Skeletal ultrastructure of the calcified red alga Galaxaura oblongata, Hainan Island, China

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Review of Palaeobotany and Palynology 104 (1999) 205–212

Skeletal ultrastructure of the calcified red alga Galaxaura oblongata, Hainan Island, China Xinan Mu a , Robert Riding b,* a

Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, 39 East Beijing Road, Nanjing 210008, China b Department of Earth Sciences, Cardiff University, Cardiff CF1 3YE, UK Received 9 January 1998; revised version received 24 August 1998; accepted 28 August 1998

Abstract Aragonite calcification in the red alga Galaxaura oblongata (Ellis et Solander) Lamouroux (Chaetangiaceae, Nemaliales) from southern China, occurs at three sites in the cortex: (1) intercellular spaces (ICS); (2) the cell wall; and (3) the inner surface of the cortex. The ICS is the principal site of calcification and contains aragonite mainly in the form of equidimensional granules ¾1–2 µm in size, although needle-like crystals up to 3 µm in length are also present locally. Granules range from densely to loosely packed. A dense and even layer peripheral to the ICS is interpreted to represent calcification of the cell wall. Coarser radial clumps of elongate crystals, forming a thin discontinuous layer on the inner surface of the cortex, resemble a cement fringe facing into the medulla, but form during the life of the alga. Calcification in these specimens shows similarities, and differences, to both coralline red algae, in which calcification is limited to the cell wall, and halimedacean green algae in which it is limited to the ICS. Some members of the Palaeozoic family Moniliporellaceae show similarities in their skeletal organization to Galaxaura.  1999 Elsevier Science B.V. All rights reserved. Keywords: algae; calcification; China; Galaxaura; Recent; Rhodophyta; Moniliporellaceae; ultrastructure

1. Introduction Although Galaxaura Lamouroux (1816) is a common benthic calcareous alga, which is widespread in present-day tropical and sub-tropical shallow marine environments (Mu, 1991, p. 337), knowledge of its calcification and ultrastructure is limited. The CaCO3 is deposited as aragonite (Meigen, 1903, p. 13) in the cortical region of the alga, and the degree of calcification varies from species to species (Svedelius, 1953). The medullary region of the alga is uncalcified. After death and decay of the organic material, Ł Corresponding

author. Fax: C44-1222-874326; E-mail: [email protected]

the calcified skeleton remains as a relatively thin external layer, <100 µm thick, in which the cortical cells are preserved as rounded outlines, surrounding the hollow interior of the cylindrical thallus. Calcification in Galaxaura has been described as extracellular (Flajs, 1977), the principal site of aragonite deposition being the intercellular space between adjacent cortical filaments (Borowitzka, 1977). According to Flajs (1977) the aragonite occurs as needles. Consequently, calcification in Galaxaura has been compared with that seen in the green algae Acetabularia (Flajs, 1977, p. 99) and Halimeda, as well as with aragonitic red algae such as Liagora and Peyssonelia (Borowitzka, 1989, p. 77). Flajs (1977, p. 99) suggested that the cell wall in Galaxaura may also

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be calcified, and this was confirmed by Okazaki et al. (1982) in studies of G. fastigiata Decaisne. Furthermore, Okazaki et al. (1982) found granular crystals less than 0.1 µm in size in the intercellular spaces and did not report the presence of aragonite needles. Galaxaura has been compared with the fossil genus Gymnocodium Pia (Pia, 1937), but the style of calcification in Gymnocodium is actually more similar to that seen in another chaetangiacean, Liagora (Mu, 1991). A Palaeozoic group, the Moniliporellaceae, has been compared with gigartinalean red algae (Gnilovskaya, 1972), but in skeletal organization exhibits some similarities with Galaxaura. In order to clarify the style and site of calcification in Galaxaura, and to assist comparisons with fossil algae, we examined present-day specimens of one species, G. oblongata, from southern China. We found that calcification occurs mainly in intercellular spaces (ICS), but also in the cell wall, and on the inner surface of the cortex. This contrasts both with coralline red algae in which calcification is limited to the cell wall, and with the green alga Halimeda in which calcification is limited to the ICS.

