Morphological traits associated to a dense cover of endolithic algae in the seven species of the genus Agaricia Research project for Biology BSc. July 2019. Universidad de Los Andes, Bogotá, Colombia.
Student: Amalia Murgueitio Calle Director: Juan Armando Sánchez
Resumen: Las algas endolíticas, en particular las del género Ostreobium, son simbiontes controversiales. Como mutualistas pueden asistir a los corales en periodos de blanqueamiento, enfermedad y adaptación a ambientes oscuros. Como parásitos, pueden afectar y disolver el esqueleto del coral. Con un incremento en la temperatura del mar, la acidificación del océano y la actividad de los microbioeroedores, comprender el rol de estos simbiontes resulta crucial. Una comparación entre áreas con y sin cobertura densa de algas endolíticas fue realizada para las siete especies del género Agaricia empleando Microscopía de Barrido de Electrones (MEB). Este estudio tuvo tres objetivos: (i) evaluar si cavidades estructurales presentes en Agaricia podrían estar siendo ensanchadas por la flora endolítica (ii) reportar en qué especies se presentan estructuras en forma de tubería que atraviesan los septos de los corales (iii) buscar, medir y cuantificar trazas de microbioerosión. Las cavidades estructurales en Agaricia son un rasgo poco explorado que no se asocia a la flora endolítica. Las ‘tuberías’ fueron reportadas por primera vez en A. fragilis y A. tenuifolia. Estas formaciones podrían estar asociadas a un intento de Ostreobium por mejorar la captura de luz en ambientes oscuros, ayudando al coral a adaptarse a ellos. El número y proporción de trazas de microbioerosión fue significativamente mayor en áreas con cobertura densa de algas endolíticas. Diferencias en la textura de la cara inferior de los corales fue evidente entre ambos tipos de muestra. Aunque los números muestrales por especie fueron bajos, hubo diferencias en la magnitud de la bioerosión entre especies. Bajo escenarios de incrementada actividad de microbioerosión, la magnitud de las respuestas de las especies podría depender de las propiedades estructurales de los esqueletos de coral. Por tanto, es importante comprender el impacto de los efectos asociados a la actividad del ensamble endolítico para una gama de especies. Abstract: Endolithic algae, in particular those of the genus Ostreobium, are controversial symbionts. Acting as mutualists they can aid corals through bleaching events, disease and adaptation to low light environments. As parasites, they could harm and mostly dissolve the coral skeleton. With rising sea temperatures, increasingly acidifying waters and an enhanced activity of microboarers, understanding the role of this symbionts seems crucial. A comparison between areas of dense endolithic algae coverage and areas without this coverage was performed for the seven species of Agaricia through Scanning Electron Microscopy (SEM). The three objectives of this study were: (i) to assess whether structural cavities present in Agaricia could be enlarged by the activity of endolithic algae colonizing them (ii) to report pipeline- like structures across coral septae and the species where they occur (iii) to search, measure and quantify traces of microbioerosion. Structural cavities in Agaricia are poorly explored traits and probably unrelated to the activity of the endolithic flora. Pipeline-like structures were reported for the first time in A. fragilis and A. tenuifolia. These structures could be associated to attempts performed by Ostreobium to enhance light-capture in low-light environments, aiding corals in adapting to them. Differences of texture in the lower plate of corals were evident between both kinds of samples. The number and proportion of microbioerosion traces was significantly higher in samples with a dense coverage of endolithic algae. Although sampling numbers per species were low, differences in the magnitude of bioerosion per species were observable. In scenarios of enhanced microbioerosion, the magnitude of bioerosion responses might depend on the structural properties of coral skeletons. Thus, it is of upmost importance to understand the extent of the effects associated with the activity of the endolithic assemblage for a wide range of species. Introduction Coral reefs are among the world’s most biodiverse ecosystems and their integrity is crucial to humanity because they provide ecosystem services such as food security, coastal protection, biochemically relevant compounds, building materials and tourism (Hughes, Barnes, et al., 2017; Lamb et al., 2018; Moberg & Folke, 1999; Weijerman et al., 2018). Unfortunately, coral reefs currently face multi-dimensional anthropogenic threats that arise from local and global pressures. Local pressures may come from the land (e.g. deterioration of water quality associated to nutrient inputs, pollutants, accumulation of plastic debris and sediments) or from the sea (e.g. habitat destruction, overexploitation, the introduction of potentially invasive species); they may arise from the spread of infectious diseases, or they may be associated to global environmental change, such as sea warming, increasing ocean acidity, sea level rise, increased ocean stratification and altered ocean circulation patterns (Doney et al., 2012; Harvell, 2002; Jackson, 2010; Weijerman et al., 2018). Together, the prospects for how these irreplaceable ecosystems might change over this century, are alarming (Hoegh-Guldberg et al., 2007; Hughes, Barnes, et al., 2017). Present conditions are more extreme than those known to have existed for the last 420,000 years, the period during which most extant marine organisms evolved (Hoegh-Guldberg et al., 2007). For example, the current CO2 concentration (~415 ppm) has significantly shifted to the right with respect to the cluster points representing the last 420 millennia. In fact, the atmosphere already contains more carbon dioxide than in any other period in the last 800 millennia (Lüthi et al., 2008; NOAA, 2019). Also, the concentration of carbonate ions (~210 µmol kg-1) has reached the lowest level for the last 420,000 years. In addition to this, seawater has become more acidic since the beginning of the industrial era (-0.1 pH units) and sea temperature is warmer (+0.7 ºC) (Salvat & Allemand, 2009; Petit et al., 1999; Hoegh- Guldberg et al., 2007). Under these novel circumstances, population-level shifts are occurring in marine ecosystems due to physiological intolerance to new environmental conditions. Also, altered dispersal patterns and interactions among species are being documented globally, leading to major shifts in community structure and diversity and making the emergence of novel ecosystems possible. The case of coral reefs is particularly concerning since they are considered the canary in the coal mine for temperature rise and ocean acidification, inasmuch as climate change effects are already noticeable (Doney et al., 2012). The ability of corals to build massive complex structures that support the whole ecosystem is vulnerable to mild shifts in pH and temperature (Doney et al., 2012; Kleypas et al., 2006). A temperature rise of 1ºC is known to cause coral bleaching, where coral tissues lose their color after releasing the symbiotic algae, zooxanthellae, that provide photosynthates to their host (Hoegh-Guldberg et al., 2007). Three massive global-scale bleaching events have been reported since the 1980s, all associated to thermal stress during marine heatwaves (Hughes et al., 2017b). While moderate bleaching is a form of stress that reduces growth rate, severe bleaching causes coral death. Bleaching events might also be related to a higher susceptibility of corals to disease. The combination of bleaching and disease may have devastating effects on living corals at a large spatial scale (Miller et al., 2009). On the other hand, for corals and other calcifying organisms, ocean acidification makes the process of secreting and maintaining a skeleton more difficult (Salvat & Allemand, 2009). Acidic conditions could mean complete dissolution of coral skeleton, as reported at pH values between 7.3 and 7.6 for two Mediterranean scleractinian species (Fine & Tchernov, 2007). Also, acidifying waters jeopardize the ability of young recruits to achieve the levels of calcifying fluid supersaturation needed to support rapid growth (Cohen, McCorkle, Putron, Gaetani, & Rose, 2009). Growth rates of adults might be reduced under more acidic conditions and coral skeletons may become less dense, increasing the likeliness of damage during storms (Doney et al., 2012). Current massive losses of coral cover in reefs indicate corals will be particularly vulnerable to future increases in temperature and acidification (Jackson, 2010; Doney et al., 2012). In the present, one third of zooxanthellate reef-building corals are under categories of high extinction risk (Carpenter et al., 2008) and the scenario for the upcoming years is expected to worsen. Between 2050 and 2100 CO2 concentration is expected to exceed 500 ppm and global temperatures will rise at least 2ºC. Under these expected conditions, carbonate accretion will be compromised, and corals will become rarer on reef ecosystems. Expected results include less diverse reefs dominated by macroalgae and noncoral communities together with carbonate reef structures that are unable to be maintained (Hoegh-Guldberg et al., 2007). Populations of several species are expected to decline with the loss of their coral-constructed habitat (Doney et al., 2012). For example, changes in fish assemblages, including declines of diversity and abundance, can occur due to coral cover loss (Pratchett, Hoey, Wilson, Messmer, & Graham, 2011). In fact, it has been suggested that fish biodiversity is threatened by reef degradation associated to climate change and the creation marine reserves isn’t enough to secure their survival (Jones, McCormick, Srinivasan, & Eagle, 2004). Invertebrates will also be threatened by the loss of coral cover. Since coral traits are related to the diversity of reef-associated invertebrates, progressive decline of coral habitat is expected to lead to losses in invertebrate diversity (Idjadi & Edmunds, 2006). Coral loss is also expected to have important socioeconomic impacts for coastal communities and regional economies. For instance, the decline in fish