Submitted 22 March 2018Accepted 22 June 2018Published 20 July 2018

Corresponding authorMalindi J Gammonmalindigammonyahooconzmalindigammonmpigovtnz

Academic editorErik Cordes

Additional Information andDeclarations can be found onpage 18

DOI 107717peerj5236

Copyright2018 Gammon et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

The physiological response of the deep-sea coral Solenosmilia variabilis to oceanacidificationMalindi J Gammon1 Dianne M Tracey2 Peter M Marriott2Vonda J Cummings2 and Simon K Davy1

1 School of Biological Sciences Victoria University of Wellington Wellington New Zealand2National Institute of Water amp Atmospheric Research Wellington New Zealand

ABSTRACTSeveral forms of calcifying scleractinian corals provide important habitat complexity inthe deep-sea and are consistently associated with a high biodiversity of fish and otherinvertebrates How these corals may respond to the future predicted environmentalconditions of ocean acidification is poorly understood but any detrimental effectson these marine calcifiers will have wider impacts on the ecosystem Colonies ofSolenosmilia variabilis a protected deep-sea coral commonly occurring throughout theNew Zealand region were collected during a cruise in March 2014 from the LouisvilleSeamount Chain Over a 12-month period samples were maintained in temperaturecontrolled (sim35 C) continuous flow-through tanks at a seawater pH that reflects theregionrsquos current conditions (788) and an end-of-century scenario (765) Impacts oncoral growth and the intensity of colour saturation (as a proxy for the coenenchymetissue that covers the coral exoskeleton and links the coral polyps) were measuredbimonthly In addition respiration rate was measured after a mid-term (six months)and long-term (12months) exposure period Growth rates were highly variable rangingfrom 053 to 3068 mm yearminus1 and showed no detectable difference between thetreatment and control colonies Respiration rates also varied independently of pH andranged from 0065 to 1756micromolO2 g proteinminus1 hminus1 A significant change in colour wasobserved in the treatment group over time indicating a loss of coenenchyme This losswas greatest after 10 months at 528 and could indicate a reallocation of energy withphysiological processes (eg growth and respiration) being maintained at the expenseof coenenchyme production This research illustrates important first steps to assessingand understanding the sensitivity of deep-sea corals to ocean acidification

Subjects Aquaculture Fisheries and Fish Science Biodiversity Ecology Marine BiologyKeywords Deep-sea Ocean acidification Physiology Scleractinian corals Stony corals Globalchange Deep-sea corals

INTRODUCTIONDeep-sea corals (Phylum Cnidaria) are an abundant and diverse group that are foundworldwide and like their shallow water counterparts several groups are characterised bytheir ability to form calcium carbonate skeletons Corals are vulnerable to environmentalchange resulting from anthropogenic disturbances such as climate change oceanacidification (OA) (Guinotte et al 2006 Turley Roberts amp Guinotte 2007) and fishing

How to cite this article Gammon et al (2018) The physiological response of the deep-sea coral Solenosmilia variabilis to ocean acidifica-tion PeerJ 6e5236 DOI 107717peerj5236

(Clark amp Rowden 2009 Clark et al 2015b) To date no research has been carried outin the New Zealand region on the impacts of climate change including OA on thisimportant group of scleractinian stony corals One study by Thresher et al (2011) in nearbyAustralian waters investigated the effects of chronic low carbonate saturation levels onthe distribution growth and skeletal chemistry of several deep-sea corals off southeasternTasmanian seamounts and found that the distribution of scleractinian corals is constrainedby low carbonate saturation levels

Deep-sea corals are generally found in water temperatures between 4 and 12 C (RobertsWheeler amp Freiwald 2006 Buhl-Mortensen amp Mortensen 2005) This largely correspondsto relatively shallow depths (between 50 and 100 m) at high latitudes and greater depths(up to 4000 m) at low latitudes (Roberts Wheeler amp Freiwald 2006) However solitary cupcorals (eg Caryophyllia antarctica Gardineria antarctica and Flabellum impensum) canbe found up to 1000 m deep in the high latitude waters of Antarctica (Parker amp Bowden2010) Compared to the large numbers of shallow-water reef building corals that havebeen described only 10 deep-sea scleractinian reef-building species have been describedglobally (Cairns 1979 Freiwald et al 2004)

The South Pacific region including New Zealand supports a broad diversity of variousdeep-sea coral fauna (Williams et al 2010) the majority of which live between depths of200 and 1200 m (Tracey et al 2011) The scleractinain corals in the region are often foundon elevated hard substrate with topographic complexity such as seamounts knolls onslope margins ridges and canyons (Tracey et al 2011) where they are in an advantageousposition to feed in high current areas on particulate organic matter (Wolankski amp Hamner1998) Branching forms of the scleractinian corals create three-dimensional reef structuresin the deep and these provide key biogenic habitat and refuge for many deep-seainvertebrates fish and sharks The community composition of various invertebratesassociated with coral-reefs is well described in the literature (eg see Henry Davies ampRoberts 2010) Fish have been seen on or in close proximity to stony and other habitat-forming deep-sea corals (Bongiorni et al 2010 Soffker Sloman amp Hall-Spencer 2011Fossaringet al 2012 Purser et al 2013 Biber et al 2013Milligan et al 2016) and the benefitsof deep-sea reef habitats to shark species have also been reported (Henry et al 2013)

Atmospheric concentrations of carbon dioxide (CO2) have increased since pre-industrialtimes due to anthropogenic emissions The ocean acts as a carbon sink absorbing this CO2but results in changes to the chemistry of seawater including a reduction in pH and theavailability of free carbonate ions (Hoegh-Guldberg et al 2007) By the end of this centuryOA is expected to cause a decline in oceanic pH by 02ndash03 pH units (IPCC 2013) This is inaddition to the pH drop of 01 units which has already been observed since pre-industrialtimes (Friedrich et al 2012) OA is enhanced at low temperatures and high pressureconditions experienced in the deep-sea (Orr et al 2005 Roberts Wheeler amp Freiwald2006 Law et al 2018) Already impacted by trawling (Clark amp Rowden 2009 Clark etal 2015a Clark et al 2015b) scleractinian corals in deep cold-water environments arepredicted to be affected by global change such as OA much sooner than corals in surfacewaters of more temperate regions (Guinotte et al 2006 Turley Roberts amp Guinotte 2007Sweetman et al 2017) By 2100 under the high CO2 RCP85 scenario (IPCC 2013) pH

Gammon et al (2018) PeerJ DOI 107717peerj5236 224

reductions of gt02ndash03 pH units from current levels are expected in 23 of deep-sea canyonregions and on 8 of seamounts the key areas where deep-water corals are typically found(Gehlen et al 2014) While the response of deep-sea corals to OA and resulting lowcarbonate saturation levels is poorly understood research such as that by Thresher et al(2011) and research investigating carbonate saturation horizons in New Zealand waters byBostock Mikaloff Fletcher amp Williams (2013) indicate that deep-sea corals will be sensitiveto such environmental changes A recent synthesis assessed the potential threat posed byOA to the diversity and productivity of New Zealand marine ecosystems including coralsand highlighted the knowledge gaps in understanding the impacts (Law et al 2018)

