Shaping up for stress: Physiological flexibility is key to survivorship in a habitat-forming macroalga

Accepted Manuscript Title: Shaping up for stress: Physiological flexibility is key to survivorship in a habitat-forming macroalga Authors: Jennifer S. Clark, Alistair G.B. Poore, Martina A. Doblin PII: DOI: Reference:

S0176-1617(18)30303-1 https://doi.org/10.1016/j.jplph.2018.10.005 JPLPH 52860

To appear in: Received date: Revised date: Accepted date:

15-6-2018 9-9-2018 4-10-2018

Please cite this article as: Clark JS, Poore AGB, Doblin MA, Shaping up for stress: Physiological flexibility is key to survivorship in a habitat-forming macroalga, Journal of Plant Physiology (2018), https://doi.org/10.1016/j.jplph.2018.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research article for consideration by Journal of Plant Physiology

Shaping up for stress: Physiological flexibility is key to survivorship in a habitat-forming macroalga

a

IP T

Jennifer S. Clarka,1, Alistair G.B. Pooreb, Martina A. Doblina

Climate Change Cluster (C3), University of Technology Sydney, P.O. Box 123 Broadway,

b

SC R

New South Wales 2007, Australia

Evolution and Ecology Research Centre, School of Biological Earth and Environmental

N

*Corresponding author: [email protected]

U

Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia

ED

M

A

Co-authors: [email protected], [email protected]

Abstract

PT

Organisms from all domains of life can have highly variable morphologies, with this plasticity suggested to increase fitness and survivability under stressful conditions. Predicting

CC E

how organisms will adapt to environmental change requires an understanding of how variable morphologies perform under environmental stress. Morphological plasticity has been documented within marine macroalgae inhabiting environmental gradients, however the

A

functional consequences of this variation has been rarely tested. In this study, form-function was assessed in the habitat-forming, intertidal macroalga Hormosira banksii. Morphological variation was quantified on two spatial scales (tidal gradient versus latitudinal gradient) and the performance tested (relative water content and photosynthetic efficiency) of morphological variants during heat and desiccation stress. At regional scales, individuals at the warm distributional edge were overall smaller in size, and had smaller vesicles (higher surface area to volume ratio; SA:VOL) than those from central populations. At local scales,

Research article for consideration by Journal of Plant Physiology

individuals high on the shore were generally shorter and had larger vesicles than those low on the shore. Vesicle morphology (SA:VOL) was found to predict relative water content and photosynthetic performance during desiccation and rehydration. Differences in SA:VOL of vesicles between heights on the shore may reflect water requirements needed to maintain tissue hydration for photosynthesis during low tide. Warm-edge populations showed increased thermal sensitivity as indicated by decreased photosynthetic yield of PSII and delays in recovery after desiccation. Sensitivities to higher temperatures amongst warm-edge

IP T

populations are potentially due to smaller fluctuations in regional temperatures as well as their morphology. This study provides a mechanistic understanding of the morphological

SC R

variation among H. banksii populations. It suggests that H. banksii has a high degree of

morphological plasticity reflecting local climate, topography and environmental conditions, with this morphological variation having functional consequences. Morphological variation across local and regional scales will be important for resilience of this species to future

N

U

climate warming.

A

Keywords: form-function, phenotypic plasticity, macroalgae; morphology; desiccation;

ED

Declaration of interest – none

M

photosynthetic efficiency; species distribution, Hormosira banksii.

PT

Introduction

Variation in morphological traits across environmental gradients has been documented in all

CC E

domains of life (Franzmann et al. 1988; Justice et al. 2008; Westoby et al. 2002) and has significant implications for organism function in both terrestrial and marine habitats (McLeod et al. 2015; Moles et al. 2007). Variation in body size, for example, is negatively correlated

A

with temperature (Chown and Gaston 2010), affecting metabolism and population growth rate. Intraspecific variation in morphology can arise through variation in selection among populations (Roberson and Coyer 2004), or through phenotypic plasticity, where a given genotype produces several different phenotypes as a response to their environment (Chapman 1995; Hopkins et al. 2008; Via and Lande 1985). For sessile organisms, moderating stress through morphological plasticity can enable them to maintain function and reproductive

Research article for consideration by Journal of Plant Physiology

output in unfavourable conditions, that can then translate to increased survivorship and affect larger-scale patterns in distribution (Helmuth et al. 2006). If changes to morphology can ameliorate stress, we would expect morphological variation along environmental gradients on small scales, with associated functional consequences for this variation. The same pattern is expected on larger scales, where the fitness of individuals commonly diminishes towards the distributional limits of a species as environmental

IP T

conditions and habitat become sub-optimal for growth and reproduction (Brown 1984). Organisms nearing their distributional limits have been shown to have reduced population

and individual sizes (Araújo et al. 2011; Zardi et al. 2015), but local selection pressures and

SC R

environmental conditions can result in species not conforming to these regional scale patterns (Sagarin and Gaines 2002). A growing body of work suggests that stress experienced at the scale of the individual organism may be more important in shaping an organism’s physiology

U

than large regional scale effects (Helmuth et al. 2010). Quantifying intraspecific variation in morphology at different spatial scales, and the relationship between morphology and

N

function, is thus needed to understand how organisms can inhabit highly variable

A

environments while maintaining physiological function.

M

As with higher plants on land, macroalgae in marine environments frequently display high levels of morphological variation along environmental gradients (Bell 1995; Kübler and

ED

Dudgeon 1996; Wernberg and Thomsen 2005). Macroalgal form is altered by a wide variety of abiotic factors including light levels (Monro and Poore 2009), water motion (Blanchette

PT

1997; Fowler-Walker et al. 2006), salinity (Falace and Bressan 2006) and depth (Roberson and Coyer 2004). This variation in morphology can then have important functional consequences. For example, in the subtidal kelp, Ecklonia radiata, wave exposure can have

CC E

an effect on the thickness of blades, which in turn affects drag resistance and the likelihood of dislodgement (Wernberg and Thomsen 2005). Intertidal macroalgae inhabit variable habitats where emersion, temperature and height on the

A

shore can all correlate with variation in algal morphology (Littler and Littler 1980). As macroalgae do not possess an active mechanism to control water loss during periods of emersion (e.g., stomates or roots to replenish evaporative water loss), morphology is fundamentally important in moderating tissue temperature and desiccation rate (Bell 1993, 1995). For example, forms with thick walls and low surface area to volume ratios reduce the rate of desiccation (Oates 1985; Bell 1993; Bell 1995). Highly branched forms can be more

Research article for consideration by Journal of Plant Physiology

effective at dissipating heat but are likely to have higher rates of desiccation (Kübler and Dudgeon 1996). Many of these observations have been linked to vertical position on the shore, suggesting that physiological function is related to desiccation and temperature tolerance during tidal cycles (Dudgeon et al. 1995; Skene 2004; Smith and Berry 1986; Williams and Dethier 2005). Intertidal macroalgae can fix between 30 and 86 % of their daily carbon while emersed (Madsen and Maberly 1990), and thus the effectiveness of carbon

IP T

fixation depends on the ability of tissues to remain hydrated. In this study, we test how the phenotype of the intertidal macroalga, Hormosira banksii (Turner) Descaisne varies with the regional and local-scale environment, and the