populations associated with loss of coral cover will have a negative impact on fisheries (Hoegh-Guldberg et al., 2007). Low income countries highly dependent on tourism might face severe consequences with a shift away from coral-dominated reefs since this will alter the perceived attractiveness of these ecosystems for tourists (Bryant, Burke, McManus, & Spalding, 1998). Countries such as Australia and the United States, where income related to tourism in coral reefs is very high, will suffer as well economic loss (Hoegh-Guldberg, 2011). Coastal protection provided by reef barriers will also be in jeopardy, leaving coastal communities and ecosystems such as mangroves, saltmarshes and seagrass meadows more vulnerable to the impact of storms and hurricanes (Barbier, 2015; Hoegh-Guldberg, 2007). This is especially worrying under a scenario of increasingly severe hurricanes and cyclones in the tropics (Webster, Holland, Curry, & Chang, 2005). The combined effect of sea level rise, the loss of beach sand attributed to storms and hurricanes together with the loss of sand production linked to coral reefs will lead to less stable beach ecosystems. This will likely have an important impact on the subsistence of coastal communities, fishing activities and tourism (Hoegh-Guldberg et al., 2007). Under a scenario of anthropogenic increase of carbon dioxide, warmer sea surface and altered ocean chemistry, there is an increasing need for developing management strategies for a large-scale crisis. Together with other strategies, a scaling-up of management practices must rely on an improved understanding of the ecological processes that underlie the resilience of coral reefs (Bellwood, Hughes, Folke, & Nyström, 2004). Since corals have important functional roles in reef ecosystems, such as the creation of three-dimensional habitats and contributing to reef growth as framework builders, understanding in detail the biology and ecology of corals is key for creating adequate strategies to cope with the current crisis (Rossi, Bramanti, Gori & Orejas, 2017; Bellwood, Hughes, Folke & Nyström, 2004). The study of scleractinian corals requires understanding the complex associations with the microorganisms that coexist within the calcium carbonate structure. The host-microbial assemblage is referred to as the coral ‘holobiont’, which consists of the coral animal, zooxanthellae, prokaryotes, fungi, protozoa and endolithic algae (Rohwer, Seguritan, Azam, & Knowlton, 2002; Thurber et al., 2009). Given its role in coral nutrition and the threat that is coral bleaching for reef ecosystems, the genus Symbiodinium is by far the most studied among coral symbionts (del Campo, Pombert, Šlapeta, Larkum, & Keeling, 2017; Glynn, 1993). However, other symbionts might also have a less studied yet very important role for understanding ecological interactions in coral reefs and predicting what can be at stake with the multiple stressors that interact synergistically in the oceans of the Anthropocene. These less studied symbionts include several kinds of endolithic organisms, both autotrophic or heterotrophic, that can live inside hard substrata such as coral skeletons. Many of them contribute to reef bioerosion, which is considered the main form of reef degradation (Tribollet & Golubic, 2011). Endoliths include euendoliths, chasmoendoliths and cryptoendoliths. Euendoliths actively penetrate the interior of hard substrates through biochemical dissolution; chasmoendoliths colonize fissures and cracks in substrates and cryptoendoliths colonize structural cavities available in porous substrates. These organisms, the boring microflora that can be found in coral substrates, include cyanobacteria, fungi together with chlorophyte and rhodophyte algae (Tribollet, 2008b). Boring algae are endolithic photothropic organisms that perforate the substrata by burrowing small cavities in which they reside (Verbruggen & Tribollet, 2011); they can also be found in preexisting cavities of skeletons or dead substrates (Schlichter, Kampmann, & Conrady, 1997). Boring algae are amongst the oldest organisms on Earth. They appeared several hundreds of millions of years before coral reefs rose to dominance 60 million years ago (Verbruggen & Tribollet, 2011). Also, Endolithic algae have a high physiological diversity as well as a cosmopolite geographical distribution (Tribollet, 2008b; Verbruggen & Tribollet, 2011). From an ecological perspective, it has been proposed that endolithic algae have a dual role as coral symbionts (del Campo et al., 2017; Gutner-Hoch & Fine, 2011; Verbruggen & Tribollet, 2011). On the one hand, metabolic interactions between endolithic algae and coral hosts suggest the existence of a mutualistic ectosymbiosis, in which the algae are involved in the cycling and saving of nutrients. Thus, endolithic algae contribute to the high productivity of photic tropical and subtropical reef ecosystems (Schlichter et al., 1997), although less than zooxanthellae. However, endolithic algae are also internal agents of microbioerosion, actively penetrating into carbon substrates by dissolving them (Tribollet & Golubic, 2011). Thus, the destabilization of the calcium carbonate skeletons of corals can be understood as a parasitic interaction where the presence of endolithic algae likely impacts reef topology and makes corals more susceptible to abrasion and further dissolution (Schlichter et al., 1997; Verbruggen & Tribollet, 2011). Endolithic algae also have an important role in the trophic chain, where they serve as food for macrobioeroders such as sea urchins or parrotfishes, that scrape the reef in order to get them (Verbruggen & Tribollet, 2011). The most common endolithic algae belong to the genus Ostreobium (Gutner-Hoch & Fine, 2011; Verbruggen & Tribollet, 2011; Vroom & Smilth, 2003), a little-known genus from a phylogenetic perspective despite its ecological importance (del Campo et al., 2017; Gutner- Hoch & Fine, 2011). There seems to be consensus about the fact that Ostreobium is an early- branching group in the Bryopsidales (Ulvophyceae, Chlorophyta) (Cremen et al., 2019; del Campo et al., 2017; Verbruggen et al., 2009; Verbruggen, Marcelino, Guiry, Cremen, & Jackson, 2017). Species of this order have a siphonous structure in which the thallus consists of a siphon, a single tubular structure with thousands of nuclei and chloroplasts (Cremen et al., 2019; Vroom & Smilth, 2003). As siphonous green algae, they lack internal cell walls and membranes compartimentalizing cell components into discrete chambers (Vroom & Smilth, 2003). However, being endolithic and microscopic, Ostreobium is an unusual genus in its order (Verbruggen et al., 2017). There seems to be consensus about what we call Ostreobium being a monophyletic group (del Campo et al., 2017; Marcelino & Verbruggen, 2016; Verbruggen et al., 2009, 2017). This clade is one of the three main branches within the Bryopsidales, together with the Bryopsidineae and the Halimedineae (Verbruggen et al., 2009, 2017). Based on a multi-locus approach, Verbruggen et al. (2009) proposed that the clade leading to the genus Ostreobium deserves a suborder-level recognition which they tentatively called Ostreobidineae. Verbruggen et al. (2017) reached the same conclusion based on chloroplast genome data, naming the suborder Ostreobineae. Internal relationships within this genus are less well understood. Lukas (1974) described O. constrictum morphologically for the first time and included filament forms previously classified as O. reinekei within O. queckettii. She also provided a key for the following species belonging to the genus Ostreobium: O. constrictum, O. brabantium, O. okamurai and O. duerdenii. By 2016, Marcelino & Verbruggen claimed that, although there were inconsistencies in the literature, the description of three species belonging to Ostreobium was available by the time they published their results. More recent approaches to the taxonomy of these endolithic algae concluded that Ostreobium is a complex formed by multiple lineages (Verbruggen, 2017), although how many lineages compose Ostreobium is still an unresolved matter. For example, Gutner-Hoch & Fine (2011) found that there are at least seven clades of Ostreobium living in two species of corals from the Red Sea. Clade distribution was variable depending on both host and depth. Del campo et al. (2017) identified three different clades of Ostreobium, each with a different geographical distribution. Sauvage, Schmidt, Suda, & Fredericq (2016) reported a large phylogenetic cryptic diversity for this genus, identifying an estimated of 85-95 species-level entities. Marcelino & Verbruggen (2016) identified more than 80 taxonomic units at the near- species level, divided into four subclades. Recently, nine new Ostreobium clades (OUT 99%) were identified in a Pocillopora coral (Massé, Dormart-Coulon, Golubic, & Tribollet, 2018). Ostreobium is a very widespread coral symbiont reported in 85% of coral species across a geographic and bathymetric range (Gutner-Hoch & Fine, 2011). These endolithic algae reside within the calcareous skeleton, where they often create a high-density green band underneath the coral tissue (Highsmith, 1981; Magnusson, Fine, & Kühl, 2007). Photosynthesis in Ostreobium is quite particular, displaying a set of adaptations that allow them to inhabit low-light environments like the limestone skeleton of a coral or deep-water habitat where light penetration can be less than 0.01% (Verbruggen & Tribollet, 2011). For instance, Ostreobium can absorb green light using xanthophyll pigments, is able to alter its chlorophyll b:chlorophyll a ratio according to water depth, and can absorb light in the red and far-red wavelengths (Fork & Larkum, 1989; Koehne, Elli, Jennings, Wilhelm, & Trissl, 1999; Magnusson et al., 2007; Schlichter et al., 1997; Shashar & Stambler, 1992). There is evidence for both a mutualistic and a parasitic interaction of Ostreobium in corals (Tribollet, 2008b; Verbruggen & Tribollet, 2011). For instance, transfer of photoassimilates from Ostreobium to the coral host has been reported for carbon (Sangsawang et al., 2017; Schlichter, Zscharnack, & Krisch, 1995) and nitrogen (Sangsawang et al., 2017). During summer bleaching events, Ostreobium receives increased photosynthetically active radiation, grows considerably in biomass