Paleobiological Perspectives on Early Eukaryotic Evolution · Department of Organismic and...

Paleobiological Perspectives on EarlyEukaryotic Evolution

Andrew H. Knoll

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138

Correspondence: [email protected]

Eukaryotic organisms radiated in Proterozoic oceans with oxygenated surface waters, but,commonly, anoxia at depth. Exceptionally preserved fossils of red algae favor crown groupemergence more than 1200 million years ago, but older (up to 1600–1800 million years)microfossils could record stem group eukaryotes. Major eukaryotic diversification �800million years ago is documented by the increase in the taxonomic richness of complex,organic-walled microfossils, including simple coenocytic and multicellular forms, as wellas widespread tests comparable to those of extant testate amoebae and simple foraminiferansand diverse scales comparable to organic and siliceous scales formed today by protists inseveral clades. Mid-Neoproterozoic establishment or expansion of eukaryophagy provides apossible mechanism for accelerating eukaryotic diversification long after the origin of thedomain. Protists continued to diversify along with animals in the more pervasively oxygen-ated oceans of the Phanerozoic Eon.

Eukaryotic organisms have a long evolution-ary history, recorded, in part, by convention-

al and molecular fossils. For the PhanerozoicEon (the past 542 million years), eukaryoticevolution is richly documented by the skeletons(and, occasionally, nonskeletal remains) of an-imals, as well as the leaves, stems, roots, andreproductive organs of land plants. Phylogenet-ic logic, however, tells us that eukaryotes musthave a deeper history, one that began long be-fore the first plant and animal fossils formed. Towhat extent does the geological record preserveaspects of deep eukaryotic history, and can thechemistry of ancient sedimentary rocks eluci-date the environmental conditions under whichthe eukaryotic cell took shape?

EXPECTATIONS FROM COMPARATIVEBIOLOGY

The diversity of eukaryotic organisms observ-able today makes two sets of predictions for thefossil record, one phylogenetic and the otherpreservational. Phylogenies suggest the relativetiming of diversification events through Earthhistory, and, when incorporated into molecularclocks, provide quantitative estimates of diver-gence times. In turn, experimental and obser-vational studies of postmortem decay indicatethat only a subset of eukaryotic clades is likely tobe represented in the geologic record and theseonly under selected environmental circum-stances. Together, insights into phylogeny and

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preservation provide an empirical guide to pa-leobiological exploration.

Molecular sequence comparisons have rev-olutionized our understanding of evolutionaryrelationships among eukaryotes, but consensuson eukaryotic phylogeny remains elusive. Mostresearchers recognize a limited number of ma-jor clades, including the opisthonkonts, amoe-bozoans, excavates, plants (sensu lato), and anSAR clade containing the stramenopiles, alveo-lates, and rhizarians (e.g., Katz 2012), and manyrecognize the potential for as-yet poorly charac-terized taxa to expand that roster (e.g., Patter-son 1999; Adl et al. 2012). Persistent uncertain-ties include the position of the root; placementof groups such as centrohelid heliozoans, hap-tophytes, and cryptomonads; and both themonophyly and relationships of photosyntheticlineages commonly grouped as Plantae.

Molecular clocks calibrated by phylogenet-ically well-constrained fossils have been used toestimate the timing of early eukaryotic diversi-fication. Choice of algorithm can strongly influ-ence these estimates (Roger and Hug 2006; Emeet al. 2014), but sensitivity tests suggest that fora given set of sampled taxa, at least some esti-mates are broadly robust to tree topology andcalibration choices (e.g., Parfrey et al. 2011).Molecular clock estimates generally agree thatmuch protistan diversification has taken placeduring the Phanerozoic Eon, paralleling the di-versification of animals, plants, and fungi. Theyalso agree on an earlier, Neoproterozoic radia-tion within the major eukaryotic clades, begin-ning perhaps 800 million years ago (Mya).Where clocks disagree is on the date for thelast common ancestor of extant eukaryotes,with some positing a long interval of eukaryoticevolution before Neoproterozoic radiation(e.g., Hedges et al. 2004; Yoon et al. 2004; Par-frey et al 2011) and others suggesting a shorterfuse (e.g., Douzery et al. 2004; Berney and Paw-lowski 2006; Chernikova et al. 2011; Shih andMatzke 2013). The differing predictions of theseclock estimates can be tested against the Prote-rozoic fossil record.

