Phycologia Volume 53 (4), 353-358 Published 16 June 2014

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

A lejandra V. G onzalez1, J essica Beltran2 and Bernabe Santelices2* 1 Departamento 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

Abstract: 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 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. Key Words: Codium bernabei, , Growth rate, Guerrilla strategy. Phalanx strategy, Phenotypic plasticity

INTRODUCTION Many marine macroalgae have a modular construction and can be considered clonal organisms because they Clonal terrestrial can exhibit different growth propagate by fragmentation and vegetative growth (Colla- strategies, from guerrilla to phalanx (Lovett-Doust 1981; do-Vides 2002a; Santelices 1999, 2004). Among , de Kroon & Hutchings 1995; Cheplick 1997). The guerrilla some species, such as Gelidiella acerosa (Forsskal) Feldmann strategy involves production of loosely arranged groups of & G. Hamel and Laurencia brachyclados Pilger, exhibit the widely spaced spreading individuals (ramets); whereas, the typical guerrilla growth (McDermid 1988, 1990); whereas, phalanx strategy involves production of compact masses of others, such as Laurencia dotyi Saito, Laurencia crustifor- mans McDermid and Ascophyllum nodosum (Linnaeus) Le closely spaced, clumping ramets (Lovett-Doust 1981; Che­ Jolis, can display phalanx growth patterns (McDermid 1988, plick 1997). The two growth strategies have ecological and 1990; Viejo & Aberg 2001). Some species, such as Aspar- evolutionary importance (Cheplick 1997). The guerrilla agopsis armata Harvey, can combine phalanx and guerrilla strategy enables clonal plants to explore and colonise new growth strategies for competition for space and as a habitats and to escape from less favourable places (e.g. low plasticity response due to a heterogeneous habitat (Monro resource levels or high competitive stress); whereas, the & Poore 2005). Among green seaweeds, the genus phalanx strategy enables clones to consolidate or maintain J.V. Lamouroux shows a guerrilla-type growth in response favourable patches (Lovett-Doust 1981; de Kroon & to light and nutrient limitation (Collado-Vides & Robledo Hutchings 1995; Cheplick 1997; Humphrey & Pyke 1998, 1999; Collado-Vides 2002b), but no other group of green 2001). Consequently, the guerrilla growth form is commonly algae has been studied with this perspective. found in early successional stages and in heterogeneous Codium is a marine macroalgal genus with cosmopolitan habitats; whereas, the phalanx growth form is more distribution (Oliveira-Carvalho et al. 2010). Some species of prominent in late successional stages and in homogeneous, this genus may exhibit a combination of guerrilla and more predictable habitats (Lovett-Doust & Lovett-Doust phalanx growth strategies at different times in their 1982). development. In Codium fragile (Suringar) Hariot and C. bernabei Gonzalez, Chacana & Silva, utricles have the * Corresponding author ([email protected]). ability to change their morphology and form elongated DOI: 10.2216/13-251.1 filaments, able to attach to the substratum, colonise new © 2014 International Phycological Society substratum and later branch into new utricles (Nanba et al.

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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. Gonzalez et al: Growth strategies in Codium bemabei and C. fragile 355

