1. 807
J. Phycol. 38, 807–820 (2002)
A PROPOSAL FOR A NEW RED ALGAL ORDER, THE THOREALES1
Kirsten M. Müller
Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
Alison R. Sherwood
Department of Botany, 3190 Maile Way, University of Hawaii, Honolulu, HI 96822, USA
Curt M. Pueschel
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York, 13901, USA
Robin R. Gutell
Institute for Cellular and Molecular Biology, Section of Integrative Biology, University of Texas at Austin, Austin, Texas, 78712, USA
and
Robert G. Sheath2
Provost’s Office, California State University San Marcos, San Marcos, California 92096, USA
Representatives of the freshwater red algal family
Thoreaceae were studied to resolve their taxonomic
and phylogenetic status. Three specimens of Nema-
lionopsis and five collections of Thorea were exam-
ined for pit plug ultrastructure and analyzed for the
sequences of the genes coding for the large subunit
of RUBISCO (rbcL) and the small subunit of rRNA
(18S rRNA). The phylogenetic trees generated from
the two genes, and a combined tree all showed the
Thoreaceae to be contained in a well-supported
monophyletic clade that is separate from the other
two families currently classified in the Batrachosper-
males, the Batrachospermaceae and the Lemanea-
ceae. In addition, secondary structure elements of
the 18S rRNA gene were observed at positions 650
and 1145 (Escherichia coli numbering system) that are
not present in other members of the Rhodophyta.
The pit plugs of the gametophytic and chantransia
stages of the Thoreaceae contain two cap layers, the
outer one of which is typically plate-like, though oc-
casionally inflated ones have been seen. No pit plug
cap membrane has been observed. These findings
indicate the Thoreaceae has been misclassified in
the Batrachospermales and should be placed in its
own order, the Thoreales. This order is character-
ized by having freshwater representatives with multi-
axial gametophytes, a uniaxial chantransia stage, and
pit plugs with two cap layers, the outer one of which
is usually plate-like.
Key index words: 18S rRNA gene; Batrachospermales;
Nemalionopsis; rbcL gene; Thorea Thoreales ord. nov.
The freshwater red algal genera Thorea and Nemalion-
opsis are currently classified in the family Thoreaceae
of the Order Batrachospermales (Sheath et al. 1993,
Pueschel et al. 1995, Necchi and Zucchi 1997, Entwisle
and Foard 1999). This family is delineated from other
freshwater Rhodophyta by having gametophytes that are
multiaxial, with a colorless central medulla and determi-
nate lateral assimilatory filaments (Starmach 1977). The
other families of the order, the Batrachospermaceae
and Lemanaceae, have uniaxial gametophytes (Sheath
1984). The thoreacean genera are largely separated
from each other based on the positioning of the mono-
sporangia. In Thorea they are on short branches and
hence are axial in their position (Sheath et al. 1993); in
Nemalionopsis they are at the tips of assimilatory fila-
ments, localized in the thallus periphery.
Our main objective is to resolve the taxonomic and
phylogenetic relationships of the Thoreaceae to other
red algal lineages. In particular, it is necessary to deter-
mine whether this family is properly classified in the
Batrachospermales because both ultrastructural and
DNA sequence analyses have raised doubt about its tax-
onomic status. For example, Schnepf (1992) noted
that Thorea hispida (Thore) Desvaux (as T. ramosissima
Bory de Saint-Vincent) contained pit plugs with a dis-
tinct plate-like outer cap layer. This observation differs
from those of Lee (1971) and Pueschel (1989) for Tho-
rea violacea Bory de Saint-Vincent (as T. riekei Bischoff)
in which the outer pit plug cap layer is domed, a char-
acteristic that is used to distinguish the Batrachosper-
males. The Batrachospermales are also delineated by
having pit plugs with no cap membrane (Pueschel
1994); however, this feature has not been observed in
Thorea or Nemalionopsis. Hence, observations of pit
plugs of the Thoreaceae using freeze substitution
methods would be helpful in resolving the classifica-
tion of this family. In terms of molecular data, Vis et al.
(1998) demonstrated, using sequence analysis of the
1Received 20 March 2001. Accepted 1 May 2002.
2Author for correspondence: e-mail rsheath@csusm.edu.

2. 808 KIRSTEN M. MÜLLER ET AL.
large subunit of RUBISCO (rbcL) and small subunit of
rRNA (18S rRNA) genes, that Thorea violacea is posi-
tioned on a branch separate from other members of
the Batrachospermales, a result that is well supported
by bootstrap resampling in the combined gene tree.
