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Pyramimonas australis sp. nov. (Prasinophyceae,
Chlorophyta) from Antarctica: fine structure and
molecular phylogeny
Isabella Moro , Nicoletta La Rocca , Luisa Dalla Valle , Emanuela Moschin , Enrico Negrisolo
& Carlo Andreoli
a Department of Biology, University of Padua, Via Ugo Bassi 58/b, 35131 Padua, Italy
Published online: 22 Jul 2011.
To cite this article: Isabella Moro , Nicoletta La Rocca , Luisa Dalla Valle , Emanuela Moschin , Enrico Negrisolo & Carlo
Andreoli (2002) Pyramimonas australis sp. nov. (Prasinophyceae, Chlorophyta) from Antarctica: fine structure and molecular
phylogeny, European Journal of Phycology, 37:1, 103-114
To link to this article: http://dx.doi.org/10.1017/S0967026201003493
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Eur.J.Phycol. (2002), 37: 103–114. #2002 British Phycological Society
DOI: 10.1017\S0967026201003493 Printed in the United Kingdom
103
Pyramimonas australis sp. nov. (Prasinophyceae, Chlorophyta)
from Antarctica: fine structure and molecular phylogeny
ISABELLA MORO, NICOLETTA LA ROCCA, LUISA DALLA VALLE,
EMANUELA MOSCHIN, ENRICO NEGRISOLO AND CARLO ANDREOLI
Department of Biology, University of Padua, Via Ugo Bassi 58\b, 35131 Padua, Italy
(Received 14 January 2001; accepted 25 July 2001)
An undescribed marine Pyramimonas,P.australis Andreoli et Moro, sp. nov., forming a bloom in a hole of Terra Nova
Bay (Ross Sea, Antarctica) sea ice, was collected, but could not be cultured. Consequently, the description of this new
species is based on light and electron microscope observations on samples that were fixed or stored at k80 mC, and its
phylogenetic position inferred from nuclear-encoded small-subunit ribosomal DNA (SSU rDNA) and chloroplast-encoded
rbcL gene sequences. This is the third Antarctic species described for this genus. The ultrastructure of the cell is consistent
with species of the subgenus Trichocystis McFadden, but differs in that it has unique body and cyst scales, and a different
encystment procedure. The outermost layer of body scales is formed by flat box scales with peripheral perforations oriented
parallel to the four edges and with a further eight central perforations oriented perpendicular to the peripheral ones. Crown
scales, which in many other species of the genus form the outermost layer over the entire cell body, were observed in this
species in the flagellar pit over the box scales. The flagella are covered by a pentagonal underlayer of scales and by limuloid
scales with two subsidiary spines, in addition to the central one. Encystment begins in the flagellate form resulting in a cyst
with an irregular wall bearing spine scales. Ultrastructural and molecular data confirm that P.australis belongs to the
subgenus Trichocystis.
Key words: Antarctica, PCR, Prasinophyceae, phylogeny Pyramimonas australis,rbcL gene, Ross Sea, SSU rDNA gene,
Terra Nova Bay, ultrastructure
Introduction
The genus Pyramimonas Schmarda comprises more
than 50 species, most of them reported from marine
plankton (McFadden et al., 1986; Moestrup & Hill,
1991; Throndsen, 1993 ; Hori et al., 1995). During
the last two decades, new species of Pyramimonas
have been found in many parts of the world and the
literature has rapidly increased (e.g. Hori et al.,
1995; Sym & Pienaar, 1999 ; Daugbjerg, 2000).
Many of the species have been found in polar
waters.
Several Pyramimonas species, belonging to the
subgenera Vestigifera McFadden, Pyramimonas
McFadden and Trichocystis McFadden (P.nansenii
Braarud, P.orientalis McFadden, Hill et
Wetherbee, P.grossii Parke, P.quadrifolia
Daugbjerg, P.aurita Daugbjerg, P.cyclotreta
Daugbjerg, P.cyrtoptera Daugbjerg, P.dichotoma
Daugbjerg, P.igloolikensis Daugbjerg and P.
