Galaxea, JCRS, 3: 51- 63 (2001) RS Jc tapanese Coral Reef ocie4y

Morphophysiological variations of symbiotic dinoflagellates in hermatypic corals from a fringing reef at Sesoko Island

E.A. Titlyanov*1,2, T.V. Titlyanova1, A. Amat2 and K. Yamazato3

1 Institute of Marine Biology, Far East Branch of Russian Academy of Sciences, Vladivostok, 690041, Russia 2 Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, 905-02, Japan 3 Research Institute for Subtropics, Asahimachi, Naha, Okinawa 900, Japan

* Corresponding author: Tel .: +7 4232-310931; Fax: +7 4232-310900; E-mail address: [email protected]

Abstract: Three types of symbiotic dinoflagellates L (large), B (brown) and G (green) found in hermatypic corals from a fringing reef of Sesoko Island (Okinawa, Japan) dif- fered morphologically, physiologically and biochemically. Colonies of the hydrocoral Millepora intricata hosted symbionts of type L only; scleractinian corals containing type B only were Pocillopora damicornis, type G only were caliendrum and S. hystrix, and both types B and G were found living together in and Echinopora lamellosa. The symbiotic dinoflagellates (SD) differed considerably in cell size, shape and structural elements in coccoid state in hospite. SD of these types also differed in photosynthetic capacities, primary production, pigment accumulation and maximum rates of cell division and degradation. Corals hosting various types of SD significantly differed in light-resistance. Scleractinian corals with symbionts of both types B and G, in the same colony, acclimated to bright light by increasing the relative number of symbionts of type G and acclimated to dim light by increasing the SD number of type B. It was shown that scleractinian corals can photo-acclimate through formation of optimal composition of SD types under various light intensities.

Key words: symbiotic dinoflagellates; zooxanthellae; hermatypic corals; symbiosis; morphology; physiology; photo-acclimation.

1982, Trench and Blank 1987), inf ectivity and INTRODUCTION repopulation (Schoenberg and Trench 1980c, A large number of investigations during the Trench 1981, Colley and Trench 1983, Berner last 20 years have show that symbiotic et al. 1993), photosynthetic pigment content, dinoflagellates (SD), resident in host cells of structure of photosynthetic system and photo- different groups of marine organisms including acclimation (Iglesias-Prieto and Trench 1994, hermatypic corals, have fundamental differ- 1997), acclimation to temperature (Gates 1990, ences (Trench 1987,1997) . The main differences Edmunds 1994) and quantity and volume of include variation in morphology (Leutenegger chromosomes (Blank and Trench 1985, Trench 1977, Shoenberg and Trench 1980a, Banaszak and Blank 1987). Recently, variations in struc- et al. 1993, Trench and Luong-Van Thinh 1995), ture of some genes of symbiotic dinoflagellates isoenzymes (Scholnberg and Trench 1980b, were also found (Rowan and Powers 1991a, Trench and Blank 1987), sterols (Withess et Baker et a1.1997, Rowan et al. 1997, Maruyama al. 1982), isoelectric forms of the peridinin- et al. 1998). However, present day nomencla- chlorophyll a-proteins (Chang and Trench ture of endosymbiotic dinoflagellates is lacking 52 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