These authors concluded that Thorea should be re-
moved from the Batrachospermales and should be
noted as incertae sedis until further studies of the Tho-
reaceae could be undertaken. Subsequent molecular
analyses using rbcL and 18S rRNA genes supported the
conclusions of Vis et al. (1998) (e.g. Acrochaetiales:
Harper and Saunders 1998, Balbianiales: Sheath and
Müller 1999). Clearly more representatives of the Tho-
reaceae need to be examined for pit plug ultrastruc-
ture and sequence analyses of the rbcL and 18S rRNA
genes before the phylogenetic status of the family can
be conclusively determined. The present work has at-
tempted to add as many samples as available for each
genus. A secondary objective of this investigation is to
ascertain whether the separation of Nemalionopsis and
Thorea, based primarily on the positioning of the
monosporangia, is supported by molecular analyses of
the rbcL and 18S rRNA genes. A third goal is to deter-
mine if the Nemalionopsis and Thorea species used in this
study are supported as being distinct taxa in molecular
trees of the rbcL and 18S rRNA genes.
materials and methods
Samples of Thorea and Nemalionopsis were obtained for study
as follows:
1. Culture Collection of Algae at the University of Göttingen
(SAG):
• 41.94 Nemalionopsis shawii Skuja f. caroliniana Howard et
Parker, Lower Barton Creek, Wake Co., NC, USA, iso-
lated by F. D. Ott, 1973.
• 42.94 Nemalionopsis tortuosa Yagi et Yoneda, Hanabusa at
Kikuchi-City, Kumamota Prefecture, Japan, isolated by F.
D. Ott, 1973 (SAG).
• 46.94 Thorea hispida (as T. ramosissima), River Thames at
Cookham, England, collected by D.M. John, isolated by
F. D. Ott, 1992.
• 45.94 Thorea violacea (as T. okadai Yamada), Kikuchi River,
Yamaga City, Kumamota, Prefecture, Japan, isolated by F.
D. Ott, 1973 (SAG 1).
• 47.94 Thorea violacea (as T. riekei), New Braunfels, Texas,
USA, isolated by F. D. Ott, 1973 (SAG 2).
2. Field collections:
• Nemalionopsis tortuosa, Tangipahoa River in Kentwood, Loui-
siana, USA, 24.iv.2000, collected by R. G. Sheath (LA).
• Thorea violacea, Hudson River near Thompson Island, Sa-
ratoga County, New York State, USA, 29.ix.1994,
29.x.1996 collected by C. M. Pueschel (NY).
• Thorea violacea, San Marcos River, San Marcos, Texas,
USA, 1.xii.1994, collected by R. G. Sheath (TX7b).
Samples for LM and EM were fixed and processed as previ-
ously described (e.g. Sheath and Müller 1999). To determine
whether a cap membrane is present in the pit plugs, the freeze
substitution method of Pueschel (1994) was followed.
Some sequences of both the rbcL and 18S rRNA genes of
Thorea and Nemalionopsis were obtained; GenBank accession
numbers are noted in Table 1. In addition, samples were
ground in liquid nitrogen, and the DNA was extracted either by
the protocol outlined by Saunders (1993) with modifications
given in Vis and Sheath (1997) or using the Qiagen Dneasy
Plant Mini Kit (Qiagen, Mississauga, Canada). The primer com-
bination F160 (5Ј-CCT CAA CCA GGA GTA GAT CC-3Ј) and
rbcLR (5Ј-ACA TTT GCT GTT GGA GTC TC-3Ј) was used to
amplify a 1272-base pair fragment of the 1467-base pair rbcL
gene (Vis et al. 1998). The reaction volume of 95 ␮L consisted
of 2 ␮L of genomic DNA; 20 ␮M each of dATP, dCTP, dGTP
and dTTP; 0.4 ␮M of each primer; 2 mM MgCl2; 10 ␮L 10ϫ re-
action buffer (Perkin Elmer, Norwalk, CT, USA); and 2 units of
Amplitaq. The 18S rRNA gene was amplified using the primer
pair G01 and G07 (Saunders and Kraft 1994). Double-stranded
PCR products were amplified in a Perkin Elmer (Norwalk, CT,
USA) Gene Amp 2400 Thermal Cycler as follows: initial dena-
turation at 95Њ C for 2 min, followed by 35 cycles of denaturation
at 93Њ C, primer annealing at 47ЊC (rbcL) or 58Њ C (18S rRNA) for
1 min, extension at 72Њ C for 4 min, and a final extension at 72Њ C
for 6 min. All PCR products were subsequently purified using the
QIAquick PCR Purification Kit (Qiagen, Mississauga, Canada) ac-
cording to the protocols of the manufacturer. The double-
stranded PCR products were sequenced using an ABI PRISM Taq
FS Cycle Sequencing Ready Reaction Kit (Applied Biosystems,
Foster City, CA, USA) and an ABI 377 Automated DNA Se-
quencer. Sequencing reactions were performed using the origi-
nal amplification primers as well as internal primers (Table 2).
Sequences of the rbcL and 18S rRNA gene sequences were
assembled using DNA sequencing software (ABI). Alignment of
the rbcL gene was easily accomplished because of the absence
of insertions or deletions. The 18S rRNA gene sequences were
manually aligned using the alignment editor AE2 (developed
by T. Macke, see Larsen et al. 1993) on the basis of primary
structure similarity and previously established eukaryotic sec-
ondary structure features (Gutell 1993). The aligned database
was then subjected to a process of comparative sequence analy-
sis (Gutell et al. 1985) as follows: 1) searches were conducted
for compensating base changes using computer programs, 2)
the resulting information was used to infer additional second-
ary structural features which refined the sequence alignment,
and 3) the revised alignment was reanalyzed and the entire pro-
cess was repeated until the proposed structures were entirely
compatible with the alignment. Secondary structure diagrams
were generated interactively with the computer program XRNA
(developed by B. Weiser and H. Noller, University of California
Santa Cruz and are available at http://www.rna.icmb.utexas.edu/).