How do protists impart a paleobiologicalsignature to sedimentary rocks? Mineralizedprotistan skeletons can form significant sedi-

mentary accumulations—the White Cliffs ofDover, for example, consist mostly of small cal-citic scales made by coccolithophorid algae.Tests and scales of calcite and silica documentPhanerozoic evolutionary histories for diatoms,chrysophytes, coccolithophoids, foraminifer-ans, and radiolarians, but do not extend intoProterozoic rocks (Knoll and Kotrc 2014). Or-ganic cell walls, tests, and scales can also survivebacterial decay, depending on molecular com-position, and, in Phanerozoic rocks, these re-mains record clades that include dinoflagellatesand prasinophyte green algae, among othergroups. As we shall see, decay-resistant organicwalls, tests, and scales also document aspects ofProterozoic protistan evolution, although it canbe challenging to relate preserved fossils to ex-tant clades.

Preservation, of course, is not the only hur-dle in paleobiological investigations of Protero-zoic rocks. There is also the challenge of recog-nition. In the first instance, how do we identifya fossil as eukaryotic rather than bacterial?Given that the record is one of morphologyand not DNA or cytology, diagnostic charactersmust be sought in the size, shape, ultrastructure,and preservational circumstances of microfos-sil populations. Eukaryotic cells are commonlylarger than bacteria and archaeons, but are notinvariably so. Conversely, cyanobacteria com-monly form extracellular sheaths and envelopesthat may encompass many cells; this being thecase, burial can preserve a 100-mm cyanobacte-rial envelope that, in life, surrounded numerousmicron-scale cells. By itself, then, size is com-monly an insufficient criterion for eukaryoticattribution. Cyst walls associated with restingstages in eukaryotic life cycles commonly havespines or other ornamentation, and they com-monly have a complex ultrastructure as observedvia TEM (e.g., Javaux et al. 2004). Bacteria canhave large envelopes (and more rarely cell size)(Schultz and Jørgensen 2001), but they rarely ifever combine large size, ornamented walls, com-plex ultrastructure, and a preservable composi-tion; thus, fossils that display all of these charac-teristics are widely regarded as eukaryotic.

Molecular fossils provide another means bywhich eukaryotic organisms can impart a sig-

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nature to the geological record. Proteins andnucleic acids have a low probability of preserva-tion, but lipids can preserve well, and sterols inparticular have been used to investigate the deephistory of eukaryotes. Abundant steranes (thegeologically stable derivatives of sterols) extract-ed from petroleum document a Phanerozoichistory of primary producers in the oceansthat, to a first approximation, parallels the his-tories inferred from microfossils and molecularclocks (Knoll et al. 2007). Molecular fossils ex-tend that record into Proterozoic and, morecontroversially, late Archean rocks.

Together then, phylogenies and preserva-tion potential furnish guides to the paleobiolo-gy of early eukaryotic evolution, providing hy-potheses of evolutionary history. How well dofossils fit these predictions?

ARCHEAN EUKARYOTES?

The three-domain view of life, predicated oncomparisons of SSU rRNA gene sequences, pos-ited that eukaryotes are sister to the Archaea(Woese et al. 1990). Given this relationshipand isotopic evidence for methanogenesis andmethanotrophy in the late Archean carbon cy-cle (Hayes 1994), logic would dictate thatthe Eukarya existed no later than �2700 Mya.This logic, however, is challenged by alternativephylogenies that nest eukaryotes within the Ar-chaea (Williams et al. 2012) and by models foreukaryogenesis that rely on archaeal–bacterialsymbiosis (Martin and Muller 1998; Moreiraand Lopez Garcia 1998). Even the most gener-ous molecular clock estimates place the lastcommon ancestor of extant eukaryotes withinthe Proterozoic Eon (Hedges et al. 2004), re-quiring that any Archean eukaryotes be stemgroups.

Unfortunately, the record of life in Archeanrocks is sparse and subject to conflicting inter-pretations. Early diagenetic cherts, a rich sourceof microfossils in Proterozoic strata, are largelybarren, perhaps reflecting the strong influenceof hydrothermal fluid flow and iron depositionon the silica cycle of Archean oceans (Fischerand Knoll 2009; Chakrabarti et al. 2012). Shales,in turn, contain abundant organic carbon, but

few structurally preserved or morphologicallydistinctive microfossils. One of the few well-documented and widely accepted fossil occur-rences in earlier Archean rocks, and perhaps theonly one that potentially bears on stem groupeukaryotes, comes from 3200-Mya shales thatcontain large (30–300 mm) spheroidal vesicles(Javaux et al. 2010). These appear to be genuinefossils, and they could, in principle, be eukary-otic; however, their simple ultrastructure andready comparison to the extracellular envelopesof some bacteria saps confidence from such aninterpretation.

