Filamentous calcareous alga provides a substrate for coral-competitive macroalgae in the degraded lagoon of Dongsha Atoll, Taiwan

Background The chemically-rich seaweed Galaxaura is not only highly competitive with corals, but also provides substrate for other macroalgae. Its ecology and associated epiphytes remain largely unexplored. To fill this knowledge gap, we herein undertook an ecological assessment to explore the spatial variation, temporal dynamics, and epiphytic macroalgae of G. divaricata on patch reefs in the lagoon of Dongsha Atoll, a shallow coral reef ecosystem in the northern South China Sea, repeatedly impacted by mass coral bleaching events. Methods Twelve spatially independent patch reefs in the Dongsha lagoon were first surveyed to assess the benthic composition in April 2016, and then revisited to determine G. divaricata percent cover in September 2017, with one additional Galaxaura-dominated reef (site 9). Four surveys over a period of 17 months were carried out on a degraded patch reef (site 7) to assess the temporal variation in G. divaricata cover. Epiphytic macroalgae associated with G. divaricata were quantified and identified through the aid of DNA barcoding. Results Patch reefs in the Dongsha lagoon were degraded, exhibiting relatively low live coral cover (5-43%), but high proportions of macroalgae (13-58%) and other substrates (rubble and dead corals; 23-69%). The distribution of G. divaricata was heterogeneous across the lagoon, with highest abundance (16-41%) in the southeast area. Temporal surveys from site 7 and photo-evidence from site 9 suggested that an overgrowth by G. divaricata was still present to a similar extend after 17 months and 3.5 years. Yet, G. divaricata provides a suitable substrate some allelopathic macroalgae (e.g., Lobophora sp.). Conclusions Our study demonstrates that an allelopathic seaweed, such as G. divaricata, can overgrow degraded coral reefs for extended periods of time. By providing habitat for harmful macroalgae, a prolonged Galaxaura overgrowth could strengthen negative feedback loops on degraded coral reefs, further decreasing their recovery potential.


Introduction
Coral-macroalgae competition is a naturally ecological process on coral reefs [1].However, anthropogenic disturbances, e.g., climate change, overfishing, and pollution, have intensified space competition of macroalgae against corals and in turn led to a phase shift from a coral-dominated to a macroalgae-dominated ecosystem [2].
The recovery of live corals on degraded reefs is strongly influenced by the types of dominant macroalgae, i.e., allelopathic versus non-allelopathic types [3,4].
Allelopathic macroalgae produce lipid-soluble secondary metabolites, e.g., loliolide derivatives or terpenes, that are poisonous to corals (known as allelochemicals).Such allelochemicals are capable of bleaching and killing coral tissue [5], decreasing the photosynthetic efficiency of zooxanthellae [6], and altering the coral microbiome, ultimately decreasing coral health [7,8].Allelopathic macroalgae are considered most detrimental for the resilience of coral reefs [12], as these types may perpetuate their dominance by deterring coral larval settlement, and inhibiting the growth and survival of juvenile recruits, key processes of coral reef recovery [9][10][11].
The red upright calcifying seaweed Galaxaura is known to be highly allelopathic against corals.Life history of the genus Galaxaura can be grouped into two morphotypes, a smooth and a filamentous type.The latter is characterized by hairy branches that are covered with fine assimilatory filaments [12].Extracts of the lipid-soluble secondary metabolites of G. filamentosa were shown to cause bleaching and death of coral tissue [13,14], and deterred coral larvae from settling [15].It has thus been suggested that high abundance of Galaxaura on degraded reefs can inhibit the recovery of live coral cover [4,15,16].
The filamentous morphotype of G. divaricata, is widely distributed in subtropical and tropical reef areas in the Pacific Ocean [17].Filamentous G. divaricata is also common on coral reefs in the shallow lagoon of Dongsha Atoll [18].Dongsha Atoll is the only large (> 500 km 2 ) coral reef atoll in the northern South China and represents a highly valuable hot-spot for marine biodiversity in this region [19].A catastrophic mass bleaching in 1998 and reoccurring bleaching events thereafter have, however, caused severe mass mortalities of corals in the Dongsha lagoon, followed by a marked increase of macroalgae [20,21].To date, little is known about the current state of recovery and dominant macroalgae in Dongsha lagoon patch reefs.The proliferation of G. divaricata on degraded reefs in the lagoon of Dongsha Atoll was first uncovered during a systematic macroalgae sampling expedition in February 2014 [18].Of interest to us was our observation that G. divaricata was highly populated by macroalgae.Habitat formation is known from other macroalgae (e.g., crustose Lobophora or canopy-forming Sargassum and Turbinaria) that provide substrate for epiphytic algae [22][23][24].The dense epiphytic community associated with G. divaricata might indicate a previously unappreciated role of Galaxaura as a habitat forming seaweed.
The goals of this study were to 1) assess the benthic composition of lagoon patch reefs, 2) document the spatial distribution of G. divaricata on patch reefs in the lagoon, 3) monitor the temporal dynamics of G. divaricata percent cover over time, and 4) quantify and identify the epiphytic macroalgae associated with G. divaricata.
The provision of new habitat for other macroalgae by G. divaricata could have several ecological implications, worth exploring.For instance, the epiphytic community on G. divaricata may enhance macroalgae biodiversity on the reef, or provide trophic support for herbivores, while a facilitation of allelopathic algal types would decrease the resilience of coral reefs.

