- Email: [email protected]
Reproductive phenology and growth rates in two species of Gracilaria from Hawaii
J. c>up. ,wI~‘. B/o/. Lk~l., 1078. Vol. 35. pp. 77i- 283 6 Elsevier:North-Holland Biomedical Press
REPRODUCTIVE TWO
PHENOLOGY
SPECIES
AND GROWTH
OF GRACZLARZA
FROM
RATES
IN
HAWAII
Abstract: Observations on wild populations ot’ Grtr~iiul-ici hl,~.s~tri”/.\rti~-l.\ (C;mclin) Silva and G. ~‘~,~~,,~~~,~,f&z J. Ag. showed significant differences in ~lnctopllyt~: tetr~~~por~~pllytc ratios from the ekpeclcd I : I ratio. As in many other perennial red aigae. the proportion of tctrasporic il~di~,id~~~ls in a poptrlation of these two G~rc,iktria spp. dominates the combined male and fimale gametophpts stage. There were significantly more male than female thalli in the G. c,o~,,r?cti”f~ii population wherca\ the gametophytes of G. hir~,sf~~~/,~/~)~;~s occurred in the expected 1: 1 ratio. In addition. there are seasonal changes 111 the proportions of tetrasporic and gametophytic individuals within the populations. Tstrasporic thalli of G. corono~~~ifolirr evinced a biphasic seasonal pattcrn with high proportions in winter and sumnu. The tetrasporic phase of G. h~rrstrpn.~/or-is. on the other hand. showed a Ion proportion in winter. Maximum biomass does not necessarily correlate wth maXimum proportion of the tetrasporophyte generation. Seasonal patterns in the proportion of malt i\tld female gametophytcs dif’fcrcd for wch stage as well as for each species. The proportion of male th:+lli in G. hrl/-.\“pi/.‘ro,,/.\ and G (,(~,,orro~~~rlr,/,lr show,ed high peaks in winter and autumn. respectively. Cystocarpic thalh were most abundant in the former in late winter and summer and in the latter in winter and spring. Iti both spccu the l’cmule gametophytcs grew significantly slower than did the malt ~~illietopliytie or tetrnsporophytic atage>. Prdcticdt ~ppli~~lioils regarding ssasonai cycles in the varioui reprodLictive siages and thclr diff~rellt~~tl growth I‘ateS arc discussed.
INTRODUCTIOh
There have been but three intensive Rao,
1973; Penniman,
investigations
1977) of seasonal
variation
(Jones,
1959; Umamaheswara
in the reproductive
stages
of
Gmcilariu species. The cycle of the male gdmetophytes
in a population remains unclear because of the rarity of male gametophytes. Studies of reproductive phenology in Gr~~il~~i~ vc~-mm~~, a temperate species (Jones. 1959), and of G. sj~~~~~t~~~~ti~ in the tropics (Umamahes~ara Rao, 1973) indicate that seasonal production of tet~dsporallgi~ and cystocarps are closely related to seasonal maxilllurn vegetative growth. Cystocarpic plants of G. vwwosu are most abundant in fall and early winter while tetrasporic plants are most abundant in July (Jones. 1959). Even though male plants were much less commonly observed than the other two stages. Jones suggested a summer maximum spermatium production. Cystocarpic and tetrasporic plants of G. sjoestrdtii were found (Umamaheswara Rao, 1973) only from November to March. again the period of maximum biomass. No male gdmetophytes of this putative tropically distributed species were ever found. The maximum occurrence of tetrasporic thalli in G. fidi$w in New Hampshire was in June and 273
MlTCHELLD.HOYLE
274
July (Penniman,
1977). Two other tfopical
no seasonal variation
in female gametophytic
the year (Umamaheswara
Rao,
species, G. cdzdis and G.,fbli~frur. showed and tetrasporophytic
1973). Spermatangial
however. found in G. edulis only in January. The tetrasporophytic stage in the life history
individuals
and cystocarpic
of the above
during
thalli
were.
species dominates
the
combined male and female gametophytic stage as is the case in many other red algae (Svedelius, 1927; Fritsch, 1945: Johnstone & Feeney, 1944; Drew. 1955: Dixon. 1965. 1970; Knaggs, 1969; Barilotti & Silverthrone. 1971; Dawes. Mathieson & 1977). In addition. tetrasporic Cheney. 1974; Hansen & Doyle, 1976; Kapraun, individuals of G. ~~JITLICOSLI have higher growth rates than the female gametophyte (Jones, 1959). Comparative growth rates among the three morphologically similar reproductive phases have recently been studied in Gtmilmia sp. from eastern Canada (Edelstein, 1977). Tetrasporic fronds were reported to increase “somewhat in weight than did gametophytic fronds. and male plants grew “somewhat
more” better”
than did female plants. The purpose of this paper is to report results on reproductive phenology and differential growth rates of the three isomorphic life history phases of two species of Gracilwiu in Hawaii.
MATERIALSAND
METHODS
G. hwsapastoris (Gmelin) Silva and G. coronop~fi~liaJ. Ag. were sampled on a monthly basis from their sublittoral habitats. growing to a depth of about 2.5 m. at Sand Island and Ft Kamehameha on the island of Oahu. Hawaii. At the time ot sampling, about one hundred 15-cm portions of large thalli were removed at random within the Gracilaria community, placed in a nylon mesh bag and returned to the laboratory where male. female (indicated by the presence of cystocarps), and tetrasporic thalli were identified with the aid of a stereoscope. and were separated and counted.
No attempt
was made to ascertain
seasonal
variation
in the production
or viability of the various reproductive products of the mature thalli. Non-reproductive thalli were not sampled as they are apparently very small and near the base ot perennial holdfasts. For growth rate experiments. whole thalli of the two species were collected at either site and immediately transported to the laboratory. The algae were maintained in an aerated holding tank under normal room temperature and lighting for two or three days. segregated according to their reproductive state and weighed for experimentation. Thalli with white patches (“ice ice” disease) were discarded. The term “ice ice” is applied by Filipino seaweed farmers to unhealthy algae with characteristic white patches. The cause of “ice ice” is as yet unknown. Branches uniform in size and shape were cut from different thalli, shaken vigorously to rcmove excess water, weighed, labelled with vinyl tags, and placed in flasks (Kimax
REPRODUCTIVE
No. 26655) containing Diamond
Head
lamps (General The fluorescent Temperature
Beach
PHENOLOGY
2 1 of filtered Park.