‘Greenland’ ; see McFadden et al., 1986 ; Daugbjerg
& Moestrup, 1992a,b, 1993; Daugbjerg et al., 1994 ;
Correspondence to: Prof. C. Andreoli. e-mail labandr!civ.
bio.unipd.it
Hori et al., 1995), have been reported from ice pools
in the Arctic. An unusual Pyramimonas sp. bloom
was also found under Arctic pack ice from August
to October 1993 (Gradinger, 1996).
The recorded occurrence of prasinophytes in the
Southern Ocean dates back to Rawlence et al.
(1987), who observed, in late November 1976, a
bloom of Pyramimonas sp. in the surface water of a
tide crack in the permanent ice of the Ross Ice Shelf
at White Island (McMurdo Sound). Subsequently,
McFadden et al. (1982) described Pyramimonas
gelidicola McFadden, Moestrup et Wetherbee, a
new species isolated from samples of sea ice, as well
as from the water of Rookery and Ace Lakes.
This species, probably endemic to Antarctica
(McFadden et al., 1982), was successively found by
several authors (Burch, 1988; Volkman et al., 1988 ;
Van den Hoff & Burton, 1989; Davidson &
Marchant, 1992; McMinn & Hodgson, 1993 ; Bell &
Laybourn-Parry, 1999). A new species of the
subgenus Vestigifera,Pyramimonas tychotreta
Daugbjerg, was described by Daugbjerg (2000) from
the Weddell Sea, from where it had already been
reported as Pyramimonas cf. gorlestonae (Buma et
al., 1992).
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104I.Moro et al.
In addition to these, an abundant, unidentified
species of Pyramimonas has been reported from
several Antarctic sites (He
!doin & Coute
!, 1992;
Ferrario & Sar, 1992; Laybourn-Parry & Marchant,
1992; Vernet, 1992 ; Brandini, 1993 ; Kopczynska et
al., 1995). Bird & Karl (1991) reported a massive
Pyramimonas sp. bloom during the austral spring
1989–90 in the northern Gerlache Strait, which was
unusual for the area because it had trichocysts.
During the austral summer 1998–99 a green algal
bloom consisting of a quadriflagellate species of
Pyramimonas developed in the surface waters of a
hole in the sea-ice of Terra Nova Bay (Andreoli et
al., 2000). Unlike other species of Pyramimonas,
such as P.gelidicola (McFadden et al., 1982), this
microalga could not be cultured in F\2-enriched
seawater medium (Guillard, 1975). Also, unlike the
two other Pyramimonas species already found in the
Southern Ocean, it bears trichocysts. The aims of
the present study were to determine by ultra-
structural and molecular analyses (of SSU rDNA
and rbcL gene sequences) whether it represented a
new species of Pyramimonas. This paper thus
provides an account of the fine structure and the
inferred phylogeny of P.australis sp. nov.
Materials and methods
Samples of surface water were taken with a Niskin bottle
on 11 December 1998 from a hole (2 m in diameter and
1n5 m in depth) in the sea ice of Terra Nova Bay
(74m41hS, 164m07hE). Some unfixed samples were stored
at k80 mC and others were fixed with 3 % glutaraldehyde
and stored at 4 mC. The fixed samples were used,
immediately after their arrival in Italy (5 months later),
for scanning electron microscopy (SEM) and transmis-
sion electron microscopy (TEM). For SEM with a
Cambridge Stereoscan 260 microscope, samples were
dehydrated in a graded series of ethyl alcohols, critical-
point dried and gold-coated. For TEM, cells were washed
three times with 0n1 M cacodylate buffer, postfixed in 1 %
Table 1. List of primers used for amplification of the SSU rDNA gene
Primer Sequence
Nucleotide position
(5h3h)
ALG1 5h-CCTGCCAGTAGTCATACGCT-3hSense j1j20
ALG3 5h-GATTCCGGAGAGGGAGCCTG-3hSense j363 j382
Oligo 3 5h-TTGGATGTGGTAGCCGTCTC-3hAntisense j403 j384
ALG6 5h-CAGAGGTGAAATTCTTGGAT-3hSense j885 j904
ALG5 5h-TGCTTTCGCAGTAGTTCGTC-3hAntisense j932 j913
Py-a1 5h-CCCCTAACTTTCGTTCTTG-3hAntisense j963 j945
Py-a2 5h-AGTATGGTCGCAAGGCTGAA-3hSense j1101 j1120
Oligo 5 5h-CACCCATAGAATCAAGAAAG-3hAntisense j1255 j1236
Oligo 8 5h-TCTGTGATGCCCTTAGATGT-3hSense j1420 j1439
ALG8 5h-AAACCTTGTTACGACTTCAC-3hAntisense j1764 j1744
ALG1, ALG3, ALG5, ALG6, ALG8, and Oligo 3, Oligo 5 and Oligo 8 were taken from Andreoli et al. (1999b) and Andreoli et al. (1999a)
respectively. Py-a1 and Py-a2 were designed from our sequence to allow for complete determination of the double-stranded DNA.
