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MEIOSPORES PRODUCED IN SORI OF NONSPOROPHYLLOUS LAMINAE OFMACROCYSTIS PYRIFERA (LAMINARIALES, PHAEOPHYCEAE) MAY ENHANCE

REPRODUCTIVE OUTPUT1

Pablo P. Leal 2

Department of Botany, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand

Catriona L. Hurd

Department of Botany, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand

Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, Australia

and Michael Y. Roleda

Department of Botany, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand

Bioforsk Norwegian Institute for Agricultural and Environmental Research, Kudalsveien 6, Bodø 8049, Norway

Different lamina of Macrocystis pyrifera sporophytes(i.e., sporophylls, pneumatocyst-bearing blades, andapical scimitars) in a wave-sheltered site were foundto be fertile. We quantified their sorus surface area,reproductive output (number of spores released) andthe viability of released spores (germination rate).Sorus area was greatest on the sporophylls, withsporangia developing on >57% of the total area andsmallest on the pneumatocyst-bearing blades with21% of the total area bearing sporangia. The apicalscimitar released the greatest number of meiospores(cells � mL�1 � cm�2) and the sporophylls the least.Meiospores produced from all types of fertilelaminae were equally viable. This reproductiveplasticity may enhance reproductive output, andcontribute to short and long-distance spore dispersaland the cryptic gametophyte propagule bank for thenext generation of sporophytes.

Key index words: germination; Macrocystis pyrifera;reproductive control; scimitar; sorus; spore viability;sporogenesis; sporophylls

The order Laminariales has phylogenetically beendivided in four families: Alariaceae, Costariaceae,Laminariaceae, and Lessoniaceae (Lane et al.2006). The family Alariaceae includes the generaAlaria, Lessoniopsis, Pleurophycus, Pterygophora, andUndaria; in the family Costariaceae are the generaAgarum, Costaria, Dictyoneurum, and Thalassiophyllum;in the Laminariaceae are the genera Cymathaere, He-dophyllum, Kjellmaniella, Laminaria, Macrocystis, Nereo-cystis, Pelagophycus, Postelsia, and Saccharina; and in

the Lessoniaceae are the genera Ecklonia, Eckloniop-sis, Egregia, Eisenia, and Lessonia (Lane et al. 2006).The life cycle of algae in the order Laminarialesconsists of an alternation of generations betweenmacroscopic sporophytes and microscopic gameto-phytes. Sporophytes form sori, the reproductive tis-sue with grouped unilocular sporangia wherehaploid biflagellated zoospores are formed (Neus-hul 1963, Graham et al. 2007, Bartsch et al. 2008,Kawai et al. 2013).Within families in the order Laminariales, sori

occur on different parts of the thallus. For generain the family Alariaceae sori are formed on the sur-face of the specialized lamina, called sporophylls,located at the base of the vegetative frond (Widdow-son 1971, Pfister 1992, Castric-Fey et al. 1999, Kraanand Guiry 2000, Silva 2009), except for the genusPleurophycus that produces sori on its blade and mid-rib (Germann 1986, Dominik and Zimmerman2006). In contrast, members of the family Costaria-ceae only produce sori on vegetative tissue (Angst1927, Sanbosunga and Hasegawa 1967, Silva 1991,Boo et al. 2011, Guiry and Guiry 2013). In the fam-ily Laminariaceae, the genera Cymathaere, Hedophyl-lum, Kjellmaniella, Laminaria, Nereocystis, Pelagophycus,Postelsia, and Saccharina form sporogenic tissue onthe surface of the vegetative frond (Herbst andJohnstone 1937, Widdowson 1965, Kain 1975,L€uning 1988, tom Dieck 1991, Blanchette 1996,L€uning et al. 2000, Bartsch et al. 2008, Mizuta andYasui 2010, Guiry and Guiry 2013) but the monospe-cific genus Macrocystis (Coyer et al. 2001, Demeset al. 2009, Macaya and Zuccarello 2010) producebasal sporophylls, like members of the family Alaria-ceae (Neushul 1963, Lobban 1978). In the familyLessoniaceae, genera Ecklonia, Eckloniopsis, andLessonia produce sori on the vegetative frond

1Received 16 July 2013. Accepted 2 November 2013.2Author for correspondence: e-mail [email protected] Responsibility: C. Amsler (Associate Editor)

J. Phycol. 50, 400–405 (2014)© 2014 Phycological Society of AmericaDOI: 10.1111/jpy.12159

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(Papenfuss 1942, Womersley 1967, Venegas et al.1992, Tsutsui and Ohno 1993, Aruga et al. 1997)but genera Egregia and Eisenia produce basal sporo-phylls (Blanchette et al. 2002, Henkel and Murray2007, Guiry and Guiry 2013). The kelp Aureophycusaleuticus, which is not classified in a laminarean fam-ily due to its exceptional features (e.g., sporophytemorphology), forms sori on its semidiscoidal hold-fast (Kawai et al. 2013).

