2000 The Paleontological Society. All rights reserved. 0094-8373/00/2603-0004/$1.00
Paleobiology, 26(3), 2000, pp. 360–385
Testate amoebae in the Neoproterozoic Era: evidence from
vase-shaped microfossils 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 testate amoebae. The fossils are tear-shaped
m long and
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 testateamoebae,
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 eukaryotes in marine
Susannah M. Porter and Andrew H. Knoll. Department of Organismic and Evolutionary Biology,Harvard
University, 26 Oxford Street, Cambridge, Massachusetts 02138. E-mail: firstname.lastname@example.org
Accepted: 1 March 2000
Molecular phylogenies indicate that the ma-
jor clades of the Eucarya, including animals,
fungi, green algae (and their descendants,
land plants), stramenopiles, red algae, and al-
veolates (ciliates, dinoﬂagellates, and apicom-
plexans), diverged during a relatively brief in-
terval of cladogenesis that substantially pre-
ceded the Cambrian diversiﬁcation of crown-
group metazoans (Gajadhar et al. 1991; Sogin
1991, 1994; Budin and Philippe 1998). The dis-
coveries of fossilized red algae (Butterﬁeld et
al. 1990, Butterﬁeld this issue) and multicel-
lular stramenopiles (German 1990; Woods et
al. 1998) in 1200–1000-Ma rocks place a min-
imum constraint on the timing of this diver-
gence and imply the existence of other eu-
karyotic clades in Neoproterozoic oceans.
Here we propose that vase-shaped microfos-
sils (genera Melanocyrillium Bloeser 1985 and
Caraburina Kraskov 1985) found widely in
Neoproterozoic rocks have afﬁnities with both
ﬁlose and lobose testate amoebae. This inter-
pretation contributes to the increasingly well
documented view that diversiﬁcation both
among and within eukaryotic clades was well
advanced by mid-Neoproterozoic time, and
provides the earliest morphological evidence
for heterotrophic eukaryotes.
Although abundant and globally distribut-
ed in Neoproterozoic rocks, vase-shaped mi-
crofossils (VSMs) have until now remained
problematic. First discovered by Ewetz (1933)
in phosphate nodules from the Visingso¨
Group, Sweden, these fossils have been vari-
ously interpreted as chitinozoans (Bloeser et
al. 1977), algal cysts (Bloeser 1985), algal spo-
rangia (Horodyski 1987, 1993), and heterotro-
phic, planktonic protists similar to tintinnids
(Fairchild et al. 1978; Knoll and Vidal 1980;
Knoll and Calder 1983). Schopf (1992) sug-
gested that VSMs may be the cysts of testate
amoebae, but did not provide detailed evi-
dence to support this conclusion. Our inter-
pretation of VSMs as testate amoebae is based
361NEOPROTEROZOIC TESTATE AMOEBAE
principally on newly discovered populations
in diagenetic dolomite nodules from the up-
permost Walcott Member of the Kwagunt For-
mation, Chuar Group, Grand Canyon. Both
abundant and exceptionally well preserved,
these fossils display hitherto unavailable (and
taxonomically informative) details of mor-
phology and test construction.
The Chuar Group, a 1600-m-thick succes-
sion of predominantly siltstone and mudstone
beds with subordinate sandstones and car-
bonates, is exposed over a
the northeastern part of the Grand Canyon,
cropping out in canyons formed by west-bank
tributaries of the Colorado River and bounded
to the east by the Butte Fault (Fig. 1A) (Ford
and Breed 1973). The group has been divided
into two formations and seven members,
based principally on distinctive carbonate and
sandstone marker beds (Fig. 1B) (Ford and
The fossiliferous Awatubi and Walcott
Members of the Kwagunt Formation are ex-
posed in the Awatubi and Sixtymile Canyons,
at the head of Carbon Canyon, and on the di-
vide between Kwagunt and Nankoweap Can-
yons, where a complete section can be fol-
lowed up Nankoweap Butte (Fig. 1A). Above
a basal bed characterized by massive stromat-
olitic bioherms, the
tubi Member consists predominantly of shales
that yield ﬁlamentous bacteria (Horodyski
1993), possible eukaryotic ﬁlaments (Horod-
yski and Bloeser 1983), acritarchs (including
abundant Chuaria [C. Downie in an appendix
to Ford and Breed 1969; Ford and Breed 1973;
Vidal and Ford 1985; Horodyski 1993]), and
VSMs (Vidal and Ford 1985; Horodyski 1993).
The overlying Walcott Member is
thick and is predominantly composed of black
shales (maximum total organic carbon [TOC]
9% [Palacas and Reynolds 1989; Cook
1991]) containing ﬁlamentous bacteria (Hor-
odyski 1993), Chuaria (Walcott 1899; Ford and
Breed 1973) and other acritarchs (C. Downie
in Ford and Breed 1969; Vidal and Ford 1985;
Horodyski 1993), and VSMs (Bloeser et al.
1977; Bloeser 1985; Vidal and Ford 1985; Hor-
odyski 1993). The base of the Walcott Member
is marked by a 3- to 10-m-thick dolomite unit
comprising three distinct lithofacies (Cook
1991): a lower dolomitic wackestone with in-
traclasts and nodular pisolitic chert; a dolo-
mite unit consisting of crinkled, folded, or
broken microbial laminations, in part siliciﬁed
(termed the ‘‘Flaky Dolomite’’ by Ford and
Breed 1973); and a wavy- to horizontally lam-
inated dolomicrite unit with cauliﬂower-like
chert in its upper 30 cm.
Directly overlying the basal Walcott dolo-
mite are Chuaria-bearing black shales (Walcott
1899; Ford and Breed 1973; this study) inter-
bedded with thin dolomicrite to dolosiltite
units containing mm-scale wavy laminae,
scattered early diagenetic chert nodules, and
local molar tooth(?) structures. The cherts pre-
serve moderately well rounded, poorly sorted
clasts of organically stained dolomicrite and
dolosiltite, cemented with isopachous silica,
and interlaminated organic-rich mats. VSMs
are abundant in the mat horizons and also oc-
cur both within the clasts and as clasts them-
selves. Overlying the dolomite beds are
m of black shales interbedded with thin pi-
solitic units. The lowermost pisolite,
thick, was described as a white ‘‘oolite’’ by
Cook (1991) owing to its grain size and the
color of its siliceous cement, but is perhaps
better understood as an oncolite in light of the
abundant cyanobacterial ﬁlaments found pre-
served within the coated grains (Schopf et al.
1973). Although VSMs are abundant in shales
associated with this unit (Bloeser 1985), they
are not observed in the oncolite itself (Schopf
et al. 1973; this study). The overlying pisolites
have a black, iron-rich siliceous cement and
are present as thin beds or lenses (Cook 1991).
