METAMORPHOSIS O F APHANIZOMENON
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Prescott, G. W. 1954. The FreshwaterAlgae. Wm. C. Brown, Iowa,
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Revsbech, N. P. & Jargensen, B. B. 1986. Microelectrodes: their
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Reynolds, C. S. & Walsby, A. E. 1975. Water blooms. Biol. Rev.
50:437-81.
J. Phycol. 25, 333-351 (1989)
MORPHOLOGY AND DEVELOPMENT OF AHNFELTIA PLICATA (RHODOPHYTA):
PROPOSAL OF AHNFELTIALES ORD. NOV.'
Christine A. Maggs2
Atlantic Research Laboratory, National Research Council of Canada, 141 1 Oxford Street, Halifax, Nova Scotia, Canada B3H 321
and
Curt M . PuescheP
Department of Biological Sciences, State University of New York at Binghamton, Binghamton, New York 13901
ABSTRACT
Ahnfeltia plicata (Hudson) Fries, the type species o j
Ahnfeltia Fries, is currently assigned to the Phyllophora-
ceae (Gigartinales). Several morphological and biochem-
ical characters distance A. plicata f r o m the Phyllophora-
ceae but, because sexual reproduction has never been
demonstrated, a n alternative placement has not been pos-
sible. A. plicata now is shown to have a heteromorphic
sexual life history. Erect branched gametophytes are dioe-
cious. I n male sori, spermatangia are cut of transversely
f r o m spermatangial mother cells. Female sori form nu-
merous terminal sessile carpogonia. Following fertiliza-
tion, several zygotes in each sorus f u s e facultatively with
undzferentiated iiztercalarj cells of the female sorus a n d
cut ofgonimoblast initials obliquely outwards. These ini-
tials give rise to branching gonimoblastjlaments thatf u s e
with apical and intercalary female sorus cells and with
each other, then grow radially outward i n the compound
external carposporophyte and terminate i n carposporan-
gia. CarPospores develop i n culture into crustose tetra-
sporophytes identical to Porphyrodiscus simulans Bat-
ters. Field-collected P. simulans tetraspores grew into erect
A. plicata axes. Tetrasporangia are formed by division
and enlargement of crust apical cells followed by sequen-
1 Received 18 July 1988. Accepted 16 February 1989.
2 Present address: Department of Biology, T h e Queen's Uni-
versity of Belfast, Belfast BT7 INN, N. Ireland.
Address for reprint requests.
333
Stewart, W. D. P., Fitzgerald, G . P. & Burris, R. H. 1967. In situ
studies on N, fixation using the acetylene reduction tech-
nique. Proc. Nut. Acad. Sci. U.S.A. 58:2071-8.
Van Liere, L. & Walsby, A. E. 1982. Interactions of cyanobac-
teria with light. I n Carr, N. G. & Whitton, B. A. [Eds.] The
Biology of Cjairobacteria. Blackwell, Oxford, pp. 9-46.
Wolk, C. P. 1982. Heterocysts. I n Carr, N. G . & Whitton, B. A.
[Eds.] The Biolog~ofCjanobacteria. Blackwell, Oxford, pp. 359-
86.
Yates, M. G. 1980. T h e biochemistry of nitrogen fixation. In
Miflin, B. J. [Ed.] The Biochemistrj ofPlants, Vol. 5. Academic
Press, New York, pp. 216-70.
tial enlargement and maturation of tetrasporocytes in a n
erosive process. Monosporangia areformed i n sori on male
garnetophjtes. Pit plugs of both gametophyte and tetra-
sporophyte phases consist of naked plug cores without cap
layers of membranes. Gametophytes exhibit both cell fu-
sions and secondary pit connections whereas tetrasporo-
phjtes f o r m cell fusions but lack secondary pit connections.
O n the basis of the unique female and postfertilization
reproductive development a n d in conjunction with the pit
plug structure which is unique amongjorideophytes, the
order Ahnfeltiales, containing thef a m i l j Ahnfeltiaceae, is
proposed.
Key index words: Ahnfeltiaceae farn. nov.; Ahnfel-
tiales ord. nov.; Ahnfeltia plicata; morphology phylog-
eny; Rhodophyta; taxonomy; ultrastructure
Ahnfeltia plicata (Hudson) Fries (1836:3 lo), based
on Fucus plicatus Hudson (1762:470), is the type
species of Ahnfeltia Fries (1836:3 10). It is one of the
world's principal commercial agarophytes, harvest-
ed mainly on the Russian coast of the White Sea
(Levring et al. 1969, Maggs et al. 1989) as a source
of high quality, low sulphate agar (Chapman and
Chapman 1980). A. plicata is distributed widely in
the North Atlantic Ocean (South and Tittley 1986)
and also occurs in the northern North Pacific and
southern South Atlantic and South Pacific Oceans
(Makienko 19'70, Maggs et al. 1989). Despite the
commercial importance and wide distribution of A.
plicata, its reproduction is poorly understood (West
334 CHRISTINE A. MAGGS AND CURT M. PUESCHEL
and Hommersand 198 l), and its taxonomic position
as a member of the Phyllophoraceae requires reas-
sessment (Farnham and Fletcher 1976, Dixon and
Irvine 1977, DeCeiv and West 1982, DeCew 1983,
Guiry et al. 1984).
In addition to '4.plicntn, the genus includes two
other nemathecium-forming species: A. fustigmtu
(Postels et Ruprecht) blakienko (1970:1086) from
the North Pacific Ocean and '4. rlopigntn Montagne
(1 852:333) from Chile. T e n carrageenophyte species
that form internal cystocarps currently are ascribed
to .4hiif~ltin (Schotter 1968), but these are related
very closely to, or conspecific irith, Gyui2ogongru.s
Martius (1828:27) (Chemin 1930, lllakienko 1970,
Maggs et al. 1989).
T h e purpose of this study of the morphology and
development in a4hilfrltin plicnta was to clarify the
taxonomic position of this and other nemathecium-
forming species classified in the Phyllophoraceae and
to reassess their familial and ordinal placement.
MATERIALS A S D METHODS
Most field collections of .-i/i)ifp/fio plicntn were made intertidall).
or by diving in Nova Scotia. Canada. .Xpproximately monthly
samples \\ere collected from earl! October 1986 to September
1987 a t Sorthivest C m e , Lunenberg County (44'32' S , 64'01'
LV). Additional collections icere made at Pubnico Point, Yar-
mouth Count) (43"36' S 65"48' LV); Peggy5 Cove, Halifax County
(44"29' S , 6 3 5 5 ' LV): Sand) Cme. Halifax Co. (44"28' N,63"34'
LV) and Broad Haven. Pembroke, IVales (5 l"46' S , 5'07' \V) (1
January 1987). S o r t h w s t Cove is a moderately wave-exposed
granite boulder shore characteriied from 0-2 m Chart Datum
bj a dense cover of -4, /i(icic/ci. Ciioudi-ur c i i 3 p m Stackhouse and
I ) p i , n l p r i i m rniurntncpn (Linnaeus) Guiry, all heavily epiphytized
b> the Trciillidln phase of Bo?iiwmniJofiicih c i i r i i f r m Hariot. Pebbles
at 3-10 m support a variet! of crustme red algae including the
P o r / ~ h ? r o d ~ i cphase
~ ~ i of .-i plrrcitn. Xdditional Porphjrodiscus sam-
ples \Yere obtained subtidall! at Fairhaven, Se\\.foundland(47"25'
S , 53"40' M') on 6 December 1986 bj R. Hooper.
Fresh specimens were examined for reproductive structures
and fixed in 4'7;formalin-seawater for anatomical investigation.
Sections icere made lcith a freezing microtome or by hand and
htained i\.ith 1 % aqueous aniline blue or 0.5% aqueous toluidine
blue (to she\\ \calls): lipids were stained with 5% Sudan 111 in
95% ethanol. Unless otherwise stated, all measurements of struc-
tures exclude wall thickness. For chromosome studies, tissue was
fixed immediately after collection from the sea or, for cultures,
1 h after the end of the photoperiod in 2:l absolute ethanol:
glacial acetic acid. Squashes were made in aceto-iron-haema-
toxylin chloral hydrate (JVittmann 1965) or DAPI (4-6-diamidi-
no-2-phenylindole)/sea\+ater(0.5pg.mL-l). These stains also were
used for hand- and freezing microtome-sections. D-XPI-stained
specimens were examined icith a Zeiss microscope fitted with an
epifluorescence condensrr: others were viewed in bright field
illumination. I'egetative anatom! \\as also studied using speci-
mens fixed in 4% glutaraldehyde, dehjdrated in ethanol, infil-
trated and embedded in Spurr's resin and sectioned 1-2 pm thick
on a Reichert ultramicrotome.
Specimens of .-ihiqdfi<i plicntn for ultrastructural studies were
collected intertidallv in Reid State Park, Sagadohoc County, Xlaine
(43"47' N. 69"45' LV) on 5 June 1985. T h e specimens of Porphj-
r o d l i c u J were from the c ~ ~ I t u r described
es below. Small pieces of
tissue were fixed in 3% glutaraldehyde. 2% formaldehyde in a
0.1 M cacodvlate buffer, pH 7.0, containing 0.2 \I sucrose to
adjust the osmolarity. .Ifter a series of dilutions and washes with
0.1 M cacodylate buffer, specimens were post-fixed in 274 OsO,,
followed by I\'ater rinses, dehydration in acetone, and infiltration
with Spurr's resin. To enhance membrane contrast, some spec-
imens were fixed in 2% KMnO,, 0.1 M NaCI; others were fixed
in aldehydes as above and then post-fixed in KMnO, rather than
osmium (Pueschel 1987). Thin-sections were stained with uranyl
acetate and lead citrate and examined on a Hitachi H-7000 TEM.
Cultures were initiated from spores or surface-sterilized apical
tips. They were grown initially at 15" C and a 16:8 h LD cycle
at an irradiance of ca. 20 pmol.m-2.s-1 in modified von Stosch's
medium (Guiry and Cunningham 1984). Large plants were aer-
ated \\-ith compressed air.
RESULTS
Habit of GainetophJte
Gametophyte axes of ilhilfrltin plicntu (Fig. 1) arise
in tufts from crustose holdfasts (Fig. 2). Holdfasts
are extensive and adhere closely to the substratum.
They a r e up to 2 cm in diameter and 300 pm thick.
Erect axes formed by a single holdfast often become
tangled and interwoven. Male and female plants a r e
indistinguishable (Fig. 1) except for reproductive
structures. Mature axes a r e usually terete, 300-800
(- 1000) pm diameter, occasionally slightly com-
pressed, e.g. 600 x 900 pm in cross-section, and the
tips often are wider than mature axes. Mature plants
vary greatly in size, from 3-2 1 cm, and in branching
pattern. Branching is generally very irregular (Fig.
l ) , ivith some axes forming a secund pattern of lat-
eral branches, whereas others are unbranched or
bear an irregular series of laterals inserted at a wide
angle and often attenuate at the point of insertion.
Secund branching usually is initiated when apices
are damaged and can be stimulated in culture when
segments of mature axes are removed and grown
separately (Fig. 3). Some subtidal plants are strictly
dichotomously branched (Fig. l), with axes slightly
compressed at dichotomies, but these plants gen-
erally produce a few irregularly branched axes.
Thalli are tough and wiry; the color varies from
almost black to light brown or yellow in bleached
specimens. Branches or whole plants of several shades
of green due to pigment mutations (Fig. 57) were
observed (e.g. six occurrences in 200 plants of one
collection).
Male plants form spermatangial sori covering the
cortex of mature axes. Sori a r e absent only from
apical and basal regions of the plants. Under a dis-
secting microscope young sori are only just visible
as slight elevations of the cortex. Toward the end
of their fertile period, male sori reach 20-25 pm in
total thickness and become more conspicuous.
Female sori a r e formed similarly only on mature
axes. Sori are linear, to 5 mm long and '70 wm high,
usually formed along one side of the axis rather than
surrounding it. Mature carposporophytes often cov-
er axes of subtidal plants, being absent only from
the extreme bases (within 2 cm of the holdfast in a
21-cm long plant) and the terminal 2.5-3 cm of
young apices. Only if growth has been retarded b y
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.) 335

