with red algal rDNAs isolated; but
distances or numbers of steps are small, and bootstrap support is less than
50%. We refer to these four groups, perhaps anthropocentrically, as the
‘crown taxa’ (after Knoll, 1992).
In an attempt to find the sister group of red algae, we inferred bootstrapped
parsimony and neighbour-joining trees (results not shown) from the essentially
complete sequence matrix. Although the addition of less-securely aligned
regions and more gaps might be expected to degrade the phylogenetic signal,
these effects should be most severe near the base of the tree (where sequences
are more divergent), while resolution could improve among closely related
groups such as the crown taxa. However, possible improvements in resolution
must be balanced against the unpredictable consequences of including in the
analyses numerous vacant (gapped) nucleotide positions.
Upon neighbour-joining analysis, bootstrap support was significantly
improved (to 92%) only for the animal-fungal clade; support for a monophyletic
Rhodophyceae actually decreased, while the cryptomonad nucleomorph
rDNAs moved outside those of the (other?) rhodophytes. The volatile Emdiana
rDNA appeared among those of plants and cryptomonads, attracting the
Glaucocystti but not the Goniomonas rDNA; and the straminipiles (SJ.VZU~,
Ochromonas, NuuicuZa, Fucus, AchZp) moved into the crown of the tree.
Bootstrapped parsimony analysis presented a rather different picture. The
animal-fungal grouping was preserved (but did not gain additional bootstrap
support) and associated somewhat more strongly with plant rDNAs. A
classical Rhodophyta remained weakly intact and isolated from other major
crown taxa. Most notably, the cryptomonad nucleomorphs were securely
attracted to the corresponding cryptomonad nuclear sequences, as were the
hitherto separate chlorarachniophyte nucleomorphs to the chlorarachniophyte
nuclear rDNAs. The determinants of these attraction effects are not obvious
from analysis of the cryptomonad (or chlorarachniophyte) rDNA sequences;
there are no stretches of pan-wise-identical nucleotides in nonconservative
regions sufficiently long to be taken as evidence for example of post-
endosymbiotic gene conversion. We interpret these results as artefacts arising
from the inclusion of numerous gapped positions.
Thus the sister-group of red algae cannot readily be identified by inference
of trees and bootstrapping. As an alternative we sought at least to rule out
some alternatives by use of nonparametric tests. Kishinb & Hasegawa (1989)
have described a test of alternative topologies under maximum likelihood; a
corresponding test under parsimony was introduced by Templeton (1983)
and modified by Felsenstein (1985). Any four independent lineages (such as
the crown taxa identified above) can be related in only 15 ways via a rooted
bifurcating tree, and we forced each of these topologies in turn, as a user
tree, on the data using the DNAML and DNAPARS programs with PHYLIP
3.53~; topologies within each of the four lineages appeared relatively robust
(Fig. 4A,B and unpublished data) and for computational reasons were not
allowed to vary. Alternative topologies were rejected if they decreased the
likelihood, or increased the number of steps, by more than 1.96 standard
deviations.

12. 92 M. A. RAGAN AND R. R. GUTELL
bdrlum
Plasmc
- Tetrahymena
1
rdlum
nas
... - - -..-
< Cryptomonas NM
Pvrenomonas NM
Figure 4. A (above), Detail from bootstrapped (n = 763) parsimony (DNAPARS) tree of SSU
rDNA sequences, based on the most-conservative 1098 nucleotide positions. Tree refit by
least squares to show approximate numbers of nucleotide changes. B, Detail from bootstrapped
(n = 1000) neighbour-joining (NEIGHBOR) tree of SSU rDNA sequences, based on the most-
conservative 1098 nucleotide positions. Width of line indicates bootstrap support: widest, 90-
100%; second widest, 70-89%, third widest, 50-690/o; narrowest, less than 50%.
The 15 alternative topologies may be classified into five groups according
to the taxa they require to be the sister group to red algae (Table 3).
Interestingly, the only sister-group relationship for red algae which could
never be rejected was that between red algae and green plants. However,
as only seven of the 15 topologies could be rejected at all (and only two
based on the core matrix), these results should not be taken as strong support
for, or strong rejection of, any of these five possibilities.

