Appendix1. Environmental conditions and summary of collection information.
Since coral reproductive activity generally demonstrates a high correlation with certain physical environmental conditions, especially sea water temperature (e.g., Fadlallah 1983;Willis et al. 1985; Harrison and Wallace 1990; Richmond and Hunter 1990), each study site is briefly characterized with respect to seasonal thermal variations. Wet and dry season differences, which have a large influence on solar insolation, water clarity, salinity, nutrients and chlorophyll a concentration in the eastern tropical Pacific are also noted. Some recent studies suggest that seasonal variations in light are also closely correlated with coral reproductive activity (Mendes and Woodley 2002; Penland et al. 2004). The following abbreviated regional treatment is largely from Glynn 1977; Kwiecinski and Chial 1983;Guzmán and Cortés 1989; Glynn et al. 1991; Fiedler 1992; Podestá and Glynn 1997; and D’Croz and O’Dea 2007.
Mean sea surface temperature (SST) off SW Costa Rica and the Gulf of Chiriquíis high (28-29oC) and relatively stable year round. Thermocline shoaling at the UvaIsland reef occasionally causes temperatures to decline to 25oC at 2 m depth (Glynn et al. 2001). Seasonal upwelling occurs during the dry season in the Gulf of Panamá, with mean surface temperature declining from 28oC in December to 24oC in March. During upwelling pulses, temperatures at 2 m depth on the Saboga coral reef often fall below 20oC and occasionally dip to 16-17oC for brief periods (Glynn et al. 2001). Near surface nutrients (NO3-, PO43-) and chlorophyll a concentrations during the mid-dry season are two to nearly 10 times higher in the Gulf of Panamá than in the Gulf of Chiriquí (D’Croz and O’Dea 2007). Galápagos mean SSTs in the eastern archipelago are highest from January through May, ranging from 24 to 26oC and are lowest (~22oC) from August to October. Upwelling of the Equatorial Undercurrent on western shores can cause SSTs to drop to 18-20oC during the warm season and to 15oC and lower during the cool season. The occurrence of El Niño and La Niña-like conditions contribute greatly to increased interannual variability in temperature.
The three nearshore study sites in Costa Rica and Panamá are markedly affected by wet and dry seasons, which are controlled by the seasonal migration of the Intertropical Convergence Zone. The dry season occurs at about the same time at these sites, from end-December or January through April, and the wet season begins suddenly in May and ends in December. Mean annual rainfall near the CañoIsland (Costa Rica) site is ~1,500 mm, and ~2,600 mm and ~1,900mm respectively in the Gulfs of Chiriquí and Panamá. Cloud cover was relatively low during the dry season in Panamá, ranging from 6.8 to 11.6% over the Gulf of Chiriquí and Panamá respectively in the early 1970s. In the wet season, cloud cover increased from 23.2 to 58.4% (see Glynn 1977). The offshore Galápagos Islands are subject to wet/warm and dry/cool seasons, but during non-El Niño years the seasonal differences in precipitation are not so pronounced as along the mainland. For example, annual rainfall at AcademyBay (Santa Cruz Island) averages about 500 mm. During the 1983 El Niño year, however, annual rainfall was 2,769 mm at AcademyBay. Light levels are reduced in the dry/cool season. With SSTs cooling in the dry season (June-December), a low cloud inversion layer forms along the shores of islands >300 m elevation, causing a moderate to heavy drizzle (“garua”).