AND GROWTH
(Whatman
The culture
No. 1 tilter paper)
vessels
were
275
~;R,~~~/l..~R/,.~ SP.
placed
sea water
under
from
fluorescent
Electric, Power Groove) providing 350 PE mm’ xc( ~2100 ft-c). lamps were on a 12 : 12 h light : dark cycle in a ‘windowless’ room.
ranged from 23 C (dark hours) to 27 28 C (light hours) due to infra-red
irradiance from the lighting system. ‘These temperature ranges do not differ from those measured across Hawaiian reefs. The culture vessels were re-positioned daily in order to reduce the effects of any light or temperature gradients that might have occurred. The laboratory experiments lasted at least seven days at the end of which time thalli were vigorously shaken and weighed. In another experiment portions 01 G. hltrstrl”i.st”/.i.v and G. ~,~)~{~~l~)~~f~li(lweighing between 6 and 10 g \x’crc tagged and transplanted to an outdoor tank at the State of Hawaii’s Anuenuc Fishcrics Research Center on Sand Island in Honolulu Harbor. Tctrasporophytes and male gametophytes were attached to bamboo poles by monotilament. The poles wet-c fixed in cinder blocks at ~0.5 m from the water surface in a circular tank (=4 m diam. and I m deep). Sea water from a subterranean well continuously flowed through the culture tank. and artificial aeration provided additional circulation. Data for the harvest or *take’ of G. hrt,.~iiprrsrot~i,~ as reported by commercial seaweed gatherers were obtained from the Division of Fish and Game of the Hawaii De~~art~ierit of L.and and Natural Resources. Growth rates were calculated as percentage compound interest fresh weight per day using the formula i = (trA:P) - 1. where i is the interest per period (growth rate per day): A. principal compounded (new weight): P. principal (old weight); and II, number of periods (days). Calculations were made with a cassette type program (Statistical Package No. 194, Wang Industries Inc.) on a Wang programmable calculator. Growth rates were transformed to arcsin values and differences among the three morphologically similar reproductive stages were tested for significance. Significant differences among three means were determined according to Duncan’s multiple range test (Duncan. 1955).
RESULTS The proportions of the tetrasporophyte and the two gametophyte stages in wild populations of G. hzwsupastoris and G. (.o1.o/1o1~jfC)iiLIat Ft Kamehameha and Sand Island are presented in Table I. While the ratio of tetrasporophyte: gametophytc generations may vary considerably from month to month, in the final analysis the proportion of the tetrasporic stqe in each species was significantly greater (P < 0.001) than that of the combined male and female thalli in the population. Tetrasporophytes of G. bz/~~~up~‘~t~~~i.s made up more than 50”,, of the population except in winter (December-parch) and late summer (Augtlst-September). On the other hand, the proportion of tetr~~s~?orophytes in G. ~~~otzt~~~if~~/i~~ dropped below
MITCIIELI,
276
D. HOYLE
50”,, only in March and April. There were significantly (P < 0.001) more male than female individuals (based on an expected 1 : 1 sex ratio) in the G. coronqC’Ji?liu population, however. there was no significant difference (P < 0.250) between the proportion of these two stages in G. ~~~r.s~i~~~.sr~~~.~ populatiotl.
Sample size
.._._-_
~
1972 2I.i Ft K~l~llel~~~le~d 24.ii Ft Kamehameha 23.iii Ft Kamehameha 17.1~ Sand Island I9.i~ Ft Kamehameha I1.v Ft Kamehaneha 17.G Ft K~rne~~rne~~ ?.vii Sand Island f5.G Ft Kamehameha 29.viii Ft Kamehameha 9,ix Sand Island 24.iw Ft Kumehameha k Ft Kamehameha 28.~1 Ft K~itneh~rneh~ xii Fr K~~~eh~irneh~ lY7.1 29.i 13.ii lti.iii Xiv v 2.G
Ft Ft Ft Ft
Kamehameha Kamehuneha Kamehameha Kamehameha
Ft Kamehameha
63 122 3x 85 63 13Y 76 IO8 43 62 100 126 112 148
124
retra4pax C’,,)
32.0 40.1 60.5 58.8 60.3 56.8 52.6 62 .ii 47.4 48.4 72.0 47.h 53.’ 54.7 48.5
Sample size
Male (“,,I
rctraspor1c I”,,)
25.0 23 I. ,I’ 35.4 77.0 32.4 24.x 36.4 ii7 71)
‘I.4 22.9
(~2.4 71.4
9s 122 114 1I14
31.7 41.0 36.8 31.4
52.6 51.3 53.5 5h.X
42.5 41 .O 46.0 57 ii
7lJ 96 92 I07
25.7 19.1 41.3 44.9
64.3 66.4 43.5 35.x
52.3
x3
41.0
43 3
52.9
55.2
The three morphologically similar reproductive phases in the life history of these two algae also show seasonal tluctuations (Fig. 1) at Ft Karneh~~rnel~~which differ for each species. The peak occurrence of G. ~~~~~~~~~~~~i~ tetrasporophytes in winter (January and February) was followed by a sudden decline in March. There was a second high peak in summer (July and August). Male gametophytes showed a definite high seasonal peak in.autumn (September-December) which gradually decreased to the annual low in February. Following this another increase in the proportion of male thalli in spring (March and April) was fofiowed by a summer low.
REPKODUC’TIVE
PHENOLOGY
AND GROWTH
C;R.4i‘IL,4RIA
SP.
277
By comparison, the proportion of female (cysto~arpic) thalli showed s~?mewhat less varjabil~ty than either of the other two stages. yet tended towards low proportiol~s in summer and early fall and high proportions in late winter and early spring. 90
[
,
J
’ 1 FMAMJJA
0’
,
,
1
,
”
~~~
”
~~
,c&?2
,
I
/
,
1’
‘1 SONPJFMA
]
:
”
,
/
”
‘I
j
~1973
Fig. I Proportwn of‘ tetrasporophytis. male and female gametaphytic thalli of Gruciiwnr /XII.J~~~XL~ OWL< and G. ~~)~~~?~~~j~~~~ju thro~igho~lt the year at Ft Kamehameha: significant differences according to Duncan‘s multiple range test are represented by different letters on the curves: any two wrves having any one letter in common do not differ signi~ca~ltly at the 95”,, level.
