S . Ward, P. Harrison J. Exp. Mar. Biol. Ecol. 246 2000 179 –221
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gonochoric Fadlallah, 1983; Harrison and Wallace, 1990. In hermaphroditic coral species, eggs and spermaries may develop on the same mesenteries as in the faviids and
mussids, on different mesenteries within the same polyp most pocilloporids and acroporids, in different polyps within the same colony, or rarely, at different times
within the same colony Fadlallah, 1983; Harrison and Wallace, 1990. Acroporids are hermaphroditic with eggs and spermaries on different mesenteries within the same
polyp.
Measures of fecundity such as the number of eggs or the number of planulae per polyp can provide a useful index of reproductive effort and, as such, are a useful
indicator of the health of a coral Kojis and Quinn, 1984; Harrison and Wallace, 1990. Consequently, changes in these measures can be an indication of sublethal stress in
corals. However, fecundity alone can underestimate the reproductive effort of the coral and is better combined with other reproductive measures such as the size of the eggs and
the volume of testes material Harrison and Wallace, 1990. It is important to understand the gametogenic cycle of the species so that sampling can occur at the most appropriate
times.
The aims of these experiments were to examine the potential effects of elevated levels of nutrients on gametogenesis and fecundity in two reef coral species, Acropora
longicyathus and Acropora aspera, transplanted into the experimental microatolls. Secondly, fecundity in colonies of Acropora aspera occurring naturally in the mi-
croatolls was quantified to determine whether these were affected by the nutrient treatments. These species were chosen as they have relatively large polyps that can be
dissected by hand so that the eggs and testes may be measured without the use of histological sections Oliver, 1979; Wallace, 1985. When measuring reproductive output
it is preferable to use polyp dissections rather than histology, as tissue shrinkage occurs during histological processes, and gametes or larvae may not occur in some sections,
hence reproductive output may be underestimated in histological sections Harrison and Wallace, 1990. A
. longicyathus is also very suitable for transplantation because as the colonies grow, the basal areas die off, so that individual sections of one or more
branches usually attach directly onto the substratum so sections of the colonies can be removed without damaging the remainder of the colony. The bottlebrush growth form
also allows easy fragmentation.
2. Methods
One Tree Reef is located in the Mackay–Capricorn section at the southern end of the Great Barrier Reef Marine Park 1528069E, 208319S and is situated 70 km east of
Gladstone on the coast of Queensland and 18 km from the edge of the continental shelf. One Tree Reef is a platform reef which measures 4.7 3 2.7 km and contains three
separate lagoons Heatwole, 1981. The unbroken emergent reef crest causes the main lagoon at One Tree Reef to be isolated from the surrounding water at low tide once the
outside tide falls below about mean surface water level generally for 5–6 h Hatcher and Frith, 1985. This restricts the time available for water exchange between the lagoon
and the ocean to half the tidal cycle. The lagoon has a complex structure with numerous
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small circular patch reefs termed microatolls. At low tide, water within these microatolls is isolated from the surrounding lagoonal water for 3–4 h and during high tide the water
rises 0.1 up to 1.2 m above the perimeter. The microatolls have walls made of coralline algae and coral and these walls drop vertically to the lagoon floor which is usually 5–8
m depth.
2.1. Experimental design Twelve microatolls in the northern end of the lagoon were chosen for the ENCORE
3
experiment. These measured 16–25 m across, had volumes of 125–446 m and had similar coral algal communities Larkum and Steven, 1994. Three microatolls were
randomly allocated to one of four treatments. Three microatolls were given no additional nutrients controls, three microatolls were dosed with ammonium chloride only
nitrogen treatment, three were dosed with potassium dihydrogen phosphate phosphor- us treatment and three microatolls were dosed with a combination of nitrogen and
phosphorus ammonium chloride plus potassium dihydrogen phosphate.
2.2. Acropora longicyathus Acropora longicyathus was chosen as the main experimental species and, conse-
quently, its gametogenesis was studied in the most detail. Sixty large colonies of Acropora longicyathus were selected within the lagoon. These colonies were located in
the lagoon, around the outside of the ENCORE microatolls and nearby patch reefs in water no more than 2 m deep. In an attempt to avoid choosing clone colonies, most
colonies were at least 10 m from each other and were selected on the basis of having different growth forms and pigmentation.
Several large pieces were taken from each parent colony and these were transplanted from the lagoon into the twelve ENCORE microatolls in February 1993, seven months
before the nutrient dosing commenced. Five colonies were randomly chosen from the sixty parents to be transplanted to each of the 12 microatolls so that there was a total of
15 A . longicyathus transplants for each treatment i.e. five colonies in three microatolls
per treatment. The pieces of each colony were transplanted onto a separate plastic rack with PVC ‘‘legs’’ to hold it above the substratum. Pieces of coral were attached to the
racks using cable ties and the racks were placed on the substratum of the microatolls. The parental colonies from which the transplants were taken were labelled so that they
could be sampled throughout the study. They received no additional nutrients or manipulation.
