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Myrionema strangulans (Chordariales,
Phaeophyceae) epiphyte on Ulva spp.
(Ulvophyceae) from Patagonian Atlantic...
Article in Journal of Applied Phycology · June 2012
DOI: 10.1007/s10811-012-9798-0
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J Appl Phycol (2012) 24:475–486
DOI 10.1007/s10811-012-9798-0
Myrionema strangulans (Chordariales, Phaeophyceae)
epiphyte on Ulva spp. (Ulvophyceae) from Patagonian
Atlantic coasts
Amira Gabriela Siniscalchi & María Cecilia Gauna &
Eduardo J. Cáceres & Elisa R. Parodi
Received: 7 April 2011 / Revised and accepted: 15 January 2012 / Published online: 18 February 2012
# Springer Science+Business Media B.V. 2012
Abstract Fronds of Ulva spp. from Patagonian Atlantic
coasts exhibited brown spots produced by the presence of
Myrionema strangulans (Chordariales, Phaeophyceae). The
occurrence of M. strangulans on Ulva spp. is widely reported
from several regions of the world, but there were no detailed
studies about the subject. In the present study, we describe the
morphology and interactions of M. strangulans with Ulva spp.
as observed under light and electron microscopes, and we
reconstruct all stages of its life cycle based upon in vitro
experiments. The prevalence of infection by M. strangulans
was 100%. In case of the strongest epiphytism, the host cuticle
exhibited perforations, massive depigmentation, cellular disorganization, and cuticle rupture. It was possible to demonstrate a purely epiphytic life strategy of the organism by
transmission electron microscopy. M. strangulans formed
discoid thalli constituted by vegetative filaments and radiating
from a central zone to a peripheral zone. Transversally, the
discs were formed by two strata: a basal monostromatic and a
filamentous erect stratum. From the monostromatic stratum,
A. G. Siniscalchi : M. C. Gauna : E. R. Parodi (*)
Laboratorio de Ecología Acuática, Departamento de Biología,
Bioquímica y Farmacia, Universidad Nacional del Sur,
San Juan 670,
B8000FTN, Bahía Blanca, Argentina
e-mail: eparodi@criba.edu.ar
A. G. Siniscalchi : M. C. Gauna : E. R. Parodi
CONICET–CCTBBca, Laboratorio GIBEA, Instituto Argentino
de Oceanografía (I.A.D.O.),
Camino Carrindanga 7.5 km,
B8000FWB, Bahía Blanca, Argentina
E. J. Cáceres
Laboratorio de Ficología y Micología, Departamento de Biología,
Bioquímica y Farmacia, Universidad Nacional del Sur—CIC,
San Juan 670,
B8000FTN, Bahía Blanca, Argentina
hyaline hairs and reproductive structures were produced. Both
plurilocular and unilocular sporangia were present. Zoids
from both plurilocular and unilocular sporangia were able to
germinate in culture. M. strangulans exhibited a haploid–
diploid, heteromorphic life cycle with thalli with three different morphologies. The haploid chromosome number was 12±
2 chromosomes.
Keywords Epiphyte . Life cycle . Myrionema strangulans .
Patagonian Atlantic coast . Ulva spp.
Introduction
Species of the genus Ulva L. are widely distributed throughout the world and are harvested for human food in several
countries, e.g., as “aonori” in Japan (Ohno 1993). Ulva is
cultured in many parts of the world in pilot commercial
systems (e.g., Parker 1981; De Busk et al. 1986; Neori et
al. 1991, 2000, 2003; Israel et al. 1993), including integrated
multi-trophic systems where Ulva cultures are combined
with aquacultures of marine animals (Cohen and Neori
1991; Jiménez del Río et al. 1996; Neori et al. 1996, 1998,
2000; Neori and Shpigel 1999; Schuenhoff et al. 2003;
Bolton et al. 2009). Bolton et al. (2009) affirmed that the
major reason for the widespread use of Ulva is that many
species of this genus can thrive unattached in sheltered
marine waters and estuaries, and have particular affinities
for growth in high nitrogen concentrations.
