Colonisation and growth strategies in two Codium species (Bryopsidales, Chlorophyta) with different thallus forms

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(1)Published 16 June 2014. Phycologia Volume 53 (4), 353-358. Colonisation and growth strategies in two Codium species (Bryopsidales, Chlorophyta) with different thallus forms A lejandra V. G onzalez 1, J essica B eltran 2. and. B ernabe Santelices 2*. 1Departamento de Ciencias Ecologicas, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile 2Departamento de Ecologia, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile. Casilla 114-D, Santiago, Chile A bstract: In clonal macroalgae, evidence of guerrilla and phalanx growth strategies has been related to a differential response due to a heterogeneous habitat. However, some species of the green algal genus Codium may exhibit different growth strategies at different times of their development. Since the crustose species C. bernabei and the erect C. fragile had different thallus forms as well as ecological and geographic distributions, we used them to test the idea that despite morphological and growth differences, both species exhibited a similar propagation strategy. We cultured, under controlled conditions, individuals of both species to determine, first, if isolated utricles can function as propagation units; second, the type of growth at different stages of development; and, third, if species show differences in specific growth rate. Our results indicated that isolated utricles could be used for propagation because they had the ability to regenerate young mat-forming thalli in both species. Thallus regeneration implied morphological modifications of utricles, which combined guerrilla and phalanx strategies. In C. bernabei, the dynamic of vegetative propagation was independent of water movement in restoring a young thallus, which combined guerrilla and phalanx growth to colonise all available substrate. In C. fragile, water movement was required to stimulate the phalanx growth strategy and to regenerate the upright thallus. However, the guerrilla phase of C. fragile grew twice as fast as that of C. bernabei. We suggest that guerrilla-type growth is required to generate the prostrate filamentous network that first colonises new substratum. Later, the phalanx-type growth develops, generating the young mat-forming thalli in both studied species. Therefore, in these species the guerrilla strategy is not only a response to environmental constraints but also an obligate early ontogenetic stage of development. Further studies are needed to explore whether both growth strategies also occur in other species, thereby conferring them adaptive plasticity in heterogeneous environments. K ey Words: Codium bernabei, Codium fragile, Growth rate, Guerrilla strategy. Phalanx strategy, Phenotypic plasticity. INTRODUCTION Clonal terrestrial plants can exhibit different growth strategies, from guerrilla to phalanx (Lovett-Doust 1981; de Kroon & Hutchings 1995; Cheplick 1997). The guerrilla strategy involves production of loosely arranged groups of widely spaced spreading individuals (ramets); whereas, the phalanx strategy involves production of compact masses of closely spaced, clumping ramets (Lovett-Doust 1981; Che plick 1997). The two growth strategies have ecological and evolutionary importance (Cheplick 1997). The guerrilla strategy enables clonal plants to explore and colonise new habitats and to escape from less favourable places (e.g. low resource levels or high competitive stress); whereas, the phalanx strategy enables clones to consolidate or maintain favourable patches (Lovett-Doust 1981; de Kroon & Hutchings 1995; Cheplick 1997; Humphrey & Pyke 1998, 2001). Consequently, the guerrilla growth form is commonly found in early successional stages and in heterogeneous habitats; whereas, the phalanx growth form is more prominent in late successional stages and in homogeneous, more predictable habitats (Lovett-Doust & Lovett-Doust 1982). * Corresponding author (bsantelices@bio.puc.cl). DOI: 10.2216/13-251.1 © 2014 International Phycological Society. Many marine macroalgae have a modular construction and can be considered clonal organisms because they propagate by fragmentation and vegetative growth (Collado-Vides 2002a; Santelices 1999, 2004). Among seaweeds, some species, such as Gelidiella acerosa (Forsskal) Feldmann & G. Hamel and Laurencia brachyclados Pilger, exhibit the typical guerrilla growth (McDermid 1988, 1990); whereas, others, such as Laurencia dotyi Saito, Laurencia crustiformans McDermid and Ascophyllum nodosum (Linnaeus) Le Jolis, can display phalanx growth patterns (McDermid 1988, 1990; Viejo & Aberg 2001). Some species, such as Asparagopsis armata Harvey, can combine phalanx and guerrilla growth strategies for competition for space and as a plasticity response due to a heterogeneous habitat (Monro & Poore 2005). Among green seaweeds, the genus Caulerpa J.V. Lamouroux shows a guerrilla-type growth in response to light and nutrient limitation (Collado-Vides & Robledo 1999; Collado-Vides 2002b), but no other group of green algae has been studied with this perspective. Codium is a marine macroalgal genus with cosmopolitan distribution (Oliveira-Carvalho et al. 2010). Some species of this genus may exhibit a combination of guerrilla and phalanx growth strategies at different times in their development. In Codium fragile (Suringar) Hariot and C. bernabei Gonzalez, Chacana & Silva, utricles have the ability to change their morphology and form elongated filaments, able to attach to the substratum, colonise new substratum and later branch into new utricles (Nanba et al. 353.

