BioEssays Volume 36 Issue 10 2014 [Doi 10.1002%2Fbies.201400060] Mentel, Marek; Röttger, Mayo; Leys, Sally; Tielens, Aloysius G. -- Of Early Animals, Anaerobic Mitochondria, And a

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Prospects & Overviews

Of early animals, anaerobicmitochondria, and a modern sponge

Marek Mentel 1) , Mayo Ro ttger 2) , Sally Leys3) , Aloysius G. M. Tielens 4)5)

and William F. Martin 2)

The origin and early evolution of animals marks an

important event in life’s history. This event is historically

associated with an important variable in Earth history

oxygen. One view has it that an increase in oceanic

oxygen levels at the end of the Neoproterozoic Era

(roughly 600 million years ago) allowed animals to

become large and leave fossils. How important was

oxygen for the process of early animal evolution? New

data show that some modern sponges can survive for

several weeks at low oxygen levels. Many groups of

animals have mechanisms to cope with low oxygen or

anoxia, and very often, mitochondria organelles usually

associated with oxygen are involved in anaerobic

energy metabolism in animals. It is a good time to refreshour memory about the anaerobic capacities of mito-

chondria in modern animals and how that might relate to

the ecology of early metazoans.

Keywords:.anaerobiosis; eukaryotes; geochemistry; hypoxia;

mitochondria; Neoproterozoic ocean chemistry;

rhodoquinone

Introduction

Humans are oxygen guzzlers. An adult human consumesabout 700L of molecular oxygen (O2) per day [1], the vastmajority of which serves ATP synthesis in our mitochondria,where about a bodyweight of ATP is produced each day to runour bodies and keep our species (and its evolution) going. If we look around at other large animals that live on land, they are all specialized to life with O2 in the atmosphere, whichshould not be too surprising, because vertebrate life on landonly goes back about 350 million years (My) [2], and arose at atime when there was roughly as much (or more) oxygen in theatmosphere as there is now: about 21% by volume [3]. Butthere was not always oxygen to breathe. Though O2 startedaccumulating in the atmosphere about 2.4 billion years (Gy)ago, its accumulation in the oceans was a much slowerprocess, and many lines of evidence indicate that theoxygenation of the oceans came to completion less than ca.600My ago [3, 4], close to the time when the first animalsappear in the fossil record [5].

The appearance of the first fossil animals 580 My ago [6],or even earlier [7], and the appearance of the first fossilsponges 535My ago [8] does not mean that those groupsarose at that time. Molecular data have it that metazoanradiation pre-dated the appearance of the first fossil animals

DOI 10.1002/bies.201400060

1) Faculty of Natural Sciences, Department of Biochemistry, ComeniusUniversity, Bratislava, Slovakia

2) Institute of Molecular Evolution, University of Du ¨ sseldorf, Du ¨ sseldorf,Germany

3) Department of Biological Sciences, University of Alberta, Edmonton, AB,Canada

4) Faculty of Veterinary Medicine, Department of Biochemistry and CellBiology, Utrecht University, Utrecht, The Netherlands

5) Department of Medical Microbiology and Infectious Diseases, ErasmusUniversity Medical Center, Rotterdam, The Netherlands

*Corresponding author:

William F. MartinE-mail: [email protected]

Abbreviations: AFDW, ash-free dry weight; DW, dry weight; FRD, fumarate reductase; gDW,

gram dry weight; GOE, great oxidation event; Gy, billion years; HMA, high-microbial abundancesponge; LECA, last eukaryotic common ancestor; LMA,

low-microbial abundance sponge; My, million years; NOE, neoproterozoicoxidation event; OMO, organelles of mitochondrial origin; PAL, presentatmospheric levels; RQ, rhodoquinone; SLP, substrate level phosphorylation;WW, wet weight.

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by many millions of years [9]. The implication is that the firstanimal groups, which evolved from unicellular eukaryotes(protists), arose at a time before the oceans were fully oxic.This makes information about the ecology and habitat of thefirst animals all the more interesting. Knowing the oxygenrequirements of the earliest animals would provide insightsinto the nature of the energy metabolism of our primitive

metazoan predecessors. At the same time, it would shed lighton the environmental conditions to which early animals wereadapted.

Has a modern sponge preservedancestral states?

