Phylogenetic investigation of the aliphatic, non-hydrolyzable biopolymer algaenan, with a focus on green algae
Introduction
Algaenan is a non-hydrolyzable, insoluble biopolymer (Tegelaar et al., 1989) that has been isolated from a variety of unicellular algae and is recognized as an important component of kerogen, the largest organic carbon sink on the planet (Berner, 1989, Hedges, 1995). Despite the importance of kerogen in the organic carbon cycle, its synthesis is incompletely understood. The discovery of an algaenan-like signature in kerogens associated with algal microfossils (Derenne et al., 1994, Derenne et al., 1992, Goth et al., 1988) led to the hypothesis that the preferential preservation of algaenan and other recalcitrant biopolymers plays a principal role in kerogen formation (Derenne et al., 1991, Gelin et al., 1996, Tegelaar et al., 1989).
Recently, a number of taphonomic studies have begun to question the selective preservation hypothesis. Taphonomic research on arthropod and leaf cuticles suggests that diagenesis creates an aliphatic signature in structures whose original composition was not aliphatic (Briggs et al., 1995, de Leeuw, 2007, Gupta et al., 2006). Another taphonomic experiment showed that soluble hydrocarbons in vegetable oil can generate an aliphatic signature like that of kerogens or biopolymers when exposed to conditions encountered during diagenesis (Versteegh et al., 2004). Additionally, it has been shown that some kerogens with an aliphatic signature were predominantly sourced by organisms that do not produce aliphatic biopolymers, implying that the aliphatic material formed during diagenesis and should be considered a geopolymer (Kuypers et al., 2002). Together, these results suggest that the formation of an aliphatic signature in kerogens and fossils is more complex than the simple selective preservation of specific biopolymers, as concluded earlier (de Leeuw, 2007, Gupta et al., 2007 and references therein). The strongest evidence that biologically produced aliphatic biopolymers could be a source of aliphatic signatures in kerogens and microfossils is the observed presence of an aliphatic biopolymer in certain microalgae.
Although algaenan is indisputably an important chemical constituent of some unicellular photosynthetic organisms, current understanding of both its chemical nature and biological function is filled with ambiguities and uncertainties. In large part, this reflects difficulties encountered in the isolation, purification and characterization of this complex material. The structure of macromolecular compounds such as algaenan cannot be fully elucidated using conventional analytical methods. Analysis has traditionally been restricted to techniques that yield information about the overall chemical nature of the biopolymer, such as Fourier transform infrared spectrometry (FTIR) or 13C nuclear magnetic resonance (NMR) spectroscopy, but do not provide an exact molecular composition. Pyrolysis–gas chromatography–mass spectrometry (py–GC–MS) has been widely used to evaluate the molecular entities comprising algaenan, but there are many aspects of the overall structure that this method cannot address, though some progress has been made using thermal decomposition models (Salmon et al., 2009a, Salmon et al., 2009b). Metzger et al. (2007) combined pulse-field gradient NMR analysis and classical chemistry to infer that the algaenan from Botryococcus braunii strain B is formed by intermolecular condensation of aliphatic polyunsaturated dialdehydes with triterpene diol (B-race), or tetraterpene diol (L-race) moieties. The polyaldehydes themselves originate from polymerization of diunsaturated α,ω-dialdehydes via an aldolization-dehydration mechanism (Metzger et al., 2008). Some structural information has also come from chemical degradation using RuO4 (Blokker et al., 1998, Blokker et al., 2006, Schouten et al., 1998), tetramethyammonium hydroxide (TMAH) and TMAH thermochemolysis (Allard and Templier, 2000, Blokker et al., 1998) and HI (Blokker et al., 1998). At present, the unifying character for the biopolymers classified as algaenan remains, however, their aliphatic nature, long carbon chains and resistance to chemical and biological attack.
