Introduction

The presence of microbial borings in fossil and modern animal skeletons has been noticed since the 19th century, attributed initially to fungi under the name Mycelites ossifragus Roux (1887). A wider spectrum of microorganisms that penetrate carbonate substrates became known after Bornet and Flahault (1888, 1889) described several new species of cyanobacteria, fungi and eukaryotic green algae in the shells of molluscs and, later on, when Ercegović (1932a, b) studied and described new cyanobacterial genera and species that penetrate coastal carbonate rocks across the intertidal and supratidal zones. The rock-penetrating habit was later found to occur as part of alternate generations in the development of various green and red algae and was studied in culture (Kornmann 1959, 1960, 1961, 1962; Kornmann and Sahling 1980).

Batters (1892) was the first to describe a carbonate-penetrating rhodophyte as a new genus and species, Conchocelis rosea, which is now referred to as the Conchocelis stage in the development of several red algae, especially Bangiales. It was Kathleen Drew (1949) who established that Conchocelis is, in fact, an endolithic phase in the development of Porphyra umbilicalis, the edible leafy red alga, which is an important part of diet in Japan (Nori). She noticed that Porphyra, which grows in England only between October and February, survives between March and September inside carbonate rocks and shells in the form of an alternating, euendolithic generation (Drew 1954, 1956). Thanks to her discoveries, the Conchocelis stages of a number of Porphyra species, including P. arasaki, P. haitanensis, P. kunidae, P. pseudolinearis, P. seriata, P. tenera and P. yezoensis, have been successfully grown in clam shells (Tseng and Borowitzka 2003). The development and alternation of generations in Porphyra that included a Conchocelis stage were then studied in culture by Tseng and Chang (1955), Kornmann (1961), Kornmann and Sahling (1980) and Bao-Fu (1984). Under natural conditions (i.e. in situ), the Conchocelis stages were first reported in various carbonate substrates from cold marine ecosystems, where they occurred down to considerable depths (70 m), so they were proposed to be used for determination of the euphotic zone; in contrast, the leafy epilithic generation of Porphyra was found mostly in the intertidal zone (Clokie et al. 1979, 1981).

Microboring organisms or microbial euendoliths (Golubic et al. 1981) evolved among cyanobacteria, green and red algae, fungi and lichens. They colonise all types of carbonate substrates in marine and freshwater ecosystems from polar to tropical environments and are major agents in marine bioerosion in coral reef ecosystems (reviewed by Tribollet et al. 2011). Green algae among phototrophic euendoliths, which are known to penetrate both dead carbonate substrates as well as live and growing scleractinian corals, are well established in modern (Odum and Odum 1955; Lukas 1973; Le Campion-Alsumard et al. 1995) and ancient (Kołodziej et al. 2012) tropical settings. In contrast, the euendolithic rhodophytes have been less frequently observed in corals or any other carbonate substrates, thus receiving much less attention. Laborel and Le Campion-Alsumard (1979) were the first to report Conchocelis in abundance in live and growing scleractinian corals (Dichocoenia sp. and Mycetophyllia sp.) in the tropical Caribbean and Brazilian reefs. They observed that filaments of Conchocelis conferred a pinkish to purplish colour to the coral skeleton and apparently excluded other euendolithic microorganisms that usually inhabit the skeletons of live corals, such as the chlorophyte Ostreobium quekettii (Odum and Odum 1955; Lukas 1973; Le Campion-Alsumard et al. 1995).

More recently, stylasterid hydroids (also known as lace corals) on Indonesian coral reefs were found to exhibit an unusual pinkish to purplish colouration, which was attributed to boring cyanobacteria in their skeletons (Puce et al. 2009), similar to those observed in cold water azooxanthellate corals (Försterra and Häussermann 2008; Tribollet et al. 2011, fig. 10). The light-dependent distribution of the red pigmentation within the lace coral colonies indicated the phototrophic nature of these microbial euendoliths (Puce et al. 2009; Pica et al. 2016). A recent microscopic analysis of stylasterid colonies from the Indo-Pacific determined that the most abundant of these microboring organisms are, in fact, representing the eukaryotic Conchocelis stages of bangialean rhodophytes, rather than euendolithic cyanobacteria (Pica et al. 2016). The taxonomy of bangialean rhodophytes is currently under revision, supported by molecular analyses (Lindstrom et al. 2015).