OsO%for 2 h and dehydrated in a graded ethanol series
followed by propylene oxide. Samples were block-stained
with uranyl acetate in the 75% ethanol dehydration
step. Samples were embedded in an Epon-Durcupan
ACM mixture. Thin sections were obtained with a
Reichert Ultracut S, poststained with lead citrate and
examined with a Hitachi HS9 microscope operating at
75 kV.
Genomic DNA was isolated from samples preserved
at k80 mC, using the DNeasy Tissue Kit (Qiagen,
Germany). The small-subunit ribosomal DNA (SSU
rDNA) gene was amplified from DNA extracts by the
polymerase chain reaction (PCR) using the terminal
primers ALG1 and ALG8 (Table 1). Amplification
conditions were : an initial denaturation step of 90 s at
95 mC followed by 45 s at 95 mC (DNA denaturation), 30 s
at 58 mC (annealing) and 90 s at 72 mC (extension for 40
cycles with a final extension step of 10 min at 72 mC. The
rbcL gene was amplified from DNA extracts by PCR
using the terminal primers RH-1S and Ce1161R (Table
2). Amplification conditions were the same as described
above, with 45 s at 72 mC for extension. The PCR
products for both genes were run on 0n8% agarose gels,
bands were excised and the DNA was extracted using the
Jetsorb Gel Extraction Kit (Genomed, Germany). The
PCR products were directly sequenced using the ABI
PRISM Dye Terminator Cycle sequencing Core Kit
(Perkin Elmer), which covered the entire length of the
genes in both directions by using the primers listed in
Tables 1 and 2. Electrophoresis of sequencing reactions
was completed with the ABI PRISM model 377, version
2.1.1 automated sequencer. The sequences of SSU rDNA
and rbcLofPyramimonas australis were aligned with
other available sequences of Pyramimonas. The complete
list of taxa used in the phylogenetic analyses including
outgroup taxa is provided in Table 3, together with
the accession numbers of comparative sequences in
GenBank. Multiple sequence alignments were performed
with the CLUSTALW computer program (Thompson et
al., 1994). Aligned sequences were analysed under the
criterion of maximum parsimony (MP) using the program
PAUP version 3.1.1 (Swofford, 1993) and according to
the neighbor-joining method (NJ) of Saitou & Nei (1987),
as implemented in the TREECON program version 1.3b
(Van de Peer & De Wachter, 1994). Bootstrap (BT)
resamplings (Felsenstein, 1985) were performed to test
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Pyramimonas australis sp.nov.from Antarctica 105
Table 2. List of primers used for amplification of the rbcL gene
Primer Sequence
Nucleotide position
(5h3h)
RH-1S 5h-ATGTCACCACAAACAGAAACT-3hSense j1j21
PRA9 5h-GGACAACAGTATGGACTG-3hSense j197 j214
PRA10 5h-GCCTTGAAACCGAATACG-3hAntisense j386 j369
PRA11 5h-ACAGGTGAAGTTAAGGGT-3hSense j694 j711
Ce800R 5h-TGCATAATAATAGGTACACC-3hAntisense j800 j781
Ce1161R 5h-CATGTGCAATACGTGAATACC-3hAntisense j1161 j1141
RH-1S, Ce1161R and Ce800R were taken from Daugbjerg et al. (1994). The other three primers were designed from our sequence to allow
for complete determination of the double-stranded DNA.