Biological investigations of the physiology, function and phylogenetic distribution of algaenan have also been limited. It has been isolated from the cell walls of chlorophycean algae from the genera Scenedesmus, Tetraedron, Chlorella (Allard et al., 2002, Goth et al., 1988), Botryococcus (Metzger et al., 2008, Templier et al., 1992) and Haematococcus (Montsant et al., 2001). Many of these genera have cell walls with a tri-laminar structure (TLS), though not all TLS-containing algae produce algaenan and not all algaenan-producing taxa have TLS (Allard et al., 2002). Beyond this ultrastructural feature, no other morphological traits seem to correlate with algaenan production or unite algaenan producers. Algaenan has also been identified in the zyogspores of Chlamydomonas monica (Blokker et al., 1999), the aplanospores of Dunaniella sp. and the akinetes of Haematococcus pluvialis (Blokker, 2000), but no other resistant algal cysts or reproductive structures have been shown to be aliphatic in nature. Connections have also been drawn between putative algal fossils and algaenan, but the cell walls of modern analogs for the fossils in question – the phycomata of Halosphaera – do not contain algaenan, calling such a correlation into question (Kodner, 2007).
Reviews have compiled the occurrences of algaenan and other non-hydrolyzable insoluble biopolymers (de Leeuw et al., 2006, Versteegh and Blokker, 2004); however, there has not been a broad scale phylogenetic assessment of algaenan-producing organisms. To date, algaenans have been reported from green algae, eustigmatophytes and a single dinoflagellate (Versteegh and Blokker, 2004) – groups that doubtfully have a common ancestor that itself synthesized algaenan.
To understand further how algaenan is distributed among eukaryotes, we have focused on the group in which the biopolymer is most abundant and diverse: the green algae. Green algae are part of a eukaryotic kingdom called the Plantae (Cavalier-Smith, 1998) or the Archeoplastida (Adl et al., 2005), that is strongly supported in most phylogenies. This clade originated with the primary endosymbiotic event between a protist and a cyanobacterium that introduced photosynthesis to the Eukarya (Keeling et al., 2005) and displays impressive diversity in cellular structure, physiology and ecology. Other groups of algaenan producers, eustigmatophytes and dinoflagellates have a much different evolutionary history. To investigate the distribution and evolutionary history of algaenan production in the Archeoplastida, we assayed its production in representatives of the three major subgroups of this clade: the green, red and glaucocystophyte algae.
Section snippets
Biomass
Algae (Table 1) were grown in batch cultures in large flasks (2 or 4 l Erlenmeyer flasks or 2 l Fernbach flasks) under air lift conditions and 24 h of light. All cultures were grown under the same temperature and light regime and on the Bolds Basal Media (BBM), all being freshwater or terrestrial strains. Cultures were harvested at the end of the log phase. Acrochaetium (a macroscopic alga), Coleochaete succata, Compsopogon sp.?, Chlamydomonas reinhardtii and Chlorella vulgaris were supplied by
Algaenan extraction
The distribution of algaenan among the organisms is summarized in Table 3. No algaenan production was found outside the Chlorophyceae or Trebouxiophyceae; in the extraction of lipid-free biomass from members of the Streptophyta, Rhodophyta, Glaucystophyta and Ulvaphyceae, all biomass was lost at some hydrolysis step. To verify that the biopolymer was not artificially assumed absent as a result of loss of small quantities during extraction, lipid-free biomass was analyzed using py–GC–MS to
Discussion
Because the algaenans found in distantly related clades doubtfully have a common evolutionary origin, “algaenan” becomes an operational term for aliphatic, non-hydrolyzable biopolymers that have similar chemical features.
Algaenan as a geomacromolecule and biomarker for green algae
The identification of algaenan in the geologic record is based mostly on the link between the aliphatic nature of the preserved organic material and the aliphatic nature of some algal biopolymer (van Bergen, 2004). Algaenan appears to have diverse and convergent biological sources, and within the green algae may be limited to a phylogenetically restricted group dominated by freshwater taxa. Considering analytical difficulties, the limited diagnostic features of algaenan, the known taphonomic
Acknowledgements
The authors gratefully acknowledge the help and advice of numerous colleagues during the course of this work. We especially thank J. de Leeuw and an anonymous reviewer for providing insightful comments. G. Love and C. Colonero provided help and advice in the laboratory. L. Lewis and Z. Cardon of the University of Connecticut prvided cultures, primers, culturing advice and helpful discussion. R.B.K. acknowledges support from the NSF GRF while A.H.K. and R.E.S. were supported by grants from the
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