Table 1. GenBank accession numbers, culture, and collection information for members of the Thoreaceae used in this study.
Taxon Collection information
GenBank accession number
rbcL 18S rRNA
Nemalionopsis shawii Skuja SAG 41.94 AF506266 AF506272
Nemalinonopsis tortuosa Yagi et Yoneda SAG 42.94 AF506267 —
Thorea hispida (Thore) Desvaux SAG 46.94 (ϭ Thorea ramosissima) AF506270 AF506273
Thorea violacea Bory de Saint Vincent (as T. violacea
[SAG1] in figures) SAG 45.94 (ϭ Thorea okadae) AF506269 AF506274
Thorea violacea (as T. violacea [SAG2] in figures) SAG 51.94 AF506271 —
Thorea violacea (as T. violacea [NY] in figures) New York State, USA, collected by C. M. Pueschel AF506268 AF506275

3. 809NEW RED ALGAL ORDER
GenBank accession numbers for previously published red algal
sequences used in the analyses are listed in Appendix 1. Mem-
bers of the Bangiophycidae were used as outgroups for the phy-
logenetic analyses, and the GenBank accession numbers for
these taxa are also given in Appendix 1.
Parsimony trees for the rbcL and 18S rRNA genes were gen-
erated with the heuristic search option under the conditions of
random sequence addition (100 replicates), steepest descent,
and tree bisection-reconnection branch swapping using the
program PAUP (version 4.0b 4a; Swofford 2000). The data were
then subjected to bootstrap resampling (1000 replicates). Anal-
yses of the rbcL gene were performed with each base weighted
equally as well as weighting first, second, and third positions of
the codon, following the criteria of Albert et al. (1993), and a par-
simony analysis of amino acid sequences was performed. Neigh-
bor-joining (NJ) trees (Saitou and Nei 1987) were constructed
using PHYLIP (Phylogeny Inference Package; Felsenstein 1993)
from a matrix of distance values estimated according to the
Kimura two-parameter model (Kimura 1980). A transition/trans-
version ratio of 2.0 and a single category substitution rate were
used. The data were also bootstrap resampled (1000 replicates)
according to PHYLIP. Number of base pair changes between se-
quences was calculated using the program MEGA (Molecular
Evolutionary Genetics Analysis; Kumar et al. 1993).
Maximum likelihood analysis of the 18S rRNA gene align-
ment was carried out using the puzzle function in PAUP (version
4.0b 4a) as described by Strimmer and von Haeseler (1996) with
1000 puzzling steps. This method applies maximum likelihood
tree reconstruction to all possible quartets that can be formed
from all sequences that serve as starting points to reconstruct a
set of optimal n-taxon trees (Strimmer and von Haeseler 1996).
This method has been shown to be equally or better able to re-
construct the true tree than NJ methods (Strimmer and von Hae-
seler 1996). The values above the branches represent the per-
centage of times that a particular cluster was found among the
1000 intermediate trees (quartet puzzling step [QPS] values).
results
Ultrastructure of pit plugs. We examined in detail the
pit plugs of T. violacea (NY) gametophyte and chantran-
sia phase, T. violacea (TX 7b) gametophyte, and Nema-
lionopsis tortuosa (LA) gametophyte (Fig. 1, A–E). In
addition, we also surveyed cultures of N. shawii, N. tor-
tuosa, T. hispida, and T. violacea, the life history phase
of which is uncertain but appears to be the chantran-
sia (not shown). All pit plugs observed (over 100) con-
tained two cap layers, the inner of which is quite thin
and typically electron translucent. The outer cap layer is
typically plate-like but more electron dense and about
twice as thick as the inner layer (Fig. 1, A and C–E).
Although most of the outer cap layers are not domed,
a small number were found to be inflated, such as
those of the N. tortuosa (LA) gametophyte (Fig. 1E).
Freeze-substituted specimens of T. violacea (NY) were
analyzed for the presence of a pit plug cap mem-
brane. Neither the gametophyte (not shown) nor the
chantransia stage (Fig. 1F) was observed to have a cap
membrane.
