Fig 1.
Cellulose formation in primary (A) and secondary plant cell walls (B). Figure republished from [67] under a CC BY license, with permission from Frontiers, Copyright 2015.
Table 1.
Summary of organic compounds known to be present or absent in coralline algae in contrast to fleshy red algae.
Fig 2.
Not drawn to scale. The pink shading represents the interfilament area. SEM images; top- example of hypothallial cell wall with PCW-only calcification, Phymatolithon laevigatum; bottom, example of perithallial cell wall with SCW calcification, Kvaleya epilaeve, scale bars- 500 nm. SEM images insets enlarged in 14B (top SEM) and 24B (bottom SEM).
Fig 3.
Proposed interaction between organic substrate, organic fluids and seawater for biomineralisation.
AHG: 3,6-anyhdrosgalactose. SXG: sulfated xylogalactan.
Fig 4.
Cell development at meristem split for Clathromorphum nereostratum, C. circumscriptum and C. compactum.
Black boxes enlarged (Fig 5).
Fig 5.
Clathromorphum deltoid interfilament and development of primary (PCW) and secondary cell wall (SCW).
(A) Deltoid interfilament calcification in Clathromorphum. Interfilament crystals grow until touching crystals growing out from opposite cell walls as in Fig 4. Attachment points- it is not known what these are, but they must be consistently present. Possibly rough spots on the external wall surface, or PCW compounds that attract the CMF. (B) Development of primary and secondary cell wall in Clathromorphum (deltoid species).
Fig 6.
(A, B) Phymatolithon investiens, (North Norway). The cell wall calcification is inconsistent. Adjacent cell walls with and without substantial calcification. (C, D) P. laevigatum, (Newfoundland). Cell wall switches from thick SCW to thin PCW for wound repair. (E) Lithophyllum kotschyanum (Ryukyu Islands, Japan). Thin cell walls of elongate PCW cells break easily. Elongate cells range in shape from straight rectangular to curved rectangular. A, B, C, D reproduced from [39] under a CC BY license, with permission from John Wiley and sons, Copyright 2017.
Fig 7.
Cell walls in Amphiroa fragilissima, (Gold Coast, Queensland, Australia).
(A) Overview. (B, C) Medullary cell walls are similar to hypothallial cell walls in CCA having a range of calcification types from a thin mineral coating on a membrane to vertically stacked micro-granules. (D, E) Rounded cortical cells do not have visible radial calcite. There is visible banding within the cell wall.
Fig 8.
Primary cell wall of medullary cells Amphiroa anceps, (Warrnambool, Victoria, Australia).
Etched for 35 minutes. (A) Overview of medullary cells. White box enlarged in B, C. Black box enlarged in D, E and F. (B) Etching has removed interfilament grains exposing organic material between cells. (C) PCW wall has irregular shaped grains encapsulated in organic fibrils (black arrows). Fibrils concentrated at external edge of cell wall forming continuous mesh. (D) Concentration of organic fibrils at cell wall edge visible rimming the cell wall (black arrows). The cell walls are thickest at the corners and there may be secondary cell wall formation (white arrows). (E) Vertical alignment of Mg-calcite grains in cell corners (black arrows). Black box enlarged in F. (F) Grains perpendicular to cell wall, possible SCW (black arrows). Laminar fibrils through cell wall (white arrow).
Fig 9.
Corallina sp., (Coffs Harbour, New South Wales, Australia).
(A) Cortical cells are only present along the long edges and not at the apical tip (growth tip). (B) In contrast to the Amphiroa, the medullary cell walls are very poorly calcified. (C, D) Cortical cell walls are thicker and more densely calcified than the medullary cells.
Fig 10.
Medullary cells in Corallina sp., (Coffs Harbour, New South Wales, Australia).
Sample etched for 35 minutes. (A) Overview. (B) Medullary PCW cell walls are thin and flexible. Black box enlarged in C. White box enlarged in D. (C) Cell walls are less than 100 nm wide. The majority of the carbonate is interfilament Mg-calcite. Interfilament grains are elongate and have fibrils attached. (D) View of cell wall from within the cell. Carbonate granules (white arrow) are encapsulated within a mesh of organic fibrils (black arrow).
Fig 11.
Jania rosea, (Tathra, New South Wales, Australia).
(A, B) Medullary cells extend to the apical tip and there is no cortical cell layer. (C) Flat rectangular Mg-calcite grains form within the surface membrane. (D, E) Medullary cell walls have dense fibrillar mesh and irregular Mg-calcite grains are enmeshed within this.
