J. Phycol. 52, 161–173 (2016) © 2016 Phycological Society of America DOI: 10.1111/jpy.12392 ARAGONITE INFILL IN OVERGROWN CONCEPTACLES OF CORALLINE LITHOTHAMNION SPP. (HAPALIDIACEAE, HAPALIDIALES, RHODOPHYTA): NEW INSIGHTS IN BIOMINERALIZATION AND PHYLOMINERALOGY1 Sherry Krayesky-Self,2 Joseph L. Richards University of Louisiana at Lafayette, Lafayette, Louisiana 70504-3602, USA Mansour Rahmatian Core Mineralogy Inc., Lafayette, Louisiana 70506, USA and Suzanne Fredericq University of Louisiana at Lafayette, Lafayette, Louisiana 70504-3602, USA New empirical and quantitative data in the study of calcium carbonate biomineralization and an All crustose coralline algae belonging in the Coral- expanded coralline psbA framework for linales, Hapalidiales, and Sporolithales (Rhodo- phylomineralogy are provided for crustose coralline phyta) are characterized by the presence of calcium red algae. Scanning electron microscopy (SEM) and carbonate in their cell walls, which is often in the energy dispersive spectrometry (SEM-EDS) form of highly soluble high-magnesium-calcite (Adey pinpointed the exact location of calcium carbonate 1998, Knoll et al. 2012, Adey et al. 2013, Diaz-Pulido crystals within overgrown reproductive conceptacles et al. 2014, Nelson et al. 2015). Besides the in rhodolith-forming Lithothamnion species from the Rhodogorgonales (Fredericq and Norris 1995), a sis- Gulf of Mexico and Pacific Panama. SEM-EDS and ter group of the coralline algae in the Corallinophy- X-ray diffraction (XRD) analysis confirmed the cidae (Le Gall and Saunders 2007) whose members elemental composition of these calcium carbonate also precipitate calcite, all other calcified red and crystals to be aragonite. After spore release, green macroalgae deposit calcium carbonate in the reproductive conceptacles apparently became form of aragonite (reviewed in Adey 1998, Nelson overgrown by new vegetative growth, a strategy that 2009). Previous studies have also shown that the may aid in sealing the empty conceptacle chamber, more stable carbonates, that is, aragonite and dolo- hence influencing the chemistry of the mite, may be present in the thallus of coralline algae microenvironment and in turn promoting aragonite filling in vegetative cells and pores (Alexandersson crystal growth. The possible relevance of various 1974, Nash et al. 2011, 2012, 2015). The pioneering types of calcium carbonate polymorphs present in study by Alexandersson (1974) using scanning elec- the complex internal structure and skeleton of tron microscopy (SEM), energy dispersive spectrom- crustose corallines is discussed. This is the first study etry (SEM-EDS), X-ray diffraction (XRD), and Feigl’s to link SEM, SEM-EDS, XRD, Microtomography and solution staining to analyze the elemental and min- X-ray microscopy data of aragonite infill in coralline eralogical condition of coralline-forming nodules algae with phylomineralogy. The study contributes to known as rhodoliths, documented that empty repro- the growing body of literature characterizing and ductive conceptacles of Lithothamnion glaciale Kjell- speculating about how the relative abundances of man rhodoliths from the Skagerrak in the North Sea carbonate biominerals in corallines may vary in become filled in with aragonite crystals, despite the response to changes in atmospheric pCO2, ocean undersaturated conditions of the local seawater acidification, and global warming. favoring dissolution of calcium carbonate crystals. Key index words: aragonite; calcite; coralline algae; Subsequently, Walker and Moss (1984), using meth- Gulf of Mexico; Hapalidiales; ocean acidification; ods similar to Alexandersson (1974), showed that Panama; phylomineralogy; psbA; X-Ray microscopy aragonite is precipitated in between the coralline crusts and their substratum, often when a space Abbreviations: BS, bootstrap value; PP, posterior occurs between them. Unfortunately, since neither probability; SEM-EDS, Energy dispersive spectrome- Alexandersson (1974) or Walker and Moss (1984) try; SEM, Scanning electron microscopy; XRD, X-ray provided empirical SEM-EDS or XRD data in their diffraction studies, no comparisons can be made between their studies and newly generated data. 1Received 1 June 2015. Accepted 18 December 2015. More recently, Smith et al. (2012) using XRD, 2Author for correspondence: e-mail [email protected]. reported the presence of aragonite in the mineral Editorial Responsibility: C. Hurd (Associate Editor) 161 162 SHERRY KRAYESKY-SELF ET AL. composition of Lithothamnion crispatum Hauck, and data in the context of phylogenetics, and utilized Nash et al. (2011) and Diaz-Pulido et al. (2014) doc- the molecular chloroplast marker psbA to establish a umented the presence of aragonite infill in cells of phylomineralogical