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Paleobiology, 26(3), 2000, pp. 360–385

Testate amoebae in the Era: evidence from vase-shaped in the Chuar Group, Grand Canyon

Susannah M. Porter and Andrew H. Knoll

Abstract.—Vase-shaped microfossils (VSMs) occur globally in Neoproterozoic rocks, but until now their biological relationships have remained problematic. Exceptionally preserved new populations from the uppermost Chuar Group, Grand Canyon, Arizona, display details of morphology and taphonomy that collectively point to affinities with the . The fossils are tear-shaped tests, ϳ20–300 ␮m long and ϳ10–200 ␮m wide, that are circular in transverse section, expand ab- orally toward a rounded or slightly pointed pole, and taper orally toward a ‘‘neck’’ that ends in a single aperture. Apertures may be circular, hexagonal, triangular, or crenulate, and may be rimmed by a distinct collar. Approximately 25% of the Chuar VSMs are curved, such that the oral and aboral poles do not lie opposite each other. Tests are preserved as mineralized casts and molds, commonly coated with organic debris or iron minerals, but they were originally composed of nonresistant organic matter. Approximately 1% have a ‘‘honeycomb-patterned’’ wall attributable to the original presence of mineralized scales whose bases were arranged regularly in the test wall. Scale-bearing testate amoebae, such as members of the Euglyphidae, are essentially identical to the honeycomb VSMs, and a close relationship between other Grand Canyon VSMs and additional testate amoebae, both lobose and filose, is likely. The VSM population therefore most likely represents a multispecies assemblage whose spatial association reflects a common habitat and/or taphonomic circumstances that favor test preservation. The assignment of these fossils to the testate amoebae strengthens the case for a major diversification of eukaryotic organisms by mid-Neoproterozoic times and, more significantly, provides the earliest morphological evidence for heterotrophic in marine ecosystems.

Susannah M. Porter and Andrew H. Knoll. Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138. E-mail: [email protected]

Accepted: 1 March 2000

Introduction filose and lobose testate amoebae. This inter- pretation contributes to the increasingly well Molecular phylogenies indicate that the ma- documented view that diversification both jor clades of the Eucarya, including , among and within eukaryotic clades was well fungi, (and their descendants, advanced by mid-Neoproterozoic time, and land ), stramenopiles, , and al- provides the earliest morphological evidence veolates (, dinoflagellates, and apicom- for heterotrophic eukaryotes. plexans), diverged during a relatively brief in- Although abundant and globally distribut- terval of cladogenesis that substantially pre- ed in Neoproterozoic rocks, vase-shaped mi- ceded the diversification of crown- crofossils (VSMs) have until now remained group metazoans (Gajadhar et al. 1991; Sogin problematic. First discovered by Ewetz (1933) 1991, 1994; Budin and Philippe 1998). The dis- in phosphate nodules from the Visingso¨ coveries of fossilized red algae (Butterfield et Group, Sweden, these fossils have been vari- al. 1990, Butterfield this issue) and multicel- ously interpreted as (Bloeser et lular stramenopiles (German 1990; Woods et al. 1977), algal cysts (Bloeser 1985), algal spo- al. 1998) in 1200–1000-Ma rocks place a min- rangia (Horodyski 1987, 1993), and heterotro- imum constraint on the timing of this diver- phic, planktonic similar to tintinnids gence and imply the existence of other eu- (Fairchild et al. 1978; Knoll and Vidal 1980; karyotic clades in Neoproterozoic oceans. Knoll and Calder 1983). Schopf (1992) sug- Here we propose that vase-shaped microfos- gested that VSMs may be the cysts of testate sils (genera Melanocyrillium Bloeser 1985 and amoebae, but did not provide detailed evi- Caraburina Kraskov 1985) found widely in dence to support this conclusion. Our inter- Neoproterozoic rocks have affinities with both pretation of VSMs as testate amoebae is based

᭧ 2000 The Paleontological Society. All rights reserved. 0094-8373/00/2603-0004/$1.00 NEOPROTEROZOIC TESTATE AMOEBAE 361 principally on newly discovered populations is marked by a 3- to 10-m-thick dolomite unit in diagenetic dolomite nodules from the up- comprising three distinct lithofacies (Cook permost Walcott Member of the Kwagunt For- 1991): a lower dolomitic wackestone with in- mation, Chuar Group, Grand Canyon. Both traclasts and nodular pisolitic chert; a dolo- abundant and exceptionally well preserved, mite unit consisting of crinkled, folded, or these fossils display hitherto unavailable (and broken microbial laminations, in part silicified taxonomically informative) details of mor- (termed the ‘‘Flaky Dolomite’’ by Ford and phology and test construction. Breed 1973); and a wavy- to horizontally lam- inated dolomicrite unit with cauliflower-like Geological Setting chert in its upper 30 cm. The Chuar Group, a 1600-m-thick succes- Directly overlying the basal Walcott dolo- sion of predominantly siltstone and mudstone mite are Chuaria-bearing black shales (Walcott beds with subordinate sandstones and car- 1899; Ford and Breed 1973; this study) inter- bonates, is exposed over a ϳ150-km2 area in bedded with thin dolomicrite to dolosiltite the northeastern part of the Grand Canyon, units containing mm-scale wavy laminae, cropping out in canyons formed by west-bank scattered early diagenetic chert nodules, and tributaries of the Colorado River and bounded local molar tooth(?) structures. The cherts pre- to the east by the Butte Fault (Fig. 1A) (Ford serve moderately well rounded, poorly sorted and Breed 1973). The group has been divided clasts of organically stained dolomicrite and into two formations and seven members, dolosiltite, cemented with isopachous silica, based principally on distinctive carbonate and and interlaminated organic-rich mats. VSMs sandstone marker beds (Fig. 1B) (Ford and are abundant in the mat horizons and also oc- Breed 1973). cur both within the clasts and as clasts them- The fossiliferous Awatubi and Walcott selves. Overlying the dolomite beds are ϳ130 Members of the Kwagunt Formation are ex- m of black shales interbedded with thin pi- posed in the Awatubi and Sixtymile Canyons, solitic units. The lowermost pisolite, ϳ1.5 m at the head of Carbon Canyon, and on the di- thick, was described as a white ‘‘oolite’’ by vide between Kwagunt and Nankoweap Can- Cook (1991) owing to its grain size and the yons, where a complete section can be fol- color of its siliceous cement, but is perhaps lowed up Nankoweap Butte (Fig. 1A). Above better understood as an oncolite in light of the a basal bed characterized by massive stromat- abundant cyanobacterial filaments found pre- olitic bioherms, the ϳ200–340-m-thick Awa- served within the coated grains (Schopf et al. tubi Member consists predominantly of shales 1973). Although VSMs are abundant in shales that yield filamentous bacteria (Horodyski associated with this unit (Bloeser 1985), they 1993), possible eukaryotic filaments (Horod- are not observed in the oncolite itself (Schopf yski and Bloeser 1983), acritarchs (including et al. 1973; this study). The overlying pisolites abundant Chuaria [C. Downie in an appendix have a black, iron-rich siliceous cement and to Ford and Breed 1969; Ford and Breed 1973; are present as thin beds or lenses (Cook 1991). Vidal and Ford 1985; Horodyski 1993]), and Two dolomite beds separated by 12 m of VSMs (Vidal and Ford 1985; Horodyski 1993). black shales form a bench approximately 130 The overlying Walcott Member is ϳ250 m m above the base of the Walcott Member thick and is predominantly composed of black (Cook 1991). The lower bed is 3.5 to 7 m thick shales (maximum total organic carbon [TOC] and has variable lithological features, includ- ϭ 9% [Palacas and Reynolds 1989; Cook ing wavy to broken algal laminations, ooids, 1991]) containing filamentous bacteria (Hor- and intraclasts (Cook 1991). The upper dolo- odyski 1993), Chuaria (Walcott 1899; Ford and mite is a massive, coarsely crystalline to mi- Breed 1973) and other acritarchs (C. Downie critic, 9- to 12-m-thick unit in which stylolites, in Ford and Breed 1969; Vidal and Ford 1985; calcite-filled fractures, vugs, and breccias are Horodyski 1993), and VSMs (Bloeser et al. common (Cook 1991). The dolomites are over- 1977; Bloeser 1985; Vidal and Ford 1985; Hor- lain by ϳ69 m of black organic-rich shales odyski 1993). The base of the Walcott Member which, at Nankoweap Butte, are directly over- 362 SUSANNAH M. PORTER AND ANDREW H. KNOLL