Though we cannot turn back time, we can look atrepresentatives from early branching lineages in the hopethat they have preserved some primitive traits. This is theapproach that Mills et al. [10] recently undertook. They collected samples of the aerobic marine invertebrate Hal-

ichondria panicea (a sponge) from well-oxygenated surfacewaters of Kerteminde Fjord in Denmark and tested theirrespiration rate, feeding rate, and overall well-being under awide range of oxygen concentrations. They showed that

H. panicea is able to respire, feed, and grow under a low-oxygen gas phase corresponding to 0.54% present atmo-spheric levels (PAL). The animals remained healthy for24 days, and the experiment was terminated before theanimals completed their life cycle. From this comparatively simple observation combined with the presumption that such“levels may have been present in the environment well before

animals originated”, they infer that “animal evolution may not

have been prohibited by the presumably low atmospheric

oxygen levels before the Neoproterozoic Era.” The strength of that conclusion hinges upon the assumption that H. panicea

has preserved the ancestral condition with regard to oxygenrequirements, as any modern taxon might exhibit uniquely derived or shared derived features that set it apart from itsancestors.

Taxonomically, H. panicea belongs to the demosponges,one of four classes of the phylum Porifera [11]. Spongestraditionally occupy earliest branches on phylogenetic trees of all animals [12]; according to some studies they are the sistergroup to all other known animal species [13]. Their anatomy isunique: They have flagellated feeding cells called choanocytesfacing internal chambers, and canals through which waterflows, supplying the organism with microscopic food-such as

the cryptophyta alga Rhodomonas sp. usedin the study for thepurpose of feeding H. panicea [10]. Choanocytes morphologi-cally resemble choanoflagellates, single-celled eukaryotesthat are the closest living relatives of all animals [14]. Relyingon phylogenetic trees showing Porifera as the earliestdiverging animal lineage, Mills et al. [10] surmise that moderndemosponges serve as analogs for early animals, and that“because the last common ancestor of all living animals likely

had a sponge-like body plan, the physiology and oxygen

requirements of modern sponges should serve as a reasonable

window into the oxygen requirements of the earliest animals.”Based on that, and given the observed tolerance of H. panicea

to low oxygen levels, they conclude that the oxygen demandsof the last common ancestor of metazoans “may have been met

well before the enhanced oxygenation of the Ediacaran Period.

Therefore, the origin of animals may not have been triggered

by a contemporaneous rise in the oxygen content of the

atmosphere and oceans.”Notwithstanding some ambiguity with regard to the word

“origin” (phylogenetic origin of the lineages? or theirappearance in the fossil record?), one might agree with theirconclusion, but there are some caveats en route to gettingthere. There are an estimated >14,000 species of sponges [11],and it would have been good to see results from representa-tives from some of the other groups. The observed oxygenrequirements of modern demosponges are one thing, but theconclusion that this reflects the unaltered ancestral state of sponge (or animal) physiology carries with it the assumptionthat nothing has changed during the 700 million years of evolution in the lineage leading to the sponge that they studied. Geochemists currently paint a picture of profoundly changing environmental conditions especially at the lateProterozoic/Phanerozoic Eons boundary [3, 15] such that the

habitat of the earliest animals, probably shallow ocean, wasfluctuating from hypoxic and very often anoxic and euxinic atbiologically productive ocean margins to well oxygenatedeven at deeper depths [3]. The environments inhabited by thefirst demosponges, the environments inhabited by the earliestanimals that left fossil traces, and the environments inhabitedby the earliest animals are not necessarily all the sameregarding their redox structure and oxygen availability [3].

And what if sponges do not represent the body plan (or thephysiology) of the last common ancestor of all living animals?Not allphylogenetic studies place sponges as the sister lineageto all other animals [12]. Recent phylogenies accompanyingthe genome sequences of the ctenophores Mnemiopsis

leidyi [16] and Pleurobrachia [17] indicated that ctenophores(comb jellies) might be basal to all animals, includingsponges. Accordingly, the nature of the metazoan commonancestor remains debated [18]. Another caveat is lineagesampling. While Mills et al. [10] looked at one species,

H. panicea, recent transcriptomic investigations of eightspecies from all four classes of the phylum Porifera uncoverunexpected genetic complexity among the sponges [19] andsupport the view that some sponges might have lost someancestral cell types, and possibly physiological processes,along their evolutionary route.

