Bianca wright

The effect of different water flow and light conditions on the growth rates of the coral species Zoanthidae, Montipora and Xenia, directly after fragmentation B.Wright

Abstract Fragmentation, caused by physical damage, plays a major part in mitigating the growth and resilience of colonial invertebrate corals in frequently disturbed environments. This study was aimed to document the short-term effects of different water flow and light conditions on coral growth directly after fragmentation. Growth rates are seen as a reflection of physiological processes, such as respiration, photosynthesis and feeding efficiency, therefore was used in the experiment to indicate the corals overall health. Despite increasing pressure on coral reefs as a result of direct contact, aquarium trades and intensive storm damage, the effects of abiotic factors to the growth of newly fragmented species has been poorly documented. To study the optimal light and flow conditions for which different coral species have maximum growth rates, three series of Montipora Capricornia, Zoanthus sociatus and Xenia elongatia, were exposed to different light levels (3500 PAR and 8000 PAR) and water flow rates (> 0.1 m/s and 10 m/s), for 28 days in a closed aquaria. Throughout the study period, the growth rates were recorded at regular intervals by measuring the surface diameter and individual polyp numbers to determine which factor may have the strongest influence. It was hypothesised that increased water flow and light levels would positively effect growth. By observation, both the X. elongata and Z. sociatus showed reduced growth in the low flow (>0.1 m/s), low light (3500 PAR) location compared to the high flow (10 m/s), low light location. The growth rates between the high (8000 PAR) and low light levels however, were relatively similar; indicating that in contrast to previous suggestions, water flow had a stronger influence on the growth of these species then light. It is possible that higher flow rates reduce disturbance of competing algae or sedimentation, Notably, other effects of increased flow, such as enhanced respiratory rates and increased nutrient uptake, might have been equally responsible, allowing maximum growth rate possible under the given environmental conditions. The montipora sp showed similar growth rates in both the high and low flow conditions, however grew almost twice the rate with higher light intensity; suggesting that light had a stronger influence then water flow. In complete contrast to the visual observations, statistical results (2-way ANOVA) showed no significant differences (P > 0.05) in the growth of the three species over time between each location. It is suggested that a more accurate statistical analysis would occur from data collected over a long-term study using a 3-way ANOVA. The research was conducted as part of a long term aquarium setup at Falmouth Marine School, contributing towards a further understanding of tropical marine species in closed captivity and towards the FMS Propagation Unit Design Project of 2012. The findings are key to understanding growth and restoration of colonial marine invertebrates, especially in cultured aquarium environments where corals undergo frequent fragmentation.

Keywords: Coral growth, Water flow, Light intensity, Fragmentation, Marine Aquaria, Abiotic factors

B.Wright 1 Kingsbury, Stratton Terrace, TR11 2SY, Falmouth, Cornwall, UK e-mail: bianca372@hotmail.com C.Baldwin Falmouth Marine School, Cornwall Collage,, TR11 2SY, Falmouth, Cornwall, UK

Introduction An extensive amount of research has supported evidence that coral reefs are one of the most productive, biologically diverse and vulnerable ecosystems on Earth (Hodgson 1998, Wilkinson 2004, Douglas 2003). Consisting of hard stony corals ‘Scleractinia’ and soft corals ‘Alcyonacea’, the intricate network of structures are typically confined to shallow tropical waters, restricted by the need for sunlight, clean water and warm temperature (Kotb 2004). Coral reefs are widely acknowledged for being among the most attractive and popular of all marine environments (Spurgeon 1992).

Not only do 25% of marine species directly rely upon coral for survival (Hanna 2008), but over 350 million people worldwide depend them for food and well-being (Serour 2004). Reefs also play an essential role in the ecosystems functioning and health, supporting water filtration, fish reproduction, shoreline protection and erosion prevention (Gomez 1997; Moberg & Folke 1999; Jameson et al. 2003 and Zaid 2000, Hughes et al. 2003, Ammar 2009) However, publications by Wilkinson (2004), Sebens (1994) and White et. al. (2000) concluded that the majority of the worlds reefs are suffering damage from the same anthropogenic activities they sustain. The studies all showed that over the last few decades, humans alone have destroyed over 20% of the world’s reefs beyond restoration. Some of the highest impacts are caused by direct contact from recreational use (Barker 2003, Hawkins & Roberts1992), destructive fishing methods (Hodgson 1999) and eutrophication due to sedimentation and pollution (Zaid 2000, Berkelmans 1999, Meesters 1994). In addition, pressures from global change and natural disturbances have resulted in escalated bleaching events and intensive storm breakage (Pittock 1999, Jackson et al. 2001, Hughes et al. 2003, Wakeford et al. 2008). Such intense and frequent stressors to coral reef habitats dramatically reduce the corals resilience and ability to regenerate adequately (Hart & Hart 2006), causing many long term, irreversible impacts to the whole ecosystem (Meesters 1994, Jackson et al. 2001). Over recent years, this concern has initiated the suggestion that future of coral reef management should be focused on restoration (Ammar 2009, Shaish et al 2010). With the growing demand for marine species in aquaria trades (Dixon 1998), coral husbandry and farming is thought to lesson the pressure on wild species, benefiting reef restoration as well as biomedical research (Kotb 2004, Baums 2008)). Evidently, an understanding of the factors and processes that determine growth and resilience of different coral species is essential for the appropriate management both for the culture and the restoration of reef ecosystems (Meesters et al.1994, Guest et al. 2010, Shaish et al. 2010) The primary architects of the reef are the hermatypic ‘reef building’ scleractinian corals, existing in symbiosis with zoozantheallae, living in the endodermal tissues of their host (Mather & Bennet 1993, Jameson et al. 2003). These unicellular dinoflagellates translocate up to 95% of the carbon fixed during photosynthesis (Berkelmans 1999, Meesters 1994), providing oxygen and nutrients to the coral polyps (Hodgson 2000. Hanna 2008,). This enables them to benefit from both heterotrophic and phototrophic carbon sources required for growth, reproduction and restoration (Serour 2004, Meesters 1994). Furthermore, the symbiosis enables rapid skeletal growth where calcium carbonate is secreted in the form of aragonite (Van-Oppen 2006), which over time, forms a structurally complex reef . In contrast, nonzooxanthellae scleractinians grow at an extremely slow rate. Unlike their counterparts, these ‘non reef-building’ Ahermaypic corals can tolerate less light and colder temperatures and can be found at depths below 500 meters (Schutter et al. 2009). Other ahermaypic corals include the soft Alcyonacea corals, which posses zooxanthellae but do not secrete a rigid calcareous skeleton (Baums 2008, Hanna 2008).