On the Patagonian Atlantic coast, the genus is widely
represented by abundant populations named as Ulva intestinalis L., Ulva compressa L., Ulva linza L., Ulva prolifera
O. F. Müller, Ulva flexuosa Wulfen, Ulva californica Wille,
Ulva rigida (C. Agardh) Thuret, Ulva lactuca L., Ulva
hookeriana (Kützing) Hayden; Maggs; Silva; Stanhope and
476
Waaland, and Ulva fasciata (Roth) Martius (Papenfuss 1964;
Bastida 1968; Kühnemann 1972; Piriz 1972; Boraso de Zaixo
1977; Rico et al. 1993; Boraso de Zaixo et al. 2004). Since the
beginning of the twenty-first century Ulva taxonomy is in a
major upheaval (Hayden et al. 2003). Difficulties in accurately
applying morphological characters have become apparent,
and DNA-based studies have revealed the many discrepancies
between morphospecies and actual taxonomic entities
(O’Kelly et al. 2010). In particular, it is remarkable that the
name “Ulva lactuca” has actually been applied to many
different species of Ulva, and that even the most commonly
accepted DNA-based concept of this species is in error because it does not match with the DNA signature of the holotype specimen of U. lactuca (O’Kelly et al. 2010). Thus, in the
absence of DNA-based studies on the Ulva species present
along the coasts of South America, almost all of these names
of European species may be misapplied to Patagonian entities.
So we apply any more precise name than “Ulva spp.” to the
individuals examined here.
Myrionema strangulans Greville is normally observed on
Ulva’s Patagonian populations (Gauna et al. 2007, 2009;
Gauna and Parodi 2008). We consider that this could be a
potential problem in future local aquaculture enterprise. The
main adverse effects of epiphytes and fouling organisms are
related to their competitive removal of nutrients and inorganic carbon from the water column (Buschmann and
Gómez 1993), and to their shading effect (Buschmann and
Gómez 1993; Kuschel and Buschmann 1991). Both factors
markedly reduce the growth performance of the host seaweeds. Another adverse factor is the increased load and drag
effect exerted on the host plants by the presence of epiphytes. This weakens the host seaweed, making it much more
vulnerable to breakage or whole thallus detachment from the
substratum, especially in intertidal systems and during periods
of increased current or wave action (Buschmann and Gómez
1993; Buschmann et al. 1990; Kuschel and Buschmann
1991). Such losses can markedly reduce biomass production
from seaweed farms (Buschmann and Gómez 1993). The
occurrence of different Myrionema species on different basiphytes, especially species of Ulva, is known from intertidal
zones around the world (Bolton et al. 2009). Several Myrionema species can grow as epiphytes on brown seaweeds such
as Petalonia fascia (O. F. Müller) Kuntze (Lindauer et al.
1961), Fucus vesiculosus Linnaeus (López Rodríguez and
Pérez-Cirera 1995), and Dictyota dichotoma (Gauna 2010)
and on green seaweeds such as U. lactuca, other Ulva spp.,
and red seaweeds as Rhodymenia spp. (Loiseaux 1967b) and
also on mollusk shells (Lindauer et al. 1961).
Myrionemataceae is a family of brown algae classified
initially by Fritsch (1945) in the order Ectocarpales sensu
lato. Later, due to both morphological characters and life
cycle, it was placed into the Order Chordariales (Asensi
1966; Wynne and Loiseaux 1976; Schneider and Searles
J Appl Phycol (2012) 24:475–486
1991). From molecular analyses, the genus Myrionema Greville is presently known to be phylogenetically related to
members of Ectocarpales (Siemer et al. 1998; Draisma et al.
