Anaerobic animals from an ancient, anoxic ecological niche

Anaerobic mitochondria: more common all the time

A look at the phylogenetic distribution of characterized anaerobic mitochondria among animal lineages shows that these are not clustered but spread across metazoan phylogeny (Figure 1). Are these recent adaptations to anaerobic habitats or are they holdovers from our more distant evolutionary past? The biochemistry and the enzyme equipment used in the facultatively anaerobic mitochondria of metazoans is nearly identical across lineages [7, 10–15], strongly indicating a common origin from the metazoan ancestor that might have lived some 600 million years (MY) ago [16]. The examples in Figure 1 cover both the Lophotrochozoa and the Ecdysozoa, where the newly-described Loricifera with their distinctive organelles belong. Although no biochemical data are yet available for these intriguing new Loriciferan mitochondria, from the electron micrographs presented by Danovaro et al. [1] the organelles look like hydrogenosomes - anaerobic forms of mitochondria that generate H₂ and adenosine triphosphate (ATP) from pyruvate oxidation [17] and which were previously found only in unicellular eukaryotes. Danovaro et al. [1] also report that the animals harbour structures resembling prokaryotic endosymbionts, reminiscent of the methanogenic endosymbionts found in some hydrogenosome-bearing protists; fluorescence of F₄₂₀, a typical methanogen cofactor [18], or lack thereof, will bring more insights as to what these structures are.

If we follow the anaerobic lifestyle further back into evolutionary history (Figure 2), beyond the origin of the metazoans, we see that the phylogenetic distribution of eukaryotes with facultative anaerobic mitochondria, eukaryotes with hydrogenosomes and eukaryotes that possess mitosomes (reduced forms of mitochondria with no direct role in ATP synthesis [19]) shows a similar picture to that seen for animals. In all six of the major lineages (or supergroups) of eukaryotes that are currently recognized [20], forms with anaerobic mitochondria have been found [21]. The newest additions to the growing collection of anaerobic mitochondrial metabolisms are the denitrifying foraminiferans [22, 23]. For this group, the underlying enzymes also have yet to be worked out. However, for the remainder of the eukaryotes summarized in Figure 2, a handful of about a dozen enzymes make the difference between a 'normal' (in the sense of 'familiar from older college textbooks') O₂-respiring mitochondrion found in mammals, and the energy metabolism of eukaryotes with anaerobic mitochondria, hydrogenosomes or mitosomes [7, 19, 21, 24]. Notably, the full complement of those enzymes, once thought to be specific to eukaryotic anaerobes, surprisingly turned up in the green alga Chlamydomonas reinhardtii [24], which produces O₂ in the light, has typical O₂-respiring mitochondria but, within about 30 min of exposure to heterotrophic, anoxic and dark conditions, expresses its anaerobic biochemistry to make H₂ [25, 26] in the same way as trichomonads, the group in which hydrogenosomes were discovered [17]. Thus, Chlamydomonas provides evidence which indicates that the ability to inhabit oxygen-harbouring, as well as anoxic environments, is an ancestral feature of eukaryotes and their mitochondria; in that sense it is a true missing link that unites mitochondria like our own and those from the anoxic world [19].

It is not yet known what role, if any, the mitochondria of the newly discovered loriciferans play in the energy metabolism of those animals and, unfortunately, the same is true for the myriad of fascinating eukaryotic protists that inhabit the very same environment where the anoxic loriciferans were found. In a series of recent papers, Stoeck and colleagues have surveyed the protistan diversity from L'Atalante and similar habitats with environmental sequencing and metagenomic techniques [3, 4, 27]. Those reports lead to a wealth of representatives from many of the same groups shown in Figure 2 being uncovered: ciliates, fungi and chromalveolates, in addition to representatives of many other eukaryotic groups, including the choanoflagellates [3], which are regarded as the unicellular sisters to the metazoan clade. The investigation of anaerobic mitochondria remains an area of rapid progress and it will be a challenge to discover what those mitochondria are doing in real-life anoxic and hypoxic environments, which are very widespread among modern habitats [28] and where eukaryotic anaerobes abound [27].

Evolutionary significance?