SC R

physiological consequences of that morphological variation. Macroalgae inhabiting areas of high temperature and desiccation stress, such as those high on the shore and close to the

equatorward limits of their distribution (warm-edge), are suggested to have a higher tolerance

U

for these stresses but exhibit retarded growth and stunted morphologies in contrast to individuals living lower on the shore or in more benign climates (Hawkins and Hartnoll 1985;

N

Stengel and Dring 1997). H. banksii is the most broadly distributed brown macroalga in

A

Australasia, with the edge of its equatorward distributional limit located at a cold-warm

M

transition zone (~28 °S). It often forms monospecific stands on wave-swept shores, occupying the entire mid to high intertidal area, having similar ecological function as species

ED

of Fucus in North America and Europe. H. banksii provides habitat, resources and modifies local environmental conditions for other organisms, enhancing biodiversity (Lilley and Schiel 2006; Schiel 2006). H. banksii ecotypes with different morphology inhabit different

PT

ecosystems from exposed rocky shores to estuaries (Macinnis-Ng et al. 2005; Mueller et al. 2015; Ralph et al. 1998) but there is little mechanistic understanding of how this variation in

CC E

morphology influences physiology. Here, we examine whether phenotypic variation of H. banksii across two spatial scales (high vs low on the shore and centre vs margin of its distribution) influences performance (photophysiology and relative water content) under thermal and desiccation stress. We predict that individuals found at the warm-edge and high

A

on the shore will have stunted morphologies and increased tolerance to stressful temperatures, compared to individuals found in more central locations and low on the shore.

Materials and methods Sample collection and study sites

Research article for consideration by Journal of Plant Physiology

H. banksii was sampled at four locations: two in the warm-edge of its distribution, Angourie (29º28’41.51” S, 153º 21’ 49.53” E) and Minnie Water (29°46’34.23” S, 153°18’07.43” E) and two in the temperate centre (central), Pearl Beach (33°32’57.70” S, 151°18’32.36” E) and Bilgola Beach (33°38’54.48” S, 151°19’39.59” E). These locations were on exposed headlands comprising rocky reef substrates where H. banksii was the dominant intertidal macroalga with percent cover of approximately 20%, 60%, 50%, and 60% respectively. Rockpool habitats were not sampled, and consequently individuals at the northern-most

IP T

location (warm edge) of the current H. banksii distribution (Flat Rock, NSW; 28º 50’ 32.79” S, 153º 36’ 29.47” E) were not used. Thalli were haphazardly collected from both high and

SC R

low on the shore two hours before maximum low tide and kept overnight at 4 ºC before analyses. Thallus and vesicle morphology

U

Each thallus (n = 10) was photographed using a digital camera to conduct morphological

N

measurements using imaging software (Image J ver. 1.48v, National Institutes of Health,

A

USA. The morphological traits measured from each thallus were length of the longest frond (excluding holdfast), number of branches (dissection) and number of vesicles. From the mid-

M

branch region of each thallus, six vesicles were chosen randomly, and their length, diameter and wall thickness measured using digital callipers (Mitutoyo Corp., Model No. CD-6”C,

ED

Japan). Total vesicle surface area, total volume, cavity volume and surface area to volume ratio (SA:VOL) were then calculated based on methods described by Ralph et al. (1998) and

PT

Macinnis-Ng et al. (2005). Entire thalli were then dried at 60 ºC for 48 h and weighed on a digital balance (NewClassic MF ML3002/01, Mettler Toledo, Switzerland).

CC E

Relative water content and photosynthetic efficiency during desiccation and re-immersion Prior to experimentation, thalli of equal length were allowed to fully hydrate in filtered 20 ºC seawater for 30 min, then blotted with tissue and weighed. After a 20 min period of dark

A

adaptation, the initial maximum quantum yield of PSII (FV/FM) was determined by applying a saturating pulse of blue light (2000 µmol photons m-2 s-1) using a pulse amplitude modulated (PAM) fluorometer (Diving-PAM, Walz, Germany). Six thalli from each height on the shore at each location were then exposed to one of three treatments. The first two treatments consisted of thalli desiccated at 20 ºC or 30 ºC under 400 µmol photons m-2 s-1 for 4 h. The third treatment was a control where thalli were submerged in filtered (0.7 µm Whatman) 20 ºC seawater under constant light exposure (400 µmol photons m-2 s-1). After the 4 h

Research article for consideration by Journal of Plant Physiology

desiccation period, thalli that were in desiccation treatments were placed in filtered 20 ºC seawater in low light (~10 µmol photons m-2 s-1) for 2 h. Thalli from all treatments were then allowed to recover a further 12 h in low light after the 2 h initial recovery (14 h total recovery). Desiccation treatments commenced when lights were turned on and thalli were placed on desiccation trays that consisted of an aluminium tray placed over a heat plate, with each tray

IP T

having metal grates to aid in maintaining air flow beneath thalli, and a mesh to ensure thalli were protected from burning. Thalli were illuminated using LED lights (custom-made LED light with a cool white spectral output) at 400 μmol photons m-2 s-1 until steady state was

SC R

reached (approx. 20 min). Experimental light intensity was determined in a pilot study (see light curves in Fig. S3) and designed to induce photoinhibition. To understand the

photosynthetic function and desiccation of H. banksii under simulated tidal emersion,

U

effective quantum yield of PSII (∆F/FM’; Y(PSII)), regulated and nonregulated

nonphotochemical quenching (Y(NPQ) and Y(NO) respectively) as well as thallus mass was

N

measured every 30 min during the desiccation period. Regulated nonphotochemical

A

quenching of light energy (Y(NPQ)) is a proxy for xanthophyll pigment cycling, which

M

responds to protect photosystems from over-excitation by dissipating excess irradiance as heat. Nonregulated nonphotochemical quenching (Y(NO)) is also an energy dissipation

ED

pathway, so any changes to the PSII antenna complex or PSII damage or thylakoid impacts will increase Y(NO). Total energy is conserved whereby Y(PSII) + Y(NPQ) + Y(NO) =1, therefore if Y(NPQ) increases relative to Y(NO) and Y(PSII) then photoprotection is

PT

occurring, however if Y(NO) increases at the cost of Y(PSII) and Y(NPQ), this suggests photoinhibition or irreversible damage to the photosynthetic apparatus (Klughammer and

CC E

Schreiber 2008). After 4 h of desiccation, recovery of the photosynthetic health of reimmersed thalli was assessed by placing thalli in the dark and measuring the maximum quantum yield (FV/FM) every 30 min for 2 h and then again 12 h later. Wet weights were recorded to the nearest 0.01 g every 30 min, and at the end of the experiment, thalli were

A

dried for 48 h at 60 °C and reweighed. Relative water content (RWC) was calculated at each time using the formula: Relative water content=

𝐷𝑒𝑠𝑖𝑐𝑐𝑎𝑡𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡−𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 𝑓𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡−𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡

× 100 %

Research article for consideration by Journal of Plant Physiology

Statistical analyses All morphological traits were contrasted among locations and heights on the shore with analysis of variance (ANOVA). Thallus traits were analysed using a two-factor mixed model design with location (random, four levels) and height on the shore (fixed, two levels). Vesicle traits were analysed using a three-factor mixed model with location (random, four levels), height on the shore (fixed, two levels) and individual (random, nested within location and

IP T

height, 10 levels) as factors. For both sets of analyses, planned comparisons among the four locations were used to contrast the two locations at the range edge with the two locations in

SC R

the range centre.

