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Paleobiological Perspectives on Early Eukaryotic Evolution

Paleobiological Perspectives on Early Eukaryotic Evolution

Paleobiological Perspectives on Early Eukaryotic Evolution

Andrew H. Knoll

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138 Correspondence: [email protected]

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

ukaryotic organisms have a long evolution- EXPECTATIONS FROM COMPARATIVE Eary history, recorded, in part, by convention- BIOLOGY al and molecular fossils. For the Phanerozoic Eon (the past 542 million years), eukaryotic The diversity of eukaryotic organisms observ- evolution is richly documented by the skeletons able today makes two sets of predictions for the (and, occasionally, nonskeletal remains) of an- record, one phylogenetic and the other imals, as well as the leaves, stems, roots, and preservational. Phylogenies suggest the relative reproductive organs of land . Phylogenet- timing of diversification events through Earth ic logic, however, tells us that eukaryotes must history, and, when incorporated into molecular have a deeper history, one that began long be- clocks, provide quantitative estimates of diver- fore the first and fossils formed. To gence times. In turn, experimental and obser- what extent does the geological record preserve vational studies of postmortem decay indicate aspects of deep eukaryotic history, and can the that only a subset of eukaryotic clades is likely to chemistry of ancient sedimentary rocks eluci- be represented in the geologic record and these date the environmental conditions under which only under selected environmental circum- the eukaryotic took shape? stances. Together, insights into phylogeny and

Editors: Patrick J. Keeling and Eugene V. Koonin Additional Perspectives on The Origin and Evolution of Eukaryotes available at www.cshperspectives.org Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016121 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121

1 A.H. Knoll preservation provide an empirical guide to pa- mentary accumulations—the White Cliffs of leobiological exploration. Dover, for example, consist mostly of small cal- Molecular sequence comparisons have rev- citic scales made by coccolithophorid algae. olutionized our understanding of evolutionary Tests and scales of calcite and silica document relationships among eukaryotes, but consensus Phanerozoic evolutionary histories for diatoms, on eukaryotic phylogeny remains elusive. Most chrysophytes, coccolithophoids, foraminifer- researchers recognize a limited number of ma- ans, and radiolarians, but do not extend into jor clades, including the opisthonkonts, amoe- Proterozoic rocks (Knoll and Kotrc 2014). Or- bozoans, excavates, plants (sensu lato), and an ganic cell walls, tests, and scales can also survive SAR clade containing the stramenopiles, alveo- bacterial decay, depending on molecular com- lates, and rhizarians (e.g., Katz 2012), and many position, and, in Phanerozoic rocks, these re- recognize the potential for as-yet poorly charac- mains record clades that include dinoflagellates terized taxa to expand that roster (e.g., Patter- and prasinophyte , among other son 1999; Adl et al. 2012). Persistent uncertain- groups. As we shall see, decay-resistant organic ties include the position of the root; placement walls, tests, and scales also document aspects of of groups such as heliozoans, hap- Proterozoic protistan evolution, although it can tophytes, and ; and both the be challenging to relate preserved fossils to ex- monophyly and relationships of photosynthetic tant clades. lineages commonly grouped as Plantae. Preservation, of course, is not the only hur- Molecular clocks calibrated by phylogenet- dle in paleobiological investigations of Protero- ically well-constrained fossils have been used to zoic rocks. There is also the challenge of recog- estimate the timing of early eukaryotic diversi- nition. In the first instance, how do we identify fication. Choice of algorithm can strongly influ- a fossil as eukaryotic rather than bacterial? ence these estimates (Roger and Hug 2006; Eme Given that the record is one of morphology et al. 2014), but sensitivity tests suggest that for and not DNA or cytology, diagnostic characters a given set of sampled taxa, at least some esti- must be sought in the size, shape, ultrastructure, mates are broadly robust to tree topology and and preservational circumstances of microfos- calibration choices (e.g., Parfrey et al. 2011). sil populations. Eukaryotic cells are commonly Molecular clock estimates generally agree that larger than and archaeons, but are not much protistan diversification has taken place invariably so. Conversely, com- during the Phanerozoic Eon, paralleling the di- monly form extracellular sheaths and envelopes versification of animals, plants, and fungi. They that may encompass many cells; this being the also agree on an earlier, Neoproterozoic radia- case, burial can preserve a 100-mm cyanobacte- tion within the major eukaryotic clades, begin- rial envelope that, in , surrounded numerous ning perhaps 800 million years ago (Mya). micron-scale cells. By itself, then, size is com- Where clocks disagree is on the date for the monly an insufficient criterion for eukaryotic last common ancestor of extant eukaryotes, attribution. Cyst walls associated with resting with some positing a long interval of eukaryotic stages in eukaryotic life cycles commonly have evolution before Neoproterozoic radiation spines or other ornamentation, and they com- (e.g., Hedges et al. 2004; Yoon et al. 2004; Par- monly have acomplex ultrastructure as observed frey et al 2011) and others suggesting a shorter via TEM (e.g., Javaux et al. 2004). Bacteria can fuse (e.g., Douzery et al. 2004; Berney and Paw- have large envelopes (and more rarely cell size) lowski 2006; Chernikova et al. 2011; Shih and (Schultz and Jørgensen 2001), but they rarely if Matzke 2013). The differing predictions of these evercombine large size, ornamented walls, com- clock estimates can be tested against the Prote- plex ultrastructure, and a preservable composi- rozoic fossil record. tion; thus, fossils that display all of these charac- How do protists impart a paleobiological teristics are widely regarded as eukaryotic. signature to sedimentary rocks? Mineralized Molecular fossils provide another means by protistan skeletons can form significant sedi- which eukaryotic organisms can impart a sig-