References:
D’Croz L and O’Dea A (2007) Variability in upwelling along the Pacific shelf of Panama and implications for the distribution of nutrients and chlorophyll. EstCoastShelf Sci 73:325-340
Fiedler PC (1992) Seasonal climatologies and variability of eastern tropical Pacific surface waters. National Oceanographic and Atmospheric Administration Tech Rpt, Nat Mar Fish Serv 109:1-65
Glynn PW (1977) Coral growth in upwelling and nonupwelling areas off the Pacific coast of Panamá. J Mar Res 35:567-585
Guzmán HM and Cortés J (1989) Growth rates of eight species of scleractinian corals in the eastern Pacific (Costa Rica). Bull Mar Sci 44:1186-1194
Mendes JM, Woodley JD (2002)Timing of reproduction in Montastrea annularis: relationship to environmental variables. Mar Ecol Prog Ser 227:241-251
Willis BL, Babcock RC, Harrison PL, Oliver JK (1985) Patterns in the mass spawning of corals on The Great Barrier Reef from 1981 to 1984. Proc 5th Int Coral Reef Congr Tahiti 4: 343-348
Table. Tubastraea coccinea. Summary of collection information from the study sites in Costa Rica, Panamá and the Galápagos Islands. The different months of the year sampled at each site are listed under “No. months collected.” Publication location refers to the sections of the study that introduce the collection and observation results. R – Results section: 1, gamete and planulae development; 2, reproductive condition; 3, seasonality; 4, lunar activity; 5, fecundity. A – Appendices: 2, 3 and 4.
Location / No. histological / No. / Planula per spawn / Fecundity collections / No. mo / Publicationcollections / colonies / observations (no. colonies) / (no. colonies) / collected / location
Costa Rica
Caño Island
(4 Aug 1985 – 25 Apr 1997) / 6 / 26 / 6 / R 1 - 5; A2,3
Panamá
Gulf of Chiriquí
Uva Island
11 Mar 1990 – 18 Mar 1998 / 18 / 73 / 8 / R 1 - 5, A2
May 1999 (planula release) / 15 / R 4,5
May 2000 (planula release) / 5 / R 4,5
Mar 2002 (planula release) / 8 / R 4,5
Mar 2003 (planula release) / 11 / R 4,5
Mar 2005 (planula release) / 17 / R 4,5
Mar 2006 (planula release) / 32 / 37 / R 3 – 5
Mar 2006 (sperm spawning) / 32 / R 4 – 5
Sep 2006 / 30 / R 3 - 5, A4
Gulf of Panamá
Saboga Island
11 Oct 1989 – 21 Mar 1998 / 18 / 73 / 8 / R 1 - 5, A 2
Taboga Island
23 Mar 1990 – 8 Aug 1994 / 25 / 70 / 12 / R 1 - 5, A 2
Ecuador
Galápagos Islands
5 May 1985- 19 May 1993 / 53 / 182 / 12 / R 1 - 5, A 2
Marchena Island
May 2006 (sperm spawning) / 26 / 36 / R 3 – 5
Appendix 2. Descriptions and development of gametes and planulae.
Stage I oocytes are rarely seen within the mesenterial gastrodermis and range from about 15 to 50 μm in diameter (Fig. a). The nucleus stains purplish-pink and has a smooth texture, with a distinctly round, cherry-red nucleolus. The cytoplasm is grainy and usually a grayish-purple to faint pink. The oocyte:nucleus diameter ratio ranges from ~1:1 to 1:2. Oocytes of 36-50 μm have been observed migrating with cytoplasmic extensions stretching outwards towards the mesoglea.
Stage II oocytes are completely bound in the mesoglea. The nucleus stains purplish-pink and displays a smooth texture. The cytoplasm is more visible and grainy, usually a grayish-purple to faint pink. Later Stage II oocytes can be distinguished by the appearance of barely visible 1-2 μm lucent vesicles (Fig. a). The oocytes stain pale pink with bluish tinges or light lavender, more darkly than in Stage I oocytes. As the cytoplasm/yolk and nucleus continue to increase in volume, the nucleolus rapidly increases in size, approximately doubling. Stage II oocytes range from approximately 50 to 160 μm in diameter. Oocyte:nucleus diameter increases to 2.5:1. One to three nucleoli of equal size have been observed in Stage II oocytes. Additional nucleoli may degenerate during further development. In Stage IV ova a minute nucleolus (3-4 μm wide) was associated with another nucleolus that appeared normal in size. Stage I oocytes were intermingled with Stage III oocytes and Stage IV ova on the same mesenteries. Possible fusion of oocytes occurred during Stages I and II. As a result, many early oocytes contained one to several cytoplasmic vacuoles, possibly remnants of a nuclear envelope. In Psammocora stellata and Pavona gigantea, several small “satellite” nucleoli appear as oogenesis progresses and are associated with the original nucleolus that remains prominent (unpub. obs.).