2. Materials and methods In April 1995, entire specimens of G. oblongata (Ellis et Solander) Lamouroux [for discussion of galaxauracean species taxonomy, see Huisman and Borowitzka (1990) and Huisman and Townsend (1993)] were collected from the shallow subtidal (<1 m depth) fringing reef flat at Xiaodonghai Bay, Sanya, on the southern extremity of Hainan Island, southern China. Specimens were air dried in the shade without rinsing in freshwater. For SEM examination, bleached (using H2 O2 or domestic NaOCl) and unbleached specimens were fractured and coated with gold-palladium. Qualitative compositional analyses were carried out using EPMA of carbon-coated specimens.

the cortex, whereas the medulla is uncalcified (Plate I, 2). In the specimens studied, both the cortical cell walls and the cortical intercellular spaces (ICS) (Plate I, 3–5) are calcified. The skeleton produced by this calcification has a honeycomb-like appearance (Plate I, 2), with open spaces (the cell interiors) separated by walls of variable thickness corresponding to the combined cell walls and ICS (Plate I, 3). In addition to this regular calcification, the inner part of the cortex is discontinuously veneered by crystals (Plate I, 6; Plate II, 1). At the outer surface of the thallus, the inner parts of the epidermal cells are calcified, but the outer parts are not (Plate II, 2). Conceptacles (Plate I, 1) and pit connections between cortical cells (Plate II, 3) are not calcified. 3.1. Intercellular space The ICS of the outermost cell layers (typically three layers in the specimens studied) of the cortical zone is calcified. The ICS is partially to completely filled by randomly packed grains of aragonite (Plate I, 3, 5). Near the margins of the ICS the grains average ¾0.1 µm (range 0.06–0.25 µm) in size, but are larger, 0.2 µm (range 0.06–0.4 µm), and generally less densely packed, towards the centre of the ICS where they appear to coalesce into aggregates (Plate III, 1). Locally, aragonite needles up to 3 µm long are also patchily present within the ICS (Plate II, 4). 3.2. Cell wall Locally, it is possible to distinguish a layer external to the ICS interpreted as calcified cell wall. This is very densely calcified and forms a well-defined even layer, ¾0.5–2.7 µm thick, that is denser than the adjacent ICS (Plate I, 3–5). Where they can be distinguished, the grains composing this layer are ¾0.1 µm in size. 3.3. Inner cortex

3. Results G. oblongata forms densely branched thalli (Plate I, 1). Calcification is observed in the walls of

Radially arranged bundles of tapering needle-like crystals, up to 0.7 µm wide and 2–5 µm long, occur as isolated fan-like clusters or closely packed layers up to ¾6 µm thick veneering the inner surface of

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PLATE I

Galaxaura oblongata (Ellis et Solander) Lamouroux. Recent, from Xiaodonghai Bay, Sanya, Hainan Island. SEM photomicrographs. Specimens unbleached except where stated. 1. Detail of whole specimen showing external appearance, branching (including fracture) and uncalcified conceptacles (pits). Scale bar D 1 mm. 2. Fractured thallus showing transverse view of calcified cortex with ovoid outlines of cells. External epidermal surface to left. Hollow uncalcified medulla to right. Scale bar D 100 µm. 3. Calcified cell walls of four adjacent cortical cells and intervening more weakly calcified intercellular space. Bleached specimen. Scale bar D 5 µm. 4. Calcified cell walls showing dense granular structure of the aragonite. Intervening space is the more weakly calcified intercellular space. Bleached specimen. Scale bar D 1 µm. 5. Densely calcified cell wall (light colour), juxtaposed against the exterior part of the intercellular space (upper right) showing coarser granular structure. Scale bar D 1 µm. 6. Contact between cortex (right) and medulla (left) showing calcified cell wall and intercellular space (lower right) with contrasting needle-like aragonite (centre left) forming an irregular crystal fringe facing into the medulla. Scale bar D 5 µm.

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PLATE II

Galaxaura oblongata (Ellis et Solander) Lamouroux. Recent, from Xiaodonghai Bay, Sanya, Hainan Island. SEM photomicrographs. Specimens unbleached except where stated. 1. Inner surface of cortex viewed from the medulla showing radial clusters of fibrous aragonite discontinuously encrusting the calcified cortex and surrounded by desiccated uncalcified organic tissue. Scale bar D 5 µm. 2. Transverse view of fractured calcified cortex showing ovoid outlines of cells. In this example it is difficult to distinguish the calcified cell wall from the intercellular space. External uncalcified surface of epidermal cells on extreme right. Medulla to left. Scale bar D 5 µm. 3. Site of cortical cell with pit connection (central pore). The calcified cell wall is just distinguishable as a thin dense regular layer contrasting with the more granular appearance of the intercellular space. Bleached specimen. Scale bar D 1 µm. 4. Calcified cortical intercellular space showing both finely granular structure (upper part) and aragonite needles (lower part). Bleached specimen. Scale bar D 1 µm.

the calcified cortex, facing into the medulla (Plate I, 6; Plate II, 1; Plate III, 2). EPMA shows that these crystals have a low Mg : Sr ratio similar to that of the ICS calcification, suggesting that they are aragonite.