Clearly, the experiments reported by Mills et al. [10] doshow that H. panicea demosponge species can survive harshhypoxic conditions (34% PAL) for relatively long periods:

24 days in their study. But it has long been known that many invertebrates, and even some vertebrates, can survive longerperiods with even less oxygen [2022]. The goldfish Carassius

auratus can survive complete anoxia for more than a week at10˚C [23] and the carp Carassius carassius can withstandanaerobiosis for months (140 days) at 2˚C [24]. These fishmaintain their vitality through ethanol fermentation, excret-ing the end products of their anaerobic metabolism, ethanoland CO2, into the surrounding water. There are severalpathways leading to ethanol fermentation in eukaryotes, butin the cases known so far they are cytosolic pathways [25], sothe carp mitochondria apparently do not participate in energy

M. Mentel et al. Prospects & Overviews....

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metabolism under those conditions. It is not known whichmetabolic end products H. panicea generated during the 24-day incubation, or whether their mitochondria were involved.Mills et al.[10] measured oxygenconsumption, but it is hard totell from the data available how much of that might have beenperformed by bacterial symbionts, and many eukaryoticorganisms with an anaerobic energy metabolism nevertheless

have oxygen consuming pathways in the cytosol that aredistinct from mitochondrial respiration.

It is difficult to compare Mills et al.’s [10] respiration datawith work on other sponges including data from the samespecies carried out previously [26] because of the differentunits of measurement used. But clearance rates reported by Mills et al. [10] of 800 algal cells/mL hour are 40% of thosefound in previous work (2,0004,000cells/mL hour) [26, 27];and respiration rates, if reported per gram dry weight (gDW)of sponge tissue (as in Table 1), are 30% of those reportedearlier (10mM/Lhour gDW, compared with 26.8mM/LhourgDW) [26]. Reduced filtration comes with reduced energy intake. At 1.75mJ/cell [28] 800 algal cells would provide thesponge with 1.4 J/hour, but a respiration rate of 10mM/Lhour

gDW means that 4.3J/hour are required for metabolism.Considering that the sponge in Mills et al. [10] experimentfiltered (a cost for other sponges of about 16% of availableoxygen) and even grew branches, there is a deficit. This couldhave been made up by filtration of bacteria (which were notcounted here). It is also possible that other factors, such asanaerobic energy metabolism, might have been involved.Transcriptomic data from the sponge they cultured wouldhave shed some light on which biochemical routes it wasemploying, because the roughly 50 enzymes involved inanaerobic energy metabolism among eukaryotic groups arereasonably well characterized [25].

What was the ancestral state of animalenergy metabolism?

In our view, the reconstruction of oxygen requirements andmetabolic traits of the earliest animals and ancestralmetazoans is best understood in the context of energy metabolism of eukaryotes, of which the animals are just one

lineage in general. Most scientists would agree that eukar-yotes originated at least 1.5 Gy ago, as fossils clearly representing eukaryotic protists are found in rocks of thatage [29, 30], and molecular estimates for the timing of eukaryotic evolution are consistent with that view [8, 31, 32].Thus, eukaryotes can be taken to have arisen about 1.6 Gy ago,at a time when anoxic environments were more widespreadthan at the dawn of animal evolution, almost a billion yearslater. What were the oxygen demands of the first eukaryotes?Currently, there are two mutually exclusive views aboutoxygen demands of early eukaryotes and their subsequentevolution.

One view has it that the earliest eukaryotes were strict

aerobes, which

at different occasions and by means of interdomain and intradomain horizontal (lateral) genetransfer-adapted to hypoxic and anoxic conditions [3335].Proponents of this view argue that early eukaryotes wereunable to survive hypoxia, but instead had to acquire genesvia lateral transfer in order to gain ecological access toanaerobic environments [3439].

We favor the alternative view that the free-living ancestorsof mitochondria were themselves facultative anaerobes with adiversity of bioenergetic enzymes [40], and that the earliesteukaryotes possessed facultatively anaerobic mitochon-dria [25]. This allowed them to extract energy from theirenvironment and surviveunder a wide range of environmental

Table 1. Respiration and filtration rates of sponges

Species

Respiration rate Filtration rate

Refs.