The variation in morphology of thalli among sites and heights on the shore was visualised using principle components ordination (PCO). Given that the parameters were measured on different scales, morphological traits were normalised before the similarity matrix using

U

Euclidean distance was constructed using PRIMER-E (v.6, Plymouth, UK). Draftsman plots

N

were used to visualise correlations among traits and showed that vesicle volume and surface

A

area were highly correlated with SA:VOL and thus only SA:VOL was used in the PCO.

M

Photosynthetic yields Y(PSII) and relative water content were contrasted between 20 °C and 30 °C using ANOVAs with location (random, four levels), height on the shore (fixed, two

ED

levels) and temperature (fixed, two levels) as factors. Separate analyses were conducted for these traits at each of six separate time points: initial (before the treatments started), after 1 h of desiccation, at the end of the desiccation period at 4 h, initial recovery after 30 min

PT

submerged, after 2 h of recovery and after 14 h of recovery. Univariate ANOVAs were conducted in the PERMANOVA+ routine of PRIMER-E (v.6), with probabilities calculated

CC E

by permutation (999) using Monte Carlo p-values. Predicting function from morphology To identify which morphological traits best predicted physiological function, a model

A

selection process was conducted (DISTLM; PRIMER-E, Plymouth, U.K.). Vesicle-level morphometrics were used as predictor variables to determine whether they regulated each of the response variables: photosynthetic efficiency of PSII and relative water content. Two critical time points were chosen after the completion of the experiment to test form-function: at the end of the desiccation treatment (4 h) and after thallus re-immersion (30 min recovery), as these demonstrated the ability of the vesicle to retain and recover moisture. Vesicle

Research article for consideration by Journal of Plant Physiology

morphometrics from each thallus were averaged to ensure data matrices matched for the DISTLM. The DISTLM was performed separately for each response variable to determine the model that best fit the variables and identified what variable or combination of variables best characterised the functional traits. Models were ranked using the Akaike Information Criterion (AIC). This was performed on morphometric and functional traits for the 20 °C treatment only, as most vesicles in the 30 °C treatment were not functional at the end of the

IP T

desiccation treatment.

SC R

Results Thallus and vesicle morphology

The morphology of H. banksii was highly variable between regions, among locations and

U

with height on the shore, based on analysis of nine morphometric parameters of vesicles and thalli. H. banksii thalli varied in form from having several long fronds with little branching

A

N

and large vesicles, to having many short, branched fronds with small vesicles (Fig. 1). Principle components ordination revealed that differences in thallus morphology between

M

tidal heights were correlated with vesicle morphometrics, primarily SA:VOL (Fig. 2a). The thallus traits of length, biomass and number of branches did not significantly differ between

ED

heights on the shore, however total length varied among locations (Fig. S1, Table S1). Vesicles found high on the shore had larger volumes and surface areas and smaller SA:VOL

PT

than those found low on the shore (Fig. S2, Table S2). Cavity volume and wall thickness differed between heights on the shore for the central populations (Fig. S2, height x region

CC E

interaction, Table S2).

Differences in morphology among locations were largely attributed to thallus level morphometrics (biomass, total length, branching and total number of vesicles) (Fig. 2b). Significant differences among locations and regions were found for thallus level

A

morphometrics (Table S1), however there was no consistent pattern of decreasing individual size with decreasing latitude (Fig. S1). The northern-most population sampled, Angourie, exhibited the smallest thalli (Fig. S1), while thalli from Minnie Water, the second marginal warm-edge population, were the largest among all locations (Fig. S1). The magnitude of the differences between thalli high and low on the shore varied among regions for all vesicle

Research article for consideration by Journal of Plant Physiology

traits, with region x height interactions only found for thallus biomass and total number of vesicles (Fig. S1, S2, Table S1, S2). Relative water content and photosynthetic efficiency during desiccation and re-immersion Overall, temperature affected the capacity of different morphologies of H. banksii to retain water during desiccation (Table S4). After the initial hour of desiccation in the 20 °C treatment, RWC of thalli varied among locations and was greater in high shore thalli than

IP T

those low on the shore (Fig. 5). In contrast, thalli exposed to 30 °C responded similarly

between heights on the shore (Fig. 5, Table 4). At the end of the desiccation period (4 h), the

SC R

effects of temperature on RWC varied among locations (location x temperature interaction, Fig. 3, 5, Table S4). All thalli exposed to 30 °C had less than 20% of their initial water content remaining, while thalli exposed to 20 °C had RWC ranging from 20–40%, which

varied among locations. The effects of temperature on RWC, however, did not differ between

U

thalli collected at different heights on the shore at the end of the desiccation period (Table

N

S4).

A

Increased temperature during desiccation reduced the ability of thalli to rehydrate during the

M

recovery period, with the magnitude of this effect varying among locations and between heights on the shore (Fig 3, Fig. 6, Table S4). In the first 30 min of recovery, RWC varied

ED

between tidal heights and among locations, with thalli recovering 25 to 75% of their RWC once re-immersed (Fig 3, 5). Significant interactions between temperature and location were present for the remainder of the recovery period, suggesting that morphologies varied in their

PT

response to temperature (Fig 5, Table S4). At the end of the recovery period (14 h), all thalli desiccated at 20 °C were able to recover ~30 % more water content than thalli desiccated at

CC E

30 °C (Fig. 5). In addition, warm-edge populations showed greater recovery of RWC at 20 °C than central populations. Conversely, thalli from central populations desiccated at 30 °C could recover more RWC than the warm-edge population thalli (Fig. 5). Amongst different tidal heights, thalli found high on the shore exposed to 20 °C were able to recover 20 % more

A

RWC at the end of the recovery period (14 h) than thalli found low on the shore (height x temperature interaction, Table S3), while thalli exposed to 30 °C showed no difference in RWC that was related to shore height (Table S3). Thalli from different regions, locations and heights on the shore varied in their photosynthetic efficiency of PSII in response to desiccation (Fig. 6, 5). Initially, FV/FM was lower in fully hydrated warm-edge populations and amongst low shore thalli (Fig. 7, Fig. 6, Table S5).

Research article for consideration by Journal of Plant Physiology

Once lights were turned on and desiccation commenced, effective quantum yield Y(PSII) was significantly reduced (60-70 % of initial FV/FM) within 1 h, compared to the submerged controls which were reduced by only ~50% (Fig. 6, Table S5). There were no interactions between temperature and locations or height on the shore. By the end of the desiccation period (4 h), initial FV/FM had declined by 95%, with Y(PSII) of thalli from different heights on the shore varying amongst locations (location x height interaction; Fig. 4, Fig. 6, Table S5). Regulated and nonregulated photochemical quenching (Y(NPQ) and Y(NO)) of thalli

IP T

from different locations and heights on the shore responded similarly within each treatment

during desiccation (Fig. S4 and S5). Y(NPQ) steadily increased during the desiccation period

SC R

with thalli in the 20 and 30 C treatments having greater Y(NPQ) than controls (Fig. S4).