In general. the peak abundance in the proportion of tetrasporic C. f?~{~.~~~~~,~~(~~~.~ thaili was antithetic to that of tetrasporic G. ~~~~~)/z(~~~~#~l~~i individuals. The proportion of the former in the population shows a winter (January and February) low peak (Fig, 1) each year. Another low peak occurred in late summer (August and September). The proportion of G. bl~rsu~)u.~torjsmale and female garnet(~pll~tes in the population roughly parallelled each other and varied little throughout the 1%month long observation
27x
MITCHELL
II. HOYLE
period. In January and February of both years the gametophyte generation constituted a greater part of the population (Table 1) than did the tetrasporic generation. Male thalli. however. constituted a greater part of the gametophyte generation in autumn and winter and a lesser part in cpring and summer. High peaks in the proportion of’ female thalli occurred in February. June. and August.
Seasonal variation in the biomass of G. ~lf~.~~i~~/.~~~~i.s during the period i~lvestigated is reflected in Fig. 2 as a low harvest of the alga in November 1972 through March 197.3.The fact that this Iow winter harvest is a good indication of the actual biomass was verified by monthly standing crop measurements (Hoyle, unpubl.) during the same period at two sites being considered within the sector which was commercially harvested. No comparable data for G. cotwwp~/7ifdi~~ are available from the Division of Fish and Game as this species is not generally harvested for commercial sale. Further data of the author (Hoyle, unpubl.) show. however, the lowest standing crops of G. ~*~~~j?~~~~~#~~~~ from December through March at Ft Kamehamcha. In both G. ~?L~~s~~~~~s~~~~~.~ and G. ~~~~~~~~p~~~~~i~i growth rates differed significantly (P < 0.025. P-c 0.005, respectively) a~tioll~ the three life history stages. Female ~dm~tophytes grew signi~~aIltly slower (Table 11) than did either the male gameto-
TABLE II Daily growth rates (as 0o increase in fresh weight per day) of the three life forms of Grucilorirr h~r~vtrptrv~o~~v and G. co~~ppifo/irr: X= mean ~s.D.; * Duncan's (1955) multiple range test: any two means not having any one letter in common differ significantly at the 95”,, level. DLiily growth Life form species
Female
Male gametophyte
.Y
gametophyte
Tetrasporophytc
2.11 1 57 -.__ I.X5 2.08 2.08 2.06
2.69 1.72 I.06 2.80 2 25 2.83
2.12 * 0.23 B
1.72 * 0.27 .A
0.70 1.04 I.29 I .oo I.18 I .6? 0.80 I.19
0.92 I .oo 0.68 0.82 0.53 0.74 0.38 0.87
I .45 1.22
I .02 * 0.29
0.65 * 0.18 B
I .47 f 0. I.3 A
A
Growth
rate (‘I,,)
I 5.3 1.51 1.37 I.37 I .45 I .66
rates (as “,> increase in fresh weight per day) for G. coro~pifolrcr in tank culture at the Anuenue Fisheries Research Center on Sand Island: .Y = mean kr 13 ; * partially bleached thallus. G. h~r,strp~~.~ro,_i,s Thallus
no,
I 2 -3 4 5 h 7 x Y IO II I2 I? I4 I5 16 I7 18 I’) 20 .v
(8 days) Il.79 IO. 17 IO.67 14.26
I I .A8 I2.70
G. c~oro/Wpr/olitr (6 days) 8.20 2.x8* Y.64 7.75* Y.40
9.20
13.41 Il.48 12.50 15.69 1 I .23 11.25 10.45 12.24 13.Y6 I?.‘)4 12.X8 Y.05 13.52 13.53
7.68 7.14 I .YX 7.X 8.40 7.74* 7.66 2.Yl I .66* 7.07 broken 7.48 6.59 1.21*
12.14 * 1.5Y
6.41 k2.77
280
MITCHELL
D. HOYLE
phytes or the te~asporophytes. Furthermore, the tetrasporic phase in both species grew faster, although not significantly so, than did the male gametophyte generation. The culturing of G. hursapastoris and G., coronop(fdi~i in large outdoor tanks at Sand Island (Table III) showed that these algae may be grown successfully (at least for short periods) on a fairly large scale under artificial conditions; G. /WSLIp~~.~iori~~grows almost twice as fast as G. c,olor?ctl_‘ifb(rct.
DISCIJSSION
Mature fertile thalli of G. f?z4r.s~~p~storisand G. coronop~fdia are found, often in the same habitat. tllroughout the year in shallow subtidal Hawaiian waters. In fact, it is rare to find a totally sterile adult plant of either of these species. Small, nonreproductive juvenile thalli may occur but these are not usually sampled. Random sampling of natural populations of these two species over an l&month period indicated a disproportionate representation of tetrasporic and combined male and female gametophytic individuals within %fild populations. All Gr~~eil~~~ifl species are presumed to possess the Po~~.sip~?ol?i~-type life history characteristic of G. IYWL~ co.w as demonstrated in vitro by Ogata. Matsui & Nakamura (1972). Indeed. observations made during the present investigation confirm that G. hur.srqmstoris and G. cororzop[fblia conform to the general tripbasic life history with alternating morphologically similar g~~metophytic and tetrasporophytic generations. ideally the tetrasporic and gametangial phases should occur in a I : 1 ratio. As in many perennial red algae and in the Gmcilmia species so fdr examined. however. the proportion of the tetrasporic stage in a population dominates the gametangial stage during most of the year. Hansen & Doyle (1976) cited various explanations that have been projected to interpret this phenomenon in other red algae. Johnstone & Feeney (1944) attributed the lower number of gametopllytic plants to the high mortality of tetraspores Dixon (1965) suggested that the extension of a species into habitats at the limits of its range might result in tetrasporophyte dominance. The sublittoral habitat (Knaggs, 1969). periodic development of tetrasporophytes from tetraspores through apomeiosis (Hansen & Doyle, 1976). and inherent advantages of diploidy over haploidy (Hansen & Doyte. 1976) have all been suggested as explanations of the phenomenon. There is no evidence that any of these hypotheses explains the dominance of Grncifuriu tetrasporophytes over gametophytes. All but Dixon’s and Knagg’s suggestions, however. appear plausible in the case at hand. Unlike other reports (Jones. 1959; Umamaheswara Rao, 1973), results of the present study show that the tetrasporic phase of G. hlasclptrsro,-i,sis not most abundant during the period of maximLlm biomass. MaxiItluln biomass in this species was in autumn while maximum tetrasporophyte abundance was in summer and winter. G. cotmop~fdia also shows two peaks in tetrasporophyte abundance: the one in winter was at the time of lowest recorded biomass and the other in summer at the
REPRODUCTIVE
PHENOLOGY
AND GROWTH
GRAC’ILARIA
SP.