(2) 354. Phycologia, Vol. 53 (4). Figs 1-16. Vegetative growth in Codium bernabei and C. fragile from isolated utricles. Fig. 1. Initial isolated utricle from C. bernabei. Scale bar = 250 (im. Fig. 2. Initial isolated utricle from C. fragile. Scale bar = 250 pm. Fig. 3. Elongation of the utricle and filament production in C. bernabei after 10 ± 2 days in culture conditions. Dotted arrow shows subapical filament (sa), and solid arrow shows basal elongation of utricles (ba). Scale bar = 250 pm..

(3) Gonzalez et al: Growth strategies in Codium bemabei and C. fragile 2000; Gonzalez & Santelices 2008; Gonzalez et al. 2012). Additionally, these utricles may achieve intercrust fusions, often overgrowing other competitors (Gonzalez & Santelices 2008). Interestingly, C. bernabei and C. fragile have different thallus forms as well as different ecological and geographic distributions. The thallus of C. bernabei is entirely prostrate; whereas, C. fragile has a prostrate base with upright branches (up to 20 cm tall). Codium bernabei is a frequent mid- to low intertidal member of wave-exposed, almost vertical rocky areas in central Chile; C. fragile is more frequent in low intertidal and shallow subtidal habitats, exposed to water currents, but infrequently exposed to wave impact. Geographically, C. bernabei is restricted to the continental coast of Chile from 25°S to about 40°S (Gonzalez et al. 2012); whereas, C. fragile is a widespread invader found along European, Asian and American coasts (Trowbridge 1998). Due to its fast growth from a single utricle, C. fragile can form full-grown spongy thalli after a few days under water movement conditions (Ramus 1972; Yotsui & Migita 1989; Park & Sohn 1992; Nanba et al. 2000).. In order to test the hypothesis that in spite of morpho logical, ecological and geographic differences both species exhibit a similar combination of propagation strategies, we cultured both species under controlled conditions to determine the growth type at different developmental stages. Additionally, we evaluated the effects of ecological factors (e.g. water movement) on the morphological differentiation of both species. Finally, we compared the growth rate between species under similar culture conditions. In spite of the large numbers of species known in the genus Codium and its widespread geographic and ecological distribution (Ver bruggen et al. 2007), we lack detailed information of its ontogeny and development patterns. In this context, this work provides a descriptive framework for understanding the effective strategies to colonise available substrata in two Codium species with different thallus forms.. 355. MATERIAL AND METHODS. Four intertidal populations of Codium bernabei were sampled from March to September 2012 along the central Chilean coast, covering approximately 500 km of its distribution: Maitencillo (32°38'S-71026,W), Horcon (32°43'S-71°30'W), Torpederas (33°0TS-71°38'W) and Pichilemu (34°22'S-72°00'W). Samples of C. fragile were collected from intertidal habitats in Calderilla Bay (27°04'S-70°50'W). For both species, three to seven crustose or erect thalli were collected from different rocks, located at least 5 m away from each other. Samples were then transported in labelled plastic bags to the laboratory at temperature of 4°C in a cooler. A total of 24 utricles were isolated from one to five thalli (crusts) of each species (Maitencillo, Horcon, Torpederas and Pichilemu for C. bernabei, Calderilla for C. fragile), following the methodology developed for Codium fragile by Nanba et al. (2000). Thallus tissue samples (approximately 2 cm2) were cleaned in beakers with filtered seawater and scrubbed to eliminate invertebrates and sand. Then the tissue samples were sonicated for 10 seconds in beakers, changing the seawater two times. From each clean tissue sample, dissociated utricles with medullary filaments were isolated using pipettes. Utricles were incubated in plastic multiplates (TPP, Techno Plastic Products AG, Trasadingen, Switzer land) under