The skeletons of deep-sea scleractinian corals are most commonly composed ofaragonite the more soluble polymorph of carbonate which makes them vulnerable to OA-induced dissolution (Anthony et al 2008) The waterrsquos suitability for carbonate depositionis determined by the carbonate saturation state () As reduces the formation ofcarbonate skeletons becomes increasingly difficult and the increased energy requirementsof calcification can ultimately threaten an organismrsquos survival The depth at which seawateris saturated with aragonite is termed the aragonite saturation horizon (ASH) Below thisdepth the ocean is under-saturated with respect to aragonite From studies of distribution(eg see Bostock et al 2015) it is suggested that most deep-sea coral species can probablytolerate some aragonite undersaturation (Ar sim08ndash09) These authors suggested thatscleractinian corals should be present in gt1 of stations down to 1800 m water depthand that some species (eg Solenosmilia variabilis) may be tolerant ofAr sim007 but theyconcluded it is unclear how deep-sea corals might respond to future OA

The rapid shoaling of the ASH over the last two decades measured at 1ndash2 m yrminus1

(Feely et al 2012) represents a significant threat to deep-sea corals as it is anticipated itwill become challenging for these ecosystem engineers to construct and maintain theirskeletons in water under-saturated with respect to aragonite (Guinotte et al 2006 Traceyet al 2013 Bostock et al 2015) Globally more than 70 of the present deep-sea coralcommunities will be subject to under-saturated conditions by the end of this century(Guinotte et al 2006) However the models used to simulate past and future changes inOA have the largest uncertainties in the Southern Ocean (eg Bopp et al 2013 Orr et al2005) Within the New Zealand region 95 of the habitat-forming scleractinian corals arefound above the ASH (Tracey et al 2013 Bostock et al 2015) Recent work suggests thatduring the present Anthropocene the ASH has already shoaled by 50 to 100m overmuch ofthe New Zealand Exclusive Economic Zone (Mikaloff-Fletcher et al 2017) This indicatesthat the proportion of the region with a carbonate chemistry favourable to aragoniticcalcifiers has already shrunk considerably (Mikaloff-Fletcher et al 2017) Further Bostocket al (2015) noted that some scleractinian corals lie below the ASH (ie in a zone whereconditions seem unfavourable for their growth) These authors hypothesised that previousshifts in the ASH could explain this unexpected result corals could have established whenthe ASH was deeper and the waters were supersaturated with aragonite at that depth andthen adapted as the ASH shoaled (Mikaloff-Fletcher et al 2017) Alternatively the patterncould indicate that these corals have some capacity to withstand or acclimate to changes inocean chemistry

Gammon et al (2018) PeerJ DOI 107717peerj5236 324

Globally most experimental work on the effects of OA on corals has been on shallowwater species and many studies note significant negative responses to OA In meta-analyses that included studies of shallow water corals Kroeker et al (2010) and Kroeker etal (2013) highlighted corals as one of the more vulnerable groups to OA For examplecalcification rates may decrease and carbonate dissolution rates may increase in shallow-water Pacific corals when pH is reduced only slightly (pH 785ndash795) with substantialimpacts when the pH is reduced to 760ndash770 (Anthony et al 2008) This pattern ofdecreasing calcification rates at lower carbonate concentrations is widely observed inshallow-water corals (Marubini et al 2008Herfort Thake amp Taubner 2008) Cellular leveleffects on shallow water corals have also been observed where the photosynthetic activityof the endosymbiont is tightly coupled with the ability of the host cell to recover fromcellular acidosis after exposure to OA (Gibbin et al 2014)

In contrast there are fewer studies on the impacts of OA on deep-sea corals A synthesisby Maier Weinbauer amp Gattuso (in press) reports that the response of only five deep-seacoral species (Madrepora oculata Lophelia pertusa Desmophyllum dianthus Dendrophylliacornigera and Caryophyllia smithii) to OA have been investigated Most of the stony coralstudies outside of the Mediterranean region have been confined to one species L pertusa(see Table 1 in Maier Weinbauer amp Gattuso in press) These studies have examined effectsof OA (throughmanipulation of pH or partial pressure of CO2 (pCO2)) after short (sim24 h)and long term (sim10ndash12 months) exposure on measures such as calcification metabolismand skeleton strength (Maier et al 2009 Hennige et al 2014 Movilla et al 2014) Herewe took a long-term approach (12 months) to assess the impacts of the projected end-of-century OA scenario on the physiology of an abundant habitat-forming scleractiniancoral species (Solenosmilia variabilis) from New Zealand and the wider southeast Pacificregion (Tracey et al 2011 Thresher et al 2011) This species is fragile long-lived and lateto mature (Thresher et al 2011 Fallon Thresher amp Adkins 2014 H Neil DM Tracey DMTracey P Marriott amp MC Clark 2010 unpublished data) and any negative impact of OAon this species could have wider ecosystem consequences

METHODSLive sampling of Solenosmilia variabilisField sampling of protected corals was approved by the Department of Conservation(permit number 35099-CAP) and coral samples were landed under the authority of theMinistry for Primary Industries (permit number B201461361)

Live colonies of S variabilis were sampled during March 2014 from the Louisville Ridge700 km east of New Zealand (Fig 1) Colonies were sampled in depths ranging from1220 to 1370 m from each of two seamount-like guyot features (referred to as seamountsthroughout) (Table 1) using an epibenthic sled deployed from the National Institute ofAtmospheric Research (NIWA) research vessel RV Tangaroa

Upon retrieval of the sled multiple live coral colonies were immediately placed in achilled bin of seawater and then transferred to an on-board aquarium with a continuousflow-rate (sim50 L hminus1) of unfiltered seawater maintained at sim5 C No feeding took place

Gammon et al (2018) PeerJ DOI 107717peerj5236 424

Figure 1 Map of the RV Tangaroa voyage track within New Zealand The map shows the LouisvilleSeamount Chain (named black dots) known seamount features in the region (small black dots) theExclusive Economic Zone (EEZ) boundary and the Extended Continental Shelf (ECS) boundary Livecolonies of Solenosmilia variabilis were sampled using an epibenthic sled from four seamount features(Anvil 39 South Ghost and Valerie) The experiment used samples from Anvil and Valerie

Full-size DOI 107717peerj5236fig-1

Table 1 A summary of data for the Louisville Ridge sample sites successfully sampled for live coralcolonies using the epibenthic sled The table presents the sample station depth range (m) bottom tem-perature (C) and position (latitude and longitude) The pH at both sample sites was 788 (calculatedfrom measured CT AT temperature and salinity)

Seamount Depth range(m)

Bottom temperature(C)

Latitude(S)

Longitude(W)

pH(calculated)

Anvil 1244ndash1370 342 374244prime 16909prime 788Valerie 1220ndash1250 330 412188prime 1642514prime 788

throughout the three-week voyage as it was assumed that the corals would obtain sufficientfood from unfiltered surface water

Conductivity Temperature and Depth (CTD) casts (Seabird 911 Seattle WA USA)and water samples were conducted at the sampling sites in order to characterise localseawater and to inform the experimental conditions (Table 1) One CTD cast was takenper site and the following water samples (one per site) 500 ml for total alkalinity (AT) and250 ml for dissolved inorganic carbon (CT) Water samples were preserved with mercuricchloride (HgCl2) The CT was determined using coulometric analysis of the CO2 stripped

Gammon et al (2018) PeerJ DOI 107717peerj5236 524

Table 2 Experiment seawater conditions pH pCO2 and carbonate parameters (averageplusmn SE) calcu-lated from measured pH alkalinity temperature and salinity on two separate dates during the experimentThe pH over the entire 12 month experiment averaged 788plusmn 00004 (control) and 765plusmn 00007 (treat-ment)