Table 3. Source of SSU rDNA and rbcL sequences analysed in this study
Subgenus Species
18S rRNA
accession no. rbcL accession no.
Pyramimonas McFadden P.cyrtoptera Daugbjerg L34819
P.octopus Moestrup et A. Kristiansen L34817
P.propulsa Moestrup et Hill AB01712 L34777
P.tetrarhynchus Schmarda L34833
Vestigifera McFadden P.cyclotreta Daugbjerg L34814
P.disomata McFadden, Hill et Wetherbee AB017121
P.mantoniae Moestrup et Hill L34810
P.mitra Moestrup et Hill L34812
P.moestrupii McFadden L34811
P.orientalis McFadden, Hill et Wetherbee L34813
P.tychotreta Daugbjerg L34778
P.‘Greenland’ (inedit) L34818
Trichocystis McFadden P.australis Andreoli et Moro AJ404886 AJ404887
P.cirolanae Pennick L34776
P.grossii Parke L34779
P.parkeae Norris et Pearson AB017124 L348164
Punctatae McFadden P.olivacea N. Carter AB017122 L348152
Pyramimonas formosa Sym et Pienaar L34834
Outgroup Cymbomonas tetramitiformis Schiller AB017126 L346876
Halosphaera sp. AB017125
Mamiella sp. AB017129 U302779
Mantoniella antarctica Marchant AB017128
Mantoniella squamata (Manton et Parke) Desikachary X73999 U30278
Micromonas pusilla (Butcher) Manton & Parke AJ010408 U30276
Nephroselmis minuta (N. Carter) Butcher U30286
Nephroselmis olivacea Stein X74754 U30285
Pterosperma cristatum Schiller AB017127 U302817
The SSU rDNA and rbcL gene sequences of Pyramimonas australis were determined in this study. The references for SSU rDNA and rbcL
of the other species were Nakayama et al. (1998) and Daugbjerg et al. (1994, 1995).
the robustness of clades. In all analyses 1000 replicates
were done. Maximum parsimony phylogenetic recon-
structions were performed only on informative
characters. A branch-and-bound and a heuristic ap-
proach were used for SSU rDNA and rbcL sequences
respectively.
Results
Pyramimonas australis Andreoli et Moro, sp. nov.
D : Cellulae conservatae 8–10 µm longae,
5–6 µm latae, longe ovatae, fauce profunda 4-
lobata. Flagella quattuor, cellulam subaequantia, in
fovea apicali inserta. Chloroplastus viridis, in lobis
quattuor profunde divisus. Pyrenoides basalis,
amylo circumcincta, 5–6 thylacoidibus parallelis
peragrata. Stigma singulum in chloroplasti lobo
prope pyrenoidem locatum, ex 4 seriebus
guttularum carotenoidearum constans, nullis thyla-
coidibus separatum. Trichocystes circa foveam
flagellorum et in sulcis longitudinalibus. Cellulae
corpus tribus squamarum stratis tectum. Stratum
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106I.Moro et al.
Figs 1, 2. Scanning electron micrographs of P.australis
cells. Fig. 1. Cell in lateral view with box scales covering
the cell body (arrows). Fig. 2. Cell in late division. Note
the longitudinal cleavage furrow (arrow).
internum ex squamis parvis quadratis constans,
iuxta plasmalemma locatum; medium ex squamis
quadratis, 16 transtra in 4 quadratis seiunctis
ostendentibus disposita; externum ex squamis
coronatis, ad foveam flagellorem limitatum.
Flagella tecta squamis parvis subiectisque penta-
gonisque, superpositis squamis limuliformibus.
Squamae per microscopium electronicum tantum
visibiles. Cellulae efferentes cystas pariete tenue
squamis spinosis instructas. Species ordine in atomo
genetico dicto ‘SSU rDNA ’ et ‘ rbcL ’ a ceteris
eiusdem generis differt.
D : Fixed cells, 8–10 µm long and 5–6 µm
wide, have an elongated oval shape. The apical part
has four rounded lobes and the antapical end is
conical and rounded. Four flagella, more or less the
length of the cell, emerge from an apical depression.