FIGURE 1. A, Geological map of the Chuar Group, northeastern Grand Canyon (modified from Link et al. 1993); NB ϭ Nankoweap Butte. B, Generalized stratigraphic column of the Chuar Group, indicating horizons where VSMs have been found (modified from C. Dehler unpublished data). Radiometric date from Karlstrom et al. 2000. NEOPROTEROZOIC TESTATE AMOEBAE 363

FIGURE 2. Thin-section of a carbonate nodule near the top of the Walcott Member, showing the abundance of VSMs. Scale bar, 1 mm. For this and all following images, sample or thin-section name and England Finder coordinates (where applicable) are given in parentheses; thin-section oriented such that the label is opposite the fixed corner. HUPC 62988 (AK10-53-13F–2B). lain by the Sixtymile Formation. Approxi- gested for Chuar Group deposition (Reynolds mately 15 m below this contact, the shales con- and Elston 1986), but several lines of evidence tain meter-scale early diagenetic dolomite indicate that marine conditions must have nodules, which in some cases preserve pre- predominated. Sedimentary structures and compacted shale laminations. An extraordi- facies relationships are consistent with marine nary abundance of VSMs (up to 4000/mm3)is deposition (Dehler and Elrick 1998). Perhaps observed within these nodules (Fig. 2), but no more compelling, Chuar sediments contain VSMs or acritarchs have been found in sur- VSMs and more than a dozen acritarch taxa rounding shales. In Sixtymile Canyon, a 12-m- (Bloeser 1985; Vidal and Ford 1985) that occur thick ‘‘karsted,’’ coarsely crystalline dolomite elsewhere in marine successions (e.g., Vidal unit occurs near the top of the Walcott Mem- 1979; Knoll and Vidal 1980; Knoll et al. 1989, ber (Cook 1991; C. Dehler personal commu- 1991). Indeed, the wide geographic distribu- nication 1999). tion of these fossils by itself suggests a marine environment, simply because most sedimen- Depositional Environment tary rocks are marine. In addition, carbon iso- Chuar Group sediments are thought to have tope data for the Chuar succession (Dehler et been deposited in a quiet marine embayment al. 1999) show high amplitude excursions sim- connected to the global ocean (Ford 1990; ilar in scale to those in the global curve con- Cook 1991; Dehler and Elrick 1998). Because structed from marine rocks of similar age of the high organic carbon content of the Wal- (Kaufman and Knoll 1995). Finally, Chuar cott Member shales, Cook (1991) envisioned a rocks locally contain high concentrations of silled basin comparable to the Black Sea, iron sulfides (Ford and Breed 1973). Walcott where restricted circulation generates anoxic black shales collected in outcrop are some- bottom waters. Alternatively, high TOC might what weathered, but they still have a mean py- be explained by high productivity related to rite iron content of 1% by weight, with indi- upwelling (Cook 1991). vidual samples containing up to 3% (Y. Shen A lacustrine environment has been sug- and D. Canfield personal communication 364 SUSANNAH M. PORTER AND ANDREW H. KNOLL

1999). Such pyrite abundances are commonly Vase-Shaped Microfossils in the Upper observed in marine sediments but are rare in Walcott Dolomite Nodules lakes (Berner and Raiswell 1984; Canfield and Morphology Raiswell 1991). The lowermost Awatubi sediments were de- Shape and Size. Like previously reported posited in shallow-subtidal to intertidal en- VSMs, the new Walcott fossils are cup- to tear- vironments, as documented by a basal stro- shaped tests, circular in transverse section, matolitic bioherm bed and the presence of that taper toward a single opening, often rimmed by a distinct collar, and flare out to- mudcracks, salt casts, and intercalated sand- ward a rounded or slightly pointed aboral stone units in overlying shales (Cook 1991; pole (Fig. 3). The population shows a wide Horodyski 1993; C. Dehler personal commu- variation in shape (Fig. 4; length/width ratios nication 1999). Relatively deeper water con- vary from 1.0/1 to 3.0/1) and in size (most in- ditions toward the end of Awatubi time are in- dividuals fall within 25 to 160 ␮m in length dicated by the absence of these features and and 15 to 105 ␮m in maximum width, al- rock types in the laminated, organic-rich though VSMs from silicified dolosiltites lower shales located higher in the member. Thinly in the Walcott Member can be up to 286 ␮min laminated black shales document subtidal de- length and 202 ␮m in maximum width). position during most of Walcott time, with mi- Symmetry. A majority of the Grand Can- nor intervals of shallow-subtidal to supratidal yon VSMs are radially symmetric, with the deposition, recorded by the ‘‘flaky dolomite’’ aperture directly opposite the aboral pole. and the ‘‘oncolite’’ beds, and massive to lam- Approximately 25%, however, are bilaterally inated and locally vuggy dolomites found symmetric, with a curved neck (Fig. 5) (Bloe- near the base and in the upper part of the suc- ser (1985), Vidal and Ford (1985), and Hor- cession (C. Dehler personal communication odyski (1993) also noted curvature in their 1999). The abundant VSMs found in the upper specimens from the Chuar Group). Both the Walcott Member appear to have accumulated general rigidity of the test (see below) and the in a quiet subtidal marine environment char- absence of any wrinkling in the concave part acterized by high rates of organic carbon burial. of the neck indicate that this curvature is bi- ological rather than the result of deformation. Age Curvature does not appear to correlate with The age of the uppermost VSM populations any other aspect of morphology—VSMs of is sharply constrained by a U-Pb zircon date widely ranging shapes and sizes are curved. of 742 Ϯ 7 Ma for an ash bed just above the Aperture and Operculum. Bloeser (1985) ob- fossiliferous dolomite nodules near the top of served distinctly shaped apertures in speci- the Walcott Member at Nankoweap Butte mens released from shales in the lower Wal- (Dehler et al. 1999; Karlstrom et al. 2000). VSMs lower in the succession are, of course, older, but probably not dramatically so. Car- bon isotopic profiles for the Awatubi and Wal- cott members support correlation with the up- per Little Dal and Coates Lakes Groups in the Mackenzie Mountains Supergroup, N.W.T., and their Shaler Group equivalents on Victo- ria Island (Link et al. 1993; Kaufman and Knoll 1995; Dehler et al. 1999)—successions constrained to be younger than 778 Ma (Rain- bird et al. 1996). Glaciogenic sediments in the Rapitan Group, which overlies the Mackenzie FIGURE 3. SEM image of a VSM, showing overall tear- Mountains Supergroup, are thought to be shaped morphology and circular aperture. HUPC 62989 Sturtian in age. (AK10-53-13A). Scale bar, 25 ␮m. NEOPROTEROZOIC TESTATE AMOEBAE 365

FIGURE 6. Triangular (A) and hexagonal (B) apertures. A, HUPC 62991 (AK10-53-13F–2A; S-61/1); scale bar, 15 ␮m. B, HUPC 62991 (AK10-53-13F–2A; T-65/1); scale bar, 25 ␮m.

cott Member. In fact, she used in part aperture FIGURE 4. Stout and elongate individuals within the upper Walcott VSM population. A, HUPC 62988 (AK10- margin shape as the basis for separating her 53-13F–2B; O-64/1). B, HUPC 62990 (AK10-53-13F–1; genus Melanocyrilllium into three species: M. G-57/3). Scale bar, 20 ␮m for both. hexodiadema (hexagonal), M. fimbriatum (trian- gular), and M. horodyskii (circular). A similar

FIGURE 5. VSMs with curved necks. A, HUPC 62990 (AK10-53-13F–1; F-56/3). B, HUPC 62988 (AK10-53-13F–2B; H-64/1). C, HUPC 62988 (AK10-53-13F–2B; H-57/4). Scale bar, 30 ␮m for all. 366 SUSANNAH M. PORTER AND ANDREW H. KNOLL