Treatment (target) pH AT (micromol kg minus1) pCO2 Ar Ca

pH 788 (control) 787plusmn 00004 2257plusmn 2871 5919plusmn 704 111plusmn 002 176plusmn 003pH 765 (treatment) 765plusmn 0001 2260 plusmn 2751 10175plusmn 1571 069 plusmn 001 109 plusmn 001

from the seawater sample after acid addition (Dickson Sabine amp Christian 2007) Theaccuracy of the method is determined by analysis of Certified Reference Material (providedby Andrew Dickson from Scripps Institution of Oceanography) with every sample batchand is estimated to beplusmn 1 micromol kgminus1 AT was determined using a closed cell potentiometrictitration (Dickson Sabine amp Christian 2007) The accuracy of the method is determinedby analysis of Certified Reference Material (provided by Andrew Dickson from ScrippsInstitution of Oceanography) with every sample batch and is estimated to be plusmn 2 micromolkgminus1

In situ pH (total scale) was calculated using measured CT AT temperature and salinityand Mehrbach equilibrium constants refit by Dickson amp Millero (1987) This calculated pHwas used to set the ambient pH conditions for the experiment

Experimental set upOnce ashore 12 coral colonies were transferred to NIWArsquos Marine EnvironmentalManipulation Facility (MEMF)Wellington where they were held in flow through seawaterat the temperature measured at the collection site (35 C) After a stabilisation period thelarge colonies were carefully broken into small portions to achieve 54 colony fragmentscomprising live polyps and adjacent branchlets The number of fragments achieved fromeach colony ranged from two up to six Each colony was kept out of the water for no longerthan 1 minute during this process Each fragment (sim4ndash6 cm in length) was then attachedin a fixed orientation to a piece of plastic mesh Colonies were then randomly assigned toone of 18 identical tanks (4 L) with three coral fragments per tank whilst also ensuring thatfragments originating from the same colony were not included in the same tanks Coralswere maintained in darkness throughout the stablisation and experimental period

The tanks were fed seawater via a continuous flow-through system (sim130 mL mminus1)Seawater pH was 788 to mimic conditions measured at the collection sites (Table 1) Thecorals were fed twice weekly with a 3 mL mixture consisting of 10 commercial coral food(JBL Koralfluid Neuhofen Germany) and 10 commercial shellfish diet (larval shellfishdiet 1800 Reed Mariculture Campbell CA USA) that was diluted to the required volumewith 1 microm filtered seawater (FSW) Corals were maintained in this tank set-up with regularfeeding for three months before the experiment began increasing the likelihood that eachcolony had a similar nutritional status at the beginning of the experiment

After three months the experiment was initiated with nine control tanks and ninetreatment tanks established Corals in the control group were exposed to ambient pH788 (pCO2519 ppm) In comparison treatment corals were exposed to low pH of 765

Gammon et al (2018) PeerJ DOI 107717peerj5236 624

(pCO2920 ppm) (Table 2) The reduced pH level was based on projected changes toseawater pH through to the year 2100 (Bopp et al 2013 IPCC 2013 Orr et al 2005)The pH in the treatment tanks was reduced gradually over three days until it reached thetreatment value Temperature was held at 35 C in all tanks

Seawater manipulation and measurementFSW from Wellington Harbour adjacent to the facility was chilled to 35 C and fedto separate header tanks before being delivered to the experimental tanks at 130 mLminminus1 in a flow-through system The pH was adjusted through the diffusion of foodgrade CO2 which was controlled using Sensorex S150C pH probes (Garden Grove CAUSA) The pH probes in each header tank were calibrated regularly with TRIS and AMPbuffers Water samples were taken from each header tank on two occasions during the12-month experiment preserved with HgCl2 and analysed for determination of AT asdescribed above These measurements of pH (on each day the water samples were taken)and AT along with temperature and salinity were used to calculate pCO2 and Ar of eachexperimental treatment using the refitted (Mehrbach et al 1973) equilibrium constants(Dickson amp Millero 1987)

Evaluating Solenosmilia variabilis responsesResponses were assessed using a variety of measures at regular intervals over the 12-monthexperiment At the beginning of the experiment all coral fragments were photographedand buoyant weighed Subsequently at bimonthly intervals over a 10-month periodmeasurements of polyp mortality (via live polyp counts) linear skeletal extension and or three-dimensional step-wise growth (referred to as linear growth throughout) andloss of coenenchyme tissue were made The coenenchyme is the outer tissue coveringthe coral skeleton that links the coral polyps and provides protection for the developingexoskeleton loss of this tissue was evaluated via changes in colour saturation (detailedbelow) Respiration rate (O2 consumption) was measured on two occasions at six and 12months

Polyp mortalityPolyp mortality was measured every two months by making a visual count of the numberof live polyps on each fragment Each tank had three fragments and polyp mortalitywas averaged for each tank to get a single average per tank (n= 9) The total percentageremaining of the initial polyp count at each time point was then calculated using thefollowing equation

100minus

[((P1minusPJ

)(P1)

)]times100

Where P1 is the polyp count taken at the first time point and PJ is the polyp count at eachof the subsequent J th time points

Linear growthEach coral fragment was photographed at bimonthly intervals to obtain a measure of lineargrowth Because fragments were cable-tied in a fixed position they remained in the same

Gammon et al (2018) PeerJ DOI 107717peerj5236 724

orientation throughout the experiment and it was possible to locate and measure the samebranch through time From the digital images linear growth was determined by selectingan easily identifiable feature on the colony fragment such as a branching point or a specificlinear growth feature Measurements were then taken from this distinctive point alongthe axis of linear growth to the area just below a live polyp where the calcification processoccurs Measurements were made using the software ImageJ ccopy (Schneider Rasband ampEliceiri 2012) Where possible a maximum of four such measurements were taken for eachfragment Where multiple measurements were taken these were then averaged to achievea single linear growth rate for each fragment Each tank had three fragments and the singlefragment linear growth rates were averaged for each tank to get a single average per tank(n= 9) Only branchlets that were sim2ndash5 cm long at the beginning of the experiment wereselected for measurement

Tissue lossImages taken to measure linear growth rates were also analysed to determine coloursaturation which was used as a proxy for the coenenchyme covering the branch and polypareas of the coral skeleton Our method used to assess colour change was based on thatof Winters et al (2009) Images taken during the experiment were cropped to remove thebackground and then colour-profiled using the colour histogram plugin on ImageJ Theentire 2D image of each coral fragment was profiled at each time point This profile providesa mean value of intensity for each of the red green and blue colour channels A pilot studywas used to confirm that a loss in intensity of the red colour channel corresponded to aloss of coenenchyme (see Supplementary Information)

The relative intensity for the red colour channel was calculated using the followingequations

T =R+G+B

Rr =RT

Gr =GT

Br =BT

where T the total intensity of an image R mean intensity of the red channel G meanintensity of the green channel B mean intensity of the blue channel and Rr Gr and Br relative intensity of the red green and blue channels respectively (Winters et al 2009)Calculating the percentage of relative brightness for the red colour channel rather thanusing the mean brightness suppresses the influence that any changes in illuminationexposure or internal camera processing may have on the brightness of each channel(Richardson et al 2009)

The percentage change in relative intensity of the red colour channel was then calculatedusing the following equation

RR= 100times(SR1minusSR2)(SJ1xT1

T2

)

Gammon et al (2018) PeerJ DOI 107717peerj5236 824

Figure 2 Solenosmilia variabilis colony in situ and S variabilis fragment in a respiration chamberThe images show a large colony of deep-sea coral Solenosmilia variabilis on a seamount flank in the NewZealand region (A) (NIWA Deep Towed Imaging System) and a fragment of S variabilis in the experi-mental respiration chamber (B) a stirrer is positioned at the top of the chamber (This figure is derived inpart from an article published in the New Zealand Journal of Marine and Freshwater Research published on25 September 2017 available online httpsdoiorg1010800028833020171374983)