2 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 of Early Eukaryotes nature to the geological record. Proteins and few structurally preserved or morphologically nucleic acids have a low probability of preserva- distinctive microfossils. One of the few well- tion, but lipids can preserve well, and sterols in documented and widely accepted fossil occur- particular have been used to investigate the deep rences in earlier rocks, and perhaps the history of eukaryotes. Abundant steranes (the only one that potentially bears on stem group geologically stable derivatives of sterols) extract- eukaryotes, comes from 3200-Mya shales that ed from petroleum document a Phanerozoic contain large (30–300 mm) spheroidal vesicles history of primary producers in the oceans (Javaux et al. 2010). These appear to be genuine that, to a first approximation, parallels the his- fossils, and they could, in principle, be eukary- tories inferred from microfossils and molecular otic; however, their simple ultrastructure and clocks (Knoll et al. 2007). Molecular fossils ex- ready comparison to the extracellular envelopes tend that record into Proterozoic and, more of some bacteria saps confidence from such an controversially, late Archean rocks. interpretation. Together then, phylogenies and preserva- In the absence of a convincing tion potential furnish guides to the paleobiolo- record, geobiologists have turned to molecular gy of early eukaryotic evolution, providing hy- fossils. Steranes of hypothesized eukaryotic or- potheses of evolutionary history. How well do igin have been reported from late Archean sedi- fossils fit these predictions? mentary rocks (Brocks et al. 1999; Waldbauer et al. 2009), potentially documenting early eu- karyotes, but raising incompletely resolved en- ARCHEAN EUKARYOTES? vironmental, phylogenetic, and geological is- The three-domain view of life, predicated on sues. The environmental concern is that sterol comparisons of SSU rRNA gene sequences, pos- biosynthesis requires molecular oxygen, yet ited that eukaryotes are sister to the Archaea geochemical data consistently indicate that the (Woese et al. 1990). Given this relationship Archean atmosphere and oceans were anoxic and isotopic evidence for methanogenesis and (Holland 2006). Sterol synthesis is possible at methanotrophy in the late Archean carbon cy- nanomolar oxygen tensions (Waldbauer et al. cle (Hayes 1994), logic would dictate that 2011), and thus a plausible but unproven solu- the Eukarya existed no later than 2700 Mya. tion holds that early cyanobacteria could have This logic, however, is challenged by alternative generated local oxygen oases within mats or phylogenies that nest eukaryotes within the Ar- sediments long before O2 began to accumulate chaea (Williams et al. 2012) and by models for in the atmosphere (e.g., Anbar et al. 2007). eukaryogenesis that rely on archaeal–bacterial The second issue is phylogenetic. A limited (Martin and Mu¨ller 1998; Moreira number of bacteria synthesize sterols (e.g., and Lopez Garcia 1998). Even the most gener- Pearson et al. 2005), raising the concern that ous molecular clock estimates place the last preserved biomarkers could be of prokaryotic common ancestor of extant eukaryotes within origin. In general, however, bacterial sterol syn- the Proterozoic Eon (Hedges et al. 2004), re- thesis is limited to simple products such as lan- quiring that any Archean eukaryotes be stem osterol, and thus it is fair to consider more com- groups. plex steranes of the type found in late Archean Unfortunately, the record of life in Archean rocks as eukaryotic until and unless complex rocks is sparse and subject to conflicting inter- sterol synthesis is shown in free-living bacteria. pretations. Early diagenetic cherts, a rich source The third concern, however, is not so easily dis- of microfossils in Proterozoic strata, are largely missed. Because steranes in late Archean rocks barren, perhaps reflecting the strong influence occur in part-per-billion concentrations, geo- of hydrothermal fluid flow and deposition logical or modern contamination must be con- on the silica cycle of Archean oceans (Fischer sidered. Fluids flow through sedimentary rocks and Knoll 2009; Chakrabarti et al. 2012). Shales, throughout their history, and biomarkers can in turn, contain abundant organic carbon, but also be emplaced during the processes of drill-

Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 3 A.H. Knoll ing and sample processing. Arguments in favor chondria of genes for anaerobic metabolism of an indigenous origin for Archean steranes (Mu¨ller et al. 2012). stress the care taken in sample preparation and Bangiomorpha pubsecens (Fig. 2E) (Butter- the varying biomarker composition of different field 2000) plays a key role in evaluating crown beds, consistent with the expectation of ecolog- group early and late hypotheses based on mo- ical heterogeneity at the time of deposition lecular clocks. An exceptionally well-preserved (Brocks et al. 2003; Waldbaueret al. 2009). Fur- population of filamentous microfossils found in ther support comes from steranes and other silicified peritidal carbonates from Arctic Can- biomarkers in fluid inclusions from quartz par- ada, Bangiomorpha displays several morphol- ticles in 2400-Mya sedimentary rocks (Dutkie- ogical features that collectively place it within wicz et al. 2006). Critics counter that biomarker the red algae. These include overall morphology, heterogeneity could reflect bed-by-bed varia- details of thallus development and reproductive tions in porosity and permeability, channeling biology, cellularly differentiated holdfasts, life later flow fluid, and note that steranes occur cycle characteristics, and details of preservation mostly near the surface of drill samples (Brocks that differ markedly from those characteristics 2011) and have a carbon isotopic composition of silicified cyanobacteria. Many specimens are distinct from the bulk of the organic matter in preserved in life position, rising vertically from the samples (Rasmussen et al 2008). The debate attachment sites on the ancient seafloor (Butter- continues, commendably fueled by new sam- field 2000). Thus, Bangiomorpha is reasonably pling programs marked by stringent protocols interpreted as a rhodophyte, although it may for drilling and sample preparation. branch earlier within the clade than extant Ban- giales. Multiple geochronological and strati- graphic constraints indicate that Bangiomorpha PROTEROZOIC ESTABLISHMENT lived 1100–1200 Mya (summarized in Knoll As noted above, molecular clocks suggest that et al. 2013). Neither its age nor its phylogenetic regardless of any deeper stem group history, attribution is likely to change markedly with crown group eukaryotes emerged during the continued study, and thus Bangiomorpha favors Proterozoic Eon. (In this discussion, the term molecular clocks that place both the last com- “crown group” is used in its broadly accepted mon ancestor of extant eukaryotes and the ac- phylogenetic sense to indicate the last common quisition of plastids before 1200 Mya. ancestor of all extant members of a clade and its Some paleontologists propose that crown descendants. Earlier, informal usage to denote a group eukaryotes can be traced further back in diverse subset of eukaryotes has been aban- time. For example, Moczydłowska et al. (2011) doned.) The last common ancestor of extant have argued that microfossils of green algae oc- eukaryotes possessed a capa- cur in rocks as old as 1800 Mya. Accepting that ble of aerobic respiration, consistent with geo- green algae (and land plants) are sister to the red chemical evidence for the permanent oxygena- algae, greens must have lived contemporane- tion of Earth’s atmosphere and surface ocean ously with 1100- to 1200-Mya Bangiomorpha, 2400 Mya (Holland 2006). Quantification of and molecular clock estimates not precluded by Proterozoic oxygen levels is difficult, but the Bangiomorpha’s age suggest that the green–red persistence of anoxic water masses beneath the split occurred up to several hundred million surface mixed layer of the oceans suggests that years before this (Wang et al. 1999; Yoon et al. pO2 remained low, perhaps no more than a few 2004; Parfrey et al. 2011). (If, as sometimes pro- percent of present-day atmospheric levels (e.g., posed, red algae are sister to greens plus glauco- Brocks et al. 2005; Canfield 2005; Scott et al. cystophytes, the green clade could have radiated 2008; Johnston et al. 2010; Frei et al. 2013). later.) The older fossils in question, however, are The chronic challenge of anoxic waters mixed simple spheroids whose affinities are not easily upward from the oxygen minimum zone is con- ascertained (Fig. 1A,B). Moczydłowska et al. sistent with the widespread occurrence in mito- (2011) maintain that only algae make resistant