In the absence of a convincing microfossilrecord, geobiologists have turned to molecularfossils. Steranes of hypothesized eukaryotic or-igin have been reported from late Archean sedi-mentary rocks (Brocks et al. 1999; Waldbaueret al. 2009), potentially documenting early eu-karyotes, but raising incompletely resolved en-vironmental, phylogenetic, and geological is-sues. The environmental concern is that sterolbiosynthesis requires molecular oxygen, yetgeochemical data consistently indicate that theArchean atmosphere and oceans were anoxic(Holland 2006). Sterol synthesis is possible atnanomolar oxygen tensions (Waldbauer et al.2011), and thus a plausible but unproven solu-tion holds that early cyanobacteria could havegenerated local oxygen oases within mats orsediments long before O2 began to accumulatein the atmosphere (e.g., Anbar et al. 2007).

The second issue is phylogenetic. A limitednumber of bacteria synthesize sterols (e.g.,Pearson et al. 2005), raising the concern thatpreserved biomarkers could be of prokaryoticorigin. In general, however, bacterial sterol syn-thesis is limited to simple products such as lan-osterol, and thus it is fair to consider more com-plex steranes of the type found in late Archeanrocks as eukaryotic until and unless complexsterol synthesis is shown in free-living bacteria.The third concern, however, is not so easily dis-missed. Because steranes in late Archean rocksoccur in part-per-billion concentrations, geo-logical or modern contamination must be con-sidered. Fluids flow through sedimentary rocksthroughout their history, and biomarkers canalso be emplaced during the processes of drill-

Paleobiology of Early Eukaryotes

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ing and sample processing. Arguments in favorof an indigenous origin for Archean steranesstress the care taken in sample preparation andthe varying biomarker composition of differentbeds, consistent with the expectation of ecolog-ical heterogeneity at the time of deposition(Brocks et al. 2003; Waldbauer et al. 2009). Fur-ther support comes from steranes and otherbiomarkers in fluid inclusions from quartz par-ticles in 2400-Mya sedimentary rocks (Dutkie-wicz et al. 2006). Critics counter that biomarkerheterogeneity could reflect bed-by-bed varia-tions in porosity and permeability, channelinglater flow fluid, and note that steranes occurmostly near the surface of drill samples (Brocks2011) and have a carbon isotopic compositiondistinct from the bulk of the organic matter inthe samples (Rasmussen et al 2008). The debatecontinues, commendably fueled by new sam-pling programs marked by stringent protocolsfor drilling and sample preparation.

PROTEROZOIC ESTABLISHMENT

As noted above, molecular clocks suggest thatregardless of any deeper stem group history,crown group eukaryotes emerged during theProterozoic Eon. (In this discussion, the term“crown group” is used in its broadly acceptedphylogenetic sense to indicate the last commonancestor of all extant members of a clade and itsdescendants. Earlier, informal usage to denote adiverse subset of eukaryotes has been aban-doned.) The last common ancestor of extanteukaryotes possessed a mitochondrion capa-ble of aerobic respiration, consistent with geo-chemical evidence for the permanent oxygena-tion of Earth’s atmosphere and surface ocean�2400 Mya (Holland 2006). Quantification ofProterozoic oxygen levels is difficult, but thepersistence of anoxic water masses beneath thesurface mixed layer of the oceans suggests thatpO2 remained low, perhaps no more than a fewpercent of present-day atmospheric levels (e.g.,Brocks et al. 2005; Canfield 2005; Scott et al.2008; Johnston et al. 2010; Frei et al. 2013).The chronic challenge of anoxic waters mixedupward from the oxygen minimum zone is con-sistent with the widespread occurrence in mito-

chondria of genes for anaerobic metabolism(Muller et al. 2012).

Bangiomorpha pubsecens (Fig. 2E) (Butter-field 2000) plays a key role in evaluating crowngroup early and late hypotheses based on mo-lecular clocks. An exceptionally well-preservedpopulation of filamentous microfossils found insilicified peritidal carbonates from Arctic Can-ada, Bangiomorpha displays several morphol-ogical features that collectively place it withinthe red algae. These include overall morphology,details of thallus development and reproductivebiology, cellularly differentiated holdfasts, lifecycle characteristics, and details of preservationthat differ markedly from those characteristicsof silicified cyanobacteria. Many specimens arepreserved in life position, rising vertically fromattachment sites on the ancient seafloor (Butter-field 2000). Thus, Bangiomorpha is reasonablyinterpreted as a rhodophyte, although it maybranch earlier within the clade than extant Ban-giales. Multiple geochronological and strati-graphic constraints indicate that Bangiomorphalived �1100–1200 Mya (summarized in Knollet al. 2013). Neither its age nor its phylogeneticattribution is likely to change markedly withcontinued study, and thus Bangiomorpha favorsmolecular clocks that place both the last com-mon ancestor of extant eukaryotes and the ac-quisition of plastids before �1200 Mya.