Two dolomite beds separated by 12 m of
black shales form a bench approximately 130
m above the base of the Walcott Member
(Cook 1991). The lower bed is 3.5 to 7 m thick
and has variable lithological features, includ-
ing wavy to broken algal laminations, ooids,
and intraclasts (Cook 1991). The upper dolo-
mite is a massive, coarsely crystalline to mi-
critic, 9- to 12-m-thick unit in which stylolites,
calcite-ﬁlled fractures, vugs, and breccias are
common (Cook 1991). The dolomites are over-
69 m of black organic-rich shales
which, at Nankoweap Butte, are directly over-
362 SUSANNAH M. PORTER AND ANDREW H. KNOLL
1. A, Geological map of the Chuar Group, northeastern Grand Canyon (modiﬁed from Link et al. 1993);
Nankoweap Butte. B, Generalized stratigraphic column of the Chuar Group, indicating horizons whereVSMs
have been found (modiﬁed from C. Dehler unpublished data). Radiometric date from Karlstrom et al. 2000.
363NEOPROTEROZOIC TESTATE AMOEBAE
2. Thin-section of a carbonate nodule near the top of theWalcott 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-
mately 15 m below this contact, the shalescon-
tain meter-scale early diagenetic dolomite
nodules, which in some cases preserve pre-
compacted shale laminations. An extraordi-
nary abundance of VSMs (up to 4000/mm
observed within these nodules (Fig. 2), but no
VSMs or acritarchs have been found in sur-
rounding shales. In Sixtymile Canyon, a 12-m-
thick ‘‘karsted,’’ coarsely crystalline dolomite
unit occurs near the top of the Walcott Mem-
ber (Cook 1991; C. Dehler personal commu-
Chuar Group sediments are thought to have
been deposited in a quiet marine embayment
connected to the global ocean (Ford 1990;
Cook 1991; Dehler and Elrick 1998). Because
of the high organic carbon content of the Wal-
cott Member shales, Cook (1991) envisioned a
silled basin comparable to the Black Sea,
where restricted circulation generates anoxic
bottom waters. Alternatively, high TOC might
be explained by high productivity related to
upwelling (Cook 1991).
A lacustrine environment has been sug-
gested for Chuar Group deposition (Reynolds
and Elston 1986), but several lines of evidence
indicate that marine conditions must have
predominated. Sedimentary structures and
facies relationships are consistent with marine
deposition (Dehler and Elrick 1998). Perhaps
more compelling, Chuar sediments contain
VSMs and more than a dozen acritarch taxa
(Bloeser 1985; Vidal and Ford 1985) that occur
elsewhere in marine successions (e.g., Vidal
1979; Knoll and Vidal 1980; Knoll et al. 1989,
1991). Indeed, the wide geographic distribu-
tion of these fossils by itself suggests a marine
environment, simply because most sedimen-
tary rocks are marine. In addition, carbon iso-
tope data for the Chuar succession (Dehler et
al. 1999) show high amplitude excursions sim-
ilar in scale to those in the global curve con-
structed from marine rocks of similar age
(Kaufman and Knoll 1995). Finally, Chuar
rocks locally contain high concentrations of
iron sulﬁdes (Ford and Breed 1973). Walcott
black shales collected in outcrop are some-
what weathered, but they still have a mean py-
rite iron content of 1% by weight, with indi-
vidual samples containing up to 3% (Y. Shen
and D. Canﬁeld personal communication
364 SUSANNAH M. PORTER AND ANDREW H. KNOLL
3. SEM image of a VSM, showing overall tear-
shaped morphology and circular aperture. HUPC 62989
(AK10-53-13A). Scale bar, 25
1999). Such pyrite abundances are commonly
observed in marine sediments but are rare in
lakes (Berner and Raiswell 1984; Canﬁeld and
The lowermost Awatubi sediments were de-
posited in shallow-subtidal to intertidal en-
vironments, as documented by a basal stro-
matolitic bioherm bed and the presence of
mudcracks, salt casts, and intercalated sand-
stone units in overlying shales (Cook 1991;
Horodyski 1993; C. Dehler personal commu-
nication 1999). Relatively deeper water con-
ditions toward the end of Awatubi time are in-
dicated by the absence of these features and
rock types in the laminated, organic-rich
shales located higher in the member. Thinly
laminated black shales document subtidal de-
position during most of Walcott time, withmi-
nor intervals of shallow-subtidal to supratidal
deposition, recorded by the ‘‘ﬂaky dolomite’’
and the ‘‘oncolite’’ beds, and massive to lam-
inated and locally vuggy dolomites found
near the base and in the upper part of the suc-
cession (C. Dehler personal communication
1999). The abundant VSMs found in the upper
Walcott Member appear to have accumulated
in a quiet subtidal marine environment char-
acterized by high rates of organic carbon burial.
The age of the uppermost VSM populations
is sharply constrained by a U-Pb zircon date
7 Ma for an ash bed just above the
fossiliferous dolomite nodules near the top of
the Walcott Member at Nankoweap Butte
(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
Mountains Supergroup, are thought to be
Sturtian in age.
Vase-Shaped Microfossils in the Upper
Walcott Dolomite Nodules
Shape and Size. Like previously reported
VSMs, the new Walcott fossils are cup- to tear-
shaped tests, circular in transverse section,
that taper toward a single opening, often
rimmed by a distinct collar, and ﬂare out to-
ward a rounded or slightly pointed aboral
pole (Fig. 3). The population shows a wide
variation in shape (Fig. 4; length/width ratios
vary from 1.0/1 to 3.0/1) and in size (most in-
dividuals fall within 25 to 160
m in length
and 15 to 105
m in maximum width, al-
though VSMs from siliciﬁed dolosiltites lower
in the Walcott Member can be up to 286
length and 202
m in maximum width).
Symmetry. A majority of the Grand Can-
yon VSMs are radially symmetric, with the
aperture directly opposite the aboral pole.
Approximately 25%, however, are bilaterally
symmetric, with a curved neck (Fig. 5) (Bloe-
ser (1985), Vidal and Ford (1985), and Hor-
odyski (1993) also noted curvature in their
specimens from the Chuar Group). Both the
general rigidity of the test (see below) and the
absence of any wrinkling in the concave part
of the neck indicate that this curvature is bi-
ological rather than the result of deformation.
Curvature does not appear to correlate with
any other aspect of morphology—VSMs of
widely ranging shapes and sizes are curved.
Aperture and Operculum. Bloeser (1985) ob-
served distinctly shaped apertures in speci-
mens released from shales in the lower Wal-
365NEOPROTEROZOIC TESTATE AMOEBAE
4. Stout and elongate individuals within the
upper Walcott VSM population. A, HUPC 62988 (AK10-
53-13F–2B; O-64/1). B, HUPC 62990 (AK10-53-13F–1;
G-57/3). Scale bar, 20
m for both.