and produces rising amounts of photoassimilates that get translocated to the host. Transfer of 14C-labelled carbon from the algae to the coral Oculina patagonica was found to be significantly higher than the transfer of these endoliths to non- bleached individuals. It has been proposed that this translocation has a potential role in assisting host’s survival through bleaching events (Fine & Loya, 2002). However, this source of energy might be insufficient for coral reproduction and it is probably limited to aiding the host’s survival and recovery from stress (Fine, Loya, & Zibrowius, 2001; Tribollet, 2008b). High blooms of euendoliths, consisting mostly of Ostreobium sp. after bleaching events have been recorded in the Great Barrier of Reef. When zooxanthellae disappear, euendoliths adjust well to high temperature and light intensities, except when they were exposed too quickly to these conditions (Fine, Meroz-Fine, & Hoegh-Guldberg, 2005; Fine, Steindler, & Loya, 2004). This might suggest that the euendolithic assemblage has an important role in reef resilience to bleaching events, but this might vary with environmental conditions and over time (Tribollet, 2008b). Blooms of euendoliths have also been reported following a white- syndrome disease event. A higher biomass of euendoliths could mean that more photoassimilates are transferred to the host, thus assisting it through the stressful event. However, a higher biomass could also mean a more pervasive dissolution of the calcium carbonate skeleton, suggesting a parasitic interaction (Fine, Roff, Ainsworth, & Hoegh- Guldberg, 2006). A parasitic interaction involving Ostreobium might be related to damage of coral tissue performed by this symbiont. Ostreobium spp. filaments have been found invading gastrodermal cells of several coral species, causing abnormalities in coral tissue (Peters, 1984). Tissue penetration by Ostreobium in acroporids has also been reported and could be related to the creation of micro-lessions which would make the coral more susceptible to pathogen attack (Fine et al., 2006). These results might, however, not be generalizable. For instance, early colonization by Ostreobium of young Pocillopora damicornis recruits didn’t seem to slow the extension rates of corals, indicating that fitness of recruits wasn’t altered by an early assembly of the symbiosis (Massé et al., 2018). The most explored parasitic role of Ostreobium is related to the dissolution of calcium carbonate structures, as this algae complex is the most pervasive and active agent of microbioerosion (Grange, Rybarczyk, & Tribollet, 2015; Tribollet, 2008b, 2008a; Tribollet, Godinot, Atkinson, & Langdon, 2009). In an experiment performed in the Great Barrier of Reef, Tribollet (2008a) found that for dead experimental blocks of Porites sp., Ostreobium -2 -1 was the main agent of microbioerosion, dissolving more than 1 kg of CaCO3 m year . Also, microbioeroders such as Ostreobium can colonize the skeleton of live corals from their base as soon as larvae settle or enter through lateral fissures. However, colonization of coral skeletons from the outside is restricted by polyps. Bioerosion can be quite pervasive in living substrates, although less strong than in dead ones (Le Campion-Alsumard, Golubic, & Hutchings, 1995; Tribollet & Golubic, 2005; Tribollet, 2008a). In fact, heavy erosion can make living corals more susceptible to breakage during storms or hurricanes (Tribollet & Golubic, 2011). The bioeroding activity of this symbiont has been found to increase under warmer and more acidic ocean conditions. Tribollet et al. (2009) placed coral blocks in seawater with 400 ppm and 750 ppm of pCO2 for three months, documenting the recruitment of an assemblage of endolithic and ephilic microorganisms. Ostreobium was the dominant microbioeroder, with filaments penetrating deeper in the high-CO2 treatment, favouring a 48% increase in dissolution of calcium carbonate with respect to the 400-ppm treatment. Another study of substrate colonization by endoliths was based on IPCC scenarios for the end of the century representing reduced (B2) and ‘business as usual’ (A1F1) green-house gas emissions (A1F1) (Reyes-Nivia, Diaz-Pulido, Kline, Hoegh-Guldberg, & Dove, 2013). A higher abundance of Ostreobium spp. was reported for high pCO2-temperature scenarios. Also, enhanced skeletal dissolution was found under enhanced pCO2-temperature scenarios and was associated with higher endolithic biomass and respiration. Thus, it is expected that under acidifying and warming waters, microbioerosion by Ostreobium will become even more pervasive This dual role associated to Ostreobium makes this symbiont a very important element for understanding the future of coral reefs under anthropogenic climate change. Ostreobium could be aiding corals to survive through bleaching and disease events, thus enhancing coral survival and recovery after stressful events. However, with decreased rates of calcification of hosts, these endoliths could also be eroding coral skeletons at a rate that might threaten the integrity of corals and other calcifying organisms. Thus, an enhanced understanding of the conditions that could favor either a parasitic or mutualistic response is very important for coral reef science and for predictions regarding the effects of acidification and warming events. Very little work has taken place in the Caribbean for assessing endolithic assemblages and the symbiosis of Ostreobium with corals. Green bands associated with endolithic assemblages have already been reported for six of the seven species of Agaricia, as well as several other species (Delvoye, 1992). Previous research performed with Agaricia undata, a depth generalist coral that colonizes mesophotic reefs as deep as 100 m, showed a greater diversity of Ostreobium (12 clades) in comparison to Symbiodinium (7 clades) (Gonzalez- Zapata et al., 2018a; González-Zapata, Gómez-Osorio, & Sánchez, 2018b). Genetic structuring of both host and Symbiodinium wasn’t found for A. undata across depths. Thus, this lack of genetic partitioning in a bathymetric gradient suggests that the identity of Symbiodinium isn’t responsible for the adaptation of the coral to a broad depth range and low light environments (Gonzalez-Zapata et al., 2018a). In contrast, most clades of Ostreobium spp. were partitioned according to reef setting (oceanic or continental siliciclastic influence). This was expected because it is likely that light conditions, dissolved particles and other factors, such as macroscopic predators, create specific associations between hosts and symbionts across a depth gradient (González-Zapata et al., 2018b). The high diversity and bathymetric structuring of Ostreobium clades, together with the lack of partitioning evidenced for hosts and Symbiodinium, seem to suggest that the presence of the endolithic algae could be involved in the ability of A. undata to colonize mesophotic ecosystems. A. undata, however, isn’t the only species in its genus to show a symbiosis with Ostreobium. Agaricia (Agariciidae) is a genus of brooding scleractinian corals present in ten ecoregions from the Caribbean and the Atlantic1. (Veron, Stafford-Smith, Turak, & Devantier, 2019). While all seven species of the genus show this symbiosis, they do not share the quality of being depth generalists. In fact, while some species like A. grahamae and A.undata are quite dominant at 60-100 m, others such as A. humilis and A. agaricites are associated with shallow waters (Bongaerts et al., 2013; Hoeksema, Bongaerts, & Baldwin, 2017). Since Ostreobium could be associated with several resilience-related processes, as well as adaptation to low- light environments for Agaricia, research should be performed in order to understand the implications for this symbiosis at a genus level. Thus, the objective of this study is to analyze which morphological traits are associated to a dense coverage of Ostreobium and discuss their possible ecological implications in the seven species belonging to the genus: A. undata, A. lamarcki, A. tenuifolia, A. agaricites, A. humilis, A. fragilis and A. grahamae (Veron, 2000). González-Zapata et al. (2018b) showed how exposed algal filaments in the surface of A. undata emerged as tubular pipeline-like structures from one coral costa to another. These authors hypothesized that these ‘pipelines’ were built by Ostreobium using residual calcium carbonate from the boring process to improve light uptake in the mesophotic zone. The first objective of this study was to search for these pipeline-like structures in the seven species of
1 Flower Garden Banks, Gulf of Mexico; Netherlands Antilles and south Caribbean; Cuba and Cayman Islands; Brazil; Bay of Campeche, Yucatán, Gulf of Mexico; Hispaniola, Puerto Rico and Lesser Antilles; Bahamas and Florida Keys; Belize and West Caribbean; Jamaica; Bermuda. the genus Agaricia and report where they are present. The second objective was to assess whether Ostreobium could enlarge cavities present in the surface of all the species of Agaricia. Ostreobium is known for its role as an euendolith, however has also been reported also as a chasmoendolith, colonizing fissures and cracks (Schlichter et al., 1997) ephilitic behavior has been reported as well (Golubic, 1973). The presence of structural cavities makes Agaricia spp. an interesting genus to test for a possible cryptoendolithic behaviour for these endolithic algae. Thus, it is possible that algae could use these cavities as cryptoendoliths and then enlarge them as a part of the boring process. The third objective was to search for traces of bioerosion in the calcium carbonate skeletons of these corals and assess whether bioerosion traces are associated with a dense cover of endolithic algae. This, in order to give a preliminary step for understanding how pervasive microbioerosion can be for this genus under current CO2 and temperature conditions.
Materials and methods Study site
Samples for this study were collected in different locations of the Colombian Caribbean, including several reef complexes in the departments of Bolívar, Magdalena and the San Andrés, Providencia and Santa Catalina Archipelago. For this study, samples were selected from the Museo de Historia Natural- ANDES without a geographical criterion and they came from the locations listed below.
