Daylength, Temperature and Nutrient Control of Tetrasporogenesis In

JEMBE 0I762

Daylength, temperature and nutrient control of tetrasporogenesisin Asparagopsisarmatq (Rhodophyta)

Michael D. Guiry and Clinton J. Dawes Depaftmenl of Botany and Martin Ryan Marine Science Institute, University College Galway, National Llniversity of lreland, Galway, Ireland; Department of Biology, University of South Florida, Tampa, Florida, USA

(Revision received 23 December 1991; accepted24 Jantary 1992)

Abstract: The formation of tetrasporangiain Falkenbergia-phasetetrasporophytes of Asparagopsisarmata Harvey (Rhodophyta : Bonnemaisoniales)isolated from introduced populations in Ireland and Italy, and from native populations in Victoria, Australia, was examinedin temperature-controlledwater-baths at 2'C intervalsfrom 11-23 " C and aI 1- or 0.5-hdaylength intervals from 8-12 h. At a daylengthof 8 h, Irish plants formed tetrasporangia only at (15-)17(-21)'C, Italian plants at 17-21'C and Australian plants at l3-17'C. However, after an incubation period of 5 wk, only the Italian strain consistentlyshowed 100/. reproduction under these conditions. At 17'C, the critical daylengthfor tetrasporogenesisis 8-9 h for the Irish and Australian strains and 9-10 h for the Italian strain. These results indicate that all three isolates differ in their responseto environmental variables.It is likely that the Mediterranean and Irish populations thus representseparate introductions into the Northern Hemisphere and that Australia was not the source of these plants. The abundance of gametophytesof this speciesat Messina, Sicily, in April and May is explained by a reproductive "window" of daylength and temperature for tetrasporogenesisfrom mid- November to late January. In Victoria, Australia, temperaturesare inductive all year but daylength at the water surface is never < 9.5 h. However, it is considered that tidal ranges of 2-3 m cause the effective daylength in winter to fall below the inductive threshold. In northern Europe, the restricted geographical distribution of gametophytes is probably determined by tidally and/or topographically induced shorter daylengthsin late October and November, and also by higher temperaturesin the intertidal on warm days. Previously reported responsesto N and P levels in enriched seawatermedia were not confirmed and may have resulted from incubation at suboptimal temperatures.Arsenic and iodine supplementationappears to be necessaryfor reproduction only in the Irish strain.

Key words: lqparagopsis armata; Control ofreproduction; Daylength and temperature effects;'Falkenbergia- phase; Introduced seaweed;Nutrient effect

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The marine red alga Asparagopsisarmeta Harvey (Bonnemaisoniaceae,Bonne- maisoniales)was endemicto the SouthernHemisphere until it was introduced into the Atlantic and the Mediterraneanin the early part of the presentcentury (Feldmann &

Correspondenceaddress: M. D. Guiry, Department of Botany and Martin Ryan Marine ScienceInstitute, University College Galway, National University of Ireland, Galway, Ireland. 198 M.D.GUIRY AND C.J. DAWES

Feldn.rann,1942). The spcciesis now u'idclydistributed in the easternnorth Atlantic from the British Islessouth to Senegal,including the Canaryand SalvageIslands, and possiblythe Azores(Price et al., 1986;South & Tittley, 1986),and is widespreadin the Mediterrancan, although gametophytesare regularlyfound only in the western basin. J. Feldmann& G. Feldmann(1939a,b,1941,1942) discovered that the separately described speciesFalkenbergia rufolanosa (Harvey) Schmitz was the tetrasporangial phase of Asparagopsisarmata by germinatingthe carposporesof the latter entity and obtaining plants that resembled the former. A similar life history for A. tariformis (Delile)Trevisan was establishedin culturefor plantsfrom Japanby Chihara(1961), who grew carpospores and obtained a Falkenbergia-phase,and for plants from Venezuelaby Rojas et al. (1982),who found that tetrasporogenesisoccurred at24oC, S:16h. Reproduction in the Falkenbergia-phaseof A. armata is reported to be rare and restrictedto autumn.The only publishedrecords for the British Islesare: Mclachlan (1967),rvho found fertile plants in Cornwall on the south coast of England in mid- October 1966; De Valcra etal. (1979)rvho collectedfertile plants at Finavarra,Co. Clare,Ircland, in October 1916,and Guiry et al. (1979)who reportedthat populations in Devon and Cornwall were forming tetrasporangiain November 1912. In northern France,J. Feldmann(195a, p. 94) remarkedthat plantsof Ihe Falkenbergia-phaseare not fertilein the irrea around Roscoff,but further south,in Portugaland Morocco, fertile plantsare found from Decemberto March (Gayral,1958, p. 288;Ardre, 1970,p. 143), and gametophytesare regularly found on the Atlantic coastsof Morocco (Gayral, 1958), Portugal(Ardr6, 1970),Spain (Seoane-Camba,1965) and France (Feldmann,1954). The perceivedlack ofreproductive tetrasporophytesin the British Isles and radically differentdistributional patternsofgametophytes and tetrasporophytesgave rise to early speculationthat both phasesreproduced independentlyby vegetativemeans (Dixon, 1964, 1965).De Valera & Folan (1964)further suggestedthat a decreasein the fertility of Irish gametophytesof l. ctrmcttamay have occurred in the period since its introduction, implying vegetativereproduction by the gametophytes,but concludedthat their materialwas not adequateto confirm this; they were also of the opinion that Irish ganretophl'ticplants r,verebecoming smallor and lessrobust. The Provisionalotlas o/ the nrurineulgueofBritainandlrelanrl(Norton, 1985,Mapsl6and 77),indicatesthatthe Folkenbergia-phaseof A. arntata is now widely distributed on western and southern coasts from Shetland to Cornwall, but the Asparagopsis-phasegametophytes are confined to restrictedareas of the west and south of Ireland. and in Britain are known only from the south-westcoast. In an attempt to establish the environmental parametersfor reproduction in the Falkenbergia-phaseof A. armata,Oza (1977,1989) grew a strain from Roscoff, Brittany (from the culture collection of H. A. von Stosch),under a range of temperature,day- lengthandnutrientconditions.Tetrasporangiawereobtainedonlyat15 t 0.5'C,6: i8 or 8 : 16 h, in media with low levels of nitrates or phosphates,and containing small amountsof As,O. and KI. Ltning ( 1981), using the samestrain, obtained reproduction TETRASPOROGENESIS IN ASPARAGOPSIS ARMATA 199 at 15'C, 8: t0h, in enrichedseawater media with low levelsof nitrate(<700prM NO.,), and found that a night-break was effectivein suppressingtetrasporangiai formation (confirming the photoperiodicnature of the response),but did not statewhether arsenicor iodinewere required for reproduction.Using the samestrain, Knappe (1985) carried out a more detailed study of the effectsof daylength at 15'C, quantifying the responseby countingspores. Casual monitoring of an intertidal population of t"heFalkenbergia-phase of A. armata in Galway Bay at 1- and 2-wk intervals from Septemberthrough December 1985 (Breeman & Guiry, unpubl. data) indicated that no sporangial production occurred during this period, even though severalwaves of reproduction took place at this time in the related,introduced speciesBonnemaisonia hamifera Hariot (Breeman & Guiry, 1989). As the temperature optimum for maximum reproduction in Bonnemaisonia hamiferais reportedto be 15"C (Oza,1971,1989;Liining, 1981;Knappe, 1985),this lack of reproduction in Asparagopsisarmata seemsto be in conflict with the published data. Severalqr.restions arc thlls apparentwith regardto the distribution and reproduction oC, of Asporagopsisarntattt in Europe:(1)If inductionoftetrasporangiais possible at 15 then why is reproduction so uncomnon in plants of the Falkenbergia-phasein the wild nearthe northern limit of distribution of A. armata?(2) Why aregametangial plants very confined in their geographicaldistribution near the northern limits of the speciesand to the Mediterranean even though the tetrasporophytesare more widely distributed? (3) Were Europeanpopulations of the speciesintroduced from Australiaand are they the result of a singleintroduction ? (4) What are the effectsof nitrate, phosphate,iodine and arseniclevels in enrichedseawater media at optimum temperatures?