Stage III oocytes stain a darker pink (Fig. c). The more numerous yolk vesicles increase in size up to ~3 μm in diameter. They are variable in size, from ~150 to 300 μm in diameter, but more uniform than in Stage IV. The nucleus is still smooth and stains purple to colorless. The nucleolus does not noticeably increase in size, but the nucleus does, elevating the nucleus:nucleolus diameter ratio slightly to ~4:1. The oocyte:nucleus diameter ratio is ~4:1.
Stage IV ova become a dark raspberry-red with large dark red vesicles. Vesicles are extremely abundant and range from ~3-10 μm. Small spheres of yolk, intermingled throughout the cell, form from the adherence of up to ~10 vesicles. Nucleoli are seldom seen, but when present appear smaller than in earlier stages. Ova:nucleus diameter ratio increases to 6.5:1, in part due to the dramatic increase in yolk production. These ova vary greatly in size due to sectioning, ranging from ~300-800 μm. A dark thick border begins to form in early Stage IV ova and continues to develop as they mature. It is grainy and orange to dark pink or raspberry, and then deepens in color to a dark red as development continues. It persists at 4-6 μm in width. This is the most distinguishing feature between late Stage III oocytes and early Stage IV ova. A grainy dark band also has been observed in Psammocora stellata and Psammocora superficialis and to a lesser degree in the agariciid corals Pavona varians and Pavona gigantea (unpub. obs. and Glynn et al. 1996). In T. coccinea the grainy appearance is due at least in part to the presence of thousands of minute, barely visible uniform dark orange to dark red vesicles concentrated along the edge of the cell. In Psammocora spp. this band is orange to crimson, and in pavonid corals it stains gray or bluish. In pavonid ova the border is less noticeable and may be apparent only at the end of oogenesis. In late Stage IV ova, the nucleus often moves to the outer edge of the cell to become flattened or saddle-shaped, lying adjacent and parallel to the egg membrane (Fig. d). Round dark yellow-gold to orange bodies, few relative to yolk vesicles, form with the central portion of the yolk and migrate to the perimeter of the cell to lay adjacent/near the border. Polar bodies were not visible in the histological sections.
Duringoocyte development in Stages II and III, endodermal cells lying adjacent to the developing oocytes began to increase dramatically in size. As these colorless, vacuolated cells expanded out from the oocytes, other inclusion-containing endodermal cell types were pushed aside or squeezed in between. These cells reached full length during early Stage III, however, they continued to develop, becoming puffier and balloon-like during Stage IV, culminating in a wide cushiony band that buffered each oocyte. They may stain a light baby blue in late developing ova andare completely deplete of cellular structure except for pinpoint green nuclei that are sometimes visible. During Stage IV development these cells are associated with masses of bubbly, pink lucent vesicles very similar in staining characteristics and morphology (light microscope) to the yolk vesicles within the oocytes (possibly nurse cells). These vesicles are no longer present outside of the ova during late Stage IV. However, no evidence of vesicular movement past the ovum plasma membrane can be discerned.
A Stage I spermary (≤45 μm in diameter), wrapped in mesoglea,consists of a group of round to tear-shaped interstitial cells which are each ~2-3 μm across (Fig. e). These cells range from a faint pink to grayish blue, but are most often a light lucent gray color. A dark nucleus is visible. Stage II spermaries become a compact sphere (~60 μm) due to numerous interstitial cells still migrating into the mesoglea. Once the interstitial cells have filled the gonad, their color changes to raspberry. These cells (spermatocytes) are approximately spherical, uniform in size, and are indicative of Stage III spermaries. The spermary then grows larger and eventually develops a lumen when ~175-200 μm in diameter (Fig. e). Smaller cells appear, filling the lumen, and these give rise to cells of different sizes as spermatocytes transition to spermatids. Stage IV spermaries range from 150 to 200 μm in diameter. When fully divided, spermatids are minute dark raspberry-red spheres. Initially these cells are not densely packed, but eventually the spermary condenses and diminishes in size until it becomes a tight packet of equal-sized spermatids (Fig. b). In histological sections, no tails were observed. However, in freshly collected field samples spermatozoa were robust in size and contained long tails.