Some paleontologists propose that crowngroup eukaryotes can be traced further back intime. For example, Moczydłowska et al. (2011)have argued that microfossils of green algae oc-cur in rocks as old as 1800 Mya. Accepting thatgreen algae (and land plants) are sister to the redalgae, greens must have lived contemporane-ously with 1100- to 1200-Mya Bangiomorpha,and molecular clock estimates not precluded byBangiomorpha’s age suggest that the green–redsplit occurred up to several hundred millionyears before this (Wang et al. 1999; Yoon et al.2004; Parfrey et al. 2011). (If, as sometimes pro-posed, red algae are sister to greens plus glauco-cystophytes, the green clade could have radiatedlater.) The older fossils in question, however, aresimple spheroids whose affinities are not easilyascertained (Fig. 1A,B). Moczydłowska et al.(2011) maintain that only algae make resistant

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cysts with ornamentation and well-defined ex-cystment structures, but resting cysts have beenwell described in various heterotrophic protists,including, for example, ciliates that fashionlarge, spheroidal, and sometimes ornamentedcysts with pylome-like excystment structures(e.g., Beers 1948, 1966; Foissner et al. 2007; Verniand Rosati 2011). Wall ultrastructure might pro-vide more definitive evidence of green algae, es-pecially should TEM reveal the distinctive tri-laminar wall structure (TLS) characteristic ofcell walls in some chlorophytes (Allard and Tem-plier 2000). TLS has been shown in Cambriangreen algae (Talyzina and Moczydłowska 2000),but not in the older microfossils under consid-eration here (Javaux et al. 2004). Biomarkermolecules might also provide insight, but ster-anes are rare in mid-Proterozoic rocks (Brockset al. 2005; Pawlowska et al. 2013) (see below),and growing evidence suggests that algaenan, analiphatic polymer known to be synthesized by a

limited diversity of green algae (Kodner et al.2009), can also form during diagenesis (Guptaet al. 2009)—molecular clocks suggest that TLSand algaenan-synthesizing green algae doubt-fully extend much below the Proterozoic–Cam-brian boundary. Thus, it remains uncertainwhether earlier Proterozoic microfossils recordcrown group green algae, stem group greens (orPlantae), another crown group clade, or stemgroup eukaryotes (Knoll et al. 2006).

In general, mid-Proterozoic sedimentaryrocks contain abundant, but only modestlydiverse fossils of probable eukaryoticorigin (Jav-aux 2011). Shales up to �1600 Mya contain mi-crofossils that combine large size (.100 mm)with complex ultrastructure, structurally com-plex ornamented or tessellated cell walls, andsurface processes of varying form (Fig. 1C,D)(Javaux et al. 2001, 2003, 2004; Xiao et al. 1997;Yin and Yuan 2007; Nagovitsin et al. 2010).Equally large vesicles with less distinctive surface

Figure 1. Late Paleo- and Mesoproterozoic fossils interpreted as eukaryotic. (A–D) Preserved spheroidal mi-crofossils interpreted as the vegetative or resting walls of unicellular protists, arranged from lowest confidence(A) to highest (C and D). (A) Unornamented spheroidal vesicle, 1400–1500 Mya Roper Group, Australia. (B)Spheroidal vesicle with corduroy-like ornamentation of vesicle wall, Roper Group. (C) Spheroidal microfossilwith surface divided into small fields and ornamented with cylindrical processes that expand distally; TEM ofwalls shows complex multilayered wall ultrastructure, .1600 Mya Ruyang Group, China. (D) Spheroidal vesiclewith asymmetrically placed cylindrical processes; TEM shows complex wall ultrastructure, Roper Group (cour-tesy of Javaux et al. 2004). (E) Macroscopic compressions assigned to the form taxon Grypania, �1400 MyaJixian Group, China (courtesy of M.R. Walter). Scale bars, 20 mm (A); 75 mm (B,D); 120 mm (C). Note 1-cmscale bar in E.