MRrpRr,qls AND METHoDS

ORIGIN OF CULTURES

Plants of the Falkenbergia-phaseof Asparagopsisarmata were isolated into unialgal culture using reproductivelysterile held-collected plants from the west coast of Ireland collectedon 6 February 1985 (Culture GALW 510, New Quay, Co Clare; 53'N, 09'13'W) and from Sicily,Italy, on 4 June 1987(Culture GALW 566,near Messina; 38"11'N, 15'33'E). Unialgal cultureswere obtained by continuouslyexcising the 2's apicesof rapidly growing plants at 16 C. 16:8tr. U-sprmol photons'm Carposporesfrom cystocarpicplants of A. armata collectedin the intertidal of Victoria, Australia,on 28 October 1987(Culture GALW 604, Sorrento;38'20'S, 144"45'W) wereinoculated into plasticdishes, grown at ll"C, 14: t0h,20-25pmol photons' m 2.s r, and a unialgal isolate of the Falkenbergia-phaseobtained. Cultures were subsequentlyrendered clonal by selectinga singlepiant of each as a stock culture, and thesewere grown at 16"C. 16 :8 h, 25 pmol photons'm-2 ' s-r with the light period digitally set to start at 0800 GMT. Plants were propagated vegetativelyby excising 2O(] M.D.GUIRY AND C.J.DAWES vegetativeapical portions of the plants, culture media and disheswere replacedevery 3-4 wk.

ENRICHED SEAWATER MEDIA

Filteredsterile seawater was enrichedusing Guiry & Cunningham's(1984) modifica- tion of von Stosch'senriched seawater medium (von Stosch,1963). Quarter-strength medium (VSr) was used in which 5 ml of stock solution (see Guiry & Cunningham, 1984) were added to each I of seawater.The final VS.-enriched seawatermedium contained 22.14mg NO3'l t (0.36mM) and 17.64mg PO4'l-' (0.19mM). Background levels of nitrate and nitrite in the medium during the experimentalperiod I t. rangedfrom 1.21to 51.04pg NO.,'l and0.22,ro 66.34 pg NO2'l The effectsof arsenic and iodine on tetrasporangialformation were assessedby supplementingthe t mediumwith AsrO,,(98 pg ' I ) and KI (16pg'1- '), basedon theconcentrations used by von Stosch(1963) and Oza (1911).

FLUORESCENT STAINING

Determination of whether tetrasporangiawere produced from new or old cells was carried out by iabellingthe cell walls of the filamentswith calcofluor,a cellulose-specifrc stain(Waaland & Waaland,1975), using 0.011" Fluorescent Brightner (Catalogue No. I CI 40622;Sigma Chemical, Poole, Dorset, UK). A concentrationof 0.2 mg'l was made by dissolving0.2 mg of the FluorescentBrightner in a few drops of distilled water and transferringto 200 ml of enrichedseawater medium. Minced fragmentsof the Irish isolate(Culture 510)were grown in this solutionfor I wk. The plants were then washed three times with fresh medium lacking the dye and plants were incubated at 17'C, 8 : 1Oh, to induce tetrasporangialformation. Plants were examined for fluorescence under an epifluorescentmicroscope (Leitz Labrofux 22D).