Y(NO) showed similar responses between treatments during desiccation except for low shore thalli from Angourie in the 30 C treatment which showed an increase in Y(NO) towards the

U

end of the desiccation period.

During rehydration, the effect of temperature on the ability of thalli to recover photosynthetic

N

efficiency varied between warm-edge and central populations (region x temperature

A

interaction, Table S5). Within the first 30 min of thalli being re-immersed, central

M

populations (Bilgola Beach and Pearl Beach) regained a higher percentage of photosynthetic efficiency (~50%) than warm-edge populations (Angourie and Minnie Water (Fig. 4, 6)).

ED

Warm-edge populations showed photoinhibition and a delay in the recovery of photosynthetic efficiency. Angourie, the northern-most population sampled, showed a reduced capacity to recover from desiccation stress and lost 60 and 70% of initial FV/FM at 20

PT

and 30 °C, respectively, while thalli from central populations and Minnie Water (warm-edge population) regained much of their photosynthetic function at 20 °C but not 30 °C (location x

CC E

height interaction, Fig. 4b, 6, Table S5). In the 30 °C treatment, less than 10 % of initial FV/FM was recovered, with high shore thalli generally unable to recover under increased temperature (Fig. 4a, Table S5). Y(NPQ) of thalli from different treatments showed variable degrees of recovery (Fig. S4). Thalli in the 30 C treatment from all locations and heights on

A

the shore demonstrated the greatest Y(NPQ) followed by thalli from 20 C and finally the controls showed the lowest level of Y(NPQ) during recovery (Fig. S4). Angourie thalli from both temperature treatments had elevated levels of Y(NPQ) compared to other locations. Thalli from all locations, heights on the shore and treatments had similar Y(NO) during recovery except for low shore thalli from Angourie which showed highest Y(NO) (Fig. S5).

Research article for consideration by Journal of Plant Physiology

Across all locations, thalli collected from high on the shore desiccated at 20 °C recovered a greater proportion of FV /FM (~20% more) than low shore thalli after 2 h of re-immersion (Fig. 6). At the end of the recovery period (14 h), desiccated thalli showed irreversible damage to photosystems across all locations, heights on the shore and temperatures, with an additional 10% reduction of FV/FM, whereas controls did not show photodamage (Fig. 7). All thalli showed 80-95% loss of initial FV/FM when desiccated at 30 °C (Fig. 7). The controls that remained immersed throughout the experiment showed similar photosynthetic efficiency

IP T

despite origin of location or height on the shore (Fig. 6).

SC R

Predicting function from morphology

Vesicle morphometrics were found to be significant predictors for RWC and photosynthetic efficiency of PSII at the two functionally important time points: end of the desiccation treatment (4 h) and initial recovery (30 min after thalli reimmersed). Surface area to volume

U

ratio (SA:VOL) was the most significant predictor variable for both time points, fitting 73%

N

of the overall variation in RWC for desiccation and 75% for recovery (Table S6). SA:VOL

A

ratio also predicted up to 70% of the overall variation in photosynthetic efficiency during desiccation, however no vesicle morphometrics were significant for recovery (Table S6).

M

Overall best solutions of the model indicated that SA:VOL ratio and cavity volume were the best predictors for recovery of RWC, with the lowest AIC values of 23.67 and highest

ED

proportion of the variation explained (R2 = 0.748) suggesting that vesicle morphology

Discussion

PT

strongly influences RWC in the H. banksii populations sampled (Table S6).

CC E

The morphology of the abundant intertidal macroalga, Hormosira banksii, varied strongly with height on the shore, and among regions, with this intraspecific variation linked to physiological performance. Under desiccation and temperature stress, up to ~70% of the overall variation in relative water content and photosynthetic efficiency could be attributed to

A

differences in morphology. Determining how morphological plasticity affects physiological functioning of species under different environmental conditions is critical for understanding species distributions now and into the future. Small scale variation in algal morphology and physiology

Research article for consideration by Journal of Plant Physiology

Morphological variation between algae at different tidal heights and variable responses to thermal and desiccation stress suggests that local environmental conditions play an important role in morphological variation, and consequently physiology and distribution (Ferreira et al. 2014; Zardi et al. 2013). In this study, the importance of the observed morphological variation in H. banksii from different heights on the shore was demonstrated as distinct differences in relative water content during desiccation and recovery. Retaining water during low tide is essential for photosynthesis and over the longer term, important for growth,

IP T

reproduction and survivorship. While the laboratory results suggest thalli are at risk of

dysfunction during low tide, mortality is generally not observed in the field, suggesting that

SC R

ameliorating factors such as local weather (e.g., high frequency variation in cloud cover and wind speed) can modify air temperatures, while humidity, wave splash, and irradiance can alter the rate of thermal and desiccation stress amongst individuals (Helmuth et al. 2006,

U

Martínez et al. 2012, Viejo et al. 2014).

The SA:VOL was the best predictor of relative water content and photophysiological

N

performance, consistent with other macroalgae (Dromgoole 1980). The ability of macroalgae

A

to tolerate desiccation is related to retaining moisture within the molecular environment

M

around the photosynthetic apparatus, which prevents membrane breakage (Kawamitsu et al. 2000), therefore photosynthesis can still occur as long as tissues remain moist. Saccate

ED

macroalgae such as H. banksii have internal reservoirs of water within each vesicle that can replenish water loss within the tissue during desiccation (Oates 1985; Osborn 1948). As H. banksii thalli from high on the shore are emersed for longer and experience higher

PT

temperatures than low shore thalli (Clark 2016), vesicles require more water to maintain physiological function during low tide. In other macroalgae, the rate of desiccation is

CC E

dependent on the thickness of thalli (Bell 1995). As there was no difference in wall thickness of H. banksii vesicles collected from different heights on the shore, physiological differences likely reflect morphological differences in vesicle water holding capacity, rather than

A

differences in the rate of water transfer through vesicle walls. The ‘critical water content’ threshold describes the desiccation tolerance of a species, where algae can photosynthetically recover once re-immersed in water (Lüning et al 1990), with desiccation beyond this point causing irreversible damage to photosynthesis (i.e. photoinhibition; Dring and Brown 1982). Thalli of H. banksii in the 30 °C treatment that reached < 20% RWC at the end of the desiccation period had a diminished photosynthetic efficiency of PSII and demonstrated a lower capacity to recover Y(PSII), while those exposed

Research article for consideration by Journal of Plant Physiology

to the 20 °C treatment, maintained RWC above 20% and were able to recover a greater Y(PSII). Additionally, thalli exposed to 30 C had greater levels of nonregulated nonphotochemical quenching (Y(NO)), suggesting photoinhibition.