FIGS.1-6. Habit and morphology of Ahnjltia plicata. FIG. 1 . Habit of male and female plants from Northwest Cove, Nova Scotia.
Both male and female gametophytes vary from irregularly to dichotomously branched. Scale bar = 12 mm. FIG. 2. Groups of young
terete axes growing from extensive crustose holdfast; lighter central area is crustose coralline alga overgrowing holdfast. Scale bar =
1 mm. FIG. 3. Excised segments of axes in culture regenerating from both ends and forming secund branch initials (arrows). Scale bar
= 1 mm. FIG.4.Female plant bearing elongate groups of paler colored carposporophytes; discrete carposporophytes (arrow) are more
spherical. Scale bar = 1 mm. FIG.5 . Vertical section through holdfast and erect axis showing horizontal growth zones in holdfast. Scale
bar = 25 wm. FIG.6. Vertical section of holdfast with numerous fused cells (arrows). Scale bar = 10 pm.

damage to the apex are carposporophytes found near
branch apices. Individual mature carposporophytes
are hemispherical and about 300 pm wide. They
may be discrete (Fig. 4) but usually are coalesced
into elongate clusters up to 5 (-12) mm long (Fig.
4), forming a flat-topped elevation 150-400 prn
above the cortex.
Monosporangial sori were observed only on male
plants. When sporangia were immature the sori were
30 pm high and not easily distinguished from sper-
matangial sori. Older monosporangial sori were up
to 45 pm thick, with a mucilaginous surface layer to
22 pm thick, and only slightly pigmented. These sori
were particularly common around the apices of lat-
eral branches that apparently had stopped growing.
Vegetative Morphology of Gametophjtes
Holdfasts. Vertical sections of holdfasts show sev-
eral (up to 7 )horizontal discontinuities (Fig. 5 ) mark-
ing growth pauses, possibly annual, and a surface
covering of mucilaginous material about 15 pm thick.
Holdfasts are pseudoparenchymatous in structure
and are composed of vertical rows of more or less
isodiametric thick-walled cells 2-3 pm wide. Con-
spicuous cell contents include the single, cap-shaped
parietal plastid, large lipid globules, and granular
cytoplasm. Cell fusions are abundant (Fig. 6 ) ; vir-
tually every cell is fused to one or more neighboring
cells in a three-dimensional network. In basal parts
of holdfasts, extensive cell fusions result in irregu-
larly-shaped cells up to 9 pm diameter, and the basic
pattern of vertical cell rows is modified. Unfused
cells are uninucleate; fused cells have variable num-
bers of nuclei. Up to four nuclei per cell were seen.
No conjunctor cells or secondary pit connections
were observed. Apical extension of groups of erect
filaments in holdfasts results in multiaxial erect axis
initials.
Erect axes. Erect axes are multiaxial. Numerous
ovate to clavate apical cells, 8-15 x 3.5-6.5 pm,
undergo transverse and oblique divisions to form
pseudodichotomous filaments (Figs. 7-1 1). Nuclei
336 CHRISTINE A. MAGGS AND C U R T M. PUESCHEL

Fics. 7-1 7. Vegetative morphology of '4hnfeltia plirnfn gametophyte. FIG. 7 . Squash of pseudodichotomously branched filaments at
apex, stained with haematoxylin to show large nuclei with prominent nucleoli; the nucleus of one cell (arrow) is dividing. Scale bar =
15 pm. FIGS.8, 9. Squash of apical filaments in DAPI, under ultraviolet and transmitted light, respectively, showing uninucleate apical
cells (nucleus of uppermost cell has divided), nucleate conjunctor cell (small arrow) and formation of second conjunctor cell by same
filament cell (large arrolv). Scale bars = 15 pm. FIG. 10. Multinucleate cells resulting from secondary pit connections. Scale bar = 15
pm. FIG. 11. Pseudodichotomously branched filaments linked in lattice arrangement by secondary pit connections. Scale bar = 15 pm.
FIG. 12. Longitudinal section (LS) of resin-embedded multiaxial apex showing formation of conjunctor cells and their fusion to neigh-
boring cells (arrows). Scale bar = 40 pm. FIG. 13. LS of apex showing elongation of apical filaments into files of medullary cells. Scale
bar = 10 pm. FIG. 14. LS of old axis s\ith elongate medullary cells, curved filaments of subcortical cells, and zones of cortical thickening,
including development of new cortical growth zone (arrow). Scale bar = 40 pm. FIG. 15. Multinucleate medullary cells. Scale bar = 20
pm. FIG. 16. LS of old medulla showing thick-walled medullary cells with lipid globules and dense granular contents; short filaments of
smaller cells (arrows) havr developed secondarily. Scale bar = 20 pm. FIG. 17. Transverse section (TS) of medulla to show numerous
secondar!- pit connections (arrows). Scale bar = 20 pm.