2001), confirming Fritsch’s original criterion. To date, a
great controversy exists regarding the taxonomy of Myrionema species. Many of them were named in accordance to
different sampling places as is the case of the morphologically similar Argentinean species Myrionema patagonicum
Scottsberg and Myrionema fuegianum Scottsberg (Asensi
1966). But several more were reported on different hosts,
i.e., Myrionema balticum (Reinke) Foslie growing on leaves
of Phyllospadix W.J. Hooker and Zostera Linnaeus, on
Macrocystis pyrifera (L.) C. Agardh, Laminaria farlowii
Setchell, and on sterile bases of Gigartina radula (Esper)
J. Agardh (Loiseaux 1970); Myrionema corunnae Sauvageau, registered on Macrocystis pyrifera, Costaria costata
(C. Agardh) De A. Saunders, Laminaria sinclairii (Harvey
ex J. D. Hooker and Harvey) Farlow, Anderson and Eaton,
Alaria marginata Postels and Ruprecht, Nereocystis luetkeana (K. Mertens) Postels and Ruprecht, and on pneumatocysts of Egregia menziesii (Turner) Areschoug (Loiseaux
1970); Myrionema incommodum Skottsberg epiphytized
Codium vermilara (Olivi) Chiaje fronds (Miravalles 2009).
The epiphyte M. strangulans Greville was formally described by Greville (1827) and was listed by several authors
as being widespread in temperate seas (Taylor 1957; Norton
1970; Guiry 1978; Lee 1980; Irvine 1982; Mol and Coppejans
1985; Fletcher 1987; Womersley 1987; Morton 2003;
Loiseaux de Goër and Noailles 2008; among others). Culture
studies in the life cycle of M. strangulans have been carried out
by Loiseaux (1967a, b, 1968, 1972) and by Kornmann and
Sahling (1983). In South America, M. strangulans has been
observed in Chile (Ramirez and Santelices 1991; Silva and
Chacana 2005) and in Argentina (Asensi 1966), together with
other Myrionema species from Patagonia’s southern regions.
Most of the morphological studies about algal epiphytism
in the world are mainly referred to red algae of commercial
interest. Brown epiphytes and brown fouling organisms as
Colpomenia sinuosa (Mertens ex Roth) Derbés and Solier,
Ectocarpus spp., Giffordia sp., and Streblonema sp. were
reported as epiphytes in several species of Gracilaria, such
as Gracilaria conferta (Schousboe ex Montagne)
Montagne, Gracilaria chilensis C.J. Bird, McLachlan, Gracilaria tenuistipitata C.F. Chang and B.M. Xia, and Gracilaria multipartita (Clemente) Harvey (Friedlander et al.
1987, 1991; Buschmann and Kuschel 1988; Friedlander
1992; Ugarte and Santelices 1992; Haglund 1992; Pickering
et al. 1993; Anderson et al. 1992; Buschmann and Gómez
1993). In Argentina, work has been done on Gracilaria
gracilis (Stackhouse) Steentoft, Irvine and Farnham but also
on Macrocystis pyrifera (Adami and Gordillo 1999; Martín
et al. 2011). A similar study in the genus Ulva has never
been undertaken neither in Argentina nor elsewhere.
J Appl Phycol (2012) 24:475–486
477
Ulva spp. fronds were obtained from intertidal and subtidal
populations along the coast of Las Grutas beach (40°51′S;
65°08′W) in the Rio Negro Province during May 2008. The
collected Ulva spp. fronds were kept on ice and retained in
labeled plastic bags until they were examined in the laboratory, within 5 h after collection. The fronds were then
brushed and rinsed under running tap water. Small portions
of infected fronds were sectioned, then immersed in fresh
0.5% solution of sodium hypochlorite for 30 s, and finally
rinsed three times, 5 min each in sterile seawater. Sonication
was subsequently applied 2 min to 5×5 mm portions in
sterile seawater, renewing the seawater after each treatment.
This cleaning procedure was followed in order to remove
diatoms as well as other epiphytes.