Macroalgae vary in their critical water contents which may relate to their position on the shore. High shore species, such as Fucus spiralis, have a high tolerance to desiccation and

IP T

can recover after losing 80-90% of their original RWC (Dring and Brown 1982). In contrast, the subtidal Mastocarpus papillatus has moderate desiccation tolerance (> 20% RWC)

needed for recovery (Bell 1993), whereas the subtidal Durvillaea willana is highly sensitive

SC R

to desiccation, failing to recover after only a 10% loss of initial RWC (Brown 1987). The

critical water content for H. banksii (20%) would suggest that it has a moderate tolerance to desiccation stress. However, the ability to recover also depends on the temperature it is subjected to. Increased temperatures can cause a delay in the time it takes for full recovery in

U

other macroalgae (Bell 1993). Temperatures that exceed upper thresholds of thermal

N

tolerance can cause breakdown of proteins and temperature sensitive enzymes needed for

A

photosynthesis, and consequently require reallocation of resources for repair instead of use in growth and reproduction (Allakhverdiev et al. 2008). This may explain the slow recovery of

M

photosynthetic efficiency and RWC after desiccation at 20 °C and the lack of recovery after exposure to 30 °C. While there was a difference in RWC of thalli from different tidal heights,

ED

there was no interaction between tidal height and temperature for photosynthetic efficiency, suggesting that the morphological phenotypes of H. banksii analysed in this study may be

PT

highly adapted to their local environment, otherwise they would not be present if they are unable to mitigate stress. The morphological differences that enable the broadly distributed

CC E

H. banksii to retain RWC and maintain photophysiology during low tide are likely an important determinant for survival and distribution at different tidal heights and latitudes.

A

Large scale variation in algal morphology and physiology

On larger spatial scales, there was a decreased tolerance to high thermal and desiccation stress in warm-edge populations compared to central populations, which contrasts with previous studies of macroalgae (Ferreira et al. 2014; Saada et al. 2016) and intertidal invertebrates (Jaramillo et al. 2017; Somero 2010). Regional clines in temperature are often predicted to result in a decrease in abundance and individual size towards the peripheral

Research article for consideration by Journal of Plant Physiology

edges of species distributions, which can adversely affect water holding capacity (Viejo et al. 2011; Zardi et al. 2015). Empirical data does not always support these predictions with local effects of abiotic factors, disturbance and gene flow overriding large scale effects (as reviewed in Sagarin and Gaines 2002; Eckert et al. 2008; Zardi et al. 2015). For example, distributional edge populations of Fucus serratus were less resilient to heat and desiccation stress due to reduced fitness and lower adaptive capacity (Pearson et al. 2009). In this study, there was no clear trend in the size of H. banksii sampled from the warm-edge to the central

IP T

populations. Population size (> 60% percent cover) and individual thallus biomass was

greatest in one of the warm-edge populations (Minnie Water, 33 km south of the northern-

SC R

most population sampled, Angourie, which nears the distributional edge). The Angourie

population demonstrated the smallest individual size, percent cover and vesicle size of all populations sampled, and this was reflected in the inability to recover from desiccation and heat stress. Our findings therefore contrast with other studies where individual size of

U

macroalgae declined towards distributional edges, attributed to both unfavourable abiotic

N

conditions and life history traits (Araújo et al. 2011; Zardi et al. 2015).

A

The timing of daily tidal regimes may contribute to differences in morphology amongst

M

locations and regions (Helmuth et al. 2002; Mislan et al. 2009; Mueller et al. 2015; Pearson et al. 2009). For example, in studies with an intertidal mussel, tidal regime was found to

ED

differ among latitudes where low tides occurred in the middle of the day in equatorward locations compared to central populations where low tides occurred at night, potentially reducing the risk of high temperature and desiccation stress (Helmuth et al. 2006; Helmuth et

PT

al. 2002). If daily tidal lows occur in the middle of the day, it may create ‘risky days’ in which thermal and desiccation stress are intensified, impacting photosynthesis and growth,

CC E

resulting in morphological variation (Helmuth et al. 2011; Mislan et al. 2009). Tidal regime has previously been found to correlate with morphological differences amongst Tasmanian and New Zealand populations of H. banksii (Bergquist 1959; Mueller et al. 2015). In this study, regions sampled are approximately 500 km apart and experience similar tidal ranges

A

and timing (Bureau of Meteorology 2017) suggesting that other local scale environmental factors likely contribute to the morphological variation found in this study. Local topography and aspect also play an important role in shaping morphology and physiology through buffering the full extent of regional climate (Helmuth et al. 2010). For instance, the warm range-edge site, Minnie Water, is characterised by large boulders where H. banksii inhabits areas between the boulders where water is trapped at low tide. Stress

Research article for consideration by Journal of Plant Physiology

otherwise experienced by thalli on flat topography could be reduced, with thalli at this location photosynthesising for longer than thalli sampled at other locations. In tropical rainforests, boulder fields were similarly found to ameliorate extreme heat stress lowering temperatures by 10 °C and creating more stable conditions (Shoo et al. 2010). Additionally, thalli with greatest branching occurred more often in warm-edge populations and amongst low shore heights compared to central populations and high shore heights. Overlapping branches are important in retaining moisture between thalli, and previous studies have found

IP T

that thalli found in aggregates prolong hydration (Bell 1992). Populations with higher percent cover and greater branching dissections would therefore be able to retain moisture between

SC R

thalli and thus photosynthesise for longer, compared to those with stunted morphologies (Bell 1993). Implications for a warming ocean

U

The effects of variation in morphology on the functional traits of H. banksii at different

N

spatial scales not only have important implications on the biology and distribution of this

A

species but are also important in providing resilience of intertidal communities to climate warming. The warm-edge population was more thermally sensitive to increases in

M

temperature than other populations, which is opposite to what was expected. Temperature variability in equatorward populations is also more stable, compared to more central or

ED

temperate populations which experience a greater fluctuation in temperatures annually (Clark 2016). With temperatures within the eastern Australian region predicted to exceed the global

PT

average, it is expected that warm-edge populations may be vulnerable to future warming (Lough and Hobday 2011). However, with the projected increase in average sea temperatures of 2-3 °C, this may not necessarily cause mortality to central populations as these species are

CC E

exposed to a wide range of daily environmental gradients and extreme temperatures, particularly in summer months. Rather, the increase in average temperatures may cause physiological effects such as damage to the photosynthetic apparatus that could have

A

downstream effects in resource allocation leading to stunted growth, decreases in fecundity and declines in long-term recruitment success. The sensitivity to higher temperatures and lack of buffering in warm-edge populations, however, may make them susceptible to discrete climate events such as heat waves (Bennett et al. 2015; Wernberg et al. 2010), however it should be noted that central populations can also be as susceptible to heat waves if temperatures exceed anomalous thresholds (Bennett et al. 2015). If temperatures surpass lethal temperatures during prolonged thermal excursions, we may see declines and

Research article for consideration by Journal of Plant Physiology

subsequent shifts in populations if species fail to recover from these events (Smale and Wernberg 2013). Further experiments are required to establish whether the morphological variation observed in this study results from underlying genotypic differences among populations, or purely phenotypic responses to local environmental conditions. If these morphological differences reflect the underlying genotype and lead to increased fitness, then factors that affect the

IP T

likelihood of local adaptation to environmental conditions (e.g., heritable variation in stress tolerance, Clark et al. 2013) will play an important role in the persistence of macroalgal

populations in the face of global climate change. In conclusion, this study found regional and

SC R

local differences in morphology that relate to the physiological function of H. banksii to

retain water for photosynthesis during desiccation and recovery and provides a mechanistic understanding of how morphological variation contributes to differences in function at

N

U

different spatial scales.