descriptions. Currently, the nomenclature list suggests that photo-acclimation of hermatypic of symbiotic dinoflagellates consists of 25 spe- corals is driven not only by changing cies in eight genera, and four orders (Trench morphophysiological parameters of polyps and 1987). This paucity of described algal their symbionts (Falkowski and Dubinsky 1981, is not due to the scarcity of species but due Zvalinsky et al. 1978, Porter et al. 1984, to the complexity and imperfection of taxo- Titlyanov et al. 1988, Titlyanov 1991) but also nomic identification (Trench 1997). The by selection of more effectively functioning classical approach to the systematics of symbiotic algae under high-light levels. This dinof lagellates is based on analysis of the free- suggestion was pointed out in connection with swimming biflagellate cells, specifically relating the explanation of coral bleaching as an adap- to the arrangement of the thecal plates, girdle, tive mechanism by Buddemeier and Fautin in and cinglulum (Trench 1997). For symbiotic 1993. dinof lagellates, taxonomic characterization In six species of hermatypic corals from the proved to be a problem as most of SD occur West Pacific (Okinawa, Japan) we found three as coccoid cells in hospite. types of symbiotic dinoflagellates (SD) that Morphophysiological distinctions between differed in morphophysiological parameters. symbiotic dinof lagellates in hospite or isolated This study described and analyzed the vari- from their host tissue are not used in the ous morphophysiological characteristics of dif- classical approach to systematics. However, ferent types of SD in hermatypic corals taken various morphological (in coccoid state), bio- from a fringing reef of Sesoko Island and chemical, physiological, and karyotypic char- acclimated in outdoor aquarium to light in- acteristics, should be referred to as genotypic tensities from 95% to 2% PAR0. differences (Trench 1997). Nevertheless, even significant morphophysiological differences in two apparent populations of symbiotic MATERIALS AND METHODS dinoflagellates in the coccoid state (stage) do The coral colonies of Stylophora pistillata not allow us to identify them as populations (Esper 1797), Seriatopora caliendrum of different species. Therefore, we are limited (Ehrenberg 1834), S. hystrix (Dana 1846), to the use of such terms as taxon, strain or Pocillopora damicornis (Linnaeus 1758), type to describe the different variations of Echinopora lamellosa (Esper 1795), and colo- SD (Trench 1987, 1997; Buddemeier and Fautin nial hydroid Millepora intricata (Milne 1993). Edwards 1857) were collected in September Morphophysiologically or genetically differ- 1997 - February 1998 from the fringing reef ent types of symbiont dinof lagellates were at Sesoko Station (Tropical Biosphere Re- more often found in different hosts (Rowan search Center), University of the Ryukyus, and Powers 1991b, Hunter et al. 1997). Okinawa, Japan. All samples were collected Recently, it was shown by restriction fragment at a depth of 2 m. Samples were stored in 6- length polymorphisms (RFLPs) that the corals m3 outdoor aquarium supplied with seawater Montastrea annularis and M. f aveolata each until the beginning of analyses and experi- host three distantly related taxa of the ments (1--3 days). dinof lagellate Symbiodinium (Rowan and Knowlton 1995). The colonies Acropora Acclimation of coral branches to 95%, 30%, cervicornis at depths of 9-18 m host 8% and 2% PARO. zooxanthellae with two RFLP genotypes in Three colonies of each coral species were constant proportions (Baker et al. 1997). used in the experiments. Coral : branches (5- Unfortunately, morphophysiological charac- cm lengths) of S. pistillata, S. hystrix, S. teristics of the different RFLP types of caliendrum, P. damicornis and M. intricata, Symbiodinium have not been investigated. and broken fragments of E. lamellosa colonies Recently, it was shown that the ratio of the were fixed with cement to ceramic the pieces RFLP types "A", "B" and "C" in the colonies in their natural orientation to light (Fig. la, of Montastrea annularis and M, f aveolata b, c, d, e). Labels were placed on the ceramic changed according to conditions of illumina- bases. These samples were placed into outdoor tion: types "A" and "B" accumulated in bright aquarium 10 cm from each other, avoiding di- light, and in dim light type "C" was more rect contact. The conditions in the aquarium abundant (Rowan et al. 1997). The latter were slightly different from the sea: the tem- Morphophysiological variations of symbiotic dinoflagellates 53

a b

C a

e f

g

Ii

Fig. 1 Branches of hermatypic corals acclimated to light intensities (95%, 30% , 8% and 2% PAR,) during 120 days. Light intensities noted in each picture. a - Millepora intricata; b - Stylophora pistillata; c - Pocillopora damicornis; d - Echinopora lamellosa; e- Seriatopora caliendrum; f - branches of S. caliendrum; magnification x4; g - polyps and connecting sheet of S. caliendrum, magnification x 16; h - polyps and connecting sheet of P. damicornis, magnification x 16. perature in the aquarium was only 1 - 29C Annual meteorological characteristics of coast- higher than in the sea during the day and 1- al observations at Sesoko Station were given 2°C lower at night. In October, the temperature by Nakano and Nakamura (1990, 1993). Dis- in the aquarium was 25-27°C during the day- solved nutrient concentrations in the surface time and 23-24°C at night. In February, the waters of the Sesoko Island were studied by temperature was 22-24°C during the daytime Crossland (1982). The water in the aquarium and 20-22°C at night. The biogenic elements was intensively aerated, water change was content and zooplankton concentration in the approximately 30% h-'. The aquarium was aquarium and in the sea were not significantly partly shaded by black plastic mesh. Light different because the seawater was pumped intensity was 95% PAR,; in shaded parts the from a depth of 2 m of the fringing reef. Input light intensity was reduced to 30%, 8% and water was not subject to filtration nor settling. 2% PAR,. 54 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