Fig 12.
Medullary cells in Jania rosea, (Tathra, New South Wales, Australia).
Etched for 35 minutes. (A) Overview. (B) Medullary PCW cells have large, broken edged cell fusions. Cell walls are thin. Black box enlarged in C, White box enlarged in D. (C) Cell walls are less than 200 nm wide. Interfilament has been removed by etching. (D) Possible development of radial Mg-calcite (in black circle) in cell wall corners.
Fig 13.
Hypothallial PCW cells in Clathromorphum circumscriptum, (Norway).
(A) Hypobasal with curved thin cell walls. (B). Cell walls are thinner than perithallial cell walls. There is no radial Mg-calcite present. Interfilament is similar to interfilament in other species and there is no deltoid interfilament as is normally present between Clathromorphum perithallial cells.
Fig 14.
Close up of typical hypothallial cell wall features.
(A) Laminar fibrils are present throughout the perithallial cell walls of both Leptophytum leave (Labrador). (B) P. rugulosum, Newfoundland. Cell walls may have vertically aligned Mg-calcite or no clear calcification pattern. Figure A reproduced from [38] Creative Commons Attribution 3.0 License, Copyright Author(s) 2018. Figure B reproduced with permission from [39]).
Fig 15.
Hypothallial PCW cells in Porolithon onkodes, (reef flat, Heron Is. Great Barrier Reef, Australia).
(A) No visible cell wall structure. Carbonate is predominantly interfilament or intrafilament. (B) Thin layer of Mg-calcite in PCW. Mg-calcite is randomly spaced and shaped grains enmeshed in a mass of fibrils. Figure reproduced with permission from [10].
Fig 16.
PCW-only cell walls in cells infilling emptied conceptacles of Phymatolithon rugulosum, (Iceland).
(A) Overview. (B) Cell walls at this scale appear to have radial vertical structure (white arrow). (C) Minimal interfilament between cell walls. White box enlarged in D. (D) Cell wall grains have well defined crystal faces and are enmeshed within a fibrous mesh. Fibrils are visible running through the interior of crystals (black arrows). Image republished from Nash and Adey [39] under a CC BY license, with permission from John Wiley and sons, Copyright 2017.
Fig 17.
Secondary cell wall in Amphiroa anceps, (Warrnambool, Victoria, Australia).
Etched for 35 minutes. (A) Overview of cortical cells with secondary cell wall and medullary cells with primary cell walls. White box enlarged in B. (B) Radial Mg-calcite in cell walls. These features were not readably visible in un-etched samples. (C) Overview of features enlarged in D, E (White box) and F (Black box). (D) Interfilament grains are generally rice grain shape with edges that have a serrated appearance. The SCW radial Mg-calcite grains are present as multiple layers of shorter radial grains. White box enlarged in E. (E) Radial grains are consistently cylindrical with smooth sides (black arrow). Laminar fibrils form dense mesh at outer perimeter of cell wall (white arrow). (F) Interfilament grain edge serrations appear to form where fibrils are attached (black arrow). Grains appear to form within the dense mesh (white arrow), although this appearance may be an artifact of the etching. Outer edge of adjacent cell wall bordered by dense mesh of fibrils (black arrowhead).
Fig 18.
Surficial cells with developing secondary cell walls in Amphiroa anceps, (Warrnambool, Victoria, Australia).
Sample etched for 35 minutes. (A) Meristem cell forming epithallial cells outwards (up) and cortical cells inwards. White box enlarged in B. (B) The beginnings of radial Mg-calcite development are visible (black arrow) within the bands of laminar fibrils.
Fig 19.
Corallina sp., (Coffs Harbour, New South Wales, Australia).
Etched for 35 minutes. (A) Overview of cortical and medullary cells. Black box enlarged in B. (B) Lower half of image shows a cortical cell with well-developed secondary cell wall and radial Mg-calcite. The upper cell SCW has radial Mg-calcite in the SCW but the grains are discontinuous. Possibly this may be an artifact of etching. There is a separate inner wall that may be a poorly-developed part of the SCW. There is no visible radial structure.
Fig 20.
Possible development of SCW and interfilament in Jania rosea, (Tathra, New South Wales, Australia).