FIGURE 7. Preservation of the VSM wall. A, In dolomite nodules, the wall is preserved as a mineralized cast coated on one or both (shown here) sides with pyrite or iron oxide. HUPC 62988 (AK10-53-13F–2B; L62); scale bar, 30 ␮m. B, In silicified carbonates, the wall is sometimes preserved as a mineralized cast coated by organic material. HUPC 62992 (AK10-60-19-1; P-62/2); scale bar, 20 ␮m. C, A VSM from the same horizon studied by Bloeser (1985). Note mineralized cast coated by organic matter (arrow points to coated wall). HUPC 62993 (BL99; N-51); scale bar, 20 ␮m. D, Siliceous cast of a VSM from the uppermost Awatubi Member. Note agglutinated appearance of the wall. HUPC 62994 (AK10-53-3); scale bar, 25 ␮m. diversity of aperture types can be found in our (from Horodyski 1993, images courtesy of B. new population (Figs. 3, 6). Most apertures are Runnegar). The vesicles photographed by within 5 and 40 ␮m in maximum width, al- Horodyski are neither uniform in size nor reg- though some can be up to 65 ␮m. Bloeser ular in shape. Instead, they appear to be crys- (1985) also interpreted small plugs in the ap- talline precipitates coated with organic matter. ertures of a few specimens as opercula, but the In the absence of specimens that unambigu- irregular shapes of these features suggest that ously show the small vesicles to be both bio- they are of sedimentary origin. None of the genic and part of the VSM life cycle, we have many thousands of Grand Canyon VSMs ex- refrained from including this feature among amined for this study possesses an opercu- our list of systematically informative charac- lum. ters. Internal Vesicles. Horodyski (1987, 1993) Wall. The new VSM populations are pre- noted numerous ϳ10-␮m organic vesicles in- served as siliceous or calcareous casts coated side rare VSMs from Grand Canyon shales internally and/or externally with a thin ve- and interpreted them as a primary feature of neer of pyrite (based on EDX analysis) (Fig. the organism. We were unable to locate such 7A). Near nodule margins, the pyritic coating specimens in Horodyski’s collections but did has been oxidized to iron oxide. Wallthickness examine color transparencies he prepared is 1.0–3.0 ␮m (mean ϭ 1.8 ␮m; SD ϭ 0.6 ␮m; NEOPROTEROZOIC TESTATE AMOEBAE 367

FIGURE 8. Honeycomb VSMs. Note constant size and regular arrangement of perforations within a single test. A, HUPC 62988 (AK10-53-13F–2B; N-42/1). B–F, HUPC 62990 (AK10-53-13F–1). B, (P-44/2). C, (R-47). D, (K-51). E, (L- 61/4). F, (P-54/2). Scale bar, 50 ␮m for all. n ϭ 71). In some specimens the wall is broken ‘‘Honeycomb-Patterned’’ Wall. The most dis- but the test retains its shape, indicating rigid- tinctive character observed in the new Grand ity at the time of deformation. The absence of Canyon material is a ‘‘honeycomb-patterned’’ crushed or flattened specimens suggests that wall found in ϳ1% of VSMs from carbonate the original wall was sturdy. nodules (Fig. 8). The pattern consists of uni- VSMs found in shales and cherts lower in formly sized holes, where pyrite is absent, the succession may be coated by a thin layer regularly distributed in a pyritized wall. The of organic matter (Fig. 7B). Bloeser (1985) re- regular arrangement of the holes and the uni- ported organic-walled VSMs from Chuar formity of their size (within a single test) sug- shales, but in our preparations from the same gest that this pattern reflects a biological trait horizons and, we believe, the specimens she il- rather than diagenetic degradation. The aver- lustrated, the fossils occur as siliceous casts age diameter of holes from different tests coated with organic debris or iron precipi- ranges from 1.0 to 11.0 ␮m (mean ϭ 5.2 ␮m, tates. In thin-section, this is particularly evi- SD ϭ 2.5 ␮m; n ϭ 99), and is not significantly dent (Fig. 7C). Thus, in all specimens that we correlated with test length. Distributions of have examined from dolomite concretions, honeycomb test length and width are not sig- bedded dolosiltite, and carbonaceous shales nificantly different (p K 0.05) from those of of the Chuar Group, the original test wall is the larger population, and the range of test preserved as a mineral cast, predominantly shapes is comparable. silica, with or without a coating of pyrite, iron oxide, or organic matter. Taphonomy A small number of VSMs, found in shales How can one account for the pattern of pres- from the top of the Awatubi Member, appear ervation observed in Chuar VSM populations? to be siliceous casts with what might be inter- Noting the rigidity of test walls, Vidal (1994) preted as an agglutinated surface texture (Fig. proposed that they may originally have been 7D). Clearly, test composition in VSMs dif- mineralized in their entirety; however, the fered substantially from that of most other mi- variable preservation of walls as silica, carbon- crofossils in the Chuar Group. ate, or phosphate (see below) favors a diage- 368 SUSANNAH M. PORTER AND ANDREW H. KNOLL

FIGURE 9. The observed variation in VSM test preservation can be explained in terms of a small number of well- established taphonomic processes (see text for discussion). netic origin for most of the mineral content of ily degradable and therefore not readily pre- preserved VSMs. Petrological observations served. The honeycomb pattern further sug- bolster this view, revealing inwardly radiating gests that in some individuals the wall con- crystal fans nucleated on the outer edge of test tained regularly distributed mineralized walls in both siliceous and phosphatic speci- scales (as discussed below, preservation of the mens (Knoll and Vidal 1980). Instead, the honeycomb pattern in VSMs from other local- combination of three-dimensional preserva- ities shows that scales, rather than holes, were tion and pyritic coating suggests that test present in the test). walls were originally composed of organic Given these features, VSM preservation can material that was mechanically strong but eas- be explained as follows (Fig. 9): NEOPROTEROZOIC TESTATE AMOEBAE 369

1. The rigid tests were entombed in surface may be circular, triangular, or hexagonal; sediments made firm by penecontempora- there is no conclusive evidence for an oper- neous lithification (nodules) or microbially culum. Most tests are radially symmetric, but produced extracellular polymeric mole- a significant proportion are curved. A small cules (Krumbein et al. 1994). Ensuing de- number are distinguished by a honeycomb- cay of wall constituents left a void that was patterned wall that reflects the regular ar- filled by early diagenetic silica or carbon- rangement of mineralized scales in the test ate, forming a mineralized cast (Fig. 9A). A wall. The presence of internal vesicles has not mineralized internal mold could form if, at been convincingly demonstrated. There exists the time of deposition, the cell was still pre- some evidence for agglutinated VSMs. sent within the test, preventing infilling of sediment (Fig. 9B). VSMs from Other Localities 2. In iron-rich sediments, sulfate reduction as- Morphology and Taphonomy sociated with decomposition of the wall would produce an iron sulfide coating (Fig. Although the Grand Canyon population is 9C) (Canfield and Raiswell 1991). VSMs variable, the fossils are sufficiently similar in coated with iron sulfide are usually found shape and size to warrant placement within a in siliciclastic sediments, consistent with single morphological group, as Bloeser did in the fact that these facies contain relatively her . Other aperturate microfossils high concentrations of iron. Iron oxide reported from localities around the world (Ta- coatings would result from oxidation of the bles 1, 2) are also indistinguishable in size and iron sulfide coat, as inferred from the re- shape from the Grand Canyon population, striction of iron oxide VSMs to the outer and therefore we have included these fossils in rind of the upper Walcott dolomite nod- our analysis of VSMs and consider their bio- ules. logical relationships to approximate those of 3. If mineralized scales were embedded with- the Grand Canyon fossils. Many other Prote- in the organic test, then during decompo- rozoic and Cambrian microfossils have shapes sition, iron sulfide precipitation would that are broadly tear- or vaselike; however, preferentially occur in association with the these have been excluded from the present organic matrix rather than with the scales analysis either because they do not resemble (Fig. 9D). In the absence of such precipita- the Grand Canyon population or because they tion, the honeycomb pattern would be dif- are not preserved or illustrated sufficiently ficult to discern. This helps to explain why well to allow confident attribution (Table 1). honeycomb walls are readily identifiable in Table 2 lists the localities and the salient upper Walcott dolomite nodules but not in characters of all confirmed (see Table 1) VSM silicified carbonates lower in the succes- populations, along with a summary of char- sion. Furthermore, because honeycomb acters for the combined, global population of VSMs account for ϳ1% of upper Walcott VSMs. (Only well-documented characters are specimens, they might be missed in the included; features such as opercula and at- much smaller sample populations recov- tached VSMs have been reported but not con- ered from silicified carbonates and shales. vincingly demonstrated.) Some features of the Grand Canyon populations are found in all Summary other populations, including size range, over- Combining morphological and taphonomic all morphology, and mode of preservation. observations, we can summarize the system- (Note that reexamination of specimens re- atically informative features of Grand Canyon portedly composed of resistant organic matter VSMs. VSMs represent degradation-prone, or- [Knoll and Calder 1983] showed them to be ganic-walled tests that vary widely in size and mineralized casts coated with organic debris.) shape. The test walls are 1–3 ␮m thick and rig- Other characters exhibited by the Grand Can- idly constructed. Apertures are small (not yon population, in particular, neck curvature flaring), may be collared, and their margins and honeycomb pattern, occur in some but not 370 SUSANNAH M. PORTER AND ANDREW H. KNOLL

TABLE 1. Localities where vasiform microfossils have been found.