In the present contribution, we analysed the microborings produced by euendolithic filaments of Conchocelis stages in the skeletons of selected species of live stylasterid corals from Indonesian reefs (see Pica et al. 2016), comparing the morphology of Conchocelis filaments with the outlines of their boreholes. The description and characterisation of the shapes, ramifications and boring patterns of Conchocelis may help to distinguish between morphologically and taxonomically different endolithic microorganisms penetrating coral skeletons, including their recognition in fossil borings as indicators of depositional depth, i.e. of the extent of the photic zone and light conditions in ancient marine environments (Clokie et al. 1979; Radtke 1993; Radtke et al. 1996; Vogel et al. 2000). It may also stimulate studies of specific bioerosion rates as reviewed by Tribollet (2008) and of a possible symbiotic or parasitic relationship between euendoliths and scleractinians (see Fine and Loya 2002), as well as stylasterid corals.

Materials and methods

From a large collection of stylasterid corals of Indonesia, microbial euendoliths were observed in 38 out of a total of 128 specimens (Pica et al. 2016). For the purpose of the present contribution, a selection of five specimens belonging to five different species of the genera Stylaster and Distichopora (see Table 1) were analysed for distribution, morphological differentiation and fine structure of microboring filaments inside their skeletons. The preceding study of euendolith abundance in the stylasterid skeletons of the same collection reported by Pica et al. (2016) showed that all specimens of the above corals harboured the same type of euendolithic filaments.

Table 1 Location and habitats of studied stylasterid corals from North Sulawesi (Indonesia)
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The samples were collected on coral reefs at North Sulawesi, Indonesia at depths from 2 to 35 m (see Pica et al. 2016; Table 1). They were preserved dry or in 70% ethanol. Small fragments of coral branches (1–2 cm long) were first gradually dehydrated in ethanol and then embedded in the Struers SpeciFix epoxy resin (Golubic et al. 1970). After complete polymerisation at ambient temperature, thin sections were cut with a diamond saw to prepare petrographic thin sections about 30–40 μm thick (Tribollet et al. 2002). One subset of sections was partially decalcified (for few seconds in 3% HCl) and stained with Toluidine blue prior to observation under a light microscope (Nikon Eclipse LV100). The second subset of partially decalcified thin sections was gold-coated for observation under a scanning electron microscope (SEM; Zeiss EVO LS15 from the Alizés platform, IRD, Bondy, France).

The dimensions of filaments (diameter of tubules, and width and length of cellular units, swellings and connections between cells) were measured from pictures taken by light microscopy and SEM using Motic software (Motic China Group, Co., Ltd.). The results were expressed as mean ± standard deviation (SD). Taxonomic determinations were consulted with Guiry and Guiry (2017).

Results

Abundant multiply branched filaments composed of eukaryotic cells, pigmented red from the auxiliary light-harvesting pigment known as phycoerythrin (e.g. Glazer 1985), were observed penetrating the skeletons of Stylaster tenisonwoodsi, Stylaster cf. eximius, Stylaster sp., Distichopora sp. and Distichopora cf. vervoorti, concentrating on the light-exposed side of coral colonies (see fig. 4 in Pica et al. 2016). They provided a red to purple colour to coral colonies, which usually appear white or differently pigmented as orange or violet due to the naturally coloured skeleton and living tissues (Fox 1972). These microboring or euendolithic filaments were made visible in sections across the coral stem after the skeletal carbonate was dissolved by dilute HCl, stained with Toluidine blue solution (Fig. 1a, c–f). The boreholes produced by these filaments inside coral skeletons were analysed separately as resin-cast replicas using SEM (Figs. 1b and 2).