Whether reproductive structures in the familyAlariaceae are limited to the sporophylls has beenexamined. Alaria nana produced sori on the vegeta-tive blade after sporophylls were experimentallyremoved (Pfister 1991). In the field, natural popula-tions of Undaria pinnatifida and Alaria crassifolia pro-duced sori on their blades, usually toward the endof their reproductive period (Stuart et al. 1999,Kumura et al. 2006). Sori have also been observedon the midrib of U. pinnatifida (Sanbosunga andHasegawa 1967), and on stipes of a LessoniaceaeLessonia nigrescens (Venegas et al. 1992). InM. pyrifera (Linnaeus) C.Agardh, the only sporo-phyll-bearing species in the family Laminariaceae,different vegetative laminae (i.e., frond initials, sur-face-canopy blades and apical scimitars) have beenreported to bear sori (Brandt 1923, Neushul 1963,Lobban 1978, Graham et al. 2007). However, thesereports are vague, short, and descriptive. Here, wedetail the morphology (surface area) of sori fromdifferent laminae (i.e., sporophylls, pneumatocyst-bearing blades, and apical scimitars) and quantifytheir reproductive output (numbers of meiosporesreleased) and the postsettlement viability (germina-tion rate) of released meiospores. The ecologicalimplications of developing reproductive structureson different parts of the sporophyte frond arediscussed.

MATERIAL AND METHODS

Seaweed collection. During low tide in spring 2012, ten adultsporophytes of M. pyrifera (4.0 � 1.5 m; hereafter Macrocystis)were collected in the upper sublittoral of a wave-sheltered sitein Hamilton Bay (45°47′ 51″ S; 170°38′ 39″ E), Otago Har-bour, New Zealand. The specimens bore sori on differentparts of their frond. Fertile laminae were classified accordingto the general classification of Lobban (1978): sporophyll, SP(Fig. 1a), specialized smooth lamina without a pneumatocyst;blade, BL (Fig. 1b), lamina with a corrugated surface andpneumatocyst; and apical scimitars, SC (Fig. 1c), lamina withunilateral divisions.

Sorus area, meiospore culture, and germination. Sporophyteswere individually processed by separating and counting allsorus-bearing laminae according to the three categories aboveand photographing each lamina with a ruler. Sorus area(Fig. 1) on different sorus-bearing laminae was measuredusing the image-analysis software ImageJ (Schneider et al.2012) and expressed as percentage of the total lamina area.Fertile laminae were gently cleaned of epiphytes by brushingthem under filtered (0.2 lm) seawater. Samples werewrapped in moist tissue paper and stored overnight at 4°C.The next day, meiospores from different sorus-bearinglaminae (n = 6, from different individual sporophytes) were

separately released by immersing 2 cm2 sorus, excised with acore sampler from the darkest sorus area (equivalent to SorusClass 2 as described by Bartsch et al. 2013), into 10 mL of0.2 lm-filtered seawater for 15 min. The sorus was thenremoved and the number of meiospores released wascounted using a hemocytometer (0.1 mm depth, bright-line,Marienfeld, Germany). Meiospore densities were adjusted to20,000–25,000 cell � mL�1 and separately dispensed ontoeach compartment of the six-well polystyrene tissue culturevessels (Costar 3516; Corning Incorporated, New York, NY,USA). Meiospores were cultivated in a temperature-controlledroom at 12°C under a 12:12 h light:dark photoperiod of50 lmol photons � m�2 � s�1 of PAR (cool-white fluorescent;Philips, Eindhoven, the Netherlands). The number of mei-ospores that germinated was counted after 3 d. At least fiverandomly chosen visual fields using a 109 objective of aninverted microscope (Olympus CK2; Olympus Optical Co.Ltd., Tokyo, Japan) were photographed using a video camera(5.1M CMOS camera, UCMOS0510KPA). Photographs wereviewed using the digital camera software ToupView 3.5 where350 meiospores were classified and counted to measure ger-mination rates according to Roleda et al. (2012).

Statistical analysis. Percentage data (sorus area and meios-pore germination) were logit transformed (Warton and Hui2011) and meiospore release data were log transformed tomeet the ANOVA assumptions. The Kolgomorov-Smirnow testwas used to test Normality and the Levene’s test to test homo-geneity of variances. One-way ANOVA (P < 0.05) was used totest lamina-specific differences in sorus size, number of mei-ospores released and the percentage germination. The posthoc Tukey test was applied when significant differences wereencountered. All statistical analyses were run in SigmaStat2.03 (SPSS Inc., Chicago, IL, USA).