of the third to fifth derivati\re cells below apical cells
divide, and small nucleate conjunctor cells are cut
off obliquely (Figs. 8, 9, 12). Each conjunctor cell
fuses laterally to a neighboring cell, which is then
binucleate (Fig. 10) and joined to the first cell by a
secondary pit connection. Cells progressively fur-
ther from the apex continue to form conjunctor
cells, so that they become linked together by sec-
ondary pit connections in a three-dimensional lattice
(Fig. 1 1). These developing medullary cells elongate
 AHNFELTZA PLZCATA (AHNFELTIALES ORD. NOV.)
in regular files (Fig. 13) and become highly multi-
nucleate (Fig. 15), containing as many as 15 equal-
sized nuclei.
The structure of mature axes in longitudinal sec-
tion (LS) (Fig. 14) consists of medulla, a transitional
subcortical layer, and cortex. Files of medullary cells
are either straight or slightly sinuous. Cells vary
greatly in dimension, both within and between dif-
ferent parts of a thallus, from 10-350 pm long and
3-20 pm wide. In older axes, some medullary cells
give rise secondarily to rhizoid-like branching fila-
ments of cells about 7 pm wide and up to 25 pm
long (Fig. 16). Medullary cells (Fig. 16) contain abun-
dant lipid globules, starch grains, and non-pig-
mented plastids; walls are laminated and increase in
thickness with age up to 8 Fm. Even young medullary
cells contain 6- 15 nuclei, presumably as a result of
fusions; nuclear division was not observed. In trans-
verse section (TS) (Fig. 17) the cell lumens are 3.5-
16.5 pm, mostly about 10 pm wide, linked together
by numerous secondary pit connections.
Medullary and cortical tissue intergrade in a nar-
row subcortical zone, which consists of filaments
curved toward the cortex (Fig. 14). Subcortical cells
are 6-1 1 x 2-10 pm, contain 2-7 nuclei and large
lipid globules, and are linked by secondary pit con-
nections. Cortical filaments also are curved initially,
and later grow perpendicular to medullary files.
Cortical cells, 1.5-4 x 1-3 pm, are uninucleate ex-
cept when fused to neighboring cells. Cells appar-
ently fuse directly as no conjunctor cells or second-
ary pit connections have been observed.
Apical cells of cortical filaments resume growth
in limited areas of cortex visible as raised patches
(Fig. 14), and they give rise to the irregularly layered
cortex seen in older axes. T h e cortex gradually in-
creases in thickness, so that a young plant with 2-3
growth rings may have a cortex 50 pm thick com-
posed of 14 cells, whereas in older axes up to 12
growth zones may be present, with a thickness of
over 300 pm composed of about 70 cells. Renewed
cortical growth may be an annual occurrence, since
new patches usually were observed in January.
Reproductive Morphology of Gametophjtes
Gametangia. Male sori (Fig. 18) develop in discrete
patches or entirely cover the cortex in TS. Sper-
matangia, borne singly on spermatangial mother
cells, are interspersed with sterile hairs. Mucilage
covers the sorus. Mature spermatangia (Figs. 18,20)
are ovoid or occasionally spherical, 4-9 x 2-4 pm,
non-pigmented, with granular contents and a single
nucleus that often stains as a haematoxylin-positive
ring. Spermatangial mother cells have little pigment
(shown by lack of autofluorescence), are 3-5(-7.5)
pm long x 2.5-3 pm wide, and often form a cup
around the base of the spermatangium. Released
spermatia deform as they squeeze through sper-
matangial walls; they are slightly ovoid initially, then
become spherical, 5-5.5 pm in diameter, and adhere
337
to hairs (Fig. 21). Spermatangial mother cells pro-
duce further spermatangia by extending narrow
projections into the old walls, which are then cut off
transversely. A layer of mucilaginous wall up to 10
pm thick over the spermatangia shows transverse
striations that stain deeply with toluidine blue (Fig.
22) and apparently represent a buildup of sperma-
tangial wall material as spermatia are released. Hair
cells (Figs. 19, 21) are elongate, 6-1 1(-20) x 1.5-
2 pm, often inflated at their apices, and contain nu-
clei about half-way along the hair. Cortical sections
of male plants show hairs buried among vegetative
cells.
Female sori (Fig. 23) develop by apical growth of
cortical filaments in extensive patches and may over-
grow the cortex at their margins (Fig. 24). Sorus
filaments are composed of normally-pigmented cells
about 2.5 pm wide and 1.5-2 times longer than wide,
between which numerous cell fusions occur (Fig.
25). Many filaments terminate in carpogonia borne
on supporting cells that are distinguished from ster-
ile sorus cells only because they are usually slightly
longer or wider, measuring 4.5-5 x 1-2 pm. The
abundant carpogonia (Figs. 26-29, 34-37) show
considerable variation in morphology, from almost
cubical to long and conical, narrowing gradually into
thick-walled trichogynes. They range in size from
4.5 x 2.5 pm to 9 x 4 pm, with a trichogyne 4.5-
14 x 1.5 pm. Carpogonia have dense contents that
stain very darkly, usually obscuring the nucleus.
Trichogynes sometimes bulge at the tips, or reflex
downwards, but usually protrude beyond the walls
of sorus apical cells, although not often beyond an
outer mucilage layer (Figs. 27, 28, 34-36). Sorus
apical cells, initially 2.5 x 2 pm, become much en-
larged in regions where carpogonia are present, in-
creasing to 9.5 pm long, and becoming vacuolate
and irregularly shaped (Figs. 27,28). T h e great ma-
jority of carpogonia remain unfertilized and become
buried (Fig. 29) by continued growth of sorus fila-
ments, which often branch after they grow past car-
pogonia, compensating for the cessation of growth
of carpogonium-bearing filaments. Female sori are
usually about 50-70 pm thick before fertilization
takes place; occasional well-developed sori (Fig. 30)
are up to 175 pm in thickness.
Postfertilization development. Trichogynes with at-
tached spermatia were not observed. T h e earliest
stage of putative postfertilization development found
was enlargement of carpogonia to more elongate
structures, 12-13 pm long and 4-5 pm wide, with
trichogyne walls still attached (Figs. 3 1, 38,39). The
zygote fuses facultatively with one or more previ-
ously undistinguished neighboring intercalary cells
of the female sorus (Fig. 41), and then cuts off 2-5
gonimoblast initials obliquely from the upper part
of the cell (Fig. 40). Gonimoblast initials usually fuse
with one or more intercalary or apical female sorus
cells and give rise to thick-walled gonimoblast fila-
ments 3-5 pm wide composed of darkly-staining cells,
338 CHRISTINE A. MAGGS AND C U R T M. PUESCHEL

FIGS.18-33. Gametangia and early postfertilization development in dhrfdtia plicnta. FIG. 18. TS of male sorus. Scale bar = 15 pm.
FIG.19. Male sorus with hair cell resembling carpogonium. Scale bar = 15 pm. FIG.20. Male sorus stained with haematoxylin showing
elongation of spermatangial mother cell (arrow) and formation of spermatangia. Nuclei in mature spermatangia stain in form of ring.
Scale bar = 10 pm. FIG.21. Released spherical spermatium adhering to inflated hair cell. Scale bar = 15 pm. FIG.22. TS of male sorus
stained with toluidine blue to show thick striated layer of wall material overlying sorus. Scale bar = 15 pm. FIG. 23. T S of cortex with
female sorus bearing numerous dark-staining carpogonia. Scale bar = 50 pm. FIG.24. Margin of female sorus including one carpogonium
(arrow) overgrowing vegetative cortex (co). Scale bar = 10 pm. FIG.25. Oblique section through female sorus showing abundant fusions
between vegetative cells. Scale bar = 15 pm. FIG. 26. Periclinal section near surface of female sorus with numerous dark-staining
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.) 339

4-17.5 pm long, that grow outward to the surface & 3 3 3

of the female sorus (Figs. 32, 41-43). Gonimoblast
filaments grow over the surface of the female sorus
but under the external mucilage (Figs. 33, 41-43)
and form irregular lobes or acute projections (Fig.
49) that fuse with intercalary or apical vegetative
cells of the female sorus. Each cell contains a single
nucleus, 1.7-2.2 pm in diameter (Figs. 45,48), mark-
edly larger than vegetative nuclei of the female so-
rus, which are 0.7-0.9 pm wide. Apical cells of young
gonimoblast filaments often dichotomize; the fila-
ments branch irregularly dichotomously.
The gonimoblast grows outward horizontally
(periclinally) (Figs. 44-50) and fuses with vegetative
cells progressively more distant from the zygote,
terminating the growth of sorus filaments. Since sur-
rounding unfused sorus filaments continue to grow,
the young gonimoblast occupies a conical depression
in the surface of the sorus, clearly visible in TS (Figs.
44, 45, 48). At this stage, the young gonimoblast
forms filaments that are branched only sparsely, in
an irregularly pinnate pattern (Fig. 51), and grows
considerable distances over the sorus (to at least 180
pm), fusing with apical vegetative cells. These long
filaments terminate in further prolific branching that
forms dense gonimoblast tissue (Fig. 52), among
which some fusions take place between gonimoblast
cells.
Several carpogonia are normally fertilized in each
female sorus; gonimoblast filaments from several zy-
gotes become confluent and form a compound car-
posporophyte. Gonimoblasts eventually develop into
a densely interwoven layer several cells thick over
the female sorus (Fig. 53), completely terminating
the growth of all sorus filaments. T h e growth form
of the gonimoblast filaments then alters, and they
grow out perpendicular to the sorus surface, i.e.
radially in T S (Figs. 53, 54). Radiating gonimoblast
filaments are 2-4 pm wide, composed of cells 6-27
pm long. Numerous fusions occur between cells of
different gonimoblast filaments (Fig. 53). Cells are
little-pigmented, highly vacuolate (Fig. 55), and uni-
nucleate, with nuclei about 1.5 pm diameter. This
gonimoblast tissue grows out marginally over the
female sorus (Fig. 54) but does not fuse with it. Fil-
aments form occasional pseudodichotomies as the
carposporophyte increases in diameter and grow to
130-1 80 pm in length before forming carposporan-
gia. No specialized sterile filaments are present be-
80
0
0
20um
4
2
- 4 3 1
I
44- /----- 4
5
-
FIGS.34-46. Carpogonia and postfertilization development
in Ahnfeltia plicata. (Abbreviations used in Figs. 34-46: c: car-
pogonium; g: gonimoblast cell; gi: gonimoblast initial; m: muci-
laginous material; tr: trichogyne; vf vegetative cell of female
sorus; z: zygote = fertilized carpogonium.) FIGS.34-36. Conical
carpogonia with short trichogynes not protruding beyond surface
mucilaginous layer. FIG. 37. Possibly abnormal, narrow carpo-
gonium. FIGS.38,39. Carpogonia after fertilization (zygotes)with
trichogyne walls still visible; in Fig. 38 unfertilized carpogonium
also is present. FIGS.40-46. Young carposporophytes. FIG. 40.
Zygote, with trichogyne still attached, has cut off gonimoblast
initials obliquely outward to sorus surface. FIG. 41. Zygote has
fused with undifferentiated intercalary cell of female sorus and
cut off gonimoblast initials, one ofwhich has fused with vegetative
cell; gonimoblast initials have formed young gonimoblast fila-
ments. FIG.42. Gonimoblast initials, but not zygote, have fused
with female sorus cells. FIG. 43. Young gonimoblast filaments
radiating outward over sorus surface from zygote. FIG.44. Young
carposporophyte, consisting of cone of branched gonimoblast
filaments radiating from zygote, occupies depression in sorus. FIG.
45. Young carposporophyte stained with haematoxylin has much
enlarged nuclei relative to vegetative cells of female sorus. FIG.
46. Margin of older carposporophyte showing development of
gonimoblast cells obliquely upward from periclinal gonimoblast
filaments.