Full-size DOI 107717peerj5236fig-2

where RR the relative intensity of the red colour channel SR1 the mean intensity of thered colour channel at time point one SR2 the mean intensity of the red colour intensity attime point two T1 time point one and T2 time point two

Respiration rateAt six and 12 months one fragment per tank was randomly selected (n = 9 foreach treatment and time point) and respiration rates measured Respiratory oxygenconsumption was measured in a 500 mL chamber sealed by an o-ring (Fig 2)

Each chamber was equipped with a magnetic stirrer to ensure homogeneity of oxygen(O2) around the coral fragments A glass vial with a Presens Pst 3 O2 sensor (RegensburgGermany) glued to its end was inserted through a hole in the chamber lid so that it madecontact with seawater in the chamber The O2 sensor was two-point calibrated beforeeach run using 0 and 100 saturated seawater 0 saturated seawater was obtainedby dissolving 1 g of sodium sulphite (Sigma-Aldrich St Louis MO USA) in seawaterand 100 saturation was achieved by bubbling air through seawater for 30 min Thechambers were placed in a 35 C water bath and kept in darkness Each coral fragmentwas left to settle in its chamber for a minimum of 20 min before the chamber was sealedThe chambers remained in the water bath for the duration of the measurement and werekept in darkness to prevent any photosynthetic activity in the seawater Total O2 used byeach coral fragment was calculated as the difference between the initial and final oxygenconcentrations measured within each chamber

Each run consisted of five incubation chambers each housing a different coral fragmentThe duration that each fragment was kept in a chamber varied depending on the coralrsquosrespiration rate a period ranging from 5 to 7 h Measurements in the chambers were

Gammon et al (2018) PeerJ DOI 107717peerj5236 924

terminated if the O2 saturation dropped below a pre-determined 90 The water volumewithin each chamber was measured at the end of each experiment

The O2 concentration in each chamber at the start and end of the experiment wasstandardised tomicrog Lminus1 and an hourly rate ofO2 consumption calculated for each individualThe protein content per individual was used to normalise the respiration rate (microg O2 mgproteinminus1 hminus1) Samples were initially frozen and the frozen tissue removed from theskeleton matrix with an airbrush and transferred to a snap-lock bag containing 5 mL ofdistilled water The protein slurry produced was then poured into a 250 mL beaker Thesnap-lock bag was rinsed into the beaker twice with 5 mL of distilled water to remove anyresidual protein The protein slurry was homogenized further using an electric homogenizer(Proxxon micropower driver Foumlhren Germany) and the total quantity of homogenizedmaterial noted A 5 microL subsample of homogenized protein slurry was transferred to a 96-well plate and analysed with the Coomassie Brilliant Blue protein assay (Bradford 1976)and a spectrophotometer (EnSpire 2300 Multilabel Plate Reader PerkinElmer WalthamMA USA) The protein concentration of each 5 microL sub-sample was then adjusted for thetotal volume of each sample and the total protein content of each individual coral fragmentcalculated

Statistical analysesStatistical analyses were carried out using the software package SPSS (Coakes amp Steed2009) Data were initially tested for normality and transformed if they did not meetassumptions A Friedman test was used to analyse data for both polyp mortality and theloss of coenenchyme (data were not normally distributed and the assumption of normalitycould not be met using log transformations) Data were categorized into 12 groups whichrepresented each of the monthly time points (zero two four six eight and 10 months ofexposure) for the treatment and control pH samples

A rm-ANCOVA was used to compare the average linear growth rate of individualsbetween the control and treatment groups The difference between the linear growthlengths of each branchlet for each sample was compared between each time point andlinear growth presented as mm linear extension per year Seamount and colony of originwere included as covariates to ensure that they had no confounding effects on the responsevariable

Respiration data were log transformed to meet the assumption of normality Thesignificant effect of treatment and interactive effect of time since exposure with treatmentwere tested using a two-way ANCOVA Respiration chamber seamount of origin andcolony of origin were included as covariates to ensure that they had no confounding effectson the response variable

RESULTSAt the end of the 12-month experiment all corals in both the treatment and controlgroups had live polyps indicating that the experimental conditions were appropriate tomaintain viable corals

Gammon et al (2018) PeerJ DOI 107717peerj5236 1024

pH was maintained at target concentrations for the duration of the experiment Theaverage pH for the treatment group was 7650 plusmn 00007 (range 7604ndash7699) and theaverage pH for the control group was 7876plusmn 00004 (range 7823ndash7920) These averagesare calculated from gt2800 pH probe measurements taken throughout the 12-monthexperiment

Polyp mortalityA visible increase in polyp mortality was noted throughout the experiment No polypmortality occurred in the first two months in either the control or treatment groupsalthough it subsequently increased over time While there was a treatment effect (Friedmantest χ2(11)= 107769 p= 0001) post hoc tests (Wilcoxon-signed rank test) showedthat these differences were between different time points of the same treatment and thatthere was no change in polyp mortality within the treatment group relative to the controlHowever from six months onward polyp mortality was consistently higher in the low pHgroup The greatest loss in polyp mortality occurred in the low pH group from four (wherecolonies still had 9204 plusmn 745 of their polyps remaining) to six months of exposure(where colonies only had 6158 plusmn 719 of their polyps remaining) The differencebetween these two time points represents a loss of 3046 of initial polyp counts over justa four-month period

Linear growthThe average linear extension rate at the control pH was 1558 plusmn 0226 mm yearminus1 and atthe reduced pH was 1603 plusmn 0260 mm yearminus1 Linear growth rates were highly variablebetween individual coral fragments ranging from 0583 to 3068 mm year minus1

Linear growth rate was also independent of time of exposure for both the reduced pHand control groups (rm-ANCOVA F440= 0481 p= 0749 and F452= 0274 p= 0893respectively) The seamount of origin also had no effect on the linear extension rate ofthe treatment colonies (rm-ANCOVA F440= 0769 p= 0552) or the control colonies(F452= 0577 p= 0681) For these reasons both time of exposure and seamount wereexcluded from the final analyses which then found no effect of reduced pH on the linearextension rate of S variabilis (rm-ANCOVA F125= 0017 p= 0899)

Coenenchyme lossWhile both the control and treatment groups lost colour throughout the experimentcolour loss was significantly greater in fragments held at reduced pH a finding that wasapparent at all time points (ie 2 4 6 8 and 10 months Wilcoxon-signed rank analysispost hoc analysis Friedman test χ2(11)= 130617 p= 0001 Fig 3) After two monthsthe colour intensity of the control group was 9761 plusmn 1933 of that measured at the startof the experiment while the low pH group retained 94396plusmn 0738 of its colour intensityBy comparison at 10 months there was on average a difference of 528 between thepercentage of initial colour remaining between the treatment and control groups

Respiration rateCoral respiration rates were higher in the control pH than in the low pH at both the six and12-month time points (Fig 1) and for all fragments were higher at the 12-month time

Gammon et al (2018) PeerJ DOI 107717peerj5236 1124

Figure 3 The effect of seawater pH on the loss of coenenchyme tissue of the coral Solenosmilia vari-abilis Images AndashB are of the same colony at control pH (pH 788) Images CndashD are of the same colony atthe treatment pH (pH 765) Images A and C were taken prior to the start of the experiment and images Band D after three months into the experiment (continued on next page )

