Seagrass proliferation precedes mortality during hypo-salinity events: a stress-induced morphometric response (NERP TE 5.3, JCU)

This dataset consists of one data file from a 10 week aquarium experiment manipulating salinity and measuring density, reproduction and growth responses of three tropical Indo-pacific seagrass species (Zostera mueller, Halodule uninervis and Halophila ovalis). 

Halophytes, such as seagrasses, predominantly form habitats in coastal and estuarine areas. These habitats can be seasonally exposed to hypo-salinity events during watershed runoff exposing them to dramatic salinity shifts and osmotic shock. The manifestation of this osmotic shock on seagrass morphology and phenology was tested.


Methods:

Hypo-salinity exposure experiments were conducted on three species of seagrass, which are ubiquitous throughout the Indo-Pacific, except Zostera muelleri Irmisch ex Ascherson, which is widespread in Australia and New Zealand only. Halodule uninervis (Forsskål) Ascherson is a tropical species that occurs throughout the Indo-West Pacific in coastal and reef habitats, while Halophila ovalis R. Brown is one of the most broadly distributed seagrass species occurring throughout the Indo-West Pacific, including temperate regions, and can be found in estuarine, reef and deepwater habitats (Waycott et al., 2004). Their habitats are periodically exposed to flood plumes of reduced salinity (Furnas, 2003). Both Z. muelleri and H. uninervis are species with linear leaf blades (blady), whereas H. ovalis has pairs of ovate leaves arising from the rhizome on petioles.

Zostera muelleri plants were collected from Pelican Banks, Gladstone (23º45.895¿S, 151º18.244¿E) during low tide three months before the experiments started. The plants were collected using a 10 cm corer, with sediment and rhizome and roots collected intact. The cores were placed in plastic-lined pots, the plastic bag sealed over the top of the seagrass with 2 ¿ 3 cm of water during transport to the experimental facility. Halodule uninervis and H. ovalis plants were collected from Cockle Bay, Magnetic Island (19º10.612S, 146º49.737E) using the same technique two months prior to the experiments. The plants were kept in 1000L aquaria at the Aquaculture facility in James Cook University on a closed circulation system in seawater piped from Bowling Green Bay seawater intake under a 30% light-reducing roof. 

The experiment consisted of 12 salinity treatments, starting from 3 PSU and increasing by 3 PSU to 36 PSU (approximate marine seawater). Salinity treatments were obtained by diluting the seawater with de-chlorinated freshwater. Every salinity treatment consisted of four replicate tanks (65L KiTab clear plastic containers) with one pot of each species per tank (i.e. n = 4). All treatments started at 36 PSU and salinity was reduced by 25% each day over four days to the target treatment salinity to mimic the more gradual decline in salinities that occur during run-off events and to minimize potential impacts from shock osmotic changes. Throughout the experiment, salinity was measured every 1-3 days using a digital salinity/conductivity/temperature meter (YSI, model 63) and salinity was adjusted when necessary to maintain salinity within 0.5 PSU of target salinity. Plant responses to these salinities were monitored for 10 weeks. Previous salinity studies indicate that seagrass changes settle down by this time (Griffin and Durako, 2012; Koch et al., 2007), and furthermore, this experimental duration is approximately equal to or more likely exceeds the length of individual hypo-salinity events in the region.

The experiments were conducted outdoors during summer/autumn months (February to April) when high ambient temperatures occur, thus chilling units were installed to moderate temperature fluctuations within the treatment tanks throughout the experiment. There were three chilled freshwater baths (1000L tanks) that were cooled using external water chillers. Each of the 12 salinity treatments had one 60L sump (60L plastic bin) that was placed randomly in one of the 3 chilling baths, each bath containing 4 sumps. The chilled baths with sumps were held underneath tables that held the experimental tanks. From each sump, water with corresponding salinity was pumped into four replicate tanks resulting in a total of 48 tanks (4 replicate tanks x 12 sumps/salinity treatments = 48 tanks in total). Each tank contained one pot of each of the three species (48 tanks x 3 species/pots = 144 pots). Temperature was recorded every 30 mins using iBCod 22L model of iBTag¿ in six randomly selected tanks for the duration of the experiment. Water temperature was 26°C on average and ranged from 22°C to 34°C reaching these temperature extremes for short periods (1-2 h) on some days. Nitrogen (N) as NH4Cl and phosphate (P) as KH2PO4 were added to the water column at very low concentrations to increase concentrations within each system by 0.05 µMol of P and 1.0 µMol of N every 2 weeks. Nutrient concentration was measured after six weeks and was found to be 0.8 µMol (±0.2) NH3, 0.4 µMol (±0.1) NOx, and 0.2 µMol (±0.8) PO4. Average light intensity under the 30% light-reducing roof was 17 mol photons m-2 d-1 of Photosynthetically Active Radiation (PAR), measured with an Odyssey 2Pi quantum sensor (Dataflow, Odyssey photosynthetic recording system) recording every 30 mins throughout the experimental period. The tanks were periodically cleaned by syphoning out sediment and organic matter accumulating at the bottom of tanks and plants were inspected every week for signs of grazing by amphipods. Amphipods were removed to prevent an outbreak, which could lead to overgrazing of the plants. Although signs of grazing were observed at times, this cleaning regime was sufficient to avoid outbreaks.