I. Microfossils that are comparable in shape and size to the Grand Canyon VSMs and to each other, and thus are included in this analysis. Visingso¨ Beds, Sweden (Ewetz 1933; Knoll and Vidal 1980) Eleonore Bay Group, East Greenland (Vidal 1979; Green et al. 1988) Jabal Rockham, Saudi Arabia (Binda and Bokhari 1980) Rysso¨ Formation, Nordaustlandet (Knoll and Calder 1983) Chatkaragai Suite, Tien Shan, Russia (Kraskov 1985; Yankaouskas 1989) Togari Group, Tasmania (Saito et al. 1988) Backlundtoppen Formation, Spitsbergen (Knoll et al. 1989) Draken Conglomerate Formation, Spitsbergen (Knoll et al. 1991) Elbobreen Formation, Spitsbergen (A. H. Knoll unpublished observations) Pahrump Group, southeast California (Horodyski 1993) II. Microfossils that do not resemble the Grand Canyon (and other VSM) populations, and the reason for their exclusion. Xihaoping Formation, China (Duan 1985):300–800 ␮m in length , China (Duan and Cao 1989):lack of stable morphology in the population Dengying Formation, China (Zhang and Li 1991; Zhang 1994):600–2400 ␮m in length Tongying Formation, China (Duan et al. 1993):300–800 ␮m in length Yuanjiaping section, China (Cao et al. 1995):no evidence for a wall, lack of stable morphology within the population III. Microfossils that are not preserved or illustrated sufficiently to allow confident attribution. Jacadigo Group, southwest Brazil (Fairchild et al. 1978) Simla Slates, Satpuli, India (Nautiyal 1978) Tanafjorden Group, Norway (Vidal and Siedlecka 1983) Vindhyan Supergroup, India (Maithy and Babu 1988) Tindir Group, northwest Canada (Allison and Awramik 1989) Uinta Mountain Group, Utah (Link et al. 1993) Upper Min‘yar Formation, southern Urals (Maslov et al. 1994) Vaishnodevi Limestone, Himalaya, India (Venkatachala and Kumar 1998) all other populations. This may in part reflect there had been regularly distributed holes in lower specimen concentrations in other local- the matrix rather than mineralized scales, ities, as these characters are exhibited by a mi- casts with the opposite relief (which have not nority of the Grand Canyon VSMs. VSMs from been observed) would be expected. the Chatkaragai Suite, Tien Shan, deserve spe- A few populations exhibit characters that cial mention because they are preserved as are not observed in the Grand Canyon speci- mineralized molds that exhibit a clear hon- mens. These include a larger length/width ra- eycomb pattern as raised circles (Fig. 10) tio of individual tests (5:1) and apertures that (Kraskov 1985; Yankaouskas 1989). Like the are crenulate in shape, both observed in the holes in pyritized honeycomb VSMs, to which Tien Shan specimens (Kraskov 1985; Yanka- they correspond, the circles are regularly ar- ouskas 1989; L. Kraskov personal communi- ranged and uniform in diameter within a sin- cation 1999) (Fig. 10A). gle individual. (VSM casts from the Eleonore Bay Group, East Greenland, also have holes, Biostratigraphic Range but their irregular size and arrangement in- No other VSM populations are as sharply dicate that they are diagenetic in origin [Vidal constrained by radiometric ages as the Chuar 1979].) The raised knobs on the Chatkaragai fossils, but all fall within the same broad molds provide particularly strong evidence stratigraphic window. VSMs have not been re- for the presence of regularly arranged scales ported from paleontologically rich and well- (rather than holes) in VSM test walls. The studied late Mesoproterozoic or early Neopro- voids left by the dissolution of the scales are terozoic successions such as the ϳ850-Ma Mi- represented in the mineralized cast by the royedikha Formation or the Ͼ1000-Ma Lak- raised knobs, and the surrounding matrix is handa and Turukhansk Groups, in Siberia cast as the depressions between those knobs (German 1990; Sergeev et al. 1997). Nor have (Kraskov 1985; Yankaouskas 1989) (Fig. 10). If VSMs been discovered in rocks that postdate NEOPROTEROZOIC TESTATE AMOEBAE 371 1983; none do. dal 1980 ser 1985; Vidal and Ford 1985; Horodyski 1993; this study 1988; S. Xiao andPorter S. unpublished M. data this study observations ouskas 1989; L. N. Kraskov personal com- munication 1999 Ewetz 1933; Knoll and Vi- Bloeser et al. 1977; Bloe- Knoll and Calder Knoll et al. 1991 Binda and Bokhari 1980 Kraskov 1985; Yanka- Knoll et al. 1989 debris, iron with coats of with iron ox- Mode of preservation References mite internal and phosphatic casts and molds ceous casts and molds organic sulfide, or iron ox- ide (some material reinterpreted) and/or molds, coated with organic debris or iron com- pounds ceous casts molds molds ide coats molds molds dolo siliceous casts Saito et al. 1988 not con- clusive not con- clusive forms Attached no iron oxide coats Vidal 1979; Green et al. netic holes yes no mineralized casts no no siliceous, calcareous, yes no calcareous and sili- m m m ␮ ␮ ␮ thick thick, sturdy thick, sturdy 2 1–3 ϳ but not conclu- sive vesicles Wall Honeycomb Internal reported yes no not noted diage- no notno noted no not notedno no noted but not noted nono nono calcareous and sili- not noted noted but not noted noyes no 1–3 no no siliceous internal siliceous cast Horodyski 1993 no not noted no no iron oxide coat A. H. Knoll unpublished no not noted yes no mineralized internal no not noted no no mineralized internal Aperture; operculum gular, and cir- cular aper- tures; opercu- lum reported but not con- clusive not noted; no operculum ture; no oper- culum not noted; operculum noted but not conclusive tures; no oper- culum not noted; operculum noted but not conclusive not noted; no operculum not noted; no operculum gular, circular, and crenulate apertures not noted; no operculum tures; no oper- culum ture; no oper- culum ture yes hexagonal, trian- no aperture shape no aperture shape no hexagonal aper- no aperture shape no aperture shape no aperture shape yes hexagonal, trian- Curva- m m m m m m m m m; m m nom circular aper- m m m m m ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ ␮ l/w ratios Size range; 3.0/1 l: 60–130 l: 40–120 w: 17–202 l: 25–286 l: 35–160 l: 34–257 l: 64–230 l: 40–140 l: 25–286 l: 50–265 l/w: 1.0/1 to l/w: 1.0–5.0/1 w: 25–62 w: 25–105 not noted yes aperture shape w: 16–119 l/w: up to 5.0/1 yes crenulate aper- not noted no hexagonal aper- w: 50–150 w: 30–80 w: 30–105 for- Spits- lomer- ¨ Beds, Swe- 2. Characters of VSM populations from eleven localities; ‘‘yes’’ means at least some members of the population have the character, ‘‘no’’ means Locality ¨ Formation, Grand Canyon, Arizona Group, East Greenland den di Arabia Formation, Spitsbergen Nordaustlandet Tien Shan, Russia bergen Formation, southeast Cali nia mania ate Formation, Spitsbergen ABLE T Chuar Group, Eleonore Bay Jabal Rockham, Sau- Elbobreen Visingso Chatkaragai Suite, Backlundtoppen Pahrump Group, Rysso Summary w: 17–202 Togari Group, Tas- Draken Cong 372 SUSANNAH M. PORTER AND ANDREW H. KNOLL

FIGURE 10. VSMs preserved as internal molds from the Chatkaragai Suite, Tien Shan. Note crenulate aperture (ar- row) molded as indentations parallel to test axis (A), and raised knobs covering specimens in both A and B. Scale bar in A, 100 ␮m; in B, 40 ␮m. Courtesy of L. Kraskov.