Fig. 1
figure 1

Networks of euendolithic Conchocelis filaments inside the coenosteum of the lace coral Stylaster sp. View of a cross-section of the colony stem after removal of skeleton material. a Network of Conchocelis filaments stained with Toluidine blue. b Scanning electron microscope (SEM) image of the three-dimensional display of resin-cast microborings produced by the Conchocelis filaments shown in a, which constitutes the ichnofossil Conchocelichnus isp. c Detail of a with spherical and droplet-shaped swellings; clusters of filament branches in the background. d Network of cylindrical filaments and series of spindle-shaped cells. e Serial arrangement of spindle-shaped cells interconnected at the narrow ends. f Branching pattern of Conchocelis filaments showing club-shaped protrusions. The scale bar in c applies to c–f

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Fig. 2
figure 2

Conchocelichnus isp. (Radtke et al. 2016), a complex trace fossil, presented as resin casts of borings that conform with the outlines of euendolithic Conchocelis filaments in shape and size (details from Fig. 1b) in the lace corals Stylaster sp. (‘white’) and Stylaster tenisonwoodsi (‘orange’). a A dense cluster of microboring casts constructed from cylindrical cells constricted at the cross-walls, flat leaf-like cells with uni-directional branching. b Lose network of borings conforming to filaments with predominantly spindle-shaped cells exhibiting constrictions at the cross-walls between cells and swellings with branches positioned at one end. c An assemblage of interconnected differently shaped borings. d Detail of a, with filaments of different shapes and sizes, cell connections in series and lateral branches. Note the exposed pit connections between cells and irregular club-shaped cell wall protrusions (centre). e A cluster of branches with exposed pit connections, located at one of the ends of a cylindrical cell unit. f Series of cellular swellings exhibiting wider and narrower interconnections and branching localised at the upper end of the cellular swelling. g Detail of c. A group of cell units with flat widening. The scale bar in b apples to a–c

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The microorganisms that formed the branched carbonate-penetrating filaments were recognised to be Conchocelis stages of bangialean rhodophytes, growing inside the coral coenosteum, without exiting into the pore space. They grew predominantly underneath and along the coenosteum interior surface. Short filament extensions were, however, observed connecting the filaments in the coenosteum interior with those outside.

The Conchocelis filaments in S. tenisonwoodsi, S. cf. eximius, Stylaster sp., Distichopora sp. and Distichopora cf. vervoorti contained membrane-bound organelles, such as chloroplasts, coloured red by the auxiliary light-harvesting pigment phycoerythrin. The filaments were composed of cells interconnected by pit connections located in the centre of cross-walls (see Li et al. 2006). The light photomicrographs (Fig. 1a) show an intricate meshwork of filaments forming branch clusters, which conform closely with the outline and orientation of the borings as replicated in polymerising resin and observed by SEM (Fig. 1b). Common types of Conchocelis filaments include series of cylindrical cells (5–10 μm in diameter) constricted at the cross-walls. The cells frequently widen upward, where they produce a cluster of branches at the apical side of the cell (Figs. 1c and 2f). The cylindrical filaments are usually replaced by series of spindle-shaped cells (9.5 ± 1.85 μm wide, 33.08 ± 7.9 μm long; n = 35), interconnected at their narrow ends (1.72 ± 0.18 μm wide; n = 18), sometimes extending for considerable distance within the skeletal carbonate (Fig. 1d, e). Spherical swellings (10–25 μm in diameter) occur solitary or in series (Fig. 1a, c). Terminal clusters of branches with club-shaped cell wall protrusions are morphological characters typical of Conchocelis (Figs. 1f and 2d).

The three-dimensional display of borings is shown by SEM images (Figs. 1b and 2). The borehole outlines maintain a close conformity with cell outlines, including details at the cell connections. Cylindrical boring traces of different shapes and branching patterns are shown in Fig. 2a–c and in more detail in Fig. 2d–g. Cylindrical tunnels with cross-wall constrictions are shown in all images of Fig. 2, which are especially clear in Fig. 2b, top and Fig. 2d–e. The traces of spindle-shaped cell units are shown in Fig. 2a, b. When viewed by SEM at higher magnification, they are seen as flat widenings (Fig. 2c, d, g), and so are the asymmetrical widenings with ramifications (Fig. 2a, b, f). Club-shaped terminal cell wall protrusions (Fig. 1f) are also well illustrated in three dimensions (Fig. 2d), as well as the clusters of branches concentrated on the apical poles of cell units (Fig. 2e, f). An important detail revealed by SEM at higher magnification is the exposed pit connections between cells (Fig. 2d, e, g).