rbcL gene and 18S rRNA gene sequence analyses. Se-
quence data for all taxa included in this study were
submitted to GenBank, and accession numbers are
given in Table 1 and in the Appendix. The rbcL gene
was successfully amplified and sequenced for six sam-
ples of the Thoreaceae. The gene contained no inser-
tions or deletions, which facilitated the construction
of a final alignment of 1201 nucleotides (out of 1467
nt). A NJ tree from analysis of the rbcL gene is de-
picted in Figure 2, and parsimony analysis of 537 phy-
logenetically informative characters resulted in three
most parsimonious trees with a length of 4275 and a
consistency index (CI) of 0.2271, one of which is
shown in Figure 3. The quartet-puzzling tree is similar
in topology, and hence the branch support is given on
the parsimony tree (Fig. 3, second value). One of the
most notable features of these trees (Figs. 2 and 3) is
that members of the Thoreaceae form a well-sup-
ported clade (maximum parsimony [MP], 99% boot-
strap; NJ, 100% bootstrap; QPS, 85%). The Tho-
reaceae forms a well-supported clade (97% bootstrap)
with two other red algal orders, the Balliales and Bal-
bianiales, in the NJ tree (Fig. 2). However, this rela-
tionship is not supported in the parsimony or quartet
puzzling analyses (Fig. 3). In fact, relationships among
the various florideophyceaen orders are not well re-
solved in any of the analyses. The exception is the as-
sociation between the Palmariales and the one se-
quence of Audouinella hermannii (Roth) Duby of the
Acrochaetiales (MP, 73% bootstrap; NJ, 90% bootstrap;
QPS, 80%). The Batrachospermales forms a moderately
supported entity (MP, 78% bootstrap; NJ, 97% boot-
strap; QPS, 61%) and the family Thoreaceae is only
weakly associated (MP, 61% bootstrap; QPS, Ͼ50%)
with this order in the parsimony and quartet puzzling
analyses (Fig. 3) and moderately associated (82% boot-
strap) along with the Balbianiales and Balliales in the
NJ analysis (Fig. 2).
Table 2. Primers used to amplify and sequence the rbcL and 18S rRNA genes.
Primer Gene Sequence (5Ј to 3Ј) Reference
G01 18S rRNA CAC CTG GTT GAT CCT GCC AG Saunders and Kraft 1994
G02.1 18S rRNA CGA TTC CGG AGA GGG AGC CTG Modified from Saunders and Kraft 1994
G04.1 18S rRNA GTC AGA GGT GAA ATT CTT GG Modified from Saunders and Kraft 1994
G06 18S rRNA GTT GGT GGT GCA TGG CCG TTC Saunders and Kraft 1994
G07 18S rRNA TCC TTC TGC AGG TTC ACC TAC Saunders and Kraft 1994
G08.1 18S rRNA GAA CGG CCA TGC ACC ACC AAC Modified from Saunders and Kraft 1994
G09 18S rRNA ATC CAA GAA TTT CAC CTC TG Saunders and Kraft 1994
F160 rbcL CCT CAA CCA GGA GTA GAT CC Rintoul et al. 1999
F949 rbcL CTG TAA GTG GAT GCG TAT GGC Rintoul et al. 1999
R897 rbcL CGA GAA TAA GTT GAG TTA CCT GC Rintoul et al. 1999
rbcLR rbcL ACA TTT GCT GTT GGA GTC TC Rintoul et al. 1999

4. 810 KIRSTEN M. MÜLLER ET AL.
Within the Thoreaceae both Nemalionopsis shawii
and N. tortuosa are closely associated, but on a branch
distinct from the Thorea samples (MP, 100% boot-
strap; NJ, 100% bootstrap, QPS, 97%). In addition, all
four collections of T. violacea (SAG1, SAG2, TX7, and
NY) and the one sample of T. hispida form a solid clus-
ter that is well supported by bootstrap and decay anal-
ysis (MP, 99% bootstrap; QPS, 99%; NJ, 97% boot-
strap) (Figs. 2 and 3). Interestingly, Thorea hispida is
strongly associated with T. violacea (SAG1) in the NJ
(100% bootstrap) and QPS (99% support, not shown)
and identical to T. violacea (SAG1) when only phylo-
genetically informative sites are considered in the par-
simony analysis. In addition, T. violacea (NY) is also
closely positioned with these two collections (MP, 99%;
NJ, 100%; QPS, 97%). The two remaining collections
of T. violacea (SAG2 and TX7), which are both from
Texas, are well supported as being distinct from T. vi-
olacea (NY, SAG1) and T. hispida; thus, T. violacea is
paraphyletic (Figs. 2 and 3).
The corrected sequence divergence values of the
rbcL gene between members of the Thoreaceae and
the other orders of red algae ranged from 16.6% (be-
tween Batrachospermum atrum and N. tortuosa) to 25.9%
(between T. violacea [TX7] and Rhodogorgon carriebowen-
sis Norris et Butcher). The Thoreaceae differed from
other members of the Batrachospermales by 16.6%–
25.0% corrected sequence divergence. Within the Thor-
eaceae the sequence divergence ranged from 5.42%
to 16.3%. In the genus Thorea, the sequence diver-
Fig. 1. Transmission electron micrographs of the pit plugs of the Thoreaceae. (A and B) Nemalionopsis tortuosa (LA) gameto-
phyte. (A) Outer cap layer is plate-like (arrowheads). (B) Outer cap layer is slightly domed (arrowheads). (C and D) Thorea violacea
(NY). (C) Gametophyte pit plug with flat plate-like outer cap layer (arrowheads). (D) Chantransia stage pit plug with a plate-like
outer cap layer (arrowheads). (E) Thorea violacea (TX7b) gametophyte with pit plug having a plate-like outer cap layer (arrowheads).