M

A

Author Statement for Research Article JPLPH_2018_157 for Journal of Plant Physiology

ED

CRediT author statement

CC E

PT

Jennifer Clark: Conceptualization, Methodology, Formal Analysis, Investigation, Data Curation, Writing – Original Draft, Visualization, Project Administration Alistair Poore: Supervision, Validation, Formal Analysis, Writing-Review & Editing Martina Doblin: Supervision, Resources, Formal Analysis, Validation, Writing- Reviewing & Editing, Funding Acquisition

A

The authors state that this research article is original work and all work/words of others have been cited.

The authors state that there are no conflicts of interest.

Acknowledgements We would like to thank Caitlin Lawson, Daniel Lewis and Charlotte Robinson for assistance with sample collection and morphometric analyses, and Kevin Davies for contributing to

Research article for consideration by Journal of Plant Physiology

Figure 1. This research was supported by the University of Technology Sydney Climate Change Cluster (C3), an Australian Postgraduate Award to JC and an Australian Research Council Discovery Project (DP14010134) awarded to MD. Samples were collected under the

A

CC E

PT

ED

M

A

N

U

SC R

IP T

Department of the Environment and Heritage Permit number P10/0057-2.0.

Research article for consideration by Journal of Plant Physiology

References Allakhverdiev, S.I., Kreslavski, V.D., Klimov, V.V., Los, D.A., Carpentier, R. and Mohanty, P. 2008, 'Heat stress: An overview of molecular responses in photosynthesis', Photosynthesis Research, vol. 98, no. 1-3, pp. 541-50. Araújo, R., Serrão, E.A., Sousa-Pinto, I. and Åberg, P. 2011, 'Phenotypic differentiation at southern limit borders: The case study of two fucoid macroalgal species with different

IP T

life-history traits', Journal of Phycology, vol. 47, no. 3, pp. 451-62. Bell, E. 1995, 'Environmental and morphological influences on thallus temperature and desiccation of the intertidal alga Mastocarpus papillatus Kützing', Journal of

SC R

Experimental Marine Biology and Ecology, vol. 191, no. 1, pp. 29-55.

Bell, E.C. 1993, 'Photosynthetic response to temperature and desiccation of the intertidal alga Mastocarpus papillatus', Marine Biology, vol. 117, no. 2, pp. 337-46.

U

Bennett, S., Wernberg, T., Joy, B.A., De Bettignies, T. and Campbell, A.H., 2015. Central and rear-edge populations can be equally vulnerable to warming. Nature

A

N

Communications, 6, p.10280.

Bergquist, P. 1959, 'A statistical approach to the ecology of Hormosira banksii', Botanica

M

Marina, vol. 1, no. 1-2, pp. 22-53.

ED

Blanchette, C.A. 1997, 'Size and survival of intertidal plants in response to wave action: A case study with Fucus gardneri', Ecology, vol. 75, no. 5, pp. 1563-78. Brown, J.H. 1984, 'On the relationship between abundance and distribution of species', The

PT

American Naturalist, vol. 124, no. 2, pp. 255-79. Chapman, A.R.O. 1995, 'Functional ecology of fucoid algae: Twenty-three years of progress',

CC E

Phycologia, vol. 34, no. 1, pp. 1-32. Chown, S.L. and Gaston, K.J. 2010, 'Body size variation in insects: A macroecological perspective', Biological Reviews, vol. 85, no. 1, pp. 139-69.

A

Clark, J.S. 2016, ‘Assessing the vulnerability of habitat-formning macroalga to climate warming: Roles of physiology, ecology and evolutionary processes in determining resilience`, PhD thesis, University of Technology Sydney, Sydney, Australia.

Dring, M. and Brown, F. 1982, 'Photosynthesis of intertidal brown algae during and after periods of emersion: A renewed search for physiological causes of zonation', Marine Ecology Progress Series, vol. 8, pp. 301-8.

Research article for consideration by Journal of Plant Physiology

Dromgoole, F. 1980, 'Desiccation resistance of intertidal and subtidal algae', Botanica Marina, vol. 23, no. 3, pp. 149-60. Dudgeon, S.R., Kubler, J., Vadas, R. and Davison, I.R. 1995, 'Physiological responses to environmental variation in intertidal red algae: Does thallus morphology matter?', Marine Ecology Progress Series, vol. 117, pp. 193- 206. Eckert, C.G., Samis, K.E. and Lougheed, S.C. 2008, 'Genetic variation across species’

Ecology, vol. 17, no. 5, pp. 1170-88.

IP T

geographical ranges: The central–marginal hypothesis and beyond', Molecular

Falace, A. and Bressan, G. 2006, 'Seasonal variations of Cystoseira barbata (Stackhouse) C.

SC R

Agardh frond architecture', Hydrobiologia, vol. 555, pp. 193-206.

Ferreira, J.G., Arenas, F., Martínez, B., Hawkins, S.J. and Jenkins, S.R. 2014, 'Physiological response of fucoid algae to environmental stress: Comparing range centre and southern populations', New Phytologist, vol. 202, no. 4, pp. 1157-72.

U

Fowler-Walker, M.J., Wernberg, T. and Connell, S.D. 2006, 'Differences in kelp morphology

N

between wave sheltered and exposed localities: Morphologically plastic or fixed

A

traits?', Marine Biology, vol. 148, no. 4, pp. 755-67.

Franzmann, P., Stackebrandt, E., Sanderson, K. et al. 1988, 'Halobacterium lacusprofundi sp.

M

Nov., a halophilic bacterium isolated from deep lake, Antarctica', Systematic and Applied Microbiology, vol. 11, no. 1, pp. 20-7.

ED

Hawkins, S. and Hartnoll, R. 1985, 'Factors determining the upper limits of intertidal canopyforming algae', Marine Ecology Progress Series, vol. 20, no. 3, pp. 265-71.

PT

Helmuth, B., Broitman, B.R., Blanchette, C.A., Gilman, S., Halpin, P., Harley, C.D.G., O’Donnell, M.J., Hofmann, G.E., Menge, B. and Strickland, D. 2006, 'Mosaic patterns of thermal stress in the rocky intertidal zone: Implications for climate

CC E

change', Ecological Monographs, vol. 76, no. 4, pp. 461-79.

Helmuth, B., Broitman, B.R., Yamane, L., Gilman, S.E., Mach, K., Mislan, K.A.S. and Denny, M.W. 2010, 'Organismal climatology: Analyzing environmental variability at

A

scales relevant to physiological stress', Journal of Experimental Biology, vol. 213, no. 6, pp. 995-1003.