Crude cultures of M. strangulans were initiated by inoculating portions of cleaned fronds of Ulva spp. in plastic
Petri dishes containing PES medium (Provasoli 1968). The
cultures were maintained at 21±1°C with an illumination
regime of 12:12 h L/D and with a photon flux density of
15 μmol photons m−2 s−1. Vegetative epiphytic filaments,
and also zoids from both plurilocular and unilocular sporangia, were isolated using the hanging-drop technique. To
avoid further diatoms contamination, a 2.5% germanium
dioxide solution was added to the culture during the first
Figs. 1–3 Myrionema strangulans on Ulva spp. 1 Brown spots
(arrows), the symptom of the presence of M. strangulans. 2 Transversal section of one Ulva spp. thallus showing M. strangulans thalli
(arrow) on its cuticle. 3 Frontal, superficial view of M. strangulans
discoid thallus, with both peripheral (black arrows) and central cells
(white arrows)
Information is scarce and little is known about the entire
range of hosts that are colonized by M. strangulans (Gauna et
al. 2007, 2009; Gauna and Parodi 2008). Therefore, the main
aims of this study were (1) to explain the association between
thalli of M. strangulans and Ulva spp. using different laboratory techniques and (2) to establish the complete life cycle of
M. strangulans in isolates coming from wild populations of
Ulva from Patagonian coasts from Argentina.
Materials and methods
478
J Appl Phycol (2012) 24:475–486
week (Lewin 1966; Christensen 1982). It was then removed
from the culture medium to avoid effects on the cell morphology of M. strangulans.
Clonal cultures were established by pipetting single
germlings or thallus fragments prior to first signs of maturity
of M. strangulans infecting portions of tissue host. Strains
were maintained for 4 weeks. M. strangulans filaments
formed small 0.2-mm spherical masses inside excised host
tissue after 4 weeks in culture. No other algae or microorganisms were present. Filaments were removed and
cultured separately for study purposes. Cytomorphometry
was carried out using a stereoscopic microscope (Nikon
SMZ 1500) and an inverted microscope (Nikon 80i), with
anoptral phase contrast and differential interference contrast
(DIC) and with an incorporated camera (Nikon DXM 1200f).
Chromosome counts were carried out in unialgal cultures
of M. strangulans derived from zoids. For this purpose, thalli
were fixed either in 1:3 glacial acetic acid/absolute ethanol or
in 6:3:1 formaldehyde/absolute ethanol/glacial acetic acid at
5°C during a period of 2–24 h. Post-fixation was carried out
Figs. 4–10 M. strangulans on Ulva spp. 4 Detail of peripheral cells of
M. strangulans’ discs. 5 SEM photomicrograph showing the general
aspect of M. strangulans thallus. 6 Erect filaments (arrows) originated
in central zones of thallus. 7 Erect filaments (arrows) under SEM.
8 General SEM photomicrograph showing the long hairs of the thallus.
9 General aspect of plurilocular sporangia. 10 SEM photomicrograph
of one plurilocular sporangium
J Appl Phycol (2012) 24:475–486
479
with 70% ethyl alcohol. The material was subsequently hydrolyzed for 30 min in 1 N HCl at room temperature and
stained with Schiff stain in darkness for 2 h (Johansen 1940),
bleached during 20 min in a 1:3:3 mixture of sodium metasulfite/1 N HCl/distilled water, washed with distilled water,
and finally mounted in a drop of 2% acetic acid solution of
ferric hematoxylin with added iron acetate (Núñez 1968).
To the observations under scanning electron microscopy
(SEM), filaments of M. strangulans on Ulva spp. fronds
were fixed in 2.5% glutaraldehyde–seawater at 5°C in cacodylate buffer pH (7.2) for 2 h. They were then mounted on
slides covered with 0.5% poly-D-lysine and dehydrated in a
graded acetone series. Samples were finally critical point
dried during 1 h, coated with gold, and observed with a LEO
EVO 40 scanning electron microscope.