(F) Thorea violacea (NY) chantransia state freeze substitution image of pit plug with no discernible pit plug cap membrane.

5. 811NEW RED ALGAL ORDER
Fig. 2. Neighbor-joining tree from analysis of 1201 nucleotides of the rbcL gene. Numbers above the branches represent bootstrap
resampling results (% of 1000 replicates). Branches lacking values had less than 50% support. Batrachospermum1 ϭ Batrachospermum
virgato-decaisneanum. Members of the subclass Bangiophycidae are used as outgroup taxa.

6. 812 KIRSTEN M. MÜLLER ET AL.
Fig. 3. One of three most parsimonious trees from analysis of the rbcL gene sequence data containing 537 phylogenetically infor-
mative characters. The first of the numbers above the branches represents bootstrap values from maximum parsimony analysis (% of
1000 replicates). The second value is the reliability value of each internal branch determined by the quartet puzzling method and in-
dicates in percent the frequency that the corresponding cluster was found among the 1000 intermediate trees. An asterisk or branch
lacking values had less than 50% bootstrap support. Members of the subclass Bangiophycidae are used as outgroup taxa.

7. 813NEW RED ALGAL ORDER
gence ranged from 5.42% (between T. violacea [SAG1]
and T. hispida) to 13.3% (between T. violacea [TX7] and
T. violacea [SAG1]). The two genera, Thorea and Nema-
lionopsis, differed by 0 (SAG1 and NY) to 8 (N. tortuosa
and T. hispida) amino acids and differed from the re-
maining Rhodophyta in the analysis by 12 to 32 amino
acids (out of a total 400 codons). Parsimony analysis
of amino acid sequences resulted in 1436 most parsi-
monious trees (not shown) (l ϭ 179, CI ϭ 0.49).
Members of the Thoreaceae again formed a well-sup-
ported (100% bootstrap) clade in the amino acid tree
in which the relationship was unresolved with the re-
maining taxa in the Batrachospermales and the other
orders (not shown).
An NJ tree from analysis of the 18S rRNA gene is
depicted in Figure 4 and parsimony analysis of 732
phylogenetically informative characters resulted in
100 most parsimonious trees with a length of 3871
and a CI of 0.4324, of which a strict consensus is
shown in Figure 5. These 100 most parsimonious trees
differed due to low resolution among the various
florideophyte orders and within the Acrochaetiales
and the Batrachospermales. The quartet puzzling tree
is similar in topology, and hence the branch support
is given on the parsimony tree (Fig. 5, second value).
As seen in the rbcL analyses, the Thoreaceae forms a
well-supported entity (MP, 100% bootstrap; QPS,
98%; NJ, 96% bootstrap) that is not strongly associ-
ated with the remaining florideophyte orders or the
Batrachospermales (Figs. 4 and 5). In fact, the Tho-
reaceae appears to be more closely associated with the
Acrochaetiales, Balbianiales, Nemaliales, and Palmari-
ales than with the Batrachospermales (Figs. 4 and 5)
(albeit this is not well supported by parsimony boot-
strap [57%] or quartet puzzling [52% QPS] and only
moderately supported by NJ bootstrap [77%]). Clus-
tering within the Thoreaceae is similar to that ob-
served in the rbcL gene sequence analyses; T. violacea
is paraphyletic with the collection T. violacea (SAG1),
being strongly associated with T. hispida. In addition,
the genus Nemalionopsis is associated with the two col-
lections of T. violacea (SAG2 and TX7). This relation-
ship is well supported by MP bootstrap (100%) and NJ
bootstrap (99%) and weakly supported by QPS (68%)
(Figs. 4 and 5).
The sequence divergence between N. shawii and N.
tortuosa was 2.88%. Within the genus Thorea, the se-
quence divergence ranged from 2.50% (T. hispida
and T. violacea [SAG1]]) to 6.36% (T. violacea [SAG2]
and T. hispida). These taxa differed from the genus
Nemalionopsis by 2.18% (T. violacea [SAG2]]) to 4.72%
(T. hispida) corrected sequence divergence.
Secondary structure signatures. Figure 6 depicts a sec-
ondary structure model diagram of the 18S rRNA
gene from T. violacea (TX7) highlighting two regions
A and B (Escherichia coli numbering: 650 and 1145 re-
gions, respectively) in which structural elements exist
that separate members of the Thoreaceae from the re-
maining Rhodophyta (based on an alignment of over
400 18S rRNA gene sequences, representing all rho-
dophyte orders, Müller et al. 2000, unpublished data).
Figure 7 depicts a gallery (collection of secondary struc-
ture elements from different samples) of the 650 re-
gion (E. coli numbering, also represented as A in Fig.
6) in which there is an additional A-U rich helix rang-
ing from 10 to 25 nucleotides in sequences of the ge-
nus Thorea. This helix is not found in either the genus
Nemalionopsis (depicted also in Fig. 7) or any other
Rhodophyta sequences examined (not shown). The
most striking example of a structural signature is the
large extended helix in the 1145 region (Fig. 8), of
which there is no homologous structure in all other
Rhodophyta 18S rRNA gene sequences examined.
