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INTRODUCTION
Porphyra is a red algal genus that comprises over 130
species, many of which form a major component of rocky
intertidal habitats (Yoshida 1997). Representatives of this
genus are cosmopolitan, spanning temperate and cold
water systems, with particularly high abundance record-
ed in the North Pacific Ocean (Mumford and Cole 1977).
Whilst most Porphyra species are thought to be regionally
confined, distribution of these species has not been thor-
oughly established (e.g. Nelson et al. 2001, 2003; Broom et
al. 2004).
The commercial importance of Porphyra has generated
much interest in its life history and systematics. Research
has focused on the effects of environmental factors such
as photoperiod, light intensity, temperature and nutri-
ents, on the morphological and reproductive develop-
ment of the species in culture (e.g. Frazer and Brown
1995; Nelson and Knight 1996; Kim and Notoya 1997;
Knight and Nelson 1999; Ruangchuay and Notoya 2003;
Niwa et al. 2004). The establishment of optimal develop-
mental conditions for Porphyra species has not only
expanded our knowledge on how to enhance cultivar
yields, but has provided a means of assessing the
impacts of harvesting wild Porphyra populations (Schiel
and Nelson 1990). Furthermore, these studies have
improved our understanding of the diversity within this
genus.
Species of Porphyra are typically recognized by a
biphasic life cycle in which there is an alternation
between a macroscopic, gametophytic leafy blade phase
and a microscopic, sporophytic filamentous phase called
the ‘conchocelis’ (Drew 1949; Kurogi 1972; Nelson et al.
2000). It is, however, well established that many varia-
tions in the life history of Porphyra species exist. Conway
and Wylie (1972) described P. subtumens (now
Pyrophyllon subtumens (J. Agardh ex R.M. Laing) W.A.
Nelson) as an asexual species, lacking the alternation of
the diploid conchocelis phase. Similarly, populations of
P. sanjuanensis V. Krishnamurthy lack the ability to sexu-
ally reproduce (Lindstrom and Cole 1990).
Algae
Volume 21(2): 1-10, 2006
Biology of Porphyra pulchella sp. nov. from
Australia and New Zealand
Jillian C. Ackland1, John A. West1, Joseph Scott2,
Giuseppe C. Zuccarello3and Judy Broom4
1School of Botany, University of Melbourne, Parkville, Victoria 3010, Australia
2Department of Biology, PO Box 8795, College of William and Mary, Williamsburg, VA 23187 USA
3School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, 6001 New Zealand
4Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand
Porphyra pulchella sp. nov. Ackland, West, Scott and Zuccarello was obtained at Mimosa Rock National Park, New
South Wales; Westgate Bridge, Victoria, Australia; and Waihau Bay, North Island, New Zealand. It occurs mainly in
mangrove habitats and is very small (± 1 mm) in field collections. In laboratory culture at 21 ± 2°C tiny blades (0.5-
3.0 mm) reproduced exclusively by archeospores liberated from vegetative cells of the upper sector of the blades.
The archeospores displayed amoeboid and gliding motility once discharged. At 14 ± 2°C the blades grew to 25 mm
and produced longitudinal spermatangial streaks mixed with ‘phyllosporangial’ streaks. The discharged ‘phyl-
lospores’ showed amoeboid motility and germinated forming asexual blades. A conchocelis phase with typical ban-
giophycidean pit connections was observed in blade cultures after 8-10 weeks at 14 ± 2°C. Conchocelis filaments
produced conchosporangia and these released amoeboid conchospores that developed into archeosporangiate
blades. Molecular data indicate that all 3 isolates are genetically identical.
Key Words: Australia, molecular phylogeny, New Zealand, Porphyra pulchella sp. nov. SSU rDNA, TEM
*Corresponding author (jwest@unimelb.edu.au)
Figure 23만컬러
Despite the current knowledge on Porphyra life histo-
ries, the taxonomy of this genus remains problematic.
Porphyra species are morphologically simple and subse-
quently have few morphological characters for species
identification (Kunimoto et al. 1999; Broom et al. 2002).
Moreover, morphological plasticity caused by environ-
mental change can hamper the identification of species
limits (Conway and Wylie 1972; Hannach and Waaland
1989). Species discrimination based on morphological
analysis is a traditional technique now thought to be
unreliable when used independently (Kunimoto et al.
1999; Nelson et al. 2003). Molecular techniques are accu-
rate indicators of phylogenetic diversity now commonly
used in combination with morphological data to identify
taxa (Broom et al. 1999; Broom et al. 2002; Kunimoto et al.
1999; Nelson et al. 2001; Nelson et al. 2003). These studies
have revealed that similarities in morphology do not nec-
essarily infer close relationships, yet morphological vari-
ation is not always an indicator of phylogenetic diver-
gence.