What is the evolutionary significance of the new findings? The L'Atalante basin has been anoxic for only about 50,000 years [1] but have all of its microbial and metazoan inhabitants only recently adapted to life in anoxic conditions during that time? Hardly. Nobody seriously considers that anaerobic prokaryotes dwelling in such anaerobic habitats, such as methanogens and sulphate reducers, have only recently adapted to anaerobic niches. The prokaryote inhabitants have existed for well over a billion years, and have reached this new habitat by dispersal, not by adaptive evolution de novo and in situ. Indeed, geochemical evidence has shown that methanogenesis and sulphate reduction, and the niches in which they occur, are truly ancient [29, 30]. (For marine environments, dispersal is not a fundamental problem because, despite vast distances, similar types of seafloor habitats often harbour similar communities, from microbes to large animals [31]). However, when it comes to eukaryotes, there is still a curious tendency to assume that eukaryotes only invaded anaerobic niches of late. Perhaps this stems from a tendency (latently anthropocentric, no doubt) to view mitochondria as obligately O₂-dependent organelles with the anaerobic forms of mitochondria being rare exceptions, even though the data (for example in Figures 1 and 2 or elsewhere [7, 8, 17, 19, 21]) tell us otherwise. In environments such as the L'Atalante basin, 'normal', O₂-dependent mitochondria are the rare exception, if they exist at all, but eukaryotes abound [1, 3, 4, 32, 33]. Hence, further study of mitochondria from such environments should be revealing. That, however, is easier said than done, since, as seen in the present study [1] as in other work on organisms from anoxic marine sediments [32], a considerable effort has to be invested in order just to demonstrate that the organisms are even alive and not just sunken carcasses. Work on organisms from these environments poses substantial technical challenges, making every new insight all the more exciting.

While Danovaro et al. opt for the term 'enigmatic' in discussing the evolutionary significance of their findings, we have a decidedly different view. Given that anaerobic forms of mitochondria are widespread throughout the eukaryotic world, we see eukaryotes in anaerobic habitats as evidence for evolution in the Darwinian sense of descent with modification, with the traits that support survival in anaerobic environments having been conserved from earlier phases of Earth's history. This view is underpinned by what geologists and geochemists have been trying to tell biologists over the last 10 years about the prevalence of anoxic and sulphidic environments during the early phase of eukaryotic and metazoan evolution, but with the biologists perhaps not taking as much notice as they should.

The bigger picture: add geological time

What are the geologists trying to tell us about anoxic and sulphidic marine habitats? A readable summary of about 10 year's progress is given in three papers [34–36]. In a nutshell, the geologists are saying that the rise in atmospheric oxygen some 2.4 billion years ago is one thing, but that the oxygen levels in the ocean, where evolution was taking place, is quite another. Several lines of isotope evidence indicate that deep ocean waters were not fully oxygenated until about 580 MY ago, about the time when the first large animals made their fossil debut. The reason for this anoxia, they say, has to do with high levels of sulphide in marine environments, from the workings of sulphate reducers [36], themselves strict anaerobes. The message for biologists is that the Earth's oceans appear to have been largely anoxic and sulphidic (like the L'Atalante basin, but not hypersaline) below the photic zone (the upper about 200 m) [34, 36] and, possibly, also in the lower photic zone [35] for the time spanning roughly 1.8 billion years ago to about the beginning of the Cambrian period 560 MY ago. That was the time during which eukaryotes arose and diversified [21]. Hence, it should hardly be surprising that the anaerobic lifestyle is widespread among eukaryotic lineages, right up into the animals [37]. One view is that the oxygenation of deep environments allowed the animals to become larger [38], which is different from saying that oxygen might in any way be causal to the Cambrian appearance of diverse animal forms [39]. Geologists have not been trying to hide their perspective from us biologists [40–42], nor have we biologists been trying to hide our progress in understanding eukaryotic anaerobes [21, 43–46]. However, it seems that it will still take some time for a new default view of deep marine environments in the Late Precambrian -- a new synthesis of sorts -- to be accepted by biologists. The scenario is one of widespread anoxic and sulphidic habitats, almost certainly (in our view) teeming with little eukaryotic creatures, all with their mitochondria well suited to life with little or no oxygen [37] and all more or less like the ones we see in anoxic and sulphidic environments today. Such environments staged and witnessed the origin of evolution's greatest early inventions [47] and the new insights emerging from them deserve our close attention.