2000; Gonzalez & Santelices 2008; Gonzalez et al. 2012). MATERIAL AND METHODS Additionally, these utricles may achieve intercrust fusions, often overgrowing other competitors (Gonzalez & Santelices Four intertidal populations of Codium bernabei were 2008). Interestingly, C. bernabei and C. fragile have different sampled from March to September 2012 along the central thallus forms as well as different ecological and geographic Chilean coast, covering approximately 500 km of its distributions. The thallus of C. bernabei is entirely prostrate; distribution: Maitencillo (32°38'S-71026,W), Horcon whereas, C. fragile has a prostrate base with upright (32°43'S-71°30'W), Torpederas (33°0TS-71°38'W) and Pi- branches (up to 20 cm tall). Codium bernabei is a frequent chilemu (34°22'S-72°00'W). Samples of C. fragile were mid- to low intertidal member of wave-exposed, almost collected from intertidal habitats in Calderilla Bay vertical rocky areas in central Chile; C. fragile is more (27°04'S-70°50'W). For both species, three to seven crustose frequent in low intertidal and shallow subtidal habitats, or erect thalli were collected from different rocks, located at exposed to water currents, but infrequently exposed to wave least 5 m away from each other. Samples were then impact. Geographically, C. bernabei is restricted to the transported in labelled plastic bags to the laboratory at continental coast of Chile from 25°S to about 40°S temperature of 4°C in a cooler. (Gonzalez et al. 2012); whereas, C. fragile is a widespread A total of 24 utricles were isolated from one to five thalli invader found along European, Asian and American coasts (crusts) of each species (Maitencillo, Horcon, Torpederas (Trowbridge 1998). Due to its fast growth from a single and Pichilemu for C. bernabei, Calderilla for C. fragile), utricle, C. fragile can form full-grown spongy thalli after a following the methodology developed for Codium fragile by few days under water movement conditions (Ramus 1972; Nanba et al. (2000). Thallus tissue samples (approximately 2 Yotsui & Migita 1989; Park & Sohn 1992; Nanba et al. cm2) were cleaned in beakers with filtered seawater and scrubbed to eliminate invertebrates and sand. Then the tissue 2000). samples were sonicated for 10 seconds in beakers, changing In order to test the hypothesis that in spite of morpho­ the seawater two times. From each clean tissue sample, logical, ecological and geographic differences both species dissociated utricles with medullary filaments were isolated exhibit a similar combination of propagation strategies, we using pipettes. Utricles were incubated in plastic multiplates cultured both species under controlled conditions to (TPP, Techno Plastic Products AG, Trasadingen, Switzer­ determine the growth type at different developmental stages. land) under controlled conditions (constant temperature 12 Additionally, we evaluated the effects of ecological factors ± 2°C, irradiance 20 ± 10 pmol photons m~2 s-1 and (e.g. water movement) on the morphological differentiation photoperiod 12:12 light:dark). During the initial 15 days, of both species. Finally, we compared the growth rate utricles were maintained without movement with enriched between species under similar culture conditions. In spite of seawater (SFC medium; Correa & McLachlan 1991), the large numbers of species known in the genus Codium and ampicillin (50 pg/ml) and germanium dioxide. its widespread geographic and ecological distribution (Ver­ After 15 days under the controlled conditions, we bruggen et al. 2007), we lack detailed information of its evaluated the state of the utricles and quantified the number ontogeny and development patterns. In this context, this of live utricles (defined as turgid, green utricles) for each work provides a descriptive framework for understanding population and species. Utricle survivorships were compared the effective strategies to colonise available substrata in two using G test with Bonferroni adjustments (Sokal & Rohlf Codium species with different thallus forms. 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 bar= 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 ar= 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 ar= 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 bar= 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. 356 Phycologia, Vol. 53 (4)

Table 1. Survival and growth strategies in C. bernabei and C. fragile under culture conditions.