This extended helix is found in all members of the
Thoreaceae sequenced thus far and ranges from 21
(N. shawii and N. tortuosa) to 124 (T. violacea [SAG2])
additional nucleotides and is characterized by a con-
served tetra-loop (GCAA) at the end of the helix and
a conserved sequence in the beginning of this helix.
The phylogenetic analyses depicted in Figures 4
and 5 excluded these regions because they would be
considered autapomorphies for the delineation of
this group. However, phylogenetic analyses were car-
ried out incorporating these regions, and there was
little or no difference in the topology or support
within the Thoreaceae or with its relationship with
other clades. Thorea violacea (SAG2) and T. violacea
(TX7) were depicted in Figures 2, 3, 4, and 5 and
form a strongly supported group; in fact, both these
taxa were found to contain longer extended helices
(121–124 nt). On the other hand, T. hispida and T. vi-
olacea (SAG1) had smaller helices (73–75 nt, respec-
tively) and the two sequences of the genus Nemalionop-
sis had identical helices of only 21 nucleotides. These
striking features, in addition to the position of the
members of the Thoreaceae on a clearly separate
branch from other members of the Batrachosper-
males, justifies the separation of the Thoreaceae from
this order into a new order, the Thoreales.
discussion
Ultrastructure of pit plugs. Ultrastructural observations
of pit plugs of members of the Thoreaceae have been
reported by several authors, nearly all of whom agree
on the existence of an outer cap layer. However, the
characterization of this layer as a plate or as a dome has
been the subject of dispute. Bischoff (1965) did not de-
scribe a plug cap in the gametophytic phase of T. viola-
cea (as T. riekei), but a thin cap layer is discernible in his
micrograph of a pit plug. By contrast, Lee (1971) dem-
onstrated domes in the cultured chantrasia phase of
the same species. In two surveys of pit plug structure
(Pueschel and Cole 1982, Pueschel 1989), the outer
cap layers of members of the Thoreaceae were catego-
rized as domes, and in a cytochemical study of the
chemical composition of outer cap layers (Trick and
Pueschel 1991), a dome-shaped cap of the Thorea
chantrasia stage was demonstrated by LM. In the only
detailed ultrastructural study of a member of the Tho-
reaceae, Schnepf (1992) clearly demonstrated pit plugs

8. 814 KIRSTEN M. MÜLLER ET AL.
Fig. 4. Neighbor-joining tree from analysis of 2486 nucleotides (including alignment gaps) of the 18S rRNA gene. Numbers
above the branches represent bootstrap resampling results (% of 1000 replicates). Branches lacking values had less than 50% support.
Members of the subclass Bangiophycidae are used as outgroup taxa.
Fig. 5. Strict consensus of 100 most parsimonious trees derived from analysis of the 18S rRNA gene sequence data containing 752
phylogenetically informative characters. The first of the numbers above the branches represents bootstrap values from maximum par-
simony analysis (% of 1000 replicates). The second value is the reliability value of each internal branch determined by the quartet
puzzling method and indicates in percent the frequency that the corresponding cluster was found among the 1000 intermediate
trees. An asterisk or branch lacking values had less than 50% bootstrap support. Batrachospermum1 ϭ Batrachospermum virgato-decais-
neanum. Members of the subclass Bangiophycidae are used as outgroup taxa.

9. 815NEW RED ALGAL ORDER

10. 816 KIRSTEN M. MÜLLER ET AL.
Fig. 6. Secondary structure model determined by comparative sequence analysis (using Ͼ 5000 eukaryotic 18S rRNA gene se-
quences, see http://www.rna.icmb.utexas.edu) of the 18S rRNA gene in Thorea violacea (TX7). (A) Region 650 (Escherichia coli num-
bering) depicted in Figure 7. (B) Region 1145 (E. coli numbering) depicted in Figure 8. ᭺, A:G pairings; ᭹, noncanonical (non-Wat-
son Crick) pairings; ؒ, G:U pairings.

11. 817NEW RED ALGAL ORDER
Fig. 7. Gallery (collection of secondary structure elements from different samples) of secondary structure diagrams of the 650 re-
gion (Escherichia coli numbering) in the 18S rRNA gene, highlighting differences among members of the Thoreaceae, (depicted as
“A” in Fig. 6). ᭺, A:G pairings; ᭹, noncanonical (non-Watson Crick) pairings; ؒ, G:U pairings.

12. 818 KIRSTEN M. MÜLLER ET AL.
with thin plates in field-collected gametophytes of Tho-
rea hispida. Our current study includes both field-col-
lected gametophytes and cultured chantransia stages of
both Nemalionopsis and Thorea, with the vast majority
having plate-like outer cap layers that are up to three
times wider than the inner layer (Fig. 1, A and C–E). In
the other members of the Batrachospermales in which
the domed outer cap layers are typical, the dome is
considerably wider than the inner cap layer by a factor
of 10–15 (e.g. Brown and Weier 1970, Aghajanian and
Hommersand 1978, Pueschel and Cole 1982, Trick and
Pueschel 1991, Sheath and Whittick 1995, Sheath et al.