The plastic meshes were cleaned of sediments the degrading SD frequency (DSDF), the pro- and algae two times a week. The corals were liferating SD frequency (PSDF), the diameters maintained in the aquarium for 120 days (from of HSD and degraded SD particles (DSDP), October 1997 to February 1998) (Fig. 1). In and the chlorophyll content. The number of parallel experiments (Titlyanov et al. 2001, in zooxanthellae was calculated either per polyp press) it was shown that complete reaccli- or per coral area (Marsh 1970). The HSD mation of corals to changes in light intensities and DSDP diameters were measured with an took 60 days. The samples were analyzed on ocular-micrometer (magnification 400x), and the parameters that follow (see Analytic pro- their sizes were calculated using the volume cedure for more details) after four months of formula for a sphere. One hundred cells were adaptation. Three branches from three dif- measured to determine the average diameter ferent colonies were used in each analysis in of algae in each analysis. Cell diameters were all variants of the experiment. The average measured for at least ten individual coral and standard deviations were calculated on colonies of the same host species. the basis of n=3. Proliferating SD frequency (PSDF) and de- Determination of growth rates of corals. grading SD frequency (DSDF) After three months of acclimation of coral Normal, dividing, and degrading branches (and fragments) to 95%, 30%, 8%, zooxanthellae were counted in tissue homogen- and 2% PAR0 the samples were detached ates under a light microscope (400x). The from ceramic the pieces, shaken to remove cells in the process of division were determined excess water, weighed, placed again onto the from the initial appearance of a division furrow same cement bases and replaced in the same in the mother cells to the formation of their aquarium. After 30 - 40 days, these samples own envelope in the daughter cells. Degraded were weighed again to determine changes in or degrading SD were identified by color, size, total branch weight. Three coral branches and shape (Titlyanov et al. 1996). A total of from three different colonies of the same spe- 500 algal cells was counted in each sample. cies were used for measurements of growth The percent of dividing cells was expressed rate for each light condition. The average as the PSDF and the percent of degrading and standard deviations were calculated on cells was expressed as the DSDF. PSDF and the basis of n=3. The growth rate of samples DSDF were examined at 09:00-10:00 h, at the was calculated using the formula: time when the number of dividing cells amounted 80% of the night maximum (that occurred at about 03:00 h) and degraded zooxanthellae numbers were highest (Titlyanov et al. 1996). In all investigated scleractinian where mo is initial weight; ml is a weight at corals, the DSDF index was an indication of the end of the experiment, z T is the time be- the daily level of degradation of SD by polyp tween the two measurements of weight, ,u is cells (Titlyanov et al. 1996). In the hydrocoral the average growth rate measured in mg Millepora intricata daily remnants of degraded • day' (Brinkhuis , 1985). It is necessary to cells (DSDP) were not completely extruded note that the error of the method is rather and, consequently, the DSDF index did not significant and reached to about 10% for the determine the level of SD degradation by polyps same coral branch of e.g. Pocillopora during the day. damicornis (by 5 replicate weightings). Chlorophyll concentration To determine chlorophyll concentration, a Analytical procedures. known number of zooxanthellae were filtered using a vacuum setup (47-mm AP Millepore SD density in coral and SD sizes filters) and stored with 90% aqueous solution of acetone in a refrigerator for two days Coral tissue was removed with a Water Pik with daily agitation of the samples. Such a (Johannes and Wiebe 1970). Using a method extracts more than 95% of chlorophylls hemocytometer, samples were examined to from the SD (Titlyanov, unpublished data). determine the numbers of healthy SD (HSD), The absorbency of acetone extracts was Morphophysiological variations of symbiotic dinoflagellates 55