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
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.
cott Member. In fact, she used in part aperture
margin shape as the basis for separating her
genus Melanocyrilllium into three species: M.
hexodiadema (hexagonal), M. ﬁmbriatum (trian-
gular), and M. horodyskii (circular). A similar
366 SUSANNAH M. PORTER AND ANDREW H. KNOLL
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
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
diversity of aperture types can be found in our
new population (Figs. 3, 6). Most apertures are
within 5 and 40
m in maximum width, al-
though some can be up to 65
(1985) also interpreted small plugs in the ap-
ertures of a few specimens as opercula, but the
irregular shapes of these features suggest that
they are of sedimentary origin. None of the
many thousands of Grand Canyon VSMs ex-
amined for this study possesses an opercu-
Internal Vesicles. Horodyski (1987, 1993)
m organic vesicles in-
side rare VSMs from Grand Canyon shales
and interpreted them as a primary feature of
the organism. We were unable to locate such
specimens in Horodyski’s collections but did
examine color transparencies he prepared
(from Horodyski 1993, images courtesy of B.
Runnegar). The vesicles photographed by
Horodyski are neither uniform in size nor reg-
ular in shape. Instead, they appear to be crys-
talline precipitates coated with organic matter.
In the absence of specimens that unambigu-
ously show the small vesicles to be both bio-
genic and part of the VSM life cycle, we have
refrained from including this feature among
our list of systematically informative charac-
Wall. The new VSM populations are pre-
served as siliceous or calcareous casts coated
internally and/or externally with a thin ve-
neer of pyrite (based on EDX analysis) (Fig.
7A). Near nodule margins, the pyritic coating
has been oxidized to iron oxide. Wall thickness
367NEOPROTEROZOIC TESTATE AMOEBAE
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.
71). In some specimens the wall is broken
but the test retains its shape, indicating rigid-
ity at the time of deformation. The absence of
crushed or ﬂattened specimens suggests that
the original wall was sturdy.
VSMs found in shales and cherts lower in
the succession may be coated by a thin layer
of organic matter (Fig. 7B). Bloeser (1985) re-
ported organic-walled VSMs from Chuar
shales, but in our preparations from the same
horizons and, we believe, the specimensshe il-
lustrated, the fossils occur as siliceous casts
coated with organic debris or iron precipi-
tates. In thin-section, this is particularly evi-
dent (Fig. 7C). Thus, in all specimens that we
have examined from dolomite concretions,
bedded dolosiltite, and carbonaceous shales
of the Chuar Group, the original test wall is
preserved as a mineral cast, predominantly
silica, with or without a coating of pyrite, iron
oxide, or organic matter.
A small number of VSMs, found in shales
from the top of the Awatubi Member, appear
to be siliceous casts with what might be inter-
preted as an agglutinated surface texture (Fig.
7D). Clearly, test composition in VSMs dif-
fered substantially from that of most other mi-
crofossils in the Chuar Group.
‘‘Honeycomb-Patterned’’ Wall. The most dis-
tinctive character observed in the new Grand
Canyon material is a ‘‘honeycomb-patterned’’
wall found in
1% of VSMs from carbonate
nodules (Fig. 8). The pattern consists of uni-
formly sized holes, where pyrite is absent,
regularly distributed in a pyritized wall. The
regular arrangement of the holes and the uni-
formity of their size (within a single test) sug-
gest that this pattern reﬂects a biological trait
rather than diagenetic degradation. The aver-
age diameter of holes from different tests
ranges from 1.0 to 11.0
99), and is not signiﬁcantly
correlated with test length. Distributions of
honeycomb test length and width are not sig-
niﬁcantly different (p
0.05) from those of
the larger population, and the range of test
shapes is comparable.
How can one account for thepattern of pres-
ervation observed in Chuar VSM populations?
Noting the rigidity of test walls, Vidal (1994)
proposed that they may originally have been
mineralized in their entirety; however, the
variable preservation of walls as silica, carbon-
ate, or phosphate (see below) favors a diage-
368 SUSANNAH M. PORTER AND ANDREW H. KNOLL
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
preserved VSMs. Petrological observations
bolster this view, revealing inwardly radiating
crystal fans nucleated on the outer edge of test
walls in both siliceous and phosphatic speci-
mens (Knoll and Vidal 1980). Instead, the
combination of three-dimensional preserva-
tion and pyritic coating suggests that test
walls were originally composed of organic
material that was mechanically strong but eas-
ily degradable and therefore not readily pre-
served. The honeycomb pattern further sug-
gests that in some individuals the wall con-
tained regularly distributed mineralized
scales (as discussed below, preservation of the
honeycomb pattern in VSMs from other local-
ities shows that scales, rather than holes, were
present in the test).
Given these features, VSM preservation can
be explained as follows (Fig. 9):
369NEOPROTEROZOIC TESTATE AMOEBAE
1. The rigid tests were entombed in surface
sediments made ﬁrm by penecontempora-
neous lithiﬁcation (nodules) or microbially
produced extracellular polymeric mole-
cules (Krumbein et al. 1994). Ensuing de-
cay of wall constituents left a void that was
ﬁlled by early diagenetic silica or carbon-
ate, forming a mineralized cast (Fig. 9A). A
mineralized internal mold could form if, at
the time of deposition, the cell was still pre-
sent within the test, preventing inﬁlling of
sediment (Fig. 9B).
2. In iron-rich sediments, sulfate reductionas-
sociated with decomposition of the wall
would produce an iron sulﬁde coating (Fig.
9C) (Canﬁeld and Raiswell 1991). VSMs
coated with iron sulﬁde are usually found
in siliciclastic sediments, consistent with
the fact that these facies contain relatively
high concentrations of iron. Iron oxide
coatings would result from oxidation of the
iron sulﬁde coat, as inferred from the re-
striction of iron oxide VSMs to the outer
rind of the upper Walcott dolomite nod-
3. If mineralized scales were embedded with-
in the organic test, then during decompo-
sition, iron sulﬁde precipitation would
preferentially occur in association with the
organic matrix rather than with the scales
(Fig. 9D). In the absence of such precipita-
tion, the honeycomb pattern would be dif-
ﬁcult to discern. This helps to explain why
honeycomb walls are readily identiﬁable in
upper Walcott dolomite nodules but not in
siliciﬁed carbonates lower in the succes-
sion. Furthermore, because honeycomb
VSMs account for
1% of upper Walcott
specimens, they might be missed in the
much smaller sample populations recov-
ered from siliciﬁed carbonates and shales.