The study of how abiotic factors effect the health and physiology of different coral species is vital in finding the optimal conditions required to grow coral (Baums 2008). Each species can differ in their need for growth, which has created a huge challenge in recent propagation efforts (Hatziolos, et al. 1998 Kuffiner 2001, Sebens et al. 2003. Guest. et al. 2011). The species chosen for this study include two different zooxanthellae scleractinians, Montipora Capricornia and Zoanthus sociatus and the soft coral, Xenia elongatia. The ‘plating cup coral’ M.Capricornia (Veron 1985), is a small polyp stony (SPS) in the family Acroporidae. They have a porous, lightweight skeletons, which develop from a flat encrusting form into a whorled cup shape as it grows larger (Sprung 2000). With a wide distribution throughout the Indo-Pacific, Red Sea, Indian Ocean and Australia, they typically favour habitats consisting of shallow clear water and abundant sunlight Mather & Bennet 1993). As a genus, Montipora sp. can reproduce sexually and asexually (Hart and Hart 2005). Most species are broadcast spawners and require environmental stimuli such as strong fluctuations in water temperature, moonlight or water movement (Zakai et al. 2000). However mortality rates of planula larvae are extremely high (Sebens 1994). Montipora typically survive by asexual reproduction, spreading quickly in the wild due to storm breakage and fragmentation (Van-Oppen & Gates 2006). Unlike other family members of Acropora, these species do not stress as easily and are more tolerant to bleaching and disease Fossa & Nilsen 1996). They are fast growing with the right conditions and can accept a wide range of lighting with moderate to high water movement. The ‘Button Polyp coral’, Zoanthus sociatus, is a soft-bodied, stoloniferous organism in the family Zooanidis (Reimer et al.

2006). This species in particular typically forms large, monospecific aggregations of colonies (Sinniger et al. 2005) found within the low intertidal and shallow subtidal zones on Caribbean and Australian coral reefs and rubble substrates (Sebens & Kenneth 1977, Karlson 1983). Subjected to wave action and intertidal desiccation, they are generally more tolerant to fluctuating conditions (Delbeek & Sprung 1997). Colonial fragments of Z. sociatus can be generated by endogenous degeneration (Koehl 1997), which indirectly facilities dispersal from the colony during disturbances, and exogenous fragmentation, caused through storm turbulence and can form large aggregations (Sebens 1977). According to a long-term study by Karlson (1984) it was found the survivorship of isolated fragments was found to be highly size dependent, yet the influences of water and light on growth and restoration have, to date, been poorly documented.

The soft coral Xenia sp. (Lamarck in 1816) in the family Xeniidae, have unbranched stalks that are short, thick and smooth. The stalk has a grouping of feathery polyps at the end, which on the X. elongata, pulse consistently. Pulsating is generally regarded as a sign of healthy Xenia sp. Although sensitivity to various fluctuations in water chemistry is thought to affect pulsing rates, to date, the specific reason for pulsing, such as feeding or respiration, and its influence to growth is still largely unknown. Due to a lack of comparative data available, the pulsing rates were not included in the study.