Varanger or Marinoan glaciation—although just before the Sturtian ice age. Several consid- their mode of preservation in shallow marine erations, however, call this correlation into environments would have become increasing- question, not least, U-Pb dates of 660 Ϯ 15 Ma ly difficult to achieve as bioturbating animals on granites that intrude immediately sub-Var- evolved. anger successions in the southern Urals (Se- VSMs are known from pre-Sturtian correl- mikhatov 1991). Sturtian glacial rocks are atives of the Chuar Group elsewhere in west- poorly dated but appear to be older than 700 ern North America (just below the tillite-bear- Ma and younger than the 742 Ϯ 7 Ma age of ing Kingston Peak Formation of the Pahrump the upper Chuar Group; the most direct age Group [Horodyski 1993]), and they occur, as constraint comes from U-Pb zircon dates of well, in Tasmanian rocks inferred to predate 723ϩ16/ Ϫ 10 Ma recently reported from a Sturtian glaciation (Saito et al. 1988; Calver tuff within the Ghubrah diamictite, Oman 1998). In northern Europe and Greenland, (Brasier et al. 2000). Sturtian glaciogenic rocks have not been iden- Alternatively, the two Varanger tillites may tified with confidence, but VSM populations in document a Marinoan and a post-Marinoan the Visingso¨ Beds, Sweden (Ewetz 1933; Knoll glaciation (Kaufman et al. 1997; see also Vidal and Vidal 1980); the Upper Eleonore Bay and Siedlecka 1983; Nystuen and Siedlecka Group, central East Greenland (Vidal 1979); 1988; Vidal and Moczydłowska 1995). The and the upper Akademikerbreen Group, VSM-bearing Draken and Backlundtoppen Spitsbergen, and its equivalents in Nordaus- Formations in Spitsbergen (which both under- tlandet (Knoll and Calder 1983; Knoll et al. lie Varanger tillites [Knoll et al. 1989; 1991]) 1989, 1991) all lie below Varanger tillites. The contain acritarch assemblages found in post- Visingso¨ Beds contain diverse acritarchs that Sturtian but pre-Marinoan successions else- permit biostratigraphic correlation with the where (although the ranges of many acritarch upper Chuar Group (Vidal and Ford 1985). taxa are long or not well-constrained [Walter Kennedy et al. (1998) proposed that lower et al. 2000]), and they overlie a ␦13C excursion Varanger tillites in northern Europe correlate interpreted as a biogeochemical proxy for with the Sturtian tillite in western North Sturtian glaciation (Kaufman and Knoll 1995; America, and that upper Varanger tillites cor- Knoll 2000). Fossils that may be VSMs (see Ta- relate with the Marinoan tillite in Australia. If ble 1) have been reported from the demon- this view is correct, most or all VSMs fall with- strably post-Sturtian Tindir Group, northwest in a relatively narrow stratigraphic interval Canada (Allison and Awramik 1989). NEOPROTEROZOIC TESTATE AMOEBAE 373

Thus, VSMs first appear in widely distrib- uted basins shortly before the Sturtian ice age, probably not much before 800 Ma. Global first appearances coincide with a shift in the ma- rine carbon isotopic record from a pattern of moderate secular variation (Ϫ1toϩ4 permil) to one of pronounced fluctuation (extreme val- ues of less than Ϫ4 and greater than 7 permil) that characterized the remainder of the Neo- Era. These fossils persist until the time of Sturtian glaciation (ϳ723 Ma) and may persist until the Marinoan ice age (ca. 610–590 Ma); they have not, however, been found in FIGURE 11. Nebela bohemica, a lobose testate , ex- uppermost Proterozoic successions. hibiting the tear-shaped test characteristic of many tes- tate amoebae. Scale bar, 20 ␮m. Courtesy of R. Meister- Biological Affinities of VSMs feld. Bloeser et al. (1977) initially interpreted VSMs as chitinozoans on the basis of similar- shales associated stratigraphically with VSMs ity in shape, but later rejected this idea be- (Summons et al. 1988), but there is no evi- cause of morphological differences between dence that the gammacerane and the micro- the two groups, including the presence of ap- fossils were produced by a single population. pendages and basal horns on many chitino- Other biomarkers and other fossils occur in zoan tests and the capacity of these younger the same unit. organisms to form chains and cocoons (Bloe- Of the gamut of opinion previously ex- ser 1985). Additionally, unlike VSMs, chitino- pressed, Schopf’s (1992) falls closest to the zoans are routinely preserved as resistant or- mark. The preserved vases are not cysts, but ganic structures—hence their name. With this we believe that their systematic affinities do lie in mind, Bloeser (1985) tentatively concluded with the lobose and filose testate amoebae that VSMs are algal cysts (phylogenetic rela- (Testacealobosea and Testaceafilosea, respec- tionships unspecified), based on the presence tively). Several lines of evidence support this of an operculum, their localized abundance, conclusion: and the absence of intermediate growth stag- es. The presence of an operculum is not con- 1. Only a limited number of protistan groups sidered here to be characteristic of VSMs, contain species that form vase-shaped tests however, and the other two features are not or loricae; of these, the testate amoebae by unique to algal cysts. Horodyski (1987, 1993) far most closely approximate the morphol- also suggested that VSMs might represent al- ogies observed in VSM populations. Like gal sporangia, but we cannot confirm the bi- VSMs, most testate amoebae have a tear- or ological nature of the internal vesicles on cup-shaped test with a relatively narrow which he based his interpretation. Morpholog- aperture (Fig. 11). Indeed, the range of ical and ecological similarities between tintin- shapes exhibited by VSMs is matched by nids and VSMs were noted by Fairchild et al. that of the testate amoebae. Short, wide (1978), Knoll and Vidal (1980), and Knoll and VSMs, for example, are closely similar to Calder (1983), but tintinnid loricae differ in members of the Difflugiidae (Testacealo- shape from VSM tests (often being most con- bosea), while pyriform VSMs are compa- stricted at the aboral pole and flaring out to- rable to members of the Nebelidae (Testa- ward the opening) and do not exhibit curva- cealobosea [Ogden and Hedley 1980; Bovee ture or aperture shapes comparable to those 1985a,b]). of VSMs (Small and Lynn 1985; Tappan 1993). 2. Although they can be as large as 600 ␮m Gammacerane, a biomarker compound linked (e.g., Difflugia pyriformis [Bovee 1985a]), tes- to ciliates, has been recovered from Chuar tate amoebae most commonly range be- 374 SUSANNAH M. PORTER AND ANDREW H. KNOLL

FIGURE 12. Triangular (A) and crenulate (B) apertures of testate amoebae. A, Trigonopyxis arcula; B, Difflugia corona. Scale bar, 20 ␮m for both. Courtesy of R. Meisterfeld.

tween 50 and 200 ␮m in length. Species [Testaceafilosea], and many members of smaller than 50 ␮m do exist and may be the Euglyphidae [Testaceafilosea] [Bovee present in high abundances (R. Meisterfeld 1985a,b]) (Fig. 13). In many species, cur- personal communication 1999; D. Patterson vature is a plastic character, occurring only personal communication 1999). We have when a physical barrier, such as a sand not observed an abundance of VSMs small- grain, obstructs test formation (R. Meister- er than 50 ␮m, but the range of VSM sizes feld personal communication 2000). is comparable to that of the testate amoe- 5. The structure and composition of testate bae. amoeban tests is comparable to that in- 3. The range of aperture shapes observed in ferred for VSMs: the tests are rigid, with VSMs is also found in testate amoebae (Fig. walls a few microns thick and composed of 12). Hexagonal apertures comparable to nonresistant (proteinaceous) organic ma- those of VSMs are exhibited by some mem- terial (Ogden and Hedley 1980). Iron and bers of the Arcellidae (Testacealobosea [R. manganese are often incorporated into the Meisterfeld personal communication 1999]), al- test, promoting pyritization in anoxic bot- though the latter possesses tests that are tom waters (Wolf 1995)—comparable to the wider and shorter than VSMs. Trigonopyxis, mode of preservation observed in the new a lobose testate amoeba, has a triangular Grand Canyon population. aperture like that found in many VSMs 6. A number of testate amoebae have agglu- (Fig. 12A; Bovee 1985a), and many mem- tinated tests, and others have tests in which bers of the Difflugiidae exhibit crenulate internally synthesized scales of silica are apertures like those found in VSMs from arrayed in a regular pattern. For example, the Chatkaragai Suite (Bovee 1985a; Kras- the tests of certain members of the Testa- kov 1985; Yankaouskas 1989) (Fig. 12B). ceafilosea (e.g., the Euglyphidae and the Many lobose and filose testate amoebae Cyphoderiidae) possess regularly ar- have circular apertures (Bovee 1985a,b). ranged imbricated siliceous scales (Fig. 14) 4. Test curvature identical to that found in whose bases are embedded in an organic VSMs is exhibited by members of both the matrix (Bovee 1985a; Ogden 1991). The ar- filose and lobose amoebae (for example, Po- rangement of these scale bases is similar to moriella in the Paraquadrulidae [Testacea- the pattern observed in honeycomb VSMs, lobosea], Cyphoderia in the Cyphoderiidae and the range of scale sizes (ϳ1 ␮mindi- NEOPROTEROZOIC TESTATE AMOEBAE 375