13. ARE RED ALGAE PLANTS? 93
Iorarachnlon 1242 NM
Chtorarachnlon 1-57 NM
-
Atexandrtum
Crypthecodlnlum
Tetrahymena
Erythrocladla’
Erythrotrlchla
Pyrenomonas NM
Cryptomonas NM
Porphyra mlniata
Porphyra leucostlcta
Porphyra purpurea
Porphyra umblllcalls
- Phycodrys
Graclllaria
Hlldenbrandia
Dixonlella
Acanthopleura
Ptacooecten
Chlamydomonas
Pyre;omonas NU
Cryptomonas NU
- Gonlomonas
Emlllana
Ochromonas
Ptasmoatum
Dlctyostellum
Entamoeba
Figure 4(B).

14. 94 M. A. RAGAN AND R. R. GUTELL
TABLE 3. Acceptability of alternative topologies of crown taxa under
nonparametric tests
Full matrix Core matrix
T-F K-H Tree T-F K-H
++
+
+
+
+
+
No
iLO
+
+
+
+
+
+
+ -WWF,R))
+ -CO-W,R))
+ -WW,R)
-WW’,W
+WC(P,R))
,-LWW’,R)
-mw-GR))
-AF(W,R))
-W,P)(C,R)
+ +
+ +
+ +
+ No
+ +
+ ++
+ +
-I-+ +
+ +
Red algae
and plants are
sister groups
Red algae
and cryptos are
sister groups
Red algae and
anima.ls+fungi are
sister groups
No
+
+
No
+
No
+ -P(RW,C)) + + Red algae are
++ -C(NM,P)) No + sister group to
+ -“wwm + + two crown taxa
+ -RV’LQ,C)) + + Red algae are
f -W(~,P)) + + more basal than
+ -WW,P)) + + three crown taxa
AF = animals+fungi C = cryptomonads P = green plants R = red algae ++best tree
We are forced to conclude that the existing SSU rDNA sequence data
have been ‘squeezed’ as much as possible using standard inferential techniques,
without revealing the precise evolutionary origin or nearest relatives of the
red algae. It is possible, although not likely, that future data may resolve
these features to produce a stable classification. Only two further lines of
analysis appear promising with the existing rDNA matrices: structure-corrected
inference, and signature analysis. In the former, covarying nucleotide
positions are downweighted to minimize effects of the violation of positional
independence. Both theoretical and practical difficulties remain, and to our
knowledge this approach has never been attempted with a data set as
extensive and varied as ours. Moreover, simplified analyses (Dixon & Hillis,
1993) suggest that improvements using this approach may be modest. The
latter approach involves identifying features of folded rRNAs characteristic
of each lineage and of putative common ancestors, and deciding among
them, e.g. by energy minimization. We hope to be able to report on such
an analysis in due course.
THE EUKARYOTE 5S rRNA TREE REVISITED
The very different phylogenetic position of red algae in our SSU rDNA
trees (Fig. 4A,B) ve7sus the 5S rRNA tree of Hori & Osawa (1986, 1987)
bears comment. As noted above, the 5S rBNA molecule presents problems
for phylogenetic inference, but the extreme difference between the two trees
invites further analysis. To this end we downloaded an aligned set of 292
5S rRNA sequences from the EMBL server (ftpembl-heidelberg.de), and
from it removed prokaryote sequences other than that of Escherichiu coli
(Migula) Cast. & Chal m., multiple sequences from individual species, sequences

15. ARE RED ALGAE PLANTS? 95
from very closely related species, and pseudogenes. From the resulting matrix
of 229 5s rRNAs we inferred trees by UPGMA and neighbour-joining,
bootstrapping each analysis 100 times.
The results (not shown) are striking. In the UPGMA tree, the red algal
5s rRNAs cluster near the base of the tree, separated from the E. cob 5S
r-RNA by only five internal nodes. In the neighbour-joining tree, the red
algal 5s rRNAs are 13 nodes removed from E. co& firmly in the crown of
the tree. (These numbers must be considered approximate, as bootstrap
support is < 15% for most nodes relating phyla or divisions). The red algal
5s rRNAs exhibit long branches; 5s rRNAs of some other eukaryotes are
as long or longer, but most are much shorter. Thus the set of 5s rRNA
sequences is in egregious violation of ultrametricity (clock-like behaviour on
all branches). Like other clustering methods, UPGMA requires, and is very
sensitive to violations of, ultrametric behaviour (Michener & Sokal, 1957;
Swofford & Olsen, 1990 pp. 440-441). UPGMA is misapplied in analysis of
these data, and this is a major reason why the global 5s rRNA tree of Hori
& Osawa (1987) is so seriously misleading.