1996a,b). Among the small number of cases in the
Thoreaceae with an inflated outer cap, the outer cap
layer is up to four times wider than the inner layer (e.g.
Fig. 1B). Hence, the outer cap layer appears to be a
good characteristic to delineate the Thoreaceae from
other members of the Batrachospermales.
Demonstrating the presence or absence of a cap
membrane is made more difficult by the fact that the
edges of the outer cap layer can produce an appear-
ance that is easily mistaken for a membrane (Pueschel
1987, 1994). Freeze substitution has clearly demon-
strated cap membranes in Cumagloia andersonii (Far-
low) Setchell et Gardner and Nemalion helminthoides
(Velley) Batters (Nemaliales) (Pueschel 1994) and
confirmed the absence of cap membranes in some
members of the Batrachospermaceae (Pueschel 1994).
In the present study, freeze substitution showed no
cap membranes in either the chantrasia or gameto-
phytic phase of Thorea violacea, a finding that is consis-
tent with an absence of cap membranes in a variety of
specimens of the Thoreaceae prepared by conven-
tional chemical fixation.
In summary, the ultrastructural study of the pit
plugs demonstrated that Nemalionopsis and Thorea are
not well classified in the Batrachospermales in that
they differ in the outer cap layer largely being a flat
plate.
Molecular analyses. All the gene trees generated
show the Thoreaceae to be a well-supported mono-
phyletic clade that is a distinct lineage from that of
the other batrachospermalean families, the Batracho-
spermaceae and Lemaneaceae. These findings con-
firm those of previous molecular studies, which in-
cluded only one member of the Thoreaceae, such as
Harper and Saunders (1998), Vis et al. (1998), and
Sheath and Müller (1999). These findings further
substantiate that Nemalionopsis and Thorea should be
removed from the Batrachospermales.
The three molecular trees generated all exhibit a
distinct separation of the two genera of the Tho-
reaceae. Hence, the current basis for distinguishing
Nemalionopsis from Thorea primarily by peripheral ver-
sus axial positioning of monosporangia (Starmach
1977, Sheath et al. 1993) is supported by this study. By
contrast, the distinction of T. hispida from T. violacea
based on degree of branching (Sheath et al. 1993) is
not supported in the gene trees. Thorea hispida
(Thore) Desvaux 1818 has precedence over T. violacea
Bory de Saint-Vincent 1808 because the basionym for
T. hispida is Conferva hispida Thore 1799. However, at
this time there are not enough samples or molecular
data to justify the synonymy of T. hispida and T. viola-
cea. This is despite the little support in the molecular
trees for the use of primary or secondary branching as
a defining taxonomic character.
The separation of the Thoreaceae from the Batra-
chospermales is further substantiated by the finding
of unique secondary structure signatures in the 18S
rRNA gene of Nemalionopsis and Thorea. Over 400 se-
quences of this gene were examined within the Rho-
dophyta, including representatives of all orders to make
this finding (Müller et al. 2000, unpublished data).
Fig. 8. Gallery (collection of secondary structure elements from different samples) of secondary structure diagrams of the 1145 re-
gion (Escherichia coli numbering) in the 18S rRNA gene, highlighting differences among members of the Thoreaceae (depicted as “B”
in Fig. 6). ᭺, A:G pairings; ᭹, noncanonical (non-Watson Crick) pairings; ؒ, G:U pairings.

13. 819NEW RED ALGAL ORDER
Woese (1987) justified using the higher order structure
from a phylogenetic perspective, and Winker and Woese
(1991) delineated three domains of organisms based
on a combination of homologous and non-homolo-
gous signature sequences of the 16S rRNA gene. The
unique helices in members of the Thoreaceae in the
positions 650 and 1145 of the 18S rRNA gene are such
signature sequences. The A:U rich helix at position 650
in Thorea has not been found in any other eukaryotic
18S rRNA gene sequences examined to date (Ͼ5000,
Gutell unpublished data). The large extended helix
in the 1145 region of both genera is not present in
other red algal 18S rRNA gene sequences (Müller et al.
2000, unpublished data). These nonhomologous sig-
natures appear to be diagnostic of members of the ge-
nus Thorea (position 650) and the family Thoreaceae
(position 1145), respectively.
Taxonomic conclusions. This study has shown that the
Thoreaceae is well separated from other members of
the Batrachospermales based on the following charac-
teristics: 1) multiaxial gametophyte thallus; 2) pit plugs
with typically plate-like outer cap layers; 3) position in
rbcL, 18S rRNA, and combined gene trees; and 4)
presence of signature elements in the 18S rRNA gene,
particularly at position 1145. Based on the fact that
the Thoreaceae is well supported as a separate mono-
phyletic grouping in the gene trees, we propose that a
new order be described for members of this clade as
follows:
Thoreales ord. nov. Müller, Sheath, Sherwood
et Pueschel
Filum multiaxialis cum gametangia, filum chanstra-
nium uniaxialialis, obturamentum lacunum cum stratum
externum planum, in aquis dulcibus.