281
same time as a high peak in biomass. Seemingly the low winter biomass at a time of peak abundance is contradictory to what might be expected inasmuch as tetrasporophytes grow faster. This suggests that in .siru growth of tetrasporic G. coronopi,ftiliu thalli is limited by winter conditions. The present investigation shows the proportion of male gametophytes in the population of a Gru~ilu~~f~ species throughout a I?-month cycle. Since there was no sig~li~cant difference between the proportion of m&e and female gametophytes in G. hursupasroris, and there was a highly significant difference between these two stages in G. coronopifbliu, apparently the ratio of male to female gametophytes varies according to the species. Another possibility is that male G. coronup~#~liu thalli appear first. the females becoming evident as cystocarpic thalli sometime after fertilization. The proportions of both male and female gametophyti~ stages and the tetrasporophytic stage in natural populations of these two algae show seasonal fluctuations. These results are contrary to the expectations of Svedelius (1927) and studies on other perennial red algae (Johnstone & Feeney, 1944: Hansen & Doyle. 1976). Svedelius’ (1927) theory of synchronous development of gametangial and tetrasporic generations is supported by the recent work of Hansen & Doyle (1976) on fri&eu corduta, in which there were no seasonal fluctuations in the densities of the gametangial and tetrasporangial phases. Earlier findings by Hasegawa & Fukuhara (1952) on I. rornucopiue and by Prince & Kingsbury ( 1973) and Mathieson & Burns (1975) on C1zondru.s crisps. show seasonal fluctuations in generations. but were not considered by Hansen & Doyle (1976) to be comparable since they had not considered sampling error. Dawes, Mathieson & Cheney (1974) using random sampling methods. however. showed that Euchruma isqfbrme shows seasonal variation in its tetrasporic phase. The seasonal variation of the tetrasporic and gametophytic stages in Gmcilariu ~ursap(~.~~oris and G. ~~roi~op~~~liu provides additional evidence that such variation may depend upon the species in question. The present investigation, however. does not provide adequate information to explain the causes of this. One may speculate that since G. hursupastoris tetrasporophytes comprise less than 50”. of the population in winter (December---March) and summer (August-September), water temperatures below andabovea tolerance range may interfere with carpospore production or development. Water temperature, which reaches its nadir in March in Hawaii. may also at least partially explain the sudden decrease in the proportion of tetrasporic G. coronopifblia in March of both years. Variations in water motion with season may also be a factor by allowing greater spore settlement during calm periods as found by Jones (1959). Whether or not seasonal environmental factors such as temperature. light intensity, photoperiod or water motion are operative in the present case has yet to be determined. Because of the titne factor required in the growth and development of mature fertile thalli, environmental factors, if involved. are sure to be ofan antecedent nature (Doty, 1971) in terms of the final results observed. The quantitative results in the present study showing that tetrasporic thalli of
282
MITCHELL
G. bursupastoris
and
G. coronop(@iu
D. HOYLE
grow
significantly
faster
under
laboratory
conditions than do cystocarpic female gdmetophytes confirm Jones’ (1959) field experiments showing that tetrasporic plants (of G. WY~ZICOS~)grow faster than do cystocarpic cantly
plants.
reduces
One could
the growth
speculate
that the parasitic
rate of the female gametophyte.
carposporophyte Contrary
signifi-
to Edelstein’s
(1977) observation, the present investigation is the first to show that there is no significant difference in the growth rate of male gdmetophytes and tetrasporophytes in this genus. The fact that male gametophytes, however, grow significantly faster than do cystocarpic plants statistically confirms Edelstein’s observation on another Gracilmin species. Knowledge of seasonal cycles and growth rates in the reproductive stages ot G. hursuppastoris and G. coronop(/diu has practical value. In Hawaii where the demand for Grucihiu as a condiment exceeds the available supply. over-harvesting by fishermen may pose a threat to the survival of the natural population. especially if the regenerative holdfasts are removed during harvest. The threat to G. coromp(folirr is, however, much less than that to G. hur.supmtori.s. Although people of native Hawaiian ancestry prefer lirnu nzanaura. i.e.. G. coronop(fblicr. the majority of the present population who eats Gracilaria are of Asim (Japanese and Filipino) ancestry and prefer the larger G. bursupmtoris. The threat to both species is partly alleviated by the fact that collection of female thalli is actively avoided because cystocarps are gritty and undesirable to the palate. Only male and tetrasporic individuals are, therefore, subject to over-harvest. Inasmuch as the least amount of G. hursqx~sto~is is harvested from November through March. and these two months are periods of relatively high occurrence of tetrasporophytes in the population, then there should be adequate opportunity for recruitment of the gametophyte generation. and thus maintenance of the population. As pointed out above. this species has a low winter biomass and. therefore, if collecting pressures were intensified in the winter months the natural
population
of the alga could be jeopardized.
ACKNOWLEDGEMENTS
I am very grateful to Drs J. Hansen and J. Stimson for critically reading a draft of the manuscript and for offering valuable recommendations for its improvement. Gratitude is also extended to the Botany Department its assistance in the final preparation of the figures.