The green chloroplast is deeply divided into four
lobes. The basal part of the chloroplast houses a
central pyrenoid, surrounded by two to three dome-
shaped starch grains and traversed by 5 or 6 parallel
thylakoids that have a lateral orientation relative to
the cell axis. The single posterio-lateral eyespot is
situated near the pyrenoid and consists of four rows
of carotenoid droplets, which are not separated by
thylakoids. Trichocysts are present around the
flagellar pit and in the longitudinal sinuses. Three
layers of scales cover the cell body: an inner layer of
small underlayer scales situated next to the plasma-
lemma; an intermediate layer of box scales, with 16
bars forming four separated squares; and crown
scales in the flagellar pit. The flagella are covered by
an underlayer of small pentagonal and limuloid
scales with two subsidiary spines, in addition to the
central one. The scales are visible only with the
electron microscope. The cells produce cysts with
thin and irregular envelopes covered by spine scales.
SSU rDNA and rbcL gene sequences were different
from those of other species of this genus.
E : The specific epithet refers to the
Southern Hemisphere.
H : Fig. 12.
H : The type material was collected from
surface waters of a hole in the sea ice of Terra Nova
Bay, Ross Sea, Antarctica (74m41hS, 164m07hE), in
December 1998. Salinity and temperature were
24n7 psu and k1n5mC, respectively.
Electron microscopy. SEM observations revealed
elongated oval cells with four flagella that are as
long as the cell, and emerge from a flagellar pit.
Longitudinal ridges are formed by the chloroplast
lobes, and scales cover the cell body (Fig. 1). Only
one cell type was observed. Asexual reproduction
occurs by longitudinal division from the posterior
pole to the anterior pole of the flagellate cell (Fig. 2).
The internal organization of the cell of P.australis
(Figs 3–10) is typical of most quadriflagellate species
of this genus. The large nucleus is located laterally,
on the side opposite to the vacuole (Figs 3, 4) near
the microbody and two of the chloroplast lobes, one
of which contains the eyespot (Fig. 4). The nucleus
contains a spherical nucleolus and heterochromatin,
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Pyramimonas australis sp.nov.from Antarctica 107
Figs 3–7. Transmission electron micrographs of P.australis cells. Fig. 3. Longitudinal section of a cell showing the nucleus
(n), vacuole (v), two dictyosomes of the Golgi complex (g), the chloroplast with starch (s) and the pyrenoid (py) traversed
by thylakoids. Fig. 4. Transverse section through the nucleus, microbody (arrow), vacuole (v) and four lobes of the
chloroplast (ch). Note the eyespot in one of the lobes of the chloroplast (double arrow). n, nucleus. Fig. 5. Location of
some trichocysts (t) around the flagellar pit. Fig. 6. Some trichocysts (t) in the basal portion of the cell. Fig. 7. Longitudinal
section through the eyespot showing four layers of lipid droplets. Note the underlayer scales (arrow) covering the cell body.
with the latter usually found close to the nuclear
envelope (Figs 3, 4). The single chloroplast is incised
to form four lobes and the large basal portion
houses a posterior pyrenoid (Figs 3, 4). The
pyrenoid is surrounded by two or three dome-
shaped starch grains and is traversed by five or six
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108I.Moro et al.
Figs 8–10. Transmission electron micrographs of P.australis cells. Fig. 8. Transverse section through the dictyosomes (g)
showing vesicles containing underlayer (arrow) and flat box (double arrow) scales, and flagellar configuration with the
synistosome (sy) located between basal bodies 1 and 2. Fig. 9. Detail of the scale reservoir (sr) directed towards the flagellar
pit and containing scales. Note the limuloid scales (arrows). Fig. 10. Longitudinal section through the anterior end showing
the transitional region (arrow), the rhizoplast (r) and crown scales (double arrow) around the flagellar pit.
