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Ocean acidification drives community shifts towards simplified non-calcified habitats in a subtropical−temperate transition zone

Rising atmospheric concentrations of carbon dioxide are causing surface seawater pH and carbonate ion concentrations to fall in a process known as ocean acidification. To assess the likely ecological effects of ocean acidification we compared intertidal and subtidal marine communities at increasing levels of pCO2 at recently discovered volcanic seeps off the Pacific coast of Japan (34° N). This study region is of particular interest for ocean acidification research as it has naturally low levels of surface seawater pCO2 (280–320 µatm) and is located at a transition zone between temperate and sub-tropical communities. We provide the first assessment of ocean acidification effects at a biogeographic boundary. Marine communities exposed to mean levels of pCO2 predicted by 2050 experienced periods of low aragonite saturation and high dissolved inorganic carbon. These two factors combined to cause marked community shifts and a major decline in biodiversity, including the loss of key habitat-forming species, with even more extreme community changes expected by 2100. Our results provide empirical evidence that near-future levels of pCO2 shift sub-tropical ecosystems from carbonate to fleshy algal dominated systems, accompanied by biodiversity loss and major simplification of the ecosystem.

at CO 2 seeps are genetically distinct 17 , and that populations of molluscs that hatch benthic larvae have adapted to chronic ocean acidification over multiple generations through dwarfism 18 .
Since the beginning of the industrial era, atmospheric CO 2 has increased from ~280 µatm to present day levels of 400 µatm 1 , and yet our understanding of the effects of ocean acidification that may have already occurred is limited; with most research focussed on potential impacts over the coming century. A recent study restored the carbonate chemistry saturation state of a coral reef flat to near pre-industrial levels and demonstrated that present-day net community calcification of coral reefs is already impaired by ocean acidification 19 . Due to the influence of the northward flowing Kuroshio Current 20,21 our study region in Japan has naturally low levels of surface seawater pCO 2 (280-320 µatm), which are near pre-industrial levels based on the global average (280 µatm; ref. 1 ). This particular chemical setting could therefore provide information on how the increase of CO 2 since the pre-industrial period has already affected ecosystems in other parts of the world.
The presence of ecosystem engineers can modify habitats, promote spatial complexity and facilitate the presence of other species 22,23 . Ocean acidification-driven changes to these habitat-forming organisms may therefore interact with the direct effects on those species residing in the habitat, and lead to lower species diversity in coral reefs, mussel beds and macroalgal habitats 5 . Previous studies in CO 2 seeps have demonstrated consistent patterns of ocean acidification impacts on the structure of marine ecosystems, with the observed ecological shifts in the acidified conditions showing a reorganisation of the community including reduced biodiversity 8,24,25 , and habitat loss (as well as structural complexity) 5,11,12 . It is currently unclear, however, how those communities located at the boundaries of biogeographic regions are likely to respond to ocean acidification; in these regions many species overlap at their range margins and may demonstrate reduced fitness and performance relative to their range centre. Here, we investigate the effects of ocean acidification at a biogeographic transition zone on the Pacific coast of Japan. This region is a global biodiversity hotspot 26,27 where temperate and subtropical communities overlap, with the co-existence of both canopy-forming fleshy macroalgae and zooxanthellate scleractinian corals. These two groups are key habitat-forming species that provide a complex three-dimensional structure that sustains a diverse ecosystem. Such a location will therefore provide information on the effects of ocean acidification on range limits at subtropical−temperate transition zones globally.
In the present study, we assessed the effects of chronic exposure to ocean acidification on intertidal and subtidal communities around a set of volcanic CO 2 seeps off Shikine Island, Japan. We examined the community composition of benthic marine life at sites with reference levels of 300 µatm pCO 2 (near pre-industrial levels) and compared them with areas exposed to increasing levels of carbon dioxide gradually up to end-of-the-century pCO 2 conditions to provide the first chemical and ecological assessment of the impact of ocean acidification at a subtropical−temperate transition zone.