morphology or ultrastructure occur in rocks asold as 1800 Mya (Fig. 1A) (Yan 1995; Lamb et al.2009). These may well be eukaryotic, especiallythose with corduroy-like, raised parallel ridgeson wall surfaces (Fig. 1B) (Yan 1995). For most,however, a lack of diagnostic features under-scores residual uncertainty at the domain level.Macroscopic impressions and compressionswhose regular morphologysuggests a eukaryoticorigin also occur in rocks of mid-Proterozoic age(Fig. 1E) (Grey and Williams 1990; Walter et al.1990; Retallack et al. 2013), with the oldest over-lapping in age with possible eukaryotic micro-fossils (Hofmann and Chen 1981; Han and Run-negar 1992). Most of this record comes frommarine rocks, but a possible glimpse of life inProterozoic lakes has been reported from 1000-to 900-Mya purportedly nonmarine beds inScotland that preserve a moderate diversity ofmicrofossils likely sourced by protists but other-wise of problematic origin (Strother et al. 2010).

From the preceding paragraphs we can drawtwo conclusions. First, although one mighthope for a clean paleobiological boundary be-tween worlds with and without eukaryotic cells,the geological record actually presents a slidingscale of certainty, from confidently interpretedprotists in 1400- to 1600-Mya rocks to moreambiguously interpreted remains at 1800 Myaand even more debated morphological and mo-lecular signatures in older successions. Paleobi-ological evidence of eukaryotic cells does not somuch bottom out as fade away. The second con-clusion is that the early eukaryotic record couldbe dominated by stem group taxa. Stem groupsare a logical necessity in biology but an empir-ical challenge for paleontologists. The fossilrecords of plants and animals contain diversestem group taxa at varying hierarchical levels,but their characters cannot always be inferredfrom comparative biology alone—what bio-logist would have predicted that stem groupbirds include quadrupeds up to 30 m long? Atpresent, the inference that early protistan fos-sils might record stem group eukaryotes (or un-recognized stem groups of major eukaryoticclades) owes more to the absence of diagnosticcharacters than it does to readily interpretedcharacter combinations.

NEOPROTEROZOIC RADIATION

Fossil diversity increased only moderately overthe first half of recorded eukaryotic history.Then, �800 Mya, things changed in the oceans:Both molecular clocks and fossils indicate pro-nounced diversification within major eukary-otic clades at this time. Organic-walled fossilspreserved as compressions in shallow marinemudstones show unprecedented taxonomicrichness, including both resting cysts and vege-tative cells with complex morphologies, as wellas an increased diversity of coenocytic and sim-ple multicellular populations (Fig. 2A–C) (But-terfield et al. 1994; Butterfield 2004, 2005a,b). Inearly interpretations, many of these fossils wereassigned to specific eukaryotic clades, includingxanthophytes, green algae, and fungi. Molecularclocks suggest that some of these attributionsreflect convergence, but morphology, molecu-lar clock estimates, and preservational potentialall support the interpretation of distinctive coe-nocytic fossils in 750- to 800-Mya rocks as Cla-dophoralean green algae (Fig. 2C) (Butterfieldet al. 1994; Graham et al. 2013). The numberof well-preserved fossil assemblages in rocksof this age remains small, and it is possiblethat continuing exploration will pull the recordof accelerating diversification deeper into thepast. At present, however, exceptionally pre-served microfossil assemblages in older rocksdo not record the diversity documented in their750- to 800-Mya counterparts (Knoll et al.2006). Nor is the increase in eukaryotic diversitymatched by a jump in observed cyanobacterialdiversity, again suggesting that the observedrecord is not simply an artifact of sampling.

Two classes of eukaryotic fossils are com-pletely unrecorded before �800 Mya. Vase-shaped microfossils comparable to tests madeby testate amoebans and some simple forami-niferans occur abundantly in mid-Neoprotero-zoic rocks around the world (Fig. 2D) (Porterand Knoll 2000). Their distinctive mode of pres-ervation, most commonly as casts and moldsconspicuous in petrographic thin sections ofshale, carbonate, and chert, lowers the proba-bility that these fossils have a deeper history yetto be discovered. More than a dozen taxa have

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been distinguished, and at least some bear closecomparison to the tests made today by arcellidamoebozoans (Porter et al. 2003). Others havebeen compared with euglyphid rhizarians, butyoung molecular clock estimates for euglyphiddiversification suggest, once again, that ob-served similarities may reflect convergence (Ber-ney and Pawlowski 2006).