REFERENCES
of the University
of Hawaii
for
REPRODUCTIVE
PHENULOC;Y
AND GROWTH
GAAC’TLA RiA
SP.
2x3
REPRODUCTIVE TWO
PHENOLOGY
SPECIES
AND GROWTH
OF GRACZLARZA
FROM
RATES
IN
HAWAII
Abstract: Observations on wild populations ot’ Grtr~iiul-ici hl,~.s~tri”/.\rti~-l.\ (C;mclin) Silva and G. ~‘~,~~,,~~~,~,f&z J. Ag. showed significant differences in ~lnctopllyt~: tetr~~~por~~pllytc ratios from the ekpeclcd I : I ratio. As in many other perennial red aigae. the proportion of tctrasporic il~di~,id~~~ls in a poptrlation of these two G~rc,iktria spp. dominates the combined male and fimale gametophpts stage. There were significantly more male than female thalli in the G. c,o~,,r?cti”f~ii population wherca\ the gametophytes of G. hir~,sf~~~/,~/~)~;~s occurred in the expected 1: 1 ratio. In addition. there are seasonal changes 111 the proportions of tetrasporic and gametophytic individuals within the populations. Tstrasporic thalli of G. corono~~~ifolirr evinced a biphasic seasonal pattcrn with high proportions in winter and sumnu. The tetrasporic phase of G. h~rrstrpn.~/or-is. on the other hand. showed a Ion proportion in winter. Maximum biomass does not necessarily correlate wth maXimum proportion of the tetrasporophyte generation. Seasonal patterns in the proportion of malt i\tld female gametophytcs dif’fcrcd for wch stage as well as for each species. The proportion of male th:+lli in G. hrl/-.\“pi/.‘ro,,/.\ and G (,(~,,orro~~~rlr,/,lr show,ed high peaks in winter and autumn. respectively. Cystocarpic thalh were most abundant in the former in late winter and summer and in the latter in winter and spring. Iti both spccu the l’cmule gametophytcs grew significantly slower than did the malt ~~illietopliytie or tetrnsporophytic atage>. Prdcticdt ~ppli~~lioils regarding ssasonai cycles in the varioui reprodLictive siages and thclr diff~rellt~~tl growth I‘ateS arc discussed.
INTRODUCTIOh
There have been but three intensive Rao,
1973; Penniman,
investigations
1977) of seasonal
variation
(Jones,
1959; Umamaheswara
in the reproductive
stages
of
Gmcilariu species. The cycle of the male gdmetophytes
in a population remains unclear because of the rarity of male gametophytes. Studies of reproductive phenology in Gr~~il~~i~ vc~-mm~~, a temperate species (Jones. 1959), and of G. sj~~~~~t~~~~ti~ in the tropics (Umamahes~ara Rao, 1973) indicate that seasonal production of tet~dsporallgi~ and cystocarps are closely related to seasonal maxilllurn vegetative growth. Cystocarpic plants of G. vwwosu are most abundant in fall and early winter while tetrasporic plants are most abundant in July (Jones. 1959). Even though male plants were much less commonly observed than the other two stages. Jones suggested a summer maximum spermatium production. Cystocarpic and tetrasporic plants of G. sjoestrdtii were found (Umamaheswara Rao, 1973) only from November to March. again the period of maximum biomass. No male gdmetophytes of this putative tropically distributed species were ever found. The maximum occurrence of tetrasporic thalli in G. fidi$w in New Hampshire was in June and 273
MlTCHELLD.HOYLE
274
July (Penniman,
1977). Two other tfopical
no seasonal variation
in female gametophytic
the year (Umamaheswara
Rao,
species, G. cdzdis and G.,fbli~frur. showed and tetrasporophytic
1973). Spermatangial
however. found in G. edulis only in January. The tetrasporophytic stage in the life history
individuals
and cystocarpic
of the above
during
thalli
were.
species dominates
the
combined male and female gametophytic stage as is the case in many other red algae (Svedelius, 1927; Fritsch, 1945: Johnstone & Feeney, 1944; Drew. 1955: Dixon. 1965. 1970; Knaggs, 1969; Barilotti & Silverthrone. 1971; Dawes. Mathieson & 1977). In addition. tetrasporic Cheney. 1974; Hansen & Doyle, 1976; Kapraun, individuals of G. ~~JITLICOSLI have higher growth rates than the female gametophyte (Jones, 1959). Comparative growth rates among the three morphologically similar reproductive phases have recently been studied in Gtmilmia sp. from eastern Canada (Edelstein, 1977). Tetrasporic fronds were reported to increase “somewhat in weight than did gametophytic fronds. and male plants grew “somewhat
more” better”
than did female plants. The purpose of this paper is to report results on reproductive phenology and differential growth rates of the three isomorphic life history phases of two species of Gracilwiu in Hawaii.
MATERIALSAND
METHODS
G. hwsapastoris (Gmelin) Silva and G. coronop~fi~liaJ. Ag. were sampled on a monthly basis from their sublittoral habitats. growing to a depth of about 2.5 m. at Sand Island and Ft Kamehameha on the island of Oahu. Hawaii. At the time ot sampling, about one hundred 15-cm portions of large thalli were removed at random within the Gracilaria community, placed in a nylon mesh bag and returned to the laboratory where male. female (indicated by the presence of cystocarps), and tetrasporic thalli were identified with the aid of a stereoscope. and were separated and counted.
No attempt
was made to ascertain
seasonal
variation
in the production
or viability of the various reproductive products of the mature thalli. Non-reproductive thalli were not sampled as they are apparently very small and near the base ot perennial holdfasts. For growth rate experiments. whole thalli of the two species were collected at either site and immediately transported to the laboratory. The algae were maintained in an aerated holding tank under normal room temperature and lighting for two or three days. segregated according to their reproductive state and weighed for experimentation. Thalli with white patches (“ice ice” disease) were discarded. The term “ice ice” is applied by Filipino seaweed farmers to unhealthy algae with characteristic white patches. The cause of “ice ice” is as yet unknown. Branches uniform in size and shape were cut from different thalli, shaken vigorously to rcmove excess water, weighed, labelled with vinyl tags, and placed in flasks (Kimax
REPRODUCTIVE
No. 26655) containing Diamond
Head
lamps (General The fluorescent Temperature
Beach
PHENOLOGY
2 1 of filtered Park.