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Pyramimonas australis sp.nov.from Antarctica 109
parallel thylakoids (Figs 3, 6). The chloroplast
contains a large amount of starch and has a posterio-
lateral eyespot (Figs 4, 7). The eyespot is composed
of one to four rows of osmiophilic globules that are
not separated by thylakoids (Fig. 7), and the number
of layers is thought to be due to the plane of section
through the eyespot. Numerous trichocysts are
visible in the cells around the flagellar pit and in the
chloroplast sinuses (Figs 5, 6). The Golgi complex,
involved in scale production, consists of opposite
dictyosomes near the basal bodies and flagellar pit,
in the region not occupied by the vacuole–
microbody–nucleus complex (Figs 3, 8). The scale
reservoir opens into the flagellar pit that contains
small body scales and flagellar scales (Figs 8, 9).
A flattened, longitudinal rhizoplast runs along-
side the microbody, separating it from the nucleus
(Fig. 10). Oblique transverse sections (Fig. 8) show
a flagellar configuration with the synistosome
located between basal bodies 1 and 2 (Moestrup &
Hori, 1989).
Scale morphology. The cell body is covered by three
kinds of scales forming inner, middle and outer
scale layers. Inner layer body scales (Figs 7, 11) are
underlayer scales similar to those seen in P.grossii
and P.cirolanae (Sym & Pienaar, 1993b). The
middle layer scales are flat box scales (Fig. 8), each
with eight peripheral perforations oriented parallel
to the four edges, and with a further eight central
perforations oriented perpendicular to the periph-
eral ones. All 16 perforations collectively form four
indistinct square subunits in the larger scale (Fig.
12). Crown scales similar to those of P.disomata
(McFadden et al., 1986) were observed near the
flagellar pit (Figs 5, 10, 13). However, we cannot
exclude the possibility that their absence in other
parts of the cell body is due to the direct fixation of
wild material. Footprint scales were not detected.
Inner and outer layer scales occur on the flagella.
The inner layer is formed by pentagonal underlayer
scales that have a conspicuous central knob (Fig.
13) and is covered by limuloid scales, each with two
subsidiary spines and a central spine (Fig. 9).
Cysts. Fixed samples also included encystment
stages and cysts. The encystment process was not
observed; however, it appears that it begins in the
flagellate cells with the production of a vesicle,
characterized by a verrucose membrane with spine
scales (Fig. 14). Subsequently, this vesicle increases
in size and then fuses with the plasmalemma,
releasing its contents to the outside, and initiating
the cyst (Figs 15, 16).
The cyst has a thin envelope, with an irregular
edge, covered by spine scales with stellate tips (Fig.
17). Mature cysts contain numerous lipid droplets, a
Figs 11–13. Transmission electron micrographs showing
details of the cell body and flagellar scales. Fig. 11.
Underlayer scales in frontal section. Fig. 12. HOLOTYPE.
Frontal view of box scale with typical perforations. Fig.
13. Longitudinal section through the flagellar pit showing
the crown scales (arrows) and flagellar underlayer scales
with central knob (double arrow).
nucleus and a chloroplast with pyrenoid and a large
amount of starch. The pyrenoid has more than two
starch grains (Fig. 17).
Phylogenetic analyses. Single trees were constructed
for cladistic and distance-based analyses for SSU
rDNA and rbcL gene sequences (Figs 18, 19).
Phylogenetic reconstructions were based on align-
ments of 1731 and 1099 positions for the SSU
rDNA and rbcL gene fragments respectively.
The phylogenies based on SSU rDNA favoured a
close relationship between Pterosperma cristatum
and members of the genus Pyramimonas (Fig. 18) as
previously reported by Nakayama et al. (1998).
However, the maximum parsimony (MP) clado-
gram showed Pyramimonas to be paraphyletic with
no significant bootstrap (BT) support. Conversely
the neighbor-joining (NJ) tree showed Pyramimonas
as monophyletic but with marginal BT support
(51%). There was strong support for the subgenus
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110I.Moro et al.
Figs 14–17. Progressive stages in cyst formation (Figs 14–16) and the mature cyst (Fig. 17). Note the mature cyst with spine
scales characterized by starry tips (in the insert, longitudinal view i29000 and cross-section through tip i19000). cv, cyst
vesicle; l, lipids; n, nucleus ; py, pyrenoid ; s, starch.
Trichocystis (P.australis and P.parkeae) (BT values
99% and 100 % in NJ and MP trees respectively).