Growth rates can be used as an overall reflection of how efficiently the corals photosynthesize, capture food, secrete calcium carbonate, resist photoinhibition, reproduce and avoid disease (Done 1982, Kuffiner 2001). As photosynthesis plays a significant role in coral growth, its evident that sunlight is key in determining the success of coral reefs. Its documented that sunlight can provide almost 95% of corals daily energy (Lewis 1977, Pearse & Musatine 1971), however a recent study by Schutter et al. (2010) found that light may not stimulate growth to the extent previously thought. Only recently has water flow been found essential to many physiological processes. Influencing; the diffusion of dissolved gasses which enables higher rates of photosynthesis and respiration (Sebens 1997), the uptake of inorganic carbon and dissolved nutrients (Lesser et al. 1994) and the capture rate of food particles (Dennison and Barnes 1988, Patterson et al. 1991). Water movement also provides the removal of sediments, algae and waste products from the corals surface (Kuffiner 2001). Despite its importance, the effects of water flow to coral biology has received considerably less attention then light (Sebens 1997, Done 1982, Graus and Macintype 1989). Kuffiner (2001) and Sebens et al. (2003) claim that although optimal flow rates vary among coral species, the majority of zooxanthellate scleractinian corals show higher calcification and growth rates with an increase in water flow. Soft corals on the other hand, have shown more hyperbolic growth patterns (Kuffner 2001. Khalesi et al. 2007). Extreme flow rates can also act as a stressor, similar to how intense light can cause bleaching, to much turbidity can break hard skeletons, causing damage to coral tissue and restricting particle capture through deformation of tentacles (Sebens et al. 1997, Jokiel 1978, Box and Mumby 2007). Corals depend on decent water flow and light for their immediate health. In optimal conditions, short-term responses of after fragmentation include; photosynthesis, respiration, polyp extension, particle capture or pulsing (Sebens et al 2003, Schutter. et al. 2009, Guest 2011) After a few weeks intermediate responses such as growth, self-attachment (Hart and Hart 2005), calcification (Van-Oppen 2006) bleaching and colony orientation (Hodgson 2000) can be observed (in terms of length, area, volume and mass). In the long-term, they adapt morphological characteristics to reflect the conditions of particular niches (Brazeu & Lasker 1992), allowing an increase in potential of gas exchange and particle capture. Enhanced growth ultimately leads to higher reproduction, resilience and survival rates (Sebens et al 2003).

Coral transplantation is seen a major tool in reef rehabilitation strategies, potentially restoring coral cover to areas of significant degradation. While it is known that fragments generate new tissue and bind to the substrate within a few weeks of artificial transplantation (Hart & Hart 2005), very little is known about the speed of natural selfattachment after physical damage (Ferse, 2010). Recently, Guest et al. (2011) claimed that considerable research is still needed into the effectiveness of self-attachment. It’s suggested that a combination of abiotic factors may determine how different coral species respond in growth, form and life history following fragmentation and transplantation. In the natural environment, fragmentation occurs constantly through predation and turbulent storms (Sebens et al 2003). In periods of stress, corals are able to release parts of their colonies for regeneration, through a process called autonomy (Shaish et al 2010). This is considered a last resort for the colony to survive. Biologically, this fragmentation is a form of asexual reproduction and is observed frequently in aquaria (Ferse, 2010). However, there are many disadvantages to this process. Not only does it prevent growth to a natural size (Van-Oppen & Gates 2006), but coral ramets produced from fragmentation results in a genetically homogeneous population (Bak & Engel 1979, Zakai et al 2000), possibly leading to weakened colonies in the long run. New colonies are unable to adapt to changing conditions, inhering the exact tolerance traits as the parent colony. These could include; sensitivity to salinity, temperature, bacterial infections, or high irradiance levels (Sebens, K.. 1994, Baums 2008). When these corals are replicated in aquaculture, it can cause significant problems for potential reef restoration efforts (Hatziolos,et al,1998) Sexual reproduction however, is key to establishing healthy and resilient coral colonies (Shaish et al 2010), resulting in a genetically heterogeneous population

(Baums 2008). The fusing of gametes allows a diverse generation to effectively adapt to changing conditions, improving tolerance levels and enhancing the colonies survival rates in disturbed environments (Meesters et al. 1994, Hall 1997). Substantial amounts of coral is fragged from the wild and exported to the European, American and Japanese aquarium trades every year (Brazeu & Lasker 1992, Delbeek & Sprung 1997, Guest et al. 2011). Therefore the culture of different coral species has become increasingly vital to ensure future sustainability (Knowlton, 1997). However establishing captive sexual reproduction is still an area of great challenge (Guest et al. 2011), limited by many intrinsic (individual) and extrinsic (environmental) factors (Stoddart 1969, Baums 2008.). Difficulty in closed systems involves the absence of environmental stimuli, proper nutrition, and suitable filtration systems to accommodate released gametes (Fossa & Nilsen 1998). Large-scale sexual coral reproduction in marine aquaculture would be a significant step in future restoration efforts. To achieve this, the optimal conditions required by different species for maximum growth needs to be understood (Dubinsky & Stambler 2006) This study is focused on the short-term growth and restoration of coral directly after fragmentation within different water flow and light regimes. Growth rates of three zoozanthellate species, M. capricornia, Z. sociatus and X. elongata are documented in order to find the optimal conditions required by each. Whilst these species thrive in the same reef aquarium, they originate from range of different habitats and therefore abiotic requirements for maximum growth may vary. Understanding abiotic influences to coral biology will contribute to the long term health and survival of colonies in closed aquariums, where frequent fragmentation impairs somatic growth, sexual reproduction and, most importantly, adaptation to surrounding conditions. The aim of this work was to study the effect of water flow on the (skeletal) growth in a short-term growth experiment. For this, a series of three genetically identical coral fragments from each species was cultured over 4 weeks (28 days) at a two defined flow speeds (0.1 m/s and 10 m/s) and two light levels (8000 PAR and 3500 PAR) in a controlled aquarium environment. The range of flow speeds was chosen to contain flow speeds higher than 0 m/s up to the highest average flow speed that was technically feasible in the experimental setup (10 m/s). Growth was measured at regular intervals, with the hypothesis that increased water flow and increased light levels would have a positive correlation to growth over time.