EXPERIMENTAL PROCEDURE

For each experiment,tufts of the Falkenbergia-phasewere taken from stock cultures and minced with an alcohol-sterilizedrazor-blade on a glass microscope slide until a homogeneousmass of cuttings 1-2 mm in lengthwas obtained.These plants were then suspendedin fresh seawatermedium and 50-60 cuttingspipetted into sterilized250-m1, wide-mouth, conical flasks containing 200-250 ml of enriched seawater. Flasks were incubated in water-baths (Compact Low Temperature,Model RMT6, Lauda, Germany) under cool-whitefluorescent bulbs (a singlePhilips PLC-20 W or two Thorn 15W)delivering 14-20 pmdrphotons'm-2's-rtothe surfaceofthewaterin the water-baths. Flasks were rotated daily to ensure an even photon dosage and to obviate any minor temperaturefluctuations. A minimum of 50 plants'flask I was assessedfor tetrasporangialformation in relation to sizeafter 5 wk + 1 d of incubation. Daylength in the water-bathswas controlled by digital timers set to begin the appro- TETRASPOROGENESIS IN ASPARAGOPSIS ARMATA 201 priate light period at 0800 GMT. Water temperatureswere monitored with submerged thermistorsand recordedevery 15 min with SquirrelData Loggers(Grant Instruments, Cambridge,UK); at no time did water temperaturesin any parts of the water-bathsvary by > t 0.2"C.Irradiances (photosyntheticailyactive radiation) were measuredusing Li-Cor light meter Model 185-Band a Li-Cor 190Squantum sensor. Contingencytables were usedto comparethe presenceor absenceoftetrasporangial productionusing the null hypothesisthat no significantdifference occurred at the 95ou confidence level (P < 0.05). x' tables were used to determine significant differences between all oaired treatments.

Rpsulrs

POSITION OF TETRASPORANGIAL PRODUCTION

Tetrasporangialformation (Fig. 1) occurred only on newly formed branchesof the Irish isolate of Asparagopsisarmata (Fig. 2A,B) as cells stained with Fluorescent Brightnerdid not form tetrasporangia.Some leaching of the FluorescentBrightner onto new cellswas apparent(Fig. 28), but this was minimal.

EFFECTS OF NITRATE AND PHOSPHATE LEVELS

Table I shows the effectsof different combinations of nitrate and phosphatelevels on reproductionin various sizeclasses of plants of the Irish strain (GALW 510).Plants wereincubated at 17'C, 8 : 16h, and tetrasporogenesisassessed after 5 wk. All media includedAsrO. (98pe'l-t) and KI (16pg'l-'). Theseresults show that the only significanteffect on reductionoftetrasporangial production occurredat the lowestlevels of nitrates and phosphates(Treatments 3, 7, 9 and 11 in Table I). When no nitrate or phosphateor very low concentrationsof thesenutrients were used (Treatments 10 and I 11), tetrasporangiawere absent.With increasingnitrate levels above 0.21mg'l (3.85pM NO; ) reproductionwas generallyin excessof 801",regardless of phosphate levelsrangingfrom0.18tol7.64mg'l-'(1.89prMto0.186mM).Plantsizeshowed no relation to tetrasporangialformation (Table I).

EFFECTS OF ARSENIC AND IODINE ADDITIONS

At optimum temperaturesand daylengthsfor the Italian strain (17-19'C, <9h), nitrate and phosphatelevels and arsenicand iodine supplementationhad no significant effect (Figs. 3,4). At the suboptimaltemperature of 2l'C, all treatmentswith arsenic and iodine addition resultedin a significantlyhigher level of tetrasporangialproduction (P < 0.05). In the Irish strain, tetrasporangialproduction was signifrcantlyhigher at 17'C, 8 : t6 h when arsenic and iodine were added (Fig. 3), but no significant differencein tetrasporangialproduction was evidenla[ 17'C. 9: 15h (Fig.3) and 19'C. 8 : 16h 202 M.D. GUIRY AND C.J.DAWES N

Figs.1-2. Tetrasporangia(t)andatetrasporocyte(tc)inAsparagopsisarmatafromlrelandinculture. areas in vegetative cells are secretory cells TETRASPOROGENESIS IN ASPARAGOPSIS ARMATA 203

TABLE I

Effectsofnitrate and phosphatelevels on tetrasporangialproduction in various size classesofplants oflrish strain of Falkenbergia-phaseofAsparagopsis armata. Plants were giown at 1?'C,8:16h,25-30pmol photons m 2 s 1, and assessedafter 5 wk. Percentagereproduction of total plants is given in final column. Y, reproduction; N, no reproduction.

Treatment Reproduction Branch size (mm)

1a No. Poo 0- I L_ J J+ | I 22.14mg' l- 17.64mg' 1- ' Y 6 50 88 (0.36mM) (0.19mM) N t 612 | t 2 22.14mg l- 1.76mg l I 6 31 9l (0.36mM) (18.spM) N 3 19 I 3 2.29mg'l 1.76mg l-' J 33 57 (38.spM) (18.spM) N ,2 22 43 0.23mg l I 1.76mg l I Y 6 44 83 (3.85pM) (18.spM) N 3 717 5 22.14mg'l I 0.l8mg' l I Y 2 50 89 (0.36mM) (1.89pM) N 711 1 6 2.29mg'l 0.18mg'l ' Y 10 36 79 (38.5mM) (1.89aM) N 2 11 21 0.23mg'l-' 0.l8mg'l-' Y t 7t4 (3.85pM) (1.8epM) N 8 42 86 I I 2.29mg'l l7.64mg'I Y 8 44 87 (38.spM) (0.19mM) N 1 713 0.23mg l-| 17.64 mg' l- | Y 3 715 (3.8spM) (0.19mM) N 29 85 0.02mg'l I 0.18mg'l I Y -0 (0.32pM) (1.89pM) N 8 46 100 u 0.00mg'l t 0.02mg'l 1 Y -0 (0.00pM) (0.2pM) N 10 36 100