Figure 1. General map of the sampled locations. All maps were created with QGIS.
The “Parque Nacional Natural Corales de Profundidad” is a marine protected area that monitors deep-sea ecosystems (Gonzalez-Zapata et al., 2018a). It was created in 2013 and has an area of 149 thousand hectares. The MPA is located 12 km away from the “Parque Nacional Natural Corales del Rosario y San Bernardo” and 32 km away from the Peninsula of Barú, the nearest point in the continent (Parques Nacionales, 2019). The depths vary between 34 and 800 m. It is estimated that 40% of the biodiversity located in the edge of the continental platform in the Colombian Caribbean is protected by this MPA. The park protects three main habitats: soft bottoms in the deep-sea, deep-water corals and mesophotic coral reefs (Maldonado & Cuervo Sánchez, 2016).
Figure 2. Parque Nacional Natural Corales de Profundidad Map. The Archipelago of San Andrés, Providencia and Santa Catalina consists of a series of oceanic islands, atolls and coral reefs located outside the continental platform of the Caribbean in Nicaragua (Díaz & García-Llano, 2010; Geister, 1973). San Andrés Island is the largest emerged portion of this oceanic archipelago. During the Miocene, it originated as a coralline atoll. The highest part of the island, the San Luis formation, consists of a calcareous crest made of Neogene lagoon and reef deposits. On the other hand, a recent reef complex formed during the Holocene covers most of its submerged part (Díaz & García- Llano, 2010). Currently, the pressure of tourism and overexploitation on San Andrés reefs coincides with documented changes in the morphofunctional cover of certain reefs (González Gamboa, 2019).
Figure 3. San Andrés and Cayo Bolívar Map. Samples were also collected from continental shelf reef banks in Cartagena, Bolívar (Velásquez & Sánchez, 2015). The coral reef system close to Cartagena is amongst the most complex siliciclastic coral reefs (Gonzalez-Zapata et al., 2018a; Sánchez, 1999). The southwestern part of Cartagena, where the sampling sites are located, has a high sediment load due to proximity to the Dique canal at the mouth of the Magdalena River. Reefs in this area have been reported to exceed turbidity threshold values for healthy coral reef waters, including reefs in the “Parque Nacional Natural Corales del Rosario y San Bernardo” (Restrepo, Park, Aquino, & Latrubesse, 2016). Turbidity in this location makes light penetration considerably lower than locations in San Andrés and PNN Corales de Profundidad. Thus, light penetration at 40 m in Cartagena can be comparable to light penetration at 70-80 m in San Andrés (Gonzalez-Zapata et al., 2018a).
Figure 4. Cartagena Map. Granate is a small cove located a few kilometers northeast from Santa Marta, Magdalena. The bay area is protected from strong trade winds by a rocky promontory, Punta de la Aguja. Reefs exist in the periphery and can be found down to a 25 m depth (Erhardt & Werding, 1975). Human impacts in the area result from a nearby city with considerable population, tourism, a harbor and industrial activity, known to affect coral reefs (Gil Agudelo & Garzón- Ferreira, 2001; Zea, 1994).
Figure 5. Santa Marta sampling region Sample collection A total of 42 coral colonies were selected for this study. Coral samples were collected by Juan Armando Sánchez, Laura Rodríguez and Adriana Rodríguez between 2015 and 2018 from the localities mentioned above (Table 1). Samples were collected between 0 to 87 m using SCUBA or mixed-gas closed circuit rebreather (CCR) below 40 m. A fragment of a live coral colony was cut for every sample using a hammer and a chisel. Samples were taken back to Biommar laboratory in Bogotá, where they were oven dried at 60°C for 36 hours. Then, they were identified using a morphological key (Rodríguez Martínez & Rodríguez Bermudez, unpublished), and an ANDES-ID code was assigned for each of them. Finally, samples were placed on a Ziploc® bag and a tagged box to be deposited in the Museo de Historia Natural-ANDES. Sample selection Different from other coral species, the endolithic assemblage living within Agaricia is usually visible at the surface, and mostly grows from the base to the borders (González-Zapata et al., 2018b). However, at times a green band is also visible mostly in thicker samples. Six individuals per species were selected from the available collection at the Museum. Samples that appeared to be well preserved were preferred. Selection of the samples was based on the criteria of having 5 corals per species with an area of visible and dense Ostreobium spp. coverage and an area without the coverage. A selection of one specimen showing no visible colonization by endolithic algae was attempted. However, it was not always possible to meet these criteria for some species showing either fully covered corals by the endolithic algae (e.g. A. agaricites) or more than one sample per species showing no visible coverage (e.g. A. tenuifolia). Some samples that looked in a poor shape were selected for A. humilis due to lack of better-preserved samples. Sample selection also aimed to be representative of the available depth gradient for every species. Area Area with without dense ANDES- Depth dense Species Location Ostreobium IM [m] Ostreobium cover cover available available 4749 38 Trampa Tortuga, SAI No Yes 4778 37 Trompadas, Cartagena Yes Yes Agaricia 4690 45 Imelda Real, Cartagena Yes Yes undata 4783 65 Trampa Tortuga, SAI Yes Yes 4729 85 Trampa Tortuga, SAI Yes Yes 4711 17 Montañita, Cartagena Yes Yes 4295 85 Nirvana, SAI Yes Yes 6418 40 a 45 Octubre Rojo, Cartagena Yes Yes Agaricia 6496 11 a 18 Octubre Rojo, Cartagena Yes Yes lamarckii 4314 29 Trompadas, Cartagena Yes Yes 4298 40 Nirvana, SAI Yes Yes 6608 25 a 30 Trompadas, Cartagena No Yes 6613 35-30 Trompadas, Cartagena Yes Yes 4108 17 Montañita, Cartagena No Yes Agaricia 4107 30 Isla Tesoro, Cartagena Yes No agaricites 4111 17 Montañita, Cartagena Yes Yes 4116 12 Nirvana, SAI Yes Yes 4101 40 Cayo Bolivar, SAI Yes No 4259 15 Nirvana, SAI Yes Yes 4255 8 Granate, Santa Marta Yes No Agaricia 4256 8 Granate, Santa Marta Yes No humilis 1090 0 Atlántico, Cartagena No Yes 4258 8 Granate, Santa Marta Yes No 4254 8 Granate, Santa Marta Yes No 4199 80 Trampa Tortuga, SAI Yes Yes 4195 66 Granate, Santa Marta Yes Yes Agaricia 6567 40-50 Trompadas, Cartagena Yes Yes fragilis 6611 25-30 Trompadas, Cartagena Yes Yes 4197 60 Nirvana, SAI No Yes 4202 65 Trampa Tortuga, SAI Yes Yes 4248 28 Trompadas, Cartagena No Yes 4243 36 Trampa Tortuga, SAI Yes Yes 4228 45 Nirvana, San Andrés Yes Yes Agaricia 4240 40 Nirvana, San Andrés Yes Yes grahamae PNN Corales de 4226 55 profundidad Yes Yes 4233 85 Nirvana, San Andrés Yes No 4671 5 La Caída, Cartagena No Yes 4672 6 La Caída, Cartagena No Yes Agaricia 4673 4 La Caída, Cartagena No Yes tenuifolia 4674 9 La Caída, Cartagena Yes No 4675 18 Montañita, Cartagena Yes Yes 4676 17 Montañita, Cartagena No Yes
Table 1. Coral samples from Museo de Historia Natural ANDES.
Figure 6. Coral samples. A. undata a. a) ANDES-IM 4690. Endolithic algae showing dense green coverage in a part of the coral. A. lamarcki b-d. b) Red circle shows green band pattern in ANDES-IM 6496. c) Dense endolithic algae coverage in ANDES-IM 6418. d) ANDES-IM 4295 with dense cover towards the base. A. humilis e-f. e) ANDES-IM 4259. Round-shaped colony with dense cover near the base. f) ANDES- IM 1090 without visible macroscopic Ostreobium cover. A. grahamae g-h. g) ANDES-IM 4228 green dense coverage in half of the sample. ANDES IM-4248 mostly without visible cover by endolithic algae. h) ANDES-IM 4228. Most of the sample shows no sense coverage of endolithic algae. A. fragilis i. i) ANDES-IM 6567 with endolithic algae cover towards the base. A. agaricites j. j) ANDES-IM-6613 with an endolithic algae cover. A. tenuifolia k-l. k) ANDES-IM 4674 showing algae cover. l) ANDES-IM 4676 without a visible cover of endolithic algae.