Ethics statement
The ecological assessments and sample collections in this study were conducted with permissions of the Dongsha Atoll National Park.

Site description
This study was conducted from April in 2016 to September 2017 in the lagoon of Dongsha Atoll (also known as Pratas Island; 20 o 40'43" N, 116 o 42'54" E), which is an isolated coral reef atoll in the northern South China Sea.The atoll covers an area of approximately 500 km 2 and is situated 450 km southwest from the coast of Taiwan and 350 km southeast from Hong Kong (Fig 1A).The climate is seasonal and varies between a northeast monsoon winter (October-April) and southwest monsoon summer (May-September) [25].Field work during the northeast winter monsoon is often restricted due to local weather conditions.The ring-shaped reef flat encircles a large lagoon with seagrass beds and hundreds of coral patch reefs [26].Channels, at the north and south of the small islet (1.74km 2 ), interrupt the reef flat, allowing for water exchange between the lagoon and the open ocean.The semi-closed lagoon is about 20 km wide with a maximum depth of 16 m near the center [20].The Lagoon patch reefs are structured into reef tops (1-5 m depth) and reef slopes (5-12 m depth), and provide important habitat and sheltered nursery grounds for numerous marine organisms, such as green sea turtles and coral reef fish, including rays and sharks [26].For background information the lagoon water temperature was measured at each survey site, every 30

Spatial variation in benthic composition and G. divaricata cover of lagoon patch reefs
To assess the benthic composition of patch reefs in the lagoon of Dongsha Atoll, 12 spatially independent reefs were initially surveyed with SCUBA in April 2016 (Fig 1B and S1 Table).A 45-m transect was laid out across each reef area: reef top (1-5 m depth) and reef slope (5-12 m depth).The two transects were 10-20 m apart from each other.The percent cover of live corals, total macroalgae (MA; all upright growing (including G. divaricata) and crustose non-coralline seaweeds, and low growing, filamentous turf algae [27]), crustose coralline algae (CCA), and other substrates was estimated using a 35 cm x 50 cm PVC sapling frame [28].Other substrates mainly constituted dead coral skeleton, rubble, and rocks covered with sediments.Estimates were done in-situ at every meter mark, with a total of 45 sampling frames analyzed per transect.The 12 sites were revisited in September 2017 to estimate the percent cover of G. divaricata and live corals only, using the same survey method described above.An additional patch reef (site 9) was included, as this site was historically shown to be dominated by G. divaricata based on photo evidence, resulting in a total of 13 survey sites (Fig 1B and S1 Table).The diameter of haphazardly selected G. divaricata thalli were measured in situ at each site and classified as small (1-5 cm diameter), medium (>5-15 cm diam.), and large (>15-30 cm diam.).