(Fig. a). The addition of nitrate and phosphate signihcantly reduced tetrasporangial productiononly at 19"C but not at the optimum temperatureof 17'C at 8:16h (Fig.3). In the Australian strain,tetrasporangial production was so low after the experimental period of 5 wk that differencesmay reflect the shorter incubation period. The addition of arsenic and iodine did result in a significantincrease in tetrasporangialproduction at 13, 15 and 17'C usingan 8 : 16-hphotoperiod (Fig. a). The additionof nitrateand phosphateresulted in a significantdecrease and thus contradictedthe aboveresults for the 15 and 17'C treatmentswhile stimulatingtetrasporangial production at 13'C, 8 : 16h if arsenicand iodinewere also added.At 17'C, 9 : 15h, only the addition of nitrate and phosphateresulted in tetrasporangialproduction of any magnitude(Fig. 3).

Fig. 1. Light micrograph of a tetrasporangium on a branch tip ( x 45). Fig.2. Bright field (A) and FluorescentBrightner-induced fluorescence (B) ofsame plant. Only newly formed branch is forming tetrasporangia as evidence by absenceof fluorescence( x 35). 204 M.D. GUIRY AND C.J.DAWES

Finavarra (Ireland)

6\80 a

660

too Sorrento (Australia)

I Nomal + fi Lo*+ @ Nomal - @ to*-

9 10 10.5 11 1r.5 t2 Daylength (h)

Fig. 3. Effects of daylength on three strains of Falkenbergia-phaseof Aspalagopsisatmata. Plants were incubated at 17'C at daylengthsshown. Normal, a normal-strengthmedium was used; low, N and P were reduced as given in Materials and Methods. + and -, presence/absenceof arsenic and iodine.

DAYLENGTH AND TEMPERATURE EFFECTS

The three geographicalstrains tested showedmarked differencesin their responseto daylengthaI lJ ' C (Fig. 3). The Italian strain showed 100% reproduction at 8 and t h, whereasthe Irish strain showed >80% at 8h and <20% reproductionat th. The Australian strain,on the other hand, showed <30% reproductionat 8h and reproduction took piace at t h only in the high-nutrientmedium with As and I supplemen- tation. Temperature differencesbetween the strains were also apparent at 8-h daylengths (Fig. a). The Italian strain showcd 1001 reproductionaL ll and 19"C, with no reproductiontaking place aI < ll 'C, reproductionbeing significantlyreduced at 2l " C TETRASPOROGENESIS IN ASPARAGO PS I S ARMATA 205

0 100 Finavarra (Ireland) ,: 80 5\ a qou

L tr 20

0 100 Sorrento (Australia)

! Nomat + ffi Low+ @ Nomat - @ to*-

13 15 11 19 21 23 Temperature ("C) Fig..tr. Effects of temperature on three strains of Falkenbergia-phaseof Asparagopsisarmata grown at a daylength of 8 h. Normal, a normal-strength medium was used; low, N and P were reduced as given in Materials and Methods. + and - , presence/absenceof arsenic and iodine. in media without arsenicand iodine additions. The Irish strain reproducedover much the same temperaturerange, but the levels of tetrasporangialproduction were much lower: at 77"C, > 801 of the plants in media containingadded arsenicand iodine formed tetrasporangia.At 19 " C, <30% of plants reproducedwith significantlyhighest tetrasporangialproduction in mediacontaining no additionalnitrate and phosphateand arsenicand iodine. AI 21 "C, a small number of plants reproducedin the low-nutrient medium with added As and I, but no reproduction was observedin the other media. Similarly, at 15 'C, < 5l" of plants in the low-nutrient medium with As and I formed tetrasporangia,and none of the other media showedany response.The Australian strain reproducedat a differenttemperature range ( 13-17 'C) than the Italian and Irish strains (11-21 'C),butinnoinstancedid >50L ofAustralianplantsformtetrasporangiaafter 5 wk of incubation. M.D. GUIRY AND C.J.DAWES

DrscussroN

Our experimentalresults confirm those of previous studies of Asparagopsisarmata (Oza, 1977,1989;Ltining, 1981;Knappe, i985; Bonin & Hawkes, 1987),viz.,IhaI tetrasporogcncsisin theFalkenbergla-phase is controlledby daylengthand temperature. Oza(1977,1989)concluded that low levelsof eitherN or P (101"normal strength)were nccessaryfor tetrasporangialproduction at 15'C. Our results,using a combinationof N and P concentrations,do not entirelysLlpport his findings; however,our investi- gations of the elfects of N and P were carried out at the optimum temperaturefor the Irish strain(17 'C; Fig. a), and it is likelythat undersuboptimal conditions (15 'C),low levelsof N and P could affectthe responsein the manner reportedby Oza (1911, 1989). This comparisonassumes that the strainhe used,which was isolatedfrom Roscoffon the north-westerncoast of France, is simiiar in its temperature,daylength and nutrient rcquirementsto the lrish strain. Dring (1974, 1984) has summarized the literature on the effects of high nutrient concentrationson reproduction in algae.It is clear from the resultspresented here that caution is necessaryin the interpretationof such effectsas they may be partly or wholly due to suboptimalincubation conditions or to other chemicalfactors in enrichedmedia. Low-nutrient effectshave alsobeen reported in tetrasporogenesisinthe Trailliella-phase of Bonnemaisoniahamifera (Ltning, 1980),but this requires reinvestigationgiven the narrow temperature requirements for tetrasporogenesisalso found in this entity (Breeman& Guiry, 1989). It is clear from our resultsthat nitrate, phosphate,arsenic and iodine additions at 17 and 19'C, 8 :16h (Fig.4) and 17'C, 9: iS h 1nig.3) in the Italian strainhave no significanteffect. At 2l " C,8 : l6 h, however,additions of As and I to the low and high N and P media significantlyincreased the numbers of tetrasporangiaformed (Fig. a). In the Irish strain at the optimum temperature(17'C), As and I additionsto low and high N and P media significantlyincreased tetrasporangial formation (Fig. a), but this was not apparentat 19'C,8: l6n, probablydue to the overalllow levelsof reproduction. A medium with low levelsof N and P to which As and I had been added was the only combination that produced even a marginal responseat 15 and 21 "C in the Irish strain(Fig. a). This supportsthe conclusionsof Oza (1977)and Luning (1981)in that the former used a very dilute medium (l0L normal-strengthvon Stosch medium with As and I added) and the latter a dilute Provasoli's enriched seawater (with r700 tM N;it is not statedwhether As and I wereadded) at 15'C, 6 : 18or 8 : t6 h. Strict comparisonof our and Oza's and Luning's resultsis not possibleas theseauthors chose to assessreproduction in terms of sporelingsformed and small numbers of sporangiatend to translateinto relativelylarge numbers of sporelings.In the Australian strain,an apparentlysuboptimal response was obtainedby us at 13, 15 and 17'C, S: i6h and at ll"C,9:15h, and, althoughAs and I additionssignificantly affected the responses,reproductive levels were generallyso low that one cannot determineif these differencesare real. TETRASPOROGENESIS IN ASPARAGOPSIS ARMATA 201