For observation under transmission electron microscopy
(TEM), filaments of M. strangulans on Ulva spp. fronds
were fixed at 4°C in 2.5% glutaraldehyde in filtered and
Figs. 11–15 M. strangulans on Ulva spp. 11 Terminal (black arrow)
and intercalar (white arrow) unilocular sporangia formed in peripheral
sites of thallus. 12 Unilocular sporangia in apical position (arrows). 13
Transversal section of an Ulva spp. thallus with M. strangulans as
epiphyte (arrow). 14 Ulva spp. thallus showing cellular disorganization
owing to the presence of M. strangulans (arrows). 15 Ulva spp. thallus
strongly infected by M. strangulans
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J Appl Phycol (2012) 24:475–486
sterilized seawater and post-fixed in 1% OsO4 in cacodylate
buffer 0.05 M. The material was subsequently dehydrated
through a graded step acetone series from 10% to absolute
acetone, embedded drop by drop in Spurr’s low-viscosity
resin (Spurr 1969), and flat-embedded (Reymond and
Pickett-Heaps 1983) between glass slides coated with dry
Teflon. Sections were cut with a Diatome 2.1-mm diamond
knife (Diatome Ltd., Switzerland), mounted on Formvarcoated grids and stained with uranyl acetate and lead citrate.
They were examined under a Jeol 100 CX-II electron microscope at the CONICET-CCT, Bahía Blanca.
observed). Finally, the degree of infection was considered
high in those cases in which thalli exhibited a percentage of
colonized area higher than 70% (i.e., strongly invaded thalli
were observed). The distinction between the categories was
arbitrary. Only those cases under the categories moderate
and high were considered diseased thalli.
Identification of epiphytes and disease symptoms
Infected fronds of Ulva spp. exhibited brown spots as symptoms of the presence of M. strangulans thalli on epidermic
hosts cells (Fig. 1). The thalli formed two systems: a monostromatic basal disk and erect uniseriate filaments (Fig. 2).
The monostromatic, discoid thalli were formed by photosynthetic filaments radiating from a central zone of cells
closely packed to the periphery (Fig. 3).Vegetative cells
were 6–17 μm in length, being shorter in the central region,
and they did not significantly vary in diameter, which was
8±1 μm (Fig. 3). Cells contained one to three discoid
chloroplasts per cell (Fig. 4). Long, erect claviform filaments originated from the monostromatic system formed
the erect stratum (Fig. 5). The erect filaments were unbranched, more than 400 μm in length when fully developed.
Prevalence and degree of infection of epiphytes were registered in each Ulva frond. In order to estimate the degree of
infection, a qualitative scale was used on 50 fronds collected
(Peters and Schaffelke 1996). This scale resulted from a
visual categorization of a dissected frond observed by light
microscopy (LM). The degree of infection was considered
low when the percentage of host’s thalli colonized by the
epiphyte organism ranged from 0 to 10% (i.e., no visible
signs of epiphytic infection were observed). The degree of
infection was categorized as moderate when the percentage
of colonized thalli varied from 11 to 70% (i.e., moderate
alterations, such as brown spots on the lamina, were
Figs. 16–18 Transmission
electron microscope images of
M. strangulans on Ulva spp. 16
General view of M. strangulans
thallus with prostrate (white
arrowhead) and erect (black
arrowheads) filaments on the
wall of Ulva spp. (WU)
(×2,000). 17 Cell of a prostrate
filament of M. strangulans on
Ulva spp. Note the amorphous
substance between both
epiphyte and host cell walls
(arrows) and the looser
disposition of the microfibrills
of M. strangulans’ cells
(×5,000). 18 Cell of a prostrate
filament of M. strangulans on
Ulva spp. Note the deformation
of the host’s cell wall by the
alteration of its normal fibrose
appearance and the bacteria in
the amorphous substance
(arrowheads) (×5,000)
Results
Morphology of thalli in nature
J Appl Phycol (2012) 24:475–486
The cell diameter in the erect filaments varied between 12±
1 μm and 20±1 μm from the apex to base, respectively (Figs. 6
and 7). Cells contained two to four discoid chloroplasts
(Fig. 6). Hyaline hairs were formed from cells of the basal
stratum (Figs. 5 and 8) with a length that varied between 100
and more than 200 μm (Fig. 8). In some cases, hyaline hairs
displayed vestiges of chloroplasts in the basal region. Erect,
uniseriate, 3–6-locular sporangia were observed (Figs. 9 and
10), 60–110 μm in length and 12–15 μm wide. Also, unilocular sporangia appeared mainly in the periphery of the
epiphytic basal disc and in few occasions in central positions (Figs. 11 and 12). They presented a length of 33±
Figs. 19–26 M. strangulans on Ulva spp. 19 Zoids released from
plurilocular sporangia. 20 Lobulated germination of zoids. 21 Zoids
germinating in the non-lobulated way. 22 Initial thallus, formed after
the lobulated germination. 23 Thallus in development. 24 Pseudodiscoid thallus with reproductive structures. 25 Later stage of
481
2 μm and a width of 16±1 μm. Sporangia were observed in
two positions: terminal and intercalar (Fig. 11). In some
thalli, many sporangia were observed, mainly in peripherical
sectors (Fig. 12).