Full-size DOI 107717peerj5236fig-3

Gammon et al (2018) PeerJ DOI 107717peerj5236 1224

Figure 3 ( continued)Note the marked reduction in the intensity of the redpink colouration to a pale colour once the coral hadbeen exposed to low pH for several months (D) Photos of S variabilis were analysed for the relative per-centage of intensity in the red colour channel (n= 17 per time-point per treatment) The mean percentageremaining (plusmn 1 SE) of the initial relative intensity is presented (E) The solid line represents the treatmentgroup and the broken line represents the control group Significant differences from the control are shownby plt 001 and plt 0001 (Wilcoxon-signed rank analysis)

Figure 4 The effects of seawater pH on the respiration rate of Solenosmilia variabilis Respiration rate(micromol O2 mg proteinminus1 hminus1) of colonies after exposure to reduced pH (pH 765 dark grey) or control pH(pH 788 light grey) for six- and 12-month exposure (n= 9 for each treatment and time-point values aremeansplusmn standard error (SE))

Full-size DOI 107717peerj5236fig-4

point For fragments in the control group (pH 788) respiration was 179 and 31 higherthan for coral colonies exposed to low pH (pH 765) after six and 12 months respectively(Fig 1) Also of note is that the respiration rates at reduced pH increased by 225 betweenthe six and 12-month time points

While the statistical analyses indicated that respiration rate was not influenced by pH(two-way ANOVA F124= 3200 p= 0086 Fig 4) there was a significant effect of timewhere respiration rates were higher for both the control and treatment groups at the12-month time point (two-way ANOVA F124= 0977 p= 0007) There was howeverno interactive effect between pH treatment and time (two-way ANOVA F124 = 0101p= 0350)

Gammon et al (2018) PeerJ DOI 107717peerj5236 1324

DISCUSSIONThis study investigated physiological responses to reduced pH in S variabilis a habitat-forming scleractinian coral species common around New Zealand and the wider southeastPacific region

S variabilis colonies were maintained for 12 months under reduced pH conditions(pH 765 Ar = 069plusmn001) and various aspects of their physiological response wereinvestigated over that time While the colonies were generally robust to OA conditions(there was no mortality) there was significant loss of coenenchyme tissue cover at lowpH (Fig 3) and indications of effects on respiration rates (Fig 4) Respiration rate wasrelatively low in the reduced pH treatment particularly at the six-month time point whenit was 179 higher in control conditions (Fig 4) although this effect was not statisticallysignificant There was no treatment effect on mortality of polyps or linear growth rates

Todate published studies onhowOAmight influence deep-sea corals have varied resultseven within different populations of the same species This is demonstrated by Georgianet al (2016) who tested the physiological response to OA of L pertusa colonies from twogeographically different populations (Gulf of Mexico USA and Tisler Reef Norway) TheGulf of Mexico corals exhibited reductions in net calcification and respiration while TislerReef corals showed only slight reductions in net calcification and elevated respiration Theauthors concluded that these differences were likely the result of environmental differences(eg depth pH food supply) between the two regions In another experiment on Lpertusa Maier et al (2009) found that incubating L pertusa for 24 h in seawater with pHlowered by 015 and 03 units relative to the ambient level resulted in calcification beingreduced by 30 and 56 respectively In another short term study Hennige et al (2014)investigated the response of L pertusa to increased CO2 conditions (750 ppm) over 21days L pertusa corals exposed to increased CO2 had significantly lower respiration ratesthan corals in control conditions but found no corresponding change in calcificationrates In a longer-term studyMovilla et al (2014) found a decline in the calcification of Ddianthus after 314 days of exposure to elevated pCO2 (800 microatm) Interestingly in anotherlong-term experiment over 12 months Hennige et al (2015) observed a decrease in thestructural integrity of dead exposed L pertusa skeleton when exposed to increased CO2

conditions Such studies provided a platform for our current studyThe Ar was lt1 in both the control and treatment waters in our experiment (Table

2) From a broad survey of New Zealand coral species and carbonate saturation Traceyet al (2013) and Bostock et al (2015) identified a strong dependency of coral distributionon Ar and Ca However many deep-sea stony corals can cope with some degree ofaragonite undersaturation (Ar sim08ndash09) with some species tolerant ofAr sim07 (Bostocket al 2015) a value lower than the Ar of 069plusmn001 in our low pH treatment While ithas been noted that some stony corals lie below the ASH (eg Bostock et al 2015 Baco etal 2017) including in the New Zealand region such as those found along the LouisvilleSeamount Chain (Bostock et al 2015) we did note reduced coenenchyme tissue cover andindications of elevated respiration rates at these levels

Gammon et al (2018) PeerJ DOI 107717peerj5236 1424

Linear growth ratesThis study found no treatment effect on the linear growth rates of corals which were seen tobe highly variable ranging from 0583 to 3068 mm yminus1 The measured linear growth rateis comparable to results from radiocarbon dating studies of S variabilis by Fallon Thresheramp Adkins (2014) who reported linear growth-rates ranging between 084ndash125 mm yminus1and by H Neil DM Tracey DM Tracey P Marriott amp MC Clark (2010 unpublisheddata) who again showed similar linear growth of 025ndash13 mm yminus1 Linear growth ratesthat are independent of pH have been found in other deep-sea corals from various inaquaria studies includingM oculata (Maier et al 2013b) and L pertusa (Form amp Riebesell2012 Maier et al 2013b) While it was found that M oculata was not affected when pHwas manipulated to end-of-century projections when the partial pressure of CO2 wasreduced to pre-industrial levels calcification rates in this species increased (Maier et al2012) This provides important information about the historical effect of OA on thecalcification of deep-sea corals and indicates that the present-day calcification rates mayhave already declined due to an anthropogenic increase in the concentration of atmosphericCO2 Although no net effect of OA on linear extension was observed in this study it isimportant to note that measurements were only taken after several months and undetectedshorter-term changes may have occurred

Respiration rateRespiration rate was highly variable ranging from 0065 microg O2 g proteinminus1 hminus1 to 1178microg O2 gminus1 proteinminus1 hminus1 These results are low compared to respiration rates found byDodds et al (2007) for the branching scleractinian L pertusa who found a respirationrate of about 05 micromol gminus1 hminus1 Here the respiration rate of S variabilis was relativelylow in the reduced pH treatment particularly at the six-month time point Interestinglythis difference decreased at the 12-month time point and this could be indicative ofacclimation A similar response was found by Maier et al (2013a) in M oculata and Lpertusa The authors attributed the observed increase in respiration rate to an increasein energy supply as a result of regular feeding thus sustaining an elevated level of coralmetabolism Regular feeding and its impact on coral metabolism can mask the effectsof OA in experimental work This was found by Buumlscher Form amp Riebesell (2017) whoconcluded that while the deep-sea coral L pertusa is capable of calcifying under elevatedCO2 and temperature its condition (fitness) ismore strongly influenced by food availabilityrather than changes in seawater chemistry With the natural habitat of S variabilis beingso inaccessible it is difficult to predict the amount of food including particulate organicmatter and sources of plankton reaching colonies in situ

Tissue lossA visible loss of coenenchyme was noted from both the control and treatment colonies inthe first two months of the experiment although this loss was significantly greater fromthe treatment colonies Tissue loss in the control group is consistent with stress and colonydeterioration which is not surprising given that deep-sea corals are difficult to maintainin a healthy state in aquaria indeed to our knowledge S variabilis has never previously

