Filament morphology has been proposed to be a factor contributing to species-specific responses within the tropical calcifying macroalgal genus Halimeda to ocean acidification (OA) (Price et al. 2011, Comeau et al. 2013, Campbell et al. 2014, 2015, Meyer et al. 2015). Halimeda thalli are composed of coenocytic (multi-nucleate) filaments that swell at their termini, resulting in the adherence of filament tips (Figure 1). The fusion of the outermost filaments or primary utricles creates compartments just anterior of the primary utricles called primary inter-utricle spaces. Because of the fusion of the primary utricles, the primary inter-utricle spaces become physically isolated from external seawater. It is within these inter-utricle spaces that calcium carbonate (CaCO3) precipitates, primarily in the form of dense aragonite crystals (Hillis-Colinvaux 1980). Thus, the potential for precipitation of aragonite crystals and their susceptibility to dissolution under future OA conditions may be influenced by the size, orientation, and degree of lateral adhesion between adjacent primary utricles. Species-specific differences in morphology of primary utricles and their adhesion likely influences the diffusion pathways and thereby uptake of ions (e.g. Ca2+, CO32−) necessary for CaCO3 precipitation (Borowitzka and Larkum 1976, Böhm et al. 1978). Further, diffusion pathways between primary utricles control H+ fluxes, either from the bulk seawater into the primary inter-utricle spaces or removal from the primary inter-utricle spaces to prevent accumulation of H+, a product of calcification. This removal of H+ from primary inter-utricle spaces would be across a shallower concentration gradient under OA, and if an active process is needed. This would correspond to a greater energetic cost to the algae leading to lower calcification rates, as in corals (Jokiel 2011, Ries 2011, Cyronak et al. 2015).
We examined morphologies of six Halimeda species to ascertain the role thalli morphology may have on susceptibility to OA in this genus. Specifically, we measured primary utricle sizes and lengths of diffusion pathways and examined how these morphological characteristics were related to tissue mineral content and crystal microstructure within primary inter-utricle spaces. Crystal microstructure investigations focused on the dimensions of aragonite needles in primary inter-utricle spaces, and micro-anhedral crystal formations at the boundary where the primary utricle meets the primary inter-utricle space. Primary utricle size and diffusion pathway length were compared across species using transmission electron microscopy (TEM) and crystal microstructure was identified by scanning electron microscopy (SEM).
Materials and methods
Species and sampling locations
A total of 120 thalli of six Halimeda species (Figure 2) were collected off the northern coast of Little Cayman Island (August 2014) from three reef habitats where each species occurs in high densities (Figure 3, Table 1). Halimeda incrassata was collected from the shallow back reef lagoon, H. tuna and H. monile were harvested from the spur and groove reef system, H. opuntia was collected on the seaward edge of a sand groove at the fore reef ledge, and H. copiosa and H. goreauii were harvested from under the ledge in deep shade. Specimens were transported in seawater from the collection sites to the Little Cayman Research Center (LCRC) where they were immediately prepared for TEM and SEM analyses (described below). Species identification was accomplished using microscopy and keys by Hillis-Colinvaux (1980) and Littler and Littler (2000).
TEM sample preparation and microscopy
Apical segments of freshly harvested thalli were immediately fixed in 2% glutaraldehyde in 0.05 m cacodylate-buffered seawater (Electron Microscopy Sciences, Hatfield, PA, USA) and stored at 4°C. Fixed segments were halved along the vertical midline, post-fixed in 1% OsO4 buffer (Electron Microscopy Sciences, Hatfield, PA, USA), and dehydrated through a series of graded ethanol concentrations (20%–100%). One half of each segment was embedded in Spurr resin and the remaining half was reserved for SEM analysis (see below). Thin sections (80 nm) were cut of resin-embedded samples (Leica EM UC7 Ultramicrotome, Leica Microsystems Inc., Buffalo Grove, IL, USA) and stained with 3% uranyl acetate (3 min) followed by lead citrate (1 min). Stained sections were examined with a TEM (FEI Tecnai G2 Spirit BioTWIN, Thermo Fisher Scientific Inc., Hillsboro, OR, USA) at 100 kV. Our observations and measurements focused on morphological characteristics of the primary region of segments consisting of primary utricles (pU) and primary inter-utricle spaces (pIUS; Figure 4). In the primary region of one representative sample from each species, measurements (≥7) of diffusion pathway lengths between adhered primary utricles and their surface diameters were made using image processing software (ImageJ v 1.47, Rasband, W.S., U.S. National Institute of Health, Bethesda, Maryland, USA).