Methods
Study Site and Carbonate Chemistry. Shikine is a volcanic island east of the Izu peninsula in Japan (34°19′9″ N, 139° 12′18″ E) with many CO 2 seeps in shallow waters that we surveyed using RV Tsukuba II. Different stations in the intertidal and subtidal zones (3-6 m below Chart datum) were surveyed and given a classification based on their mean pCO 2 levels. Those stations with a similar pCO 2 level were then grouped together for subsequent analysis. The groupings used were 300, 400, 1100 and 1800 µatm pCO 2 for the intertidal, and 300, 400, 700, 900, and 1500 µatm pCO 2 for the subtidal. The intertidal stations included eight '300 µatm' stations, one '400 µatm' station, two '1100 µatm' stations and one '1800 µatm' station. The subtidal included three '300 µatm' stations, one '400 µatm' station, one '700 µatm' station, one '900 µatm' station and one '1500 µatm' station (see Fig. 1 for station locations). The abundance and distribution of rocky shore communities varies greatly in both space and time, even along the same stretch of coast 28 . Following the suggestion of refs 29,30 we used multiple reference stations (eight '300 µatm' pCO 2 in the intertidal, and three '300 µatm' pCO 2 in the subtidal) to assess the variability of 'normal' rocky shore communities to compare with our high CO 2 stations.
To describe the carbonate chemistry of the survey stations, pH, temperature, salinity and total alkalinity (TA) were measured through in situ measurements and/or discrete sampling at the respective stations using both a YSI sensor (YSI Pro Plus, USA) and a TOA-DKK multisensor (WQ-22C, TOA-DKK, Japan) in June 2015. Intertidal stations were surveyed by fixing the sensors to the shore (50 cm below the low water mark), with discrete samples collecting surface water. Subtidal stations were surveyed by fixing the sensors to the seafloor at 5-6 m depth. Discrete samples in the subtidal surveys were taken by SCUBA divers close to the bottom (5-6 m depth). Long term monitoring of pH, temperature, conductivity and dissolved oxygen of the bottom water at the '300 µatm' and '900 µatm' pCO 2 stations was carried out in June 2016 using durafet pH sensors (Seafet, Sea-Bird Scientific, Canada) calibrated on the total scale, Hobo conductivity loggers (U24-002-C) and Hobo dissolved oxygen data loggers (U26-001) (Bourne, Onset, USA) with the sensors fixed 30 cm above the seafloor at a depth of 5-6 m. A Horiba multi-parameter meter (U-5000G, Horiba Ltd, Kyoto Japan) coupled with a GPS (eTrex30x, Garmin) was used to document the spatial variation in carbonate chemistry, where we mapped the spatial distribution of pCO 2 using the nearest neighbour interpolation algorithm in ArcGIS (ESRI, New York, USA), see Fig. 1.
In order to assess differences in species richness between the CO 2 levels (since taxonomic groups were used to evaluate community changes across stations), species diversity was assessed during 30-minute searches in the intertidal zone at a '300 µatm' station and a '1100 µatm' station. Species richness in the subtidal zone at '300 µatm' , '400 µatm' and '900 µatm' stations was assessed by identifying the different species observed in the photoquadrats. The taxonomic groups that were assigned to the species observed in the intertidal and subtidal zones are shown in Tables S1 and S2.
The variation in habitat complexity along pCO 2 gradients was assessed based on the abundance of sessile taxa, along with a rank (between 0 and 5) for that taxon representing the biogenic habitat complexity provided. These ranks were: Minimum habitat complexity = 0, e.g. all encrusting algae; Very low complexity = 1, e.g. small spirorbids and small barnacles; Low complexity = 2, e.g., turf and low-profile fleshy algae, sponges and non calcifying anthozoans; Moderate complexity = 3, e.g. branched coralline algae and sparse oysters; High complexity = 4, e.g. canopy-forming algae, clumps of mussels; Exceptionally high habitat complexity = 5, e.g. hard corals (see Supporting Information, Table S3). The habitat complexity was calculated as follows: the abundance of each taxonomic group was normalised (using the decostand function, vegan package) and then had the rank (0-5 score) applied, providing a habitat complexity score for each quadrat. In order to provide a relative measure across the pCO 2 sites, the habitat complexity score was normalised to between 0 and 1, where the quadrat showing the maximum complexity had a score of 1. These scores were then used to calculate the mean habitat complexity and its variability (standard error) for the different pCO 2 stations.