Infection of M. strangulans on Ulva spp
Discs of M. strangulans appeared in all collected fronds
of Ulva spp., thus the prevalence of infection was
100%. Nevertheless, the degree of infection was different in different thalli: 45% of the thalli showed evidence of a low degree of infection, 38% of a moderate
development, showing erect filaments (black arrow) and unilocular
sporangia, some of them already empty (white arrows) by the releasing
of zoids. 26 Pseudodiscoid thallus showing both empty (white arrows)
and fully (black arrows) plurilocular sporangia
482
degree of infection, and 17% of a severe degree of
infection. In the cases of low degree, the hosts’ cuticle
remained intact (Fig. 13) but in thalli with high degrees
of infection the cuticle exhibited perforations, sometimes
accompanied by massive depigmentation and cellular disorganization (Figs. 14, 15, and 18).
M. strangulans–Ulva spp. interaction under TEM
M. strangulans prostrate thallus cells did not penetrate into
host’s cells by any degree (Fig. 16). Only the presence of an
amorphous, medium electron dense adhesive was apparent
between them (Figs. 17 and 18). In the contact sector, the
host’s cell wall altered its normal fibrose appearance
(Figs. 17 and 18) and in turn M. strangulans cells had their
own cell walls with a looser disposition of the microfibrills
(Fig. 17).
Figs. 27–35 M. strangulans on
Ulva spp. 27 Pseudodiscoid
adult thallus under culture
conditions. 28 Initial thalli
formed by non-lobulated germination. 29 Late developmental stage showing the first
vegetative filaments with thick
walls. 30 General aspect of a
filamentous thallus. 31 Initial
thallus generated by the germination of zoids from unilocular
sporangia. 32 Fertile diploid
thallus with plurilocular sporangia (white arrows) and hyaline hairs (black arrows). 33
Fusion of zoids from unilocular
sporangia, resulting in zygotes
with two stigmas (arrows). 34
Metaphase plate showing 12
chromosomes. 35 Graphic
representation of Fig. 30
J Appl Phycol (2012) 24:475–486
M. strangulans in culture
Biflagellated zoids from plurilocular sporangia were 10–
12 μm in length and exhibited two to four ribbon-shaped
plastids and one to two stigmata (Fig. 19). Under cultivation,
they germinated in two different ways after a very fast settlement. One way consisted in the formation of germlings with
normally four and rarely five lobes, each of which finished as
a cell (Fig. 20). Alternatively, germlings maintained initially
an oval form and then they elongated (Fig. 21).
Star-shaped, lobed germlings experienced successive cellular divisions resulting in small discs (Fig. 22). These small
discs developed into large discs by cellular synchronic divisions in their marginal cells. They also formed the first filaments by development of hair-like protrusions (Figs. 23 and
24). Eventually, the discs became fertile with the development
of reproductive structures typical of the species. In a few days,
several sessile unilocular and plurilocular sporangia were
J Appl Phycol (2012) 24:475–486
483
observed (Figs. 25 and 26). These sporangia measured 15–
30 μm, with sizes similar to those of the epiphytic thalli. Zoids
released from these sporangia were able to generate new discs.