Materials and methods Three different corals species were used for the study; Zoanthidae ‘button polyp coral, Cup plate Montipora Capricornia, and pulsing Xenia Elongata. Three fragments from each species where propagated from their colonies and grown within a mature closed-circuit reef aquarium (450 Liters) where a variety of tropical marine organisms were already present. The reason for using one mature aquarium, rather then separate tanks, is to provide a stable environment in which all factors, apart from light and flow, are consistent throughout. Therefore any fluctuations in the physical or chemical conditions, such as temperature, nitrogen, salinity or pH, would be the same for all the study species. In addition, a tank in which marine life is already present provides conditions more similar to the natural environment, therefore results can later be compared to data collected from the wild.

Tank set up The tank system used for the study consisted of; A V2 400 protein skimmer, four Aqua Medic 1 Hydor Powerheads (10 times water turnover per hour), two 150 watt, double ended Metal Halide light bulbs and two 18W LED Bluemoon bulbs. The lighting system was set to a consistent light photo period of 12 hours. Longer time periods can contribute to algae growth and eutrophication, higher temperatures and even bleaching. Seawater was made up from Tetra Marine Seasalt (Synthetic salt mix) and filtration was achieved through matured live rock. Temperature was maintained at 22 24 C, salinity at 35 g/L and pH at 7.9 – 8.4. Water quality was measured at routine intervals using Tretatest Kit (Marine test) as part of general maintenance to the mature reef aquaria. The Tretatest kits were carried out as per instructions, following both the industry standards and the methods used by Falmouth Marine school. During the experiment, calcium concentration in the system 350 – 400 mg l-1, magnesium concentration 1,200 mg l-1, nitrate concentration NO3, > 0.1 mg l-1 and PO4 3- phosphate concentration around 0.02 mg l-1 .Each of the nine fragments was fed indirectly by daily feeding of 4 g frozen enriched artemia to the entire coral system.

Experimental setup A fragment from each species was assigned to three different locations depending on different water flow and light conditions that were already present within the tank. The three locations included the combinations of High Light and High Flow (HLHF), Low Light and High Flow (LLHF), and Low Light and Low flow (LLLL). These combinations allow for an analysis of which factor, if any, has a greater effect on the growth of the coral fragments. The tank and the conditions were set up prior to propagation, ensuring that each of the fragged species could be placed directly in the three locations directly after breakage.

The different flow rates were provided by four Hydor powerheads, two fixed at the top end of the tank and adjusted with strong laminar flow towards the centre. Flow velocity was measured by placing a portable SENSA RC-2 electromagnetic velocity meter (Aquadata) with its sensor probe submerged at the three proposed locations (without disturbing other organisms). Water exiting the power head had the highest flow, measuring 10 m/s. Low flow was measured at > 0.1 m/s and was found furthest away. Light levels were measured using a PAR Meter. High intensity, towards the top of the tank, was measured at 8000 PAR, and 3500 PAR at the lowest levels, near the bottom. To determine the three proposed locations for the study, the high and low light levels were measured within the areas of the high and low flow previously stated.

Fragging of Study species The M. capricornia was fragged from a larger ‘parent’ colony. Handled with gloves, three separate pieces around 5cm long, where broken off to replicate physical damage that may occur in the natural environment. These were fixed to small rocks using underwater epoxy glue (Cultured aragonite or plugs can also be used as a base), and placed securely in each of the three locations. However as the coral will exude mucus, they were be placed away from other sessile organisms. The parent colony was kept in pristine conditions in a separate aquarium following the damage to allow healthy recovery.

Zoanthus corals frags are initially broken from the parent colony within the tank. Due to the connecting mat like nature of the colony, it was fragged by carefully cutting beneath the polyps grip. When removed from the water they are able to tolerate some exposure without stress. The debris on the bottom of the polyp was dried and stuck onto the rock. Strong reef glue or 2-way epoxy is most suitable to connect these species. After a few seconds when the frag is securely set, they were placed into the experimental tank. Another method often used, uses a rubber band to secure the frags onto a rock. When the rubber band naturally breaks away, the zoanthids should be connected. However the first method is recommended and was used for this experiment.

X. elongata are particularly sensitive to handling, therefore propagation needs to be done with care. They produce a toxin mucous as a defense mechanism which presents a potent smell when removed from the water, therefore gloves are necessary. The parent colony was removed only briefly from the water. Propagation was achieved through a longitudinal cut between the stalks with a further separation into three small frags. These were individually fixed using epoxy putty on rock and carefully placed into the three different locations. The mucous produced may attract bacteria or disturb surrounding corals. They have also be known to ‘climb’ towards more favorable conditions, therefore the frags were placed in the tank with a decent amount of space around them.