Combining morphological and taphonomic
observations, we can summarize the system-
atically informative features of Grand Canyon
VSMs. VSMs represent degradation-prone, or-
ganic-walled tests that vary widely in size and
shape. The test walls are 1–3
m thick and rig-
idly constructed. Apertures are small (not
ﬂaring), may be collared, and their margins
may be circular, triangular, or hexagonal;
there is no conclusive evidence for an oper-
culum. Most tests are radially symmetric, but
a signiﬁcant proportion are curved. A small
number are distinguished by a honeycomb-
patterned wall that reﬂects the regular ar-
rangement of mineralized scales in the test
wall. The presence of internal vesicles has not
been convincingly demonstrated. There exists
some evidence for agglutinated VSMs.
VSMs from Other Localities
Morphology and Taphonomy
Although the Grand Canyon population is
variable, the fossils are sufﬁciently similar in
shape and size to warrant placement within a
single morphological group, as Bloeser did in
her taxonomy. Other aperturate microfossils
reported from localities around the world (Ta-
bles 1, 2) are also indistinguishable in size and
shape from the Grand Canyon population,
and therefore we have included these fossilsin
our analysis of VSMs and consider their bio-
logical relationships to approximate those of
the Grand Canyon fossils. Many other Prote-
rozoic and Cambrian microfossils haveshapes
that are broadly tear- or vaselike; however,
these have been excluded from the present
analysis either because they do not resemble
the Grand Canyon population or because they
are not preserved or illustrated sufﬁciently
well to allow conﬁdent attribution (Table 1).
Table 2 lists the localities and the salient
characters of all conﬁrmed (see Table 1) VSM
populations, along with a summary of char-
acters for the combined, global population of
VSMs. (Only well-documented characters are
included; features such as opercula and at-
tached VSMs have been reported but not con-
vincingly demonstrated.) Some features of the
Grand Canyon populations are found in all
other populations, including size range, over-
all morphology, and mode of preservation.
(Note that reexamination of specimens re-
portedly composed of resistant organic matter
[Knoll and Calder 1983] showed them to be
mineralized casts coated with organic debris.)
Other characters exhibited by the Grand Can-
yon population, in particular, neck curvature
and honeycomb pattern, occur in some but not
370 SUSANNAH M. PORTER AND ANDREW H. KNOLL
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
Xihaoping Formation, China (Duan 1985):300–800
m in length
Doushantuo Formation, 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
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
lower specimen concentrations in other local-
ities, as these characters are exhibited by a mi-
nority of the Grand Canyon VSMs. VSMs from
the Chatkaragai Suite, Tien Shan, deservespe-
cial mention because they are preserved as
mineralized molds that exhibit a clear hon-
eycomb pattern as raised circles (Fig. 10)
(Kraskov 1985; Yankaouskas 1989). Like the
holes in pyritized honeycomb VSMs, to which
they correspond, the circles are regularly ar-
ranged and uniform in diameter within a sin-
gle individual. (VSM casts from the Eleonore
Bay Group, East Greenland, also have holes,
but their irregular size and arrangement in-
dicate that they are diagenetic in origin [Vidal
1979].) The raised knobs on the Chatkaragai
molds provide particularly strong evidence
for the presence of regularly arranged scales
(rather than holes) in VSM test walls. The
voids left by the dissolution of the scales are
represented in the mineralized cast by the
raised knobs, and the surrounding matrix is
cast as the depressions between those knobs
(Kraskov 1985; Yankaouskas 1989) (Fig. 10). If
there had been regularly distributed holes in
the matrix rather than mineralized scales,
casts with the opposite relief (which have not
been observed) would be expected.
A few populations exhibit characters that
are not observed in the Grand Canyon speci-
mens. These include a larger length/width ra-
tio of individual tests (5:1) and apertures that
are crenulate in shape, both observed in the
Tien Shan specimens (Kraskov 1985; Yanka-
ouskas 1989; L. Kraskov personal communi-
cation 1999) (Fig. 10A).
No other VSM populations are as sharply
constrained by radiometric ages as the Chuar
fossils, but all fall within the same broad
stratigraphic window. VSMs have not been re-
ported from paleontologically rich and well-
studied late Mesoproterozoic orearly Neopro-
terozoic successions such as the
royedikha Formation or the
handa and Turukhansk Groups, in Siberia
(German 1990; Sergeev et al. 1997). Nor have
VSMs been discovered in rocks that postdate
371NEOPROTEROZOIC TESTATE AMOEBAE
2. Characters of VSM populations from eleven localities; ‘‘yes’’ means at least some members of the population have the character, ‘‘no’’ means none do.
vesicles Wall Honeycomb
l/w: 1.0/1 to
yes hexagonal, trian-
gular, and cir-
but not con-
yes no calcareous and sili-
ceous casts and
molds with coats of
organic debris, iron
sulﬁde, or iron ox-
ide (some material
Bloeser et al. 1977; Bloe-
ser 1985; Vidal and
Ford 1985; Horodyski
1993; this study
Visingso¨ Beds, Swe-
no aperture shape
not noted; no
no no siliceous, calcareous,
casts and molds
Ewetz 1933; Knoll and Vi-
m no circular aper-
ture; no oper-
no not noted diage-
no iron oxide coats Vidal 1979; Green et al.
1988; S. Xiao and S. M.
Porter unpublished data
Jabal Rockham, Sau-
no aperture shape
noted but not
no not noted no noted but
molds with iron ox-
Binda and Bokhari 1980
no hexagonal aper-
tures; no oper-
no not noted no no calcareous and sili-
Knoll and Calder 1983;
l/w: up to 5.0/1 yes crenulate aper-
tures; no oper-
no not noted yes no mineralized internal
Kraskov 1985; Yanka-
ouskas 1989; L. N.
Kraskov personal com-
Togari Group, Tas-
no aperture shape
noted but not
no not noted no noted but
siliceous casts Saito et al. 1988
not noted no hexagonal aper-
ture; no oper-
no not noted no no mineralized internal
Knoll et al. 1989
no aperture shape
not noted; no
no not noted no no siliceous internal
Knoll et al. 1991
no aperture shape
not noted; no
no not noted no no siliceous cast Horodyski 1993
not noted yes aperture shape
not noted; no
no not noted no no iron oxide coat A. H. Knoll unpublished
Summary w: 17–202
yes hexagonal, trian-
yes no mineralized casts
coated with organic
debris or iron com-
372 SUSANNAH M. PORTER AND ANDREW H. KNOLL
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
their mode of preservation in shallow marine
environments would have become increasing-
ly difﬁcult to achieve as bioturbating animals
VSMs are known from pre-Sturtian correl-
atives of the Chuar Group elsewhere in west-
ern North America (just below the tillite-bear-
ing Kingston Peak Formation of the Pahrump
Group [Horodyski 1993]), and they occur, as
well, in Tasmanian rocks inferred to predate
Sturtian glaciation (Saito et al. 1988; Calver
1998). In northern Europe and Greenland,
Sturtian glaciogenic rocks have not been iden-
tiﬁed with conﬁdence, but VSM populations in
the Visingso¨ Beds, Sweden (Ewetz 1933; Knoll
and Vidal 1980); the Upper Eleonore Bay
Group, central East Greenland (Vidal 1979);
and the upper Akademikerbreen Group,
Spitsbergen, and its equivalents in Nordaus-
tlandet (Knoll and Calder 1983; Knoll et al.