Species identifications based on nuclear SSU (18S)
rDNA have unveiled a greater diversity of Porphyra than
was previously recognised using morphological criteria
alone (Broom et al. 1999; Nelson et al. 1998, 2001, 2003;
Oliveira and Ragan 1994; Oliveira et al. 1995). The SSU
rDNA locus is a slowly evolving gene (Hillis and Dixon
1991) that has been used to infer systematic relationships
of red algal specimens at an ordinal and familial level
(Saunders and Kraft 1993; Bailey and Freshwater 1997;
Ragan et al. 1994). Whilst it is too conservative to be used
to distinguish organisms at an interspecies level, the final
third (3( end) of the SSU rDNA locus in Porphyra com-
prises variable domains which differ amongst species.
These regions have proved useful for the discrimination
of Porphyra entities at a species level (Broom et al. 1999;
Kunimoto et al. 1999). Moreover, small subunit rDNA
sequencing analyses have been used to reassess the taxo-
nomic status of Porphyra species previously identified on
the basis of morphological characters. Nelson et al. (2003)
used this technique to establish that Pyrophyllon subtu-
mens, P. cameronii (W.A. Nelson) W.A. Nelson and
Chlidophyllon kaspar (W.A. Nelson and N.M. Adams)
W.A. Nelson, originally placed in the genus Porphyra
(order Bangiales), are members of the order
Erythropeltidales. The results of this study signify that
the morphological and anatomical characters originally
used to identify these Porphyra species are a result of evo-
lutionary convergence.
In this paper, we have described a new species of
Porphyra (P. pulchella) based on morphology and life his-
tory in culture, TEM of the conchocelis phase and molec-
ular analysis of the nuclear SSU rDNA locus. Two
Australian populations of P. pulchella (isolates 3924 and
4422) were morphologically and genetically contrasted to
a morphotype from New Zealand and previously
described Porphyra entities, to confirm the taxonomic sta-
tus of this species. The Australian isolates are especially
distinct from most other Porphyra species because of the
very small blade size (1-2 mm) seen in field collections
and the unusual habitat, i.e., mangroves of temperate
coastlines.
MATERIALS AND METHODS
Field collections
Foliose thalli (1-2 mm) of Porphyra (holotype isolate
3923) were collected on 16th December, 1998 from pneu-
matophores of the mangrove Avicennia marina (Forsk.)
Vierh. at Nelson’s Lagoon, Mimosa Rock National Park,
New South Wales, Australia (36°41’ S, 149°59°E). A sec-
ond collection (paratype isolate 4422) was made 6th April
2004 from a band of Caloglossa vieillardii (Kützing)
Setchell on wood pilings at Westgate Bridge, Port Phillip
Bay, Victoria, Australia (37°49’S, 144°53’E). A morpho-
type also examined, was collected 1st August 2003 from
Waihau Bay east, North Island, New Zealand (37°37’S,
177°55’E). Terminology for reproductive structures and
life history phases of Porphyra is in accordance with
Nelson et al. (1999).
Culturing the blade phase
Thalli were given two mild osmotic shocks to kill vari-
ous colourless flagellates. Blades were placed in a 60 ×15
mm Petri dish containing deionised water for 10 seconds,
then transferred to Modified Provasoli’s Medium
[MPM/2 (West 2005), 10 ml of enrichment per litre of
sterilised 30 psu natural seawater] for one minute, before
receiving a second osmotic shock for ten seconds. Blades
were then incubated in a 60 ×15 mm Petri dish contain-
ing MPM/2 treated with 25
µ
g/ml antibacterial
ciprofloxacin hydrochloride (ciprofloxacin, Sigma
Chemical Co. Pty. Ltd, St Louis, MO) for 48 hours to
reduce bacterial contamination. Petri dishes were main-
tained in a controlled environment culture room at 21 ±
2°C, 12:12 LD photoperiod and 10-20
µ
mol photons m–2
s–1 of cool white fluorescent lighting. Clean blades were
transferred into PyrexTM (#3250, Corning Glass Works,
Corning, NY, USA) 500 ml storage dishes and 60 ×15
2 Algae Vol. 21(2), 2006
mm Petri dishes containing 250 ml and 10 ml of medium,
respectively, and maintained in the above conditions.
Archeospore discharge was induced by incubating
blades with archeosporangia in fresh medium.
Archeospores were pipetted into 500 ml storage dish-
es, 60 ×15 mm Petri dishes and 10 ml plastic six-welled
plates (Iwaki SciTech Div., Asahi Techno Glass,
Funabashi, Japan) containing MPM/2 medium. A cover-
slip (22 ×22 mm or 22 ×50 mm, #1) was placed in each
dish as a substratum for archeospore settlement.
Dishes were maintained in a controlled environment
culture room or controlled environment E-36L plant
growth chambers (Percival Scientific Inc., Perry, Iowa,
USA) set to the desired photoperiod and temperature.
Desired irradiance levels were obtained by covering fluo-
rescent lamps with dark plastic window screen (1mm
mesh) and using cardboard boxes to elevate dishes closer
to the light source. Dishes were placed on New
BrunswickTM Model G2 rotary shakers (New Brunswick
Scientific Co., New Brunswick, New Jersey, USA) set at
approximately 80 rpm.