AND GROWTH
(Whatman
The culture
No. 1 tilter paper)
vessels
were
275
~;R,~~~/l..~R/,.~ SP.
placed
sea water
under
from
fluorescent
Electric, Power Groove) providing 350 PE mm’ xc( ~2100 ft-c). lamps were on a 12 : 12 h light : dark cycle in a ‘windowless’ room.
ranged from 23 C (dark hours) to 27 28 C (light hours) due to infra-red
irradiance from the lighting system. ‘These temperature ranges do not differ from those measured across Hawaiian reefs. The culture vessels were re-positioned daily in order to reduce the effects of any light or temperature gradients that might have occurred. The laboratory experiments lasted at least seven days at the end of which time thalli were vigorously shaken and weighed. In another experiment portions 01 G. hltrstrl”i.st”/.i.v and G. ~,~)~{~~l~)~~f~li(lweighing between 6 and 10 g \x’crc tagged and transplanted to an outdoor tank at the State of Hawaii’s Anuenuc Fishcrics Research Center on Sand Island in Honolulu Harbor. Tctrasporophytes and male gametophytes were attached to bamboo poles by monotilament. The poles wet-c fixed in cinder blocks at ~0.5 m from the water surface in a circular tank (=4 m diam. and I m deep). Sea water from a subterranean well continuously flowed through the culture tank. and artificial aeration provided additional circulation. Data for the harvest or *take’ of G. hrt,.~iiprrsrot~i,~ as reported by commercial seaweed gatherers were obtained from the Division of Fish and Game of the Hawaii De~~art~ierit of L.and and Natural Resources. Growth rates were calculated as percentage compound interest fresh weight per day using the formula i = (trA:P) - 1. where i is the interest per period (growth rate per day): A. principal compounded (new weight): P. principal (old weight); and II, number of periods (days). Calculations were made with a cassette type program (Statistical Package No. 194, Wang Industries Inc.) on a Wang programmable calculator. Growth rates were transformed to arcsin values and differences among the three morphologically similar reproductive stages were tested for significance. Significant differences among three means were determined according to Duncan’s multiple range test (Duncan. 1955).
RESULTS The proportions of the tetrasporophyte and the two gametophyte stages in wild populations of G. hzwsupastoris and G. (.o1.o/1o1~jfC)iiLIat Ft Kamehameha and Sand Island are presented in Table I. While the ratio of tetrasporophyte: gametophytc generations may vary considerably from month to month, in the final analysis the proportion of the tetrasporic stqe in each species was significantly greater (P < 0.001) than that of the combined male and female thalli in the population. Tetrasporophytes of G. bz/~~~up~‘~t~~~i.s made up more than 50”,, of the population except in winter (December-parch) and late summer (Augtlst-September). On the other hand, the proportion of tetr~~s~?orophytes in G. ~~~otzt~~~if~~/i~~ dropped below
MITCIIELI,
276
D. HOYLE
50”,, only in March and April. There were significantly (P < 0.001) more male than female individuals (based on an expected 1 : 1 sex ratio) in the G. coronqC’Ji?liu population, however. there was no significant difference (P < 0.250) between the proportion of these two stages in G. ~~~r.s~i~~~.sr~~~.~ populatiotl.
Sample size
.._._-_
~
1972 2I.i Ft K~l~llel~~~le~d 24.ii Ft Kamehameha 23.iii Ft Kamehameha 17.1~ Sand Island I9.i~ Ft Kamehameha I1.v Ft Kamehaneha 17.G Ft K~rne~~rne~~ ?.vii Sand Island f5.G Ft Kamehameha 29.viii Ft Kamehameha 9,ix Sand Island 24.iw Ft Kumehameha k Ft Kamehameha 28.~1 Ft K~itneh~rneh~ xii Fr K~~~eh~irneh~ lY7.1 29.i 13.ii lti.iii Xiv v 2.G
Ft Ft Ft Ft
Kamehameha Kamehuneha Kamehameha Kamehameha
Ft Kamehameha
63 122 3x 85 63 13Y 76 IO8 43 62 100 126 112 148
124
retra4pax C’,,)
32.0 40.1 60.5 58.8 60.3 56.8 52.6 62 .ii 47.4 48.4 72.0 47.h 53.’ 54.7 48.5
Sample size
Male (“,,I
rctraspor1c I”,,)
25.0 23 I. ,I’ 35.4 77.0 32.4 24.x 36.4 ii7 71)
‘I.4 22.9
(~2.4 71.4
9s 122 114 1I14
31.7 41.0 36.8 31.4
52.6 51.3 53.5 5h.X
42.5 41 .O 46.0 57 ii
7lJ 96 92 I07
25.7 19.1 41.3 44.9
64.3 66.4 43.5 35.x
52.3
x3
41.0
43 3
52.9
55.2
The three morphologically similar reproductive phases in the life history of these two algae also show seasonal tluctuations (Fig. 1) at Ft Karneh~~rnel~~which differ for each species. The peak occurrence of G. ~~~~~~~~~~~~i~ tetrasporophytes in winter (January and February) was followed by a sudden decline in March. There was a second high peak in summer (July and August). Male gametophytes showed a definite high seasonal peak in.autumn (September-December) which gradually decreased to the annual low in February. Following this another increase in the proportion of male thalli in spring (March and April) was fofiowed by a summer low.
REPKODUC’TIVE
PHENOLOGY
AND GROWTH
C;R.4i‘IL,4RIA
SP.
277
By comparison, the proportion of female (cysto~arpic) thalli showed s~?mewhat less varjabil~ty than either of the other two stages. yet tended towards low proportiol~s in summer and early fall and high proportions in late winter and early spring. 90
[
,
J
’ 1 FMAMJJA
0’
,
,
1
,
”
~~~
”
~~
,c&?2
,
I
/
,
1’
‘1 SONPJFMA
]
:
”
,
/
”
‘I
j
~1973
Fig. I Proportwn of‘ tetrasporophytis. male and female gametaphytic thalli of Gruciiwnr /XII.J~~~XL~ OWL< and G. ~~)~~~?~~~j~~~~ju thro~igho~lt the year at Ft Kamehameha: significant differences according to Duncan‘s multiple range test are represented by different letters on the curves: any two wrves having any one letter in common do not differ signi~ca~ltly at the 95”,, level.