controlled conditions (constant temperature 12 ± 2°C, irradiance 20 ± 10 pmol photons m~2 s-1 and photoperiod 12:12 light:dark). During the initial 15 days, utricles were maintained without movement with enriched seawater (SFC medium; Correa & McLachlan 1991), ampicillin (50 pg/ml) and germanium dioxide. After 15 days under the controlled conditions, we evaluated the state of the utricles and quantified the number of live utricles (defined as turgid, green utricles) for each population and species. Utricle survivorships were compared using G test with Bonferroni adjustments (Sokal & Rohlf 1995). The surviving utricles were maintained for an. Fig. 4. Elongation of the utricle and filament production in C. fragile after 10 ± 2 days in culture conditions. Arrow shows basal elongation of utricles (ba). Scale bar = 250 pm. Fig. 5. Numerous primordia (buds) emerge in several points along the elongated utricles of C. bernabei after 15 ± 2 days in culture conditions. Scale bar = 300 pm. Fig. 6. Close-up view of primordia emerging from elongated utricles in C. bernabei after 15 ± 2 days in culture conditions. Scale b a r= 100 pm. Fig. 7. Close-up view of primordia emerging from elongated utricles in C. fragile after 15 ± 2 days in culture conditions. Scale b a r= 100 pm. Fig. 8. Network with numerous intersecting branchlets in C. bemabei after 45 ± 2 days in culture conditions. Scale bar = 500 pm. Fig. 9. Network with numerous intersecting branchlets in C. fragile after 15 ± 2 days in culture conditions. Scale bar = 500 pm. Fig. 10. Buds that differentiate into cylindrical to clavate utricles in C. bernabei after 60 ± 2 days in culture conditions. Scale b a r= 1000 pm. Fig. 11. Close-up view of cylindrical to clavate utricles in C. bernabei with rounded apices after 60 ± 2 days in culture conditions. Scale bar = 500 pm. Fig. 12. Growth pattern of C. bernabei with water movement after 60 ± 2 days in culture conditions. Scale bar = 250 mm. Fig. 13. Growth pattern of C. fragile without water movement after 45 ± 2 days in culture conditions. Scale bar = 250 mm. Fig. 14. Growth pattern of C. fragile with water movement after 45 ± 2 days in culture conditions. Scale bar = 250 mm. Fig. 15. Erect and dichotomous thalli of C. fragile formed with water movement after 60 ± 2 days in culture conditions. Scale b a r= 1000 pm. Fig. 16. Close-up view of dichotomous thalli of C. fragile formed by numerous mucronate utricles and grown with water movement after 60 ± 2 days in culture conditions. Scale bar = 500 pm..

(4) 356. Phycologia, Vol. 53 (4). Table 1. Survival and growth strategies in C. bernabei and C. fragile under culture conditions. Species. P o p u la tio n. Survival (%). C. bernabei. M aitencillo. 97.2 ± 2.4. H orcon. 90.0 ± 6.9. T orpederas. 87.5 ± 0.0. Pichilem u. 79.0 ± 0.0. C alderilla. 100 ± 0.0. C. fragile. No. T re a tm e n t1 WO WM WO WM WO WM WO WM WO WM. utricles 11 12 11 11 12 9 12 7 12 12. ± ± ± ± ± ± ± ± ± ±. 1 0 1 1 1 1 1 1 0 0. U tricles w ith guerrilla grow th (after 15 days) 11 12 11 11 12 9 12 7 12 12. ± ± ± ± ± ± ± ± ± ±. 1 0 1 1 1 1 1 1 0 0. U tricles w ith p h alan x grow th (after 60 days) 11 12 11 11 12 9 12 7. ± ± ± ± ± ± ± ± 0 12 ±. 1 0 1 1 1 1 1 1 0. 1 WO, without water movement; WM, with water movement. additional 45 days under the controlled conditions previ ously described. Enriched seawater (SFC medium) was exchanged three times a week. Fifty percent of the surviving utricles of each species and populations were maintained in culture conditions without water movement; whereas, the remaining 50% of utricles were subjected to shaker movement in a beaker shaker (100 rpm, SOI orbital shaker, Stuart Scientific, Stone, Staffordshire, UK). All morpholog ical modification of the utricles were monitored and photographed once a week using a Cool Snap-Pro Camera (Media Cybernetics, Inc., Rockville, Maryland USA) mounted on stereoscopic Lupe Nikon SMZ-10A microscope (Nikon Corp., Tokyo, Japan). In order to compare differences in growth rate between species and among populations, 