SEM sample preparation and microscopy
The remaining halves of fixed apical segments were prepared for SEM using the same procedure as above through dehydration in 100% ethanol. The dehydrated samples were then dried using hexamethyldisilazane (HMDS; Electron Microscopy Sciences, Hatfield, PA, USA). Dried samples were fractured vertically on the mid-plane to expose interior surfaces and mounted on aluminum stubs using carbon adhesive tabs. Stub-mounted samples were sputter coated with palladium and viewed with an SEM (FEI XL 30 Field Emission ESEM/SEM, Thermo Fisher Scientific, Inc., Hillsboro, OR, USA).
Tissue mineral content
Tissue mineral content (assumed to be primarily CaCO3) was determined for aboveground portions of whole thalli. Fresh aboveground tissues were rinsed in distilled water to remove salts, dried at 60°C to constant weight and acidified (95:5% of DI:12N HCl vol:vol) until no effervescence formed following acid addition. Post-acidified samples were rinsed with distilled water, dried at 60°C and weighed. The differences in pre-acidified and post-acidified weights were assumed to be the mineral proportion of the thalli tissue and reported as % CaCO3.
Calcium carbonate content data were log transformed to meet assumptions of normality (Shapiro-Wilk’s test) and homogeneity of variance (Levene’s test), and compared among species using an ANOVA followed by a priori contrasts (SAS University Edition, SAS Institute Inc., Cary, NC, USA). Linear regression analyses were used to determine relationships between average primary utricle size, diffusion pathway length, and CaCO3 content across the species (SigmaPlot 11.0, Systat Software Inc., San Jose, CA, USA). All statistical analyses were considered significant at p<0.01. Measurements of primary utricle surface diameters and diffusive pathway lengths within individual samples were not independent and violated the assumptions of ANOVA. Therefore, individual sample means were compared to evaluate similarities and differences across the six species examined for these measurements.
Primary utricle size
The average sizes of primary utricles determined by surface diameters differed among the single representative samples of the six species examined (Table 2, Figure 4). Halimeda tuna and H. goreauii had the largest and smallest primary utricles with average surface diameters of ~41 and 19 μm, respectively. Halimeda incrassata and H. monile primary utricle surface diameters were intermediate between those of H. tuna and H. goreauii, ranging from ~31 to 32 μm. Halimeda copiosa and H. opuntia also had similar primary utricle surface diameters averaging ~26 and 24 μm, respectively, and were on the low end of the range for the species examined.
Diffusion pathway length
Diffusion pathway lengths measured as adhesion length of adjacent primary utricles also varied by species (Figure 5, Table 2). The longest diffusion pathway observed was ~19 μm in Halimeda tuna. Halimeda incrassata and H. monile also had relatively long diffusion pathways of ~10–13 μm. The shortest diffusion pathways up to ~6 μm in length were similar in H. copiosa, H. goreauii, and H. opuntia. We also found a positive linear relationship between average diffusion pathway length and primary utricle surface diameter across the examined species (Figure 6A).