‘‘plugged’’ with sedimentary material (Og- den and Hedley 1980; S. Bamforth personal communication 1999), consistent with the interpretation, favored here, that the ‘‘oper- cula’’ observed in some VSMs (Bloeser 1985) represent sedimentary debris. 8. While the biogenic origin of the internal vesicles observed by Horodyski (1993) is in doubt, it nevertheless should be noted that, during adverse conditions, testate amoebae can form a spheroidal internal cyst (Ogden and Hedley 1980).

In summary, living testate amoebae can ac- count for the full range of morphological and taphonomic features observed in VSM popu- lations from the Chuar Group and elsewhere.

FIGURE 13. Testate amoebae with curved necks. A, Cy- Indeed, a number of living testate amoebae phoderia ampulla, and B, Nebela retorta. Scale bar in A, 20 would produce fossils indistinguishable from ␮m; in B, 30 ␮m. Courtesy of R. Meisterfeld. VSMs. Thus, testate amoebae provide strong candidates for VSM attribution. To determine whether the testate amoebae provide a unique- ly close fit with VSMs, it is necessary to look at other protists that produce aperturate tests, cysts, or loricae (Table 3). Clathrulina (Desmothoracida) possesses a comparably sized organic test, but the test is round, is covered with pores, lacks a differ- entiated aperture region, and attaches to the substrate by a stalk (Febvre-Chevalier 1985). Trachelomonas () possesses an aper- turate test, but the apertural opening is very small, and the test is round to elliptical in shape, lacks a differentiated ‘‘neck’’ region, FIGURE 14. The test of Euglypha tuberculata. Note reg- and exhibits neither curvature nor the variety ularly arranged siliceous scales. Scale bar, 20 ␮m. Cour- of aperture shapes seen in VSMs. In addition, tesy of R. Meisterfeld. Trachelomonas is smaller than most VSMs (gen- erally 20–50 ␮m), lacks mineralized scales, ameter in Cyphoderia ampulla [Bovee 1985a; and is often ornamented with pores, spines, or D. Patterson personal communication warts (Leedale 1985; Graham and Wilcox 1999]; to Ͼ10 ␮minSpenoderia lenta [Ogden 2000; D. Patterson personal communication and Hedley 1980]) matches that observed 1999). Some stramenopiles, such as Mallomon- for the honeycomb VSMs. Species of Nebela, as and Mallomonopsis, are covered with imbri- a lobose testate amoeba, also possess reg- cated siliceous scales, but the tests are usually ularly arranged siliceous scales, which they covered with long spines, lack a collar, do not acquire by consuming scale-producing fi- display curvature, have apertures much small- lose testate amoebae, sequestering the er in diameter than those of VSMs, and com- scales of their prey, and then incorporating monly break apart upon death (Hibberd and those scales into their own test (R. Meister- Leedale 1985; D. Patterson personal commu- feld personal communication 2000). nication 1999; P. Siver personal communica- 7. The tests of testate can become tion 1999). The tests of single-chambered fo- 376 SUSANNAH M. PORTER AND ANDREW H. KNOLL b,c m m ␮ ␮ ϩ 100 Peritrichs Ͻ with a stalk ) indicates that Ϫ a,c Graham and Wilcox 2000. j m 10–50 800 ␮ Ͻ ϪϪ m ␮ 200 to Folliculunids m ␮ c,e m 50–125 ␮ ?? ? 100–300 elaborate Tintinnids commonly collar often P. Siver, all personal communications 1999; i d,h m m 20–100 ␮ ␮ Ϫ 200 ϳ 1–80 D. Patterson, ϳ h Single-chambered feld, c,h,i m commonly ␮ R. Meister ? m g ␮ long spines 100 1 Mallomonas Ͻ ϳ usually with S. Bamforth, f c,h,j ) indicates that at least some members of the taxon possess the character, a ( m ϩ ␮ Ϫϩϩ ϩ m ␮ warts or spines on test 5 Tappan 1993; Trachelomonas Ͻ often with e c 6–10 ϳ m 20–50 ? ? ␮ Clathrulina m ␮ differentiated aperture 75 Loeblich and Tappan 1988; ϳ with a stalk; no d c,f,g,h m m pores are Lee et al. 1985; ␮ c ␮ ϩ Ϫϩ ϩ ϪϩϩϪ ϩϩ ϩ Ϫ ϪϪϪϪ ϩϩ ϩ Ϫϩ Ϫϩ ϪϪϪϪ ϩ ϪϪ ϩϪϪϩ ϩϩ ϪϩϪϪ ϪϪ Testate 200 amoebae 5–50 commonly 60– Hamilton 1952; b m ␮ VSM m characters 3. A comparison of VSMs with modern candidates. A ( ␮ ´-Fremiet 1936; Faure 160 mostly 5–40 organic scales contradict an affini- ty with VSMs a ABLE none do. Sac- or tear-shaped Apertural shapes T Length commonly 25– Nonresistant wall Rigid wall Curved neck Aperture diameter Collar Honeycomb: non- Other features that NEOPROTEROZOIC TESTATE AMOEBAE 377 raminifera, such as Allogromia and Lagynis, are Evolutionary Implications comparable in shape and size to Grand Can- What Does This Assemblage Represent? yon VSMs and are composed of a nonresistant organic material, but the tests are flexible, and Given the preceding comments, we might none have a curved neck, a collar, comparable interpret VSMs in several possible ways. Con- aperture shapes, or mineralized scales (Loe- ceivably, they could represent an extinct pro- blich and Tappan 1988; D. Patterson personal tist group that independently acquired attri- communication 1999). butes similar to those of the testate amoebae. Many ciliates possess loricae; those groups The strong similarity between the honeycomb most similar to VSMs are the tintinnids, the VSMs and the scale-bearing testate amoebae, folliculinids, and the peritrichs. Tintinnid lo- however, suggests that at least these VSMs ricae are commonly larger, however, and are represent testate amoebae, and it is not unrea- sonable to interpret the other VSMs as crown- differently shaped, with a flaring aperture or stem-group testate amoebae as well. If lo- and no curvature. In addition, they often have bose and filose testate amoebae constitute a elaborated collars (Small and Lynn 1985). The monophyletic group (a hypothesis defended tests of some folliculinids are comparable in by Cavalier-Smith [1993]), then at least some shape to VSMs and some are curved, but they VSMs could represent a stem group related to are flexible (Faure´-Fremiet 1936), lack com- the ancestors of both lobose and filose forms. parable aperture shapes, are without miner- If we use the taxonomy of modern testate alized scales, and are, for the most part, much amoebae as a guide, however, the morpholog- larger than VSMs (200 to Ͼ800 ␮m [Small and ical variation observed in the Chuar VSMs Lynn 1985]). The tests of the peritrichs Vagin- would suggest that the assemblage is com- icola and Cothurnia are similar in shape to posed not of one species, but a dozen or more VSMs, are composed of nonresistant organic that includes crown-group taxa (a diversity matter, and may be curved. However, they are only hinted at in the current taxonomic clas- Ͻ ␮ small ( 100 m) and flexible, lack mineral- sification of VSMs [Bloeser 1985]). ized scales, and attach to the substrate by a The most likely interpretation of these fos- stalk (Hamilton 1952; Small and Lynn 1985; D. sils, then, is that they are a multispecies as- Patterson personal communication 1999). semblage that includes both lobose and filose No single character uniquely links VSMs to testate amoebae, and, conceivably, other the testate amoebae; however, the combination groups as well. This is consistent with the ob- of characters exhibited by these fossils is served variability within the assemblage, and found only in the testate amoebae. We have with the fact that diverse filose and lobose tes- particular confidence that specimens marked tate amoebae commonly occur together in by honeycomb walls are related to scale-bear- modern environments (Bovee 1985b). Preser- ing testate amoebae, such as Euglypha. More vational association presumably would be the broadly, the range of morphologies found in result of a common habitat and taphonomic the upper Walcott dolomite nodules is selection for the test composition and/or matched by living species of both lobose and structure. filose amoebae (for example, Nebela, Cyphod- eria, and several genera in the Hyalosphenidae Implications for the Evolution of Testate [Bovee 1985a,b]). We cannot rule out the pos- Amoebae sibility that some of the most generalized mor- Several independent lines of evidence indi- phologies represent additional, unrelated di- cate that the Chuar Group is marine. Most versity, such as single-chambered foraminif- modern testate amoebae, however, live in era, but testate amoebae appear to dominate freshwater or terrestrial habitats. Thus, if our the assemblage. By extension, we believe that interpretations of both systematics and pa- morphologically comparable VSM popula- leoenvironment are correct, then testate amoe- tions found elsewhere are systematically com- bae must once have been more conspicuous in parable. marine environments than they are today. 378 SUSANNAH M. PORTER AND ANDREW H. KNOLL