Multiaxial gametophytic filaments, alternating with
uniaxial chantransia stage, pit plugs with plate-like
outer cap layers, freshwater habitats.
Type family: Thoreaceae (Reichenbach) Hassall
with same characteristics as order.
Type genus: Thorea Bory de St.-Vincent
Supported by NSERC grant 0183503 to R. G. S., OGS scholar-
ship to K. M. M., NSERC PGSB scholarship to A. R. S. and NIH
GM48207 and the University of Texas start-up funds to R.R.G.
We thank the Culture Collection of Algae at the University of
Göttingen (SAG) for supplying five cultures used in this study
and Dana Couture, Gary Sullivan, and John Titus for help in
field collections and Jaime Cannone for assitance with rRNA se-
quence and structural analyses. Technical assistance in DNA se-
quencing by Angela Holliss and in manuscript preparation by
Toni Pellizzari is gratefully acknowledged.
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Appendix 1
rbcL: Ahnfeltia plicata U04168, Amphiroa fragilissima U04039, Antithamnion sp. X54532, Audouinella hermannii U04033, Balbiania
investiens AF132293, Ballia callitricha AF149029, Bangia atropurpurea AF043370, B. fuscopurpurea AF043366, Batrachospermum atrum
AF029139, B. gelatinosum AF026045, B. helminthosum AF029142, B. louisianae AF029144, B. macrosporum AF029145, B. turfosum
AF029147, B. virgato-decaisneanum AF029148, Bonnemaisonia hamifera U04044, Chondrus crispus U02984, Compsopogon coeruleus
AF087116, Dumontia contorta U26823, Erythrotrichia carnea AF087118, Gelidium elegans AB030623, Gracilaria tikvahiae U04172,
Gracilariopsis sp. U04170, Grateloupia filicina AB038612, Halosaccion glandiforme U04173, Halymenia dilatata AB038604, Lemanea
fluviatilis AF029150, Nothocladus nodosus AF029152, Palmaria palmata U04186, Paralemanea catenata AF029154, Porphyra miniata
AF168663, Porphyridium aerugineum X17597, Psilosiphon scoparum AF029155, Ptilophora subcostata U16835, Rhododraparnaldia
oregonica AF029156, Rhodogorgon carriebowensis U04183, Rhodymenia pseudopalmata U04182, Sirodotia suecica AF029158, Thorea
violacea(TX7) AF029160, Tuomeya americana AF029352.
18S rRNA gene: Ahnfeltia plicata Z14139, Amphiroa fragilissima U60744, Antithamnionella floccosa AF236788, Audouinella amphiroae
AF079785, A. arcuata AF079784, A. asparagopsis AF079795, A. caespitosa AF079787, A. dasyae L26181, A. daviesii AF079788, A.
endophytica AF079789, A. hermannii AF026040, A. macrospora (1) AF199505, A. macrospora (2) AF199506, A. pectinata AF079790, A.
proskaueri AF079791, A. rhizoidea AF079792, A. secundata AF079784, A. tenue AF079796, A. tetraspora AF079793, Balbiania investiens
AF132294, Ballia callitricha AF236790, Bangia atropurpurea AF043365, B. fuscopurpurea AF043365 AF043355, Bangiopsis subsimplex
AF168627, Batrachospermum boryanum AF026044, B. gelatinosum AF026045, B. helminthosum AF026046, B. louisianae AF026047, B.
macrosporum AF026048, B. turfosum AF026049, B. virgato-decaisneanum AF026050, Bonnemaisonia hamifera L26182, Bostrychia
moritziana AF203893, Camontagnea oxyclada AF079794, Champia affinis U23951, Chantransia sp. AF199507, Chondrus crispus Z14140,
Chroodactylon ornatum AF168628, Compsopogon coerulus AF087124, Corallina elongata U60946, Dumontia contorta AF317099,
Erythrotrichia carnea L26189, Galaxaura marginata AF006090, Gelidium elegans AB017670, Gracilaria gracilis L26205, Gracilariopsis sp.
L26208, Grateloupia filicina U33132, Halosaccion glandiforme L26193, Halymenia plana U33133, Hildenbrandia rivularis AF208830, H.
rubra AF108413, Lemanea fluviatilis AF026051, Meiodiscus spetsbergensis U23814, Nemalion helminthoides L26196, Nemalionopsis tortuosa
AF342743, Nothocladus nodosus U23815, Palmaria palmata Z14142, Paralemanea catenata AF026052, Plocamiocolax pulvinata U09618,
Plocamium cartilagineum U09619, Porphyra miniata AF175540, Porphyridium aerugineum L27635, Psilosiphon scoparum AF026041,
Ptilophora subcostata U60348, Rhodochorton purpureum U23816, Rhododraparnaldia oregonica AF026043, Rhodogorgon carriebowensis
AF006089, Rhodymenia leptophylla U09621,Sirodotia suecica AF026053, Sirodotia huillensis AF026054, Thorea violacea (SAG2)
AF342744, Thorea violacea (TX7) AF026042, Tuomeya americana AF026055.