Gammon et al (2018) PeerJ DOI 107717peerj5236 1524

been maintained for more than a few weeks in this state The significantly greater effect ofreduced pH on the rate of tissue loss highlights that other physiological mechanisms werealso playing a part A loss of tissue when exposed to OA conditions has also been observedin tropical corals (eg Pocillopora damicornis and Oculina patagonica) but the tissuesof these two species regenerated when the corals were returned to ambient pH (Kvittet al 2015) For these shallow warm-water corals reduced pH induced tissue-specificapoptosis a breakdown of coenenchyme and a subsequent loss of the colonial form Thetissue loss of S variabilis seen here could represent the early stages of a similar responseand warrants a longer-term study Interestingly the same rate of polyp mortality overtime was observed in both the treatment and control colonies while coenenchyme losswas greater in the treatment group The coenenchyme has a function in connecting eachneighbouring polyp and protecting the growing skeleton A loss of the coenenchyme couldmean a shift away from the coralrsquos ability to produce a colonial three-dimensional matrix(Hennige et al 2015) Reverting to solitary and non-calcifying polyps has been proposed asan evolutionary mechanism which has allowed corals to survive through geological periodsof unfavourable calcification conditions (Kvitt et al 2015) and could explain several lsquolsquoreefgapsrsquorsquo in the geological records (Wood 1999)

Alternatively the observed loss of coenenchyme could represent a reallocation ofenergy That is corals in the treatment group may have been diverting energy away fromthe maintenance of tissues allowing them to maintain other metabolic requirements(eg linear growth respiration and reproduction) For this reason tissue loss in corals isconsidered a better indicator of physiological stress than skeletal linear growth (AnthonyConnolly amp Willis 2002)Maier et al (2016) show that the energy required for calcificationin M oculata is a small fraction (sim1-3) of overall metabolic requirements Assumingthat the energy requirements for calcification in S variabilis are similar this substantiatesour comment that tissue loss may be a better indicator of physiological stress than lineargrowth and partly explains why this study found no treatment effect on the linear growthrates of corals

CONCLUSIONDeep-sea corals are typically difficult to study due to their poor survival rate in laboratoryconditions For this reason physiological studies of their responses to environmental changehave been limited to date The data presented here for S variabilis represent an importantfirst-step towards understanding the biology of this ecologically important species and toour understanding of the sensitivity of deep-sea corals to OA In New Zealand specificallythe lack of knowledge of organism responses is well recognised the potential threat posedby OA to the diversity and productivity of marine ecosystems (including to corals) isclassed as medium for vulnerability low to medium for knowledge of established responseand low for understanding mechanistic response ecosystem interaction and interaction ofother stressors (Law et al 2018) This study found that S variabilis lost tissue in responseto OA and we hypothesize that this could represent a reallocation of energy with coralsdiverting energy away from the maintenance of non-essential tissue It is assumed however

Gammon et al (2018) PeerJ DOI 107717peerj5236 1624

that an organism would not continue to break down tissues to help support skeletalthree-dimensional linear andor step-wise growth as a threshold will ultimately be reachedwhere the animal becomes seriously compromised If this were to happen then there wouldbe major changes to the structure and function of this species as an important ecosystemengineer in the deep-sea

This study has signposted the need to better understand the long-term implications andmechanisms of OA on colony tissue loss the most notable effect of decreased pH observedTo our knowledge this study is the first to apply a technique of measuring tissue loss to adeep sea coral in an experiment designed to measure the coralsrsquo response to OA Studiessuch as this which find a limited response in those physiological variables which aretypically measured (eg respiration and linear growth) should consider what the potentialcost of maintaining those parameters may be Here we demonstrate an additional measureof tissue loss which could be routinely included in future studies to gain a more holisticunderstanding of the organismsrsquo response We also recommend that future studies assessthe impact of OA on skeletal morphology and density which were not assessed here Suchimpacts have the potential to change colony integrity and survival Combined with ongoingand more refined modelling work to inform future projections of the ASH and CSH in theSouth Pacific this study nevertheless improves our knowledge on the impacts of OA onthis important and ecologically vulnerable coral group in the New Zealand region

ACKNOWLEDGEMENTSThe sampling of live coral specimens for shore-based laboratory observation formed aSecondary Objective of the TAN1402 Voyage to the Louisville Seamount Chain We thankPrincipal Scientists Ashley Rowden and Malcolm Clark for providing support and theplatform for collecting the live samples We acknowledge scientists and crew on boardTangaroa Voyage TAN1402 in particular Rob Stewart and Steve Parker (NIWA) JohnGuinotte (previously of the Marine Conservation Institute) and Sonia Rowley (Universityof Hawaii Manoa and Bishop Museum Hawaii US) for their help with the live coralsamples and Kim Currie (NIWAUniversity of Otago Research Centre for Oceanography)for analysing the water samples Neill Barr and Graeme Moss (NIWA) were vital inhelping prepare the equipment for all aspects of the experiment at sea and on land and inmanaging the Marine Environmental Manipulation Facility We also thank Stephen Cairns(Smithsonian Institute DC US) andMarcelo Kitahara (Universidade de Satildeo Paulo Brazil)for confirming species identification and defining morphological terms On-going advicewas received from Conny Maier (last affiliation Microbial Ecology and BiogeochemistryGroup Laboratoire drsquoOceacuteanographie de Villefranche surMer France)We are very gratefulto Kate Sparks and Miles Lamare (Department of Marine Science University of Otago)for loaning us the respiration chambers Ron Thresher (CSIRO) and Paal Buhl Mortensen(IMR) advised on various practicalities and procedures of the experiment

Gammon et al (2018) PeerJ DOI 107717peerj5236 1724

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis work was supported by Mary Livingston and Martin Cryer New Zealand Ministryfor Primary Industries (MPI ZBD201041) The funders had no role in study design datacollection and analysis decision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsNew Zealand Ministry for Primary Industries MPI ZBD201041

Competing InterestsDianne M Tracey Peter MMarriott and Vonda J Cummings are employed by the NationalInstitute of Water amp Atmospheric Research

Author Contributionsbull Malindi J Gammon conceived and designed the experiments performed theexperiments analyzed the data contributed reagentsmaterialsanalysis tools preparedfigures andor tables authored or reviewed drafts of the paper approved the final draftbull Dianne M Tracey conceived and designed the experiments performed the experimentscontributed reagentsmaterialsanalysis tools authored or reviewed drafts of the paperapproved the final draftbull Peter M Marriott conceived and designed the experiments performed the experimentscontributed reagentsmaterialsanalysis tools authored or reviewed drafts of the paperbull Vonda J Cummings and Simon K Davy conceived and designed the experimentscontributed reagentsmaterialsanalysis tools authored or reviewed drafts of the paper

Field Study PermissionsThe following information was supplied relating to field study approvals (ie approvingbody and any reference numbers)

Field sampling of protected corals was approved by the Department of Conservation(permit number 35099-CAP) and coral samples were landed under the authority of theMinistry for Primary Industries (permit number B201461361)

Data AvailabilityThe following information was supplied regarding data availability

The raw data are provided in a Supplemental File

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj5236supplemental-information

Gammon et al (2018) PeerJ DOI 107717peerj5236 1824

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Bongiorni L MeaM Gambi C Pusceddu A Taviani M Danovaro R 2010 Deep-water scleractinian corals promote higher biodiversity in deep-sea meiofaunalassemblages along continental margins Biological Conservation 143(7)1687ndash1700DOI 101016jbiocon201004009

Bopp L Resplandy L Orr JC Doney SC Dunne JP GehlenM Halloran P HeinzeC Ilyina T Seacutefeacuterian R Tjiputra J Vichi M 2013Multiple stressors of oceanecosystems in the 21st century projections with CMIP5 models Biogeosciences106225ndash6245 DOI 105194bg-10-6225-2013

Bostock H Mikaloff Fletcher SEWilliamsMJ 2013 Estimating carbonate parametersfrom hydrographic data for the intermediate and deep waters of the SouthernHemisphere Oceans Biogeosciences 106199ndash6213 DOI 105194bg-10-6199-2013