Culturing the conchocelis phase
Conchocelis filament tufts were treated in the same
way as blades to eliminate colourless flagellates and
reduce bacterial contamination, and cultured in the same
light and temperature regime.
Sterilised mollusc shell pieces (area 1.0-1.5 cm2, thick-
ness 0.5-1.0 mm) were inoculated with five conchocelis
filament tufts (0.2-0.5 mm in diameter) and placed in a 60
×15 mm Petri dish containing MPM/2. Conchocelis fila-
ments were secured to shells using a coverslip (22 ×22
mm, #3) and 2mm glass rods. The coverslip and glass
rods were removed after conchocelis filaments attached
to the shell pieces.
All Petri dishes were sealed with parafilm to minimise
evaporation. The medium was renewed every 3-10 days.
Blades and conchocelis filaments were examined using a
Zeiss GFL bright field compound microscope and a Zeiss
dissecting microscope with a Zeiss LCD 1500 fiber optic
light source. Photomicrographs were taken with a Zeiss
MC 100 35 mm camera using Ektachrome 200 colour
film. The transparencies were scanned with an Epson
FilmScan 200 using Photoshop 5.0 software on a
Macintosh G4 computer.
Transmission electron microscopy (TEM)
Conchocelis filaments were removed from culture dish
bottoms and fixed for 90 minutes at ambient temperature
in 2.5% glutaraldehyde or 1.5% paraformaldehyde in a
0.1 M phosphate buffer solution (pH 6.8) with 0.25 M
sucrose. Following buffer rinses, samples were post-
fixed in the same buffer for 90 min in 1% OsO4at ambi-
ent temperature, rinsed thoroughly in distilled H2O, left
in 50% acetone for 30 minutes and stored in a 70% ace-
tone-2% uranyl acetate solution at ambient temperature
for 4 hours. Samples were then dehydrated in a graded
acetone series, infiltrated and embedded in EmBed 812
(Electron Microscopy Sciences, P.O. Box 251, 321 Morris
Rd., Fort Washington, PA 19034) and polymerized at
70°C for 3 days. Thin sections were cut with an RMC
MT6000-XL ultramicrotome, stained with lead acetate
and viewed with a Zeiss EM 109 electron microscope.
Molecular phylogeny
DNA extraction followed a modified Chelex extraction
method (Zuccarello et al. 1999). Amplification of an
approximately 900 bp region of the nuclear-encoded
small subunit of ribosomal RNA (SSU), corresponding to
the final third of the molecule (Saunders and Kraft
1994;Broom et al. 1999) followed the procedure in Broom
et al. (1999). All PCR products were electrophoresed in 1-
2% agarose to check product size and sequenced follow-
ing procedures in Zuccarello et al. (1999).
Sequences were assembled using the computer soft-
ware supplied with the ABI sequencer, and aligned with
Clustal X (Thompson et al. 1997). All sequences were
compiled in Se-Al version2a11 (Rambaut 1996).
Phylogenetic relationships were inferred with PAUP*
4.0b10 (Swofford 2002). Outgroups and related sequences
used were selected from GenBank deposits and the
accession numbers are indicated in Fig. 26. Outgroups
used were Erythrocladia sp., Erythrotrichia carnea
(Dillwyn) J. Agardh and Smithora naiadum (C.L.
Anderson) G.J. Hollenberg.
Maximum-parsimony trees (MP) were constructed in
PAUP*, using the heuristic search option, 500 random
sequence additions, TBR branch swapping, unordered
and unweighted characters, gaps treated as missing data.
The program Modeltest version 3.6 (Posada and
Crandall, 1998) was used to find the model of sequence
evolution that best fits each data set by a hierarchical
likelihood ratio test (
α
= 0.01) (Posada and Crandall,
2001). When the best sequence evolution model had been
determined, maximum-likelihood was performed in
PAUP* using the estimated parameters (substitution
model, gamma distribution, proportion of invariable
sites) (5 random additions).
Ackland et al.: Biology of Porphyra pulchella sp. nov. 3
Support for individual internal branches was deter-
mined by bootstrap analysis (Felsenstein 1985), as imple-
mented in PAUP*. For MP bootstrap analysis, 1000 boot-
strap data sets were generated from resampled data (5
random sequence additions), for ML bootstrap analysis
100 bootstrap data sets were generated (1 random
sequence addition).
RESULTS
All observations were made on specimens (isolates
3923 and 4422) grown in unialgal culture.
Porphyra pulchella J.C. Ackland, J.A. West, J. Scott and
G.C. Zuccarello sp. nov.
Description: Laminae roseo-rubrae, stipiti carentes,
rotundae vel ovatae (0.5-3 mm ad 21 ± 2°C) aut lineari-
lanceolatae (10-25 mm ad 14 ± 2°C), in maturitate mar-
ginibus modice undulatis praebentibus thallis aspectum
leviter undulatum; laminae monostromaticae 19-21
µ
m
crassae. Divisiones cellularum diffusae marginales.