t
carpogonia among vegetative filaments. Scale bar = 15 pm. FIGS. 27, 28. Carpogonia, terminal on undifferentiated filaments, with
trichogynes reaching or penetrating surface mucilaginous layer. Scale bars = 10 wm. FIG.29. TS of carposporophyte showing unfertilized
carpogonia with long trichogynes buried by continued growth of sorus filaments. Scale bar = 20 pm. FIG.30. Carposporophyte formed
on massive female sorus containing several layers of buried carpogonia (arrow). Scale bar = 100 Pm. FIG. 31. Zygote, with trichogyne
wall still attached (arrow). Scale bar = 10 pm. FIG. 32. Zygote has cut off gonimoblast initials upward (arrows); these have fused to
nearby intercalary cells of female sorus and divided transversely to form apical cells of gonimoblast filaments. Scale bar = 10 pm. FIG.
33. Apical cells of young gonimoblast filaments radiating out over surface of sorus from buried zygote. Scale bar = 10 pm.
340 CHRISTINE A . MAGGS AND C U R T M. PUESCHEL

FIGS.47-56. Carposporophytes and monosporangia in .Ih)lfZtto plirntn. FIG.47.TS of female sorus with carpogonia and young
carposporophyte (arrow). Scale bar = 20 pm. FIG.48. Young carposporophyte, with wide gonimoblast filaments fused to vegetative
cells, occupies depression in female sorus. Scale bar = 10 pm. FIG.49. Liargin of young carposporophyte; branched gonimoblast filaments
forming lateral projections that fuse with vegetative cells (arrows). Scale bar = 20 pm. FIG.50. Young carposporophyte forms oval area
of gonimoblast filaments originating from zygote. Scale bar = 40 pm. FIG.3 1 . Periclinal section just below surface of female sorus, with
older carposporophyte forming long, little-branched gonimoblast filaments. Scale bar = 20 pm. FIG.52. Periclinal section through
interuoven gonimoblast tissue, showing fusions betFveen gonimoblast cells (arrow). Scale bar = 20 Wm. FIG.53. T S of part of mature
carposporophyte shoicing intencoven periclinal gonimoblast filaments that have formed radiating gonimoblast filaments with cell fusions
between them (arrow). Scale bar = 20 pm. FIG.54. T S of axis with mature carposporophyte showing carposporophyte borne on female
sorus. Scale bar = 100 pm. FIG.55. I'acuolate gonimoblast filaments terminate in cells bearing carposporangia. Scale bar = 15 pm. FIG.
56. T S through monosporangial sorus \\.ith thick mucilaginous covering. Scale bar = 20 pm.

tween carposporangial filaments. Carposporangial posporangia (Fig. 5 5 ) are ovate to pyriform, 10-23
supporting cells (Fig. 5 5 ) are pigmented, 10-15 Pm Prn x 3-6.5 wm. They a re uninucleate with two
long x 2-3 Prn wide, and often partly- enclose the ribbon-like plastids sometimes visible in the granular
carposporangia in a cup-shaped depression. Car- cytoplasm. Carposporangial supporting cells regen-
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.) 34 1

FIGS.57-60. Mutant and wild-type pigmentation in Ahnfeltia plicata. FIG.57. Two green and one yellow pigmentation mutants from
one collection of A. plicata. Yellow tissue is partly streaked with wild-type brown coloration. Scale bar = 1 mm. FIG. 58. T S of part of
wild-type red carposporophyte formed by green female plant showing irregular interface between green tissue of female sorus and
reddish-brown carposporophyte. Scale bar = 50 hm. FIG.59. TS of red carposporophyte borne on green female plant with thick cortex.
Scale bar = 50 hm. FIG.60.T S of carposporophyte with wild-type red pigmentation borne on wild-type female plant. Scale bar = 50 pm.

erate carposporangia by upward extension into the
old walls left by a discharged carpospore. The nu-
cleus divides, a constriction appears in the middle
of the cell, and the new carposporangium is cut off
from the supporting cell. This apparently occurs
repeatedly, as nests of up to four walls have been
seen around carposporangia. A mucilaginous wall
layer 7-1 5 pm thick covers the carposporangia.
A mature carposporophyte thus consists in TS of
three distinct layers: (1) cones of gonimoblast tissue,
growing out of zygotes in the female sorus, that
spread outwards horizontally and become interwo-
ven, (2) radiating gonimoblast filaments, terminat-
ing in (3) carposporangial supporting cells and car-
posporangia.
Evidence that gonimoblast tissue results from fer-
tilization of carpogonia was provided by observa-
tions on two bright green mutant female plants that
bore reddish carposporophytes. Transverse sections
of the carposporophytes (Figs. 58, 59) showed that
the interface between green and red tissue consisted
of cones of red tissue growing out among the green
female sorus filaments and covering them. T h e ra-
diating gonimoblast filaments were pigmented only
slightly as in carposporophytes borne on red females
(Fig. 60) but were discernibly red in color (Figs. 58,
59); carposporangia were pigmented normally. Re-
leased carpospores grew into red crusts.
Monosporangia. Monosporangial sori (Fig. 56) de-
velop on male thalli. Apical cells in the cortex elon-
gate and divide to produce filaments of 2-5 cells
that are clearly distinguished from the compact cor-
tex by their weak pigmentation and loose coherence.
Filaments terminate in cup-shaped supporting cells
342 CHRISTINE A . MAGGS A N D CURT M. PUESCHEL
that bear single monosporangia. Sporangia (Fig. 56)
are usually ovoid, occasionally spherical, (5)- 10-20
pm long x 3.5-6 pm wide, pigmented, uninucleate,
with granular contents and ribbon-like plastids. Re-
leased monospores are spherical.
T t t rasporophjte
Tetrasporophytes (Fig. 6 1) are crustose, growing
very closely appressed to hard, usually quartz, sub-
strata. Individuals are about 3 cm across but often
become confluent. Growing margins are extremely
thin; older parts of the thallus usually show concen-
tric growth rings indicated by variations in crust
thickness. Plants are violet-bro1r.n to yellowish in
color.
Younger parts of crusts (Fig. 62) consist of reg-
ularly arranged vertical files of cells 2.5-4 pm wide,
isodiametric to 2 times longer than \+vide,tvith walls
about 1.5 pm thick bet\veen cells. Young cells a re
uninucleate (Fig. 62) with the nucleus about 1.5 prn
diameter. In the basal regions of sections, numerous
cell fusions obscure the pattern of vertical filaments.
Fusions (Fig. 63) occur between neighboring cells
in all directions, forming irregularly shaped cells
that are usually multinucleate and contain a nucleus
from each of the fused cells. Large cells u p to 12 x
8 pm can contain 3-4 nuclei. Secondary pit connec-
tions a r e absent. Buried hairs, superficially resem-
bling carpogonia, terminated some vegetative fila-
ments in one crust (Fig. 64): no hairs were seen at
the crust surface.
Tetrasporangial sori arise by differentiation of all
apical cells (Fig. 65) in round o r oval patches 175-
300 pm wide. N o sterile filaments are present in the
sori. Differentiation initially involves elongation of
the usually isodiametric apical cells to cells 2-3 times
longer than ivide. Transverse divisions (Fig. 66) re-
sult in loosely coherent short filaments about 3-4
cells above the dense vegetative tissue. Apical cells
of these filaments enlarge to 20 x 4 pm, becoming
clavate or spindle-shaped tetrasporocytes with gran-
ular contents. T h e first tetrasporocyte division is
oblique or transverse and the second divisions also
can be either oblique o r transverse. T h e second di-
visions usually occurred synchronously in both cells,
but some, possibly abnormal, 3-celled sporangia were
also seen. Mature tetrasporangia (Fig. 67) are clavate
or ovoid, almost regularly zonate to irregularly
obliquely zonate, and measure 17.5-25 x 5-7.5 pm.
While the apical tetrasporocyte in a sorus filament
is maturing, the subtending cell begins to differen-
tiate into another tetrasporocyte. Following dis-
charge of tetraspores, the old sporangium wall re-
mains attached to the apex of the developing
tetrasporocyte (Figs. 68, 69). This second tetra-
sporocyte matures and releases tetraspores, while its
subtending cell develops in turn into a third tetra-
sporangium. After release of these spores, it appears
that the next cell of the filament, now apical and
bearing a long series of walls, divides. T h e process
of sporangial filament formation and differentiation
is then repeated. Some unusual structures observed
in squashes of sori provided evidence that sporangial
differentiation has been correctly interpreted as an
erosive process. Pairs of fused tetrasporocytes (Fig.
70) could not have developed from apical cells since
fusions d o not occur in dividing apical cells but in-
stead must have been derived from the subtending
cells, among which cell fusions a r e common. A tetra-
sporocyte bearing another tetrasporocyte laterally
and old walls terminally (Fig. 71), indicating that it
was once in the position of a pseudodichotomy, could
onl) have developed by erosion of a short dichoto-
mous filament. Older sori (Fig. 72) occupy a pit in
the crust surface, with a total sorus depth of about
40 pm, and a re covered by mucilaginous wall ma-
terial 20-35 pm thick. This feature is visible in Por-
p1ij i o d i sci t s simulnns type material (Dixon a nd Irvine
1977, fig. 77A).
Cltrastructu)e
T h e ultrastructure of vegetative cells of both ga-
metophytic and tetrasporophytic phases is typically
florideophycean in nature (Figs. 73, 74). Chloro-
plasts lack pyrenoids and generally have a single
peripheral thylakoid that surrounds several plate-
like thylakoids. Mitochondria a re found at the cis
face of Golgi bodies (Figs. 73, 74). Microbodies are
abundant near nuclei of tetrasporophytes (Fig. 74).
Pit plugs in both gametophytes and tetrasporo-
ph) tes are cylindrical, 0.2-0.3 pm in diameter and
in length, with only a slight waist (Figs. 75-77). There
is no indication of cap layers; the pit plugs are ho-
mogeneously electron-dense. All but a few of the pit
connections examined had deep invaginations of the
plasmalemma at each end of the pit plug (Figs. 75-
77). T h e invaginations varied in depth and width,
but they occurred in all fixation regimes tested: glu-
taraldeh) de followed by osmium, glutaraldehyde
followed by permanganate, and permanganate alone.
T h e ) were more pronounced in the last two pro-
cedures.
Determination of the presence or absence of a cap
membrane is complicated by the invaginations of
the cell surface. A section through the invagination
could be misinterpreted as a layered cap (Fig. 76).
If a cap membrane were present on the pit plug side
of the invaginating plasmalemma, it would be ap-
pressed to the end of the plug by the plasmalemma.
N o membrane was found in this position (Fig. 7 7 ) .
If a cap membrane were on the cytoplasmic side of
the invagination, it would be even more conspicuous
(Figs. 75,76). Permanganate was used in the fixation
o r post-fixation process to enhance the contrast of
membranes (Pueschel 1987). These procedures also
gave no indication of a cap membrane in ilhizfeltia
p l ICCI t o .
PhP~lolog~
T h e Northwest Cove '4. plicnta population had
spermatangia from July-August to January, carpo-
gonia from July to November, developing carpo-
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.) 343