The other novel class of microfossils is 10- to30-mm scales preserved in �800-Mya rocksfrom northwestern Canada (Fig. 2F–H). Origi-nally reported by Allison and Hilgert (1986),

the fossils were observed in thin sections ofchert nodules and interpreted as siliceous scalesbroadly comparable to those of extant chryso-phytes. The discovery, however, that the scalesare preserved by mineral phosphate (Cohenet al. 2011) prompted a restudy in which thou-sands of specimens were recovered by the dis-solution of encompassing limestones in weakacid. Some 38 distinctive scale types have beendocumented in exceptional morphological de-tail (Cohen and Knoll 2012), making these themost diverse eukaryotic fossils known before

Figure 2. Late Mesoproterozoic and Neoproterozoic fossils interpreted as eukaryotic. (A) Irregularly spheroidalmicrofossil with long cylindrical processes, 750–800 Mya Svanbergfjellet Formation, Spitsbergen. (B) Largemicrofossil with opaque inner wall bearing small spines and longer cylindrical processes, within encompassingsmoothly spheroidal vesicle, Svanbergfjellet Formation. (C) Cladophora-like branching filamentous microfossilwith apparently coenocytic subunits, Svanbergfjellet Formation. (D) Three-dimensionally (3D) preserved min-eral replicate of testate eukaryote, Chuar Group, Grand Canyon (courtesy of Porter et al. 2003). (E) Bangio-morpha, interpreted as an early-branching red alga, 1100–1200 Mya Hunting Formation, Arctic Canada (cour-tesy of Butterfield 2000). (F–H). Scale microfossils preserved three-dimensionally in �800-Mya carbonaterocks of the Fifteenmile Group, Yukon Territory, Canada (courtesy of Cohen and Knoll 2013). Scale bars,60 mm (A,C); 120 mm (B); 10 mm (F); 14 mm (G,H). Note scale bars in D and E.



the Ediacaran diversification of animals. Thefossils are assuredly eukaryotic and bear func-tional comparison to organic or siliceous scalessynthesized by diverse protists today. Phyloge-netically, however, it is challenging to place anyof these taxa within specific eukaryotic clades.Uncertainty remains, as well, as to the originalcomposition of the scales. Does the observedphosphate record biomineralization (Cohenet al. 2011) (interesting if correct, becausephosphatic biomineralization is extremely rareamong living protists) or early diagenetic phos-phate replication within sediments (Cohen andKnoll 2012)? In either event, the scale assem-blage from northwestern Canada is, for now,unique.

Fossils, then, record an apparent burst ofNeoproterozoic diversification. This paleon-tological expansion is mirrored by molecularclock estimates, but not, intriguingly, by molec-ular biomarkers in Neoproterozoic rocks (Paw-lowska et al. 2013). Should we interpret thedearth of steranes in pre-Ediacaran sedimentaryrocks as evidence of absence or an absence ofevidence? Given the close stratigraphic corre-spondence between the microfossil and bio-marker records of Phanerozoic primary pro-ducers (Schwark and Empt 2006; Knoll et al.2007), the lack of eukaryotic biomarkers inolder strata has commonly been taken to indi-cate bacterial (especially cyanobacterial) domi-nance of primary production. Pawlowska et al.(2013), however, argue that this pattern owesmore to preservation than production.

In the view of Pawlowska et al. (2013), mi-crobial mats that covered Proterozoic seafloorswere the primary sources of sedimentary organ-ic matter preserved in Proterozoic shales. More-over, they note that the aggressively oxidizingenvironments generated diurnally by cyanobac-terial oxygen production within mats (e.g.,Gingras et al. 2011) would have destroyed lipidssourced from the overlying water column. Inconsequence, the lack of steranes in most Pro-terozoic shales may simply reflect preservationalcircumstances common before the EdiacaranPeriod, when evolving animals dramatically re-stricted the distribution of benthic mat com-munities.

Without question, mats were major contrib-utors of organic matter to Proterozoic sedi-ments, and regardless of other influences, abun-dant cyanobacteria and other bacteria wouldhave diluted molecular signatures of eukaryotesliving in mats or the water column. That said,many of the organic-rich shales sampled forbiomarker analysis come from relatively deepbasins in which bottom waters were anoxic,mooting the destructive impact of oxygen-richmat interiors. Petrological examination of Pro-terozoic shales also suggests the need for a morenuanced view of Proterozoic sediment accumu-lation. In many Proterozoic shales, mat hori-zons are separated by variably thick event bedsthat record pulses of mud deposition. Thesemud layers are rich in organic matter, providingan avenue for phytoplankton to evade the mat-seal effect of Pawlowska et al. (2013). Sedimen-tological observations also suggest that the ae-rial extent of benthic mats began to decline wellbefore the Ediacaran Period; the case is bestmade for carbonates, where microbial lamina-tion is much less common in later Neoproter-ozoic beds than it is in older successions (Knolland Swett 1990).