Temporal variation in G. divaricata cover
To assess variations in the G. divaricata cover over time, we selected the slope area of a degraded patch reef (site 7) that was considerably overgrown by G. divaricata (14-18%) and had relatively low coral cover (13-19%).Percent cover of G. divaricata, and live corals were estimated in April 2016, the last month of the winter monsoon season, and three times in the summer monsoon season in July, and September 2016, and in September 2017, spanning a period of 17 months.At each time 45 photographs were taken in 1 m intervals along a 45 m fixed transect with an Olympus Stylus-TOUGH TG4 digital camera (25-100 lens, 35mm equivalent) mounted onto a PVC-quadrat (height = 0.64 cm) above a 35 cm x 50 cm sampling frame.Cover estimates were obtained from photographs using ImageJ software, and a superimposed 10 x 10 reference grid, where 1 square represented 1 % of the total grid area.G. divaricata cover estimates were arbitrarily ranked into four different categories: very low (0-1.5%),low (>1.5 -5%), high (>5-20%), and very high (>20%).

Epiphytic macroalgae associated with G. divaricata
This study was carried out in September 2017.Thirty thalli of G. divaricata were collected from a degraded reef (site 7) with relatively high percent cover of G. divaricata (14-18%).G. divaricata thalli were haphazardly collected along a 45-m transect at 5 m depth.Epiphytic macroalgae were removed and identified to the closest identifiable taxonomic unit, using either the Dongsha seaweed guide book [18] or DNA barcoding.The presence and absence of each taxonomic unit was recorded, and the occurrence frequency (f) was calculated as follow: f = c (taxonomic unit i )/n, where c (taxonomic unit i ) stands for the count number of thalli that have the epiphyte taxonomic unit i, and n equals 30, the total number of thalli analyzed.For DNA barcoding, macroalgae samples were preserved in silica gel after collection, and the total genomic DNA of samples was extracted with Quick-DNA TM Plant/Seed Miniprep Kit (Zymo Research Co., USA).Primers for the plastid gene specific amplifications were used as follows: rbcL F7/R753 for red algae [29], rbcL F68/R708 for brown algae [30], and tufA F210/R1062 for green algae [31].The newly generated sequences were deposited in GenBank and searched using BLASTn against the GenBank database (S2 and S3 Tables).Sequence similarities of >98% were considered for species identification.

Statistical analysis
Spatial variations in the percent cover of major benthic categories (corals, total macroalgae, crustose coralline algae, and other substrates) and G. divaricata were compared between two reef areas (top and slope) among sites using a two-way ANOVA, with area and site as fixed factors.Similarly, a two-way ANOVA was applied to evaluate the temporal variations in the cover of two major benthic categories (live corals and G. divaricata) among four time points, with benthic category and time as fixed factors.A significant difference was considered for pvalues lower than 0.05.Maps and statistical graphs were done using R software.

Benthic composition
Our spatial survey showed that both live coral cover and total macroalgae cover significantly varied between reef top (1-5 m) and reef slope (5-10 m) and among sites (area × site: F 11, 1056 = 17.601-26.27,P < 0.05; Figs 2A and 2B, and S4 Table ).Percent cover for corals, macroalgae, and CCA were generally higher on the reef top, while other substrates were slightly higher on the slope (area: F 1, 1056 = 6.617-62.725,P < 0.05; Figs 2A-2C and S4 Table ).We found that the macroalgae cover generally exceeded live coral cover on patch reefs in the Dongsha lagoon (Figs 2A and 2B).Using an arbitrary cutoff of 25%, we observed a higher coral cover in the west of the lagoon, in between the North and South channel, where water exchange is more efficient, i.e., sites1-3, 8, 11, and 13 (Fig 2A).In contrast, no clear spatial pattern of the macroalgae cover was observed.Using a 50% cutoff, it, however, appeared that the shallow and calm area in the southeast lagoon showed a higher macroalgae cover than other areas (i.e., sites 7 and 10; Fig 2B).Compared with live corals and total macroalgae, the CCA cover was relatively low (range: 1-3%; Fig 2C ), was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