Bostock HC Tracey DM Currie KI Dunbar GB Handler MR Mikaloff Fletcher SESmith AMWilliamsMJM 2015 The carbonate mineralogy and distribution ofhabitat-forming deep-sea corals in the Southwest Pacific region Deep-sea researchPart I Oceanographic Research Papers 10088ndash104 DOI 101016jdsr201502008

BradfordMM 1976 A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding AnalyticalBiochemistry 72248ndash254

Buhl-Mortensen L Mortensen 2005 Distribution and diversity of species associatedwith deep-sea gorgonian corals off Atlantic Canada Cold-water Corals and Ecosys-tems 1849ndash879 DOI 1010073-540-27673-4_44

Buumlscher JV Form AU Riebesell U 2017 Interactive effects of ocean acidificationand warming on growth fitness and survival of the cold-water coral Lopheliapertusa under different food availabilities Frontiers in Marine Science 4101DOI 103389fmars201700101

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ClarkMR Althaus F Schlacher TAWilliams A Bowden DA Rowden AA 2015a Theimpacts of deep-sea fisheries on benthic communities a review ICES Journal ofMarine Science 73(suppl_1)i51ndashi69 DOI 101093icesjmsfsv123

ClarkMR Anderson O Bowden D Chin C George S GlasgowD Guinotte J HererraS Osterhage D Pallentin A Parker S Rowden AA Rowley S Stewart R Tracey DWood S Zeng C 2015b Vulnerable marine ecosystems of the Louisville Seamountchain voyage report of a survey to evaluate the efficacy of preliminary habitatsuitability models New Zealand aquatic environment and biodiversity Report No149 86 p

ClarkMR Rowden AA 2009 Effect of deepwater trawling on the macro-invertebrateassemblages of seamounts on the Chatham Rise New Zealand Deep Sea ResearchPart I Oceanographic Research Papers 561540ndash1554 DOI 101016jdsr200904015

Coakes SJ Steed L 2009 SPSS analysis without anguish using SPSS version 140 forWindows New York John Wiley amp Sons Inc

Dickson AG Millero FJ 1987 A comparison of the equilibrium constants for the disso-ciation of carbonic acid in seawater media Deep Sea Research Part A OceanographicResearch Papers 34(10)1733ndash1743 DOI 1010160198-0149(87)90021-5

Dickson AG Sabine CL Christian JR (eds) 2007Guide to best practices for ocean CO2measurements Vol 3 North Pacific Marine Science Organization PICES SpecialPublication 191

Dodds LA Roberts JM Taylor AC Marubini F 2007Metabolic tolerance of the cold-water coralLophelia pertusa(Scleractinia) to temperature and dissolved oxygenchange Journal of Experimental Marine Biology and Ecology 349(2)205ndash214DOI 101016jjembe200705013

Fallon S Thresher R Adkins J 2014 Age and growth of the cold-water scleractinianSolenosmilia variabilis and its reef on SW Pacific seamounts Coral Reefs 3331ndash38DOI 101007s00338-013-1097-y

Feely RA Sabine CL Byrne RH Millero FJ Dickson AGWanninkhof R MurataA Miller LA Greeley D 2012 Decadal changes in the aragonite and calcitesaturation state of the Pacific Ocean Global Biogeochemical Cycles 261ndash15DOI 1010292011GB004157

Form AU Riebesell U 2012 Acclimation to ocean acidification during long-termCO2 exposure in the cold-water coral Lophelia pertusa Global Change Biology18843ndash853 DOI 101111j1365-2486201102583x

Fossaring JH Kutti T Helle K Bergstad OA 2012 Associations and functional linksbetween tusk and cold water coral and sponge habitats examined by experimentallong-line fishing In Theme 1 ABSTRACT and PROGRAM BOOK international deep-sea coral symposium Amsterdam The Netherlands 1ndash6 April

Freiwald A Fossa J Grehan A Koslow T Roberts J 2004 Cold-water coral reefs out ofsight no longer out of mind Cambridge United Nations Environment ProgrammemdashWorld Conservation Monitoring Centre

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Friedrich T Timmermann A Abe-Ouchi N Bates M ChikamotoM Church J DoreD Gledhill M Gonzalez-Davila M Heinemann T Ilyina J Jungclaus E McLeodA Santana-Casiano JM 2012 Detecting regional anthropogenic trends in oceanacidification against natural Variability Natural Climate Change 2167ndash171DOI 101038nclimate1372

GehlenM Seacutefeacuterian R Jones DO Roy T Roth R Barry J Joos F 2014 Projected pHreductions by 2100 might put deep North Atlantic biodiversity at risk Biogeosciences116955ndash6967 DOI 105194bg-11-6955-2014

Georgian SE Dupont S KurmanM Butler A Stroumlmberg SM Larsson AI CordesEE 2016 Biogeographic variability in the physiological response of the cold-watercoral Lophelia pertusa to ocean acidificationMarine Ecology 37(6)1345ndash1359DOI 101111maec12373

Gibbin EM PutnamHM Davy SK Gates RD 2014 Intracellular pH and its response toCO2-driven seawater acidification in symbiotic versus non-symbiotic coral cells TheJournal of Experimental Biology 2171963ndash1969 DOI 101242jeb099549

Guinotte J Orr J Cairns S Freiwald A Morgan L George R 2006Will human-induced changes in seawater chemistry alter the distribution of deep-seascleractinian corals Frontiers in Ecology and the Environment 4141ndash146DOI 1018901540-9295(2006)004[0141WHCISC]20CO2

Hennige SJ Wicks LC Kamenos NA Bakker DCE Findlay HS Dumousseaud CRoberts JM 2014 Short-term metabolic and growth responses of the cold-watercoral Lophelia pertusa to ocean acidification Deep Sea Research Part II TopicalStudies in Oceanography 9927ndash35 DOI 101016jdsr2201307005

Hennige SJ Wicks LC Kamenos NA Perna G Findlay HS Roberts JM 2015Hiddenimpacts of ocean acidification to live and dead coral framework Proceedings of theRoyal Society B Biological Sciences 282(1813)1ndash10 DOI 101098rspb20150990

Henry LA Davies AJ Roberts JM 2010 Beta diversity of cold-water coral reef commu-nities off western Scotland Coral Reefs 29427ndash436 DOI 101007s00338-009-0577-6

Henry LA Navas JM Hennige SJ Wicks LC Vad J Roberts JM 2013 Cold-watercoral reef habitats benefit recreationally valuable sharks Biological Conservation16167ndash70 DOI 101016jbiocon201303002

Herfort L Thake B Taubner I 2008 Bicarbonate stimulation of calcification andphotosynthesis in two hermatypic corals Journal of Phycology 44(1)91ndash98DOI 101111j1529-8817200700445x

Hoegh-Guldberg O Mumby PJ Hooten AJ Steneck RS Greenfield P Gomez EHatziolos ME 2007 Coral reefs under rapid climate change and ocean acidificationScience 3181737ndash1742 DOI 101126science1152509

IPCC 2013 Climate change 2013 the physical science basis In Stocker TF Qin DPlattner GK Tignor M Allen SK Boschung J Midgley BM eds Contribution ofworking group I to the fifth assessment report of the intergovernmental panel on climatechange Cambridge Cambridge University Press DOI 101017CBO9781107415324

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Kroeker KJ Kordas RL Crim RN Singh GG 2010Meta-analysis reveals negativeyet variable effects of ocean acidification on marine organisms Ecology Letters13(11)1419ndash1434 DOI 101111j1461-0248201001518x