4. Discussion 4.1. Crystal-grain shape It has been suggested that the skeletons of some Galaxaura species have a needle-like struc-

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PLATE III

Galaxaura oblongata (Ellis et Solander) Lamouroux. Recent, from Xiaodonghai Bay, Sanya, Hainan Island. SEM photomicrographs. Unbleached specimens. 1. Calcified cortical intercellular space showing outer finely granular and inner more coarsely granular parts. Scale bar D 1 µm. 2. Detail of Plate II, 1, showing cluster of fibrous aragonite crystals encrusting the inner surface of the calcified cortex and facing towards the medulla. Scale bar D 1 µm.

ture (Lowenstam, 1955). Flajs (1977) was the first to use scanning electron microscopy to examine the ultrastructure of Galaxaura. In the three species (G.

cylindrica, G. fragilis, G. lapidescens) that he examined, he reported that the skeleton consisted of aragonite needle aggregates, which he designated

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the Galaxaura type (Flajs, 1977, p. 99). According to Flajs (1977, p. 99) the aragonite in Galaxaura occurs as parallel arrangements of orientated or unorientated needles, which he compared with those seen in the dasycladalean green alga Acetabularia. However, although needles occur occasionally, the specimens of G. oblongata examined consist fundamentally of densely packed microgranular aragonite. This is consistent with the observations of Okazaki et al. (1982, fig. 18) who studied calcification in G. fastigiata by TEM and SEM and found that calcification was initiated in the intercellular space (ICS), and consists of granular aragonite crystals ¾0.1 µm in diameter. They do not mention the presence of aragonite needles. In contrast, Flajs (1977, pl. 12, figs. 6–7; pl. 13, figs. 1–2) illustrates needle fabrics in G. lapidescens and G. cylindrica that are distinctly different to the dense microgranular structure in G. oblongata. Possibly the illustration of G. cylindrica by Flajs (1977, pl. 12, fig. 4) also shows microgranular wall structure, but the magnification is too low to decide this. Our conclusion is that the skeleton of G. oblongata is, despite the local presence of aragonite needles, distinctly different from the well-developed interlocking meshwork of needles shown by Flajs (1977, pl. 12, figs. 6–7; pl. 13, figs. 1–2) and distinguished as the ‘Galaxaura-type’ of structure (Flajs, 1977, p. 99, fig. 15). It is difficult to explain how granular and needlelike crystals coexist in the same individuals of this alga. Flajs (1977, pl. 17, figs. 1–3) records grain diminution of aragonite in the dasycladalean green algae Dasycladus and Cymopolia. Similar phenomena have been reported by Macintyre and Reid (1994) in the green alga Halimeda incrassata. They found that even in the living plant, both aragonite granules (minimicrite) and needles are present in the same individual. They considered that the granules are diagenetic products from the alteration of aragonite needles and they found that in some segments of the alga, aragonite needles alter into small equant crystals (Macintyre and Reid, 1994, fig. 3a– c). However, in our specimens of G. oblongata, we have observed no clear sign of alteration, and the grains in Galaxaura appear smaller than those in Halimeda. A tentative interpretation proposed here is that the existence of both granular and needle-like grains

in the same specimens of G. oblongata could be a result of the microenvironmental conditions within the algal thallus. The different morphology of the aragonite crystals may reflect differing rates of crystallization which may be controlled by the Ca2C ions in solution or by variations in pH, which in turn may be controlled by the physiological activity of the alga; for example, the intensity of photosynthesis (see Borowitzka, 1986). We conclude that the granular aragonite in G. oblongata is primary calcification rather than a diagenetic product such as that seen in Halimeda. This is consistent with the observations of Okazaki et al. (1982) of the form of initial CaCO3 in Galaxaura fastigiata. However, we cannot rule out the possibility of some diminution and alteration of needle size and shape in Galaxaura. 4.2. Cell wall calcification The layer rimming the cells is interpreted here to represent calcification of the cell wall itself. Its even thickness, very finely granular structure and absence of needles are consistent with its being calcified cell wall, and in these respects it resembles that observed in coralline algae (cf. Bosence, 1991). Furthermore, its thickness (¾0.5–2 µm) is closely comparable with the thickness of the cell wall in G. fastigiata figured by Okazaki et al. (1982, figs. 11–12) (0.5– 1.6 µm). 4.3. Galaxaura-style calcification The presence of cell wall calcification in Galaxaura, suspected by Flajs (1977, p. 99) and confirmed by Okazaki et al. (1982), and the predominantly finely granular nature of aragonite in the ICS, demand reassessment of the style of calcification in this alga. Previous comparisons, emphasizing extracellular calcification and needle-like fabrics, were with green algae, such as Acetabularia (Flajs, 1977) and Halimeda (Borowitzka, 1989, p. 77). It now appears that Galaxaura is in fact unusual in combining ICS and cell wall calcification. This complicates comparisons with the Halimeda model of calcification. In contrast, Galaxaura is unusual in exhibiting a combination of both cell wall and ICS calcification that would have parallels with both coralline red algae and with green algae. This in turn raises com-