Spatial variations in G. divaricata cover
The percent cover of Galaxaura divaricata was significantly different between reef tops and reef slopes, showing a higher percent cover on the slope (area: F 1, 1144 = 6.574,P < 0.05; S5 Table ).There was a significant statistical interaction between area (slope and top) and site (e.g., top > slope at site 5 and slope > top at site 7; area × site:  ).
Survey sites in the south, center, west, and north of the lagoon were characterized by very low cover of G. divaricata (range: 0-1.4%;Fig 3 and S6 Table).During our survey, we observed that the thallus shape and size of G. divaricata varied across sites (S1 Fig).Small ball-shaped or slender thalli were dominant on patch reefs in the northeast lagoon, while medium ball-shaped and large, carpet-like thalli were exclusively present in the southeast lagoon.To further rule out the possibility of cryptic species, our DNA barcoding analyses confirmed that all samples across sites were 100% identical in their rbcL sequences, indicative of conspecificity (S3 Table ).

Temporal dynamics of G. divaricata cover
Our temporal survey at a Galaxaura-dominated reef (the slope area of site 7) revealed that the percent cover of G. divaricata did not vary significantly among the surveys conducted at four time points (April 2016, July 2016, September 2016, and September 2017) over a period of 17 months (time: F 3, 352 = 0.632, P = 0.595; Fig 4 and S7 Table).The percent cover between live corals and G. divaricata did not differ significantly (benthic category: F 1, 352 = 0.086, P = 0.770; Fig 4 and S7 Table).
Overall, there was no significant statistical interaction between the percent cover of G.  ).Across four time points the mean G. divaricata cover remained relatively high (16.45+ 1.17%), while mean coral cover was low (15.91 + 0.6%).In addition, we provide photo-evidence from an additional patch reef (site 9, 3-

Epiphytic macroalgae associated with G. divaricata
We identified 21 taxonomic groups of macroalgae, including macroscopic filamentous cyanobacteria, in association with G. divaricata (Table 1 and S2 Table).Among these, 15 were identified to the species level, with seven species of red algae, three species of brown, and five species of green algae (Table 1 and S2 Table)