This is not an unreasonable proposition, and Implications for Eukaryote Evolution it calls to mind groups such as ony- The Structure of the Eukaryotic Tree. A wide- chophorans and chelicerate arthropods that ly accepted view of eukaryote evolution has have similar histories. Testate amoebae are not been one in which a rapid crown radiation of absent from the marine realm today; several the major taxa was preceded by a longer his- groups, including members of both the Tes- tory during which more primitive taxa, such taceafilosea (Golemansky 1974; Bovee 1985a; as microsporidia, diplomonads, slime molds, Sudzuki 1979) and the Testacealobosea (Sud- and trichomonads, gradually diverged (Sogin zuki 1979; Bovee 1985b) inhabit marine envi- et al. 1986, 1989; Sogin 1991, 1994). That the ronments such as tidal pools and beach sands. earliest branching taxa lacked mitochondria, In molecular phylogenies of silica-secreting fi- chloroplasts, and, in the case of diplomonads, lose clades, the earliest branching species, Pau- even well-developed Golgi bodies supported linella chromatophora, lives in fresh- and brack- their basal position in the tree, and suggested ish-water conditions, but other spe- cies are marine (R. Meisterfeld personal com- that the principal metabolic organelles were munication 1999). acquired gradually, long after the origin of the Modern testate amoebae tend to live in hab- clade (Sogin et al. 1986, 1989). Recently, how- itats that are rich in organic matter (e.g., peat ever, this view, derived from phylogenies con- bogs, forest litter, humus [Bovee 1985a,b]), en- structed using small-subunit (SSU) rRNA se- vironments that would not have been repre- quences, has been questioned. Phylogenetic sented to any great extent on land before the trees based on other gene sequences place mid- radiation of land plants. Thus, some or all of these putatively basal taxa well like other heterotrophic groups that radiated within the crown-group eukaryotes (Baldauf on land, testate amoebae may have originated and Doolittle 1997; Philippe and Adoutte in marine environments and later expanded 1998). Comparison of rates of sequence evo- into terrestrial niches as land plants diversi- lution between and within lineages indicates fied. The narrow test aperture of testate amoe- the reason for the incongruence: SSU rRNA bae is considered to be an adaptation for life sequences in many of the early-branching taxa in episodically dry terrestrial habitats (Haus- have evolved at an unusually high rate (Phi- mann and Hu¨ lsmann 1996); the Chuar fossils, lippe and Adoutte 1998; Stiller et al. 1998; however, imply that this is an exaptation that Stiller and Hall 1999), suggesting that their facilitated but did not originate with the in- basal position may be an artifact of long- vasion of nonmarine environments. branch attraction (Felsenstein 1978). Further- VSMs are preserved in association with more, the discovery of mitochondria-like high concentrations of organic matter in tidal- genes in microsporidia (Germot et al. 1997; flat, lagoonal, and subtidal facies (Knoll and Hirt et al. 1997), diplomonads (Roger et al. Vidal 1980; Knoll et al. 1991; this study). By 1998), and trichomonads (Bui et al. 1996; Ger- analogy with modern testate amoeban ecolo- mot et al. 1996) may indicate that mitochon- gy, it is reasonable to assume that the tidal-flat dria were secondarily lost in these taxa, in- and lagoonal specimens lived in those envi- validating their status as primitively amito- ronments. It is unclear, however, whether the chondriate groups. (Similar genes occur in the VSMs from the subtidal facies are preserved in nuclear genome of the entamoebae, a group situ; there are examples of modern testate widely accepted as having lost its mitochon- amoebae that live in deep-water conditions dria [Clark and Roger 1995]). When correc- (Bovee 1985a,b), but the high concentration of tions for long-branch attraction are made, VSMs in these facies suggests that they may however, the free-living, amitochondriate Pe- have been transported. Indeed, transportation lobiontida (represented by the genus Masti- and winnowing of modern testate amoebae gamoeba) remains below the enlarged crown of tests can result in concentrations comparable the eukaryotic rDNA tree (Stiller et al. 1998; in magnitude to those observed for VSMs (R. Stiller and Hall 1999). Giardia and other diplo- Meisterfeld personal communication 1999). monads also retain their status as early NEOPROTEROZOIC TESTATE AMOEBAE 379