Scanning Electron Microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) Areas of dense coverage of endolithic algae were identified and then determined by visual estimation of the endolithic algae in the coral dry fragment using a stereoscope. Areas without such dense coverage were also identified and used as controls only when no visual signs indicated the presence of algal filaments under the stereoscope. Three different types of cuts were made for areas with and without endolithic algal coverage: cuts for observing the upper plate of the corals, cuts for observing the lower plate of the corals (for unifacial samples, A. tenuifolia excluded) and transversal cuts. For surface and base samples, a motor-tool was used to cut dry fragments of around 1 cm2. For transversal samples, a notch was made using the motor-tool and a fracture of the coral was made using two pliers. Organic matter attached to the coral surface was removed first with water pressure using a waterpik, and then by 5- minute exposure to sodium hypochlorite 5.25% (weight). Samples were then washed with distilled water and placed for 10 minutes in a beaker with hydrogen peroxide. Finally, they were washed with distilled water and fixed in ethanol 40%. When samples were completely dry, they were taken to the Centro de Microscopía, Uniandes for sample preparation. Coins of COP $50 or $100 (metallic alloys) were covered with aluminium foil. SEM conducting double-sided carbon tape was used to attach samples to the covered coin. A brushstroke of graphite carbon paint was applied on the corner of the coin and the border of the samples. Samples were coated with gold before SEM and EDS were executed. Sample observation was performed using a JEOL JSM-6490LV Scanning Electron Microscope. Occasionally a Tescan Lyra 3 SEM Dual Beam SEM-FIB was employed for observation and EDS. Photos of transversal samples were taken without a protocol since thickness of the corals is so variable. Bioerosion traces were sought upon the carbonate surface and when they were absent a picture of the smooth calcium carbonate structure was taken. For upper plate and lower plate samples, a photo of the whole sample was taken (10- 20X approximately). Five areas of the sample were selected for 30X and 100X pictures. A detailed picture was included at a convenient magnification (350-500X). EDS was performed when pipeline or structures were found on samples. EDS was performed on the calcium carbonate structure of the same coral as a control. Image analysis Photos were analysed with ImageJ. Counts were done using the Multi-point tool after applying a grid. Measurements (e.g. pore diameter and pipeline length) were performed using the Straight-line tool. Pipeline length includes strictly the section of the pipeline not embedded in the coral skeleton. Pipeline diameter was measured at the thinnest point in the centre and the thickest point in the extremes. Distance between pipelines was measured only for parallel pipelines in the same septa when they were available. Adobe® Photoshop was used for improving shine and contrast of the pictures and for building picture mosaics. Statistical analyses All statistical analyses were performed using the software R version 3.5.3. Welch’s t-tests were used when the data did not meet the assumption of homoscedasticity. For all tests α=0.05. Boxplots and mosaic plots were also obtained using the same version of R. Structural cavities Paired t-tests were performed between samples with and without a dense cover of endolithic algae, to assess if cavity diameter was different between both kinds of samples for upper and lower plates. To avoid bias, only samples with both an area with dense endolithic coverage and without were considered. Traces of bioerosion Paired Welch’s t-tests were performed between samples with both an area with and without dense endolithic algae coverage, in order to document the number of traces of bioerosion present in 1 cm2. To avoid bias, only samples with both an area with dense endolithic coverage and without were considered. In both upper and lower plates of the corallum, Pearson’s Chi-squared contingency tests were used for assessing if the proportion of samples showing traces of bioerosion was different between the two kind of areas. A two-sample t- test was used for assessing differences in the diameter of the pores between upper and lower plate samples.
Results Structural cavities Cavities were found at both plates of the corallum, except in A. tenuifolia, which is a bifacial (Wells, 1973) and only one plate was assessed. Cavities at the upper plate where located in the valleys and had a very variable diameter, ranging from 17 to 296 μm. These cavities also had a highly variable shape, from very large and thin apertures to fully round shapes. Distance between cavities also seemed quite variable. While at times they were rare morphological traits, some corals seemed to have a narrower space between cavities than the diameter of a sole structure. Cavities at the lower plate were smaller in diameter and seemed to follow a straight line, although sometimes curves were also found. I assessed whether the presence of a dense cover of endolithic algae could enlarge these structural cavities since beside inhabiting inside limestone, Ostreobium, has been reported to inhabit structural fissures and cracks (Schlichter et al., 1997) but is also capable of chemical dissolution of CaCO3 (Reyes-Nivia, Diaz-Pulido, & Dove, 2014; Verbruggen & Tribollet, 2011). Results, however, show a significant on the opposite direction of what expected for the upper plate and a null effect for the lower plate.
Figure 7.1 Structural Cavities. Cavities in the surface of corals. Figure 7.2 Structural Cavities. Cavities in the surface of corals. A. agaricites a1-b3. a1-a3) cavities in samples with dense endolithic algae cover A. grahamae e1-f3. e1-e3) cavities in samples with dense endolithic algae cover. from ANDES-IM 4116. b1-b3) cavities in samples without dense endolithic algae e1) ANDES-IM 4240. e2-e3) ANDES-IM 4228. f1-f3) cavities in samples without dense endolithic algae cover. f1) ANDES-IM 4248. f2) ANDES-IM 4228. f3) cover from ANDES-IM 4116. A. fragilis c1-d3. c1-c3) cavities in samples with dense endolithic algae cover from ANDES-IM 4248. ANDES-IM 6567. d1-d3) cavities in samples without dense endolithic algae cover A. humilis g1-h3. g1-g3) cavities in samples with dense endolithic algae cover. from ANDES-IM 6567. g1) ANDES-IM 4254. g2) ANDES-IM 4258. g3) ANDES-IM 4259. h1-h3) cavities in samples without dense endolithic algae cover. h1) ANDES-IM 1090.
h2-h3) ANDES-IM 4259.
A. lamarcki i1-j3. i1-i3) cavities in samples with dense endolithic algae cover. i1) ANDES-IM 6418. i2) ANDES-IM 6496. i3) ANDES-IM 6418. j1-j3) cavities in samples without dense Ostreobium cover. j1) ANDES-IM 6418. j2) ANDES-IM 4314. j3) ANDES-IM 6496. A. undata l1-m3. cavities in samples with dense endolithic algae cover. l1) ANDES-IM 4729. l2-l3) ANDES-IM 4778. m1-m3) cavities in samples without dense Ostreobium cover. m1) ANDES-IM 4729. m2) ANDES-IM 4729. m3) ANDES-IM 4690. A. tenuifolia m1-n3. m1-m3) cavities in samples with endolithic algae cover. m1-m2) ANDES-IM 4675. m3) ANDES-IM 4674. n1-n3) cavities in samples without dense Ostreobium cover. n1) ANDES-IM 4675. n2) ANDE S-IM 4671. n3) ANDES-IM 4675.
Presence of Number Mean traces of Species ANDES-IM of cavities diameter SD Ostreobium counted [μm] (Yes/No) Yes - - - 4199 No - - - 4195 Yes - - - Yes 11 90.187 30.716 6567 No 21 108.053 21.517 A. fragilis Yes 6 69.747 32.739 6611 No 10 105.671 32.739 4197 No 5 150.707 32.873 Yes - - - 4202 No - - - 4749 No 7 65.092 35.766 4778 Yes 30 29.558 11.436 Yes 12 56.341 21.686 4690 No 8 44.402 13.666 Yes 9 35.360 10.912 A. undata 4783 No 6 57.842 30.641 Yes 17 52.306 12.055 4729 No 11 54.079 15.659 Yes - - - 4711 No 12 36.029 13.606 Yes 6 23.004 5.151 6613 No 8 142.342 22.648 4108 No 3 45.098 4.599 4107 Yes 6 46.618 7.42 A. agaricites Yes 9 54.706 16.708 4111 No 9 66.696 8.886 Yes 8 38.604 9.94 4116 No 6 23.138 15.772 4101 Yes 5 61.356 6.426 Yes 7 144.241 25.182 4259 No 7 155.981 50.688 4255 Yes 8 66.109 14.524 A. humilis 4256 Yes 4 111.035 34.613 1090 No 7 69.505 23.921 4258 Yes 6 73.384 10.468 4254 Yes 10 96.762 14.877 4248 No 7 72.546 8.664 Yes 10 181.177 54.572 4243 No 9 134.763 18.961 A. grahamae Yes 9 67.204 16.677 4228 No 6 66.085 15.317 Yes 5 63.967 12.787 4240 No 4 51.454 5.904 4226 Yes 9 101.654 22.513 4233 Yes 4 107.865 25.431 Yes - - - 4295 No - - - Yes 6 75.871 40.358 6418 No 5 247.373 36.895 Yes 7 163.194 28.689 6496 A. lamarckii No 6 202.053 43.698 Yes 6 50.381 11.977 4314 No 9 44.270 21.663 Yes - - - 4298 No 5 194.964 43.679 6608 No 25 143.278 37.475 4671 No 8 149.146 47.030 Yes 14 96.124 30.859 4672 No 8 89.294 37.415 4673 No 8 85.038 38.638 A.tenuifolia 4674 Yes 8 38.801 20.492 Yes 8 110.768 62.613 4675 No 12 153.313 23.930 4676 No 8 111.490 30.582
Table 2. Measurements of upper plate cavities
Mean Presence of Number of diameter of ANDES- traces of traces of Species traces of SD IM Ostreobium bioerosion bioerosion (Yes/No) counted [μm] Yes - - - 4199 No - - - Yes - - - 4195 No 10 30.842 9.583 A. fragilis Yes 9 24.397 12.849 6567 No 9 25.566 5.559 4197 No 18 27.507 7.419 Yes - - - 4202 No 7 30.834 7.085 4749 No 10 42.434 14.055 4778 No 11 28.008 17.054 Yes 5 26.859 8.639 4690 No 6 30.153 7.67 A. undata 4783 Yes 8 27.786 9.803 Yes 9 30.873 7.475 4729 No 9 30.321 7.373 Yes 19 30.983 6.370 4711 No 7 32.367 5.581 Yes 6 23.829 3.408 A. 6613 No 7 21.285 7.000 agaricites 4116 Yes 10 21.014 4.449 1090 No 5 15.954 4.535 A. humilis 4255 Yes 5 21.931 9.939
4243 Yes 7 44.497 5.247 Yes 12 19.981 6.408 A. 4240 No 11 30.275 6.274 grahamae Yes 18 31.548 10.280 4226 No 7 25.411 11.785 Yes 7 15.673 6.757 4295 No 10 18.685 4.667 Yes 3 25.937 3.042 6418 No 5 16.646 0.831 Yes 6 16.925 6.142 A. lamarcki 6496 No 8 14.620 5.057 Yes 6 50.381 11.977 4314 No 9 15.940 4.875 Yes 9 33.031 9.498 4298 No 5 27.169 7.784 Table 3. Measurements of lower plate cavities
Figure 8. Diameter of cavities in upper plate for Figure 9. Diameter of cavities in lower plate for samples with and without cover of endolithic samples with and without cover of endolithic algae. algae. Samples without a dense cover have a (mean samples with: 26.7 μm; without: 26.6 μm. P- significantly higher mean diameter (99.8 vs. 72.1 value for t-test: 0.9) μm; t.test p-value: 4.193e-06).