FIGS.61-72. Vegetative and reproductive morphology of Porphyrodiscus simulans phase of Ahnfeltia plicata. FIG. 61. Habit of crust
with concentric growth rings on quartz pebble. Scale bar = 2 mm. FIG. 62. Vertical section (VS) through upper part of crust stained
with haematoxylin showing filaments of uninucleate, thick-walled cells. Scale bar = 10 pm. FIG. 63. VS of lower crust with numerous
cell fusions resulting in large, irregularly-shaped cells. Scale bar = 10 pm. FIG.64. VS of crust with buried hair cells (arrows). Scale bar
= 10 pm. FIG. 65. VS of crust surface forming tetrasporangial initials. Scale bar = 40 pm. FIG. 66. Division of clavate tetrasporangial
initials yields ovoid tetrasporocytes and smaller subtending cells. Scale bar = 10 bm. FIG. 67. VS of mature sorus with nearly regularly
to very irregularly zonate tetrasporangia and dark-staining subtending cells. Scale bar = 20 fim. FIG. 68. Squash of older sorus stained
with toluidine blue showing walls of discharged sporangia (arrows) attached apically to subtending cells. Scale bar = 20 pm. FIG. 69.
Tetrasporangial subtending cells with old walls attached apically (arrow) enlarge sequentially to form tetrasporocytes. Scale bar = 20
pm. FIG.70. Pair of fused tetrasporocytes (arrow) resulting from sequential transformation of fused vegetative cells into tetrasporocytes.
Scale bar = 10 pm. FIG. 71. Tetrasporocyte (arrow) with another tetrasporocyte attached laterally resulting from the transformation
of a pseudodichotomous vegetative filament into tetrasporocytes. Scale bar = 20 pm. FIG. 72. VS of mature tetrasporangial sorus in
surface pit formed by erosion of vegetative filaments as they are transformed into sporangia and possibly also from continued apical
growth of surrounding vegetative filaments. Scale bar = 40 pm.

sporophytes from September to November and ma-
ture carposporophytes from October to May-July.
Carposporophytes and old female sori decayed from
May to August so that some temporal overlap often
occurred between female sori and occasionally car-
posporophytes from two different annual crops.
Monosporangia were not observed in subtidal col-
lections, but in the intertidal zone they were found
from November to January.
Male and female gametophytes in the subtidal
population at Northwest Cove exhibited a sex ratio
of almost exactly 1:1. In a collection of 200 game-
tophytic thalli, there were 96 males and 104 females.
In intertidal habitats, however, monosporangial
males sometimes predominated: samples from a
Welsh intertidal population consisted of 69 mono-
sporangial males and 8 females with carposporo-
phytes.
Spore Development i n Culture
Carpospores. Carpospores were cultured from three
collections of carposporophytic Nova Scotian plants.
344 CHRISTINE A . MAGGS A N D C U R T M. PUESCHEL

FIGS.73-77. Thin-section electron microscopy. FIG. 73. l’egetative cell of gametophyte showing Golgi bodies (G) associated with
mitochondria (M) (C: chloroplast). Scale bars for Figs. 73 and 74 = 0.5 pm. FIG. 74. Vegetative cell of tetrasporophyte. Golgi body is
associated t\.ith mitochondrion (SI):microbodies (Sfb) are in proximity to nucleus (N). FIG. 75. Pit plug (P) of gametophyte consisting
only of plug core. Plasmalemma is invaginated near both ends of pit plug (arrows), but cap membranes are absent (W: cell wall). Scale
bar, for Fig$. i 5 - 7 5 = 0.2 pm. FIG.76. Gametophyte. Section through deep invagination (arrow) can give false appearance of layered
cap. FIG.77. Pit plug o f terrasporophyte. ;\gain plasmalemma is deep1)- invaginated (arrows), but cap membranes are absent.