Intertidal habitat complexity was provided by barnacles, mussels and oysters, and coralline algae (which formed a thick crust) at '300 µatm' . These calcifying groups drastically decreased in abundance with an overall shift in the communities as pCO 2 levels rose (Figs 3 and 5b). These shifts lead to a decrease in the complexity of the habitat (K-W: H = 48.50, p < 0.001), reducing two-fold from '300 µatm' to '1100 µatm' , and more than 6-fold to '1800 µatm' (Fig. 5a). At the high CO 2 levels, the main habitat was low-profile fleshy algae (Figs 3 and 5a).
Despite the increasing abundance of fleshy algae with rising pCO 2 , there was a 56% reduction in algal species richness from '300 µatm' (18 spp.) to '1100 µatm' (8 spp.) with little overlap in species composition between these two CO 2 levels ( Fig. 5c and Table S1). The '300 µatm' stations had a diverse community of Rhodophyta with 16 species compared to only six species at '1100 µatm' . There were 33 and 32 macrofaunal taxa at '300 µatm' and '1100 µatm' respectively, but the community composition was very different, with only seven species common to both sets of stations ( Fig. 5c and Table S1). Subtidal zone. Changes in the subtidal benthic community were remarkably similar to those observed intertidally, with reduced abundances of calcifying organisms as CO 2 levels increased from '300 µatm' to '400 µatm' and again to '700 µatm' and beyond (Figs 3 and 6). The cover of coralline algae (K-W: H = 42.46, p < 0.001; Fig. 6b) and hard corals (K-W: H = 19.67, p < 0.001; Fig. 6a) was significantly reduced as CO 2 levels rose. Hard corals were common at '300 µatm' , where they had 11 ± 22% cover, however, they were only sporadically found at '400 µatm' with just two colonies observed accounting for 0.7 ± 1.6% cover, and absent from more highly elevated CO 2 stations (Figs 3 and 6a). Soft corals and anemones were not recorded in the elevated CO 2 stations corresponding to the end-of-the-century projections ('700 µatm' , '900 µatm' and '1500 µatm') and were rare at '300 µatm' and '400 µatm' . Due to their low abundance at '300 µatm' and '400 µatm' , the soft corals and the anemones did not significantly differ with CO 2 level (K-W: H = 6.32, p = 0.18 and H = 5.09, p = 0.27 respectively) (Fig. S4).
The cover of non-calcifying macroalgae was high at all subtidal stations yet there were major shifts in community composition (Figs 3 and 6c-f). Large canopy forming macroalgae had significantly reduced abundance at '400 µatm' and higher pCO 2 end-of-the century projections (K-W: H = 18.53, p < 0.001, Fig. 6c) whereas low-profile algae and turf algae increased in cover as CO 2 levels rose, with their cover significantly higher at '400 µatm' and higher CO 2 level stations compared to '300 µatm' stations (K-W: H = 23.41, p < 0.001 and H = 44.81, p < 0.001 Fig. 6d,e). Due to the overall reduction in the percentage of calcified and non-calcifying macroalgae, the proportion of biofilm encrusted substrata significantly increased from 2 ± 3% to 20 ± 14% at '300 µatm' and '1500 µatm' respectively (K-W: H = 32.88, p < 0.001; Fig. S5).
Both hard corals and canopy forming macroalgae formed a biogenically complex habitat in the subtidal zone at '300 µatm' CO 2 (Fig. 3). The sharp decrease of these two groups lead to significantly reduced habitat complexity (K-W: H = 50.48, p < 0.001) at CO 2 levels corresponding to the mid-('400 µatm') and end-of-the-century projections ('700 µatm' , '900 µatm' and '1500 µatm') (Figs 3 and 5c). The communities radically changed as pCO 2 rose with distinct communities observed at each pCO 2 site (Fig. 5d) with less complex low-profile and turf algae dominating at the highest pCO 2 (Fig. 3).