Pyramimonas propulsa,P.olivacea and P.disomata
were differently grouped in the MP tree and in the
NJ tree.
The phylogenetic reconstructions based on rbcL
sequences showed Pyramimonas as monophyletic
and supported by moderately high BT values (Fig.
19). Cymbomonas tetramitiformis formed the sister
group to Pyramimonas as previously suggested
(Daugbjerg et al., 1994, 1995). The subgenera
Trichocystis,Vestigifera and Pyramimonas were
each monophyletic in both analyses, although not
always supported by high BT values. The subgenus
Punctatae McFadden, represented here by Pyrami-
monas formosa Sym et Pienaar and P.olivacea, did
not appear as monophyletic in the MP and NJ trees.
The positioning of Pyramimonas formosa was, in
fact, controversial, being linked to the subgenus
Trichocystis in the NJ tree but as a sister group to
the clade containing Punctatae McFadden and
Pyramimonas in the MP tree. Neither topology
received support from BT values.
Pyramimonas australis sp. nov. belonged to the
subgenus Trichocystis, as a sister group to the clade
P.grossii and P.cirolanae. Finally, the more basal
nodes of the ingroup in both the MP and the NJ
trees were poorly supported by BT, making res-
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Pyramimonas australis sp.nov.from Antarctica 111
Fig. 18. Phylogeny of the genus Pyramimonas based on SSU rDNA sequences. (A) Maximum parsimony (MP) method.
Statistical analyses of the most parsimonious cladogram : Tree length l381 steps; Consistency Index l0n66; Retention
Index l0n73 ; Rescaled Consistency Index l0n48. (B) Neighbor-joining (NJ) method. Numbers above branches are
bootstrap values expressed as the percentage after 1000 replicates; only values 50 % are reported.
Fig. 19. Phylogeny of the genus Pyramimonas based on rbcL gene sequences. (A) Maximum parsimony (MP) method.
Statistical analyses of the most parsimonious cladogram : Tree length l1382 steps; Consistency Index l0n38; Retention
Index l0n47 ; Rescaled Consistency Index l0n18. (B) Neighbor-joining (NJ) method. Numbers above branches refer to the
bootstrap values expressed as the percentage after 1000 replicates; only values 50 % are reported.
olution of the phylogenetic relationships between
the various subgenera of Pyramimonas weak.
Discussion
The inability to maintain P.australis in culture and
the limited amount of available material prevented
us from obtaining a more detailed characterization
of this organism (i.e. the configuration of the
flagellar apparatus, the distribution and the mor-
phology of the crown scales and the absence\
presence of hair scales on the flagellar surface).
However, our results are sufficient to classify this
microalga as a new species of the subgenus
Trichocystis, adding it to the other seven species
reported by Hori et al. (1995). This is due, above all,
to the successful DNA amplification from wild
samples of P.australis. This result, in addition to
those of Moon-van der Staay et al. (2000) and
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112I.Moro et al.
Table 4. Occurrence of some cytological characteristics in Pyramimonas australis and species belonging to the subgroups 1
and 2 of subgenus Trichocystis (sensu Sym & Pienaar, 1993b)
Features considered P.australis
Trichocystis
Subgroup 1 Subgroup 2
Many trichocysts jk j
Body underlayer scales without central knob jj k
Box scales without side walls jj k
Limuloid scales with two ancillary posteriorly directed spines jj k
Scale reservoir simple jj k
Chloroplast incised jj j
Eyespot multilayered jk j
Pyrenoid surrounded by two or three starch grains jj j
Cyst present jj j
j, feature present; k, feature not present.
Edvardsen et al. (2000), confirms that molecular
data from natural samples can be utilized.
The Trichocystis subgenus, characterized by the
presence of trichocysts, has been considered het-
erogeneous, leading Sym & Pienaar (1993b)to
suggest that two subgroups exist. As with P.
oltmannsii Schiller (Zingone et al., 1995), ultra-
structural characters of P.australis do not conform
to those of either of these subgroups (Table 4).
Moreover, in the trees inferred from the rbcL gene
(Figs 18, 19), P.australis is interpolated between the
representatives of these two subgroups. As a conse-
quence the molecular data currently available can
neither resolve the positioning of P.australis relative
to the two subgroups nor be used to evaluate the
validity of the subgroups.