1989, 1991) all lie below Varanger tillites. The
Visingso¨ Beds contain diverse acritarchs that
permit biostratigraphic correlation with the
upper Chuar Group (Vidal and Ford 1985).
Kennedy et al. (1998) proposed that lower
Varanger tillites in northern Europe correlate
with the Sturtian tillite in western North
America, and that upper Varanger tillites cor-
relate with the Marinoan tillite in Australia. If
this view is correct, most or all VSMs fall with-
in a relatively narrow stratigraphic interval
just before the Sturtian ice age. Several consid-
erations, however, call this correlation into
question, not least, U-Pb dates of 660
on granites that intrude immediately sub-Var-
anger successions in the southern Urals (Se-
mikhatov 1991). Sturtian glacial rocks are
poorly dated but appear to be older than 700
Ma and younger than the 742
7 Ma age of
the upper Chuar Group; the most direct age
constraint comes from U-Pb zircon dates of
10 Ma recently reported from a
tuff within the Ghubrah diamictite, Oman
(Brasier et al. 2000).
Alternatively, the two Varanger tillites may
document a Marinoan and a post-Marinoan
glaciation (Kaufman et al. 1997; see also Vidal
and Siedlecka 1983; Nystuen and Siedlecka
1988; Vidal and Moczydłowska 1995). The
VSM-bearing Draken and Backlundtoppen
Formations in Spitsbergen (which both under-
lie Varanger tillites [Knoll et al. 1989; 1991])
contain acritarch assemblages found in post-
Sturtian but pre-Marinoan successions else-
where (although the ranges of many acritarch
taxa are long or not well-constrained [Walter
et al. 2000]), and they overlie a
interpreted as a biogeochemical proxy for
Sturtian glaciation (Kaufman and Knoll 1995;
Knoll 2000). Fossils that may be VSMs (see Ta-
ble 1) have been reported from the demon-
strably post-Sturtian Tindir Group, northwest
Canada (Allison and Awramik 1989).
373NEOPROTEROZOIC TESTATE AMOEBAE
11. Nebela bohemica, a lobose testate amoeba, ex-
hibiting the tear-shaped test characteristic of many tes-
tate amoebae. Scale bar, 20
m. Courtesy of R. Meister-
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 (
to one of pronounced ﬂuctuation (extremeval-
ues of less than
4 and greater than 7 permil)
that characterized the remainder of the Neo-
proterozoic Era. These fossils persist until the
time of Sturtian glaciation (
723 Ma) and may
persist until the Marinoan ice age (ca. 610–590
Ma); they have not, however, been found in
uppermost Proterozoic successions.
Biological Afﬁnities of VSMs
Bloeser et al. (1977) initially interpreted
VSMs as chitinozoans on the basis of similar-
ity in shape, but later rejected this idea be-
cause of morphological differences between
the two groups, including the presence of ap-
pendages and basal horns on many chitino-
zoan tests and the capacity of these younger
organisms to form chains and cocoons (Bloe-
ser 1985). Additionally, unlike VSMs, chitino-
zoans are routinely preserved as resistant or-
ganic structures—hence their name. With this
in mind, Bloeser (1985) tentatively concluded
that VSMs are algal cysts (phylogenetic rela-
tionships unspeciﬁed), based on the presence
of an operculum, their localized abundance,
and the absence of intermediate growth stag-
es. The presence of an operculum is not con-
sidered here to be characteristic of VSMs,
however, and the other two features are not
unique to algal cysts. Horodyski (1987, 1993)
also suggested that VSMs might represent al-
gal sporangia, but we cannot conﬁrm the bi-
ological nature of the internal vesicles on
which he based his interpretation. Morpholog-
ical and ecological similarities between tintin-
nids and VSMs were noted by Fairchild et al.
(1978), Knoll and Vidal (1980), and Knoll and
Calder (1983), but tintinnid loricae differ in
shape from VSM tests (often being most con-
stricted at the aboral pole and ﬂaring out to-
ward the opening) and do not exhibit curva-
ture or aperture shapes comparable to those
of VSMs (Small and Lynn 1985; Tappan 1993).
Gammacerane, a biomarker compound linked
to ciliates, has been recovered from Chuar
shales associated stratigraphically with VSMs
(Summons et al. 1988), but there is no evi-
dence that the gammacerane and the micro-
fossils were produced by a single population.
Other biomarkers and other fossils occur in
the same unit.
Of the gamut of opinion previously ex-
pressed, Schopf’s (1992) falls closest to the
mark. The preserved vases are not cysts, but
we believe that their systematic afﬁnities dolie
with the lobose and ﬁlose testate amoebae
(Testacealobosea and Testaceaﬁlosea, respec-
tively). Several lines of evidence support this
1. Only a limited number of protistan groups
contain species that form vase-shaped tests
or loricae; of these, the testate amoebae by
far most closely approximate the morphol-
ogies observed in VSM populations. Like
VSMs, most testate amoebae have a tear- or
cup-shaped test with a relatively narrow
aperture (Fig. 11). Indeed, the range of
shapes exhibited by VSMs is matched by
that of the testate amoebae. Short, wide
VSMs, for example, are closely similar to
members of the Difﬂugiidae (Testacealo-
bosea), while pyriform VSMs are compa-
rable to members of the Nebelidae (Testa-
cealobosea [Ogden and Hedley 1980; Bovee
2. Although they can be as large as 600
(e.g., Difﬂugia pyriformis [Bovee 1985a]), tes-
tate amoebae most commonly range be-
374 SUSANNAH M. PORTER AND ANDREW H. KNOLL
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
smaller than 50
m do exist and may be
present in high abundances (R. Meisterfeld
personal communication 1999; D. Patterson
personal communication 1999). We have
not observed an abundance of VSMs small-
er than 50
m, but the range of VSM sizes
is comparable to that of the testate amoe-
3. The range of aperture shapes observed in
VSMs is also found in testate amoebae (Fig.
12). Hexagonal apertures comparable to
those of VSMs are exhibited by some mem-
bers of the Arcellidae (Testacealobosea [R.
Meisterfeld personal communication 1999]), al-
though the latter possesses tests that are
wider and shorter than VSMs. Trigonopyxis,
a lobose testate amoeba, has a triangular
aperture like that found in many VSMs
(Fig. 12A; Bovee 1985a), and many mem-
bers of the Difﬂugiidae exhibit crenulate
apertures like those found in VSMs from
the Chatkaragai Suite (Bovee 1985a; Kras-
kov 1985; Yankaouskas 1989) (Fig. 12B).
