See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/257577035 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 CITATIONS READS 0 97 4 authors, including: Cecilia Gauna Eduardo JORGE Cáceres 15 PUBLICATIONS 21 CITATIONS 53 PUBLICATIONS 213 CITATIONS Centro Cientifico-Tecnologico Bahia Blanca SEE PROFILE Universidad Nacional del Sur SEE PROFILE Elisa R. Parodi Universidad Nacional del Sur 64 PUBLICATIONS 369 CITATIONS SEE PROFILE All content following this page was uploaded by Cecilia Gauna on 26 May 2015. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. 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 480 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). 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