Ultrastructural data on P.australis show it to be
morphologically and structurally similar to other
quadriflagellate species of Pyramimonas. The main
difference, apart from the distinctive box scales, is
the cyst scales.
Unlike the other seven species of the subgenus
Trichocystis, but like P.gelidicola (Van den Hoff &
Burton, 1989), P.australis produces cyst scales,
which are morphologically different from the five
scale types of the motile cells. These scales are spine-
like and have stellate tips and are deposited in a
vesicle (cyst vesicle) that coalesces with the cell
membrane, being released from the vesicle by
reverse pinocytosis, though on a grander scale than
reported by Melkonian et al. (1986) and Van den
Hoff & Burton (1989). The synthesis of cyst scales
probably occurs in the Golgi cisternae (Moestrup &
Walne, 1979) as is the case for flagellar and body
scales.
P.australis represents the third species of
Pyramimonas known to produce cyst scales (Van
den Hoff & Burton, 1989; Daugbjerg et al., 2000),
but this is a novelty in the subgenus Trichocystis.
Cysts have been reported in many Pyramimonas
species (Sym & Pienaar, 1993a,b; Daugbjerg, 2000)
but the process of cyst formation in P.australis is
different from those already described in P.
amylifera Conrad (Hargraves & Gardiner, 1980),
P.gelidicola (Van den Hoff & Burton, 1989) and P.
pseudoparkeae Pienaar et Aken (Pienaar & Aken,
1985). To date, the three Antarctic species of this
genus have been found to produce cysts (Van den
Hoff & Burton, 1989; Daugbjerg, 2000 ; this study).
The MP phylogenetic reconstruction, based on
SSU rDNA, failed to recognize the genus
Pyramimonas as monophyletic. It must be noted,
however, that most of the nodes within the clades
containing Pyramimonas and Pterosperma are not
supported by BT values; moreover less than 10 % of
the positions in the alignment proved informative
for cladistic analysis due to the slow rate of
evolution of the region under consideration. As a
consequence it was not possible to solve the phylo-
genetic relationship within the clades containing
Pyramimonas and Pterosperma as previously found
by Nakayama et al. (1998). In both SSU rDNA
analyses, Cymbomomas tetramitiformis formed a
sister group to Halosphaera sp. rather than to the
genus Pyramimonas, as suggested by rbcL gene
analyses (Daugbjerg et al., 1994). As a consequence,
the identity of the sister group to Pyramimonas
remains controversial. All the analyses to date
(Daugbjerg et al., 1994; Nakayama et al., 1998 ; this
study) show good BT support for both competing
hypotheses. Present phylogenetic analyses based on
rbcL agree with a previous analysis using the same
data but a different approach (maximum likelihood :
Daugbjerg et al., 1994). The genus Pyramimonas
is monophyletic, as are the subgenera Vestigifera
and Pyramimonas, all with very high BT values.
The monophyly of Trichocystis is also well sup-
ported although the BT values are lower than those
of the previous clades. However, the phylogenetic
relationships between the different subgenera of
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Pyramimonas australis sp.nov.from Antarctica 113
Pyramimonas are far from resolved, with the more
basal nodes being poorly supported by BT values
in both MP and NJ trees.
An aim of future expeditions to Antarctica is to
isolate and culture P.australis to clarify its life cycle
and the morphology of the crown scales, to provide
further evidence for the assumption based on limited
current observations that the process of cyst form-
ation is novel in this species.
Acknowledgements
The authors wish to thank Dr Maria Chiara
Chiantore (Istituto di Scienze Ambientali, Uni-
versity of Genoa, Italy) for finding the bloom
of Pyramimonas australis during the 1998–9
expedition. Prof. E. Nardi (Department of Plant
Biology, University of Florence, Italy) and Prof. E.
Pianezzola (Department of Antiquity Science, Uni-
versity of Padua, Italy) very kindly provided the
Latin diagnoses. The authors acknowledge the
financial assistance of the Italian National Pro-
gramme of Antarctic Research (PNRA). The manu-
script was improved following comments from two
anonymous referees.
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