Survival No. Utricles with guerrilla Utricles with phalanx Species Population (%) Treatment1 utricles growth (after 15 days) growth (after 60 days) C. bernabei Maitencillo 97.2 ± 2.4 WO 11 ± 1 11 ± 1 11 ± 1 WM 12 ± 0 12 ± 0 12 ± 0 H orcon 90.0 ± 6.9 WO 11 ± 1 11 ± 1 11 ± 1 WM 11 ± 1 11 ± 1 11 ± 1 Torpederas 87.5 ± 0.0 WO 12 ± 1 12 ± 1 12 ± 1 WM 9 ± 1 9 ± 1 9 ± 1 Pichilemu 79.0 ± 0.0 WO 12 ± 1 12 ± 1 12 ± 1 WM 7 ± 1 7 ± 1 7 ± 1 C. fragile Calderilla 100 ± 0.0 WO 12 ± 0 12 ± 0 0 WM 12 ± 0 12 ± 0 12 ± 0 1 WO, without water movement; WM, with water movement. additional 45 days under the controlled conditions previ­ fragile (Calderilla population = 100%) vs all the populations ously described. Enriched seawater (SFC medium) was of C. bernabei [C. fragile vs C. bernabei (Maitencillo): G test exchanged three times a week. Fifty percent of the surviving = 3.92, v= 1, P = 0.048; C. fragile vs C. bernabei (Horcon): G utricles of each species and populations were maintained in test = 14.39, v = 1; P < 0.0001; C. fragile vs C. bernabei culture conditions without water movement; whereas, the (Torpederas): G test= 18.6, v= 1, P < 0.0001; C. fragile vs C. remaining 50% of utricles were subjected to shaker bernabei (Pichilemu): G test = 31.5, v = 1, P < 0.0001], movement in a beaker shaker (100 rpm, SOI orbital shaker, Utricles from different populations and species exhibited a Stuart Scientific, Stone, Staffordshire, UK). All morpholog­ similar growth pattern, starting with the elongation of the ical modification of the utricles were monitored and basal portion of the utricles. After 10 days of culture, the photographed once a week using a Cool Snap-Pro Camera elongation reached up to three to four times the initial utricle (Media Cybernetics, Inc., Rockville, Maryland USA) length. In a few cases (8-10% of the cases in C. bernabei), mounted on stereoscopic Lupe Nikon SMZ-10A microscope filament production occurred at the apical or subapical (Fig. (Nikon Corp., Tokyo, ). 3, dotted arrow) portion of utricles. In all other cases (100% In order to compare differences in growth rate between in C. fragile and 88-90% in C. bernabei), filament production species and among populations, 30 utricles attached to started at the base or between the base of the utricle and the substratum from four genetically different populations medullary filament (see arrow in Fig. 3 for C. bernabei and (Gonzalez 2007) were cultured for 60 days (Maitencillo, Fig. 4 for C. fragile). Torpederas and Pichilemu for Codium bernabei', Calderilla After 15 ± 2 days, the elongated utricles of both species, for C. fragile). They were monitored every week and initially unattached in the petri dish, adhered to the substrate maintained under controlled conditions with or without and continued a prostrate growth, increasing to 6-10 times water movement. Enriched seawater (SFC medium) was their initial length. Once adhered, numerous discrete exchanged three times a week. Utricle growth was measured by comparing pictures from day 1 (Tiday) versus day 60 primordia formed from the swollen tip of medullary (Teodays) under these culture conditions using a 2 X 2-cm filaments (buds) and emerged at several points along the quadrat with hundreds of points (considered as 100%). Data elongated utricles (Figs 5-7). Numerous branches arose from were transformed to percent coverage (PC) of utricles within these projections that adhered to the substratum and the quadrats. Finally, the specific growth rate (SGR) was elongated, forming new branches similar to the medullary filaments. The bud production and the prostrate growth calculated as SGR = In (PC60days/PCiday)/T60days (Ramus & Venable 1987). Differences among populations and between occurred several times, forming third- and fourth-order species were compared using one-way analysis of variance branches. After 30 days, the filaments of both species formed (Sokal & Rohlf 1995). 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 RESULTS the formation of cellular projections. After 60 ± 2 days of growth, the filamentous network in Utricles from Codium bernabei and C. fragile incubated C. bernabei produced buds in close proximity. Later they under laboratory conditions (Figs 1, 2) showed 79-100% differentiated as cylindrical to clavate utricles with rounded survival. Utricle survival (Table 1) was similar among the apices (Fig. 10). This portion of the prostrate, filamentous Torpederas, Horcon and the Pichilemu populations of C. thalli full of utricles gradually acquired the shape of a small bernabei. Significant differences in utricles survival were crust of C. bernabei, similar to those often found in the field found between Maitencillo and the other three populations (Fig. 11). New projections then emerged from the new of C. bernabei (97.2% Maitencillo vs 90% Horcon: G test = utricles and attached to the substrate, forming a new 4.5, v= 1, P = 0.01; 97.2% Maitencillo vs 87.5% Torpederas: network next to the initial young thallus, which was thus G test = 7.5, v = 1, P = 0.007; 97.2% Maitencillo vs 79% filled with new utricles, filling up the substrate with new Pichilemu: G test = 17.0, v = 1, P < 0.0001) and between C. projections. After numerous events of filament elongation, Gonzalez et al.: Growth strategies in Codium bernabei and C. fragile 357