Per ml of sponge Per gDW Removal efficiency Per ml of sponge Per gDW

( mmol O2 /hour) (%) ( mM) (L/hour)

Neogombata magnificaa 2.22 14.88 0.63 4.2 [78]

Theonella swinhoei 1.38 4 2 9 0.156 [79]

Mycale sp.b 1.965 46.58 1.2 2.23 0.756 10.75 [80]

Tethya cryptac 0.893 7.42 1.1 1.78 0.158 1.31 [80]

Verongia gigantead 3.036 31.34 5.6 10.72 0.191 1.908 [80]

Verongia fistularise 4.736 33.58 5.3 10.6 0.44 3.12 [81]

Halichondria panicea 1.936 26.8f 0.12h 1.7 [26]

Halichondria panicea 7.93f 0.048i 0.68i [10]

1013g

aOne milliliter sponge volume¼1 g wet weight (WW); DW¼15% WW; AFDW¼12% WW.bOne milliliter sponge volume¼70 mg DW; AFDW¼70.3% DW.cOne milliliter sponge volume¼100 mg; AFDW¼65% DW.dOne milliliter sponge volume¼121 mg; AFDW¼37.8% DW.eOne milliliter sponge volume¼1.2g WW¼0.141 g DW¼0.103g AFDW.f Saturated oxygen.gReduced oxygen (11, 4, and 3% PAL ¼27.5, 10, 7.5mM).hReflecting clearance of 2,000 Rhodomonas cells/L hour.iRatio of reported clearance of 800 Rhodomonas cells/Lhour to rate reported by Thomassen and Riisga ˚ rd [26].

....Prospects & Overviews M. Mentel et al.

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Figure 1. Schematic phylogeny of protists and animals harboring anaerobic mitochondria, hydrogenosomes, or mitosomes, schematically

indicating the higher order phylogeny of eukaryotes that groups all known eukaryotic diversity into six major clades called “supergroups” [9,

74]. Internal branching order within the clade of animals is based on Dunn et al. [12]. Note that regardless of whether the position of the root

of the tree is as shown, or is basal to opisthokonts [8, 74], or is on the excavate branch as new findings suggest [75], the distribution of

organelles of mitochondrial origin with anaerobic capabilities spans groups on either side of the root. Also note that the many lineages with

aerobic mitochondria (occurring in all supergroups) are not shown in the figure. Organelle classification as in Muller et al. [25]. The nature of

energy metabolism of sponge mitochondria (bold) is uncertain, highlighted by the question mark. Encircled “DNA” indicates the presence of a

genome in the respective mitochondrial type. LECA, last eukaryotic common ancestor.



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conditions, including different concentrations of oxygen andsulfide [25, 41, 42]. Under this view, the ability of eukaryotesto survive in anaerobic environments was present in the

eukaryote common ancestor. This would directly account forthe findings that (i) anaerobes are interspersed all across thetree of eukaryotic life, interleaving with their aerobic relatives,and (ii) that to survive hypoxic and anoxic conditions,different eukaryotes use overlapping subsets of a set of about50 enzymes that were present in the eukaryote commonancestor [25]. Because enzymes for aerobic and anaerobicenergy metabolism were present in the eukaryote commonancestor, their vertical inheritance into the common ancestorof animals is not surprising, nor is the differential loss of unneeded enzymes in lineages that have specialized tostrictly aerobic and anaerobic habitats, respectively. In animal

lineages that are regularly confronted withanoxia, the enzymes have been preserved incases studied so far [25].

How do eukaryoteanaerobes do it?

When the endosymbiotic theory for theorigin of organelles was being revived in the1970s, oxygen was seen as the factor thattriggered the origin of mitochondria [43]. Atthat same time, hydrogenosomes werebeing discovered [44], which turned out

to be anaerobic forms of mitochondria from eukaryotes thatinhabit anoxic environments [45]. Today we know fivedifferent functional classes of mitochondria, all descended

from one and the same endosymbiotic event [25]: classicalaerobic mitochondria (class 1) such as those found in rat liver;anaerobic mitochondria (class 2) as are found among severalgroups of invertebrates [46, 47]; hydrogen-producing mito-chondria (class 3), which have so far only been found inciliates, but might also be present in Blastocystis [4851];hydrogenosomes (class 4), which occur in anaerobic groupsof fungi, ciliates, trichomonads, and other protists; andmitosomes (class 5), which are being found in an increasingnumber of eukaryotic groups [45, 52]. Of the five classes of organelles of mitochondrial origin (OMOs), all but class 5generate ATP from the breakdown of carbon compounds, but