measured at 630 and 663 nm by a Hittachi U- thesis (Pgross)was calculated by summing the 2000 spectrophotometer. Chlorophylls a and Pnet and Rd values. The rate of Pgrosswas nor- C2 of SD were determined using the spectro- malized per mm3 SD volume. photometric equations of Jeffrey and Hum- phrey (1975). Chlorophyll content in corals Statistical analysis was calculated in ,a g per 1000 polyps or per A `Student's t-test was used to analyze the cm2 of coral surface and per mm3 of the data and to evaluate differences between zooxanthellae volume. means. The difference between means with p<0.05 was considered significant. Measurement of photosynthesis and dark res- piration rates Closed chamber incubations were performed RESULTS to measure net 02 production and 02 con- Morphological characteristics of different sumption rates. Six branches of each species types of symbiotic dinoflagellates (SD) were incubated in a 1.41 sealed plexiglas cham- Figure 2 represents three apparent popula- ber. The water in the chamber was stirred tions of SD which differ under light micro- by a magnetic stirring bar. The chamber was scope magnification. The isolated populations held in a thermostatic bath to maintain the of SD exhibited noticeably different coloration, temperature at 25°C and lit with a halogen dimensions, and shape and were subsequently lamp. Oxygen variations were recorded with categorized as L (large), B (brown) and G a Clark Oxygen Electrode (TOA Electronics (green) types of SD. The type L was found Model). The 02 electrode was calibrated before only in the colonial hydroid Millepora intricata. each measurement according to Green and Scleractinian corals contained types B and G Carritt (1967). Quantum flow of PAR in the as in initial variant as after maintenance in respirometer, equaled 1200 ii mol•m-2•sl which aquarium under 95% and 30% PAR0. While was enough to saturate the photosynthetic Pocillopora damicornis harbored only type B process in branches corals (Titlyanov et al. and and S. caliendrum 1988). The light intensity in the respirometer had only type G. Stylophora pistillata and could be reduced by neutral filters. PAR in- Echinopora lamellosa colonies always con- tensity was measured with a Li-Cor radiation tained both types B and G, although their sensor (Model Li-192 SB). The corals were relative proportions varied. Morphological exposed to both light and dark conditions for characteristics of the three SD types from 2 hours. The rates of oxygen expulsion in five coral species are presented in Table 1 the light (Pnet) and oxygen consumption in the and in Figures 3d, 6d. dark (Rd) were normalized per cm2 of the Type L had the largest (on average more coral surface. The rate of gross photosyn- than 15 ,um) cell diameter (Table 1). The shape

Table 1. Morphological characteristics of symbiotic dinoflagellates (SD) freshly isolated from hermatypic cor- als from a fringing reef at Sesoko Island (initial variant), n = 3 56 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

of these algae was spherical, the color varied erably larger (p<0.05) than DSDP diameters from deep-to light-brown (Fig. 2a, b, c). De- of B and G types cells. graded symbiotic particles (DSDP) possessed Type B cells were of smaller size than that a round shape and were red-brown in color of type L, with diameters varying from 8 to (Fig. 2a, b). The average diameter of DSDP 14 ,im (Table 1; Fig. 2d, e, f, g). The average was 7.2±1.2 /t m (Table 1) and was consid- cell diameter in the colonies of P. damicornis