Sample etched for 35 minutes. (A) Overview of surficial cells. White box enlarged in B. Black box enlarged in C. Black dashed box enlarged in D. (B) Epithallial cell appears to have a poorly-developed radial structure. It is not clear if this is a typical SCW radial Mg-calcite. (C) Partial growth of Mg-calcite perpendicular to cell wall. This may be poorly-developed SCW. (D) Interfilament grains are visible through the cell wall fibrillar mesh. These grains are comparable to the rice-shaped grains in other species but are generally longer (~ 500 nm compared to ~200 nm in most species) similarly to the elongate flat grains on surficial cells (Fig 11).
Fig 21.
Perithallial cellular calcification features in Porolithon onkodes, (Heron Is., Great Barrier Reef, Australia [10]).
(A) Perithallial cell immediately beneath epithallus. (B) Radial grains within cell wall have semi-regularly spaced organic fibrils threaded through and around the Mg-calcite radial grains. This location in the crust had been naturally etched by micro-boring activity. (C) The boundary between the cell wall and interfilament has both a fibrillar mesh and patches of organic membrane. Sample was etched for 50 minutes in deionised water then sonic cleaned for 2 minutes. Most of the interfilament carbonate is removed by this preparation processes. (D) This site is near the base of the crust and has been exposed to seawater. The cell wall and interfilament has undergone remineralisation. However, the cell wall fibrils appear to have coalesced and formed distinct laminar bands. Panels A, B, D reproduced with permission from [10].
Fig 22.
Porolithon onkodes, (Heron Is. Great Barrier Reef, Australia [10]).
Cell wall exposed by internal bioerosion etching the surface. (A) Radial Mg-calcite is segmented smaller grains instead of a long continuous grain as in previous image. Possibly the joint lines are the site of fibrils removed by the etching. (B) Grains appear fibrous as if bundles of smaller thinner grains. (C) View into vertically-facing radial Mg-calcite grains. A mass of fibrils enmesh the Mg-calcite grains. Spaces between radial grains are filled with irregularly shaped Mg-calcite nano-granules. Figure adapted with permission from [10].
Fig 23.
Perithallial cell wall in Clathromorphum compactum, (Greenland).
Sample etched 20 minutes in deionised water, sonic cleaned for 2 minutes. Approximately 300 microns below surface. (A) Radial Mg-calcite in cell wall. There is a patch of membrane between the cell wall and interfilament. White box enlarged in B. (B) Organic fibrils thread between and through the Mg-calcite grains. (C) Laminar banding of regularly spaced holes where fibrils presumably were present prior to etching.
Fig 24.
Northern boreal CCA species with radial calcite and microfibrils.
(A) Phymatolithon rugulosum (Newfoundland). (B) Kvaleya epilaeve (Labrador). (C) Phymatolithon investiens (North Norway). (D) Leptophytum leave (Labrador). The radial Mg-calcite in K. epilaeve and L. leave is thicker (100 nm and 60 nm) in diameter than the P. onkodes (20–25 nm) and Clathromorphum (40–50 nm). Images for Phymatolithon species republished from Nash and Adey [39] under a CC BY license, with permission from John Wiley and sons, Copyright 2017. K. epilaeve and L. leave Nash and Adey [38] under a CC BY license, with permission from Copernicus, Copyright 2017.
Fig 25.
Coralline epiphytes (Mediterranean, samples from [94]).
(A) The epiphytes form a single cell layer on the seagrass substrate. (C) There is no clear boundary between a calcified cell wall and interfilament. (B, D) The cell wall appears not to be calcified and elongate Mg-calcite, similar in form to CCA interfilament, Mg-calcite fills space between cell wall and seagrass surface.
Fig 26.
Coralline epiphytes (Mediterranean, samples from [94]).
(A, B) Calcification within the primary cell wall and interfilament. (C, D) Parts of the coralline epiphyte have well developed PCW calcification comparable to CCA PCW.
Fig 27.
Mg-calcite and fibrils in the interfilament.
(A) Lithothamnion glaciale (Scotland, sample from [18]). (B) Porolithon onkodes (Heron Is. Great Barrier Reef, Australia). (C) Phymatolithon rugulosom (Newfoundland). (D) Kvaleya epilaeve (Labrador). (E, F) Clathromorphum compactum (Greenland) with distinctive deltoid interfilament made of clumps of elongate thin cylindrical Mg-calcite. Images for Phymatolithon species republished from Nash and Adey [39] under a CC BY license, with permission from John Wiley and sons, Copyright 2017. K. epilaeve and L. leave Nash and Adey [38] under a CC BY license, with permission from Copernicus, Copyright 2017.