Cellulae marginales uni- vel biseriatae, quadratae (20-24
µ
m) vel elongatae, 20-24
µ
m latae, 40-48
µ
m longae; cel-
lulae centrales vegetativae demum irregulariter disposi-
tae, polygonales (19-24
µ
m); cellulae basales irregulariter
dispositae, polygonales et demum elongatae, cellulis cen-
tralibus vegetativis plerumque majores, chloroplastis sin-
gulis stellatis pyrenoide unica. Archaeosporangia secus
marginem superiorem laminae prodientia, regulariter
disposita, aliquantum quadrata (19-22
µ
m). Archaeosporae
liberatae 10-14
µ
m in diametro, motum amoeboideum et
prolabentem exhibentes. Planta monoica; spermatangia
strias conspicuas elongatas pallidas secus laminam supe-
riorem se praebentia cum striis phyllosporangiorum
putatorum mixtas; 32 divisione [a/4, b/4, c/2] facta, 4-6
µ
m in diametro, dimissa immota. Phyllosporangia
obscure roseo-rubrae, cellulis spermatangialibus majora,
singula vel bina terna quaternave aggregata; phyllospo-
rae 14-16
µ
m in diametro, motum amoeboideum
exhibentes.
Conchocelis, praebens se caespitem densum e filamen-
tis tenuibus ramosis uniseriatis constantem, includere se
in conchas potest. Divisiones cellularum intercalares.
Cellulae breves vel elongatae, 9-12
µ
m latae 32-40
µ
m
longae, conjunctionibus primariis fovearum junctae;
chloroplasti singuli elongati torti parietales pyrenoide
unica vel pluribus. Conchosporangia obscure roseo-
rubra, filamenta uniseriata et fasciculos 2-12-cellulares
facta, a superficie visa isodiametra, brevia vel elongata,
10-27
µ
m lata 7-27
µ
m longa. Conchosporae liberatae 10-
14
µ
m in diametro, motum amoeboideum exhibentes.
Blades pinkish-red, lacking stipe, round to ovate (0.5-3
mm at 21 ± 2°C) or linear-lanceolate (10-25 mm at 14 ±
2°C). Margins of mature blades moderately undulate giv-
ing thalli a slightly ruffled appearance. Blades monostro-
matic, 19-21 (m thick. Cell divisions diffuse and margin-
al. Marginal cells in one or two rows, quadrate (20-24
µ
m) to elongate, 20-24
µ
m wide and 40-48
µ
m long; cen-
tral vegetative cells becoming irregularly arranged,
polygonal (19-24
µ
m); basal cells irregularly arranged,
polygonal and becoming elongate, mostly larger than
central vegetative cells, chloroplasts single and stellate
with one pyrenoid. Archeosporangia along the upper
blade margin, regularly arranged, relatively quadrate
(19-22
µ
m). Liberated archeospores 10-14
µ
m in diame-
ter, exhibiting amoeboid and gliding motility.
Monoecious; spermatangia conspicuous, elongate, pale
streaks along the upper blade, intermixed with streaks of
presumed phyllosporangia; 32 spermatangia formed by
division [a/4, b/4, c/2], 4-6
µ
m in diameter, immotile
when discharged. Phyllosporangia dark pinkish-red,
larger than spermatangial cells, single or divided into
groups of 2-4; phyllospores 14-16
µ
m in diameter and
exhibiting amoeboid motion. Conchocelis pinkish-red
dense tuft of fine, branched, uniseriate filaments with the
ability to embed in mollusc shells. Cell divisions inter-
calary. Cells short to elongate, 9-12
µ
m wide and 32-40
µ
m long, connected by primary pit connections; chloro-
plast single, elongate, twisted and parietal with one or
more pyrenoids. Conchosporangia dark pinkish-red,
formed as uniseriate filaments and in clusters of 2-12
cells, isodiametric in surface view, short to elongate, 10-
27
µ
m wide and 7-27
µ
m long. Liberated conchospores
10-14
µ
m in diameter, exhibiting amoeboid motion.
Holotype specimen: Blades (NSW 722255), conchocelis
(NSW722445), Royal Botanic Gardens, Mrs. Macquaries
Road, Sydney, NSW 2000, Australia. Collection data: 16
xii 1998, on pneumatophores of the mangrove Avicennia
marina at Nelson’s Lagoon, Mimosa Rock National Park,
New South Wales (36°41’ S, 149°59’E).
Holotype culture: CCAP 1379/3. Culture Collection of
Algae and Protozoa, SAMS Research Services Ltd.,
Dunstaffnage Marine Laboratory, Dunbeg, Argyll, PA37
1QA, UK. Blade and conchocelis phases of isolate 3923
are included.
Paratype: NSW 722446, Royal Botanic Gardens, Mrs.
Macquaries Road, Sydney, NSW 2000, Australia.