RESULTS

Sorus area. Sori occurred in each of the threetypes of lamina (Fig. 1). Sorus area was greatest onthe sporophylls (Fig. 2a) with sporangia developingon >57% of the total area and smallest on the pneu-matocyst-bearing blades with 21% of the total area

FIG. 1. Sorus-bearing lamina in Macrocystis pyrifera. (a) Sporo-phyll, (b) pneumatocyst-bearing blade, and (c) apical scimitar;scale bar = 2 cm. Corresponding line illustrations show the typi-cal location where sori (S) are found in each lamina-type.

SORI ON NONSPOROPHYLLOUS LAMINAE 401

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becoming fertile. This difference in sorus size wasstatistically significant (ANOVA: F2,108 = 12.029,P < 0.001) and a post hoc test (Tukey, P < 0.05)revealed SP > SC = BL.Meiospore release. Meiospores released from differ-

ent types of fertile laminae ranged from 5.15 9 103

to 6.35 9 104 cells � mL�1 � cm�2. The number ofmeiospores released varied between the fertile lami-nae (ANOVA: F2,26 = 605.903, P < 0.001) with thescimitar releasing the most meiospores and the spo-rophylls the least (Tukey, P < 0.05; SP < BL < SC;Fig. 2b).

Meiospore germination. Meiospore germination after3 d post cultivation ranged from 39% to 66% (Fig. 2c)and there was no significant difference in the develop-ment of meiospores from the different laminae(ANOVA: F2,17 = 1.547, P = 0.245).

DISCUSSION

It seems that reproduction in nonsporophylloustissue among species in the sporophyllous familyAlariaceae (Sanbosunga and Hasegawa 1967, Pfister1991, Stuart et al. 1999, Kumura et al. 2006) andfor the sporophyllous Macrocystis (Brandt 1923,Neushul 1963, Lobban 1978, this study) is not un-usual. Results of our study show that meiosporesproduced from sporophyllous and nonsporophyl-lous (blade and apical scimitar) lamina are equallyviable, and germination rates are within the rangepreviously reported (Roleda et al. 2012).The question of why sporophyll-bearing Laminari-

ales usually do not reproduce on vegetative parts ofthe frond had been asked previously. Pfister (1992)suggested three hypotheses: (i) confining reproduc-tion to the sporophylls permits vegetative fronds toremain a fast growing, “photosynthetic organ.” Ifthis hypothesis is correct, a decrease in vegetativegrowth prior to reproduction is predicted; in sup-port of this idea sporogenesis in the Laminarialesgenerally occurs when the growth rate decreases(Kain 1975, Bartsch et al. 2008), although this idearequires testing for Macrocystis. (ii) Sporogenesis onnonsporophyllous laminae is selected againstbecause they are removed or damaged by waves inwave-exposed sites. This idea may explain why weencountered fertile apical scimitars only inwave-sheltered sites. In a demographic survey ofMacrocystis in Southern New Zealand, all sporo-phytes from wave-exposed sites have a torn apicalscimitar (P. Leal, unpublished data). (iii) “Physio-logical constraints maintain reproduction on thesporophylls” (Pfister 1992) whereby internal chemi-cal cues mediate sporogenesis. Growth substanceshave been related to sori formation in Laminaria dig-itata (Hudson) J.V.Lamouroux, for which the pres-ence of sporangium inhibitor substances keep theyoung frond free of sori during the season of rapidgrowth (Buchholz and L€uning 1999, L€uning et al.2000). Similarly, the application of high externalconcentrations of indole-acetic acid induced vegeta-tive growth and delayed sori formation in Saccharinajaponica (Areschoug) C.E. Lane, C. Mayes, Druehl &G.W. Saunders [= L. japonica; = L. ochotensis] (Kaiet al. 2006). The exposure to exogenous abscisicacid produced an opposite reaction, at high concen-tration it suppressed growth and promoted the spo-rogenesis of the same species (Nimura and Mizuta2002). Putative growth substances have beendetected in Macrocystis (de Nys et al. 1991) but anyrole in controlling the onset of sporogenesis isunknown (Stirk et al. 2003).

FIG. 2. Macrocystis pyrifera (a) sorus area, expressed as percent-age of the total surface area of the bearing-sorus laminae, (b)number of spores released per sorus area per individual sporo-phyte, and (c) corresponding percentage germination. Sorus-bearing laminae are sporophylls (SP), pneumatocyst-bearingblades (BL), and apical scimitar (SC). Bars represent themean � SD. Different letters above the bar indicate a significantdifference (Tukey test, P = 0.05).