Growth parameters M. capricornia and Z.sociatus are hard corals and therefore growth was measured by surface diameter (mm), using a vernier calliper carefully placed between two chosen points on surface edge. Since tentacle extension of scleractinian can be variable over time, any tentacles extending beyond the skeleton were not included. The growth of the X. elongata was measured visually by counting individual stalks and polyps. Only live polyps were counted and the newly formed polyps were recorded once they projected from the basal stalk.The surface diameter of each fragment was measured (mm) before the experimental period and at each observation date (once a week) the same diameter was measured. The total surface growth was calculated using the formula: D n - D n-1 where D n is the is the surface area at the end of a growth interval and D n-1 is the surface diameter recorded at the start of the growth interval. The same was done for polyp number using the formula: P n - P n-1 and basal stalk number, BS n - BS n-1. The measurements recorded were written up into tables and converted into charts. As the experiment was conducted on a short-term basis, over 28 days, the corals had not yet formed full reattachment to the substrate, therefore were very carefully moved out of the aquarium for measurements. Methods were non destructive, however to reduce the amount of handling and interference, recordings were only taken once a week.

Data analysis Statistical analysis was performed using Minitab 15.0. For each species, With suggestions from staff at Falmouth Marine School, a repeated measures two-way analysis of variance (ANOVA) was used to test effects of flow and light treatments on growth rates over time. Results were considered statistically significant when P-values were below 0.05.

Results Observations All the coral fragments showed increased growth between the start and end of the study period (Figure 1). The surface diameter of the two Scleractinian frags, M. capricornia and Z. sociatus, showed a more consistent growth rate then the polyp increase of the X. elongata. However overall patterns in growth rates as a result of different light and flow conditions varied over time. Observational findings suggest that not only does light and flow strongly influence growth rates after fragmentation, but each species responds differently to different abiotic levels.

Total Growth (mm/polyps)

20 19

16 14

12 8 4 0

6

6 3

3

6 4

3

M. Capricornia Z sociatus Species

X. elongata

HL,HF LL.HF LL,LF

Fig 1. The total growth of the 9 fragments achieved from days 0 - 28. in each location, Z. sociatus and M capricornia are measured by surface diameter (mm) and X elongata in polyp number.

The surface diameters (D) of all Z. sociatus fragments (referred to as S1, S2, S3) increased within all three conditions (Fig. 2a,b). The highest rate of growth was presented by S1 in the HLHF conditions, with a sum of 6 mm, increasing from 40 mm to 46 mm (D n - D n- 1). No growth was observed for any fragments within the first week (day 7). Both S2 in LLHF and S3 in LLLF, showed a total 3 mm increase in diameter, however showed difference in growth patterns between days 14 - 28. The first differences between light and flow regimes became apparent in week 2 (day 14). The S1 and S2 conditions grew at 2 mm and 1 mm, respectively, whilst S3 showed no growth. This rate was consistent for S1 and S2 throughout days 14 - 28. S3 however, showed no growth until day 28, where surface diameter was recorded at an increase of 3 mm having occurred between days 21- 28. Observations of the effects of light on growth, show that from day 14, S1 grew at twice as fast at high light levels (8000 PAR) compared to S2 under low light conditions (3500 PAR) where water flow was 10 m/s for both . The difference in growth between High Flow (10 m/s) and Low flow (>0.1 m/s), with the same low light levels (3500 PAR), showed an inconsistent growth rate in the Low flow regime. It’s possible that this spurt in growth could be due to stress levels and slower adjustment speeds.

Time of growth (weeks/days)

FRAG (S)

Location

Total Surface Diameter, mm. (D)

Growth per observation

Day 0 (n-1)

S1

HL, HF

40 mm

0 mm

S2

LL, HF

46 mm

0 mm

S3

LL, LF

45 mm

0 mm

S1

HL, HF

40 mm

0 mm

S2

LL, HF

46 mm

0 mm

S3

LL, LF

45 mm

0 mm

S1

HL, HF

42 mm

2 mm

S2

LL, HF

47 mm

1 mm

S3

LL, LF

45 mm

0 mm

S1

HL, HF

43 mm

2 mm

S2

LL, HF

48 mm

1 mm

S3

LL, LF

45 mm

0 mm

S1

HL, HF

45 mm

2 mm

S2

LL, HF

49 mm

1 mm

S3

LL, LF

48 mm

3 mm

S1

HL, HF

(D n - D n-1 ) = 45 - 40

6 mm

S2

LL, HF

(D n - D n-1 ) = 49 - 46

3 mm

S3

LL, LF

(D n - D n-1 ) = 48 - 45

3 mm

Week 1 (7 days)

Week 2 (14 days)

Week 3 (21 days)

Week 4 (28 days) (n)

Sum of total growth ( n -

n-1 )

Surface area (Increase in Surface diameter (mm)

Fig 2a. Growth of Z. sociatus within the three locations (1,2,3) over a period of 28 days. The surface diameter (D)and was measured before and after the study (D n - D n-1 ) and growth was recorded per observation (mm).