Collection data: 6 iv 2004, in a band of Caloglossa vieil-
lardii (Kützing) Setchell on wood pilings at Westgate
4 Algae Vol. 21(2), 2006
Bridge, Port Phillip Bay, Victoria (37°49’S, 144°53’E).
New Zealand material: Collection date. 1 viii 2003,
dense patches in sand on rocks in stream, Waihau Bay,
North Island, (37°37’S, 177°55’E), collected by Tracy Farr.
The collection number of the herbarium sheet and DNA
sample is ASD 154. It is deposited and registered at Te
Papa (with a WELT number).
Etymology: Latin pulchellus = small and beautiful
(Stern 1973).
Development of the gametophytic blade phase in cul-
ture
Young blades were round to ovate and matured into
ovate blades at 21 ± 2°C (Fig. 1) and to linear-lanceolate
blades at 14 ± 2°C (Fig. 2) that were moderately ruffled
at the margin. In culture, blades were pinkish-red in
colour. Thalli were monostromatic, each cell containing a
single, stellate chloroplast with one pyrenoid, a vacuole
and a nucleus. The small size of blades was a distin-
guishing feature of this species. They grew up to 3 mm in
diameter at 21 ± 2°C and up to 25 mm in length at 14 ±
2°C. Blades attached to a substratum by a mass of
branched rhizoids (Fig. 3). Rhizoids developed from
irregularly arranged, slightly elongated polygonal basal
cells at the base (Fig. 4). Vegetative cells were smaller
than basal cells and mostly polygonal and irregularly
arranged, becoming quadrate to rectangular and regular-
ly aligned along the thallus margin (Fig. 5). In mature
thalli, marginal vegetative cells were commonly grouped
in pairs.
The development of archeosporangia and subsequent
mass release of archeospores readily occurred in blades
maintained at 21 ± 2°C. Thalli ranging from 1-3 mm in
length and as young as 16 days developed archeosporan-
gia from up to 14 rows of vegetative cells located at the
blade apex. During sporangium development, the vac-
uoles of differentiating cells became reduced in size.
Archeosporangia thus appeared a darker pinkish-red
than vegetative cells (Fig. 6). Prior to discharge, the cell
wall enclosing each archeosporangium disintegrated.
Archeospores remained surrounded only by a plasma
membrane and were loosely positioned in the extracellu-
lar polysaccharide matrix. The entire mass release of
archeospores occurred in approximately 25 minutes,
leaving behind the colourless cell wall with depressions
where the archeospores were originally positioned (Fig.
7). Extracellular matrix was extruded from the thallus
during spore release. Discharged archeospores displayed
a pulsing amoeboid motility, whereby fat pseudopodia
randomly extended and retracted from the cell (Fig. 8).
Amoeboid archeospores travelled at < 8
µ
m min–1 and
remained dynamic for up to 24 hours. Mucilage was
secreted from all amoeboid cells (Fig. 8). Occasionally
archeospores exhibited gliding motility. These cells
remained in contact with the slide as they translocated at
a relatively constant velocity of 0.27-0.4
µ
m s–1 (Fig. 9).
Archeospores settled and developed a single digitate rhi-
zoid within 24 hours of release. All germinating spores
developed into asexual blades. Development of
archeosporangia and subsequent mass release of
archeospores was induced by transferring blades from 14
± 2°C to 21 ± 2°C, 16L:8D or 12L:12D hours and 40
µ
mol
m–2 s–1, for 48-72 hours.
Male sexual reproductive structures developed in 10-
20 mm long monoecious thalli, after 5-6 weeks at 14 ±
2°C. Spermatangial sori were conspicuous, as pale, gold-
en streaks 3-7 cells across, up to 3 mm long at the blade
apex (Fig 10). Spermatangial packets were 16-20
µ
m wide
and 22-28
µ
m long, and were divided into 32 cells (4 x 4 x
2). These packets were intermixed with larger, dark pink-
red phyllosporangia. Phyllosporangia were commonly
undivided but did occur in packets of two and four, 16-
22
µ
m wide and 20-28
µ
m long (Figs 10 and 11).
Spermatia (4-6
µ
m in diameter) and phyllospores (14-16
µ
m in diameter) were released simultaneously following
the degeneration of the surrounding cell walls (Fig. 12).
Discharged spermatia were slightly deformed in shape
but remained immotile.
Phyllospores were amoeboid following their release.
All phyllospore germlings developed into blades (Fig
13). Conchocelis filaments occasionally developed in cul-
tures of blades bearing spermatangia and phyllosporan-
gia, after 8-10 weeks at 14 ± 2°C. Whilst this indicates the
presence of female reproductive structures (carpogonia),
we did not observe any carpogonia or zygotosporangia
between phyllosporangia and spermatangia.
Development and TEM observations of the conchocelis
phase in culture
The conchocelis comprised a densely branched net-
work of narrow, uniseriate filaments in tight, spherical
tufts that were free floating or anchored to a substrate.