The skeletons of the five stylasterid corals under study are all coloured red by Conchocelis filaments. The filaments of the siphonal chlorophyte Ostreobium quekettii and very fine tunnels probably belonging to fungi were recognised in Distichopora cf. vervoorti, although they were rare. The concentration of Ostreobium filaments was too low to confer a green colour to stylasterid skeletons as it is known for scleractinian corals (e.g. Odum and Odum 1955; Lukas 1973; Le Campion-Alsumard et al. 1995).

Discussion

Puce et al. (2009) identified microboring organisms in the skeletons of Stylaster species as cyanobacteria. After the study of more stylasterid colonies, it was determined that the prevalent euendoliths in stylasterid corals are eukaryotic Conchocelis stages of bangialean red algae (Pica et al. 2016). The colonisation of stylasterid skeletons seems to be relatively widespread, as euendoliths were observed in at least two distinct genera of lace corals in the present study. The interrelationship between euendolithic microorganisms and their host corals opens several ecologically and taxonomically interesting questions. For example: (1) Is this relationship symbiotic, parasitic or commensal? (2) How does Conchocelis cope with the coral growth? (3) What is the longevity of the Conchocelis euendolithic residing in stylasterids? (4) What is the distribution of Conchocelis in space and time, and how does it relate to water clarity? (5) To which rhodophyte species do these developmental Conchocelis stages belong?

  1. (1)

    Puce et al. (2009) suggested that phototrophic euendoliths in stylasterid corals may be symbiotic, if they exchange metabolites as it was proposed for the relation between the euendolithic chlorophyte Ostreobium and its scleractinian host corals by Schlichter et al. (1995) and Fine and Loya (2002), which may be especially beneficial in the event of coral bleaching (e.g. van Oppen and Lough 2009). Since stylasterid corals do not host zooxanthellae, such an arrangement may be beneficial throughout the life of the coral, despite some losses to its skeleton density due to dissolution by euendolithic microorganisms (see Tribollet 2008 for a review). How efficient are Conchocelis filaments in dissolving lace coral skeletons and what is the impact on their structural resilience remains to be determined. The filament abundance of Conchocelis certainly varies among different stylasterid coral species but also among colonies of a same species (Pica et al. 2016). The potential benefit to the Conchocelis is to live under the protection of their hosts.

  2. (2)

    The pink colouration in stylasterids has been observed to be particularly intensive in the lower parts of the colony, while gradually fading towards the tips, indicating a certain lag between the Conchocelis growth behind the accretion of the coral skeleton. This pattern may suggest that the Conchocelis filaments were present but “diluted” by the high rates of the lace coral growth (Pica et al. 2016), which would be similar to conditions observed in scleractinian corals (Godinot et al. 2012). According to such a hypothesis, the Conchocelis filaments would have colonised the stylasterid skeleton early, starting from its base while in contact with the substratum that may already have been colonised by microborers. An inversion of this condition has resulted in green banding produced by Ostreobium in massive scleractinian corals; as coral growth slowed down (see Fine and Loya 2002), it permitted intense ramification of the euendolith, which produced a green band (Lukas 1973; Le Campion-Alsumard et al. 1995). However, equivalent red bands in stylasterid corals were not observed. The stylasterids are known for relatively slow accretion rates of a few mm/year, especially in deep waters (Wisshak et al. 2009), while the penetration rates of Conchocelis filaments require further studies.