5 4 3 2 1 0 Day 0

Day 7

Day 14

Day 21

Time in days (After fragmentation)

Day 28

HL,HF LL.HF LL,LF

Fig 2b. The effect of flow and light regimes on surface area per observation during the experimental period

The surface diameters (D) of all M. capricornia, fragments (referred to as M1, M2, M3) also increased within all the three locations (Figure 3a,b). The total growth measured at the end of the period was the same as the Z. sociatus, however the growth rates showed a different pattern with time. The highest rate of growth was presented by M1 in the HLHF conditions, with sum growth of 6 mm, increasing from 65 mm to 71 mm (D n - D n- 1). This frag showed the only growth in the first week (day 7), with an increased diameter of 2 mm. Measurements from day 14 showed an increase of 2 mm. However, measurements at days 21 and 28, showed an unexplained reduction in growth rate, with an increase of only 1 mm at both observations. Both M2 in LLHF and the M3 in LLLF showed the same pattern and rate of growth throughout the study. Both showed the first growth at day 14, with an increase of 1 mm. This was consistent at each observation for the rest of the study period, resulting in a total 3 mm diameter increase. Like growth patterns of the Z. sociatus fragments, the first differences between light and flow regimes became apparent in week 2 (day 14). M1 with high light levels grew at 2 mm whilst both M2 and M3, with low light, showed no growth. In comparison, both M2 and M3 both presented the same, slow but steady increase throughout the period, under different flow rates but the same

light (3500 PAR). By observation, its possible that light have had a stronger influence on the rate of growth compared to the water flow.

Time of growth (weeks/ days) Day 0 (n-1)

Week 1 (7 days)

Week 2 (14 days)

Week 3 (21 days)

Week 4 (28 days) (n)

Sum of total growth ( n -

n-1 )

FRAG

Location

Total Surface Diameter, mm.

(D)

Growth per observation

M1

HL, HF

65 mm

0 mm

M2

LL, HF

48 mm

0 mm

M3

LL, LF

54 mm

0 mm

M1

HL, HF

67 mm

2 mm

M2

LL, HF

48 mm

0 mm

M3

LL, LF

54 mm

0 mm

M1

HL, HF

69 mm

2 mm

M2

LL, HF

49 mm

1 mm

M3

LL, LF

55 mm

1 mm

M1

HL, HF

70 mm

1 mm

M2

LL, HF

50 mm

1 mm

M3

LL, LF

56 mm

1 mm

M1

HL, HF

71 mm

1 mm

M2

LL, HF

51 mm

1 mm

M3

LL, LF

57 mm

1 mm

M1

HL, HF

(D n - D n-1 ) = 71 - 65

6 mm

M2

LL, HF

(D n - D n-1 ) = 51 - 48

3 mm

M3

LL, LF

(D n - D n-1 ) = 57 - 54

3 mm

HL,HF LL.HF LL,LF

Increase in Surface diameter (mm)

Fig 3a. Growth of M. capricornia within the three locations (1,2,3) over a period of 28 days. The polyp and stalk number per observation and the sum of total polyps are recorded.

5 4 3 2 1 0 Day 0

Day 7

Day 14

Day 21

Day 28

Time in days (After fragmentation) Fig 3b. The effect of flow and light regimes on surface diameter of M. capricornia, per observation during the experimental period

The X. elongata fragments (E) exhibited a fast growth rate of individual polyps per week however no new basal stalks were observed (fig. 3a, 3.b). The highest polyp increase was shown by E2, in the LLHF conditions, with total of 19 new polyps by the end of the study. Compared with a total of 14 new polyps for E1, and 6 for E3. LLLF showed less polyp growth then the LLHF conditions, with a difference of 13 polyps observed. Both these locations however resulted inconsistent growth patterns. E2 showed a rapid increase with 10 new polyps within 7 days of fragmentation. However this was inconsistent with the following observations, where growth rates was reduced greatly, developing 3 polyps in week 2, 4 polyps in week 3 and then only 2 in the 4th week. H3 had a consistent increase with 6 new polyps growing between weeks 1-3. however like H2, there was a decrease observed in week 4 where only 1 new polyp had formed.

The only growth pattern that had a positive correlation of increased growth over time, was represented by E1 in HLHF. This increased by 2, 2, 4 and 7 new polyps observed in weeks 1,2,3 and 4, respectively. If H2 in week 1 was a result of observational error, its possible HLHF that the results may be concluded as inaccurate and lead to incorrect understandings of the fragments growth rates.

Time of growth (weeks/days)

Day 0 (n-1)

Week 1 (7 days)

Week 2 (14 days)

Week 3 (21 days)

Week 4 (28 days) (n)

Sum of total growth ( n - n-1 )

Frag.

Location

Total Polyps (P)

Polyp Growth per observation

Total Basal Stalks (BS)

Stalk Growth per observation

E1

HL, HF

35

0

3

0

E2

LL, HF

29

0

3

0

E3

LL, LF

33

0

3

0

E1

HL, HF

37

2

3

0

E2

LL, HF

39

10

3

0

E3

LL, LF

34

1

3

0

E1

HL, HF

38

2

3

0

E2

LL, HF

42

3

3

0

E3

LL, LF

36

2

3

0

E1

HL, HF

42

4

3

0

E2

LL, HF

46

4

3

0

E3

LL, LF

39

3

3

0

E1

HL, HF

49

7

3

0

E2

LL, HF

48

2

3

0

E3

LL, LF

39

0

3

0

E1

HL, HF

(P n - P n-1 ) = 49 - 35

14

(BS n -BS n-1 ) = 3 -3

0

E2

LL, HF

(P n - P n-1 ) = 48 - 29

19

(BS n -BS n-1 ) = 3 -3

0

E3

LL, LF

(P n - P n-1 ) = 39 - 33

6

(BS n -BS n-1 ) = 3 -3

0

Fig 4a. Growth of X. elongata (E) within the three locations (1,2,3) over a period of 28 days. The polyp and stalk number per observation and the sum of total polyps are recorded.