Tubastraea coccineacolonies are brooders with fertilized ova (embryos) or planulae released into the gastrovascular cavity. They are held there until the oral pore and several mesenterial filaments are formed. Stage I planulae and Stage IV ova appear similar. Planulae are mostly vesicular and are devoid of a nucleus. The dark red border of Stage IV ova has disappeared. The yolk appears to migrate peripherally and channels into finger-like projections (Fig. f). When released into the gastrovascular cavity, Stage I planulae are a bubbly amorphous mass. A small layer of cells begins to form around the outer edge. Additional cell rows continue to form progressively toward the interior. The outermost cells become ciliated as other cell types also differentiate. An invagination of the periphery begins. The inner yolk vesicles and cytoplasm clump into spheres and move peripherally. Furrows form and progress towards the interior as the planula continues to develop. This may represent incomplete cleavage, possibly a type of superficial cleavage, resulting from the large volume of yolk and cell formation. The center of the planula now becomes more spacious and interstitial cells form along both sides of the cleavage plane.
Stage II planulae are defined by the formation of a thin concentric circle of mesoglea ~ 50μm from the larva’s edge. The mesoglea (staining light gray to blue) separates the ciliated cells, mucous cells and cnidoblasts from the centrally located yolk and locus of future gastrodermal cell formation. The forming epidermis becomes highly differentiated with cell types while the interior of the planula is still highly vesicular with no special features (Fig. g). This inner yolky material becomes progressively less crowded through the gradual movement of vesicles towards the periphery of the cell. These vesicles diminish in size, becoming smaller and smaller until once beyond the mesoglea they completely vanish upon reaching the outer ciliated cells. The vesicles appear intermingled between the different cell types within the epidermis.
During the loss of internal vesicular material, grainy grayish strings of cells form near the center of Stage III planulae. Short, stubby blue mesenteries form where these strings touch the mesoglea. A central lumen forms in the interior as the vesicular material migrates (Fig. h). This seems to be a brief transitory period and may aid in pushing the remaining yolk vesicles outwards. When the lumen disappears, most vesicles have departed from the center and are replaced by a matrix of large colorless cells with gray nuclei. A fully formed oral pore is now present and all 12 mesenteries have begun to form. Mesenteries radiate toward the oral pore like the longitudinal demarcations on a globe. The mesoglea wraps around the oral pore and appears continuous with the mesenteries. In Stage IV planulae, very short mesenterial filaments begin to develop on stubby mesenteries (Fig. i).
Figure. Tubastraea coccinea. Reproductive stages (a) Stage I oocyte (IO) partially enveloped by mesoglea. Lone arrow points to enlarged interstitial cell (oogonium). (b) Hermaphroditic mesentery with Stage IV spermaries (IVS), Stage III spermaries (IIIS) and two Stage II oocytes (IIO) with a few dark yolk granules beginning to form. (c) Female mesenteries with nine oocytes representing later Stage III development. (d) Late Stage IV ovum with triangular nucleus that has completely migrated towards the plasma membrane. (e) Two Stage III spermaries (IIIS) with well formed lumen. One early Stage I spermary (IS) at lower right. (f) Stage I planula (embryo) displaying migration of yolk vesicles and the formation of first cells at peripheral edge. (g) Cross section of a Stage III planula through the oral pore (center). (h) Transitional space formed during Stage III planula development. Note dark vesicles still present in yolk. (i) Stage IV planula revealing at least 5 mesenterial filaments with formed gastrodermis.