Pipeline-like Structures
Pipeline-like structures across the costae of corals were found in only 11.9% of the specimens. They were observed in 4 individuals of A. fragilis and one of A. tenuifolia. No pipelines were found in A. undata, A. grahamae, A. lamarcki, A. humilis or A. agaricites. Pipeline shape was variable, but mostly thicker at the extremes and thinner at the centre. Some pipelines showed a thick layer of calcification while others had a smoother aspect. These structures were found on samples both with and without a dense coverage of endolithic algae. However, in the three samples where comparison was possible, the number of pipelines per 1 cm2 was higher in samples with dense coverage. Pipeline length varied between 33 and 136 μm. In general, the mean length of the pipelines was 72.53 μm, mean diameter for the centre was 9.86 μm, the mean diameter for the extremes of the pipeline was 15.84 μm and mean distance between parallel pipelines in the same septa was 254.80 μm.
EDS analysis showed differences in composition between the pipeline and the coral skeleton. Coral skeleton is composed of carbon (C), calcium (Ca) and oxygen (O) but pipelines seem to show additional elements. Although in different proportions, pipeline samples showed sulphur (S) from very high to quite low proportions. Magnesium (Mg) and Barium (Ba) were detected in smaller proportions. Sodium (Na) and Chloride (Cl) were detected at times, probably due to residual salt in the sample.
Figure 10. a-b) A. fragilis. Several pipeline-like structures in ANDES- Figure 11. A. fragilis. f) detail of highly calcified rough-looking IM 4199 in sample with macroscopic dense coverage of endolithic pipeline-like structure in ANDES-IM 4199 in area without dense algae. c) Pipelines present in sample of A. tenuifolia ANDES-IM 4674 endolithic algae coverage. g) The only pipeline-like structure found with dense coverage of endolithic algae. in ANDES-IM 4197, a sample without dense endolithic algae cover of endolithic algae. h) Detail of pipeline-like structure in sample with dense endolithic algae cover, ANDES-IM 4202. i) Detail of pipeline- like structure in sample without dense endolithic algae cover in ANDES-IM 4202.
Andes Area Mean Diameter Diameter Mean IM with/without Length of Number of of distance dense of SD pipelines SD SD SD pipelines/cm2 pipelines between coverage of pipelines at center at base pipelines Ostreobium (μm) (μm) 4197 1 With 82.23 0 8.65 0 10.38 0 - - 4199 222 With 73.4 19.82 9.07 3.78 16 6.14 268.92 172.64 4199 81 Without 73.58 25.89 1325 4.21 23.5 8.6 435.77 279.91 4202 79 Without 96.2 35.83 10.69 6.66 15.57 7.99 448.51 253.28 4202 91 With 73.06 24.2 8.31 4.2 12.56 6.25 224.56 328.6 6567 101 With 49.64 9.92 10.95 3.2 16.74 5.86 78.02 40.65 6567 28 Without 78.92 8.39 9.73 2.6 20.18 7.82 63.95 51.57 4674 49 With 53.24 15.23 8.23 5.33 11.8 6.32 249.92 183.56 Table 4. Pipeline-like structures
Traces of bioerosion
Traces of bioerosion were found in the upper plate of all the species and the lower plate for every species except for A. tenuifolia (or base in some cases for A. humilis). They were also found in a lateral view of the samples. Traces were more common on the lower plate. Traces in the upper plate were mostly located in broken septa, where round pores were found. They didn’t seem to follow a distribution pattern, but ratter appeared in scattered areas around samples and many times weren’t present. One sample, A. humilis ANDES-IM 4256 seemed to be particularly eroded in the surface, showing persistent traces of bioerosion not located in a broken area but the surface of the septa. The bioerosion pattern in lower plates was more complex. Samples without dense colonization of endolithic algae showed robust round-looking or sheat-looking crystals of calcium carbonate. However, they seem to disappear or corrode in samples with endolithic algae (this is particularly clear in ANDES-IM 4202 were even the lower cavities seemed to disappear). Thus, the surface looks flatter in samples were the endolithic algae is present at high densities and the natural rugosity of the coral practically disappears. Pores were also present in lower plates, in a 100% of the samples with a cover of the endolithic area. The distribution of the pores wasn’t uniform, and it was concentrated in several areas, specially over protuberances at the lower plate. Traces of bioerosion were found in most of the lateral samples, although at different densities. This time, traces of bioerosion came in two directions, possibly because filaments of endoliths followed a network pattern. Some of the filament traces were long and thin while others looked like round pores similar to those of upper and lower plate samples. Traces of bioerosion don’t seem to be as likely to appear in every species. For instance, way more traces and a higher proportion of pores were found in A. humilis in comparison to A. tenuifolia. Paired Welch’s t-tests were performed between samples with both an area with and without dense endolithic algae coverage, comparing the number of traces of bioerosion present in 1 cm2. For both upper and lower plate samples the differences were significative. Pearson’s Chi-squared contingency tests also showed significative differences in the proportion of samples showing traces of bioerosion between the two kind of areas. An unexpected result was a significatively higher diameter in pores from upper plates.
Fig 13. Traces of a b c d bioerosion on transversal and longitudinal samples. a, b, f, h, j & o. Filamentous traces of microbioerosion found in samples with dense coverage of endolithic algae from transversal cut.
e f g h e, i, l. Filamentous traces of microbioerosion in samples without dense coverage of endolithic algae from transversal cut. c & d. Round-shaped traces of microbioerosion visible in longitudinal cut. j i k l g, k, p. Smooth-looking surfaces of samples without visible traces of microbioerosion from areas without dense coverage of endolithic algae.
m n o p Figure 14. Examples of traces of bioerosion in upper-plate photos of corals. A. humilis. a) Figure 15. Examples of changes in texture in lower plate samples. Round-like and sheet- Sample without dense cover of endolithic algae. b-f) heavily eroded surface, sample with like crystals looked dissolved or absent. Traces of bioerosion are also present. Red circle dense coverage of endolithic algae. A. lamarki g) broken septum (sample with dense shows lower plate cavity and the blue circle shows round-shaped calcium carbonate coverage of endolithic algae) showing activity of bioerosion. h) Broken septum of a sample with dense coverage of endolithic algaefrom ANDES-IM 6418. A. agaricites i) Close-up to crystals. a broken septum of ANDES-IM 6613. Sample with dense coverage of endolithic algae.