A main difference observed between these in vitro discs and
the epiphytic discs is their premature fertility. They showed a
high production of both unilocular and plurilocular sporangia
and their durations were very short under controlled culture
condition. Finally, the thalli became pseudodiscoid, exhibiting
an irregular shape with entangled filaments (Fig. 27).
Non-lobulated germlings increased in size after the initial
elongation and then they formed a germination tube. After
several transversal cell divisions a filament was formed
(Fig. 28). Two weeks later, small, branched thalli had developed
(Fig. 29). Their cells were 10–15 μm long and presented a thick
wall (Fig. 30). These filamentous thalli formed both endogenous hairs and plurilocular sporangia (Fig. 30). Released zoids
germinated to form thalli with the same morphology.
measured 4–5 μm in length and presented one chloroplast
with a stigma. A few hours after release, they were able to
adhere to an artificial substratum.
Normally, the zoids germinated without conjugation and
generated small, irregular thalli (Fig. 31). Under culture
conditions, the thalli became fertile in a few days, producing
plurilocular sporangia (Fig. 32). In a few days, they also
formed endogenous hairs together with reproductive structures (Fig. 32). Gametic zoids also conjugated (Fig. 33) to
form zygotes. Inside zygotes, two stigmas and two chloroplasts were observed (Fig. 33). Afterwards, by transversal
cellular divisions zygote germinated generating thalli identical to those formed by the zoids formed in unilocular
sporangia, and also to those generated by lobulated germination from plurilocular sporangia on epiphytic thalli.
Development of gametes from unilocular sporangia
Gametophytic chromosomes were so small that observations were difficult. Nonetheless, they were observed on
several metaphasic plates, which exhibited 12±2 chromosomes as haploid number (n) (Figs. 34 and 35).
Gametic zoids released from unilocular sporangia were
smaller than those released by plurilocular sporangia. They
Fig. 36 M. strangulans on
Ulva spp. Summary of the
major events of the M.
strangulans in vitro
life cycle
Karyology of M. strangulans
Zoids
Zoids
Plurilocular sporangia
Apomeiotic Unilocular sporangia
Zoids
Filamentous
thalli (2n) (C)
Pseudodiscoid
thalli (2n) (B)
Lobulated
Plurilocular sporangia
Non-lobulated
Germination
Zoids
Plurilocular sporangia
Epiphytic disc on host
(2n) (A)
Meiotic unilocular sporangia (R!)
Gametic zoids (n)
Fusión of zoids
Pseudodiscoid
thalli (n) (D)
Zoids
Zigote (2n)
Plurilocular sporangia
Pseudodiscoid
thalli (2n) (E)
Unilocular sporangia
484
Discussion
M. strangulans interactions with Ulva spp
We agree with Robertson-Andersson (2007), who stated that
M. strangulans is an exclusively epiphytic organism on
Ulva thalli. From TEM micrographs, it became evident that
cells of the prostrate system of M. strangulans thallus did
not show any apparent attaching structure in contact with
the Ulva cell wall. So the epiphyte clearly did not penetrate
into the host tissue affecting cellular morphology or ultrastructure, excluding the external deformation of the cell wall
and the alteration of its normal fibrous appearance. The only
clearly apparent evidence of a relationship of both partners
was the presence of an amorphous material between cell
walls, with a putative adhesive function.
Also in agreement with Robertson-Andersson et al.
(2008), our observations show that the host infected sites
in direct contact with this epiphyte are sites of rupture
because of the disintegration of the circular M. strangulans
discs and a later perforation. Also, a perforation disease of
Ulva has been reported previously in Israel by Colorni
(1989), but in that case there was no biological agent that
appeared to be associated with the disease symptoms.
Several authors such as Loiseaux (1967b), Schneider and
Searles (1991), and Lindauer et al. (1961) have suggested
certain host specificity between M. strangulans and Ulva.