Vegetative filament cells were 9-12
µ
m in diameter and
32-40
µ
m in length (Fig. 14). The single chloroplast in
each cell was rose to purple coloured, elongate, twisted
and parietal with one or more pyrenoids.
Conchosporangia were variable in form. Entire vegeta-
tive filaments commonly differentiated into conchospo-
Ackland et al.: Biology of Porphyra pulchella sp. nov. 5
6 Algae Vol. 21(2), 2006
Fig. 1. Small 2 mm orbiculate blade formed at 14°C. Scale bar = 0.2 mm.
Fig. 2. Lanceolate 20 mm blade with spermatangia and phyllosporangia in linear streaks at the apical end. Scale bar = 5 mm.
Fig. 3. Basal attachment with multiple rhizoids. Scale bar = 20
µ
m.
Fig. 4. Rhizoids (arrowheads) developing from basal ends of cells near the blade base. Scale bar = 15
µ
m.
Fig. 5. Elongate meristematic cells at blade margin and more quadrate meristematic cells away from the margin. Scale bar = 15
µ
m.
rangial branches (Fig. 14). These branches were 10-27
µ
m
in diameter with transverse to oblique cross walls.
Conchosporangia also occurred in large clusters of up to
12 sporangia, irregularly distributed amongst vegetative
filaments (Fig. 14, 15). The cells were usually shorter (7-
27
µ
m) than those of vegetative cells (32-40
µ
m),
appeared a darker pinkish-red and contained reduced
vacuoles and a single chloroplast that occupied a large
portion of the cell. Prior to conchospore discharge, the
cell wall at the apex of conchosporangial filament broke
down. Conchospores appeared slightly elongated as they
successively passed through the filament before dis-
charging at the filament apex. Discharged conchospores
were pinkish-red, approximately 10-14 um in diameter
and displayed an amoeboid motion similar to that of
archeospores and phyllospores. All germinating con-
chospores developed into blades. The development of
these blades was often aberrant. Many conchospore
germlings underwent irregular cell divisions and exhibit-
ed a high cell death rate. Blades consequently developed
into irregular shapes (Fig. 16). Some thalli produced
patches of vegetative cells that were lighter in colour
Ackland et al.: Biology of Porphyra pulchella sp. nov. 7
Fig. 6. Transition zone of larger vacuolate vegetative cells below and somewhat smaller developing archeosporangial cells with
reduced vacuoles above. Scale bar = 10
µ
m.
Fig. 7. Discharged archeospores and empty sporangial walls visible. Scale bar = 30
µ
m.
Fig. 8. Freshly discharged archeospores have short fat pseudopodia (arrow) that move continuously in all directions. Faint strands of
mucilage (arrowhead) are visible. Scale bar = 8
µ
m.
Fig. 9. Archeospores in contact with a slide displaying smooth gliding movement through the medium. Scale bar = 10
µ
m.
8 Algae Vol. 21(2), 2006
Fig. 10. Linear streaks of alternating spermatangia (arrowhead) and phyllosporangia (arrow) in blade grown at 14°C. 16 spermatan-
gia are formed in two layers (32 total) from each vegetative cell. Single or double phyllospores are formed from each vegetative
cell. Scale bar = 30
µ
m.
Fig. 11. Spermatangial packet (bracket) with 16 spermatia. Phyllosporangial packets of 2 (single arrow, arrowhead) or 4 (double
arrow). Scale bar = 20
µ
m.
Fig. 12. Discharged spermatia (arrowhead) and phyllospores (arrow). Spermatia are non-motile and phyllospores are amoeboid.
Scale bar = 30
µ
m.
Fig. 13. Phyllospores still in the old blade matrix and showing the bipolar germination (arrowheads) forming new blades (not con-
chocelis). Scale bar = 12
µ
m.
compared to other vegetative cells of the same blade.
Despite this unusual development, mature blades repro-
duced asexually by archeospores when grown at 20-
22°C.
As is typical of Porphyra species, the conchocelis fila-
ments had the ability to burrow into calcium carbonate
shell matrices. Eleven days after placing the conchocelis
filaments on mollusk shell fragments, filaments had
attached and begun penetration. By six weeks, vegetative
filaments appeared on the same plane as calcium carbon-
ate crystals of the shell matrix, indicating that the fila-
ments had burrowed into the shell. After three months,
Ackland et al.: Biology of Porphyra pulchella sp. nov. 9
Fig. 14. Conchocelis filaments growing free in culture. Vegetative filaments (arrowhead), intercalary conchosporangial filaments
(arrow) and conchosporangial clusters (bracket) are seen. Scale bar = 30
µ
m.
Fig. 15. A cluster of conchosporangial cells. Scale bar = 15
µ
m.
Fig. 16. Conchosporeling blade with two colour and cell patterns in upper and lower sectors. Scale bar = 35
µ
m.
Fig. 17. Conchocelis filaments growing in a mollusk shell matrix. Scale bar = 50
µ
m.
an intricate network of filaments bearing conchosporan-
gia had developed throughout the shell matrices (Fig.