The species richness of the benthic flora and fauna was reduced by 71% as CO 2 levels rose, with a total of 49, 20 and 14 species at '300 µatm' , '400 µatm' and '900 µatm' , respectively ( Fig. 5f and Table S2). This change in faunal species richness included seven hard coral species, a sea anemone, a soft coral species and a sponge, which were only observed at '300 µatm' . In addition, small gastropods were abundant at '300 µatm' , but not at '400 µatm' or '900 µatm' . Other mobile benthic fauna that were only found in the '300 µatm' stations were sea cucumbers and coral boring serpulids and barnacles. Algal diversity was greatly reduced shifting from a diverse Figure 5. Changes in habitat complexity (mean ± SE), communities, and species richness with increasing pCO 2 for intertidal (a-c) and subtidal (d-f) habitats. (a,d) A significant difference between pCO 2 groups is indicated with a different letter (Kruskal-Wallis with Bonferroni-adjusted Fisher's least significant difference). (b,e) The change in communities are illustrated by an nMDS plot based on Bray Curtis distance. The colour of each point represents the pCO 2 : green: '300 µatm' , black: '400 µatm' , light blue: '1100 µatm' and orange: '1800 µatm' for the intertidal and green: '300 µatm' , black: '400 µatm' , blue: '700 µatm' , red: '900 µatm' and pink: '1500 µatm' for the subtidal. Ellipses represent the 95% interval confidence. (c,f) Algal (blue) and faunal (red) species richness are shown with darker colours used for species only found in that site, and lighter shades for species that overlap across two sites. For the subtidal, the species overlap are graduated (from darkest to lightest) in the following order: 300-400 µatm, 300-900 µatm and 400-900 µatm (no species were common to all three sites).

Discussion
Our comparisons of intertidal and subtidal rocky reef communities along natural gradients in CO 2 have revealed that ocean acidification is a threat to many marine organisms, as it can drive fundamental shifts in coastal marine ecosystems towards simplified, low diversity communities. Abrupt changes in subtidal and intertidal communities were revealed from present-day to near-future levels of CO 2 (300 µatm to 400 µatm), and then again to future levels (400 µatm to 700 µatm) and beyond. In natural coastal ecosystems, mean pCO 2 levels predicted for as soon as the mid-century will have periods of such low aragonite saturation and high availability of inorganic carbon that this will cause biodiversity loss driven by a decline in habitat-forming species (e.g. coralline algae, canopy-forming macroalgae, scleractinian corals, and barnacles) and an increase in low-profile and turf algae. Our observations suggest that ocean acidification will shift ecosystems at subtropical−temperate transition zones from complex calcified biogenic habitats towards less complex non-calcified habitats.
Increases in dissolved CO 2 provide a resource for algae that cannot use bicarbonate ions for their photosynthesis 37 and is expected to increase the prevalence of macroalgae 8,9,12,38 . The significantly increased occurrence of low-profile fleshy algae with increasing pCO 2 aligns with results from other shallow marine carbon dioxide seeps. However, not all macroalgae species respond in the same manner to the effects of elevated CO 2 , with some species gaining a relative advantage over their counterparts 3 . The resulting pattern is that ocean acidification alters successional development due to competition for space by a few highly tolerant species 39,40 . The prevalence of low-profile fleshy algae in our elevated pCO 2 sites may contribute towards the observed decline in canopy-forming macroalgae and corals 41 . In this context, natural analogues offer opportunities to assess competitive interactions and the effects of ocean acidification on ecological functions 40 .