4 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 Paleobiology of Early Eukaryotes

Figure 1. Late Paleo- and 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, . (B) Spheroidal vesicle with corduroy-like ornamentation of vesicle wall, Roper Group. (C) Spheroidal microfossil with surface divided into small fields and ornamented with cylindrical processes that expand distally; TEM of walls shows complex multilayered wall ultrastructure, .1600 Mya Ruyang Group, China. (D) Spheroidal vesicle with 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 , 1400 Mya Jixian Group, China (courtesy of M.R. Walter). Scale bars, 20 mm(A); 75 mm(B,D); 120 mm(C). Note 1-cm scale bar in E. cysts with ornamentation and well-defined ex- limited diversity of green algae (Kodner et al. cystment structures, but resting cysts have been 2009), can also form during diagenesis (Gupta well described in various heterotrophic protists, et al. 2009)—molecular clocks suggest that TLS including, for example, that fashion and algaenan-synthesizing green algae doubt- large, spheroidal, and sometimes ornamented fully extend much below the Proterozoic–Cam- cysts with pylome-like excystment structures brian boundary. Thus, it remains uncertain (e.g., Beers 1948, 1966; Foissneret al. 2007; Verni whether earlier Proterozoic microfossils record and Rosati 2011). Wallultrastructure might pro- crown group green algae, stem group greens (or vide more definitive evidence of green algae, es- Plantae), another crown group clade, or stem pecially should TEM reveal the distinctive tri- group eukaryotes (Knoll et al. 2006). laminar wall structure (TLS) characteristic of In general, mid-Proterozoic sedimentary cell walls in some chlorophytes (Allard and Tem- rocks contain abundant, but only modestly plier 2000). TLS has been shown in diversefossilsofprobableeukaryoticorigin(Jav- green algae (Talyzina and Moczydłowska 2000), aux 2011). Shales up to 1600 Mya contain mi- but not in the older microfossils under consid- crofossils that combine large size (.100 mm) eration here (Javaux et al. 2004). Biomarker with complex ultrastructure, structurally com- molecules might also provide insight, but ster- plex ornamented or tessellated cell walls, and anes are rare in mid-Proterozoic rocks (Brocks surface processes of varying form (Fig. 1C,D) et al. 2005; Pawlowska et al. 2013) (see below), (Javaux et al. 2001, 2003, 2004; Xiao et al. 1997; and growing evidence suggests that algaenan, an Yin and Yuan 2007; Nagovitsin et al. 2010). aliphatic polymer known to be synthesized by a Equally large vesicles with less distinctive surface

Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 5 A.H. Knoll morphology or ultrastructure occur in rocks as NEOPROTEROZOIC RADIATION old as 1800 Mya (Fig. 1A) (Yan1995; Lamb et al. 2009). These may well be eukaryotic, especially Fossil diversity increased only moderately over those with corduroy-like, raised parallel ridges the first half of recorded eukaryotic history. on wall surfaces (Fig. 1B) (Yan 1995). For most, Then, 800 Mya, things changed in the oceans: however, a lack of diagnostic features under- Both molecular clocks and fossils indicate pro- scores residual uncertainty at the domain level. nounced diversification within major eukary- Macroscopic impressions and compressions otic clades at this time. Organic-walled fossils whose regular morphologysuggests aeukaryotic preserved as compressions in shallow marine origin also occur in rocks of mid-Proterozoic age mudstones show unprecedented taxonomic (Fig. 1E) (Grey and Williams 1990; Walter et al. richness, including both resting cysts and vege- 1990; Retallack et al. 2013), with the oldest over- tative cells with complex morphologies, as well lapping in age with possible eukaryotic micro- as an increased diversity of coenocytic and sim- fossils (Hofmann and Chen 1981; Han and Run- ple multicellular populations (Fig. 2A–C) (But- negar 1992). Most of this record comes from terfield et al. 1994; Butterfield 2004, 2005a,b). In marine rocks, but a possible glimpse of life in early interpretations, many of these fossils were Proterozoic lakes has been reported from 1000- assigned to specific eukaryotic clades, including to 900-Mya purportedly nonmarine beds in xanthophytes, green algae, and fungi. Molecular Scotland that preserve a moderate diversity of clocks suggest that some of these attributions microfossils likely sourced by protists but other- reflect convergence, but morphology, molecu- wise of problematic origin (Strother et al. 2010). lar clock estimates, and preservational potential From the preceding paragraphs we can draw all support the interpretation of distinctive coe- two conclusions. First, although one might nocytic fossils in 750- to 800-Mya rocks as Cla- hope for a clean paleobiological boundary be- dophoralean green algae (Fig. 2C) (Butterfield tween worlds with and without eukaryotic cells, et al. 1994; Graham et al. 2013). The number the geological record actually presents a sliding of well-preserved fossil assemblages in rocks scale of certainty, from confidently interpreted of this age remains small, and it is possible protists in 1400- to 1600-Mya rocks to more that continuing exploration will pull the record ambiguously interpreted remains at 1800 Mya of accelerating diversification deeper into the and even more debated morphological and mo- past. At present, however, exceptionally pre- lecular signatures in older successions. Paleobi- served microfossil assemblages in older rocks ological evidence of eukaryotic cells does not so do not record the diversity documented in their much bottom out as fade away. The second con- 750- to 800-Mya counterparts (Knoll et al. clusion is that the early eukaryotic record could 2006). Nor is the increase in eukaryotic diversity be dominated by stem group taxa. Stem groups matched by a jump in observed cyanobacterial are a logical necessity in biology but an empir- diversity, again suggesting that the observed ical challenge for paleontologists. The fossil record is not simply an artifact of sampling. records of plants and animals contain diverse Two classes of eukaryotic fossils are com- stem group taxa at varying hierarchical levels, pletely unrecorded before 800 Mya. Vase- but their characters cannot always be inferred shaped microfossils comparable to tests made from comparative biology alone—what bio- by testate amoebans and some simple forami- logist would have predicted that stem group niferans occur abundantly in mid-Neoprotero- include quadrupeds up to 30 m long? At zoic rocks around the world (Fig. 2D) (Porter present, the inference that early protistan fos- and Knoll 2000). Their distinctive mode of pres- sils might record stem group eukaryotes (or un- ervation, most commonly as casts and molds recognized stem groups of major eukaryotic conspicuous in petrographic thin sections of clades) owes more to the absence of diagnostic shale, carbonate, and chert, lowers the proba- characters than it does to readily interpreted bility that these fossils have a deeper history yet character combinations. to be discovered. More than a dozen taxa have

6 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 Paleobiology of Early Eukaryotes

Figure 2. Late Mesoproterozoic and Neoproterozoic fossils interpreted as eukaryotic. (A) Irregularly spheroidal microfossil with long cylindrical processes, 750–800 Mya Svanbergfjellet Formation, Spitsbergen. (B) Large microfossil with opaque inner wall bearing small spines and longer cylindrical processes, within encompassing smoothly spheroidal vesicle, Svanbergfjellet Formation. (C) Cladophora-like branching filamentous microfossil with apparently coenocytic subunits, Svanbergfjellet Formation. (D) Three-dimensionally (3D) preserved min- eral replicate of testate , Chuar Group, (courtesy of Porter et al. 2003). (E) Bangio- morpha, interpreted as an early-branching red alga, 1100–1200 Mya Hunting Formation, Arctic (cour- tesy of Butterfield 2000). (F–H). Scale microfossils preserved three-dimensionally in 800-Mya carbonate rocks 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. been distinguished, and at least some bear close the fossils were observed in thin sections of comparison to the tests made today by arcellid chert nodules and interpreted as siliceous scales amoebozoans (Porter et al. 2003). Others have broadly comparable to those of extant chryso- been compared with euglyphid rhizarians, but phytes. The discovery, however, that the scales young molecular clock estimates for euglyphid are preserved by mineral phosphate (Cohen diversification suggest, once again, that ob- et al. 2011) prompted a restudy in which thou- served similarities may reflect convergence (Ber- sands of specimens were recovered by the dis- ney and Pawlowski 2006). solution of encompassing limestones in weak The other novel class of microfossils is 10- to acid. Some 38 distinctive scale types have been 30-mm scales preserved in 800-Mya rocks documented in exceptional morphological de- from northwestern Canada (Fig. 2F–H). Origi- tail (Cohen and Knoll 2012), making these the nally reported by Allison and Hilgert (1986), most diverse eukaryotic fossils known before

Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 7 A.H. Knoll the diversification of animals. The Without question, mats were major contrib- fossils are assuredly eukaryotic and bear func- utors of organic matter to Proterozoic sedi- tional comparison to organic or siliceous scales ments, and regardless of other influences, abun- synthesized by diverse protists today. Phyloge- dant cyanobacteria and other bacteria would netically, however, it is challenging to place any have diluted molecular signatures of eukaryotes of these taxa within specific eukaryotic clades. living in mats or the water column. That said, Uncertainty remains, as well, as to the original many of the organic-rich shales sampled for composition of the scales. Does the observed biomarker analysis come from relatively deep phosphate record (Cohen basins in which bottom waters were anoxic, et al. 2011) (interesting if correct, because mooting the destructive impact of oxygen-rich phosphatic biomineralization is extremely rare mat interiors. Petrological examination of Pro- among living protists) or early diagenetic phos- terozoic shales also suggests the need for a more phate replication within sediments (Cohen and nuanced view of Proterozoic sediment accumu- Knoll 2012)? In either event, the scale assem- lation. In many Proterozoic shales, mat hori- blage from northwestern Canada is, for now, zons are separated by variably thick event beds unique. that record pulses of mud deposition. These Fossils, then, record an apparent burst of mud layers are rich in organic matter, providing Neoproterozoic diversification. This paleon- an avenue for phytoplankton to evade the mat- tological expansion is mirrored by molecular seal effect of Pawlowska et al. (2013). Sedimen- clock estimates, but not, intriguingly, by molec- tological observations also suggest that the ae- ular biomarkers in Neoproterozoic rocks (Paw- rial extent of benthic mats began to decline well lowska et al. 2013). Should we interpret the before the Ediacaran Period; the case is best dearth of steranes in pre-Ediacaran sedimentary made for carbonates, where microbial lamina- rocks as evidence of absence or an absence of tion is much less common in later Neoproter- evidence? Given the close stratigraphic corre- ozoic beds than it is in older successions (Knoll spondence between the microfossil and bio- and Swett 1990). marker records of Phanerozoic primary pro- Phanerozoic examples show that bacterial ducers (Schwark and Empt 2006; Knoll et al. primary production was transiently high dur- 2007), the lack of eukaryotic biomarkers in ing episodes of widespread subsurface anoxia older strata has commonly been taken to indi- in the world’s oceans—for example, at the be- cate bacterial (especially cyanobacterial) domi- ginning of the TriassicPeriod (Grice et al. 2005). nance of primary production. Pawlowska et al. Thus, we might expect that in Proterozoic (2013), however, argue that this pattern owes oceans with persistent subsurface anoxia, cya- more to preservation than production. nobacteria and other photosynthetic bacteria In the view of Pawlowska et al. (2013), mi- would dominate primary production (Johnston crobial mats that covered Proterozoic seafloors et al. 2009), in part because of low fixed-nitro- were the primary sources of sedimentary organ- gen abundances in surface waters (Fennel et al. ic matter preserved in Proterozoic shales. More- 2005). Indeed, Boyle et al. (2013) have argued over, they note that the aggressively oxidizing that, in Proterozoic oceans, sulfidic subsurface environments generated diurnally by cyanobac- water masses could only develop beneath sur- terial oxygen production within mats (e.g., face waters dominated by nitrogen-fixing pri- Gingras et al. 2011) would have destroyed lipids mary producers. Conversely, biogeochemical sourced from the overlying water column. In study of Silurian microbial mats shows a strong consequence, the lack of steranes in most Pro- presence of eukaryotic biomarkers (Bauersachs terozoic shales may simply reflect preservational et al. 2009), suggesting that mat-seals provide circumstances common before the Ediacaran imperfect barriers to the burial of eukaryotic Period, when evolving animals dramatically re- lipids. stricted the distribution of benthic mat com- Thus, although Pawlowska et al.’s (2013) hy- munities. pothesis usefully urges caution in the interpre-