30 utricles attached to substratum from four genetically different populations (Gonzalez 2007) were cultured for 60 days (Maitencillo, Torpederas and Pichilemu for Codium bernabei', Calderilla for C. fragile). They were monitored every week and maintained under controlled conditions with or without water movement. Enriched seawater (SFC medium) was exchanged three times a week. Utricle growth was measured by comparing pictures from day 1 (Tiday) versus day 60 (Teodays) under these culture conditions using a 2 X 2-cm quadrat with hundreds of points (considered as 100%). Data were transformed to percent coverage (PC) of utricles within the quadrats. Finally, the specific growth rate (SGR) was calculated as SGR = In (PC 60days/PCiday)/T60days (Ramus & Venable 1987). Differences among populations and between species were compared using one-way analysis of variance (Sokal & Rohlf 1995).. RESULTS Utricles from Codium bernabei and C. fragile incubated under laboratory conditions (Figs 1, 2) showed 79-100% survival. Utricle survival (Table 1) was similar among the Torpederas, Horcon and the Pichilemu populations of C. bernabei. Significant differences in utricles survival were found between Maitencillo and the other three populations of C. bernabei (97.2% Maitencillo vs 90% Horcon: G test = 4.5, v = 1, P = 0.01; 97.2% Maitencillo vs 87.5% Torpederas: G test = 7.5, v = 1, P = 0.007; 97.2% Maitencillo vs 79% Pichilemu: G test = 17.0, v = 1, P < 0.0001) and between C.. fragile (Calderilla population = 100%) vs all the populations of C. bernabei [C. fragile vs C. bernabei (Maitencillo): G test = 3.92, v = 1, P = 0.048; C. fragile vs C. bernabei (Horcon): G test = 14.39, v = 1; P < 0.0001; C. fragile vs C. bernabei (Torpederas): G test= 18.6, v= 1, P < 0.0001; C. fragile vs C. bernabei (Pichilemu): G test = 31.5, v = 1, P < 0.0001], Utricles from different populations and species exhibited a similar growth pattern, starting with the elongation of the basal portion of the utricles. After 10 days of culture, the elongation reached up to three to four times the initial utricle length. In a few cases (8-10% of the cases in C. bernabei), filament production occurred at the apical or subapical (Fig. 3, dotted arrow) portion of utricles. In all other cases (100% in C. fragile and 88-90% in C. bernabei), filament production started at the base or between the base of the utricle and the medullary filament (see arrow in Fig. 3 for C. bernabei and Fig. 4 for C. fragile). After 15 ± 2 days, the elongated utricles of both species, initially unattached in the petri dish, adhered to the substrate and continued a prostrate growth, increasing to 6-10 times their initial length. Once adhered, numerous discrete primordia formed from the swollen tip of medullary filaments (buds) and emerged at several points along the elongated utricles (Figs 5-7). Numerous branches arose from these projections that adhered to the substratum and elongated, forming new branches similar to the medullary filaments. The bud production and the prostrate growth occurred several times, forming third- and fourth-order branches. After 30 days, the filaments of both species formed a network with numerous intersecting branchlets (Figs 8, 9). These utricle transformations were possible by protoplasm movement and migration of numerous plastids, which led to the formation of cellular projections. After 60 ± 2 days of growth, the filamentous network in C. bernabei produced buds in close proximity. Later they differentiated as cylindrical to clavate utricles with rounded apices (Fig. 10). This portion of the prostrate, filamentous thalli full of utricles gradually acquired the shape of a small crust of C. bernabei, similar to those often found in the field (Fig. 11). New projections then emerged from the new utricles and attached to the substrate, forming a new network next to the initial young thallus, which was thus filled with new utricles, filling up the substrate with new projections. After numerous events of filament elongation,.