a rill,

d f g

h i I k

Z to 11 0

Fig. 2 Different types of symbiotic dinoflagellates from hermatypic corals acclimated to 95% and 30% PARo: a - SD of type L from Millepora intricata (95% PAR0), dividing cell (above) , degraded SD particles DSDP (below); b - SD of type L from M. intricata (30% PAR0), adult cells (above) , two DSDP (below); c - SD of type L from M. intricata (30% PARo), host cell with four adult algae; d - Pocillopora damicornis (algae of type B, 95% PAR.), dividing cell (above), DSDP (below); e - P. damicornis (algae of type B, 95% PARo) - host cell containing four adult algae; f - P. damicornis (30% PARo) adult cell, (cell surface in focus , above picture), host cell with two DSDP; g - SD of type B from Stylophora. pistillata (30% PAR0) adult cell. with large accumulation body (inner part of the cell in focus), common DSDP; h - SD of type G from Seriatopora caliendrum (95% PAR0) - adult cells with well-visible pyrenoids and accumulation bodies; i - SD of type G from S. caliendrum (95% PARo) - adult cell with large accumulation body; j - S. caliendrum (algae of type G) (30% PAR.) - host cell containing three SD; k - SD of type G from S. caliendrum (30% PARo) - three adult cells; 1 - SD of type G from S. pistillata (95% PARo) - adult cell (above), DSDP (below); m - SD of type G from S. pistillata (30% PAR0) - dividing and adult cells (above), DSDP (below); n - SD from S. pistillata (95% PAR0) - adult cell of type B (above), adult cell of type G (below); o - SD from from S. pistillata (30% PARo) - two adult cells of type B (above), adult cell of type G (below). Morphophysiological variations of symbiotic dinoflagellates 57

and S. pistillata was 11.4±1.1 ,um (Table 1). example, by chlorophyll accumulation in unit Type B cells were spherical with a golden- of volume of symbiotic cells. For instance, brown color (Fig. 2e, f, g). DSDP of this the SD of M. intricata, type L, accumulated type were different from that of type L in the greatest quantity of pigments, to 19.8 ,u g that they had smaller sizes, irregular shape per mm3 cell volume. The lowest capabilities and a light-brown color (Fig. 2d, f, g). The in accumulation of pigments were found in type average diameter of degraded cells was 5.7± G in S. hystrix (1.3 1ag per mm3), and inter- 1.0 ,u m (Table 1). Accumulation bodies were mediate capabilities were found in type B (4.1 pale, yellowish in color and were the primary to 14.8 1ug per mm3) (Table 2). The lowest constituent of the DSDP (Fig. 2g). level of pigment accumulation in SD of S. The cells of type G were the smallest, their pistillata (the natural mixture of types B and sizes varied from 7 to 11 ,u m and were sig- G) was close to that of type G, and the upper nificantly smaller than cells of type B (Fig. level was close to cells of type B (Table 2). 2h, i, j, k; Table 1) and they were different The coral branches, acclimated to various light from SD of types L and B in that they were intensities differed from each other in colora- shaped as spheres or bean-shaped cells and tion (Fig. 1). were an olive-green color. Moreover, these The comparison of photosynthetic capaci- features were constant in the colonies accli- ties of all three types of algae in hospite, in mated to the different light conditions (Fig. corals acclimated to 30% PARo, showed that 2h, i, j, k). The size of degraded particles of the highest photosynthetic rate Pgmos was pos- type G were small (an average 4.2±0.9 ,um), sessed by type G with 211 1ag02 per mm3 SD deep olive-green color and of irregular shape, volume per h and the least was type L, with similar to that of the type B (Fig. 21, m; Table 61 ,ugO2 per mm3 of SD volume per h. The 1). Several distinct morphological character- differences in the photosynthetic capacities istics were observed in SD of the type G, for between various algal types were significant. example accumulation bodies and pyrenoids, Photosynthetic capacities of SD types B and with a bluish starch sheath (Fig. 2h, i, k). G declined significantly under acclimation to bright light, at the same time SD of type L Physiological characteristics of different types did not lose their photosynthetic capacities of SD under the light. Types B and G from corals All physiological characteristics were meas- incubated under 30% PARo , reached their ured in corals acclimated to different light peak photosynthetic capacities in 380 ,u mob intensities (Table 2; Figs 3, 4, 5, 6, 7, 8). The m'2• while algae of type L did not reach types of SD are significantly (p<0.05) different their maximum photosynthetic capacities under by several physiological characteristics : for this light intensity (Table 2).