Phanerozoic examples show that bacterialprimary production was transiently high dur-ing episodes of widespread subsurface anoxiain the world’s oceans—for example, at the be-ginning of the Triassic Period (Grice et al. 2005).Thus, we might expect that in Proterozoicoceans with persistent subsurface anoxia, cya-nobacteria and other photosynthetic bacteriawould dominate primary production (Johnstonet al. 2009), in part because of low fixed-nitro-gen abundances in surface waters (Fennel et al.2005). Indeed, Boyle et al. (2013) have arguedthat, in Proterozoic oceans, sulfidic subsurfacewater masses could only develop beneath sur-face waters dominated by nitrogen-fixing pri-mary producers. Conversely, biogeochemicalstudy of Silurian microbial mats shows a strongpresence of eukaryotic biomarkers (Bauersachset al. 2009), suggesting that mat-seals provideimperfect barriers to the burial of eukaryoticlipids.

Thus, although Pawlowska et al.’s (2013) hy-pothesis usefully urges caution in the interpre-

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tation of Proterozoic steranes (or their absence),its applicability to the broad Proterozoic recordremains uncertain. To date, few analyses havetargeted 850- to 650-Mya black shales in whichmolecular biomarkers might be expected tomirror evident microfossil diversification. Bythe Ediacaran Period, however, steranes are rel-atively abundant in carbonaceous shales (Knollet al. 2007). In fact, microfossils suggest a diver-sification of green algal phytoplankton at aboutthe time when C29 sterols (principally sourcedby green algae) (Kodner et al. 2008) becameabundant constituents of sedimentary organicmatter. Some 20% of the microfossil taxa inEarly Cambrian rocks are confidently interpret-ed as the phycomata of prasinophyte greens,and additional chlorophyte diversity may be re-corded by other preserved cysts (e.g., Moczyd-łowska 2010).

What might have driven the observed Neo-proterozoic diversification of marine eukary-otes? Perhaps we can take a lesson from Cambri-an animal radiation. Molecular clocks suggestthat animals began to diverge �800 Mya; fossils,in turn, indicate the presence of metazoans 40–100 million years before the Cambrian explo-sion (Erwin et al. 2011). Although both geneticsand environmental change played a role in ani-mal diversification, the evolution of carnivory isthought to have set off an ecological arms racebetween predators and prey that fueled the ob-served Cambrian diversification of animals(Stanley 1973; Bengtson and Conway Morris1992; Sperling et al. 2013) and algae (Knoll1994; Vidal and Moczydłowska-Vidal 1997).Might the evolution of eukaryophagy have hada broadly comparable effect on Neoproterozoicecosystems?

The ability to phagocytose bacteria and oth-er small particles appears to be plesiomorphicamong the Eukarya. Predation on large cells,however, is commonly derived and focusedwithin a relatively small number of clades. Pre-liminary analysis of molecular clocks suggeststhat eukaryophagy evolved in several clades (in-cluding ciliates, dinoflagellates, amoebozoans,and rhizarians) during the NeoproterozoicEra, and the same logic that underpins ecologi-cal amplification of Cambrian animal diversifi-

cation applies to this event. Indeed, Stanley’s(1973) early formulation of the predation hy-pothesis can be applied at least as well to eukar-yophagy in protists as it has been to carnivory inanimals. Experiments indicate that protistanand micrometazoan grazers both result inincreased growth rates and biomass for eukary-otic phytoplankton, but not cyanobacteria(Trommer et al. 2012; Ratti et al. 2013); thus,eukaryophagy could, in principle, have facilitat-ed the rise of eukaryotic phytoplankton to eco-logical prominence.

Porter (2011) was the first to propose thatprotistan predation might have driven the ob-served Neoproterozoic expansion of eukaryoticfossils. Does this hypothesis make specific pre-dictions that might be tested against the record?The vase-shaped tests introduced earlier pro-vide three lines of evidence consistent with theeukaryophagy hypothesis. The first is phyloge-netic; some of the testate microfossils in 750- to800-Mya rocks can be allied with a eukaryopha-gic amoebozoan clade (Porter et al. 2003). Thenthere is functional evidence; by their nature,these tests would have provided protectionagainst eukaryophagic predators, so the mid-Neoproterozoic radiation of such structures is,again, consistent with the eukaryophagy hy-pothesis. Moreover, some preserved tests haveregular half-moon perforations, thought to re-flect attack by vampyrellid or other protistanpredators (Porter et al. 2003).