EVIDENCE FROM NUCLEAR GENES
The non-rRNA molecular biology of red algae is in its infancy, and that
of red algal nuclear genes even more so. Sequences are known for P-tubulin
genes of Porphyra purpurea (R. M. MacKay and J. W. Gallant, unpublished)
and the cc-tubulin gene of Cyanidium culdarium (Roth) C.Ag. (Pearson &
Burns, 1993), while those of the p- and y-tub&n genes of C. culdarium are
nearing completion (B. R. Oakley & R. G. Burns, pers. comm. 1994). A
polyubiquitin cDNA of Aghothmnion neglecturn Feldmann-Mazoyer (Apt &
Grossman, 1992) and the polyubiquitin gene of Gruciluriu uerrucosu (Huds.)
Papenf. (Zhou & Ragan, 1995a) have also been sequenced. Although the
highly conserved tubulin and ubiquitin sequences provide too few informative
characters for use in inference of global phylogenies, the presence of a
polyubiquitin gene suggests that red algae are not among the oldest eukaryote
lineages (Krebber, Wostmann & Bakker-Grunwald, 1994). Gene sequences
are available for mitochondrial aconitase (Zhou & Ragan, 1995d) and a
calmodulin-like protein from G. uerrucosu (Y.-H. Zhou & M. A. Ragan,
unpublished), but the former is only the fourth eukaryote aconitase so
characteri?ed, while paralogy complicates inference of phylogenetic trees from
the latter.
Sequences of the nuclear-encoded NAD- and NADP-linked glyceraldehyde-
3-phosphate dehydrogenase genes (gupc and gupA respectively) of Chondrus
crispus Stackh. (Liaud et al, 1993, 1994) and GruciZuriu uerrucosa (Zhou &
Ragan, 1993, 1994, 1995b) are known. We inferred six most-parsimonious
GAPDH protein trees using PROTPARS (100 independent addition series),
and calculated a consensus using CONSENSE in PHYLIP version 3.53. As
gupA and gupC have apparently arisen via duplication of a single ancestral
gene, we rooted the consensus tree (Fig. 5) on the point of duplication
(Dayhoff et a& 1972; Gogarten et uk, 1989; Iwabe et al, 1989). As with
other GAPDH protein trees, some features of Figure 5 are not readily
explicable, and conclusions must be drawn cautiously.

16. 96 M. A. RAGAN AND R. R. GUTELL
-
I
Aspergillus nidulans
Cochliobolus heterostrophus
-
Gracilaria verrucosa GapC
Chondrus crispus GapC
- Escherichia coli gap1
Trypanosoma brucei cytosolic
- Chlamydomonas reinhardtii GapC
Ttypanosoma brucei glycosomal
Anabaena variabilis gap1
Nicotiana tabacumGapA
Chondrus crispus GapA
- Anabaena variabilis gap2
Bacillus stearothermophilus gap
Bacillus megaterium gap
Escherichia coli gap2
Zymomonas mobilis gap
Anabaena variabilis gap3
Figure 5. Protein parsimony tree of glyceraldehyde3phosphate dehydrogenase sequences.
Majority-rule consensus (CONSENSE) of the six most-parsimonious trees (2728 steps each)
inferred using PROTPARS with 500 independent random addition orders. All positions were
weighted equally. When positions were weighted inversely (i.e. by (n-I)-’ where n = number
of different amino acids at that position among fully sequenced GAPDHs), no changes were
observed in the topology of the GAPA subtree, while support for the red alga-green plant
clade was weakened (Y.-H. Zhou & M. A. Ragan, unpublished)
The gupA gene, whose mature product is plastid-localized, appears to have
entered the eukaryote lineage endosymbiotically (Brinkmann et al, 1987;
Liaud, Zhang & Cerff, 1990; Martin et al, 1993) before the divergence of
red algae and green plants (Zhou & Ragan, 1993), and subsequently to have
been transferred to the nucleus and duplicated within the green plant lineage.
In agreement with these results, the position of the Anabaena gap2 gene in
Figure 5 supports a cyanobacterial origin for gupA.
~The ga..C branch of Figure 5 clearly supports sistergroup relationships
between red algae and green plants, and between animals and fungi (Zhou
& Ragan, 1995b). Six equally parsimonious trees were found (each 2728

17. ARE RED ALGAE PLANTS? 97
steps in length), and both sister-group relationships appear in all six trees.