Mean Presence of Number of % of pores diameter of ANDES- traces of traces of ranging Species traces of SD IM Bioerosion bioerosion between 1-20 bioerosion (Yes/No) counted [μm] [μm] Yes 22 2.004 0.590 100 4199 No - - - - Yes 28 10.920 3.644 89.286 4195 No - - - - A. fragilis Yes 4 6.584 5.295 100 6567 No 5 8.826 2.731 100 4197 No - - - - Yes 11 9.148 8.982 81.818 4202 No - - - - 4749 No - - - - 4778 No - - - - Yes 14 7.764 3.465 100 4690 No - - - - A. undata 4783 Yes 11 2.021 0.777 81.818 Yes 7 1.200 0.225 100 4729 No - - - - Yes 16 7.477 2.055 100 4711 No - - - - Yes 23 2.528 0.825 100 6613 A. No - - - - agaricites 4116 Yes 6 6.798 2.104 100
1090 No 29 3.372 2.988 93.103 A. humilis 4255 Yes 29 3.027 1.943 89.655
4243 Yes 22 3.881 3.443 81.818 Yes 21 1.626 0.780 90.476 A. 4240 No - - - - grahamae Yes 10 1.880 0.965 80.000 4226 No 13 1.994 0.657 100 Yes 40 1.737 0.854 80.000 4295 No 15 1.622 0.530 93.333 Yes 13 8.253 2.389 100 6418 No - - - - Yes 8 7.787 0.775 100 A. lamarcki 6496 No - - - - Yes 20 3.189 1.125 100 4314 No - - - - Yes 20 1.906 1.147 90.000 4298 No - - - -
Table 5. Lower plate traces of microbioerosion Mean Presence of Number of % of pores diameter of traces of traces of ranging Species ANDES-IM traces of SD Bioerosion bioerosion between 1-20 bioerosion (Yes/No) counted [μm] [μm] Yes 15 3.675 1.223 100 4199 No - - - - Yes 9 10.920 3.644 100 4195 No - - - - Yes 12 11.934 4.024 100 6567
fragilis No - - - -
A. Yes - - - - 6611 No - - - - 4197 No - - - - Yes - - - - 4202 No - - - - 4749 No - - - - Yes 50 4.066 2.564 100 4778 No - - - - Yes 24 3.817 1.073 100 4690 No - - - - Yes 11 3.742 1.593 100 4783 No - - - - Yes 7 7.003 2.603 100 4729 No - - - -
Yes 61 5.338 1.869 100 A.undata 4711 No - - - -
Yes 7 2.247 0.824 100 6613 No - - - - 4108 No - - - - 4107 Yes 11 4.850 1.569 100 Yes - - - - 4111 No - - - -
A.agaricites Yes 15 4.402 2.064 100 4116 No - - - - 4101 Yes 29 3.367 2.511 100
A. tenuifolia A. lamarcki A. grahamae 4676 4675 4674 4673 4672 4671 6608 4298 4314 6496 6418 4295 4233 4226 4240 4228 4243 4248
Table Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No No No No No No No 6
.
Upper platemicrobioerosiontraces of 19 24 25 22 15 12 22
6 4 ------
12.724 13.247 18.623 42.705 15.604 7.906 4.391 1.921 3.301 ------
13.071 2.234 3.028 1.929 6.791 0.736 2.058 9.870 9.352 ------
84.000 75.000 77.273 100 100 100 100 100 0 ------
Mean Presence of Number of % of pores diameter of ANDES- traces of traces of ranging Species traces of SD IM Bioerosion bioerosion between 1-20 bioerosion (Yes/No) counted μm [μm] No 13 5.189 2.194 100 Fragilis 6611 Yes 13 1.482 0.508 84.615 No 24 8.051 12.224 83.333 Undata 4690 Yes 26 6.122 2.469 100 4108 No 36 2.094 0.479 100 Agaricites 6613 Yes 14 1.349 0.339 78.571 1090 No 9 4.013 1.199 100 Humilis 4256 Yes 16 4.522 1.338 100 4248 No 11 1.828 0.419 90.909 Grahamae 4240 Yes 25 5.239 2.469 100 4295 No 9 1.988 0.443 100 Lamarckii 4298 Yes 13 15.119 24.176 76.923 4676 No 2 3.202 1.514 100 Tenuifolia 4675 Yes 12 4.724 3.200 100
Table 7. Traces of microbioerosion in lateral samples
Figure 16. Number of counted pores in 5 random areas of a 1 cm2 samples for samples with and without a dense cover of endolithic algae. Areas with a dense cover of endolithic algae have a significatively higher mean number of pores (14.3 vs 2.1). P-value Welch’s t.test: 0.002444.
Figure 17. Mosaic plot showing how for lower plates every sample with a dense endolithic cover showed presence of pores at the base, while most of the samples (75%) without a dense cover had no presence of pores. Pearson’s chi-squared contingency test showed proportions are significantly different (P-value 4.537e-05).
Figure 18. Number of counted pores in samples with and without Figure 19. Mosaic plot showing how for upper plates most samples a dense cover of endolithic algae in the surface of the upper plate with a dense endolithic cover of endolithic algae showed presence of the seven species of Agaricia. of pores (68,4%) while just a small percentage of samples without a dense cover presented them (15.6%). Pearson’s chi-squared contingency test showed proportions are significantly different (P- value: 6.366e-06).
Figure 20. Diameter of pores at the upper plate and lower plate. Mean dia is significantly higher in the upper plate (P-value: 0.0078) in comparison with the lower plate.
Table 8. General measurements of pipeline-like structures and samples where they were found.
Discussion General remarks: An important limitation of this study is the lack of a quantitative approach for measuring the density and genetic identity of the endolithic algae living within the coral tissue sampled. A dense cover of endolithic algae that can be assessed macroscopically and with the aid of a stereoscope should be a good indicator of the presence of Ostreobium spp. in the sample. However, it does not mean that areas lacking this dense cover have absolutely no presence of Ostreobium nor that the genetic identity of what inhabits the dense coverage belongs exclusively to this genus. In fact, Ostreobium is not limited to colored zones and it can be present in most regions of the skeleton at densities insufficient to be visible (Kühl, Holst, Larkum, & Ralph, 2008; Le Campion-Alsumard et al., 1995; Massé et al., 2018). Thus, it is possible that morphological traits found in ‘control’ samples, for example pipelines or traces of bioerosion, were indeed created by Ostreobium. On the other hand, the greenish layer commonly reported in the literature as the “Ostreobium band” may also contain a variety of other kinds of algae, for example species of Eugomontia, Phaeophila, Entocladia and Codiolum (Glynn & Manzello, 2015). Thus, it is possible that some of the traces of bioerosion that coincide with the 1-20 μm measures, which correspond to a range of diameter values for Ostreobium spp. filaments (Kobluk & Risk, 1977; Schroeder & Ginsburg, 1971), were caused by other agents of bioerosion able to create similar-looking pores. Besides from other algae, the role of bioeroding sponges, for example, could be an important source of calcium carbonate dissolution for Agaricia, since spicules composed of silicium were found both in the surface and inside the skeleton of a small amount of the samples. However, the presence of a green band or dense coverage of endolithic algae should be indicative for an important presence of Ostreobium. First, 12 clades of Ostreobium spp. have already been reported in A. undata from samples with an overlapping geographic and bathymetric distribution to the ones from this study (González-Zapata et al., 2018b). Second, several studies have shown Ostreobium to be dominant in endolithic assemblages (Chazottes, Campion-Alsumard, & Peyrot-Clausade, 1995; Che et al., 1996; Reyes-Nivia et al., 2013). Third, Ostreobium has an impressive cosmopolite and bathymetric distribution, having been found at depths as extreme as 300 m and is able to colonize tropical settings as well as cold temperate environments, such as Chilean fjords in Patagonia (Försterra & Häussermann, 2008; Stjepko Golubic et al., 2019; Vogel, Gektidis, Golubic, Kiene, & Radtke, 2000). Finally, it is to be expected that most traces of bioerosion, especially in areas of dense cover, are created by Ostreobium spp. since it has been shown that this agent of microbioerosion can be responsible for 70-90% of carbonate removal among an euendolithic community (Tribollet, 2008a). Thus, it is very likely that many of the morphological traits sampled in here are associated to Ostreobium spp. Structural cavities To the extent of my knowledge, cavities in the upper and lower plates of the corallum haven’t been described for Agaricia. However, these cavities do seem to be present in at least one sample of the detailed description provided by Wells (1973; Fig. 9) for an individual of A. lamarcki. The fact that they are consistently present; seem to be distributed in a relatively organized way, following linear patterns most times; and seem to have a great variability in shape between samples and species, but seem fairly regular within the same coral; suggests that that they are a part of the morphology of the coral. If this is the case, how these cavities are formed and whether they have any sort of function in the coral physiology could be addressed in future studies. Here, I attempted testing whether the size of this cavities could be enlarged by the colonization of fissures and further dissolution of the walls by endolithic algae, making the diameter broader. What was found was a statistically significative larger size for cavities in areas without the presence of a dense endolithic algae assemblage for the upper plate and no relation for the lower plate. This, probably, has no relation to the presence of endolithic algae, but likely it has to do with the area of the coral were samples were taken. Most samples showed a thick coverage towards the base and a lack of visible coverage towards the end of leaves or sheets. Thus, it is likely that most corals have increasingly larger cavities with distance from the base. However, this is yet to be assessed in a more careful way. Pipeline-like structures This study is the first report of pipeline-like structures in A. fragilis and A. tenuifolia. Most specimens sampled showed no pipelines. Specifically, no pipelines were found in A. undata, where they were identified for the first time (González-Zapata et al., 2018b). This suggests that pipelines are likely to be associated with either certain clades of Ostreobium or are more likely to occur under specific environmental conditions. To evaluate whether these pipelines could be built by Ostreobium, molecular identification was attempted unsuccessfully. However, González-Zapata, Gómez-Osorio & Sánchez, 2018 had previously found Ostreobium DNA in similar-looking pipeline-like structures in A. undata corresponding to clades N and H2. A comparison of diameter measurements from this study with values reported in the literature and published microscopic images was done to assess whether pipelines found in these samples correspond to calcified Ostreobium filaments. Pipeline diameter of samples from this study ranged between 2.6 and 37 μm. Schlichter et al. (1997) measured endolithic algal filaments that ranged between 1.2 and 4.2 μm from uncalcified filaments living inside corals. González-Zapata, Gómez-Osorio & Sánchez (2018) published images with networks of uncalcified Ostreobium filaments that ranged between 2.8 and 17.6 μm in A. undata. Also, several studies have shown that exposed filaments of endolithic algae can undergo a process of precipitation of calcium carbonate on and within them which result in a complete or partial encrustation