Kroeker KJ Kordas RL Crim R Singh GG 2013 Impacts of ocean acidification onmarine organismsquantifying sensitivities and interaction with warming GlobalChange Biology 191884ndash1896 DOI 101111gcb12179

Kvitt H Kramarsky-Winter E Maor-Landaw K Zandbank K Kushmaro A RosenfeldH Tchernov D 2015 Breakdown of coral colonial form under reduced pHconditions is initiated in polyps and mediated through apoptosis Proceedings ofthe National Academy of Sciences of the United States of America 1122082ndash2086DOI 101073pnas1419621112

Law CS Bell JJ Bostock HC Cornwall CE Cummings VJ Currie K Davy SK GammonM Hepburn CD Catriona LH LamareMMikaloff-Fletcher SE NelsonWAParsons DM Ragg NLC Sewell MA Smith AM Tracey DM 2018 Ocean acidifi-cation in New Zealand waters trends and impacts Journal of Marine and FreshwaterResearch 52(2)155ndash195 DOI 1010800028833020171374983

Maier C Bils F Weinbauer MGWatremez P PeckMA Gattuso JP 2013a Res-piration of Mediterranean cold-water corals is not affected by ocean acidifi-cation as projected for the end of the century Biogeosciences 105671ndash5680DOI 105194bg-10-5671-2013

Maier C Hegeman J Weinbauer MG Gattuso JP 2009 Calcification of the cold-watercoral Lophelia pertusa under ambient and reduced pH Biogeosciences 61671ndash1680DOI 105194bg-6-1671-2009

Maier C Popp P Sollfrank NWeinbauer MGWild C Gattuso JP 2016 Effects ofelevated pCO2 and feeding on net calcification and energy budget of the Mediter-ranean cold-water coral Madrepora oculata Journal of Experimental Biology Articlejeb-127159 DOI 101242jeb127159

Maier C Schubert A Berzunza-SagravenchezMMWeinbauer MGWatremez P GattusoJ-P 2013b End of the century pCO2 levels do not impact calcification in Mediter-ranean cold-water corals PLOS ONE 8(4)e2655 DOI 101371journalpone0062655

Maier CWatremez P Taviani MWeinbauer MG Gattuso JP 2012 Calcification ratesand the effect of ocean acidification on Mediterranean cold-water corals Proceedingsof the Royal Society B 279(1734)1716ndash1723 DOI 101098rspb20111763

Maier CWeinbauer MG Gattuso JP Fate of Mediterranean cold-water corals as aresult of global climate change A synthesis In Orejas C Jimeacutenez C edsMediter-ranean cold-water corals past present and future New York Springer In Press

Marubini F Ferrier-Pages C Furla P Allemand D 2008 Coral calcification respondsto seawater acidification a working hypothesis towards a physiological mechanismCoral Reefs 27(3)491ndash499 DOI 101007s00338-008-0375-6

Mehrbach C Culberson CH Hawley JE Pytkowicx RM 1973Measurement of theapparent dissociation constants of carbonic acid in seawater at atmospheric pressureLimnology and Oceanography 18(6)897ndash907 DOI 104319lo19731860897

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Mikaloff-Fletcher SE Bostock HCWilliamsM Forcen A 2017 Modelling the effectsof ocean acidification in New Zealand New Zealand aquatic environment andbiodiversity report 21 p

Milligan RJ Spence GJ Roberts JM Bailey DM 2016 Fish communities associated withcold-water corals vary with depth and substratum type Deep Sea Research Part I11443ndash54 DOI 101016jdsr201604011

Movilla J Orejas C Calvo E Gori A Loacutepez-Sanz Agrave Grinyoacute J Domiacutenguez-CarrioacuteC Pelejero C 2014 Differential response of two Mediterranean cold-water coralspecies to ocean acidification Coral Reefs 33675ndash686DOI 101007s00338-014-1159-9

Orr JC Fabry VJ Aumont O Bopp L Doney SC Feely RA Yool A 2005 Anthro-pogenic ocean acidification over the twenty-first century and its impact on calcifyingorganisms Nature 437681ndash686 DOI 101038nature04095

Parker SJ Bowden DA 2010 Identifying taxonomic groups vulnerable to bottomlongline fishing gear in the Ross Sea Region CCAMLR Science 17105ndash127

Purser A Orejas C Gori A Tong R Unnithan V Thomsen L 2013 Local vari-ation in the distribution of benthic megafauna species associated with cold-water coral reefs on the Norwegian margin Continental Shelf Research 5437ndash51DOI 101016jcsr201212013

Richardson AD Braswell BH Hollinger DY Jenkins JP Ollinger SV 2009 Nearsurface remote sensing of spatial and temporal variation in canopy phenologyEcological Applications 19(6)1417ndash1428 DOI 10189008-20221

Roberts JMWheeler AJ Freiwald A 2006 Reefs of the deep the biology and geology ofcold-water coral ecosystems Science 312(5773)543ndash547 DOI 101126science1119861

Schneider CA RasbandWS Eliceiri KW 2012 NIH Image to ImageJ 25 years of imageanalysis Nature Methods 9(7)671ndash675 DOI 101038nmeth2089

Soffker M Sloman KA Hall-Spencer JM 2011 In situ observations of fish associatedwith coral reefs off Ireland Deep Sea Research I 58818ndash825DOI 101016jdsr201106002

Sweetman AK Thurber AR Smith CR Levin LA Mora CWei CL Gooday AJ JonesDOB RexM Yasuhara M Ingels J Ruhl HA Frieder CA Danovaro RWuumlrzbergL Baco A Grupe BM Pasulka A Meyer KS Dunlop KM Henry L-A Roberts JM2017Major impacts of climate change on deep-sea benthic ecosystems ElementaScience of the Anthropocene 51ndash23 DOI 101525elementa203

Thresher RE Tilbrook BD Fallon S Wilson NC Adkins J 2011 Effects of chroniclow carbonate saturation levels on the distribution growth and skeletal chemistryof deep-sea corals and other seamount megabenthosMarine Ecology Progress Series44287ndash99 DOI 103354meps09400

Tracey D Bostock H Currie K Mikaloff-Fletcher S WilliamsM Hadfield M NeilH Guy C Cummings V 2013 The potential impact of ocean acidification ondeep-sea corals and fisheries habitat in New Zealand waters New Zealand aquaticenvironment and biodiversity Report No 117 p 101

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Tracey DM Rowden AA Mackay KA Compton T 2011Habitat-forming cold-watercorals show affinity for seamounts in the New Zealand regionMarine EcologyProgress Series 4301ndash22 DOI 103354meps09164

Turley CM Roberts JM Guinotte JM 2007 Corals in deep-water will the unseenhand of ocean acidification destroy cold-water ecosystems Coral Reefs 26445ndash448DOI 101007s00338-007-0247-5

Williams A Schlacher TA Rowden AA Althaus F ClarkMR Bowden DA StewartR Bax NJ Consalvey M Kloser RJ 2010 Seamount megabenthic assemblagesfail to recover from trawling impactsMarine Ecology 31(Suppl 1)183ndash199DOI 101111j1439-0485201000385x

Winters G Holzman R Blekhman A Beer S Loya Y 2009 Photographic assessmentof coral chlorophyll contents implications for ecophysiological studies and coralmonitoring Journal of Experimental Marine Biology and Ecology 38025ndash35DOI 101016jjembe200909004

Wolankski E HamnerWM 1998 Topographically controlled forces in the ocean andtheir biological influence Science 241177ndash181 DOI 101126science2414862177

Wood R 1999 Reef evolution Oxford Oxford University Press 165ndash198

Gammon et al (2018) PeerJ DOI 107717peerj5236 2424