Tissue mineral content
Mineral content (% CaCO3) of above-ground tissue for whole thalli significantly differed among the examined species (F5,59=54.2, p<0.01), and statistically distinct groups were identified (Table 2). The highest average CaCO3 content (95%) was found in Halimeda copiosa and H. goreauii. Calcium carbonate content was 5%–9% lower, on average, in H. monile and H. opuntia than in H. copiosa and H. goreauii. Halimeda incrassata and H. tuna were the least calcified species with average CaCO3 contents of 79 and 72%, respectively. Linear regression analyses between average CaCO3 content compared to diffusion pathway length for all species revealed a strong inverse relationship (Figure 6B). There was also a strong inverse relationship between average CaCO3 content and primary utricle surface diameter (Figure 6C).
Aragonite crystal microstructure
Apical segment SEM analyses revealed both similarities and differences in the aragonite crystal microstructure of the primary inter-utricle spaces of the six species examined (Figure 7). Each species’ primary inter-utricle space was densely filled with aragonite crystals predominantly in the form of aragonite needles up to 5 μm in length and 0.1–0.5 μm in width. In the primary inter-utricle spaces of Halimeda incrassata and H. tuna, the dominant crystals were slender needles not exceeding 0.2 μm in width. For H. copiosa and H. goreauii, needles ranged from 0.2 to 0.4 μm in width in the primary inter-utricle space interior and at the boundary where primary utricle filaments retracted from the primary inter-utricle space leaving an imprint in the crystals. A similar observation of dense needle (0.2–0.3 μm in width) infill in the interior and boundary of the primary inter-utricle space was also observed for H. opuntia. In H. monile, needles 0.2 μm in width, on average, also populated the primary inter-utricle space interior, but these were not the dominant crystal form observed at the primary inter-utricle space boundary. Rather than needles, a thick (4–12 μm) rim of micron-sized (<1 μm) anhedral crystals was observed adjacent to the primary utricles in H. monile. Although the other species examined did not form a dense rim of micro-anhedral crystals, small crystal aggregates could be seen on top of short needles in the vicinity of the primary utricle. In some cases, these small crystal aggregates were prominent where the diffusion pathway enters the primary inter-utricle space (Figure 7).
Micro-anhedral crystal aggregates were observed where the diffusion pathway between adhered primary utricles is adjacent to the primary inter-utricle space, primarily in species (H. copiosa, H. goreauii, H. opuntia) with the smallest primary utricles (<27 μm diameter) and shortest diffusive path lengths (<6 μm). In contrast, micro-anhedral crystal aggregates were less apparent where the diffusive pathway joins the primary inter-utricle space in species (H. incrassata, H. tuna) with the widest primary utricles (>32 μm) and longest diffusive pathways (>13 μm). Halimeda monile was the exception, with complete micro-anhedral crystal infill in the primary inter-utricle space even though its morphology was comparable to H. incrassata.
There was an inverse relationship between tissue mineral content and diffusion pathway length (r2=0.87; Figure 6B) that was potentially influenced by primary utricle size (r2=0.70; Figure 6C) since size and pathway length were highly correlated (r2=0.92; Figure 6A) in the six Halimeda species examined. These results imply that primary utricle size and/or diffusion pathway length may influence the degree of calcification within the Halimeda genus. For example, average CaCO3 content was 95% in H. copiosa and H. goreauii, species with 4.5 to 6-fold shorter diffusion pathways compared to H. tuna (72% CaCO3). Shorter diffusion pathways may lower diffusive resistance for the influx of Ca2+ and potentially HCO3− or CO32− from seawater to the primary inter-utricle space, increasing the potential for CaCO3 formation (Borowitzka and Larkum 1976). Higher CaCO3 content in species with shorter pathways could also promote greater efflux of H+, a by-product of calcification whose build-up in the calcifying region inhibits CaCO3 formation and/or promotes dissolution (Jokiel 2011, 2013, Ries 2011, Cyronak et al. 2015).