Many lobose and ﬁlose testate amoebae
have circular apertures (Bovee 1985a,b).
4. Test curvature identical to that found in
VSMs is exhibited by members of both the
ﬁlose and lobose amoebae (for example, Po-
moriella in the Paraquadrulidae [Testacea-
lobosea], Cyphoderia in the Cyphoderiidae
[Testaceaﬁlosea], and many members of
the Euglyphidae [Testaceaﬁlosea] [Bovee
1985a,b]) (Fig. 13). In many species, cur-
vature is a plastic character, occurring only
when a physical barrier, such as a sand
grain, obstructs test formation (R. Meister-
feld personal communication 2000).
5. The structure and composition of testate
amoeban tests is comparable to that in-
ferred for VSMs: the tests are rigid, with
walls a few microns thick and composed of
nonresistant (proteinaceous) organic ma-
terial (Ogden and Hedley 1980). Iron and
manganese are often incorporated into the
test, promoting pyritization in anoxic bot-
tom waters (Wolf 1995)—comparable to the
mode of preservation observed in the new
Grand Canyon population.
6. A number of testate amoebae have agglu-
tinated tests, and others have tests in which
internally synthesized scales of silica are
arrayed in a regular pattern. For example,
the tests of certain members of the Testa-
ceaﬁlosea (e.g., the Euglyphidae and the
Cyphoderiidae) possess regularly ar-
ranged imbricated siliceous scales (Fig. 14)
whose bases are embedded in an organic
matrix (Bovee 1985a; Ogden 1991). The ar-
rangement of these scale bases is similar to
the pattern observed in honeycomb VSMs,
and the range of scale sizes (
375NEOPROTEROZOIC TESTATE AMOEBAE
13. Testate amoebae with curved necks. A, Cy-
phoderia ampulla, and B, Nebela retorta. Scale bar in A, 20
m; in B, 30
m. Courtesy of R. Meisterfeld.
14. The test of Euglypha tuberculata. Note reg-
ularly arranged siliceous scales. Scale bar, 20
tesy of R. Meisterfeld.
ameter in Cyphoderia ampulla [Bovee 1985a;
D. Patterson personal communication
minSpenoderia lenta [Ogden
and Hedley 1980]) matches that observed
for the honeycomb VSMs. Species of Nebela,
a lobose testate amoeba, also possess reg-
ularly arranged siliceous scales, whichthey
acquire by consuming scale-producing ﬁ-
lose testate amoebae, sequestering the
scales of their prey, and then incorporating
those scales into their own test (R. Meister-
feld personal communication 2000).
7. The tests of testate amoebas can become
‘‘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.
Indeed, a number of living testate amoebae
would produce fossils indistinguishable from
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 (Euglenozoa) 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,
and exhibits neither curvature nor the variety
of aperture shapes seen in VSMs. In addition,
Trachelomonas is smaller than most VSMs (gen-
m), lacks mineralized scales,
and is often ornamented with pores, spines, or
warts (Leedale 1985; Graham and Wilcox
2000; D. Patterson personal communication
1999). Some stramenopiles, such as Mallomon-
as and Mallomonopsis, are covered with imbri-
cated siliceous scales, but the tests are usually
covered with long spines, lack a collar, do not
display curvature, have apertures much small-
er in diameter than those of VSMs, and com-
monly break apart upon death (Hibberd and
Leedale 1985; D. Patterson personal commu-
nication 1999; P. Siver personal communica-
tion 1999). The tests of single-chambered fo-
376 SUSANNAH M. PORTER AND ANDREW H. KNOLL
3. A comparison of VSMs with modern eukaryote candidates. A (
) indicates that at least some members of the taxon possess the character, a (
) indicates that
Length commonly 25–
Sac- or tear-shaped
1 2 2112 11
1 2 2222 11
m pores are
1 2 2222 22
1 2 1221 11
1 1 2122 22
Other features that
contradict an afﬁni-
ty with VSMs
with a stalk; no
with a stalk
Lee et al. 1985;
Loeblich and Tappan 1988;
P. Siver, all personal communications 1999;
Graham and Wilcox 2000.
377NEOPROTEROZOIC TESTATE AMOEBAE
raminifera, such as Allogromia and Lagynis, are
comparable in shape and size to Grand Can-
yon VSMs and are composed of a nonresistant
organic material, but the tests are ﬂexible, and
none have a curved neck, a collar, comparable
aperture shapes, or mineralized scales (Loe-
blich and Tappan 1988; D. Patterson personal
Many ciliates possess loricae; those groups
most similar to VSMs are the tintinnids, the
folliculinids, and the peritrichs. Tintinnid lo-
ricae are commonly larger, however, and are
differently shaped, with a ﬂaring aperture
and no curvature. In addition, they often have
elaborated collars (Small and Lynn 1985). The
tests of some folliculinids are comparable in
shape to VSMs and some are curved, but they
are ﬂexible (Faure´-Fremiet 1936), lack com-
parable aperture shapes, are without miner-
alized scales, and are, for the most part, much
larger than VSMs (200 to
m [Small and
Lynn 1985]). The tests of the peritrichs Vagin-
icola and Cothurnia are similar in shape to
VSMs, are composed of nonresistant organic
matter, and may be curved. However, they are
m) and ﬂexible, lack mineral-
ized scales, and attach to the substrate by a
stalk (Hamilton 1952; Small and Lynn 1985; D.
Patterson personal communication 1999).
No single character uniquely links VSMs to
the testate amoebae; however, the combination
of characters exhibited by these fossils is
found only in the testate amoebae. We have
particular conﬁdence that specimens marked
by honeycomb walls are related to scale-bear-
ing testate amoebae, such as Euglypha. More
broadly, the range of morphologies found in
the upper Walcott dolomite nodules is
matched by living species of both lobose and
ﬁlose amoebae (for example, Nebela, Cyphod-
eria, and several genera in the Hyalosphenidae
[Bovee 1985a,b]). We cannot rule out the pos-
sibility that some of the most generalizedmor-
phologies represent additional, unrelated di-
versity, such as single-chambered foraminif-
era, but testate amoebae appear to dominate
the assemblage. By extension, we believe that
morphologically comparable VSM popula-
tions found elsewhere are systematically com-
What Does This Assemblage Represent?
Given the preceding comments, we might
interpret VSMs in several possible ways. Con-
ceivably, they could represent an extinct pro-
tist group that independently acquired attri-
butes similar to those of the testate amoebae.