Other evidence for protective armor comesfrom the �800-Mya scale microfossils intro-duced earlier (Cohen and Knoll 2012). Andthen there is the expansion of multicellularand coenocytic fossils. Both theory and experi-ment suggest that multicellularity provides pro-tection against protistan predators (e.g., Boraaset al. 1998). Intriguingly, as noted above, mo-lecular clocks suggest that animals date from themid-Neoproterozoic Era as well (Erwin et al.2012), perhaps implicating eukaryophagy inthe origin of animal multicellularity. At present,the idea that the establishment or expansion ofeukaryophagy drove mid-Neoproterozoic eu-karyotic diversification in the oceans remainsa hypothesis to be tested by the careful integra-tion of function and phylogeny, as well as con-



tinuing paleontological research. It does, how-ever, have the merit of accounting for a broadspectrum of paleontological observations.

Last, we can ask about the environmentalcontext of Neoproterozoic eukaryotic diversifi-cation. Increased microfossil diversity immedi-ately preceded an interval of global glaciations,popularly known as the Snowball Earth (Hoff-man et al. 1998). Changes in both export fluxesand mean Redfield ratios of an increasingly eu-karyotic phytoplankton have been implicatedin the CO2 drawdown that initiated glaciation(Tziperman et al. 2011), and decreasing pCO2

has, in turn, been postulated to drive adaptiveevolution in Rubsico, the key enzyme in CO2

fixation by algae and cyanobacteria (Young etal. 2012). Tectonic changes also characterizedthe later Neoproterozoic Earth, and these alsoinfluenced atmospheric chemistry and climate.The key point for ongoing research is that anexpanding ecological presence of eukaryotes inmarine ecosystems may have provided impor-tant new feedback in the integrated Earth sys-tem, both facilitating and reflecting changes inthe physical environment.

CONCLUDING REMARKS

Of course, eukaryotic diversification did notend with the close of the Proterozoic Eon. In-deed, most eukaryotic diversity is a product ofPhanerozoic evolution. Fossils conspicuouslyrecord the radiations of complex multicellu-

lar clades, first animals in the oceans and laterembryophytic land plants, land animals, andmorphologically complex fungi (e.g., Knoll2011). The chemistry of sedimentary rocks in-dicates that the transition from Proterozoic toPhanerozoic ecosystems also involved environ-mental change. Animals radiated in Cambrianoceans richer in oxygen than their Proterozo-ic counterparts, with pO2 increasing to levelsthat matched or exceeded those of the presentduring the later Paleozoic Era (Berner 2009;Dahl et al. 2010). Protists diversified as well.Mineralized skeletons document the Paleozo-ic diversification of radiolarians and benthic fo-raminiferans followed by Mesozoic radiationsof coccolithophorid algae, dinoflagellates, anddiatoms, not to mention the expansion of fo-raminiferans into the planktonic realm (Lipps1993).

Those radiations, however, are only the lat-est chapter in a much longer history of eukary-otic evolution. Careful field and laboratory in-vestigations of Proterozoic sedimentary rocksare yielding increasing evidence of earlier eu-karyotic diversification and its environmentalcontext (Fig. 3). Uncertainties abound, butpresent evidence suggests that crown group eu-karyotes radiated into a world quite distinctfrom today’s, with moderately oxic surfaceoceans and, commonly, anoxia in subsurfacewater masses. As phylogenies, molecular clocks,paleoenvironmental reconstructions, and geo-chronological calibration all continue to im-

Marine redox

Major diversificationof eukaryotes Land plants

Animals

Crown group eukaryotes

Total group eukaryotes

Bacteria/Archaea

Archean Proterozoic Phan

4567 2500 542 (Mya)

No O2 Low O2/subsurface anoxia High O2

Figure 3. A summary of early eukaryotic evolution. Solid bars denote confident interpretation of geologic record;dashed bars indicate uncertain or controversial extensions of the record. Phan, Phanerozoic Eon (literally, theage of visible animal life). See text for references.

A.H. Knoll


prove, our interpretations of the early fossil re-cord will become richer and better integratedwith inferences from comparative biology.

ACKNOWLEDGMENTS

My thinking on eukaryotic evolution has bene-fitted from discussions with S. Porter, P. Cohen,M. Giordano, S. Ratti, P. Falkowski, L. Katz,L. Parfrey, and especially D. Lahr. I thank theNASA Astrobiology Institute for support, E.Javaux for comments on an earlier draft, andM. Walter, N. Butterfield, E. Javaux, S. Porter,and P. Cohen for images in Figures 1 and 2.

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