The effectsof arsenic and iodine additions on induction of tetrasporogenesisin the Irish strain areparticularly interesting. Von Stosch( I 963) showedthat in culture arsenic and iodine were necessary elements for the development of gametophytes of Asparagopsiswith a normal morphology and Codomier et al. (1979)found that concentrations of 5 pM KI or KIO. were optimal for growth of gametophytes,but that initial concentrationsin excessof 15prM inhibitedgrowth. Knappe & Werner (1975)later indicated that the gametophyteswere up to 10 times more efhcient than the tetrasporophytesin scavengingiodine from culture media. Arsenic is present in various forms in aquaticenvironments (Sanders, 1979; Anderson & Bruland, 1991;Lee et al., 1991)and, in the natural environment,there shouldnot be any problemswith availability in the small quantitiesrequired. It is not clear, however,how arsenic and iodine levels could affect the photoperiodic mechanism except perhaps by means of enzyme inhi- bition or promotion. In previous studies of tetrasporogenesisin the Falkenbergia-phaseof Asparagopsis armatavariouscombinations of temperatureand daylengthwere used. Oza(1977,1989) o experimentedwith daylengthsof 4, 6, 8, 10, 12, 74 and 16h at 9, 12, 15, 18 and 2l C, oC, but only obtainedtetrasporangia in appreciablenumbers at 15 6: 18h and 15'C, 8 : 10h. Ltining (1981) usedtemperatures of 5, 10, 15 and 20'C in combinationwith daylengthsof 8, 12 and 16h, and his plantsonly reproducedat 15'C, 8 : l6 h. Knappe (1985)examined the effects of daylengthar3,4,5,6, 8,9 and 10h at 15'C, quantifying the responseby countingspores; after 28 d, sporeshad formed at daylengthsof4-9 h, but not at 3 and 10h; maximal numbersof sporeswere formed at 6h. After 42d, sporangiawere alsoformed at 3-h daylengths.Our resultsare consistent with thoseof Oza(1911,1989),LLining (1981) and Knappe( i985)with regardto thecritical daylength for tetrasporogenesisand lead to the conclusionthat the Irish and French strainshave the same requirementsand that they are of similar genetic origin. In our studies,tetrasporogenesis occurred over relatively narrow temperaturebands: 13-17'C in the Australianstrain, (15-)17(-21)'C in the Irish strainand 17-21'Cin the Italian strair-r,and then only in daylengthsof <9 h. It is clear that the temperature requirementsin thc Irish and Italian strain, althoughdifferent, are more similar to each other than they nrc to the Australian strain. A.tpurugopsisumtute rvas first described from Garden lsland, Western Australia (W.H.Harvey. 1855),and is apparcntlynative to New South Wales, Victoria, Tasrnania,South Australiaand WesternAustralia (W.H.Harvey, 1862; Lucas & Perrin,1947; Levring. 1953 ; Huisman& Walker.1990), New Zealandand the Chatham Islands(Levring, 1955 : Chapman,1969; Bonin & H arvkes,1987), and Chile(S antelices & Abbott, 1978; Santelices,i988). In Europe, both the Falkenbergiaand Asparagopsis phasesof A.tptrragopsisonncrttt were discoveredat four centres,i.e.. Algeria, C6te des Alberes (Mediterranean France), Biarritz (Bay of Biscay, Atlantic) and on the CherbourgPeninsula in the English Channel,at about the sametime in the early 1920s (seeFeldmann & Feldmann, 1942,for a literature review of the sequenceof eventsup to 1941).These almost simultaneousoccurrences gave rise to early speculationsthat 208 M.D.GUIRY AND C.J. DAWES the populationsr-night bc the result of separateintroductions (e.g., Westbrook, 1930; Svedelius,1933). Of course,there is alrvaysthe possibility that thetime of theirdetection was a function of the observers;in other words, field-workers were searchingfor the speciesafter being made aware of the possibilityof its presence(see Feldmann & Feldmann,1942, p. i05). It must be admitted,nevertheless, that a largeand distinctive entity such as the gametophytesof Asparagopsrscould not easily be overlooked. At present, Lhe Falkenbergia-phaseof A. armata is widely distributed in the Mediterranean(e.g., Feldmann & Feldmann, 1942,Fig. 8; Giaccone,1978; Furnari & Scammacca,1970; Giiven & Oztig, 197l; Meffez& Mathieson,198 1; Gallardo et al., 1985;Giacconc etal., 1985;Athanasiadis, 1987), but gametophytesare known only from the westernMediterranean | (e.g..Feldmann & Feldmann,1942,Fig.8;Gayral, 1958;Meiiez& Mathieson,198 l;Aranda eta1.,1984; Giaccone eta1., 1985). Dixon (1964, i965) and Dixon & Irvine (1911)concluded (iargely, it seems,on the basis of the work of Schiffner, 193 1 ) that distinctionsbetween l. armata andA. taxiformiswere not entirelyclear, despite the opinion of J. Feldmann(1939, pp.277-278) that some of the herbarium material cited by Schiffner was badly prepared and thus misleading. Asparagopsistaxiformis is widespread in the warmer waters of the Northern and SouthernHemispheres, and in the easternAtlantic is known from Madeira, the Salvage and Canary Islands southwards(Levring, 1974 Price et al., 1986).This speciesis restricted to the south-easternMediterranean (Feldmann, 1942),and there appear to be very few literature records of gametophytesother than the original descriptionfrom AlexandriainEgypt(De1ile,1813,p. 115; 1824,p.a01).Aleem(1945)reportedthatat Alexandria "The speciesflourishes during July and August particularlyin 2-3 m depth." Aleem (1951, pers. comm.) later reportedbolhlhe Asparagopsisand Falkenbergra phases at 10-30 m in May and June. Gametophytesare alsoreported from Libya (Nizamuddin eLa\., 1919,citing an earlyrecord by De Toni). Various opinions on the conspecificityof the two speciesof Asparagopslshave led to Falkenbergia-phaseplants being referred to A. armata as if this were the only species occurringin the Mediterranean(e.g., Diapoulis & Verlaque,1981;cf. Athanasiadis, 1987, p.29). Thcre are, however, some records of Falkenbergiafrom the western Mediterranean prior to 1920and the type locality of Falkenbergiahillebrandii (Bornet in Ardissone)Falkenberg, widely regardedas the tetrasporophyteof A. taxiformls,is the island of Elba, off the north-westcoast of Italy (Ardissone,1883)2. Withregardtothepossibleconspecificityof,4.arnlatoandA.taxiformis, Bonin& Hawkes (1987,p.587) have concludedthat the two entitiesare morphologicallyand