Fig 28.
Mg-content of PCW, SCW and interfilament of Phymatolithon Rugulosum, P. borealis, P. investiens, P. laevigatum.
There is a consistent offset in Mg-content between the SCW (perithallial cell wall) and interfilament. PCW (hypothallial cell wall) Mg-content is higher but the offset from PCW is not consistent. P. cell wall: perithallial cell wall. Hyp. cell wall: hypothallial cell wall. Image republished from Nash and Adey [39] under a CC BY license, with permission from John Wiley and sons, Copyright 2017).
Fig 29.
SCW and Mg-content in Porolithon onkodes (Heron Is. Great Barrier Reef, Australia).
(A, B) Cell wall in P. onkodes with internal banding possibly from an interruption in growth resulting in segmentation of the radial Mg-calcite. White box is image in Fig 21A. (C, D) SEM (carbon coated as this is best for showing the contrast between Mg and Ca whereas platinum is better for imaging). (C) Dark bands are associated with the internal edges of the cell wall segmentations. Dark bands have elevated Mg. Bands are ~0.6 to 1 micron apart. (D) Site for EDS lines, letters are matching mol% MgCO3 values in E. (E) EDS lines across the cell wall in D. Highest Mg bands labeled. Peaks of high Mg are ~ 300–400 nm apart. Lines 4, 5, 6 are top, middle and bottom EDS lines in D.
Fig 30.
Leptophytum laeve (sample from Nash and Adey [38]) SEM-EDS line.
Mg content range 7.4–16.9 mol% MgCO3. Highest Mg content at internal edges and near external edges of cell walls. Yellow arrow- EDS line, 14 sample points.
Fig 31.
Meristem split in Clathromorphum compactum (Gulf of Maine, USA).
(A) Overview. Cell layers above the split break off. (B) Absence of radial calcification in cell wall nearest to meristem split, organics leak into interfilament region. (C) Split forms laterally across cell and interfilament. Black box enlarged in D. (D) Growth features mirror both up and down from split.
Fig 32.
Meristem cell layers without split.
(A) P. leavigatum, (sample from [39]) meristem visible between the change from thinner cell walls of the epithallus to thicker perithallial cell walls. (B) Porolithon onkodes (Heron Is. Great Barrier Reef, Australia). The epithallus peels off, but there is no split along the meristem cell layers. Reproduced with permission from [10].
Fig 33.
Location and development of radial cell wall in Clathromorphum.
(A, B, C) C. compactum (Gulf of Maine, USA). Radial calcification present ~1 μm from split. (D) C. nereostratum (Bering Sea). View up into perithallial cells above meristem split.
Fig 34.
Formation of the interfilament and delayed development of SCW at meristem split.
Clathromorphum compactum, (Gulf of Maine, USA). At the split there is no calcified material. Organic material leaks through the split into the interfilament area outside of the cell. Calcification commences with the formation of interfilament grains vertically orientated. The perpendicular radial Mg-calcite forms ~1 micron above the split and is longer, further from the split. Fibrils are visible stretching between the cell wall radial Mg-calcite crystals. In this image there is no readily visible PCW carbonate.
Fig 35.
Interfilament Mg-calcite in Clathromorphum compactum (Greenland).
Carbonate along meristem split is coated with organic material.
Fig 36.
Rice-grain interfilament and response to etching in Clathromorphum compactum (Greenland) perithallial and hypothallial growth.
(A) Deltoid interfilament is not removed by etching. (B) Rice-grain interfilament in hypothallial is removed by etching. (C) Intact hypothallial interfilament. (D) Close-up from B, showing fibrils throughout the hypothallial interfilament, visible after etching. The Clathromorphum hypothallial interfilament is comparable to hypothallial interfilament in other CCA genera.
Fig 37.
Interfilament in Clathromorphum circumscriptum, (Norway).
(A) Transition from deltoid to non-deltoid interfilament corresponds with change to higher mol% MgCO3. (B) Corresponding with the absence of deltoid interfilament is thicker side cell wall and shorter cell length.
Fig 38.
Rows of carbonate clumps within a portion of damaged crust in Clathromorphum compactum, (Arctic Bay).
The regularity of their size and spacing suggests a strong bio-controlled process. Possibly these are cellulose synthase complexes (or terminal complex) that normally would be active within the cell wall and have reactivated after damage to the crust.
Fig 39.
Transition from SCW to PCW cells in Lithothamnion sp., and Mg content change (Panama, sample from [26]).