FIGURE 15. Summary of earliest eukaryotic fossil record (see text). B ϭ body fossils (preserved morphology), M ϭ molecular fossils (biomarkers). Sources of data noted in text. branching in a number of other gene trees (Drouin et al. 1995; Bhattacharya and Weber (Klenk et al. 1995; Baldauf and Doolittle 1997; 1997). Trees based on SSU rRNA indicate that Hashimoto et al. 1997; Stiller and Hall 1997; the filose testate amoebae (represented by Eu- Hilario and Gogarten 1998). Therefore, a re- glypha rotunda and Paulinella chromatophora) are vised view of the eukaryotic tree still supports derived from sarcomonads (Cavalier-Smith a rapid radiation, but with an evolving sense and Chao 1996/7) and that this group is close- of the nature and character of subtending ly related to the Chlorarachniophyta (Philippe branches. and Adoutte 1995; Cavalier-Smith and Chao The Position of Testate Amoebae in the Eukary- 1996/7; Medlin et al. 1997). These analyses do otic Tree. Testate amoebae are divided into not agree, however, on the relationship be- two groups on the basis of pseudopod shape: tween this larger clade and the other eukary- the filose testate amoebae (Testaceafilosea) otic lineages. Philippe and Adoutte (1995) and the lobose testate amoebae (Testacealo- place it in a sister relationship with the hap- bosa). The relationship between these two tophytes, basal to a clade containing the Chlo- groups is uncertain. Conventionally, they have robionta (green algae, plants), the Fungi, and been regarded as two distinct lineages that in- the Metazoa. Analyses by Cavalier-Smith and dependently acquired tests (e.g., Schuster Chao (1996/7) alternatively indicate that the 1990), but the common possession of rami- clade is nested high within the tree, as a sister cristate mitochondria (mitochondria with tu- to the , or that the clade is sister to bular cristae that branch) suggests a connec- a group that includes the Fungi, the Metazoa, tion between the two (Patterson 1994, 1999). the Chlorobionta, the , and the The relationships between the filose and lo- . Medlin et al. (1997) concluded that bose testate amoebae and other eukaryotes the clade constitutes a sister group of the het- are also unresolved. On the basis of their ram- erokonts and , and that together icristate mitochondria, Patterson (1999) united those clades are sister to the Chlorobionta. all testate amoeba into a phylum-level clade Thus, the details of phylogenetic relation- that also includes other amoebans, acantham- ships are yet to be confirmed, but all phylog- oebans, plasmodial slime molds, leptomyxids, enies place the testate amoebae well within and ; sequence comparisons of actin the ‘‘crown’’ of the eukaryotic tree. genes provide some support for this clade Constraining the Timing of Major Events in Eu- 380 SUSANNAH M. PORTER AND ANDREW H. KNOLL karyote Evolution. (See Fig. 15.) The earliest advanced by the mid-Neoproterozoic (Butter- morphological evidence for eukaryotes in- field et al. 1994); leiosphaerid acritarchs that cludes the spirally coiled megascopic alga go back to the beginning of the era may also from the ϳ1850-Ma (Hoffman 1987, be the phycomata of green phytoflagellates personal communication 1999) Negaunee (Tappan 1980). Iron-Formation, Michigan (Han and Runne- More generally, support for an early Neo- gar 1992), and large (40–200 ␮m) sphero- proterozoic/late Mesoproterozoic eukaryote morphic acritarchs from the 1900–1800-Ma radiation comes from the dramatic increase in Chuanlinggou Formation, China (Zhang the diversity of acritarchs (Vidal and Knoll 1986). Steranes, biomarker compounds char- 1983; Knoll 1994; Xiao et al. 1997) and carbo- acteristic of eukaryotes, are found in Ͼ2700- naceous compression fossils (Hofmann 1994) Ma shales from the Fortescue Group, north- at this time. Cellularly preserved remains like western Australia (Brocks et al. 1999). Neither the 1200-Ma bangiophytes are rare, but acri- the fossils nor the biomarkers can be assigned tarchs and carbonaceous compressions are to modern taxa, however, and some or all may suitably common to cast doubt on the inter- represent extinct stem-group eukaryotes. pretation of this record as a preservational ar- They nevertheless suggest that the eukaryote tifact. Exceptionally well-preserved fossil as- clade had originated by 2700 Ma, and that semblages have been found in Mesoprotero- some diversification had occurred by Paleo- zoic rocks, but none contains the diversity of proterozoic times. eukaryotic morphological or biomarker fossils By the late Mesoproterozoic/early Neopro- known from Neoproterozoic rocks. The diver- terozoic eras the divergence of modern eu- sification of multiple crown taxa at this time karyotic clades had begun. This is document- therefore reflects a real event, driven by in- ed by a growing inventory of fossils inter- trinsic innovations (sexual reproduction is preted as , including ban- commonly invoked) and/or extrinsic causes giophyte red algae from the 1204 Ϯ 22-Ma (e.g., an increase in atmospheric oxygen or (Kah et al. 1999; L. Kah personal communi- oceanic nitrate levels). cation 1999) Hunting Formation in arctic Can- Heterotrophy in Early Eukaryotic Evolution. ada (Butterfield et al. 1990; Butterfield this is- From the summary in the previous para- sue) and the stramenopile Paleovaucheria from graphs, it is evident that the Proterozoic fossil the Ͼ1000-Ma Lakhanda Group, eastern Si- record of eukaryotes is dominated by photo- beria (German 1990; Woods et al. 1998). Di- autotrophic groups. Nonetheless, in the over- nosterane, a biomarker whose parent sterol is all scheme of eukaryotic diversity, photosyn- synthesized by dinoflagellates, has been re- thetic clades are far outnumbered by respiring ported from the ϳ1100-Ma Nonesuch Forma- and, to a lesser extent, fermenting taxa. Pat- tion (Pratt et al. 1991), the ϳ800-Ma Bitter terson (1999) recognized 71 phylum-level Springs Formation, and the ϳ570-Ma Perta- clades of eukaryotic organisms. Of these, 62 tataka Formation (Summons and Walter 1990; contain only heterotrophs, 6 contain mostly or Summons et al. 1992; Moldowan et al. 1996); exclusively photosynthetic organisms, and 3 and microfossils with polygonal excystment include both autotrophic and heterotrophic structures in the 780–750-Ma Wyniatt For- subclades. Moreover, all algal protists ac- mation, arctic Canada, are plausibly if not un- quired their chloroplasts via endosymbiosis ambiguously interpreted as dinoflagellates (Gibbs 1992), so even the eukaryotic capacity (Butterfield and Rainbird 1998). Gammacera- for photosynthesis depends on the preexisting ne in shales of the Chuar Group indepen- ability to swallow particles. Indeed, it is the dently supports the presence of alveolates in physiological ability to ingest particles—born Neoproterozoic ecosystems, in this case cili- of a flexible membrane and cytoskeleton—that ates (Summons et al. 1988). Proterocladus, a gave eukaryotes an ecological foothold in bac- Cladophora-like alga from the ca. 750–700-Ma terial/archeal ecosystems; cell-ingesting preda- Svanbergfjellet Formation of Spitsbergen sug- tors added new dimensions to microbial eco- gests that green algal diversification was well systems (Knoll and Bambach in press). NEOPROTEROZOIC TESTATE AMOEBAE 381

If eukaryotic heterotrophs existed more cussed in Medioli et al. 1990a; Medioli et al. than 1200 million years ago (as inferred from 1990b; Poinar et al. 1993; Waggoner 1996; the presence of red algae in rocks of this age), Boeuf and Gilbert 1997), with questionable why are their earliest fossil representatives representatives found in Carboniferous rocks not found until ca. 800 Ma? Older hetero- (Vasicek and Ruzicka 1957; as discussed in trophs may not have had preservable struc- Medioli et al. 1990a; Wolf 1995). tures that were sufficiently diagnostic for us to The presence of testate amoebae in Neo- recognize them—many heterotrophic clades proterozoic rocks supports the inference, have no fossil record, and, conceivably, some drawn from molecular phylogenies in combi- Mesoproterozoic or earliest Neoproterozoic nation with the fossil record, that the major acritarchs could be the remains of heterotro- clades of eukaryotic organisms diverged from phic eukaryotes. one another and began to diversify during late Alternatively, older heterotrophic protists Mesoproterozoic/early Neoproterozoic time. may not have been diverse. A strong case can It also confirms the existence of additional eu- be made that global primary productivity was karyote clades in Neoproterozoic oceans. limited in Mesoproterozoic oceans and in- Most significantly, it provides the earliest creased thereafter (Brasier and Lindsay 1998; morphological evidence for heterotrophic eu- Anbar and Knoll 1999). The first appearance karyotes in marine ecosystems, thereby indi- of recognizable heterotrophs during the early cating that complex (multi-tiered) ecosystems to mid-Neoproterozoic Era coincides with an were in place by Neoproterozoic time. increase in acritarch diversity (Knoll 1994) as well as a major shift toward increased C-iso- Acknowledgments topic variability in the marine carbon cycle We thank the Grand Canyon research team, (Kaufman and Knoll 1995; documented local- and in particular K. Karlstrom, C. Dehler, M. ly in the Grand Canyon by Dehler et al. 1999). Timmons (all from the University of New Thus, while VSMs are not a proxy for the or- Mexico), and A. Weil (University of Michigan) igin of heterotrophy in eukaryotes, they may for many useful discussions about Chuar reflect a diversification of eukaryotic preda- Group geology. C. Dehler is also thanked for tors facilitated by increasing primary produc- sharing unpublished data and for many help- tivity—an event that may also have included ful comments on the manuscript. S. Bamforth other groups such as the microscopic ances- (Tulane University), W.Foissner (University of tors of fungi and animals. Salzburg), V. Golemansky (Bulgarian Acade- my of Sciences), J. Lipps (University of Cali- Conclusions fornia at Berkeley), R. Meisterfeld (Institute for New morphological observations and taph- Biology II, Aachen), F. Medioli and D. B. Scott onomic inferences derived from exceptionally (Dalhousie University), D. Patterson (Univer- preserved VSM populations in the upper sity of Sydney), L. Rothschild (NASA/Ames Chuar Group, Grand Canyon, support the hy- Research Center), P. Siver (Connecticut Col- pothesis that testate amoebae were wide- lege), M. Sogin and colleagues (Marine Bio- spread and relatively diverse constituents of logical Laboratories), and Anna Wasik (Nen- mid- to late Neoproterozoic marine ecosys- cki Institute of Experimental Biology, Warsaw) tems. The fossils appear to represent a multi- all provided useful advice and/or informa- species assemblage of filose and lobose testate tion on protistan biology. We also thank S. amoebae; VSMs with a distinct honeycomb- Awramik (University of California at Santa patterned wall are nearly identical to scale- Barbara), T. Fairchild (University of Sa˜ o Pau- bearing testate amoebae, such as Euglypha. lo), and B. Runnegar (University of California The presence of testate amoebae in marine at Los Angeles) for providing fossil material rocks of this age more than doubles their or photographs for comparative study, L. N. stratigraphic range, which until now extended Kraskov (VSEGEI, Russia) for providing pho- from the Recent to the Triassic (Cushman tographs for publication, D. Lange (Harvard 1930; Bradley 1931; Frenguelli 1933; as dis- University) for technical help, and S. Xiao 382 SUSANNAH M. PORTER AND ANDREW H. KNOLL

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