The high prevalence of M. strangulans found in the present
paper supports this conjecture. M. strangulans had been
detected on U. lactuca wild populations by Kornmann
and Sahling (1983), in False Bay (South Africa) in an
abalone farm in GansBaai. Recently, M. strangulans
was also observed in wild populations of Ulva capensis
Areschoug (Robertson-Andersson 2003). M. strangulans
was found persisting on Ulva throughout the year, with
a slight indication of a seasonal pattern to infection
rates. This author affirmed that the infection coincides with
a general weakening of the thalli, with lowered nitrogen
content.
Life cycle and morphology M. strangulans
During the life cycle in culture (Fig. 36), M. strangulans
presented different types of thalli: (a) epiphytic diploid
thalli, corresponding to the description of the species in
nature; (b) pseudodiscoid diploid thalli and (c) filamentous
diploid thalli, both observed under the same culture conditions; (d) pseudodiscoid haploid thalli had a which similar
morphology as in (b) and were also observed under similar
culture conditions; and (e) thalli similar to (d) but diploid.
All the observations allow inferring that the studied population of M. strangulans has a diploid–haploid life cycle
with alternations of generations in both phases.
J Appl Phycol (2012) 24:475–486
Loiseaux (1967b) was the first who observed that M.
strangulans displayed three diploid generations with different
morphological characteristics. We observed these different
morphotypes in our cultures too, as well as two ways of
germination of the zoids released from plurilocular sporangia.
We hypothesize that the pseudodiscoid, diploid thalli originated from the fusion of zoids from unilocular sporangia and
must generate on the field thalli with epiphytic morphology,
but this morphology did not develop in culture conditions. It is
probable that these differences were related to different conditions, mainly to the different substrata in both situations.
Two main differences with Loiseaux (1967b) were detected:
(a) we did not observe plurilocular sporangia in diploid pseudodiscoid thalli that originated from the fusion of zoids from
unilocular sporangia; (b) we did not observe fusion between
zoids that originated from haploid pseudodiscoid thalli. Moreover, Loiseaux (1967b) affirmed that none of the nuclear
colorations were useful in her culture to count the M. strangulans chromosomes. In contrast, we successfully used Schiff
staining, which allowed us to corroborate the haploid phase.
Similar to us, Kornmann and Sahling (1983) also showed that
1.5 h after germination the spores formed star-shaped lobulated germlings, and these young thalli developed plurilocular
sporangia and developed hairs from a central area after 11 days
in culture.
The life cycle of M. strangulans can be compared with
those of other Myrionema species such as Myrionema feldmannii Loiseaux, Myrionema magnusii (Sauvageau) Loiseaux, and Myrionema orbiculare J. Agardh. (Loiseaux
1967b). The main difference occurs with M. magnusii and
M. orbiculare, which are both without unilocular sporangia
and thus with an asexual reduced life cycle. On the contrary
the haploid–diploid life cycles of M. strangulans and M.
feldmannii are very similar with (1) zooids formed from
unilocular sporangia that also can merge and (2) discoid
and filamentous thalli. The only difference resides in the
size, form, and position of unilocular sporangia that are
highest, lateral, and pedicelated in M. feldmannii.
Chromosome counts
Haploid numbers varying between 18 and 21 have been
reported in several species as M. orbiculare, M. feldmannii,
and M. magnusii (Loiseaux 1964a, b, 1967a). Consequently,
in the studied population of M. strangulans, the haploid
number 12±2 was lower than in all these species. Our
results are in fact more in agreement with the chromosome
numbers reported to other species of Chordariaceae (Cole
1967; Kawai 1986; Caram 1961, 1965).
Acknowledgments MCG is a post-doctoral research fellow from the
National Council of Scientific and Technical Research of Argentina
(CONICET) and ERP is a researcher of CONICET. EJC is a researcher
J Appl Phycol (2012) 24:475–486
of the Commission of Scientific Research of the Province of Buenos
Aires, Argentina (CIC). Support was provided by grants from the
Secretaría de Ciencia y Tecnología de la Universidad Nacional del
Sur (PGI CSU-24/B145) and CONICET (PIP-11220100100503). We
are also grateful to Bch. Croce, María Emilia for her participation in the
first instances of this work.
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