In general. the peak abundance in the proportion of tetrasporic C. f?~{~.~~~~~,~~(~~~.~ thaili was antithetic to that of tetrasporic G. ~~~~~)/z(~~~~#~l~~i individuals. The proportion of the former in the population shows a winter (January and February) low peak (Fig, 1) each year. Another low peak occurred in late summer (August and September). The proportion of G. bl~rsu~)u.~torjsmale and female garnet(~pll~tes in the population roughly parallelled each other and varied little throughout the 1%month long observation
27x
MITCHELL
II. HOYLE
period. In January and February of both years the gametophyte generation constituted a greater part of the population (Table 1) than did the tetrasporic generation. Male thalli. however. constituted a greater part of the gametophyte generation in autumn and winter and a lesser part in cpring and summer. High peaks in the proportion of’ female thalli occurred in February. June. and August.
Seasonal variation in the biomass of G. ~lf~.~~i~~/.~~~~i.s during the period i~lvestigated is reflected in Fig. 2 as a low harvest of the alga in November 1972 through March 197.3.The fact that this Iow winter harvest is a good indication of the actual biomass was verified by monthly standing crop measurements (Hoyle, unpubl.) during the same period at two sites being considered within the sector which was commercially harvested. No comparable data for G. cotwwp~/7ifdi~~ are available from the Division of Fish and Game as this species is not generally harvested for commercial sale. Further data of the author (Hoyle, unpubl.) show. however, the lowest standing crops of G. ~*~~~j?~~~~~#~~~~ from December through March at Ft Kamehamcha. In both G. ~?L~~s~~~~~s~~~~~.~ and G. ~~~~~~~~p~~~~~i~i growth rates differed significantly (P < 0.025. P-c 0.005, respectively) a~tioll~ the three life history stages. Female ~dm~tophytes grew signi~~aIltly slower (Table 11) than did either the male gameto-
TABLE II Daily growth rates (as 0o increase in fresh weight per day) of the three life forms of Grucilorirr h~r~vtrptrv~o~~v and G. co~~ppifo/irr: X= mean ~s.D.; * Duncan's (1955) multiple range test: any two means not having any one letter in common differ significantly at the 95”,, level. DLiily growth Life form species
Female
Male gametophyte
.Y
gametophyte
Tetrasporophytc
2.11 1 57 -.__ I.X5 2.08 2.08 2.06
2.69 1.72 I.06 2.80 2 25 2.83
2.12 * 0.23 B
1.72 * 0.27 .A
0.70 1.04 I.29 I .oo I.18 I .6? 0.80 I.19
0.92 I .oo 0.68 0.82 0.53 0.74 0.38 0.87
I .45 1.22
I .02 * 0.29
0.65 * 0.18 B
I .47 f 0. I.3 A
A
Growth
rate (‘I,,)
I 5.3 1.51 1.37 I.37 I .45 I .66
rates (as “,> increase in fresh weight per day) for G. coro~pifolrcr in tank culture at the Anuenue Fisheries Research Center on Sand Island: .Y = mean kr 13 ; * partially bleached thallus. G. h~r,strp~~.~ro,_i,s Thallus
no,
I 2 -3 4 5 h 7 x Y IO II I2 I? I4 I5 16 I7 18 I’) 20 .v
(8 days) Il.79 IO. 17 IO.67 14.26
I I .A8 I2.70
G. c~oro/Wpr/olitr (6 days) 8.20 2.x8* Y.64 7.75* Y.40
9.20
13.41 Il.48 12.50 15.69 1 I .23 11.25 10.45 12.24 13.Y6 I?.‘)4 12.X8 Y.05 13.52 13.53
7.68 7.14 I .YX 7.X 8.40 7.74* 7.66 2.Yl I .66* 7.07 broken 7.48 6.59 1.21*
12.14 * 1.5Y
6.41 k2.77
280
MITCHELL
D. HOYLE
phytes or the te~asporophytes. Furthermore, the tetrasporic phase in both species grew faster, although not significantly so, than did the male gametophyte generation. The culturing of G. hursapastoris and G., coronop(fdi~i in large outdoor tanks at Sand Island (Table III) showed that these algae may be grown successfully (at least for short periods) on a fairly large scale under artificial conditions; G. /WSLIp~~.~iori~~grows almost twice as fast as G. c,olor?ctl_‘ifb(rct.
DISCIJSSION
Mature fertile thalli of G. f?z4r.s~~p~storisand G. coronop~fdia are found, often in the same habitat. tllroughout the year in shallow subtidal Hawaiian waters. In fact, it is rare to find a totally sterile adult plant of either of these species. Small, nonreproductive juvenile thalli may occur but these are not usually sampled. Random sampling of natural populations of these two species over an l&month period indicated a disproportionate representation of tetrasporic and combined male and female gametophytic individuals within %fild populations. All Gr~~eil~~~ifl species are presumed to possess the Po~~.sip~?ol?i~-type life history characteristic of G. IYWL~ co.w as demonstrated in vitro by Ogata. Matsui & Nakamura (1972). Indeed. observations made during the present investigation confirm that G. hur.srqmstoris and G. cororzop[fblia conform to the general tripbasic life history with alternating morphologically similar g~~metophytic and tetrasporophytic generations. ideally the tetrasporic and gametangial phases should occur in a I : 1 ratio. As in many perennial red algae and in the Gmcilmia species so fdr examined. however. the proportion of the tetrasporic stage in a population dominates the gametangial stage during most of the year. Hansen & Doyle (1976) cited various explanations that have been projected to interpret this phenomenon in other red algae. Johnstone & Feeney (1944) attributed the lower number of gametopllytic plants to the high mortality of tetraspores Dixon (1965) suggested that the extension of a species into habitats at the limits of its range might result in tetrasporophyte dominance. The sublittoral habitat (Knaggs, 1969). periodic development of tetrasporophytes from tetraspores through apomeiosis (Hansen & Doyle, 1976). and inherent advantages of diploidy over haploidy (Hansen & Doyte. 1976) have all been suggested as explanations of the phenomenon. There is no evidence that any of these hypotheses explains the dominance of Grncifuriu tetrasporophytes over gametophytes. All but Dixon’s and Knagg’s suggestions, however. appear plausible in the case at hand. Unlike other reports (Jones. 1959; Umamaheswara Rao, 1973), results of the present study show that the tetrasporic phase of G. hlasclptrsro,-i,sis not most abundant during the period of maximLlm biomass. MaxiItluln biomass in this species was in autumn while maximum tetrasporophyte abundance was in summer and winter. G. cotmop~fdia also shows two peaks in tetrasporophyte abundance: the one in winter was at the time of lowest recorded biomass and the other in summer at the
REPRODUCTIVE
PHENOLOGY
AND GROWTH
GRAC’ILARIA
SP.