(5) Gonzalez et al.: Growth strategies in Codium bernabei and C. fragile. (Calderilla). (Maitencillo). (Torpederas). (Pichilemu). Fig. 17. Comparison of specific growth rates among different populations of C. bernabei and C. fragile after 60 ± 2 days in culture conditions with water movement. Different letters indicate significant differences (P < 0.001).. followed by generations of erect utricles, the young thallus covered the entire available surface. The above development patterns, guerrilla-type growth (bud formation, filament elongation and invasion of new areas) alternating with phalanx-type growth (massive differ entiation o f erect utricles) was shown by C. bernabei under the two water movement treatments. W ater movement did not make any difference in the growth strategy of this species (Fig. 12). In the case of C. fragile, two kinds of responses were shown after 60 days of culture. The treatm ents without water movement indefinitely maintained the guerrilla-type growth (Fig. 13). In contrast, only treatm ents of C. fragile with water movement showed phalanx growth with numer ous upright utricles arising from the prostrate filament network (Fig. 14). The cylindrical to clavate utricles with the characteristic m ucronate apices of C. fragile differentiated at several precise points. From there, they grew intermingled helically, forming the erect and dichotom ous thalli charac teristic of this species (Figs 15, 16). Com parisons of the vegetative growth rate between species and among populations were made only in the guerrilla-type grow th. A fter 60 ± 2 days in culture conditions, the species and populations showed significant differences in their specific growth rate (Fig. 17; F3>23 = 48.04, P < 0.001). Even though both species showed similar guerrilla-type growth, the highest daily growth rate was observed in C. fragile, which grew 1.7 to 2.9 times more than any population of C. bernabei. Moreover, within C. bernabei, the Maitencillo population had a significantly lower growth rate than either the Torpederas or the Pichilemu population (Fig. 17).. DISCUSSION Although utricle survival varied am ong the populations and species of Codium tested, the isolated utricles may function as propagation units in all populations studied. Utriclebased growth and propagation in both species involved initial utricle elongation followed by adhesion to the substratum , then bud form ation acting as initial branches,. 357. and later consolidation o f the p ro strate filam entous network. These coenocytic transform ations were made possible by cytoplasmic streaming and migration of plastids, which led to the form ation of cellular projection. N anba et al. (2002) described a similar pattern of protoplasm movement in C. fragile, suggesting that the protoplasm plays an im portant role in the vegetative propagation of the isolated utricles and filamentous thalli. Moreover, studies on C. fragile have described the form ation of a filamentous stage in which the thalli remain viable long term and can develop into full-grown spongy thalli (Ramus 1972; Park & Sohn 1992). Similar to descriptions of clonal growth in land plants, we observed a guerrilla and phalanx pattern of vegetative propagation in both species of Codium. In macroalgae, the change from phalanx to guerrilla growth form has been explained as a strategy to escape environmental stress, including limiting levels of light intensity, nutrients, and sedimentation (e.g. Collado-Vides 2002b; M onro & Poore 2005). In our study, we found that guerrilla-type growth is first required to generate the prostrate filamentous network colonising the new substratum. Later, phalanx-type growth develops in both species. Thus, the guerrilla growth strategy is not only a response to environmental constraints but also an obligate early ontogenetic stage of development in these species. Studies in clonal species with different organisation levels are required to evaluate the generalisation of guerrilla growth in early stages of development. In general, inform ation is scarce on the factor(s) that in seaweed trigger changes from guerrilla- to phalanx-type growth. In the two species of Codium tested here, the mode of vegetative propagation of C. bernabei was independent of w ater movem ent, and combined repeated patterns of guerrilla- and phalanx-type growth resulted in colonisation of all available substrata. By contrast, in C. fragile, water movement was required to stimulate phalanx growth and regenerate the erect thallus. These results are consistent with previous findings for this species which indicate that photon flux intensities above 60 pmol photons nT 2 s-1 and water movement are needed to stimulate phalanx-type growth (Ramus 1972; Park & Sohn 1992; N anba et al. 2005). From an evolutionary perspective, the guerrilla-phalanx continuum can be interpreted as an adaptive response of m odular plants to patchy resource supply, thereby ensuring resource acquisition. Compact, densely branched m orphol ogies (i.e. phalanx growth strategy) have the ability to exploit patches of high resource supply; whereas, elongated, sparsely branched m orphologies (i.e. guerrilla growth strategy) rapidly explore new environments or escape less suitable environments (Lovett-Doust 1981). Seaweed studies have focussed mostly on describing one or the other type of growth form but seem to have missed the biological consequences of exhibiting both strategies in the same generation. So far, this has been described in Asparagopsis armata and the two species of Codium studied here. Further studies are needed to explore if a double growth strategy also occurs in a species with two phases of generation (haplodiplontic life cycles), conferring to both phases and taxa adaptive plasticity in a heterogeneous environment..

(6) 358. Phycologia, Vol. 53 (4). ACKNOWLEDGEMENTS We thank A. D. Mann for the grammatical improvements and valuable comments and suggestions by the associate editor (F. Leliaert) and two anonymous reviewers. Financial support for this study was obtained from FONDECYT 1120129 (B. Santelices) and FONDECYT 11110120 (A.V. Gonzalez).. REFERENCES C heplick G.P. 1997. Responses to severe competitive stress in a. clonal plant: differences between genotypes. Oikos 79: 581-591. C ollado-V ides L. 2002a. Clonal architecture in marine macro. algae: ecological and evolutionary perspectives. Evolutionary Ecology 15: 531-545. C ollado-Vides L. 2002b. Morphological plasticity of Caulerpa proiifera in relation to growth form in a coral reef lagoon. Botanica Marina 45: 123-129. C ollado-V ides L. & R obledo D. 1999. Morphology and photo synthesis of Caulerpa (Chlorophyta) in relation to growth form. Journal o f Phycology 35: 325-330. C orrea J.A. & M c L achlan J. 1991. 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