lDuetothepracticeofgivingthenameAsparagopsisarmatatothetetrasporophytephase,itissometimes not clear from various floristic accounts whether authors are referring to the gametophyte or tetrasporophyte phase.

2 AslheFalkenbergiaphasesofAsparagopsisarmataandA.taxiformisaresaidtobeindistinguishable(e.g., Dixon, 1964),it is conceivablethat, as to type, Falkenbergiahillebrandii represents the former rather than the latter species and the original collection from Elba representsan early record of the introduction of A. drmata into the Mediterranean. TETRASPOROGENESIS IN ASPARAGOPSIS AKMATA 209

ecologicallydistinct: A. urmatais primarily an epiphyte,attaching to other plants by meansof modifiedbranches referred to asbarbs;l . taxiformisis usuallyassociated with sand-coveredhabitats, having a well-developedrhizomatous system for anchorage,and Iacksbarbs. Other authors (Santeiices & Abbott, 1978;Santelices, 1988;Huisman & Walker, 1990)have maintainedthe two specieson the basisof similardistinctions. In the British Isles,the Falkenbergia-phaseof Asparagopsisarmata was first found in the vicinity of Galway Harbour in 1939(De Valera, 1942;Drew,l950; De Valera& Folan, 1964),and Asparagtpsis-phasegamctophytes were discoveredtwo yearslater at Muigh Inis (Mweenish),at thehead of GalwayBay (De Vaiera,19421,Drew,1950). The Falkenbergitt-phasewas subsequentlydiscovered in Britain by C. C. Harvey & K. M. Drew (1949)on Lundy Island off the coast of north Devon, and the gametophyteswere locatedat a nurnberof siteson the Lizard Peninsulain Cornwallin 1950(Drew, 1950). The tetrasporophytehas spread independently,probably by vegetativefragmentation, in the BritishIsles since these initiai introductions(Dixon, 1964,1965 Dixon & Irvine, 1977 Tittley in Irvine et al., 1975;Farnham, 1980; Norton, 1985).However, it is very unlikely that the gametophytesregularly perennate, as implied by various authors (e.g., Dixon, 1964,1965; De Valera& Folan, 1964),becaus e Asparagopsrs-phaseplants have not at any time been found in Ireland from October-May. If such perennationwere possible,it seemsreasonable to supposethat gametophyteswould be as widespreadas the tetrasporophytes. With regard to the incubation period for tetrasporogenesisin our strains of the Falkenbergia-phaseof Asparagopsisarmata, it shouldbe noted that 5 rather than 6-8 wk is a more appropriateincubation period for the assessmentof a responsein an autumn- reproducing speciesbecause of the major changesin daylength and temperaturethat rapidly take place at this time of year. Temperatureand daylengthgraphs for Messina, Sicily, Italy, Galway Bay, Ireland, and Sorrento, Victoria, Australia, are shown in Figs. 5-7, respectively.The temperaturesavailable for most localities are the maxima recordedfor surface,open water, and take no accountof smallerbodies of inshorewater or tidal pools,which, as we shall seeiater, may be more relevant.Similarly, the daylength curvesare those at the surfaceof the water and assumethat the effectivedaylength starts at a soiar elevationof 0' ("daylight", but not including "civil twilight" in Dring, 1984). Figs. 5-7 also show the maximum and minimum temperaturesat which reproduction may take place in the tetrasporophytesand the maximum inductive daylength. At Messina (Fig. 5), seawatertemperatures are within the inductive range from late Septemberuntil early July, and daylengthsare inductive from mid-November until the beginningof February.Although water temperaturesare only marginallyinductive from Januaryuntil May, a reproductive"window" (LUning, 198l;Guiry & Cunningham, 1984)for this speciesoccurs from early November until late January.At Messina, ganretophyticplants of l. ornlota are common in spring and becomedominant in the upper subtidal in April and May (Tripodi, pers. comm.) and this correlateswell with an autumnalinductive "window" aspredicted by our results.Aranda et al.(1984, Fig. 9) reportedfour wavesoftetrasporangial reproduction in piants ofthe Falkenbergia-phase M.D. GUIRY AND C.J.DAWES