Discussion
Our study shows that most patch reefs in the lagoon of Dongsha Atoll are degraded.Many reefs have a low live coral cover (below 25%) and high proportions of macroalgae, dead corals, and rubble, all of which are signs of reef degradation [34].This is consistent with previous surveys that reported degraded conditions of lagoon patch reefs at Dongsha [35,36].The filamentous form of Galaxaura divaricata overgrows degraded patch reefs in the southeast lagoon.This area is sheltered by a 2 km-wide reef flat, harboring shallow (1-5 m) and calm waters that may provide suitable growth conditions for G. divaricata.The proliferation of macroalgae is likely the consequence of an initial coral decline [37,38].The synergistic effects of thermal stress, overfishing, and typhoon damage may have caused the decline of the once pristine corals in the Dongsha lagoon, followed by a proliferation of G. divaricata, among other macroalgae.Thermal stress on corals has increased over the past decades, with waters surrounding Dongsha Atoll warming at a faster rate than other areas of the South China Sea [35,39,40].Recurrent bleaching events have caused high coral mortality and eradicated thermo-sensitive coral genera from the lagoon [41].
Overfishing and the extensive use of dynamite and cyanide, prior to the establishment of the Dongsha Atoll National Park in 2007 reduced fish, and destroyed large areas of coral framework [20,42].Insufficient grazing after disturbance can lead to the establishment and full outgrowth of macroalgae beyond their initial stages [43].
Galaxaura is known to be largely unpalatable for various herbivorous fishes due to its calcareous thallus and low nutritional content [44][45][46].Local herbivorous fish population in the Dongsha lagoon may not be effective to control the outgrowth of Galaxaura in certain areas.
Semi-closed lagoons are highly vulnerable to eutrophication and hypoxia, especially under the backdrop of climate change [47,48].Reoccurring events of hypoxia during hot summers in 2014 and 2015 have caused substantial mass-die offs of the coral associated fauna and flora in the Dongsha lagoon [49].Particularly, densities of macroinvertebrates, including echinoids, sea cucumbers, lobsters, and giant clams are extremely low (Table S4).Galaxaura appears to be well adapted to hypoxic conditions.For instance, G. filamentosa was one of the few algae to proliferate after a mass-die off caused by hypoxia in an atoll lagoon in French Polynesia [50].
Although the filamentous G. divaricata is a common allelopathic seaweed in subtropical and tropical waters, it has never been reported as a nuisance in overgrowing coral reefs.Our observations are the first to report a prolonged G. divaricata overgrowth in degraded coral reefs.For instance, the G. divaricata cover was equally high on a degraded reef after 17-months.We further provide photoevidence from another patch reef showing that the same G. divaricata overgrowth was present to a similar extend after 3. Expanding temporal surveys in the future will be worth of doing to confirm the longterm persistence of G. divaricata overgrowth.
A prolonged overgrowth of filamentous G. divaricata may have profound implications for the recovery potential of degraded reefs at Dongsha Atoll.Owning to its allelopathic effects on corals long-standing canopies of G. divaricata are likely to hamper coral recruitment ultimately preventing coral recovery [15,51].As a caveat of this study, it is important to note that we did not attempt to isolate and identify allelopathic chemicals in G. divaricata.But, previous studies have identified lipidsoluble terpenoid compounds from filamentous Galaxaura cell extracts as allelochemicals that were capable of bleaching and killing of coral tissue [13].It is also known that Galaxaura can change the chemical microclimate on degraded reefs with adverse effects on fish feeding behavior [4].For instance, butterflyfish and other corallivores avoid corals in close association with Galaxaura, making it potentially difficult for these trophic guilds to find food [52,53].Unlike other calcifying algae, such as coralline algae, Galaxaura does not stabilize the reef matrix.Thus, a prolonged Galaxaura overgrowth may contribute to the erosion and flattening of the reef structure, which negatively impacts biodiversity, and trophic support for coral associated organisms [54].
The filamentous G. divaricata is used as habitat by a variety of macroalgae.

The availability of new habitat for epiphytic macroalgae provided by a prolonged
Galaxaura overgrowth could have several implications for the ecology and recover potential of the reef.For instance, nutrient rich epiphytes could provide trophic support for herbivorous fishes and invertebrate, such as crustaceans and mollusks [24,55,56].On the other hand, the association with the unpalatable Galaxaura may provide a refuge from herbivory for certain palatable algae [38,57], and facilitate their establishment on the reef, increasing macroalgae biodiversity [58].The facilitation of harmful, allelopathic algal types could decrease the resilience and promote alternative stable states on coral reefs [59] .Some of the identified G. divaricata epiphytes, such as cyanobacteria [10], Dictyota [60], and Lobophora [9,61] are widely shown to overgrow corals after disturbance, and are known for their allelopathic inhibition of coral larvae recruitment.Here, we firstly report that an undescribed species Lobophora sp.(as Lobophora sp28 in [32]), the third most abundant macroalga on G. divaricata, overgrows and kills corals in the Dongsha lagoon through epizoism (Fig 7 and S2).Moreover, the microscopic filaments of G. divaricata may facilitate the attachment of macroalgae spores, while the calcified branches may provide structural support for fine, filamentous macroalgae.Considering that an increased substrate availability can promote macroalgae biomass on coral reefs, we hypothesize that, by providing a habitat for epiphytic macroalgae, G. divaricata may facilitate the diversity and abundance of macroalgae on degraded reefs.This study is merely observational and does not provide experimental evidence for the facilitation of macroalgae diversity and abundance by G. divaricata.However, the abovementioned hypotheses would be of great interest awaiting future validation.