Figure 2. Generalized metabolic scheme of major pathways in anaerobic mitochondria

(class 2). Inside the mitochondrion malate dismutation resulting in the production of

acetate, succinate, and propionate is shown together with the corresponding anaerobic

electron-transfer chain, while for comparison the Krebs cycle and corresponding aerobic

electron-transfer chain are shown in gray. The propionyl-CoA cycle that is used to

convert succinate to propionate is simplified and shown as a linear pathway (see [25] fordetails). Modified after [25, 55, 63]. For other examples of anaerobic energy metabolism

in animal mitochondria, see [20, 23]. CI to CIV, respiratory complexes I to IV; UQ,

ubiquinone; RQ, rhodoquinone; C, cytochrome c; A, ATPase; FRD, fumarate reductase;

1, phosphoenolpyruvate carboxykinase (ATP dependent); 2, malate dehydrogenase; 3,

malic enzyme; 4, pyruvate dehydrogenase complex; 5, acetate:succinate CoA-transfer-

ase; 6, succinyl-CoA synthetase; 7, fumarase. Note that the NADH-dependent fumarate

reductase of protists and the quinone-dependent fumarate reductase of animal

mitochondria are different enzymes (see [25] for a discussion).



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Figure 3. Distribution of genes

for anaerobic energy metabolism

in sponges and eukaryotes. The

right portion of the figure is for

reference and is slightly modified

from Mu ¨ ller et al. [25]. The left

portion of the figure was prepared

as described in [25] by BLAST-

ing [76] the query sequences

against sponge EST data from

Riesgo et al. [19], downloaded

from http://thedata.harvard.edu/

dvn/dv/spotranscriptomes. To

check for bacterial or protistan

sequences, the sponge ESTs

meeting the criteria for inclusion

in the figure (Blast e-value of

1010 and >25% local amino

acid identity) were reblasted to

GenBank; sequences from the

sponge EST data hitting prokar-

yotes only were scored as absent(a list of the sequences so scored

is listed in Supplementary

Table S1). Note that two forms of

fumarate reductase exist in eu-

karyotic anaerobes: the soluble

NADH-dependent enzyme, for ex-

ample from Trypanosoma [25],

and the membrane bound rhodo-

quinone-utilizing enzyme, for ex-

ample from from Ascaris

mitochondria [77].



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http://thedata.harvard.edu/dvn/dv/spotranscriptomes








atmospheric oxygen levels correspond to about 250mMdissolved O2, and the Pasteur point (the concentration atwhich facultative anaerobic eukaryotes start to respire O2) isabout 2mM dissolved O2. The oxygen dependence of sterolsynthesis indicates that eukaryotes (and animals) arosesubsequent to the origin of oxygenic photosynthesis, notthat they arose in a fully oxic world.

Conclusion

And what are the sponge mitochondria in the report by Millset al. [10] doing? No end products or gene expression datawere reported, so we can only guess, and it is possible that themitochondria were doing nothing cytosolic fermentationsmight have been involved, and it is not clear how much of theoxygen consumption they measured was attributable tobacteria associated with the sponge, rather than the spongeitself. Indeed, another strategy that animals use to survivehypoxia is metabolic arrest: the animal simply bringsmetabolism close to a halt and waits for better times [73].

Whatever future studies of the anaerobic metabolism of sponges reveal, it seems likely that we will not see afundamentally new form of anaerobic animal energy metabolism, rather just more variations on a theme thatwas present in the common ancestor of animals. Whethersponges are indeed the first animals is a phylogeneticquestion. Whether they have preserved ancestral metabolictraits is a biochemical issue that hinges on knowledge aboutthe specifics of energy metabolism in their mitochondria.The mitochondria of diverse invertebrate lineages can respireoxygen at presently available levels and can perform malatedismutation under anaerobic conditions [25, 46]. This clearly suggests that the first animals could do the same and thus

possessed facultatively anaerobic mitochondria with fumaratereductase and RQ. Hence, the ability of sponges to survivelow oxygen does not come as a surprise, and knowing whatsponge mitochondria do under anaerobic conditions willimprove our understanding of energy metabolism in an early-branching animal lineage.

The authors have declared no conflict of interest.

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