281
same time as a high peak in biomass. Seemingly the low winter biomass at a time of peak abundance is contradictory to what might be expected inasmuch as tetrasporophytes grow faster. This suggests that in .siru growth of tetrasporic G. coronopi,ftiliu thalli is limited by winter conditions. The present investigation shows the proportion of male gametophytes in the population of a Gru~ilu~~f~ species throughout a I?-month cycle. Since there was no sig~li~cant difference between the proportion of m&e and female gametophytes in G. hursupasroris, and there was a highly significant difference between these two stages in G. coronopifbliu, apparently the ratio of male to female gametophytes varies according to the species. Another possibility is that male G. coronup~#~liu thalli appear first. the females becoming evident as cystocarpic thalli sometime after fertilization. The proportions of both male and female gametophyti~ stages and the tetrasporophytic stage in natural populations of these two algae show seasonal fluctuations. These results are contrary to the expectations of Svedelius (1927) and studies on other perennial red algae (Johnstone & Feeney, 1944: Hansen & Doyle. 1976). Svedelius’ (1927) theory of synchronous development of gametangial and tetrasporic generations is supported by the recent work of Hansen & Doyle (1976) on fri&eu corduta, in which there were no seasonal fluctuations in the densities of the gametangial and tetrasporangial phases. Earlier findings by Hasegawa & Fukuhara (1952) on I. rornucopiue and by Prince & Kingsbury ( 1973) and Mathieson & Burns (1975) on C1zondru.s crisps. show seasonal fluctuations in generations. but were not considered by Hansen & Doyle (1976) to be comparable since they had not considered sampling error. Dawes, Mathieson & Cheney (1974) using random sampling methods. however. showed that Euchruma isqfbrme shows seasonal variation in its tetrasporic phase. The seasonal variation of the tetrasporic and gametophytic stages in Gmcilariu ~ursap(~.~~oris and G. ~~roi~op~~~liu provides additional evidence that such variation may depend upon the species in question. The present investigation, however. does not provide adequate information to explain the causes of this. One may speculate that since G. hursupastoris tetrasporophytes comprise less than 50”. of the population in winter (December---March) and summer (August-September), water temperatures below andabovea tolerance range may interfere with carpospore production or development. Water temperature, which reaches its nadir in March in Hawaii. may also at least partially explain the sudden decrease in the proportion of tetrasporic G. coronopifblia in March of both years. Variations in water motion with season may also be a factor by allowing greater spore settlement during calm periods as found by Jones (1959). Whether or not seasonal environmental factors such as temperature. light intensity, photoperiod or water motion are operative in the present case has yet to be determined. Because of the titne factor required in the growth and development of mature fertile thalli, environmental factors, if involved. are sure to be ofan antecedent nature (Doty, 1971) in terms of the final results observed. The quantitative results in the present study showing that tetrasporic thalli of
282
MITCHELL
G. bursupastoris
and
G. coronop(@iu
D. HOYLE
grow
significantly
faster
under
laboratory
conditions than do cystocarpic female gdmetophytes confirm Jones’ (1959) field experiments showing that tetrasporic plants (of G. WY~ZICOS~)grow faster than do cystocarpic cantly
plants.
reduces
One could
the growth
speculate
that the parasitic
rate of the female gametophyte.
carposporophyte Contrary
signifi-
to Edelstein’s
(1977) observation, the present investigation is the first to show that there is no significant difference in the growth rate of male gdmetophytes and tetrasporophytes in this genus. The fact that male gametophytes, however, grow significantly faster than do cystocarpic plants statistically confirms Edelstein’s observation on another Gracilmin species. Knowledge of seasonal cycles and growth rates in the reproductive stages ot G. hursuppastoris and G. coronop(/diu has practical value. In Hawaii where the demand for Grucihiu as a condiment exceeds the available supply. over-harvesting by fishermen may pose a threat to the survival of the natural population. especially if the regenerative holdfasts are removed during harvest. The threat to G. coromp(folirr is, however, much less than that to G. hur.supmtori.s. Although people of native Hawaiian ancestry prefer lirnu nzanaura. i.e.. G. coronop(fblicr. the majority of the present population who eats Gracilaria are of Asim (Japanese and Filipino) ancestry and prefer the larger G. bursupmtoris. The threat to both species is partly alleviated by the fact that collection of female thalli is actively avoided because cystocarps are gritty and undesirable to the palate. Only male and tetrasporic individuals are, therefore, subject to over-harvest. Inasmuch as the least amount of G. hursqx~sto~is is harvested from November through March. and these two months are periods of relatively high occurrence of tetrasporophytes in the population, then there should be adequate opportunity for recruitment of the gametophyte generation. and thus maintenance of the population. As pointed out above. this species has a low winter biomass and. therefore, if collecting pressures were intensified in the winter months the natural
population
of the alga could be jeopardized.
ACKNOWLEDGEMENTS
I am very grateful to Drs J. Hansen and J. Stimson for critically reading a draft of the manuscript and for offering valuable recommendations for its improvement. Gratitude is also extended to the Botany Department its assistance in the final preparation of the figures.
REFERENCES
of the University
of Hawaii
for
REPRODUCTIVE
PHENULOC;Y
AND GROWTH
GAAC’TLA RiA
SP.
2x3