The strong similarity between the honeycomb
VSMs and the scale-bearing testate amoebae,
however, suggests that at least these VSMs
represent testate amoebae, and it is not unrea-
sonable to interpret the other VSMs as crown-
or stem-group testate amoebae as well. If lo-
bose and ﬁlose testate amoebae constitute a
monophyletic group (a hypothesis defended
by Cavalier-Smith ), then at least some
VSMs could represent a stem group related to
the ancestors of both lobose and ﬁlose forms.
If we use the taxonomy of modern testate
amoebae as a guide, however, the morpholog-
ical variation observed in the Chuar VSMs
would suggest that the assemblage is com-
posed not of one species, but a dozen or more
that includes crown-group taxa (a diversity
only hinted at in the current taxonomic clas-
siﬁcation of VSMs [Bloeser 1985]).
The most likely interpretation of these fos-
sils, then, is that they are a multispecies as-
semblage that includes both lobose and ﬁlose
testate amoebae, and, conceivably, other
groups as well. This is consistent with the ob-
served variability within the assemblage, and
with the fact that diverse ﬁlose and lobose tes-
tate amoebae commonly occur together in
modern environments (Bovee 1985b). Preser-
vational association presumably would be the
result of a common habitat and taphonomic
selection for the test composition and/or
Implications for the Evolution of Testate
Several independent lines of evidence indi-
cate that the Chuar Group is marine. Most
modern testate amoebae, however, live in
freshwater or terrestrial habitats. Thus, if our
interpretations of both systematics and pa-
leoenvironment are correct, then testateamoe-
bae must once have been more conspicuous in
marine environments than they are today.
378 SUSANNAH M. PORTER AND ANDREW H. KNOLL
This is not an unreasonable proposition, and
it calls to mind animal groups such as ony-
chophorans and chelicerate arthropods that
have similar histories. Testate amoebae are not
absent from the marine realm today; several
groups, including members of both the Tes-
taceaﬁlosea (Golemansky 1974; Bovee 1985a;
Sudzuki 1979) and the Testacealobosea (Sud-
zuki 1979; Bovee 1985b) inhabit marine envi-
ronments such as tidal pools and beach sands.
In molecular phylogenies of silica-secreting ﬁ-
lose clades, the earliest branching species,Pau-
linella chromatophora, lives in fresh- and brack-
ish-water conditions, but other Paulinella spe-
cies are marine (R. Meisterfeld personal com-
Modern testate amoebae tend to live in hab-
itats that are rich in organic matter (e.g., peat
bogs, forest litter, humus [Bovee 1985a,b]), en-
vironments that would not have been repre-
sented to any great extent on land before the
mid-Paleozoic radiation of land plants. Thus,
like other heterotrophic groups that radiated
on land, testate amoebae may have originated
in marine environments and later expanded
into terrestrial niches as land plants diversi-
ﬁed. The narrow test aperture of testate amoe-
bae is considered to be an adaptation for life
in episodically dry terrestrial habitats (Haus-
mann and Hu¨lsmann 1996); the Chuar fossils,
however, imply that this is an exaptation that
facilitated but did not originate with the in-
vasion of nonmarine environments.
VSMs are preserved in association with
high concentrations of organic matter in tidal-
ﬂat, lagoonal, and subtidal facies (Knoll and
Vidal 1980; Knoll et al. 1991; this study). By
analogy with modern testate amoeban ecolo-
gy, it is reasonable to assume that the tidal-ﬂat
and lagoonal specimens lived in those envi-
ronments. It is unclear, however, whether the
VSMs from the subtidal facies are preserved in
situ; there are examples of modern testate
amoebae that live in deep-water conditions
(Bovee 1985a,b), but the high concentration of
VSMs in these facies suggests that they may
have been transported. Indeed, transportation
and winnowing of modern testate amoebae
tests can result in concentrations comparable
in magnitude to those observed for VSMs (R.
Meisterfeld personal communication 1999).
Implications for Eukaryote Evolution
The Structure of the Eukaryotic Tree. A wide-
ly accepted view of eukaryote evolution has
been one in which a rapid crown radiation of
the major taxa was preceded by a longer his-
tory during which more primitive taxa, such
as microsporidia, diplomonads, slime molds,
and trichomonads, gradually diverged (Sogin
et al. 1986, 1989; Sogin 1991, 1994). That the
earliest branching taxa lacked mitochondria,
chloroplasts, and, in the case of diplomonads,
even well-developed Golgi bodies supported
their basal position in the tree, and suggested
that the principal metabolic organelles were
acquired gradually, long after the origin of the
clade (Sogin et al. 1986, 1989). Recently, how-
ever, this view, derived from phylogenies con-
structed using small-subunit (SSU) rRNA se-
quences, has been questioned. Phylogenetic
trees based on other gene sequences place
some or all of these putatively basal taxa well
within the crown-group eukaryotes (Baldauf
and Doolittle 1997; Philippe and Adoutte
1998). Comparison of rates of sequence evo-
lution between and within lineages indicates
the reason for the incongruence: SSU rRNA
sequences in many of the early-branching taxa
have evolved at an unusually high rate (Phi-
lippe and Adoutte 1998; Stiller et al. 1998;
Stiller and Hall 1999), suggesting that their
basal position may be an artifact of long-
branch attraction (Felsenstein 1978). Further-
more, the discovery of mitochondria-like
genes in microsporidia (Germot et al. 1997;
Hirt et al. 1997), diplomonads (Roger et al.
1998), and trichomonads (Bui et al. 1996; Ger-
mot et al. 1996) may indicate that mitochon-
dria were secondarily lost in these taxa, in-
validating their status as primitively amito-
chondriate groups. (Similar genes occur in the
nuclear genome of the entamoebae, a group
widely accepted as having lost its mitochon-
dria [Clark and Roger 1995]). When correc-
tions for long-branch attraction are made,
however, the free-living, amitochondriate Pe-
lobiontida (represented by the genus Masti-
gamoeba) remains below the enlarged crown of
the eukaryotic rDNA tree (Stiller et al. 1998;
Stiller and Hall 1999). Giardia and other diplo-
monads also retain their status as early
379NEOPROTEROZOIC TESTATE AMOEBAE
15. Summary of earliest eukaryotic fossil record (see text). B
body fossils (preserved morphology), M
molecular fossils (biomarkers). Sources of data noted in text.
branching in a number of other gene trees
(Klenk et al. 1995; Baldauf and Doolittle 1997;
Hashimoto et al. 1997; Stiller and Hall 1997;
Hilario and Gogarten 1998). Therefore, a re-
vised view of the eukaryotic tree still supports
a rapid radiation, but with an evolving sense
of the nature and character of subtending
The Position of Testate Amoebae in the Eukary-
otic Tree. Testate amoebae are divided into
two groups on the basis of pseudopod shape:
the ﬁlose testate amoebae (Testaceaﬁlosea)
and the lobose testate amoebae (Testacealo-
bosa). The relationship between these two
groups is uncertain. Conventionally,they have
been regarded as two distinct lineages that in-
dependently acquired tests (e.g., Schuster
1990), but the common possession of rami-
cristate mitochondria (mitochondria with tu-
bular cristae that branch) suggests a connec-
tion between the two (Patterson 1994, 1999).