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parisons between Galaxaura and both red and green algae in terms of details of its reproductive structures and overall morphology. Galaxaura has reproductive structures (conceptacles) within the thallus (as pitlike bodies), but its overall vegetative appearance is reminiscent of green algae such as Halimeda. The presence of cortical ICS probably favours and localizes calcification, as it does in Halimeda (Borowitzka, 1989) but is further supplemented by cell wall calcification. 4.4. Coarse internal crystals Clusters or layers of relatively coarse fibrous, probably aragonitic, crystals on the inner surface of the cortex (Plate I, 6; Plate II, 1; Plate III, 2) have also been recorded by Flajs (1977, pl. 12, fig. 4) in G. cylindrica as a ‘turf-like’ layer. These crystals form during the life of the alga, as show by the fact that in G. oblongata they are intimately associated with the soft tissue at the boundary between the medulla and cortex (Plate I, 6; Plate II, 1). However, they differ considerably in size and shape from the grains and needles of the intercellular spaces in the calcified cortex. They appear to represent a deposit which has the appearance of a cement-like veneer, but which must, at least to some degree, depend for its formation on microenvironmental conditions occurring within the alga during its life. Somewhat similar ‘early diagenetic deposits’ are known in coralline algae (see Cabioch and Giraud, 1986, p. 152). 4.5. Ancient analogues Members of the Gymnocodiaceae Elliott, 1955, an extinct group mainly ranging from Permian to Cretaceous, were originally placed in the green algae (Pia, 1927). Following comparison of Gymnocodium with Galaxaura (Pia, 1937; Elliott, 1955, 1961), they have widely been regarded as red algae (Riding and Guo, 1991). Knowledge of the ecology and phytogeography of extant chaetangiaceans (such as Galaxaura and Liagora) has been used to interpret the palaeoecology and palaeobiogeographical distribution of Cretaceous gymnocodiaceans (Mu, 1993). It has been suggested that the style of calcification in Gymnocodium is actually more similar to that seen in Liagora rather than in Galaxaura (Mu, 1991). An

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obscure group of fossil algae, the Moniliporellaceae Gnilovskaya, principally known from the Ordovician of Kazakhstan, has been compared with gigartinalean red algae (Gnilovskaya, 1972). However, some moniliporellaceans show similarities in skeletal appearance to Galaxaura. For example, Moniliporella, Contexta, Texturata, and Villosoporella (see Gnilovskaya, 1972) all exhibit calcification of a relatively thin cortical zone, although in most cases the cells preserved are elongate rather than rounded. Future studies of these problematic Palaeozoic algae should explore comparisons with extant chaetangiaceans such as Galaxaura.

5. Conclusions (1) Intercellular space calcification in G. oblongata in the cortical region is generally very finely microgranular aragonite. Locally there are traces of longer needle-like crystals, and the inner part of the ICS locally is more weakly calcified. (2) A dense calcified layer with even thickness and up to 2 µm thick probably represents the cell wall itself. The structure of this layer is even more finely granular than that of the ICS and lacks needlelike crystals. (3) Together, ICS and cell wall calcification create a honeycomb-like skeleton defining the cortical cells of the alga. This combination of sites is unusual and modifies previous views which emphasized comparisons with calcification in green algae such as Halimeda. Calcification in Galaxaura shows a combination of intracellular and extracellular processes which are comparable with both coralline algae and Halimeda, respectively. (4) On the inner surface of the cortex, relatively coarse fibrous, probably aragonite, crystals form individual radial clusters that locally unite to form a layer ¾5 µm thick facing inwards towards the centre of the thallus. These cement-like crystals form during the life of the alga. (5) Some members of the Palaeozoic family Moniliporellaceae possess a thin-walled calcified cortex, with rounded cells, that resembles that seen in Galaxaura.

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Acknowledgements Bangmei Xia kindly identified Galaxaura oblongata. This work was supported by the Chinese National Science Foundation and the Laboratory of Palaeobiology and Stratigraphy, Nanjing Institute of Geology and Palaeontology. The South China Sea Institute of Oceanography is thanked for assisting field-work, and the Academia Sinica-Royal Society exchange programme for facilitating a visit by XM to Cardiff. We are grateful to Michael A. Borowitzka and Daniel W.J. Bosence for helpful comments on the manuscript.

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