402 PABLO P. LEAL ET AL.

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An alternative explanation is that the develop-ment of sorus on blades and apical scimitars couldbe a trait that enhances long distance meiosporedispersal in area of slow flows and such a trait mightbe inheritable. However, meiospores released intothe water column may take longer time to swim andsettle into the benthos and could therefore beexposed to high irradiance and UVR compromisingtheir viability (i.e., germ tube, gametophyte, andsporophyte developments; Edwards 1998, Roledaet al. 2006, Cie and Edwards 2008). Therefore, meio-spores originating from distal blades and apicalscimitars may play an integral role in successfulrecruitment only if they are released and disperseduring periods of low irradiance, e.g., at night, dur-ing cloudy or overcast days (Amsler and Neushul1989, Cie and Edwards 2008) or during low tidewhen the whole Macrocystis frond lay prostrate andcloser to the benthos (P. Leal, personal observation)away from the holdfast where sporophylls arelocated. Fertile scimitars and blades released morecells per unit area compared to the sporophylls, butthe latter have more biomass per unit sporophyte.Therefore, sporophylls may generate more meio-spores settling proximate to the parental sporo-phyte. The overall meiospore output from eachtissue type to the population’s banks of microscopicspores (cf “seed banks”; Schiel and Foster 2006) andthe next generation of sporophytes requires furtherstudy.

We know surprisingly little about the controls ofsporogenesis in Macrocystis compared to other mem-bers of the Laminariales and the environmentaland/or endogenous triggers that control the devel-opment of sori on nonsporophyllous lamina of Mac-rocystis are currently unknown. Sorus production insporophylls of Macrocystis can continuously occurwith an appropriate translocation of photosynthatesfrom the surface canopy (Neushul 1963, Reed 1987,Reed et al. 1996, Dayton et al. 1999, Graham 2002).Control of reproduction in kelps has often beenattributed to physico-chemical factors such as photo-period, temperature, and nutrients (tom Dieck1991, Bartsch et al. 2008). For example, in S. latiss-ima and Laminaria setchellii short photoperiodinduced cessation of blade growth followed by theformation of sori (L€uning 1988, tom Dieck 1991).Nutrient availability also strongly affects the repro-duction of Laminariales. Nutrient-rich media (i.e.,phosphorous and nitrogen) enhanced sorus forma-tion in S. angustata (Mizuta et al. 1999, Nimuraet al. 2002), A. crassifolia and U. pinnatifida (Kumuraet al. 2006). All these species have a higher internalcontent of phosphorous and nitrogen in sporoge-nous tissue than in vegetative tissue, indicating astrong influence of nutrients on reproduction ofLaminariales (Nimura et al. 2002, Kumura et al.2006, Bartsch et al. 2008). Additionally in S. japon-ica, the interaction of low water temperature, longphotoperiod, and low nutrients delayed the onset of

sorus formation (Mizuta et al. 1999). Accordingly,the formation of sori in natural populations of Lam-inariales seems to be restricted to periods of low orno growth in autumn to winter, coinciding withdecreasing day-length and temperature and increas-ing nutrient availability (Kain 1975, Bartsch et al.2008). However, for Macrocystis sori may be presentthroughout the year, as observed and reported inthe southeast and northeast Pacific coast of bothhemispheres (Reed et al. 1996, Buschmann et al.2004, 2006), and southeastern coasts of NewZealand (P. Leal, unpublished data).One reason that we know so little about develop-

mental regulation in Macrocystis is its size – unlikeLaminaria listed above, it cannot be easily cultured toa mature reproductive state in the laboratory,thereby limiting our understanding of the factorscausing sporogenesis. If sporogenesis can be inducedon isolated discs of different Macrocystis laminae (i.e.,sporophyll, pneumatocyst-bearing blade, and scimi-tar) as in other Laminariaceae species (e.g., Buch-holz and L€uning 1999, Gruber et al. 2011), someoutstanding questions may be answered on the envi-ronmental (physico-chemical cues) and physiological(e.g., age and size class, and growth substances) fac-tors, that control of sporogenesis in this species.

Pablo P. Leal is supported by a scholarship from BECASCHILE-CONICYT. Catriona L. Hurd and Michael Y. Roledawere supported by a Royal Society of New Zealand Marsdengrant (UOO0914). We are grateful to Pamela Fern�andez andRoc�ıo Su�arez for assisting in field sampling. We also thankthe reviewers for the critical and helpful comments.

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