of filaments by calcium carbonate crystals (Jones & Goodbody, 1982; Kobluk & Risk, 1977; Lukas, 1974; Schroeder, 1972; Schroeder & Ginsburg, 1971). Schroeder (1972) reported calcified Ostreobium filaments of 15-300 μm. Kobluk & Risk (1977) described filaments of diameter 8-30 μm. Schroeder & Ginsburg (1971) found filaments of diameter 1-20 μm being enveloped by crystals of 5-150 μm giving rise to calcified filaments that ranged from 20 to 300 μm in diameter. The great range of diameters of calcified and uncalcified Ostreobium filaments reported in the literature makes plausible that pipelines found on this study correspond to endolithic algal filaments calcified to a certain extent. The previous identification of Ostreobium DNA in similar-looking pipeline structures in A. undata points in the same direction (González- Zapata, Gómez-Osorio & Sánchez, 2018). Pipeline-like structures reported in A. undata showed additional elements from the calcium carbonate coral skeleton. In particular, sulfur (S) and potassium (K) (González-Zapata, Gómez-Osorio & Sánchez, 2018). Pipeline-like structures from this study showed also sulfur (S), yet no potassium (K) was found. Sulfur can be related to small cysteine-rich proteins (SCRiPs) that are found in the membranes of calcifying organisms (González-Zapata et al., 2018b; Sunagawa, DeSalvo, Voolstra, Reyes-Bermudez, & Medina, 2009). EDS analysis from this study reported the presence of barium (Ba) and magnesium (Mg) in pipelines. Magnesium has also been found in previous analyses of calcified filaments of Ostreobium, which showed crystals in cores and crusts of the filaments to be composed of magnesium calcite (Jones & Goodbody, 1982; Schroeder, 1972; Schroeder & Ginsburg, 1971). González-Zapata et al., (2018b) hypothesized that Ostreobium could be using the calcium carbonate waste of the boring process to make pipeline-like structures across host’s costae. Another, non-exclusive possibility, is for the endolithic algae’s filaments to project into the sea away from one costa until they reach the other costa, where they will get calcified after a period of exposure. The exposure of endolithic filaments living as ephilits has been described (Golubic, 1973) and the observation of Ostreobium sp. filaments projected out of a substrate and further getting calcified has been recorded (Kobluk & Risk, 1977). Also, the qualitative observation that some pipeline-like structures seemed thinner and smoother while others seemed thicker and rougher could suggest that the calcification process is progressive. Thus, it’s possible than more recent pipelines have a thinner crust that becomes thicker with time of exposure. The presence of magnesium in pipelines, the similarity in the diameter of pipelines and calcified filaments reported in the literature and the observation of different degrees of calcification in the pipelines, suggests there is a possible link between pipeline-structure formation and studies performed in the 1970’s on submarine cementation. This could be tested in further investigations. It has been proposed that pipeline-like projections from costae improve light uptake in the mesophotic zone (González-Zapata et al., 2018b). Matching this prediction, here, all the A. fragilis samples occur in the twilight zone (>30 m) and three of them in the lower mesophotic zone (>60 m) (Pyle, 1998). However, a sample from 9 m for Agaricia tenuifolia also showed pipeline-like structures. If this sample occurred in a cave, for example, pipeline projections across costae could be proposed to have first appeared as means to aid light uptake in general under low-light situations. Future studies could try to test whether pipelines associate with a specific ecologic niche in order to achieve a better understanding of their possible functionality. If it is the case that these projections increase light uptake under low light situations, pipeline structures could be related to the mutualistic interaction between Ostreobium and corals, helping zooxanthellate corals to colonize low-light environments. Traces of bioerosion This study shows clear evidence that the dense cover of endolithic algae at the lower plate of corals is associated to morphological changes in the texture and general appearance of coral surfaces in the genus Agaricia. The qualitative observation that round or sheet-like shaped crystals of calcium carbonate seemed to have dissolved or gone absent at the lower plate of unifacial species is of interest. This could be produced directly by Ostreobium and/or other endolithic algae capable of biochemical dissolution of CaCO3 due to the production of acidic or chelating substances (Golubic et al., 1984; Le Campion-Alsumard, 1975; Ricci & Davidde, 2012; Tribollet, 2008b). According to Tribollet (2008b), the species composition of eudolithic assemblages in live substrates is a result of a selection process that favors oligotrophic, positively phototrophic and fast-growing taxa. These taxa can infest the host at early stages of its development and growth starts from the point of fixation to the substrate. In Agaricia, dense cover of euendoliths is usually concentrated towards the base of the coral and become less frequent with increasing distance from this point of fixation. Probably, the process behind the observed pattern is an early dissolution of calcium-carbonate round or sheat-shaped crystals by the euendolithic assemblage near the base. But since the assemblage hasn’t colonized the margins of the corals, well-developed crystals are still visible in this area. The seventh column of tables 5 and 6 shows that, for most samples, the majority of traces of bioerosion are compatible with measurements reported for the diameter of Ostreobium spp. Traces of microbioerosion are morphologically very similar to those reported in the literature for Ostreobium. Round-shaped pores resemble in size and shape those reported by Tribollet (2008b) in dead and live-coral substrates (fig 2. a and b). Filamentous traces, mostly present in lateral samples but at times at upper and lower plate samples, resemble the shape of traces reported for Ostreobium in oysters (Che et al., 1996; fig 5.a) and carbonate allochem (Jones & Goodbody, 1982; figs 7, 8). Thus, it is very likely that a vast number of the traces of microbioerosion are indeed created by Ostreobium. Mean significative differences in the diameter of pores at the upper and lower plate is an unexpected result probably related with differences in roughness between both areas that allow only the thicker filaments to perforate the upper plate substrate. However, there is very little information on the distributions of filaments across a living coral and the explanation could be more complex. Although sampling numbers per species are low, this study shows tentative differences in the presence of traces of microbioerosion and the number of pores found between species that could be further explored. For instance, specimens of A. humilis showed highly eroded surfaces while there was lack of traces of microbioerosion in most of the sampled specimens of A. tenuifolia. Reyes-Nivia et al., (2013) found that, although future warming and acidification will lead to increased rates of microbioerosion, the magnitude of bioerosion responses will also depend on the structural properties of coral skeletons. Thus, the range of implication for reef carbonate losses is also highly depending on the species. Studying these differences in a broad range of corals could lead to having a clearer picture about the processes underlying coral reef resilience in the Anthropocene. Conclusions and perspectives Although structural cavities are not enlarged by the presence of endolithic flora, they seem to be a part of coralline morphology as a highly variable morphological trait. Similar pipeline- like structures to those found in A. undata (González-Zapata et al., 2018b) were found only in four individuals of A. fragilis and one of A. tenuifolia. The function of this structures is still poorly understood but their presence is probably indicative of attempts performed by Ostreobium spp. to enhance light-harvesting in low-light environments. I propose as a hypothesis to be tested that filaments are able to cross one costa to reach the other, since these algae are capable of ephilitic behavior. Further calcification then takes place with exposure to the environment as has been recorded for Ostreobium in other contexts. Further studies could assess whether the presence of pipeline-like structures is associated with a certain ecological niche or could be related exclusively to particular clades of endolithic algae. This study shows evidence for alterations in coralline surface associated with the presence of a dense cover of endolithic algae in Agaricia. A higher number of traces of microbioerosion and a higher proportion of samples with these traces was found for areas with a dense coverage of endolithic algae for both the upper and the lower plate, although differences are clearer for the lower plate. The lower plate of areas with dense coverage looks smooth in comparison with the rugosity of samples without this coverage. This pattern could be explained by chemical dissolution performed by endolithic algae as a part of the boring process. Given the dual role of Ostreobium in particular and the endolithic flora in general, an enhanced understanding of the effects of endolithic assemblages living within different species of corals could lead to better projections for understanding the future of coral reefs in the Anthropocene. Also, it could be informative for conservation as well as for selection of more resilient species for reef-restoration practices. In the case of some species of Agaricia this is also important given their extinction risk status. A. lamarcki has a vulnerable status with a decreasing population trend and A. tenuifolia is near-threatened. A lamarcki is particularly susceptible to bleaching and a dramatic increase in the occurrence of white plague has been reported. A. tenuifolia has also suffered in particular due to hurricane damage (Aronson, Bruckner, Moore, Precht, & Weil, 2008; Kramer & Kramer, 2002). In this sense, further investigations could assess the resistance of these coral species to the enhanced activity of microboarers. Also, what boring processes documented for samples of living corals could mean in terms of resilience of reefs to a multiplicity of stressors may be addressed from different perspectives, in order to gain a clearer scene on the balance between parasitic and mutualistic interactions of endolithic flora and corals. References
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