Helmuth, B., Harley, C.D.G., Halpin, P.M., O'Donnell, M., Hofmann, G.E. and Blanchette, C.A. 2002, 'Climate change and latitudinal patterns of intertidal thermal stress', Science, vol. 298, no. 5595, pp. 1015-7. Helmuth, B., Yamane, L., Lalwani, S., Matzelle, A., Tockstein, A. and Gao, N. 2011, 'Hidden signals of climate change in intertidal ecosystems: What (not) to expect when you are

Research article for consideration by Journal of Plant Physiology

expecting', Journal of Experimental Marine Biology and Ecology, vol. 400, no. 1-2, pp. 191-9. Hopkins, R., Schmitt, J. and Stinchcombe, J.R. 2008, 'A latitudinal cline and response to vernalization in leaf angle and morphology in Arabidopsis thaliana (Brassicaceae)', New Phytologist, vol. 179, no. 1, pp. 155-64. Jaramillo, E., Dugan, J.E., Hubbard, D.M., Contreras, H., Duarte, C., Acuña, E. and Schoeman, D.S. 2017, 'Macroscale patterns in body size of intertidal crustaceans

IP T

provide insights on climate change effects', Plos One, vol. 12, no. 5, pp. 1-24.

Justice, S.S., Hunstad, D.A., Cegelski, L. and Hultgren, S.J. 2008, 'Morphological plasticity

SC R

as a bacterial survival strategy', Nature Reviews Microbiology, vol. 6, no. 2, pp. 1628.

Kawamitsu, Y., Driscoll, T. and Boyer, J.S. 2000, 'Photosynthesis during desiccation in an intertidal alga and a land plant', Plant and Cell Physiology, vol. 41, no. 3, pp. 344-53.

U

Klugghammer, C. and Schreiber, U. 2008, ‘Complementary PS II quantum yields calculated

N

from simple fluorescence parameters measured by PAM fluorometry and the

A

saturation pulse method’, PAM Application Notes, 1:27-35. Kübler, J.E. and Dudgeon, S.R. 1996, 'Temperature dependent change in the complexity of

M

form of Chondrus crispus fronds', Journal of Experimental Marine Biology and Ecology, vol. 207, no. 1, pp. 15-24.

ED

Lilley, S.A. and Schiel, D.R. 2006, 'Community effects following the deletion of a habitatforming alga from rocky marine shores', Oecologia, vol. 148, no. 4, pp. 672-81.

PT

Littler, M.M. and Littler, D.S. 1980, 'The evolution of thallus form and survival strategies in benthic marine macroalgae: Field and laboratory tests of a functional form model', American Naturalist, pp. 25-44.

CC E

Lough, J.M. and Hobday, A.J. 2011, 'Observed climate change in australian marine and freshwater environments', Marine and Freshwater Research, vol. 62, no. 9, pp. 98499.

A

Macinnis-Ng, C.M., Morrison, D.A. and Ralph, P.J. 2005, 'Temporal and spatial variation in the morphology of the brown macroalga Hormosira banksii (Fucales, Phaeophyta)', Botanica Marina, vol. 48, no. 3, pp. 198-207. Madsen, T.V. and Maberly, S.C. 1990, 'A comparison of air and water as environments for photosynthesis by the intertidal alga Fucus spiralis (Phaeophyta)', Journal of Phycology, vol. 26, no. 1, pp. 24-30.

Research article for consideration by Journal of Plant Physiology

McLeod, I., McCormick, M., Munday, P. et al. 2015, 'Latitudinal variation in larval development of coral reef fishes: Implications of a warming ocean', Marine Ecology Progress Series, vol. 521, pp. 129-41. Mislan, K.A.S., Wethey, D.S. and Helmuth, B. 2009, 'When to worry about the weather: Role of tidal cycle in determining patterns of risk in intertidal ecosystems', Global Change Biology, vol. 15, no. 12, pp. 3056-65. Moles, A.T., Ackerly, D.D., Tweddle, J.C., Dickie, J.B., Smith, R., Leishman, M.R.,

IP T

Mayfield, M.M., Pitman, A., Wood, J.T. and Westoby, M. 2007, 'Global patterns in seed size', Global Ecology and Biogeography, vol. 16, no. 1, pp. 109-16.

SC R

Mueller, R., Fischer, A.M., Bolch, C.J.S. and Wright, J.T. 2015, 'Environmental correlates of phenotypic variation: Do variable tidal regimes influence morphology in intertidal seaweeds?', Journal of Phycology, vol. 51, no. 5, pp. 859-71.

Oates, B.R. 1985, 'Photosynthesis and amelioration of desiccation in the intertidal saccate

U

alga Colpomenia peregrina', Marine Biology, vol. 89, no. 2, p. 109119.

N

Osborn, J.E. 1948, 'The structure and life history of Hormosira banksii (Turner) Decaisne',

A

Transactions of the Royal Society of New Zealand, vol. 77, J. Hughes, Printer, pp. 4771.

M

Pearson, G.A., Lago-Leston, A. and Mota, C. 2009, 'Frayed at the edges: Selective pressure and adaptive response to abiotic stressors are mismatched in low diversity edge

ED

populations', Journal of Ecology, vol. 97, no. 3, pp. 450-62. Ralph, P.J., Morrison, D.A. and Addison, A. 1998, 'A quantitative study of the patterns of

PT

morphological variation within Hormosira banksii (Turner) Decaisne (Fucales: Phaeophyta) in south-eastern Australia', Journal of Experimental Marine Biology and Ecology, vol. 225, no. 2, pp. 285-300.

CC E

Roberson, L.M. and Coyer, J.A. 2004, 'Variation in blade morphology of the kelp Eisenia arborea: Incipient speciation due to local water motion?', Marine Ecology Progress Series, vol. 282, pp. 115-28.

A

Saada, G., Nicastro, K.R., Jacinto, R., McQuaid, C.D., Serrão, E.A., Pearson, G.A. and Zardi, G.I. 2016, 'Taking the heat: Distinct vulnerability to thermal stress of central and threatened peripheral lineages of a marine macroalga', Diversity and Distributions, pp. 1-9. Sagarin, R.D. and Gaines, S.D. 2002, 'The 'abundant centre' distribution: To what extent is it a biogeographical rule?', Ecology Letters, vol. 5, no. 1, pp. 137-47.

Research article for consideration by Journal of Plant Physiology

Schiel, D.R. 2006, 'Rivets or bolts? When single species count in the function of temperate rocky reef communities', Journal of Experimental Marine Biology and Ecology, vol. 338, no. 2, pp. 233-52. Shoo, L.P., Storlie, C., Williams, Y.M. and Williams, S.E. 2010, 'Potential for mountaintop boulder fields to buffer species against extreme heat stress under climate change' International Journal of Biometeorology, vol. 54, no. 4, pp. 475-8. Skene, K.R. 2004, 'Key differences in photosynthetic characteristics of nine species of

IP T

intertidal macroalgae are related to their position on the shore', Canadian Journal of Botany, vol. 82, no. 2, pp. 177-84.

SC R

Smith, C.M. and Berry, J.A. 1986, 'Recovery of photosynthesis after exposure of intertidal algae to osmotic and temperature stresses: Comparative studies of species with differing distributional limits', Oecologia, vol. 70, no. 1, pp. 6-12.

Somero, G.N. 2010, 'The physiology of climate change: How potentials for acclimatization

U

and genetic adaptation will determine 'winners' and 'losers'', Journal of Experimental

N

Biology, vol. 213, no. 6, pp. 912-20.