Table 2. Physiological characteristics of symbiotic dinoflagellates (SD) in hospite (n=3) 58 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

Physiological differences among types of tained type G algae (8.4 and 7.2 mg per g per symbiotic dinof lagellates were also displayed day, respectively). Stylophora pistillata har- in the intensity of division and degradation of bored algae of types B and G, and had growth symbionts (Figs 3c, 4c, 5c, 6c). The highest rates of 3.8 mg per g per day. The colonies average level of prolif erous SD frequency P. damicornis (containing type B only) had (PSDF) was (6.1%) found in algae of type G growth rates of 1.8 mg per g per day. The in the coral S. hystrix maintained during 120 lowest growth rate (1.1 mg per g per day) days under 95% PARo (Fig. 5c). The lowest was detected in the hydrocoral M. intricata average maximum level was found in algae of which harbored type L. Difference in the rate type L (M. intricata) - 1.8% (Fig. 3c). The of growth between two coral species containing highest level of degraded SD frequency (DSDF) different SD types was significant (p<0.05) . (3.8%) was found in algae of type G (Fig. Under extremely low light (8% PARo), M. 6c), the least - in algae of type B (Fig. 4c). intricata displayed reductions in coral branch PSDF means significantly differed in L, B and weight. Pocillopora damicornis grew very G types of algae acclimated as to 95% as to slowly with a rate of 0.3 mg per g per day. 30% PARo (Figs 3e, 4c, 5c and 6c). Under The growth rates of the coral branches of S. 95% PARo, an average PSDF level in algae of caliendrum and S. hystrix were around 2 mg type G was higher approximately 3-fold than per g per day. in SD of types L and B. In light variants (8% PARo) this difference was insignificant (p> Photo-acclimation of hermatypic corals (con- 0.05). taining the same or various types of SAD) to Coral species, containing different types of different light intensities (Figs 3-8) SD, were different in growth rates (Table 2). Millepora intricata colonies containing SD Under 95% PARo light intensities the highest of type L were able to acclimate to light in- growth rates were found in the corals S. tensities from 95% to 8% PARo (Fig. 3). Under caliendrum and S. hystrix, which both con-

Pocillopora damicornis Millepora intricata a b a h

c d c d

Fig. 4 Physiological state of the coral Pocillopora Fig. 3 Physiological state of branches of the hydro- damicornis after 120 days of acclimation to light in- coral Millepora intricata after 120 days of acclimation tensities 95%, 30% 8% and 2% PARo: a - SD population to light intensities 95%, 30% and 8% PARo: a - SD density; b - chlorophyll content per mm3 of SD volume population density; b - chlorophyll contents per mm3 (chl per mm) ; c - proliferous SD frequency (PSDF) of SD volume (chl per mm) ; c - proliferous SD fre- and degrading SD frequency (DSDF) ; d - average quency (PSDF) ; d - average volume of SD and relative volume of SD and relative number of large (10-13 number of large (14-15 ,u m in diameter) and small ,u m in diameter) and small (7-9 u m in diameter) (12-13 ;um in diameter) algal cells. algal cells. Morphophysiological variations of symbiotic dinoflagellates 59

2% PARo light intensity, colonies bleached algae of type G, had similar photoacclimative and died. The acclimation to low (30% PARo) capabilities (Figs 5, 6). These species accli- and extremely low (8% PARo) intensity led to mated to light intensities from 95% to 2% PA a 2-fold increase in population density of SD R0. However, the population density of SD in branches of M. intricata compared to that increased only in S. caliendrum, under 30% of algal density under bright light (95% PARo). PARo , and symbiont density significantly Chlorophyll concentrations were significantly dropped with further reductions in light in- higher in symbiont cells acclimated to 30% tensity. Chlorophyll concentration in type G and 8% PARo. The PSDF level in dim light alga, like SD of type B, increased when low- was significantly lower than in bright light. ering the light intensity from 95% to 8% and Zooxanthellae volume reduced from 1480 ,a m3 even to PARo 2%. With the low light levels in (under 95% PARo) to 1080 ,a m3 (under 8% aquarium, intensity of cell division (PSDF) and PARo). degradation (DSDF) dropped. Cell sizes in SD of type B acclimated to the light inten- samples acclimated to different light levels sities from 95% to 2% PARo in P. damicornis changed insignificantly (Figs 5, 6). (Fig. 4). SD population density increased 1.5 The corals S. pistillata and E. lamellosa times subsequent to lowering the light intensity harbored algae of both types B and G, and from 95% to 8% PARo. With further decreases were acclimated in a similar manner (Figs 7, in light intensity to 2% PARo, the algal density 8). As in the coral P. damicornis, with algae was significantly reduced. Under shade, chlo- of type B, density of symbionts increased with rophyll concentrations in symbionts gradually a reduction in light intensity to 8% PARo but increased. PSDF and DSDF levels, and SD sizes declined with further reductions to 2% PARo. decreased significantly under shade. Chlorophyll concentrations in SD increased Both S. caliendrum and S. hystrix, harboring under acclimation to extremely low light in-