All folloived the same developmental pattern: re- cal, with granular cytoplasm, ribbon-like plastids,
sults are given here for spores from plants collected and the single nucleus visible as a clear spot. Spores
at Sandy Cove on 18 November 1986. Released car- germinated by the formation of a hyaline protuber-
pospores (Fig. 78) were 9-13 pm diameter, spheri- ance (Fig. 79) that grew out as a germination tube
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.)
(Fig. 80) into which the spore contents were evac-
uated, leaving an empty spore wall. The germination
tube was then cut off by a transverse division from
the empty wall. Within 5 days, irregular divisions
gave rise to multicellular sporelings with empty spore
walls still attached (Fig. 81). Cell fusions were ob-
served in the basal layer after 8 days, and hairs up
to 60 pm long with dense cytoplasm at the tips were
present. Sporelings grew by divisions of the margin-
al meristem, reaching 300 pm in diameter after 1.5
months and 1.8 mm after 3 months. By 4 months
crusts (Fig. 82) were up to 2.6 mm diameter and
showed concentric growth rings. Cells were isodia-
metric (4 pm) or wider than high (Fig. 83). Numer-
ous fusions between cells of the erect filaments ob-
scured the vertical files, forming cells up to 11 pm
wide. Crusts had a thick surface cuticle.
One replicate culture was transferred to 10”C and
8:16 h LD and in still culture remained sterile for
12 months, at which time the crusts were mostly
confluent and up to 10 mm in diameter. This culture
then was aerated, and 3 months later it formed tet-
rasporangial sori about 100 pm wide (Fig. 84) with
a thick mucilaginous covering. Elongate tetraspo-
rocytes (Fig. 85) divided to form irregularly zonate
tetrasporangia 12-2 1 x 5-6 pm that released spores.
Monospores. Monospores, from a male plant col-
lected at Sandy Cove on 27 November 1986, ger-
minated (Fig. 86) by evacuation of the contents into
a germ-tube that was then cut off by a cross-wall as
in carpospores. Sporeling discs were up to 250 pm
diameter after 1.5 months and to 1.3 mm after 3
months; they formed abundant hairs. At 3 months,
erect axes (Fig. 87) were initiated by a few discs,
particularly on the edges of the slides and where
discs adjoined. Following transfer of the culture to
aeration, all discs formed erect axes that 2.5 months
later were 4 mm high and branched (Fig. 88). Abun-
dant monosporangia developed on all erect thalli,
often terminating the growth of lateral branches.
Released monospores germinated freely, covering
the interior of the culture vessel. Sporelings formed
basal discs that gave rise to erect axes after 3 months
when they were 1-2 mm in diameter. No other re-
productive structures were observed in these cul-
tures.
Tetraspores. T h e germination pattern of tetra-
spores from a Porphyodiscus plant collected at Fair-
haven, Newfoundland, on 6 December 1986, was
identical to that of carpospores and monospores.
Discs were up to 1.1 mm diameter after 2 months
and had formed numerous hairs. By 3 months discs
resembled plants grown from carpospores and
showed concentric growth rings (Fig. 89). After
transfer to aeration, discs grew to 3.5-4.1 mm in
diameter and initiated rings of erect axes. Two and
a half months later these axes were 10 mm high,
500 pm wide and irregularly branched (Fig. 90). All
thalli (which presumably included some female
plants) formed abundant monosporangia that re-
345
leased spores. Monosporelings gave rise to discs that
initiated further erect axes. After 14 months in cul-
ture, axes were up to 50 mm in length (Fig. 91).
Four months after transfer to 10” C and 8: 16 h LD,
under aeration, they had failed to form any repro-
ductive structures other than monosporangia.
Chromosome Counts
Nuclei in dividing apical cells of field-collected
gametophytes are 2.5-4.5 pm diameter with prom-
inent nucleoli 2-2.5 pm wide. Numerous nuclei at
metaphase and anaphase were found in Wittmann’s-
stained squashes, but chromosomes could be distin-
guished in only a few nuclei. These nuclei had 15-
23 minute chromosomes surrounding the nucleoli.
Photographic documentation was not possible and
counts were approximate due to the extremely small
chromosome size.
Marginal cells of gametophytic holdfasts grown in
culture from tetraspores measured 7.5-13 x 3.5-
6.5 pm, and nuclei were up to 5 pm diameter with
the nucleolus to 3 Km (Fig. 92). In dividing cells (Fig.
93), chromosomes were much larger than in apical
cells, and counts of 24-31 were obtained (Fig. 94).
The best estimate was of about 30 chromosomes. In
marginal cells of crusts grown in culture from car-
pospores there was usually a high frequency of di-
viding cells, but no successful chromosome counts
were made in either these or in dividing tetraspo-
rocytes.
DISCUSSION
Historical Background
T h e reproductive morphology of Ahizfeltia plicata
long has been the subject of speculation and debate.
T h e initial description by Hudson (1762) did not
mention reproduction, but Smith and Sowerby
(1803: 1089) referred to “small, dark, prominent tu-
bercles.” C. A. Agardh (1822:310) reported that the
reproductive structures, termed nemathecia, were
composed of articulated filaments. Their function
was still unknown when J. Agardh (1851:3 11) spec-
ulated that the cells of these filaments might be re-
leased as tetraspores, like those of Gjmnogongrus
species. Kutzing (1849:789) regarded the filaments
as chains of spores (“Kettensporen”), as did J. Agardh
(1876:206). Buffham (1893) and Schmitz (1893) re-
alized that only the terminal cells of the nemathecial
filaments were released but differed in their inter-
pretation of these bodies. Buffham believed that they
were asexual spores since they were too large to be
spermatia (“pollinoids”). Schmitz interpreted the
nemathecia as a parasite, Sterrocolax decipiens Schmitz
(1893:394), and the spores as those of the parasite.
Schmitz’s (1893) view of the parasitic nature of
the nemathecia was soon challenged by Brebner
(1896) who observed that cultured monospores
formed discs resembling holdfasts of A. plicata.
Chemin (1930, 1937), Gregory (1930, 1934) and
346 CHRISTINE A. MAGGS AND C U R T M. PUESCHEL