A

Stengel, D. and Dring, M. 1997, 'Morphology and in situ growth rates of plants of Ascophyllum nodosum (Phaeophyta) from different shore levels and responses of

M

plants to vertical transplantation', European Journal of Phycology, vol. 32, no. 2, pp. 193-202.

ED

Via, S. and Lande, R. 1985, 'Genotype‐environment interaction and the evolution of phenotypic plasticity', Evolution, vol. 39, no. 3, pp. 505-22.

PT

Viejo, R.M., Martínez, B., Arrontes, J., Astudillo, C. and Hernández, L. 2011, 'Reproductive patterns in central and marginal populations of a large brown seaweed: Drastic

CC E

changes at the southern range limit', Ecography, vol. 34, no. 1, pp. 75-84. Wernberg, T. and Thomsen, M.S. 2005, 'The effect of wave exposure on the morphology of Ecklonia radiata', Aquatic Botany, vol. 83, no. 1, pp. 61-70.

A

Westoby, M., Falster, D.S., Moles, A.T., Vesk, P.A. and Wright, I.J. 2002, 'Plant ecological strategies: Some leading dimensions of variation between species', Annual Review of Ecology and Systematics, vol. 33, no. 1, pp. 125-59.

Williams, S.L. and Dethier, M.N. 2005, 'High and dry: Variation in net photosynthesis of the intertidal seaweed Fucus gardneri', Ecology, vol. 86, no. 9, pp. 2373-9. Zardi, G.I., Nicastro, K.R., Ferreira Costa, J., Serrão, E.A. and Pearson, G.A. 2013, 'Broad scale agreement between intertidal habitats and adaptive traits on a basis of

Research article for consideration by Journal of Plant Physiology

contrasting population genetic structure', Estuarine, Coastal and Shelf Science, vol. 131, pp. 140-8. Zardi, G.I., Nicastro, K.R., Serrão, E.A., Jacinto, R., Monteiro, C.A. and Pearson, G.A. 2015, 'Closer to the rear edge: Ecology and genetic diversity down the core-edge gradient of

A

CC E

PT

ED

M

A

N

U

SC R

IP T

a marine macroalga', Ecosphere, vol. 6, no. 2, pp. 1-25.

Research article for consideration by Journal of Plant Physiology

FIGURE CAPTIONS

IP T

Figure 1: Example of H. banksii phenotypes found across different locations sampled in this study: Angourie and Minnie Water (warm edge), Pearl Beach and Bilgola Beach (central). Orange box (top) shows phenotypes from edge populations, while blue box (bottom) shows phenotypes from central populations. Sea surface Temperature (SST) data reflect average sea surface temperature (MODIS –Aqua SST) taken from 2003-2015 from the Integrated Marine Observing System (IMOS).

SC R

Figure 2: Principle coordinates ordination of thalli from four locations (Angourie ANG, Minnie Water MW, Pearl Beach PB and Bilgola Beach BB) and two tidal heights (low and high on the shore) based on all morphometric traits. In a) thalli are labelled by height on the shore and in b) thalli are labelled by location and region (filled symbols = warm-edge, open symbols= centre). The first two principal coordinate axes describe 66.8% of variation amongst all thalli.

A

N

U

Figure 3: Mean (± SE) relative water content (%) of thalli collected from (a) two heights on the shore, and (b) at four locations at the end of the desiccation period (4 h) and after 30 minutes of recovery when thalli were reimmersed in seawater. Samples are grouped according to whether they were desiccated at 20 or 30 °C.

ED

M

Figure 4: Mean (± SE) of photosynthetic efficiency of thalli from (a) two heights on the shore and (b) at four locations. Photosynthetic yield at three time points: 1) initial, 2) the end of the desiccation period (4 h), and 3) 30 minutes after thalli were resubmerged. The controls remained submerged while the 20 °C and 30 °C temperature treatments were emergent during the desiccation period.

CC E

PT

Figure 5: Changes in mean (± SE) relative water content (%) with time for high and low shore thalli under two temperature treatments 20 °C (filled) and 30 °C (unfilled) in four populations: Angourie, Minnie Waters, Pearl Beach, and Bilgola Beach. Control treatments shown as inverted triangles. Shaded area represents when thalli were re-immersed in seawater (10 μmol photons m-2 s-1, 20 °C) while white area represents desiccation periods where thalli were heated under high light (400 µmol photons m-2 s-1).

A

Figure 6: Mean (± SE) of photosynthetic efficiency of PSII over time of thalli from high and low on the shore under two temperature treatments: 20 °C (filled) and 30 °C (unfilled) in four populations Angourie, Minnie Waters, Pearl Beach, and Bilgola Beach. Control treatments shown as inverted triangles. Shaded area represents when thalli were re-immersed in seawater (10 μmol photons m-2 s-1, 20 °C) while white area represents desiccation periods where thalli were heated under high light (400 µmol photons m-2 s-1). Figure 7: Mean (± SE) of percent loss of maximum quantum yield (FV/FM) between initial and final time point at a) 20 °C and b) 30 °C for thalli collected from two heights on the shore and four locations.

Research article for consideration by Journal of Plant Physiology

ED

M

A

N

U

SC R

IP T

FIGURES

A

CC E

PT

Figure 1

SC R

IP T

Research article for consideration by Journal of Plant Physiology

CC E

PT

ED

M

A

N

U

b)

A

Figure 2

o

o

30 C

20 C

SC R

b) Location

Angourie Minnie Water Pearl Beach Bilgola Beach

U o

20 C

o

30 C

ED

After 4 h desiccation

A

CC E

PT

Figure 3

o

30 C

IP T

o

20 C

Low shore High shore

N

100 90 80 70 60 50 40 30 20 10 0

a) Height on shore

A

100 90 80 70 60 50 40 30 20 10 0

M

Relative Water Content (%)

Relative Water Content (%)

Research article for consideration by Journal of Plant Physiology

o

20 C

o

30 C

After 30 min reimmersed

Research article for consideration by Journal of Plant Physiology

Low shore High shore

a) Height on shore

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

o

o o Control 20 C 30 C Control 20 C

Angourie Minnie Water Pearl Beach Bilgola Beach

0.6

U

0.5 0.4

N

0.3

A

0.2

M

0.1

CC E

o

o

o o Control 20 C 30 C Control 20 C 30 C Initial After 4 h desiccation After 30 min reimmersed

PT

Figure 4

A

SC R

0.7

ED

Photosynthetic efficiency (YPSII)

b) Location 0.8

0.0

o

30 C

IP T

Photosynthetic efficiency (YPSII)

0.8

A

N

U

SC R

IP T

Research article for consideration by Journal of Plant Physiology

A

CC E

PT

ED

M

Figure 5

A

N

U

SC R

IP T

Research article for consideration by Journal of Plant Physiology

A

CC E

PT

ED

M

Figure 6

Research article for consideration by Journal of Plant Physiology

120

Low shore High shore

a)

100

60 40 20

SC R

80 60 40

U

20

ch

N

Location

A

CC E

PT

ED

Figure 7

a ol Bi lg

lB ea

ar

A

M

Pe

W e ni M in

An g

ou

at

er

rie

0

h

100

b)

ac

120

IP T

0

Be

FV/FM Percent Loss (%)

80