Number of individual polyps

10 9 8 7 6 5 4 3 2 1 0 Day 0

Day 7

Day 14

Day 21

Time in days (After fragmentation)

Day 28

HL,HF LL.HF LL,LF

Fig 4b. The effect of flow and light regimes on X. elongata, showing polyp number per observation during the experimental period

Statistical data, 2-way ANOVA The 2-way ANOVA showed no significant differences in any of the coral species over time or location. M. capricornia, showed no significant differences between different light and water flow conditions location (P =0.168) or between the start and end of the study (P = 0.095) (appendix 1a,b). The confidence intervals for the means of condition types strongly overlapped, suggesting that the difference between these means are not statistically significant. Similar results were found for the Z. sociatus fragments (appendix 2a, 2b), with no significant different between locations (P=0.410) and time (P=0.062) or the X elongata (appendix 3a, 3b); between locations (p= 0.345) and time (p= 0.402). The confidence intervals for the means of condition types strongly overlapped in all the results, suggesting that the difference between these means are not statistically significant. The residual plots for growth (normal probability graphs) show that theres an overall trend in growth over time, (appendix 1c, 2,c 3c) however none of the fragments show a correlated increase within any of the locations. The histograms of the residuals shows the same unusual trend as the normal probability plot, however it is more evident that the data doesn’t fit. For the data, evidence of both skewness and outliers exist. As these don’t fit the normal ‘bell shaped distribution, it can be assumed that the residuals are not normal and the data doesn’t fit the model used.

Discussion This experiment was aimed to determine if various water flow and light conditions effected the growth of fragmented coral species. By investigating if these species responded differently to abiotic factors, further research can be made towards coral captivity and fragmentation, where optimal conditions for growth can be provided. As this was a shortterm study based on limited data previously collected at Falmouth Marine School, various problems occurred with the accuracy of results. Like many previous studies, this experiment was conducted through two ‘low’ and ‘high’ light and flow conditions. These were measured at 3500 PAR and 8000 PAR and >0.1 m/s and 10 m/s respectively. Although providing a generalised view of abiotic effects on growth, they did not characterise the regimes with a meaningful and varied number. However, due to the limited tank size and funding, these were the only conditions that were technically feasible in the experimental setup. Conclusions could only assumed by observation and were not found to be statically accurate. For a longer and more detailed study, a much wider range of speeds and light combinations needs to be carried out, extending on the data collected for this experiment.

Long-term varied data would allow for statistical analysis to be performed correctly. In complete contradiction to each other, the observed results showed a general increase in measured growth over the period of the experiment for all fragments. The 2-way Anova however, showed no significant differences in data or confidence means between growth, species and location. Although a 2 way-ANOVA was suggested by staff members for the data provided, it can be concluded that the data did not satisfy the assumptions for ANOVA testing. In future experiments, the error terms of the ANOVA analyses should be tested for homogeneity of variances (P > 0.05) using Levene’s test and normality (P> 0.05) Shapiro–Wilk test. In addition, the data that did not satisfy the assumptions for ANOVA should have been subsequently tested using the non-parametric Kruskal–Wallis test to in order to find if there was infact statistical

differences between conditions and growth rates. This could then be followed by a comparisons using the Mann– Whitney U test. These statistical tests were not recommended for the data available in this experiment, however, could be used if the experiment is continued for a longer period. Over a long-term study, not only would data provide a more detailed analysis of growth rates, but important physiological processes could also be measured. With access to a larger equipment set up, measurements could include; photosynthesis, respiration and scope for growth as well as buoyant weight, surface area and skeletal density. These processes would indicate physiological and morphological changes and the influences on growth rates under different abiotic factors. These were not measured in this small scale experiment due to equipment availability and risk of disturbance to other marine organisms in the matured reef aquaria. An example of further measurements includes, Schutter et al. (2010). Specific growth rates (µ) were calculated of Galaxea fascicularis using the formula: µ = (1nSA n -1nSA n-1 )Dt [day -1]. Where, µ represents the specific growth rate (day-1), SAn-1 is the surface area at the start of a growth interval and SAn is the surface area at the end. Dt is time between measurement. The same can be done for both polyp number and Buoyant Weight (BW).

The observations found in this experiment, showed that growth of both M. capricornia and Z. sociatus had an increase with time in all flow and light treatments. Since differences in growth between treatments only started to become apparent at week 2 and 3 for the majority of fragments, growth experiments using should last at least 12 weeks for scleractinian corals. Previous research by Shaish et al. 2010 showed that scleractinian fragments take longer to adjust to new environments after physical damage, where energy goes into physiological repairs and self-attachment. A very minimum of 12 weeks would provide a more accurate correlation between the effects of light and flow on growth.