2.3. Acropora aspera Portions of five colonies of Acropora aspera were also transplanted into the
microatolls. This species forms very large colonies and the five parent colonies which were chosen were large, situated at least 50 m apart and were different colours. One
piece of each of these parent colonies was transplanted into each of the 12 microatolls, which provided 15 transplants for each treatment i.e. five colonies in three microatolls
S . Ward, P. Harrison J. Exp. Mar. Biol. Ecol. 246 2000 179 –221
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per treatment. Transplanted pieces were attached to plastic racks and placed into the microatolls in the same manner as those of Acropora longicyathus.
The five A . aspera parent colonies were not monitored.
2.4. Non-transplanted A. aspera In addition, colonies of Acropora aspera were already growing naturally in nine of
the microatolls and 18 of these naturally occurring colonies were labelled and mapped for sampling.
There were two fertilization periods; the initial dosing commenced in September 1993 and continued until January 1995. Microatolls were dosed twice a day, once every low
tide when the microatolls were isolated from the surrounding lagoon. Nutrients were added to achieve concentrations within the microatolls of 10 mM nitrogen and 2 mM
phosphorus at the start of each fertilization. The nutrients were rapidly dispersed within the microatolls so that nutrient concentrations were 2 mM ammonium and 0.2 mM
phosphorus within minutes following addition, and were close to background levels within 1–3 hours after dosing K. Koop, pers. comm.. The rate of nutrient dissipation
was influenced by weather conditions with concentrations dropping fastest on windy days.
During the second fertilization phase, from January 1995 to February 1996, con- centrations were increased to achieve loadings of 20 mM ammonium and 4 mM
phosphorus. Microatolls were dosed three times every low tide rather than once to ensure a more constant concentration. Actual concentrations of nutrients during the low
tides in this period were much closer to the intended concentrations than those achieved in the first period of fertilization. Concentrations of approximately 20 mM ammonium
and 4 mM phosphorus were maintained for much of the low tide period. Over the 430 days of fertilization total loading of the microatolls ranged between 2097 and 5977
moles of NH –N and 245 to 1380 moles PO –P. The lower figures for each represent
4 4
loadings between the low tide dosing periods. This experiment used short term pulsed additions which resulted in comparatively small increases in nutrient availability.
2.5. Sampling procedure The 60 Acropora longicyathus transplants and 60 parental colonies were sampled at
approximately three monthly intervals from February 1993 to November 1995. Sixty Acropora aspera transplants were sampled approximately one month before spawning
and a few days prior to the predicted spawning period each year. The 18 colonies of A .
aspera naturally occurring in the microatolls were sampled a few days before the predicted spawning period each year.
For all groups of corals, pieces of coral branches between 7 and 10 cm long were broken from the colonies using long nosed pliers and placed into 10 formaldehyde in
seawater. In the laboratory these pieces were decalcified using 0.5–1 M hydrochloric acid and 2 formaldehyde. Decalcification took 1–2 weeks and generally required one
change of acid. The decalcification process was continued until no skeletal material remained. Samples were then placed into 70 alcohol in plastic vials.
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2.6. Processing of samples Five polyps were scored for each sample. Individual polyps were dissected out from
the decalcified tissue while viewed under a stereo dissecting microscope after Oliver, 1979; Wallace, 1985. Each polyp was dissected to expose the eight mesenteries. Four
mesenteries contained strings of eggs and four mesenteries developed testes. For each polyp, the number and the size of the eggs were scored. Two measurements were taken
from each egg, a ‘‘length’’ and ‘‘width’’ measurement using a calibrated eyepiece micrometer in the dissecting microscope. The egg size was the mean of these two
measures. The egg size of the polyp was the mean of the individual egg sizes. ‘‘Egg total’’ was calculated by adding all the individual egg sizes or by multiplying the polyp
egg size by the polyp egg number.
Testes were counted for the testes number. The length and width of each testis was measured using a calibrated eyepiece micrometer. The length and width of each testis
were multiplied together and the sum of these calculations for each polyp was called the ‘‘testes total’’ for that polyp.
2.7. Statistical analyses The egg number, polyp egg size, egg total and testes total were analysed for Acropora
longicyathus and Acropora aspera transplants using orthogonal two way nested analyses of variance Underwood, 1981. The main factors were the presence or absence of
nitrogen and the presence or absence of phosphorus. For the nitrogen analyses, microatolls exposed to elevated nitrogen nitrogen only and nitrogen plus phosphorus
microatolls were compared with those which did not receive nitrogen controls and phosphorus only microatolls. For the phosphorus analyses, microatolls exposed to
phosphorus phosphorus only and nitrogen plus phosphorus microatolls were compared with those which did not receive phosphorus controls and nitrogen only. Microatolls
were nested within the nitrogen by phosphorus interaction. Heterogeneity of variance was tested using Cochran’s test.
The data for the colonies of Acropora aspera naturally occurring in the microatolls were analysed using two way orthogonal analyses of variance without the microatoll
nesting as these colonies were only present in nine of the twelve microatolls. Fecundities of transplanted colonies of Acropora longicyathus in each treatment were
compared with those of their parental colonies using one way analyses of variance.
3. Results