The number of shoots in each pot for Z. muelleri and H. uninervis, or the number of leaf pairs for H. ovalis were counted prior to the experiment and then weekly during the first four weeks of the experiment and fortnightly from the sixth week up to and including the tenth week. Change in shoot density (deltaSht) was calculated as a percentage change in each week relative to pre-treatment for each individual replicate:
              deltaSht=[(Sht tx-Sht t0 )/Sht t0 ]×100							Eq 1
where deltaSht is change in shoot density, Sht tx is shoot density at time x (weeks 1 through to 10) and Sht t0 is shoot density at week zero (pre-treatment).

Leaf morphometrics (width and height) of Z. muelleri, H. uninervis and H. ovalis and number of leaves per shoot for the two blady species were measured after 10 weeks at treatment salinity. These data were used to calculate foliar surface area (SA) as follows:
                SA=shoot density × (leaves per shoot-0.5)  × leaf length × leaf width		Eq 2
for blady species (H. uninervis and Z. muelleri); and,
                SA=leaf density × pi × ((leaf length)/2)  × ((leaf width)/2)					Eq 3
for the ovate species H. ovalis where: 
SA is the foliar leaf area (cm2), 
shoot density are leaves per experimental pot,
leaves per shoot are the mean number of leaves (usually 1 to 4) per seagrass shoot and 
leaf length (cm) and leaf width (cm) of the youngest fully mature leaf. 

A half leaf was subtracted from the total number of leaves per shoot in calculating SA of blady species to account for one leaf on each shoot being in development and therefore not full sized (Collier et al., 2012). 

Halophila ovalis was the only species to flower throughout the experimental period. Flowering had commenced prior to the initiation of the experiment and continued throughout. New leaf pairs are produced in H. ovalis every 3 or 4 days at experimental water temperatures (~26 - 27°C McMahon, 2005) and H. ovalis typically had 4 to 5 leaf pairs per branch. Flowering is initiated in young leaf pairs, with more advanced reproductive structures away from the growing apex. Assuming a leaf pair production rate of 4 days, we conservatively assumed that all reproductive structures present after 4 weeks (28 d) were initiated under treatment conditions. We counted all reproductive structures (male and female flowers, as well as fruits) in each pot at weeks 4, 6, 8 and 10. 

Leaf growth rate was measured in week 10 on the two blady species (Z. muelleri and H. uninervis) using the leaf hole punch method (McMahon, 2005). Holes were punched using a hypodermic needle in the top of the sheath of each shoot, and after 5 ¿ 7 days we measured the distance between the mark in the sheath and the mark on the leaves. We aimed to measure up to 10 shoots per replicate pot, though the actual number measured in each pot was variable depending on shoot density and visibility of marks.
 
For more details see the publication:

Collier, C. J., Villacorta-Rath, C., van Dijk Kj, T. M., & Waycott, M. (2014). Seagrass proliferation precedes mortality during hypo-salinity events: a stress-induced morphometric response. PloS one, 9(4), e94014. DOI: 10.1371/journal.pone.0094014


References:

Collier, C.J., Waycott, M., and Giraldo-Ospina, A. (2012). Responses of four Indo-West Pacific seagrass species to shading. Mar Pollut Bull 65, 342-354.
Furnas, M. (2003). Catchment and corals: terrestrial runoff to the Great Barrier Reef (Townville Queensland: Australian Institute of Marine Science).

Griffin, N.E., and Durako, M.J. (2012). The effect of pulsed versus gradual salinity reduction on the physiology and survival of Halophila johnsonii Eiseman. Marine Biology 159, 1439-1447.

Koch, M.S., Schopmeyer, S.A., Kyhn-Hansen, C., Madden, C.J., and Peters, J.S. (2007). Tropical seagrass species tolerance to hypersalinity stress. Aquat Bot 86, 14-24.

McMahon, K.M. (2005). Recovery of subtropical seagrasses from natural disturbance. In Centre for Marine Studies (Brisbane: The University of Queensland), pp. 198.

Short, F.T., and Duarte, C.M. (2001). Methods for the measurement of seagrass growth and production. In Global seagrass research methods, F.T. Short, and R. Coles, eds. (Amsterdam: Elsevier Science), p. 473.

Waycott, M., McMahon, K., Mellors, J., Calladine, A., and Kleine, D. (2004). A guide to tropical seagrasses of the Indo-West Pacific (Townsville: James Cook University).


Format:

A 97 KB xlsx file, Seagrass_salinity.xlsx


Data Dictionary:

There are two tables in the spreadsheet, Ongoing Measures and Single time point measures (week 10).

Ongoing Measures

- Date: the date on which the measure was taken
- Week: weeks since the start of the salinity treatment
- Salinity (practical salinity units): The salinity treatment level
- Tank#: Replicate tank A,B,C, or D
- Species: Zm for Zostera muelleri, Hu for Halodule uninervis, Ho for Halophila ovalis
- Shoot count (shoots/pot): The shoot or leaf pair (for Ho) in each of 4 replicate pots
- Reproductive structures (total/pot): count of total flowers and fruits per pot

Single time point measures (week 10)

- Salinity (practical salinity units): The salinity treatment level
- Rep: Replicate tank A,B,C, or D
- Species: Zm for Zostera muelleri, Hu for Halodule uninervis, Ho for Halophila ovalis
- Leaf area (cm2): The total leaf surface area in the pots A=leaf density × ? × ((leaf length)/2)  × ((leaf width)/2)
- Leaf extension (mm/sht/d): The rate of new leaf growth produced per shoot per day

Tags: marine
Organization: Centre for Tropical Water & Aquatic Ecosystems Research (TropWater)