17). Some conchosporangial filaments emerged from the
shell surface and eventually liberated conchospores that
germinated, forming blades. Conchocelis filaments also
reproduced asexually by fragmentation.
TEM preparation often resulted in the disruption of
conchocelis filament cell walls. Cell wall breakage per-
mitted a greater influx of fixatives and embedding resins.
The cellular content of disrupted cells was thus better
preserved than that of cells with intact walls. TEM of
conchocelis vegetative filament cells revealed that the
cells were connected by a primary pit connection (Figs.
18 and 19). The nucleus of each cell contained a promi-
nent nucleolus and condensed chromatin (Fig. 20).
Although mitochondria and Golgi bodies were scarce the
cis-region of each Golgi complex was always associated
with a mitochondrion (Fig. 21). Floridean starch granules
were relatively small and abundant in the cytoplasm of
each cell (Fig. 21). The chloroplast thylakoids were typi-
cally unstacked and covered by disk-shaped phycobili-
somes (Fig. 22). The pyrenoids had inconspicuous, phy-
cobilisome-free thylakoids winding through their matrix
and small plastoglobuli frequently occurred at the
periphery of pyrenoids and within several regions of the
chloroplast stroma (Fig. 22).
Figure 23 represents a diagram summarising the life
cycle of Porphyra pulchella in culture as we currently
understand it.
Phylogenetic analysis
The partial sequence of the 18S gene consisted of 840
characters, 165 of which were parsimony-informative.
The evolutionary model least-rejected by the hierarchical
likelihood test was: TrN (Tamura and Nei 1993) (substi-
tution rate matrix: a = 1.0, b = 1.65, c = 1.0, d = 1.0, e =
5.07, f = 1.00) plus proportion of invariable sites set to
0.599 and a gamma distribution of 0.432. The MP analy-
sis produced 60 MP trees of 453 steps. Maximum
Likelihood (ML) analysis produced one tree of -ln L score
of 3502.2473. Both tree topologies were similar and the
MP strict consensus topology is shown in Fig. 24.
Sequences of ribosomal DNA PCR amplification prod-
ucts from the Australian and New Zealand Porphyra
samples were compared. Sequences from the blades and
‘conchocelis’ filaments of isolate 3923 were identical,
confirming that both phases were of the same species.
Blades of isolates 3923 and 4422 shared identical
sequences. These samples form a very supported clade
with a sample from Waihau Bay, North Island, New
Zealand. The tree topology is congruent with other stud-
ies showing a polyphyletic Porphyra (Broom et al. 2004;
Nelson et al. 2005). No sequences in GenBank had
sequences identical to P. pulchella. Porphyra pulchella
forms a sister group relationship with P. pseudolinealis,
although this relationship is only poorly supported (<
70%).
DISCUSSION
The conchocelis phase in the one culture isolate (3923)
of Porphyra pulchella occurred without any evidence of
sexual reproduction in the blades grown for many
months at 20 ± 2°C. Molecular data (Fig. 25) clearly veri-
fies its genetic link with the blade phase. Conchospores
developed frequently and spore germination produced
blades that reproduced by archeospores. The early stages
of conchospore germination often showed a pattern of
cell shape and size aberrant in the lower and upper sec-
tors of the blade. We have no evidence of what is occur-
ring but the pattern suggests that meiotic segregation
occurred in a manner similar to that in P. yezoensis Ueda
(Miura and Ohme-Takagi 1994).
Porphyra pulchella displayed a striking difference in
blade size and reproduction at different temperatures. At
the higher temperature (20 ± 2°C) in two weeks, small
blades (0.5-3 mm) developed that reproduced with asex-
ual archeospores. At 14 ± 2°C the blades take 5 weeks to
mature reaching 20-25 mm before reproducing with
spermatangia and phyllosporangia. This indicates that P.
pulchella may be adapted to warmer coastal waters that
allow more rapid reproduction and spore dissemination.
Kim and Notoya (2003) investigated the life history of
Porphyra koreana M.S. Hwang and I.K. Lee, observing that
archeospore formation began at 2 weeks when blades
were 1.5 mm long in 15-25°C in long days (14:10 LD) and
in short days (10:14LD) archeospore formation began at 4
weeks in 20-25°C and at 5 weeks in 15°C. At 20°C sper-
matia and zygotospores began release at 7-8 weeks in
long days. In short days at 15-25°C no sexual structures
developed for the entire culture period of 18 weeks.
Blades reached only 4 mm at 20-25°C but grew to 60 mm
in 5-10°C. Archeospores were formed in all conditions.
In contrast to P. koreana, at higher temperatures only
archeospores formed in P. pulchella and archeospores,
spermatia and phyllospores were present in lower tem-
peratures.