8 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 Paleobiology of Early Eukaryotes tation of Proterozoic steranes (or their absence), cation applies to this event. Indeed, Stanley’s its applicability to the broad Proterozoic record (1973) early formulation of the predation hy- remains uncertain. To date, few analyses have pothesis can be applied at least as well to eukar- targeted 850- to 650-Mya black shales in which yophagy in protists as it has been to carnivory in molecular biomarkers might be expected to animals. Experiments indicate that protistan mirror evident microfossil diversification. By and micrometazoan grazers both result in the Ediacaran Period, however, steranes are rel- increased growth rates and biomass for eukary- atively abundant in carbonaceous shales (Knoll otic phytoplankton, but not cyanobacteria et al. 2007). In fact, microfossils suggest a diver- (Trommer et al. 2012; Ratti et al. 2013); thus, sification of green algal phytoplankton at about eukaryophagy could, in principle, have facilitat- the time when C29 sterols (principally sourced ed the rise of eukaryotic phytoplankton to eco- by green algae) (Kodner et al. 2008) became logical prominence. abundant constituents of sedimentary organic Porter (2011) was the first to propose that matter. Some 20% of the microfossil taxa in protistan predation might have driven the ob- Early Cambrian rocks are confidently interpret- served Neoproterozoic expansion of eukaryotic ed as the phycomata of prasinophyte greens, fossils. Does this hypothesis make specific pre- and additional chlorophyte diversity may be re- dictions that might be tested against the record? corded by other preserved cysts (e.g., Moczyd- The vase-shaped tests introduced earlier pro- łowska 2010). vide three lines of evidence consistent with the What might have driven the observed Neo- eukaryophagy hypothesis. The first is phyloge- proterozoic diversification of marine eukary- netic; some of the testate microfossils in 750- to otes? Perhaps we can take a lesson from Cambri- 800-Mya rocks can be allied with a eukaryopha- an animal radiation. Molecular clocks suggest gic amoebozoan clade (Porter et al. 2003). Then that animals began to diverge 800 Mya; fossils, there is functional evidence; by their nature, in turn, indicate the presence of metazoans 40– these tests would have provided protection 100 million years before the Cambrian explo- against eukaryophagic predators, so the mid- sion (Erwin et al. 2011). Although both genetics Neoproterozoic radiation of such structures is, and environmental change played a role in ani- again, consistent with the eukaryophagy hy- mal diversification, the evolution of carnivory is pothesis. Moreover, some preserved tests have thought to have set off an ecological arms race regular half-moon perforations, thought to re- between predators and prey that fueled the ob- flect attack by vampyrellid or other protistan served Cambrian diversification of animals predators (Porter et al. 2003). (Stanley 1973; Bengtson and Conway Morris Other evidence for protective armor comes 1992; Sperling et al. 2013) and algae (Knoll from the 800-Mya scale microfossils intro- 1994; Vidal and Moczydłowska-Vidal 1997). duced earlier (Cohen and Knoll 2012). And Might the evolution of eukaryophagy have had then there is the expansion of multicellular a broadly comparable effect on Neoproterozoic and coenocytic fossils. Both theory and experi- ecosystems? ment suggest that multicellularity provides pro- The ability to phagocytose bacteria and oth- tection against protistan predators (e.g., Boraas er small particles appears to be plesiomorphic et al. 1998). Intriguingly, as noted above, mo- among the Eukarya. Predation on large cells, lecular clocks suggest that animals date from the however, is commonly derived and focused mid-Neoproterozoic as well (Erwin et al. within a relatively small number of clades. Pre- 2012), perhaps implicating eukaryophagy in liminary analysis of molecular clocks suggests the origin of animal multicellularity. At present, that eukaryophagy evolved in several clades (in- the idea that the establishment or expansion of cluding ciliates, dinoflagellates, amoebozoans, eukaryophagy drove mid-Neoproterozoic eu- and rhizarians) during the Neoproterozoic karyotic diversification in the oceans remains Era, and the same logic that underpins ecologi- a hypothesis to be tested by the careful integra- cal amplification of Cambrian animal diversifi- tion of function and phylogeny, as well as con-

Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 9 A.H. Knoll tinuing paleontological research. It does, how- lar clades, first animals in the oceans and later ever, have the merit of accounting for a broad embryophytic land plants, land animals, and spectrum of paleontological observations. morphologically complex fungi (e.g., Knoll Last, we can ask about the environmental 2011). The chemistry of sedimentary rocks in- context of Neoproterozoic eukaryotic diversifi- dicates that the transition from Proterozoic to cation. Increased microfossil diversity immedi- Phanerozoic ecosystems also involved environ- ately preceded an interval of global glaciations, mental change. Animals radiated in Cambrian popularly known as the (Hoff- oceans richer in oxygen than their Proterozo- man et al. 1998). Changes in both export fluxes ic counterparts, with pO2 increasing to levels and mean Redfield ratios of an increasingly eu- that matched or exceeded those of the present karyotic phytoplankton have been implicated during the later Era (Berner 2009; in the CO2 drawdown that initiated glaciation Dahl et al. 2010). Protists diversified as well. (Tziperman et al. 2011), and decreasing pCO2 Mineralized skeletons document the Paleozo- has, in turn, been postulated to drive adaptive ic diversification of radiolarians and benthic fo- evolution in Rubsico, the key enzyme in CO2 raminiferans followed by radiations fixation by algae and cyanobacteria (Young et of coccolithophorid algae, dinoflagellates, and al. 2012). Tectonic changes also characterized diatoms, not to mention the expansion of fo- the later Neoproterozoic Earth, and these also raminiferans into the planktonic realm (Lipps influenced atmospheric chemistry and climate. 1993). The key point for ongoing research is that an Those radiations, however, are only the lat- expanding ecological presence of eukaryotes in est chapter in a much longer history of eukary- marine ecosystems may have provided impor- otic evolution. Careful field and laboratory in- tant new feedback in the integrated Earth sys- vestigations of Proterozoic sedimentary rocks tem, both facilitating and reflecting changes in are yielding increasing evidence of earlier eu- the physical environment. karyotic diversification and its environmental context (Fig. 3). Uncertainties abound, but present evidence suggests that crown group eu- CONCLUDING REMARKS karyotes radiated into a world quite distinct Of course, eukaryotic diversification did not from today’s, with moderately oxic surface end with the close of the Proterozoic Eon. In- oceans and, commonly, anoxia in subsurface deed, most eukaryotic diversity is a product of water masses. As phylogenies, molecular clocks, Phanerozoic evolution. Fossils conspicuously paleoenvironmental reconstructions, and geo- record the radiations of complex multicellu- chronological calibration all continue to im-

No O2 Low O2/subsurface anoxia High O2 Marine Major diversification of eukaryotes Land plants Animals Crown group eukaryotes Total group eukaryotes Bacteria/Archaea

Archean Proterozoic Phan 4567 2500 542 (Mya)

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, the age of visible animal life). See text for references.

10 Cite this article as Cold Spring Harb Perspect Biol 2014;6:a016121 Paleobiology of Early Eukaryotes prove, our interpretations of the early fossil re- trol on euxinia in the mid-Proterozoic ocean. Nat Com- cord will become richer and better integrated mun 4: 1533. Brocks JJ. 2011. Millimeter-scale concentration gradients of with inferences from comparative biology. hydrocarbons in Archean shales: Live-oil escape or fin- gerprint of contamination? Geochim Cosmochem Acta 75: 3196–3213. ACKNOWLEDGMENTS Brocks JJ, Logan GA, Buick R, Summons RE. 1999. Archean molecular fossils and the early rise of eukaryotes. Science My thinking on eukaryotic evolution has bene- 285: 1033–1036. fitted from discussions with S. Porter, P.Cohen, Brocks JJ, Buick R, Summons RE, Logan GA. 2003. A re- M. Giordano, S. Ratti, P. Falkowski, L. Katz, construction of Archean biological diversity based on molecular fossils from the 2.78 to 2.45 billion--old L. Parfrey, and especially D. Lahr. I thank the Mount Bruce Supergroup, Hamersley Basin, Western NASA Astrobiology Institute for support, E. Australia. Geochim Cosmochim Acta 67: 4321–4335. Javaux for comments on an earlier draft, and Brocks JJ, Love GD, Summons RE, Knoll AH, Logan GA, Bowden S. 2005. Biomarker evidence for green and pur- M. Walter, N. Butterfield, E. Javaux, S. Porter, ple sulfur bacteria in an intensely stratified Paleoproter- and P. Cohen for images in Figures 1 and 2. ozoic ocean. Nature 437: 866–870. Butterfield NJ. 2000. Bangiomorpha pubescens n. gen., n. sp.: Implications for the evolution of sex, multicellularity, REFERENCES and the Mesoproterozoic/Neoproterozoic radiation of eukaryotes. Paleobiology 26: 386–404. ÃReference is also in this collection. Butterfield NJ. 2004. 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