The same result is obtained with inverse weighting (i.e. when the relative
weight is (n-l)-‘, where n is the number of different amino acids at that
site). When the GAPDH protein trees is bootstrapped (n = loo), however,
support for the red alga-green plant clade is found to be weak (52%).
The triosephosphate isomerase gene of Grucilaria uerruco.ru has recently
been sequenced (Zhou & Ragan, 199%). The G. verruc~~a TPI tends to
branch deeply among eukaryote TPIs, but because the protein is relatively
small, bootstrap support for many topological features is poor. Inverse
weighting resolves the G. verrucosa and green plant TPIs as sister groups,
although with modest bootstrap support (Zhou & Ragan, 1995c).
The red alga Por$$~ru purpureu contains at least two nuclear genes encoding
elongation factor (EF) la, one expressed in both the conchocelis (diploid
sporophyte) and leafy-thallus (haploid gametophyte) phases, the other expressed
only in the conchoecelis (Q Y. Liu, S. L. Baldauf & M. E. Reith, unpublished).
The protein parsimony tree (inferred using PAUP 3.1: Swofford, 1993) shows
the Porphyra EF- la sequences to branch near that of Dictyostelium, just basally
to EF-la’s of animals and fungi but less basally than those of green plants.
This result tends to support red algae being a crown taxon; but caution
must again be exercised, because the ~rypanosoma EF-lcr behaves anomalously
in this tree.
EVIDENCE FROM MITOCHONDRIAL AND PLASTID GENES
Neighbour-joining analysis of the mitochondrial gene encoding subunit 3
of cytochrome oxidase (~0x3) from the red alga Chondrzls crispus suggests that
red algae are the sister group to green plants (Boyen et al, 1994). Data on
other mitochondrial genes from other red algae are becoming available (B.
F. Lang & G. Burger, pers. comm.) but have not yet been published.
Sequences are also available for genes encoding several phycobilisome
subunits (Bernard et al, 1992; Apt & Grossman, 1993; Grossman et aL, 1993;
Roe11 & Morse, 1993), and the entire plastid genome of Par-hyra purpurea
has recently been completed (Reith & Munholland, 1993; M. E. Reith, pers.
comm.). Like gapA sequences, however, these data are of limited utility in
global phylogenies because the corresponding genes are typically absent from
nonphotosynthetic eukaryotes.
EVIDENCE FROM NONSEQUENCE DATA
Rothschild (1985: 93) noted a much stronger immunological crossreactivity
between ribulose 1,5-bisphosphate carboxylase large subunits of pea and two
red algae than between those of pea and chromophytes. Apt, Hoffman &
Grossman (1993) h ave shown that in transgenic constructs the peptide
translated from the plastid-transit-encoding region of the Aglaothamnion neglecturn
gene encoding small subunit of ribulose 1,5-bisphosphate carboxylase is
functional in targeting the corresponding peptide of pea across the pea
chloroplast membrane. Zhou and Ragan (in press) have recently demonstrated
that the polyadenylation in the red alga Gracilaria verrucosa more closely
resembles that of green plants than that of fungi or animals; multiple

18. 98 M. A. RAGAN AND R. R. GUTELL
polyadenylated transcripts can be produced from individual nuclear genes,
the AATAAA polyadenylation signal is not strictly conserved, and GT-rich
clusters downstream from the poly(A) addition site are absent from some
genes.
INDIRECT EVIDENCE FROM STUDIES OF PLASTID ORIGIN
Most molecular-s;quence evidence indicates that plastids arose
endosymbiotically from cyanobacterial ancestors (Douglas & Turner, 199 1;
Douglas, 1994). Mereschkowsky (1905: 602 and 1910) initially proposed that
plastids of red, brown and green algae arose independently from different
groups of cyanobacteria, and some molecular-sequence data do appear to
support this idea (Scherer, Lechner & Boger, 1993). Most molecular data,
however, are more consistent with plastids having arisen from a single point
within the cyanobacteria (Morden et al, 1992; Douglas, 1994; Wolfe et al,
1994). The location of intron 1 in the, transit-peptide-encoding region of gu&4
in green plants and G. verrucosa (Zhou & Ragan, 1994) may likewise suggest
that gupA entered the eukaryote lineage only once, hence that red algae and
green plants share a common photosynthetic eukaryotic ancestor.