and later consolidation of the prostrate filamentous network. These coenocytic transformations were made possible by cytoplasmic streaming and migration of plastids, which led to the formation of cellular projection. Nanba et al. (2002) described a similar pattern of protoplasm movement in C. fragile, suggesting that the protoplasm plays an important role in the vegetative propagation of the isolated utricles and filamentous thalli. Moreover, studies on C. fragile have described the formation 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). (Calderilla) (Maitencillo) (Torpederas) (Pichilemu) Similar to descriptions of clonal growth in land plants, we Fig. 17. Comparison of specific growth rates among different observed a guerrilla and phalanx pattern of vegetative populations of C. bernabei and C. fragile after 60 ± 2 days in propagation in both species of Codium. In macroalgae, the culture conditions with water movement. Different letters indicate change from phalanx to guerrilla growth form has been significant differences (P < 0.001). explained as a strategy to escape environmental stress, including limiting levels of light intensity, nutrients, and followed by generations of erect utricles, the young thallus sedimentation (e.g. Collado-Vides 2002b; Monro & Poore covered the entire available surface. 2005). In our study, we found that guerrilla-type growth is The above development patterns, guerrilla-type growth first required to generate the prostrate filamentous network (bud formation, filament elongation and invasion of new colonising the new substratum. Later, phalanx-type growth areas) alternating with phalanx-type growth (massive differ­ develops in both species. Thus, the guerrilla growth strategy entiation of erect utricles) was shown by C. bernabei under is not only a response to environmental constraints but also the two water movement treatments. Water movement did an obligate early ontogenetic stage of development in these not make any difference in the growth strategy of this species species. Studies in clonal species with different organisation (Fig. 12). In the case of C. fragile, two kinds of responses levels are required to evaluate the generalisation of guerrilla were shown after 60 days of culture. The treatments without growth in early stages of development. water movement indefinitely maintained the guerrilla-type In general, information is scarce on the factor(s) that in growth (Fig. 13). In contrast, only treatments of C. fragile trigger changes from guerrilla- to phalanx-type with water movement showed phalanx growth with numer­ growth. In the two species of Codium tested here, the mode ous upright utricles arising from the prostrate filament of vegetative propagation of C. bernabei was independent of network (Fig. 14). The cylindrical to clavate utricles with the water movement, and combined repeated patterns of characteristic mucronate apices of C. fragile differentiated at guerrilla- and phalanx-type growth resulted in colonisation several precise points. From there, they grew intermingled of all available substrata. By contrast, in C. fragile, water helically, forming the erect and dichotomous thalli charac­ movement was required to stimulate phalanx growth and teristic of this species (Figs 15, 16). regenerate the erect thallus. These results are consistent with Comparisons of the vegetative growth rate between previous findings for this species which indicate that photon species and among populations were made only in the flux intensities above 60 pmol photons nT2 s-1 and water guerrilla-type growth. After 60 ± 2 days in culture movement are needed to stimulate phalanx-type growth conditions, the species and populations showed significant (Ramus 1972; Park & Sohn 1992; Nanba et al. 2005). differences in their specific growth rate (Fig. 17; F3>23 = 48.04, From an evolutionary perspective, the guerrilla-phalanx P < 0.001). Even though both species showed similar continuum can be interpreted as an adaptive response of guerrilla-type growth, the highest daily growth rate was modular plants to patchy resource supply, thereby ensuring observed in C. fragile, which grew 1.7 to 2.9 times more than resource acquisition. Compact, densely branched morphol­ any population of C. bernabei. Moreover, within C. bernabei, ogies (i.e. phalanx growth strategy) have the ability to exploit the Maitencillo population had a significantly lower growth patches of high resource supply; whereas, elongated, sparsely rate than either the Torpederas or the Pichilemu population branched morphologies (i.e. guerrilla growth strategy) (Fig. 17). 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 DISCUSSION growth form but seem to have missed the biological consequences of exhibiting both strategies in the same Although utricle survival varied among the populations and generation. So far, this has been described in Asparagopsis species of Codium tested, the isolated utricles may function armata and the two species of Codium studied here. Further as propagation units in all populations studied. Utricle- studies are needed to explore if a double growth strategy also based growth and propagation in both species involved occurs in a species with two phases of generation (haplodi- initial utricle elongation followed by adhesion to the plontic life cycles), conferring to both phases and taxa substratum, then bud formation acting as initial branches, adaptive plasticity in a heterogeneous environment. 358 Phycologia, Vol. 53 (4)

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