Messina .,

16P 16g

(15"c) rA 6 b a t2E o daylength(10 h) ti l0

Jan-l Mar-l May-l Jul-l Aus-30 Oct-30 Dec-30

Fig. 5. Temperature (-o-) and daylength curves for Messina Harbour, Sicily. Temperature data are monthly maxima for March 1976-March 1977 (De Domenico et al., 1979).Daylength curve (for a solar elevatiou of0') was calculated usir.rga spreadsheetincorporating formulae given by Dring (1984). of A. armata in the Straits of Gibraltar, but this seemsto be contradictedby our results on the strain from Messina as induction would not be possiblein spring or summer. In Galway Bay(Fig. 6),seawater temperatures appear to be inhibitory( < 15'C) from mid-Septcmberto mid-June,with open-watertcmperatures in the Bay only risingabove 15'C for a short period in July to September.Inductive daylengths are only present

Galway / \ uppe.temperature (2loC) 22 20 18 5.,) () 16 k P 14 i.) t2 F 10 / ,rr"rdaytength(9 h) 8

Jan-l Mar-1 May-l Jul-1 Aug-30 Oct-30 Dec-30

Fig. 6. Temperature (-o-) and daylength curves for Galway Bay, Ireland. Temperature data are maxima for 1987at Shellfish ResearchLaboratory, Carna, Co Galway (unpubl. data). Daylength curve (for a solar elevation of0') was calculated using a spreadsheetincorporating formulae given by Dring (1984). TETRASPOROGENESI S IN A SPA R4 G OP S I S AR],TATA 21.1

24 Sorrento 22

Uppertemperature ( 19"C) 20 - t8 \ /. \ EIJ .\ 16 8.

ho t4'

