Phylogenetic investigation of the aliphatic, non-hydrolyzable biopolymer algaenan, with a focus on green algae

Abstract

Algaenan, an aliphatic biopolymer found in various microalgae, has been implicated as the source of a sizable proportion of the aliphatic refractory organic matter in sedimentary rocks. Because of its recalcitrant nature, algaenan is thought to be preserved selectively in the formation of kerogen and microfossils. Its taxonomic distribution in organisms has not been studied in detail or in a phylogenetic context. Here, we evaluate the distribution and phylogenetic relationships of algaenan-producing organisms from a broad, eukaryote-wide perspective down to the level of genus and species. We focus on the kingdom Plantae, as most described algaenan producers belong to this superkingdom. The phylogenetic distribution of algaenan producers within the Plantae is actually quite limited and a detailed phylogenetic analysis of the two classes that include all green algal algaenan producers suggests that there is no finer-grained pattern of phylogenetic distribution to the production of this biopolymer. Our results suggest that the biopolymer is not widespread ecologically or phylogenetically, is not found abundantly in marine organisms and likely represents a functional description of molecular class, rather than a biomarker for green algae. This adds to a growing body of literature that questions the selective preservation hypothesis for insoluble organic matter and calls for a more detailed chemical and structural analysis of algaenan.

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.