  3. (3)

    The observations by Pica et al. (2016) on stylasterid corals also include abrupt terminations of the colour and of the presence of Conchocelis. This suggests that Conchocelis may have terminated its growth by the release of spores to start an epilithic phase. The leafy portion of bangialean rhodophytes is known to be seasonal, as determined by photoperiodic control of spore release (Dixon and Richardson 1969), although both foliose and euendolithic phases can be perpetuated independently, thus able to “short-circuit” the alternation of generations (Campbell and Cole 1984).

  4. (4)

    The leafy, epilithic phase of bangialean rhodophytes is separated from the euendolithic growth of Conchocelis in space as well as in time. Conchocelis has been found in deep cold waters of the North Sea down to a depth of 78 m, while the leafy epilithic thallus was observed only in the intertidal zone (Clokie et al. 1981). The means of transportation of the propagules that connect the two phases remain an unresolved problem. The ability of Conchocelis as a rhodophyte to photosynthesise at low light levels (Vadas and Steneck 1988) accounts for its considerable depth distribution. Clokie et al. (1981) proposed, therefore, that Conchocelis may serve as a palaeobathymetric indicator for determining depositional depths in ancient oceans. Because the microborings of Conchocelis preserve well as fossils and can be recognised by their complex morphology, as shown in the present contribution, they may play an important role in determination of the ancient photic zone, together with traces of other phototrophic euendoliths among chlorophytes and cyanobacteria. In oligotrophic tropical and subtropical waters, the chlorophyte Ostreobium has been recorded at 200- and 300-m depths (LeCampion-Alsumard et al. 1982; Vogel et al. 2000). This euendolithic chlorophyte is known to have red-shifted pigments (chlorophylls), allowing life in poorly illuminated environments (Koehne et al. 1999; Magnusson et al. 2007). Tropical euendolithic cyanobacteria may also harbour specific chlorophyll pigments (chl d) to survive in low light regimes (Behrendt et al. 2011). Specialised pigments in Conchocelis require further study.

    Euendolithic habit in the development of red algae has a long geological history. Palaeoconchocelis starmachii (Kazmierczak and Golubic 1979) penetrated crinoid ossicles during the upper Silurian over 425 million years ago and remained organically preserved inside its borehole, including pit connections between its cells (Campbell 1980). Fossil of the foliose phase of Bangialean rhodophyte Bangiomorpha pubescens is about 1000 million years old (Butterfield 2000, 2015). Other fossil findings refer exclusively to Conchocelis boring traces, recently described as ichnospecies Conchocelichnus seilacheri (Radtke et al. 2016). They were recorded along different Phanerozoic strata from the Silurian (Bundschuh 2000); Triassic (Schmidt 1992), Jurassic, Lower Cretaceous (Glaub 1994) and Lower Tertiary deposits (Radtke 1991).

  5. (5)

    The species distinction in bangialean rhodophytes was traditionally based on the morphology of the foliose generation. The Conchocelis phase was identified indirectly while grown from carpospores of different Porphyra species identified by their leafy thalli. However, the molecular methods of gene sequencing have recently redefined the diversity of bangialean rhodophytes, so as to follow more closely the phylogenetic history (Lindstrom and Fredericq 2003; Wang et al. 2013; Lindstrom et al. 2015). Accordingly, the redefined genus Porphyra is presently restricted to five described species and a number of undescribed ones (see Müller et al. 2005; Lindstrom et al. 2015), while other species that used to be classified as Porphyra are now classified among seven other genera (Sutherland et al. 2011).

    The Conchocelis observed in stylasterid corals from Indonesia most likely belong to a tropical bangialean taxon, such as Porphyra marcosii P.A. Cordero or Pyropia vietnamensis (Tak. Tanaka & Pham-Hoàng Ho) J.E. Sutherland & Monotilla (see Ruangchuay and Notoya 2003) or some other taxon found south of the Philippines (see Ame et al. 2010).

    The discovery of Conchocelis stages of Bangiales in tropical hydrozoan lace corals expands our knowledge of the distribution of these rhodophytes in tropical reefs and stimulates further investigations of rhodophyte diversity and ecology, including those that colonise and penetrate the skeletons of live and growing corals.