Diffusive ion transport associated with the primary inter-utricle space, and subsequent localized changes in [H+] and [CO32−] may have contributed to the different densities and locations of micro-anhedral crystals among the six Halimeda species examined. The occurrence of micro-anhedral crystals primarily in Halimeda species with short diffusive pathways (H. copiosa, H. goreauii, H. opuntia), as well as higher CaCO3 content in this study, suggests that greater exchange with external seawater may positively affect calcification, but also alter the primary inter-utricle space aragonite needle crystal form.
In contrast to Halimeda species with short diffusive pathways, those with long pathways (H. incrassata, H. tuna) were dominated by slender needles and lacked micro-anhedral crystal aggregates. A long path length for diffusion would isolate the primary inter-utricle space from the external seawater and limit cellular metabolism effects, although this interaction and its effect on calcification is presently unclear. The low CaCO3 content associated with long diffusion path lengths found in the present study may be attributable to reduced crystal formation, perhaps limited by Ca2+ and/or inorganic carbon availability. Alternatively, greater build-up of H+ and CO2 in the primary inter-utricle space from calcification and cellular respiratory processes, respectively, may have contributed to greater dissolution of aragonite needles leading to more narrow forms. Aragonite needles in H. opuntia and H. tuna have been shown to narrow in response to elevated partial pressure of CO2 (pCO2) (Robbins et al. 2009, Wizemann et al. 2015). Narrow needles in new segments grown in CO2-enriched (>650 μatm) seawater were suggested to be formed under rapid calcification, potentially catalyzed by an increase in external carbonic anhydrase activity at low pH (Hofmann et al. 2014, Wizemann et al. 2015). Although a few studies observed thinner needles, in our previous work on seven Halimeda species we found no changes in crystal morphology under year 2100 (~1000 μatm) compared to ambient pCO2 levels (Peach et al. 2016, 2017).
The relationships among primary utricle morphology, degree of calcification and crystal forms among the six Halimeda species examined corresponded to evolutionary lineages. For example, species of the Opuntia lineage (H. copiosa, H. goreauii, and H. opuntia) exhibited more slender primary utricles, shorter diffusion pathways and higher CaCO3 content in comparison to species belonging to the Halimeda (H. tuna) and Rhipsalis (H. incrassata and H. monile) lineages (Hillis 2001). These morphological lineage characteristics are likely the result of long-term evolutionary adaptations to abiotic (e.g. irradiance, wave energy) and biotic (e.g. grazing pressure) conditions at natural growth sites (Hillis-Colinvaux 1980, Kooistra et al. 2002, Verbruggen and Kooistra 2004). Increased pliability and reduced drag in some species (e.g. H. tuna and H. incrassata) with low CaCO3 content and swollen utricles may have allowed this evolutionary lineage to become established primarily in shallow back reef and lagoon sites (Kooistra et al. 2002). In contrast, high tissue CaCO3 content in H. goreauii and H. copiosa may be an adaptation to reduce herbivorous fish grazing in structurally complex habitats such as the reef ledge where these species were collected (Hay et al. 1983, Paul and Hay 1986, Kooistra et al. 2002).