(A) Banding. (B) Overview of Mg-content change. (C) EDS spot analyses on PCW-only cell walls. Values range from VHMC (25–36 mol% MgCO3) to dolomite composition (>37 mol% MgCO3). (D) EDS line (black arrow) across transitioning cell wall. Blue plot is the relative change Mg content by weight percent, mol% values plotted in F. (E) EDS line across perithallial SCW cell wall, mol% values plotted in F. (F) Mol% MgCO3 values for the EDS lines in D and E. The interior edge of the transition cell wall has elevated Mg values, comparable to PCW, the remainder of the cell wall with developing radial Mg-calcite has values comparable to the SCW-only perithallial cell wall.
Fig 40.
Hypothallial tissue in Phymatolithon investiens, (North Norway).
(A) Overview (BSE). Basal hypothallus along field of view (white arrow) and into field of view (black arrow). CCA is growing over old CCA crust that has been transformed to aragonite. Black box enlarged in B. (B) Transition from PCW hypothallial cells to perithallial cells with typical radial cell wall and minimal interfilament. Gradual transition from PCW hypothallial to SCW perithallial. This transition is typical for CCA. Black box enlarged in C. (C) Hypothallial cell wall grains appear vertically stacked (white arrows) and second bands of vertical structure walls are forming (white arrowhead). Image republished from Nash and Adey [39] under a CC BY license, with permission from John Wiley and Sons, Copyright 2017.
Fig 41.
Experimentally induced abrupt shifts from SCW perithallial cells to PCW-only cells.
(A, B) Lithophyllum cabiochae. Collected from the Mediterranean and placed in aquaria, not stained (sample from [97]). (A) The base of the switch is equivalent to the time of transfer. The switch comprises a row of 3–4 elongate, PCW-only cells followed by a return to perithallial SCW cells. (B) Highest mol% MgCO3 of cell walls below and above transfer line from an EDS line transect. (C, D) Clathromorphum compactum. Collected from the Gulf of Maine and placed in aquaria, not stained (supplied by J Halfer, University of Toronto). A row of 2–3 elongate, PCW-only cells. The return to cell structure comparable to pre-transfer takes ~10 cells. Average mol% MgCO3 of cell walls below and above transfer line (S2 Table). (E, D) Porolithon onkodes collected from Heron Island, Great Barrier Reef, transferred to aquaria, embedded in resin and returned to the reef slope within 7 days (sample from [33]). This particular sample was recollected every three months over the following 15 months, kept in aquaria for ~7 days, then returned to the reef. The elongate cell row is only present the first time it was collected. Higher Mg content in elongate cells indicated by darker grey shades. Values ranged up to 80 mol% MgCO3.
Fig 42.
(A) Experimental crust of Lithophyllum cabiochae, (Mediterranean, sample from [97]). Where the crust switches to elongate cells photosynthetic pigment is mostly absent. (B, C) Rhodolith Lithothamnion glaciale, (Scotland, sample from [18]). Pigment concentrates with increased SCW-only cells at the edge of the bands. The abrupt shift to PCW-only cells corresponds with reduction in pigment, which gradually builds up again as the cells shift back to perithallial SCW cells.
Fig 43.
Transition from SCW to PCW cells in Lithothamnion glaciale, and Mg-content change (Scotland [18]).
(A) Banding in L. glaciale, white box enlarged in B. (B) Abrupt switch from perithallial cells with SCW to hypothallial-style cells with PCW-only. (C, E) PCW densely calcified, no clear radial calcite structure. (D, F) SCW with radial Mg-calcite. (E, F) SEM-EDS transects across the cell walls (black arrows). (G) EDS mol% MgCO3 measurements across the cell walls. The PCW has values up to dolomite composition, with lower values equivalent to SCW radial Mg-calcite. Wall int.: interior edge of cell wall. Wall ext.: exterior edge of cell wall.
Fig 44.
XRD pattern for Lithothamnion glaciale, (Scotland, sample from [18]).
Black line is L. glaciale. Red line is symmetrical scan made by mirroring the left side of the Mg-calcite peak. Area between the red and black lines is the asymmetry attributable to higher Mg phases. Based on this and the EDS, the PCW mineral ranges from 30–50 mol%, assuming this is disordered dolomite: % ordered: mol% calculated if using calibration for ordered dolomite. % disordered: mol% calculated if using calibration for disordered dolomite (Methods).
Table 2.
Summary of calcification features.