framework. The present study the reef-building coralline Porolithon onkodes (Hey- answers the call by Smith et al. (2012) to provide drich) Foslie using EDS and XRD. Diaz-Pulido et al. empirical and quantitative data in the study of cal- (2014) also illustrated empty conceptacles of cium carbonate biomineralization in corallines and P. onkodes filled in with aragonite. Torrano-Silva to add psbA sequences in an expanding coralline et al. (2015) used micro-CT or 3D X-ray imaging on psbA framework for phylomineralogy. various coralline samples and concluded that the system could be applied to 3D models and that materials of different density could be individually MATERIALS AND METHODS analyzed. They stated that this tool would be appli- Field collections. Specimens of the biogenic rhodolith-form- cable to the study of aragonite in coralline algae ing crustose coralline Lithothamnion sp. 1 and Lithothamnion sp. 3 were collected in the vicinity of the Florida Middle since it is very dense and can be distinguished from ° 0 ° 0 the other forms of calcium carbonate. Grounds, 84 02.7 W, 28 10.27 N, between 43 and 42 m depth, on July 7, 2006 offshore in the NE Gulf of Mexico; In the northern Gulf of Mexico, subtidal rhodo- and in the vicinity of Ewing Bank, 28° 05.8450 N; 91° 01.8170 lith beds harbor a diverse community of rhodolith- W, between 54 and 58 m depth, on November 16, 2012 off- forming coralline algae (Felder et al. 2014, shore in the NW Gulf of Mexico (Table 1) with an Hourglass- Fredericq et al. 2014, Richards et al. 2014) that have design box dredge using minimum tows (usually 10 min or less; Fredericq et al. 2014, Felder et al. 2014). A Lithothamnion yet to be fully described and characterized. Prelimi- nary SEM imaging of a biogenic rhodolith-forming sp. 2 specimen from the Gulf of Chiriquı, Pacific Panama, was collected by hand at a depth of 16 m while SCUBA diving coralline species identified on the basis of morpho- (Table 1). Specimens were preserved in silica gel on site. logical and molecular evidence as belonging to the Vouchers are deposited in the Algal Herbarium of the genus Lithothamnion, revealed aggregates of needle- University of Louisiana at Lafayette (LAF). like crystals present in their empty reproductive Light microscopy. Specimens were examined under a Zeiss conceptacles. The specimens may conform to the Stemi 2000-C stereomicroscope to remove all visible epi- species concept of Lithothamnion occidentale (Foslie) phytes. Habit protuberances were removed using a single- Foslie, described from Cruz Bay, St. John Island, US edged razor blade and forceps, and longitudinally hand-sec- tioned. No portions of the vouchers were decalcified. Photos Virgin Islands, and reported for the Gulf of Mexico of the habit were taken with a Canon PowerShot A3300 (Fredericq et al. 2009, Mateo-Cid et al. 2014). How- camera. ever, the type specimen of L. occidentale needs to be SEM. Five protuberances comprising five individuals each carefully examined before this species epithet can were selected for SEM, with each protuberance cut longitudi- be assigned with confidence to the material exam- nally into two approximately equal-sized pieces using a razor ined. Likewise, a second species of Lithothamnion blade. The resulting sections were mounted using liquid gra- from the Gulf of Chiriquı, Pacific Panama, showed phite and coated with 11.5 nm of gold. SEM micrographs were produced using a Hitachi S-3000N SEM at a voltage of the same needle-like crystals in empty conceptacles. 15 kV. The SEM is housed in the Microscopy Center at UL In typical light microscopy-based morphological and Lafayette. taxonomic treatments of corallines, hand-and micro- SEM-EDS. The same samples selected for SEM were used tome sections are made after decalcification of the for SEM-EDS analyses using the Hitachi S-3000N and an IXRF thallus parts, hence removing all traces of the cal- system EDS 2000. One nonreproductive sample of Lithotham- cium carbonate mineralogy. In contrast, the present nion sp. 1 (LAF6521) was excluded from SEM-EDS measure- study did not decalcify the samples enabling the ments. To ensure accuracy in element concentration measurements, the input count rate was maintained at 1,000 provision of empirical evidence to confirm the nat- c/s or higher. The working distance was between 15 and ure of the crystals in the complex internal structure 25 mm and the voltage was at 25 kV. The live time for each and skeleton of the coralline rhodoliths, and to measurement was 25–28 s. Element concentrations measuring quantify their elemental composition with SEM- less than 1% (by weight or mole %) were not included in the EDS, XRD, and ZEISS X-ray microscopy, which is a analyses. Individual crystals were quantified as often as possi- new state-of-the