l', o h lt Lower temperature(l l'C) \ o lu ti

Jul-1 Aug-30 Oct-31 Dec-31 Mar-1 May- 1 Jun-30

Fig.7. Temperature (-o-) and daylength curves for Sorrento, Victoria, Australia. Temperature data are maximum valuesrecorded by King ( 1970).Daylength curve (for a solar elevationof 0 " ) was calculatedusing a spreadsheetincorporating formulae given by Dring (1984). from early November to mid-February, during which time open-water temperatures would be inhibitory. However, Breeman & Guiry (1989, Fig. 8) have shown that temperaturesin the intertidal of Galway Bay are occasionallyhigher than those found in open water, due largely to emersion,particularly during mild autumns and periods of spring tides. At two sites on the southern shores of Galway Bay, these authors showedthat the maximumin situ seawatertemperatures during 1985were > 15'C in late Septemberand early October. Daylengthsat the water surface(Fig. 6) are still too long at this time (xl2h); however,this daylengthcurve does not take account of the effect of light reduction in the water column at times of high water, particularly during spring tides. It has been shown that the effects of large tidal ranges,particularly in inshore waters, and of high waters of spring tides occurring at dawn and dusk, are sufficientto reduce the effectivedaylength perceived by the plants by at least an hour (Dring, 1987; Breeman & Guiry, 1989).Additionally, there may be further effects of position: plants in tidal pools with steepsides or in positions with elevatedground to the west or to the east would experiencefurther reductions in effective daylengths. Certain habitats,due to aspectand topology, may also experiencehigher temperatures at low water. Thus, a combination of local daylength and temperature effects may explainthe restricteddistribution of Asparagopsisarmata gametophytes on the west and south coasts of Ireland and on the south coast of England. Temperaturemay be the primary rangeJimitingfactor with local tidal and topographicaleffects determining local distribution and the completion of the sexual life-history cycle. The more restricted distribution of A. armata gametophytes as compared to the gametophytes of Bonnemaisoniahamifera (Norton, 1985, Maps 78 and 79) is likely to result from a 212 M.D. GUIRY AND C,J.DAWES narrower reproductivewindow for tetrasporogenesisin Asparagopsls.This is a conse- quenceof threefactors: lower temperaturelimits of 15'C rn Asparagopslsvs. 12'C in Bonnemaisonia;critical daylengths of t9 vs. 11h, respectively; and longer tetra- sporangialinduction requirementsin Asparagopsrs.These differences probably account for the reportednorthern bmitof Asparagopsrsgametophytes on the west coast of Ireland as comparedto the west coastof Norway for gametophytesof B. hamifera(see Breeman & Guiry, 1989). On the south-westerncoast of Australia, where Asparagopsisarmata is a native species,the temperaturecurves in the area of Port Philip Heads, Victoria (Fig. 7) are such that the specieswould have the potential to reproduce most of the year if there were no daylengthcontrol of tetrasporogenesis.However, the curve shown in Fig. 7 for daylengthsat the surface of the water at this latitude indicates that daylength never reachesinductive levels as it is never <9.25h. As Asparagopstsgametophytes occur commonlyon theseshores in the australspring, it is clearthat conditionsmust at some time becomeinductive. Tidal and, perhaps,topographical effects probably come into operation in May and June (austral autumn). Decreasingthe effectivedaylength by an hour at thesetimes would result in inductiveconditions (13-14 "C, 8.25:tS.ZStr.). Al1 three strains examined in this study differ in their daylength and temperature requirementsfor tetrasporogenesis.Of the three strains, the Australian isolate is the most different,and it seemsvery unlikely that Australia was the origin of eitherthe Irish or Sicilian strains,particularly as the south coast of Australia in one of the few coasts in the world wherethe coastlinefollows latituderather than longitude,and it is probable that the daylengthand temperatureresponses of Asparagopsisare very similar all along the south coast of Australia. Latitudinal ecotypesmay not exist in Australia, except perhapson the Indian Oceancoast. In reality,the temperatureand daylengthresponses of the Australian strain are more suited to European coasts than the strains that presently occur there and Asparagopsrsgametophytes would be more abundant and more widespreadif it were an Australian strain similar to that from Victoria which had been introduced. According to Bonin & Hawkes (1987), A. armata is common throughout New Zealatd, exceptfor the Kermadec Islands (30" S, 178'W) and, taking into accountthe latitudinal spreadofthe islands and the consequentpotential occurrenceoftemperature and daylength ecotypes,this seemsto be a more plausible origin for the European strains. However, Bonin & Hawkes (1987) reported that tetrasporangiawere formed in a New Zealand strain incubated at 13'C, 12 : 12h ("spring conditions"; see Hawkes, 1986).In northern New Zealand, tetrasporogenesisoccurred in wild plants from March to September(Bonin & Hawkes, 1987),which is consistent with their experimentalresults. Bonin & Hawkes's resultsdiffer radically from those we obtained with our Australian strain in regard to the critical daylength for reproduction. A temperatureof 13'C is within the reproductiverange of the Australian strain, but is cleariy outside the rangesof the European strains. However, both the Australian and European strains do not reproduce at daylengths of > t h. Nevertheless,given the TETRASPOROCENESIS IN A SPA K{GO PSI S A RMATA 213 longitudinal spreadof New Zealand (34-41 'S), it is possiblethat latitudinal ecotypes existthere, but further investigationsare necessary to establishthis as a potential source of the European strains. Santelices& Abbott (1978)and Santelices(1988) described A. armataas occurring in Chile from Iquique(20'S) to Coquimbo(30"S). Althoughthese authors described the speciesas "tropical to subtropical", open-watertemperatures on the Chilean coast at 20 ' S vary from l6 oC in winter to 22 " C in summer.However, Chilean populations would have a longer critical daylengththan European or Australian populations as the minimum daylengthat 20'S in winter would be :11h. Apparently, therefore,none of the known distributional areasof nativeAsparagopsis armatain the SouthernHemisphere are readily identifiableas the origin of the European strainsof this adventivespecies, except perhaps for the southernpart of New Zealand. However, it is clear that the widely cited assumption of Australia as the origin of the European strains is not consistent with results presentedhere. Although the Irish and Italian strains are more similar to eachother in their temperature and daylengthresponses (Figs. 3,4), there are sufficientdifferences to suggestthat these strains resulted from separateintroductions. If they were derived from a single introduction, one would expectthat their physiologicalresponses would not have had time to adapt to local conditions in the intervening 65 yr. That such physiological changesdo not occur rapidly is supportedby the fact that strainsof the Trailliella-phase of Bonnemaisoniahamifera (also an introduced species)from Ireland and the Canary Isles had the samedaylength and temperatureresponses in culture (Breeman& Guiry, unpubl. data). The possibility that the Mediterranean and Atlantic populations representseparate introductions lends some support to early speculationson the subject (e.g.,Westbrook, 1930; Svedelius,1933), but further studiesof other strains from, for example,Biarritz (France), Spain, Algeria and the Mediterraneancoast of France, are necessary. In conclusion, tetrasporogenesisin Irish, Italian and Australian strains of the Falkenbergia-phaseof Asparagopsr.rshow clearresponses to daylengthand temperature. The Italian strain respondsmore rapidly and, perhaps,more completelythan the Irish strainwithin roughly similar temperaturelimits (11-21 'C). The Australian strain shows a slower and lesscompiete response, similar to the responsetime of the Irish strain but over quite a differenttemperature range (13-17'C). Previouslyreported responses to nitrate and phosphatelevels in enriched seawatermedia were confirmed, but only for the Irish strain, and thereis evidencethat arsenicand iodine supplementsare necessary for reproduction in this strain. The effectsof arsenic and iodine need to be separated and identified,and further studiesof the responsesto the various forms of arsenicfound in aquaticenvironments (see Anderson & Bruland, 1991;Lee et al., 1991)would be of interest. The possible day-time temperature effects extrapolated here from previous work on Bonnemaisoniahamifera need to be verified experimentally,particularly as previous studies of temperature effects on photoperiodic responsesin higher plants suggestthat it is the night-time temperaturesthat are critical (Breeman et a1.,1988). 214 M.D. GUIRY AND C.J.DAWES

ACKNowLEDGEMENTS

This studywas aidedby fundingfrom the Irish marine subprogrammeof STRIDE, a EuropeanCommunity programme in Scienceand Technology.We are also grateful to: the FulbrightInternational Exchange Program, which allowedC. J. Dawesto spend a sabbaticalyear in Ireland;the DevelopmentFund, UniversityCollege, Galway, which provided funding for equipment; F. Ardr6, Laboratoire de Cryptogamie, Mus6um National d'Histoire Naturelle, Paris, who searchedfor original material of Fucu.stari- formis Delile and who provided assistancewith the literature;the Schoolof Botany, University of Melbourne, for the award of a ResearchFellowship allowing M. D. Guiry to spenda sabbaticalyear in Australiain 1987-88 and to G.T. Kraft, A.J.K. Millar and R. Wetherbeefor facilitating his work there; ProfessorG. Tripodi, for providing temperaturedata for the Straitsof Messina;and to A. M. Breeman,E. M. Cunningham and D.J. Garbary for their helpful commentson the manuscript.We are particularly indebted to Wendy Guiry for culture maintenance.

RgpERpNces

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