This study demonstrated the importance of water flow to the growth of the Z. sociatus. Absence of flow resulted in a much slower growth rate whereas an increase in growth was found at 10 m/s. Assuming these observations were accurate, it agrees with the findings of Jokiel (1978) who documented that the growth of Pocillopora sp correlated with increased velocity from 2-15 m/s. Although no significant difference was found in growth between the flow and light conditions, it was clear by the observation it has a relatively small effect. Algae and sedimentation may have also pay a role in reduced growth in flow areas of >0.1 m/s. As this was a live reef tank, marine organisms could pay a role in predation or disturbing substrates. The M. capricornia was observed to with an increased rate of growth between the start and end of the study. Although the HLHF conditions accelerated initial growth of its fragment in the first 2 weeks, it showed a reduced growth in weeks 3 and 4. This could be for a number of reasons. Firstly, it could be assumed a human error in observations. As recordings was measured over the surface diameter (mm) and the edge of the coral was jagged, misplacement of to either side of the fixed points could easily result in a 1 mm difference. Another reason for this variation could be the result of respiratory rates. Sebens et al (2003) documented that reduction in the diffusive boundary layer from increased water flow resulted in higher respiratory rates. It was hypothesised that respiration increases metabolic CO2 of which 70% of energy provided is used calcification, therefore may limit the energy supplies to growth. The results gained for the Pulsing Xenia sp. are difficult to determine due to the inconsistency in data at LLHF conditions in week two, with a sudden burst of 10 new polyps. The most likely assumption for this occurring is a human area in observation, where polyp numbers were miscounted, Although theres a chance it may have been due to other factors, another study would have to be conducted in order to make an accurate comparison. By doing this, accurate conclusions can be made.

Although statistically accurate results were not achieved in this particular experiment, the methods discussed would contribute to continued research in this area. This proposal can be put forward for the FML Propagation Unit Design Project of 2012. By establishing an ‘industry standard’ experiment, results would help gain a much needed understanding of the effects abiotic conditions on the physiology as well as growth in a larger range marine coral species. This would help achieve health and survival of colonies, benefiting coral aquaculture in the future, where corals are strongly impaired by frequent fragmentation.

Acknowledgments This study was conducted as part of long-term research for Falmouth Marine School, with the aim of improving coral husbandry techniques and gaining a further understanding of coral biology. I thank Craig Baldwin and the staff of Falmouth Marine School for the experimental set up and data provided.

This work was funded by Falmouth Marine School, Cornwall Collage.

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Appendix 1: Montipora. Capricorna 1a: Two-way ANOVA: Growth versus Week, Location Source Week Location Error Total

DF 4 2 8 14

S = 0.5164

Week 0 1 2 3 4

Mean 0.00000 0.66667 1.33333 1.00000 1.00000

Location HLHF LLHF LLLF

SS 3.06667 1.20000 2.13333 6.40000

MS 0.766667 0.600000 0.266667

R-Sq = 66.67%

F 2.88 2.25

P 0.095 0.168

R-Sq(adj) = 41.67%

Individual 95% CIs For Mean Based on Pooled StDev +---------+---------+---------+--------(---------*---------) (---------*--------) (---------*---------) (---------*---------) (---------*---------) +---------+---------+---------+---------0.70 0.00 0.70 1.40

Mean 1.2 0.6 0.6

1b) Residual Plots for Growth

Individual 95% CIs For Mean Based on Pooled StDev ---------+---------+---------+---------+ (----------*----------) (----------*----------) (----------*----------) ---------+---------+---------+---------+ 0.50 1.00 1.50 2.00

Appendix 2: Z. sociatus, 2a: Two-way ANOVA: Growth versus Week, Location Source DF SS MS F P Week 4 8.4 2.1 3.50 0.062 Location 2 1.2 0.6 1.00 0.410 Error 8 4.8 0.6 Total 14 14.4 S = 0.7746 R-Sq = 66.67% R-Sq(adj) = 41.67%

Individual 95% CIs For Mean Based on Pooled StDev Week Mean ---------+---------+---------+---------+ 0 0 (--------*--------) 1 0 (--------*--------) 2 1 (-------*--------) 3 1 (-------*--------) 4 2 (--------*-------) ---------+---------+---------+---------+ 0.0 1.2 2.4 3.6

Individual 95% CIs For Mean Based on Pooled StDev Location Mean ---+---------+---------+---------+-----HLHF 1.2 (------------*------------) LLHF 0.6 (------------*------------) LLLF 0.6 (------------*------------) ---+---------+---------+---------+-----0.00 0.60 1.20 1.80

2b: Residual Plots for Growth

Appendix 3: Xenia.elongata 3a) Two-way ANOVA: Growth versus Week, Location Source DF SS MS F P Week 4 33.333 8.33333 1.14 0.402 Location 2 17.733 8.86667 1.22 0.345 Error 8 58.267 7.28333 Total 14 109.333 S = 2.699 R-Sq = 46.71% R-Sq(adj) = 6.74%

Individual 95% CIs For Mean Based on Pooled StDev Week Mean --+---------+---------+---------+------0 0.00000 (-----------*-----------) 1 4.33333 (-----------*-----------) 2 2.33333 (-----------*-----------) 3 3.66667 (-----------*-----------) 4 3.00000 (-----------*-----------) --+---------+---------+---------+-------3.0 0.0 3.0 6.0

Individual 95% CIs For Mean Based on Pooled StDev Location Mean ------+---------+---------+---------+--HLHF 3.0 (----------*----------) LLHF 3.8 (----------*----------) LLLF 1.2 (----------*----------) ------+---------+---------+---------+--0.0 2.5 5.0 7.5

3b) Residual Plots for Growth