The plastids of cryptomonads and photoautotrophic straminipiles are now
widely thought to have arisen via secondary associations between eukaryotic
hosts and eukaryotic symbionts (Douglas, 1994). In the case of cryptomonads,
this symbiont appears to have been a red alga (Douglas et al 1991) or a
close relative thereof, and the eukaryotic host a Goniomonas-like flagellate
(Kugrens & Lee, 1991; McFadden, Gilson & Hill, 1994). Numerous
observations appear consistent with this scenario, including the numbers of
extraplastidal membranes in cryptomonads and photoautotrophic straminipiles
(Tomas & Cox, 1973; Gibbs, 1981; Whatley, John & Whatley, 1979; Whatley,
1981), the presence of and biosynthetic capability within the nucleomorph
compartment of cryptomonads (McFadden, Gilson & Douglas, 1994), and the
nonphotosynthetic nature of the most basal lineages within the straminipiles
(Leipe et al, 1994). This scenario is further supported by the specific
relationship observed between rDNAs of Goniomonas and cryptomonad nuclei
(Fig. 4A,B, and McFadden, Gilson & Hill, 1994).
Evidence is also accumulating that secondarily nonphotosynthetic eukaryotes
can retain molecular evidence of the lost plastid genome for evolutionarily
significant periods of time (e.g. Plasmodium: Howe, 1992; Gardner et al,
1993). TO our knowledge there is no evidence that animals or fungi descend
from a photosynthetic ancestor. If red algae, green plants, fungi and animals
are the crown taxa in the sense argued above, and if red algae and green
plants do in fact descend from a common photosynthetic eukaryote, and if
animals and fungi were never photosynthetic, then red algae and green plants
must be sister groups.
As a consequence of this result, the presence of two membranes surrounding
rhodoplasts and chloroplasts can be interpreted not only as evidence of the
origin of these plastids in a primary endosymbiotic event involving a
cyanobacterium but also, most parsimoniously, as a synapomorphy. Storage
of starch in the cytoplasm and the absence of phagotrophy might likewise
be synapomorphies in red algae and green plants, and a Kingdom Plantae

19. ARE RED ALGAE PLANTS? 99
TABLE 4. Plantae sensu Cavalier-Smith
(1981)
Kingdom Plantae
Subkingdom Viridiplantae
Division Chlorophyta
Division Charophyta
Division Embryophyta
Subkingdom Biliphyta
Division Glaucophyta
Division Rhodophyta
sel2su Cavalier-Smith (1981) (Table 4) might in fact be founded on
synapomorphic character states (see also Cavalier-Smith, 1993: 956-957).
This classification implies a relationship between glaucophytes (e.g.
Cyanophora, Glaucocystk, Glaeochaete) and red algae, and between glaucophytes
and green plants. Cavalier-Smith (1987: 75-76) interprets ultrastructural
features as indicating that “(t)he transition between Glaucophyceae and
Rhodophyceae is so gradual that they might almost be treated as a single
class”, and both Cavalier-Smith (1986: 323) and O’Kelly (1992) comment
upon ultrastructural features linking glaucophytes with green plants. The SSU
rDNA data do not resolve these issues; other types of analyses (above) and
sequence data from more-variable genes or gene regions will probably be
required.
ACKNOWLEDGEMENTS
It is a pleasure to acknowledge the contribution our colleagues Carolyn J.
Bird, Ellen Rice Kenchington, Colleen A. Murphy and Rama K. Singh in
sequencing red algal SSU rDNAs. Sandie L. Baldauf, Debashish Bhattacharya,
Roy G. Bums, Detlef D. Leipe, Qng Yan Liu, Ron M. MacKay, Geoff I.
McFadden, Michael E. Reith and Mitchell L. Sogin graciously allowed us
access to unpublished data and(or) to manuscripts prior to publication. We
thank Tom Cavalier-Smith, Georges E. Merinfeld and Lynn J. Rothschild for
helpful comments. Joe Felsenstein and Gary J. Olsen supplied source
code for PHYLIP and fastDNAm1 respectively; Burkhard Plache compiled
DNAPARS and fast DNAml for the AVX-2, and Optimax Software (Halifax,
N.S.) donated computer time. R.R.G. acknowledges support from U.S.
National Institutes of Health Grant GM 48207 and from the W. M. Keck
Foundation.
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