Seriatopora hystrix Seriatopora caliendrum

a b a b

c d c d

Fig. 5 Physiological state of the coral Seriatopora Fig. 6 Physiological state of the coral Seriatopora hystrix after 120 days of acclimation to light inten- caliendrum after 120 days of acclimation to light in- sities 95%, 30% 8% and 2% PARo: a - SD population tensities 95%, 30%, 8% and 2% PARo: a - SD popu- density; b - chlorophyll content per mm3 of SD volume lation density; b - chlorophyll content per mm3 of SD (chl per mm) ; c - proliferous SD frequency (PSDF) volume (chl per mm) ; c - prolif erous SD frequency and degrading SD frequency (DSDF) ; d - average vol- (PSDF) and degrading SD frequency (DSDF); d - av- ume of SD and relative number of large (10-13 u m erage volume of SD and relative number of large in diameter) and small (7-9 ,u m in diameter) algal (10-13 ,um in diameter) and small (7-9 ,um in di- cells. ameter) algal cells. 60 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

tensity (2% PARo). PSDF and DSDF indices pistillata. Under extremely low light (2% PA of SD in S. pistillata and E. lamellosa signifi- R0) the number of type G algae slightly in- cantly dropped with shading (Figs 7, 8). The creased, but there was a decline in type B changes in the average size of SD of S. pistillata algae (Fig. 7). In corals that contained only and E. lamellosa, exposed to gradually lowered one type of SD, the lowering of light intensity light intensities, differed from all investigated caused the number of large cells to decrease, corals. With a reduction in light intensity and the number of small cells to increase (Figs from 95% to 6% PARo algal sizes were sig- 3-6). nificantly increased. Algal sizes were slightly differed when corals were exposed to low light (8% PARo) and extremely low light (2% PARo). DISCUSSION The relative number of large cells, 10-13 / m Morphophysiological analyses of symbiotic in diameter (presumably type B) increased dinof lagellates in six species of hermatypic together with an increase in an average SD corals from a fringing reef at Sesoko Island sizes, and the number of small-sized algae, 7- (Okinawa, Japan) showed that scleractinian 9 ,um in diameter (presumably algae of type corals and the colonial hydroid Millepora G), declined. Under acclimation to extremely intricate, contain three morphophysiological low light the ratio of large and small cells types of symbiotic dinoflagellates (SD) which changed in the opposite direction. Under ac- we called type L (large), B (brown) and type climation to lowered light a number of golden- G (green). Morphological and physiological brown colored algae of type B increased and differences in these types of algae, living in a number of olive-green cells of type G de- various coral species, remained stable under creased. This was especially obvious in S. photo-acclimation to the range of light condi- tions. Stable differences such SD shape, size

Stylophora pistillata Echinopora lamellosa a b a b

c d c d

Fig. 7 Physiological state of the coral Stylophora pistillata after 120 days of acclimation to light in- Fig. 8 Physiological state of the coral Echinopora tensities 95%, 30%, 8% and 2% PARo: a - SD popu- lamellosa after 120 days of acclimation to light in- lation density; b - chlorophyll content per mm3 of SD tensities 95%, 30%, 8% and 2% PARo: a - SD popu- volume (chi per mm) ; c - prolif erous SD frequency lation density; b - chlorophyll content per mm3 of SD (PSDF) and degrading SD frequency (DSDF); d - av- volume (chl per mm) ; c - prolif erous SD frequency erage volume of SD and relative number of large (PSDF) and degrading SD frequency ( DSDF) ; d - av- (10-13 ,u m in diameter) and small (7-9 u m in di- erage volume of SD and relative number of large ameter) algal cells; relative number of golden-brown (10-13 ,u m in diameter) and small (7-9 ,u m in di- SD cells (B type). ameter) algal cells. Morphophysiological variations of symbiotic dinoflagellates 61