Conclusions
Our observations illustrated that the allelopathic and unpalatable filamentous seaweed, Galaxaura divaricata, can become dominant on degraded reefs in shallow, sheltered, and calm environments.We show that G. divaricata provides suitable substrate for a variety of macroalgae, further facilitating macroalgae growth and abundance on degraded reefs.Thus, a prolonged proliferation of Galaxaura could potentially enhance negative feedback loops, thereby perpetuating reef degradation.
Several common epiphytic macroalgae on Galaxaura are allelopathic and known to frequently overgrow corals.Macroalgal assemblages, such as the Galaxaura-epiphyte system, warrant further investigation to better understand their ecological implications on the resilience of coral reefs, especially of shallow atoll lagoons.There are 439 listed coral reef atolls on earth; among them are 335 with semi-enclosed lagoons [62].
Atoll lagoons are highly productive and serve as valuable nursery habitat for marine life; however, they are most vulnerable to the effects of climate change [48,63].
Results from our study can be informative for the management and conservation of lagoons and shallow, inshore coral reef ecosystems, especially in the South China Sea and the Pacific Ocean, where filamentous Galaxaura is very common.

Valonia ventricosa Green
Cyanobacteria (filamentous > 1cm) Cyanobacteria a Identification of species and taxonomic groups according to [18].557 b GenBank number in parentheses based on species identifications through DNA barcoding.558 c Denomination according to [32].559 d Denomination according to [33].560 min from March 2016 to September 2017 using HOBO Pendant® Temperature/Light 8K Data Loggers (UA-002-08, Onset Computer Corporation, USA).Water temperatures were highest during the summer monsoon, averaging 30.1 o C, and lowest during the winter monsoon, averaging 24.8 o C. Maximum temperatures from July to August reached 34 o C on reef tops and 32.7 o C on reef slopes.

F 12 ,
1144 = 7.460, P < 0.05; Fig 3 and S5 Table).G. divaricata cover was significantly different among the 13 sites (site: F 12, 1144 = 179.278,P < 0.05; Fig 3 and S5 Table), showing highest cover in the southeast area of the lagoon, i.e., site 9 (41%) and the slope of site 7 (16%) (Fig 3).Patch reefs in the northeast lagoon exhibited moderate, low, and very low cover of G. divaricata (range: 0.21-5.7%)(Fig 3 and S6 Table divaricata and live corals among time points (benthic category × time: F 3, 352 = 0.363, P = 0.780; Fig 4 and S7 Table 5 m) overgrown by G. divaricata.Photographs of the site were taken in February 2014 and in September 2017, showing that the same G. divaricata overgrowth was present after 3.5 years (Figs 5A and 5B).G. divaricata frequently grew on live corals, where the holdfast penetrated the calcium-carbonate structure, creating a strong was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.The copyright holder for this preprint (which this version posted December 24, 2018.; https://doi.org/10.1101/363796doi: bioRxiv preprint attachment to the corals (Fig 5C).In several cases we observed a fluorescent pink discoloration and bleaching of the coral tissue at the contact zone with G. divaricata, strongly indicative of allelopathic inhibition by G. divaricata (Fig 5D).
5 years.The photos clearly show that G. divaricata dominated the reef substrate of the site in both, the cooler northeast monsoon (winter) season (Fig 5.A, water temperature: 22.5 o C), and the warmer southwest monsoon (summer) season (Fig 5.B, water temperature: 29 o C).Due to challenging weather conditions, we were only able to conduct our quantitative temporal survey in April, the last month of the winter season, and therefore we cannot rule out potential variations in G. divaricata cover over the full length of that season.

Fig 1 .
Fig 1. Study site.A) Geographical location of

Fig 2 .
Fig 2. Spatial variation in benthic categories in the Dongsha lagoon.

Table. Information and Genbank numbers of Galaxaura divaricata samples from various locations in the lagoon of Dongsha Atoll that were used 624 for DNA barcoding in this study. 625
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Table. Percent cover of Galaxaura divaricata on 13 patch reef sites in the lagoon of Dongsha Atoll, South China Sea.
Data are relative cover expressed as mean + SD (%) of 45 replicate quadrats for reef top and reef slope.

S8 Table. Paucity of macrobenthic invertebrates in the Dongsha lagoon. 645
Data derived from a belt transect survey of 13 patch reefs and seven seagrass beds (10,000 m 2 total area 646 surveyed) across the lagoon of Dongsha Atoll in September 2017.647 648