In conclusion, inter-species’ differences in primary utricle morphology accounted for variability in mineral content and aragonite crystal forms in six Halimeda species. The characteristics generally corresponded to the evolutionary lineages (Halimeda, Opuntia, Rhipsalis) to which they belong. Halimeda copiosa, H. goreauii, and H. opuntia (Opuntia lineage) had small primary utricles and short diffusion pathways leading to the primary inter-utricle spaces, where aggregates of micro-anhedral crystals were observed on top of needles (0.2–0.4 μm in width). This was in contrast to the minimal micro-anhedral formations and more narrow (0.1–0.2 μm) needles observed in H. tuna (Halimeda lineage) and H. incrassata (Rhipsalis lineage), species with larger primary utricles and longer diffusion pathways. Although morphologically similar to H. incrassata, primary inter-utricle spaces in H. monile were heavily cemented with micro-anhedral crystals and aragonite needles were virtually absent. The present study revealed ultra- and micro-structural distinctions among species that have been suggested to cause mixed responses of Halimeda species to OA (Price et al. 2011, Comeau et al. 2013, Campbell et al. 2014, 2015, Meyer et al. 2015). Species of the Opuntia lineage have been shown in several multi-species investigations to be more susceptible to OA relative to species of other lineages, including Halimeda and Rhipsalis. For example, Price et al. (2011) found that H. opuntia (Opuntia lineage) exhibited a more negative calcification response to elevated pCO2 when compared to H. taenicola, a less calcified species of the Halimeda lineage with larger utricles. Campbell et al. (2014, 2015) also found that exposure to elevated pCO2 led to significant reductions in net calcification for H. opuntia, in contrast to H. incrassata, a less calcified species of the Rhipsalis lineage whose net calcification rates were unaffected. Based on these experimental results, we hypothesize that weakly calcified species with large primary utricles and long diffusion pathways, and therefore species with smaller and more isolated inter-utricle spaces, may be more tolerant to long-term acidified seawater exposure. Further microscopy work on additional samples is needed to verify our findings, and may also assist in clarifying the role of morphology in OA tolerance for the Halimeda genus over the long-term.
Funding for SEM and TEM work was provided by a Dissertation Year Award (K.E.P.) and Climate Change Initiative Grant (M.S.K.) awarded by Florida Atlantic University. We thank the Cayman Islands Marine Conservation Board and Department of Environment for permitting collection and exportation of specimens, and are grateful to Carrie Manfrino and the Little Cayman Research Centre staff for logistic support on Little Cayman Island. We also thank Elizabeth Dutra for her assistance with field collections and Chris Johnson for his assistance with figures.
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About the article
Katherine E. Peach
Katherine E. Peach is an Assistant Professor of Biology at Broward College in Pembroke Pines, Florida, USA. She earned her PhD in Integrative Biology in May 2016 from Florida Atlantic University, an MS in Oceanography at Nova Southeastern University, and dual BS degrees in Marine Science and Biology from Coastal Carolina University. Her research interests include determining whether dominant reef macroalgae and corals have the capacity to acclimate and adapt to future changes in environmental conditions, with an emphasis on ocean acidification.
Marguerite S. Koch
Marguerite S. Koch is a Professor in the Biological Sciences Department of Florida Atlantic University. She received her PhD in Marine Biology and Fisheries from the University of Miami, an MS in Marine Science from Louisiana State University, and a BS in Biology from Tulane University. She studied coral reef ecology at Fairleigh Dickinson University, fisheries and estuarine ecology at the University of Washington, and was a graduate exchange scholar investigating nutrient cycling in coastal marine ecosystems at the University of Exeter. Her present research examines biogeochemical changes in tropical marine ecosystems and ecophysiological responses of marine plants under climate change.
Patricia L. Blackwelder
Patricia L. Blackwelder is an Associate Professor at Nova Southeastern University and Supervisor of the University of Miami Center for Advanced Microscopy (UMCAM). Dr. Blackwelder received her PhD in 1976 at the University of South Carolina, Columbia SC, USA. Her research focuses on the application of electron microscopy to ultrastructure/biomineralization in marine invertebrate organisms and biomedical studies. She has been co-author on 55 publications in these fields.
Debbie Guerrero-Given is a research scientist in the Electron Microscopy (EM) Facility at the Max Planck Florida Institute for Neuroscience. She enjoys the morphological analysis of the variety of samples that are brought in the EM facility and every challenge to produce high quality images that relate to the different functions of the brain.
Naomi Kamasawa heads the Electron Microscopy (EM) Facility in the Max Planck Florida Institute for Neuroscience. She has long-term experience in electron microscopy studying different kinds of biological specimens including bacteria, yeasts, plants, cultured cells, nervous tissues from invertebrates and vertebrates using various EM techniques and applications. She is very enthusiastic about visualizing high-resolution morphology in order to understand their physiological function.
Published Online: 2017-01-12
Published in Print: 2017-02-01