of •healthy cells, shape and average size of sity of cell division (PSDF) and level of degra- degraded SD particles (DSDP), color of dation (DSDF), by host cells, increased under healthy-looking SD and DSDP, levels of maxi- acclimation to high light, which is an indirect mum and minimum concentration of chloro- confirmation of stimulation of SD metabolism phyll, calculated per cell volume of SD, in the by bright light. The reductions in the level of rate of photosynthesis in light optimum 30% mitotic activity, by reducing light intensity, PAR0), and in maximum levels of PSDF and was detected before in Porites astreoides DSDF. On the basis of morphophysiological (Wilkerson et al. 1988) and in S. pistillata analyses we propose that these types of algae (Titlyanov et al. 2000). have not only phenotypic but genotypic dif- The other acclimative reactions in all inves- ferences. tigated corals appeared to depend on symbiont However, the above mentioned stable mor- composition. Thus, if corals had only one type phological differences in SD are not enough of algae (L or G), under acclimation to 8% for determining SD types distinguished as PAR4, the population density was reduced. In species or subspecies (Trench 1997). However, the corals P, damicornis (with algae of type in the opinion of Iglesias-Prieto and Trench B), S. pistillata and E. lamellosa (SD of both (1994, 1997) we can distinguish types (L, B, G) B and G types), in the same conditions, the as a separate taxa. This was confirmed by a number of algae increased or were stable. series of analyses on lipids and fatty acids Our investigations showed that the average and genetic-molecular methods (Hidaka et al. algal volume depends on light intensity. With in preparation; Zhukova and Titlyanov, in shade, cell volumes of all SD types were re- preparation). In the present study we showed duced. However, in corals, that contained a that some species of scleractinian corals (near mixture of B and G symbiont types, reducing Sesoko Island) contain a mixture of two types light intensity resulted in an increase in average B and G symbionts. We showed for the first cell size in dim light (8% PARA) and dropped time that the same coral contains two dis- under extreme shade (2% PARO). There were tinct (in morphology and physiology) types changes in the number of different types of of symbionts. Earlier, Rowan with co-authors SD in favour of the more low light tolerant (1997) showed that some coral species from type (type B). Under adaptation to bright light Atlantic Ocean contained three genetically de- the relative number of algae more tolerant to termined types of zooxanthellae. extreme high irradiance (type G) increased. In all probability, such photo-acclimation by Ecological importance of existence of different selection of more productive (or more toler- types of SD in hermatypic corals ant) SD types to light allows the majority of The presented material and our following hermatypic coral species to dwell in a wide investigations give basis to consider that algae range of light intensities from 100% to 0.5% of type L are not resistant to bright light PAR0 (Titlyanov and Latypov 1991). and are not able to acclimate to low light The redistribution of the three types of SD (lower than 6% PAR0). The SD of type G were ("A", "B", "C") because of light, was found tolerant to bright light, however were incapa- earlier by Rowan et al. (1997) in two Caribbean ble of acclimating to light lower than 2% PAR0. coral species Montastera annularis and M. In contrast, SD of type B from P. damicornis f aveolata. These species could harbour two were less tolerant to bright light (branches or three types of SD together in the same partly bleached) but were able to acclimate colony. Under adaptation of the whole colony, to extremely low light (to 0.8% PARO,) or only parts, to low light, the redistribution (Titlyanov et al, in preparation). In all inves- of RFLP types changed to a predominance of tigated corals the acclimation to high light was type "C". Rowan et al. (1997) concluded that possible by reductions in zooxanthellae popu- the type "C" is more tolerant to low light, and lation densities and reductions in chlorophyll types "A" and "B" to bright light. Unfortu- concentrations. These common acclimative nately, all conclusions on tolerance and sensi- reactions to bright light, for some hermatypic tivity of three RFLP types of SD to light by corals, were detected earlier by Zvalinsky et Rowan et al. (1997) were made without meas- al. (1978), Titlyanov et al. (1980), Falkowsky urements of light intensity in microhabitat of and Dubinsky (1981) and Dustan (1982). corals or for different parts of the corals, and Moreover, in all investigated corals the inten- without revealing specific photo-acclimative 62 E.A. Titlyanov, T.V. Titlyanova, A. Amat and K.Yamazato

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