FIGS.78-94. Development in culture of spores of .4ht@tia plicafa and Porphjrodiscus sirnulans. FIGS.78-85. Carpospore development.
FIG.78. Released carpospore with single nucleus (arrow). Scale bar = 10 pm, FIG. 79. Early germination stage; carpospore has formed
hyaline protuberance (arrow). Scale bar = 10 pm. FIG. 80. After 2 days cytoplasm has partly evacuated into germination tubes leaving
empty spore walls. Scale bar = 20 pm. FIG. 81. After 5 days discoid spores have attached empty spore walls. Scale bar = 20 fim. FIG.
82. Crusts show- concentric groivth rings. Scale bar = 1 mm. FIG. 83. VS through vegetative crust showing complete fusions (arrows)
between neighboring cells. Scale bar = 10 gm. FIG. 84. VS through crust with tetrasporangial sorus bearing undivided tetrasporocytes
and zonate tetrasporangium (arrow). Scale bar = 50 pm. FIG. 85. Tetrasporocyte showing oblique metaphase plate of first division.
Scale bar = 5 pm.FIGS.86-88. Development of monospores released by male gametophyte. FIG. 86. Germinating monospores showing
sporelings attached to empty spore walls. Scale bar = 20 pm. FIG. 87. Discs from monospores have produced erect axes. Scale bar = 2
mm. FIG. 88. Older monospore culture \vith irregularly branched axes growing from extensive holdfast. These plants were forming
abundant monosporangia. Scale bar = 2 mm. FIGS.89-91. Development of field-collected P. sirnulans tetraspores. FIG. 89. Young discs
showing concentric growth rings. Scale bar = 1 mm. FIG. 90. Erect axes of A. pIirata growing from holdfasts. Scale bar = 5 mm. FIG.
91. Older, terete, irregularly branched axes. Scale bar = 10 mm. FIGS.92-94. Dividing marginal cells of gametophyte discs derived
from tetraspores. Scale bars = 5 pm. FIG. 92. Cell with large nucleus and nucleolus adjacent to dividing cell with chromosomes. FIG.
93. Cell in anaphase of mitotic division. FIG.94. Cell with about 20 of 30 chromosomes visible.
 AHNFELTIA PLZCATA (AHNFELTIALES ORD. NOV.)
Rosenvinge (193 1) concurred with Brebner (1896)
that the “tubercles” were the reproductive organs
of A. plicata. Detailed morphological studies failed
to elucidate the life history. Chemin (1930) believed
that monospores represented a form of asexual re-
production, similar to tetraspores: they could not be
carposporangia because carpogonia had not been
observed. Rosenvinge (193 1) found that nemathecia
were composed of two zones, between which ra-
diating filaments were not continuous. He reported
that both these zones and the vegetative thallus were
of the same ploidy. A layer of “generative cells,”
regarded as reduced procarps, gave rise to the outer
zone which might be interpreted as either (1) tetra-
sporangia that remained undivided because they
were mitotic rather than meiotic, or (2) haploid gon-
imoblast filaments terminating in carpospores, thus
a unique type of carposporophyte. Gregory ( 1 934)
reported spermatangial sori on separate male plants
but found no evidence of carpogonia.
More recently, Schotter (1968) favored Rosen-
vinge’s second hypothesis, that nemathecia were car-
posporophytes, but he concluded that only chro-
mosome counts could establish beyond doubt the
reproductive cycle of A. plicata. Magne (1972) also
regarded monospores as carpospores and speculated
that plants bearing them might be free-living car-
posporophytes that cycled independently of other
phases. This suggestion was refuted by the discovery
(Farnham and Fletcher 1974, 1976) that the life
history of A. plicata involved the crustose tetraspo-
rophyte Porphjrodiscus sirnulans Batters ( 1 897:439),
although an obligate relationship between the two
phases was considered unlikely. Chen (1977) dem-
onstrated that A. plicata undergoes a heteromorphic
life history in which nemathecial monospores grow
into tetrasporangial crusts and tetraspores from these
give rise to erect axes. Fertilization and meiosis nev-
er have been demonstrated.
New Observations
We have shown that the life history of Ahnfeltia
plicata involves the production of gametangia by
dioecious gametophytes, fertilization, and the de-
velopment of a carposporophyte (Fig. 95). We now
can confirm speculations by DeCew and West (1982),
DeCew (1983) and Guiry et al. (1984) that A. plicata
has a sexual heteromorphic life history. Previous
studies of the life history of A. plicata (Schmitz 1893,
Chemin 1930, Gregory 1930, 1934, Rosenvinge
1931) misinterpreted the extreme simplicity of the
female reproductive structures, which consist only
of sessile carpogonia, and the very complex and un-
usual features of carposporophyte development.
The only previous description of gametangia was
the identification by Gregory (1934) of spermatan-
gial sori in plants from Wales. T h e reference in
Dixon and Irvine (1977) to “apparently abortive
carpogonial branches” and “auxiliary cells” results
from an adaptation of Kylin’s (1956:3 15) description
347
of infertile procarps (L. M. Irvine, pers. comm.).
This in turn was presumably based on Rosenvinge’s
interpretation of young gonimoblasts as reduced
procarps. Although the structure of female sori was
studied in detail by Rosenvinge (193 1) and Gregory
(1934), it was incorrectly interpreted. Both authors
observed “flask-shaped cells” that stained deeply with
haematoxylin, but Rosenvinge decided that they
were not carpogonia and played no role in nemathe-
cia1 development. Rosenvinge and Gregory also made
careful observations on the development of goni-
moblast tissue, but again misinterpreted them.
Our cultures of Ahnfeltia plicata and Porphjrodiscus
sirnulans followed a heteromorphic life history in
culture, carpospores giving rise to P. simulans crusts
and tetraspores to A. plicata erect axes. This is the
first report of the development in culture of field-
collected P. siinulans spores, previous studies having
been made on A. plicata carpospores (Farnham and
Fletcher 1976, Chen 1977). Canadian plants of P.
sirnulans correspond well with type material illus-
trated by Dixon and Irvine (1977) and Porphjrodiscus
siinulans Batters (1 897:439) should be reduced to
synonymy with Ahnfeltia plicata. Chromosome counts
of approximately 30, which we presume to be the
haploid number, were obtained in gametophytes.
This does not correspond with Rosenvinge’s (193 1)
report of four chromosomes in dividing gonimoblast
cells (as “nemathecial filaments”). We were unable
to make counts in gonimoblast cells, but the chro-
mosomes were quite unlike the structures illustrated
by Rosenvinge. T h e only successful counts we made
were in cells of the holdfast margins. We do not
have conclusive evidence that meiosis takes place in
tetrasporangia. However, observations on the tet-
rasporophyte of A. fastigiata in culture (Maggs et al.
1989) included some indications of meiosis in divid-
ing tetrasporocyte nuclei. These contain 30 large
bodies, probably pairs of chromosomes. T h e 1:1 ra-
tio of males and females in subtidal populations is
also consistent with a meiotic origin of gameto-
phytes. That fertilization occurs prior to gonimo-
blast development is strongly suggested by obser-
vations of mutant green female plants bearing red
carposporophytes.
In addition to this heteromorphic life history in-
volving meiosis and syngamy, monospores recycle
erect male gametophytes (Fig. 95). Farnham and
Fletcher (1976) were correct in their hypothesis that
the relationship between gametophyte and sporo-
phyte is not obligate. T h e occurrence of intertidal
populations with 90% male plants bearing mono-
sporangia attests to the effectiveness of this form of
reproduction, which might be important in habitats
that are unsuitable for the crustose phase. Tetra-
sporophytes are common on pebbles, but mature
gametophytes are found only on more stable sub-
strata probably because their erect growth habit is
more easily damaged by mechanical disturbance.
A similar heteromorphic life history was observed
348 CHRISTINE A. MAGGS AND C U R T M. PUESCHEL
@ @
00
tetraspores
(I,
0
-Q-
w
2
tetrasporangia
FIG. 95. Life history of .4hr+ltia plicata
in cultures of .4h1zfpltinfnstigintn from British Colum-
bia, but the life history of the South American species
.I elongntn is unknown (Maggs et al. 1989). Game-
tangial sori and carposporophyte structure in A. f a s -
tigintn and '4. elongntci closely resemble those of ;i.
plicatn, but monosporangia are known only in '4.
plicatn. Species of .4hnjeltio with external carpospo-
rophytes are remarkably homogeneous in both veg-
etative and reproductive morphology and in ultra-
structural features. Doubts about the affinities of '4.
plicnta with the Phyllophoracae (e.g. Guiry et al.
1984) have been borne out fully by the present in-
vestigation. Morphological and biochemical fea-
tures consistent with distancing '4.plicatn from both
the Phyllophoraceae and the order Gigartinales in-
clude the transverse division of spermatangial moth-
er cells to form spermatangia, sessile carpogonia,
lack of auxiliary cells, direct development of a com-
pound external carposporophyte from the diploid-
ized carpogonium, true monosporangia, an appar-
ently unique method of tetrasporangial development,
spore germination by evacuation, and production of
agarocolloids. T h e configuration of pit plugs of . 4 h ~
fpltin, a plug core \vithout caps or cap membranes,
is the most primitive type found in florideophyte
red algae. Correlated \+.ith this simple plug config-
uration is an unspecialized female reproductive ap-
paratus and an unspecialized pattern of postfertili-
zation development. Both types of ch a ra c te rs ,
reproducti1.e features and pit plug structure, indi-
zygote
carpoTorophyte
I
tetrasporophyte
cate that .1h)feltin should be removed from the
Gigartinales and placed in a n separate order. Ac-
cordingly, a new family and order of the Florideo-
phycidae, based on Ah~zfeltiaplicata, a r e proposed as
follo\vs:
Ahnfeltiaceae Maggs e t Pueschel fam. nov.
Tlialli gametophytici ex hnptero extendente et axibus
Prectis v i illtiasin libusque coizsta ntes, jilamenta uegetativa
cell it lns con iu t i ctiiw s f o rma n tia qua e eflciun t foveo-col-
ligntiones inferiores et quoque demonstrant fusiones di-
rectas cellularum; spermatangia singulatim abscissa
diT'isionibus transilersis n cellulis matricalibus sperma-
taligifieris, cnrpogonia terminalin, sessilia in jilamentis
i i o ~rnaii festis sori feminei qui ex cortice rlegetativo ex-
t rixsecus ei~olrlit;post fecu nda tionem ca rpogonia cum cel-
l ulis covi vi unibus ilegetatiiisqzte facultative conuingunt
pxtrinsecits nbscidentia initia gonimoblasti; complures ZJ-
gotn e in om ii i so rofein in Po eficiu ntjila menta ramlJicantia
gotiitnoblnsti crescentin extus in soro, conuingentia cum
cellulis sterilibus et nliis cellulis gonimoblasti, jilamenta
goninoblnsti coifertiin intexta, extrinsecus radiantia in
CCI rposporophjtis coinpositis exterioribusque atque termi-
H O ntin i,i carpospornngiis; monosporangia fortasse for-
inn tn per cell u las tn u ta tas co rtica lesque; tet rasporophq'ta
crustacen, fiisionibus directis cellulae prnedita sed foveo-
colligationibus iTzfPrioribus carentin, tetrasporangza in soris
p e r diiisiones npicales formantes jilamenta breuia atque
p e r discriminntionen sequentialem in tetrasporophq'ta
deorsuni n celluln npicnli ezrolentia, tetrasporangia mat-