Notoya et al. (1993) investigated the life histories of
10 Algae Vol. 21(2), 2006
Ackland et al.: Biology of Porphyra pulchella sp. nov. 11
Figs 18-22. TEM of conchocelis phase. Fig. 18. Low magnification TEM of several cells connected by pit plugs (arrowheads). Scale bar
= 4.5
µ
m. Fig. 19. High magnification TEM of pit plug between two vegetative filament cells. Although difficult to see in this
image, pit plugs of Porphyra and Bangia possess a singe, thin cap but no cap membrane. Scale bar = 0.25
µ
m. Fig. 20. Nucleus
with prominent nucleolus (large asterisk) and electron-dense chromatin (arrowheads). Mitochondria (small asterisks). Scale bar
= 0.11
µ
m. Fig. 21. Mitochondrion (M) is associated with the cis-region of the Golgi complex (arrowhead) in conchocelis cells.
Abundant starch granules (S) can be seen in the cytoplasm of cells. Scale bar = 0.37
µ
m. Fig. 22. Unstacked chloroplast thylakoids
covered by disc-shaped phycobilisomes (arrowheads). The phycobilisome-free pyrenoid thylakoids and plastoglobuli (arrows)
are more readily apparent in this micrograph. Scale bar = 1.32
µ
m.
Porphyra lacerata A. Miura and P. suborbiculata Kjellman.
Archeospores formed within 1-3 weeks at higher temper-
atures (25°C) and blades remained small without sexual
reproduction whereas at lower temperature (15-20°C)
spermatia and zygotospores developed on blades in 4-5
weeks. These patterns appear similar to that seen for P.
pulchella.
In the paper describing P. koreana as a new species
Hwang and Lee (1994) provide a table of general charac-
ters showing the basic differences among the species, P.
koreana, P. kinositae, P. tenera, P. lacerata and P. kuniedae.
The apparent lack of carpogonia in P. pulchella sug-
gests that other conditions may be needed to have fully
functional sexual blades. Certainly the spermatia and the
phyllosporangia look normal and are released normally.
We observed over 2000 phyllospore germlings that
12 Algae Vol. 21(2), 2006
Fig. 23. Life history diagram of P. pulchella.
Blade
Gametophyte
Apica l
Spor e line
Archeospore
discharge
Archeospore
Archeospore
germlin gs
Young blade
Spermatia & phyllospore
release
Fer tilisat ion
?
Zygotospor e
Con chocelis
Spor ophyte
Conchosporangia Conchospore
sper ma tia
phyllospore
?
Car pogonia
f
Spermatangia &
intercalary phyllosporangia
�
formed only blades (no conchocelis filaments). These
were clearly not obtained from sexual fertililization.
Phyllospores, archeospores and conchospores display
a distinct amoeboid movement. This has been reported
briefly in Porphyra dioica (Holmes and Brodie 2004) and
in Porphyra yezoensis (Ueki et al. 2005). Spore motility is
common to most red algae that have been investigated
with time lapse videomicroscopy (Pickett Heaps et al.
2001). Ackland et al. (2006) provides evidence that
pseudopodia involved in amoeboid movement are regu-
lated by the cytoskeletal components actin and myosin.
Other workers have seen amoeboid motility of mono-
spores in Colaconema caespitosum (J. Agardh) Jackelman,
Stegenga and J.J. Bolton [as Audouinella botryocarpa
(Harvey) Woelkerling)] (Guiry et al. 1987), monospores
of Erythrotrichia (Rosenvinge 1927), carpospores and
tetraspores of Liagora harveyana Zeh and Helminthora
stackhousei (Clemente) Cremades and Pérez-Cirera
Ackland et al.: Biology of Porphyra pulchella sp. nov. 13
Fig. 24. Strict consensus of Maximum Parsimony trees based on partial 18S data of various Porphyra samples. Genbank samples given
with their accession numbers. Branches showing bootstrap support ≥50% shown (before diagonal MP analysis; after diagonal
ML analysis). Erythropeltidales selected as the outgroup.
(Cunningham et al. 1993, Guiry 1990). Although it is a
relatively slow process, spore movement appears neces-
sary for substrate selection and attachment prior to ger-
mination. The mechanism of amoeboid movement in red
algae has not been fully investigated.
The complete lack of spermatium motility in Porphyra
pulchella corresponds to our observations on other red
algae such as Bostrychia and Murrayella (Pickett-Heaps et
al. 1998, McBride and West 1999b, Wilson et al. 2002,
2003).
Molecular phylogeny of Porphyra is being slowly
investigated to resolve the species complexes. Porphyra
suborbiculata, P. koreana, P. lacerata and several other
smaller species have morphological and reproductive
features similar to those of P. pulchella but insufficient
molecular data are available to clearly indicate their
overall relationships.
ACKNOWLEDGEMENTS
This research is partially supported by Australian
Research Council grants SG0935526 (1994), S19812824
(1998), S19917056 (1999-2001), S0005005 (2000), a grant
from the Australian Biological Resources Study (2002-
2005) as well as a grant from the Hermon Slade
Foundation (2005-2007) to JAW and GCZ.
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Received 28 April 2006
Accepted 20 May 2006
16 Algae Vol. 21(2), 2006