Appendix 3. Ovum and polyp sizes, and number of ova polyp-1 at five localities.
Table. Tubastraea coccinea. Mean (SE) Stage IV ovum diameters and volumes, polyp volumes, ova polyp-1, ova mesentery-1, and ova polyp-1 at five localities. All measurements from histological sections. Sample mean differences were tested employing Kruskal-Wallis (K-W) analyses and Dunn’s (D) method for a posteriori multiple comparisons (p < 0.05). Parametric analyses employed ANOVA tests, and Tukey (T) and Student-Newman-Keuls (SNK) a posteriori multiple comparisons procedures. Egg volume was calculated from the mean egg diameter using the formula for a sphere; eggs polyp-1 are the product of eggs mesentery-1 at each site and 24, which is the overall mean number of mesenteries polyp-1 at all sites. Can Caño Isl.; Uva Uva Isl.; Sab Saboga Isl.; Tab Taboga Isl.; Gal Galápagos Isl.
Attribute / Location / SignificanceCaño Isl., / Uva Isl., / Saboga Isl., / Taboga Isl. / Galápagos Isl.
Costa Rica / Panamá / Panamá / Panamá / Ecuador
Diameter, µm ± SE / 458.6 ± 91.6 / 466.7 ± 15.1 / 462.6 ± 27.9 / 525.6 ± 75.3 / 503.0 ± 12.6 / p < 0.001, K-W
(n col, ova) / (19, 191) / (26, 311) / (17, 217) / (18, 246) / (41,741) / p < 0.05, D
Gal > Uva, Can
Tab > Uva, Can, Sab
Max diameter / 800 / 910 / 920 / 770 / 1010
Stage IV ova, μm
Polyp area, cm2 ± SE / 1.04 ± 0.09 / 0.72 ± 0.02 / 0.80 ± 0.02 / 0.96 ± 0.05 / 0.82 ± 0.02 / p < 0.001, K-W
(n col, polyps) / (5, 13) / (11, 87) / (21, 111) / (8, 27) / (14, 116) / p < 0.05, D
Sab = Uva, Gal
Volume, mm3± SE / 0.071 ± 0.004 / 0.069 ± 0.003 / 0.081 ± 0.005 / 0.092 ± 0.004 / 0.088 ± 0.003 / p < 0.001, K-W
(n ova) / (191) / (309) / (217) / (246) / (741) / p < 0.05, D
Tab > Uva, Can, Sab
Gal > Uva, Can
Mesenteries polyp-1 / 23.4 ± 2.6 / 23.4 ± 0.6 / 21.5 ± 1.2 / 26.3 ± 1.7 / 26.3 ± 1.3 / p > 0.05
no ± SE / (8) / (27) / (22) / (15) / (24)
(n polyps)
Ova mesentery-1 / 1.3 / 3.7 ± 0.4 / 1.9 ± 1.9 / 3.2 ± 0.5 / 2.0 ± 0.3 / p = 0.02, ANOVA
no ± SE / (1, 6) / (9, 52) / (2, 9) / (9, 51) / (21, 140) / NS Tukey test
(n pol, mes)
Ova polyp-1 / 31.2 / 88.1 ± 10.7 / 45.6 ± 45.6 / 77.9 ± 14.4 / 48.3 ± 7.2 / p = 0.02, ANOVA
no ± SE
(n polyps) / (1) / (9) / (2) / (7) / (22) / NS Student - Newman Keuls
Appendix 4. Seasonal and lunar reproductive activity of Tubastraea coccinea at Caño Island, Costa Rica.
Figure. Reproductive activity at Caño Island, Costa Rica, as function of season (a) and lunar phase (b), based on 6 collections and 26 colonies examined (1985-1997). Top halves of both plots refer to percent colonies with spermaries, bottom halvesto percent colonies with planulae. Percentage values noted beside collections indicate the proportions of spermaries or planulaein Stage IV development. Abscissas Julian and lunar days (full moon occurred near Lunar Day 15).