 AHNFELTIA PLICATA (AHNFELTIALES ORD. NOV.)
ura irregulatiin zonata; obturamenta foveae stratis pyxi-
datis et mernbranis pjxidatis carentia; paries cellulae
agarocolloidia contenentes.
Tjpus speciei: Ahnfeltia plicata (Hudson) Fries (1836:
310), basionym Fucus plicatus Hudson (1 762:470).
Gametophytic thalli consisting of spreading hold-
fast and erect multiaxial branched axes, vegetative
filaments forming conjunctor cells that give rise to
secondary pit connections, also exhibiting direct cell
fusions; spermatangia cut off singly by transverse
divisions from spermatangial mother cells, carpo-
gonia terminal, sessile on undistinguished filaments
of female sorus that develops outward from vege-
tative cortex; following fertilization, carpogonia fuse
facultatively with non-specialized vegetative cells and
cut off gonimoblast initials outwardly; several zy-
gotes in each female sorus give rise to branching
gonimoblast filaments that grow externally on sorus,
fusing with sterile cells and other gonimoblast cells,
gonimoblast filaments becoming densely interwo-
ven, then radiating outward in the compound ex-
ternal carposporophytes and terminating in carpo-
sporangia; monosporangia may be formed by
modified cortical cells; tetrasporophytes crustose,
with direct cell fusions but lacking secondary pit
connections, tetrasporangia developing in sori by
apical divisions that form short filaments and by
sequential differentiation into tetrasporocytes
downward from the apical cell, mature tetraspo-
rangia irregularly zonate; pit plugs lacking cap layers
and cap membranes; cell walls containing agarocol-
loids.
Type species: Ahnfeltia plicata (Hudson) Fries
(1836:3 lo), basionym Fucus plicatus Hudson (1762:
470).
Ahnfeltiales Maggs et Pueschel ord. nov.
Carpogonia terininalia, sessilia i n j l a m e n t i s n o n m a n -
festis sorzfeminei qui extrinsecus e cortice vegetativo evolvit;
post fecundationem carpogonia cum cellulis communibus
vegetativisque facultative conuingunt extrinsecus absci-
dentia inztza gonimoblasti; filamenta ramijicantia goni-
rnoblasti in soro femineo extus crescunt, cum cellulis ve-
getativis et cum aliis cellulis gonimoblasti conuingentia,
tuin extrinsecus radiant et in carposporangiis terminant;
obturainenta foveae stratis pyxidatis et membranis pyxi-
datis carentia; paries cellulae agarocolloidia contenentes.
Tjpusfamiliae: Ahnfeltiaceae fam. nov.
Carpogonia terminal, sessile on undistinguished
filaments of female sorus that develops outward from
vegetative cortex; following fertilization, carpogo-
nia fuse facultatively with non-specialized vegetative
cells and cut off gonimoblast initials outwardly;
branching gonimoblast filaments grow externally on
female sorus, fusing with vegetative cells and other
gonimoblast cells, then radiate outward and termi-
nate in carposporangia; pit plugs lacking cap layers
and cap membranes; cell walls containing agarocol-
loids.
Type family: Ahnfeltiaceae fam. nov.
349
Relationships of Ahnfeltiales
The two most significant reproductive features
that relate Ahnfeltiales to other florideophyte red
algae are the sessile carpogonia and the lack of dif-
ferentiated auxiliary cells. Sessile carpogonia that
undergo post-fertilization development without the
involvement of auxiliary cells are found in Acro-
chaetiales and Palmariales. In species of Acrochae-
tiales in which a sexual life history is known, the
zygote gives rise directly to a filamentous gonimo-
blast or carposporangia, or is transformed into a
tetrasporangium (e.g. Stegenga and van Wissen
1979, Abdel-Rahman and Magne 1984, Abdel-Rah-
man 1985). Neither the zygote nor gonimoblast cells
fuse with any non-differentiated gametophytic cells.
In addition to the sessile carpogonia, Acrochaetiales
are characterized by their simple filamentous growth
form (Garbary and Gabrielson 1987). In Palmari-
ales, which are multiaxial, the zygote either grows
out directly into the tetrasporophyte as in Palmaria
(van der Meer and Todd 1980) or divides first to
form a generative stalk cell and a tetrasporocyte in
Rhodophjsema (DeCew and West 1982). No fusions
are formed and carposporophytes are absent. Ac-
rochaetiales and Palmariales are united with Ne-
maliales, Batrachospermales and Corallinales, which
form carpogonial branches but not auxiliary cells,
by the occurrence of two pit-plug cap layers (Pues-
chel and Cole 1982, Gabrielson and Garbary 1986).
Reproduction in Gelidiales shows some similari-
ties with Ahnfeltia, the most significant being the
absence of differentiated auxiliary cells, but carpo-
gonia are intercalary rather than sessile in this order
(Hommersand and Fredericq 1988). The zygote fuses
with undifferentiated cortical cells and gives rise to
gonimoblast filaments that fuse randomly with ter-
minal cells of nutritive filaments. Like Ahnfeltia,
members of Gelidiales have transverse division of
spermatangial mother cells, germinate by spore
evacuation and form agarocolloids (Hommersand
and Fredericq 1988). However, sporelings form rhi-
zoids as well as apical derivatives, an elaborate sys-
tem of nutritive filaments is formed, carposporo-
phytes develop internally (Hommersand and
Fredericq 1988), and pit plugs have a cap layer and
cap membrane (Pueschel and Cole 1982).
Some authors have suggested affinities between
the Porphjrodiscus phase of A. plicata and Hilden-
brandia based on anatomical similarity (DeCew 1983)
and tetrasporangial morphology (Denizot 1968).
However, a number of features distinguish Hilden-
brandiales from Ahnfeltiales. These include the ap-
parent absence of direct cell fusions in Hildenbran-
diales, the presence in this order of secondary pit
connections formed without conjunctor cells (Ca-
bioch and Giraud 1982, Pueschel 1988), and the
presence of cap membranes (Pueschel and Cole
1982). Although erosive tetrasporogenesis is found
in both groups, the superficial sori in which tetra-
350 CHRISTISE >I.
5IAGGS A S D CURT M . PUESCHEL
sporogenesis of .4h t f p l t i n begins are not found in the
Hildenbrandiales. Tetrasporangial development is
solely an erosive process in Hildmbm tzdicc, leading
to the formation of deep pits (Pueschel 1982),
whereas in A.lh+~ltin, cell division and erosion are
combined so that only shallow pits are formed.
T h e pit plug structure of Ahnfeltiales most re-
sembles that of tivo of the most primitive genera of
red algae, Rliodochnptr (Rhodochaetales) (Pueschel
and Magne 1987) and Cotnpsopogoti (Compsopogon-
ales) (Scott et al. 1988). Even plug diameters a re
similar, being less than 0.5 Fm. Pit plugs of Bangi-
ales, the other traditional bangiophyte order with
pig plugs, can be over 1 .O Pm in diameter, have one
cap layer but no cap membrane (Pueschel 1987,
Pueschel and Cole 1982), and are found only in the
filamentous, normallv diploid phase of the life his-
tory. Other than .\hr;feltiales, Corallinales and pos-
sibly Batrachospermales are the only florideophyte
orders that might be characterized by absence of
cap membranes (Pueschel 1987). However, in Cor-
allinales and Batrachospermales, tivo-layered caps
are present. All florideoph!-te orders lacking plug
caps typically have cap membranes. Although the
simplicity of pit plug ultrastructure in .ihnjdtin is
more like that of bangiophytes than florideophytes,
.Ihttfdtin does not show other ultrastructural traits
associated with some bangiophytes, such as absence
of peripheral encircling thylakoids and absence of
Golgi body/mitochondrion associations (Pueschel
and hlagne 1987).
It is becoming clear that there are several possible
lines of descent from the plexus of orders ivith rel-
atively unspecialized female reproductive systems to
the traditional higher florideophyte orders, those
ivith auxiliary cells and strongly differentiated car-
pogonial branches. Sorting out the lower florideo-
phyte orders, a task made difficult by the current
paucity of characters, is crucial to understanding the
phylogeny o f red algae. Only lvhen this is accom-
plished rvill \ve be able to answer such questions as
whether the higher florideophytes arose monophy-
letically from a comnion lower florideophyte ances-
tor or independently from different progenitors in
this plexus.
We are grateful for helpful discussions \\.ith 5f. H. Hommersand,
G. \V. Saunders, J. SfcLachlan and L. 5f, Irvine. J . P. van der
Sfeer kindly read and made helpful comments on the manuscript.
We thank R . Hooper for collecting some of the material and
Angela Shipman for the Latin diagnoses. This stud! was partly
supported by Sational Science and Engineering Research Coun-
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INFRAGENERIC T A X O N O M Y OF AHA'FELTIA (AHNFELTIALES, RHODOPHYTA)'
Christirw A. i\.laggs,2 Jack L. i2.1cLachlan3ccud Gary W. Saunclers4
Atlantic Research Laboratory, National Research Council of Canada, 141 1 Oxford Street, Halifax, Nova Scotia, Canada B3H 321
ABSTRACT
Ahnfeltia plicata (Hudson) Fries, the type species of
Ahnfeltia Fries, is reported to be a widespread alga which
is a n important source of agar. However, after character-
izing type material and representative populations of A.
plicata, we f o u n d that several taxa usually considered to
be synonyms o f A. plicata instead represent separatp species
with dzfferinggeographic distributions. Ahnfeltia plicata
sensu strict0 is characterized by the development of ex-
ternal carposporophytes on female sori that are present
only on mature axes. Pseudocarposporophytes bearing
monosporangia and cortical inonosporangial sori occur on
both young and old axes. Characteristic vegetativefeatures
include long medullary cells and cortical growth rings.
Ahnfeltia plicata S.S. is circumpolar in both hemispheres
and is harvested for agar i n the White Sea. Ahnfeltia
Received 18 July 1988. Accepted 16 February 1989.
* Present address: Department of Biology, T h e Queen's Uni-
versity of Belfast, Belfast BT7 l N N , N. Ireland.
Address for reprints.
Present address: Department of Biological Sciences, Simon
Fraser University, Burnaby, B.C., Canada V5A 1%.
351
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Gymnogongrus comosus Kutzing f r o m Chile and
Gymnogongrus filiformis Kutzing f r o m southern Ar-
gentina appear to be synonymous wzth A. plicata. Ahn-
feltia fastigiata (Postels et Ruprecht) Makienko, f r o m the
North Pacijic, dzffers frovn A. plicata principally i n the
development offemale sori and carposporophytes only near
apices; medullary cells are shorter and cortzcal groulth
rings, cortical monosporangza and pseudocarposporo-
phytes are not formed. I t is dioecious and its lqe historj
imlolves the detlelopment f r o m carpospores of a crustose
Porphyrodiscus tetrasporophyte. W e suggest that A. pli-
cata var. tobuchiensis K a n n o et Matsubara, the prin-
cipal f o r m of Ahnfeltia utilized for agar production in
the North Pacijic, represents a n unattached ecad of A.
fastigiata. Ahnfeltia elongata Montagne, known onlj
f r o m Chile and with carposporophytes restrzcted to rnazn
axes, is more closely related to A. plicata than to A.
fastigiata. Thalli are large and regularly dichotovnous,
with long medullary cells, and do not f o r m pseudocar-
posporophytes or monosporangia.
Key index words: agar; Ahnfeltia elongata; Ahnfeltia
fastigiata; Ahnfeltia plicata; Ahnfeltia plicata vur.