The presence of highly calcified communities at all of our reference sites reflects the high carbonate saturation levels that typify this region due to naturally low background pCO 2 levels 20 . At the highest CO 2 stations the exposed shells and skeletons of calcifying organisms had visible signs of dissolution, as seen in other field studies worldwide [42][43][44][45] . The decrease in the abundance of calcifying macrofauna from our reference sites to '400 µatm' sites, where even the lowest Ω aragonite remains higher than values typically observed nowadays in many parts of the ocean 46,47 , suggests that ocean acidification is already impairing the growth and survival of calcifiers 19 . This is a concern and provides an insight into the effects of ocean acidification in other parts of the world that have already experienced increases in pCO 2 from 300 µatm to 400 µatm during the last century since the Industrial Revolution. Communities of zooxanthellate scleractinian corals currently thrive at high latitude (here 34° N) in East Asia due to warm, northward flowing currents which bring low pCO 2 , high carbonate saturated waters into the region 48 . These communities are an important reservoir of diversity for hermatypic corals, and a number of species found at our study site are endemic to the region 49 . We observed an abrupt decline in their abundance and diversity as CO 2 levels rose and CaCO 3 saturation state fell. Despite differences in biogeography, the major ecosystem changes we recorded along the CO 2 gradients are broadly consistent with findings from other naturally acidified tropical coral reef settings 12,13,50 . Moreover, these patterns are comparable to those seen on tropical reefs in Florida, where present-day seasonal reductions in saturation state are contributing to reef dissolution, the die-back of scleractinians and an increase in low-profile fleshy algal growth 44,51 .
The Japanese subtropical-temperate transition zone is highly diverse due to a mix of subtropical and temperate species, which allows for the coexistence of diverse macroalgae with scleractinian zooxanthellate corals. This zone is at the leading-edge for subtropical species and the trailing-edge for temperate species, and this biogeographic boundary is likely to undergo fundamental shifts with future climate change 52,53 . With increased temperature threatening corals in the tropics 54 , it could be expected that higher latitudes will act as refugia, but this would require the loss of other ecologically important species that typically dominate these latitudes 55 . Our results support the notion that ocean acidification may constrain the shift of coral to higher latitudes [56][57][58] .
Biogenic complexity promotes the provisioning of habitats, allowing high levels of biodiversity to be sustained within an ecosystem 5 . Reductions in habitat complexity cause a reduction in biodiversity 5,59 . We found that as CO 2 levels rose, there was a shift from structurally complex canopy-forming fleshy algae and corals to less complex low-profile fleshy algae and an absence of corals. This reduction in habitat complexity may have contributed towards the reduced species richness in our elevated pCO 2 sites, as well as the minimal overlap in observed species among the different sites as many marine organisms rely on a particular habitat (e.g. ref. 60 ). As the effect of ocean acidification could cause a simplification of the ecosystems, we can expect ocean acidification to also alter the delivery and the quality of the ecosystem services associated with these marine communities 61 and this should be a focus of future work.
Carbon dioxide seeps are open systems that allow recruitment from outside and this hinders genetic adaptation 62 . Whilst organisms that survive at such seeps may upregulate genes to acclimate to high pCO 2 levels 63 , only species with very limited genetic dispersal can be expected to evolve to cope with the local conditions 64 . The CO 2 seep systems described in this report can nevertheless provide insights into how marine ecosystems have been changing under increased anthropogenic CO 2 and into the near future. Thus, reference sites showed pre-industrial levels of CO 2 and sites on the fringe of the CO 2 gradient showed present-day and mid-century CO 2 levels with minimum variations of these levels on short periods of time. Extreme variations of pCO 2 concentrations at natural analogues are commonly observed 11,13 and may bias the observed response of organisms 65 . Such extreme variations in pCO 2 levels were also observed at the highest pCO 2 sites of the Shikine CO 2 systems, yet we also located stations with small increases and variations in pCO 2 that are well suited to projected levels of ocean acidification.
In conclusion, we found that an increase in CO 2 resulted in profound community-level changes in a biodiverse subtropical-temperate transition zone. Both intertidal and subtidal communities became highly simplified, with reduced biogenic habitat complexity and biodiversity. We highlight that ocean acidification may constrain tropical coral range expansion. Our findings suggest that a threshold for macroalgal and coral habitats at the subtropical-temperate transition zone is likely to be exceeded by 2050, with even more extreme changes expected by the end-of-the-century. Overall, ocean acidification is expected to simplify coastal marine communities throughout East Asia.