The relationships between the ﬁlose and lo-
bose testate amoebae and other eukaryotes
are also unresolved. On the basis of their ram-
icristate mitochondria, Patterson (1999) united
all testate amoeba into a phylum-level clade
that also includes other amoebans, acantham-
oebans, plasmodial slime molds, leptomyxids,
and Gromia; sequence comparisons of actin
genes provide some support for this clade
(Drouin et al. 1995; Bhattacharya and Weber
1997). Trees based on SSU rRNA indicate that
the ﬁlose testate amoebae (represented by Eu-
glypha rotunda and Paulinella chromatophora) are
derived from sarcomonads (Cavalier-Smith
and Chao 1996/7) and that this group is close-
ly related to the Chlorarachniophyta (Philippe
and Adoutte 1995; Cavalier-Smith and Chao
1996/7; Medlin et al. 1997). These analyses do
not agree, however, on the relationship be-
tween this larger clade and the other eukary-
otic lineages. Philippe and Adoutte (1995)
place it in a sister relationship with the hap-
tophytes, basal to a clade containing the Chlo-
robionta (green algae, plants), the Fungi, and
the Metazoa. Analyses by Cavalier-Smith and
Chao (1996/7) alternatively indicate that the
clade is nested high within the tree, as a sister
to the heterokonts, or that the clade is sister to
a group that includes the Fungi, the Metazoa,
the Chlorobionta, the Amoebozoa, and the
Chromista. Medlin et al. (1997) concluded that
the clade constitutes a sister group of the het-
erokonts and alveolates, and that together
those clades are sister to the Chlorobionta.
Thus, the details of phylogenetic relation-
ships are yet to be conﬁrmed, but all phylog-
enies place the testate amoebae well within
the ‘‘crown’’ of the eukaryotic tree.
Constraining the Timing of Major Events in Eu-
380 SUSANNAH M. PORTER AND ANDREW H. KNOLL
karyote Evolution. (See Fig. 15.) The earliest
morphological evidence for eukaryotes in-
cludes the spirally coiled megascopic alga
Grypania from the
1850-Ma (Hoffman 1987,
personal communication 1999) Negaunee
Iron-Formation, Michigan (Han and Runne-
gar 1992), and large (40–200
morphic acritarchs from the 1900–1800-Ma
Chuanlinggou Formation, China (Zhang
1986). Steranes, biomarker compounds char-
acteristic of eukaryotes, are found in
Ma shales from the Fortescue Group, north-
western Australia (Brocks et al. 1999). Neither
the fossils nor the biomarkers can be assigned
to modern taxa, however, and some or all may
represent extinct stem-group eukaryotes.
They nevertheless suggest that the eukaryote
clade had originated by 2700 Ma, and that
some diversiﬁcation had occurred by Paleo-
By the late Mesoproterozoic/early Neopro-
terozoic eras the divergence of modern eu-
karyotic clades had begun. This is document-
ed by a growing inventory of fossils inter-
preted as crown eukaryotes, including ban-
giophyte red algae from the 1204
(Kah et al. 1999; L. Kah personal communi-
cation 1999) Hunting Formation in arctic Can-
ada (Butterﬁeld et al. 1990; Butterﬁeld this is-
sue) and the stramenopile Paleovaucheria from
1000-Ma Lakhanda Group, eastern Si-
beria (German 1990; Woods et al. 1998). Di-
nosterane, a biomarker whose parent sterol is
synthesized by dinoﬂagellates, has been re-
ported from the
1100-Ma Nonesuch Forma-
tion (Pratt et al. 1991), the
Springs Formation, and the
tataka Formation (Summons and Walter 1990;
Summons et al. 1992; Moldowan et al. 1996);
and microfossils with polygonal excystment
structures in the 780–750-Ma Wyniatt For-
mation, arctic Canada, are plausibly if not un-
ambiguously interpreted as dinoﬂagellates
(Butterﬁeld and Rainbird 1998). Gammacera-
ne in shales of the Chuar Group indepen-
dently supports the presence of alveolates in
Neoproterozoic ecosystems, in this case cili-
ates (Summons et al. 1988). Proterocladus, a
Cladophora-like alga from the ca. 750–700-Ma
Svanbergfjellet Formation of Spitsbergen sug-
gests that green algal diversiﬁcation was well
advanced by the mid-Neoproterozoic (Butter-
ﬁeld et al. 1994); leiosphaerid acritarchs that
go back to the beginning of the era may also
be the phycomata of green phytoﬂagellates
More generally, support for an early Neo-
proterozoic/late Mesoproterozoic eukaryote
radiation comes from the dramatic increase in
the diversity of acritarchs (Vidal and Knoll
1983; Knoll 1994; Xiao et al. 1997) and carbo-
naceous compression fossils (Hofmann 1994)
at this time. Cellularly preserved remains like
the 1200-Ma bangiophytes are rare, but acri-
tarchs and carbonaceous compressions are
suitably common to cast doubt on the inter-
pretation of this record as a preservational ar-
tifact. Exceptionally well-preserved fossil as-
semblages have been found in Mesoprotero-
zoic rocks, but none contains the diversity of
eukaryotic morphological or biomarker fossils
known from Neoproterozoic rocks. The diver-
siﬁcation of multiple crown taxa at this time
therefore reﬂects a real event, driven by in-
trinsic innovations (sexual reproduction is
commonly invoked) and/or extrinsic causes
(e.g., an increase in atmospheric oxygen or
oceanic nitrate levels).
Heterotrophy in Early Eukaryotic Evolution.
From the summary in the previous para-
graphs, it is evident that the Proterozoic fossil
record of eukaryotes is dominated by photo-
autotrophic groups. Nonetheless, in the over-
all scheme of eukaryotic diversity, photosyn-
thetic clades are far outnumbered by respiring
and, to a lesser extent, fermenting taxa. Pat-
terson (1999) recognized 71 phylum-level
clades of eukaryotic organisms. Of these, 62
contain only heterotrophs, 6 contain mostly or
exclusively photosynthetic organisms, and 3
include both autotrophic and heterotrophic
subclades. Moreover, all algal protists ac-
quired their chloroplasts via endosymbiosis
(Gibbs 1992), so even the eukaryotic capacity
for photosynthesis depends on the preexisting
ability to swallow particles. Indeed, it is the
physiological ability to ingest particles—born
of a ﬂexiblemembraneand cytoskeleton—that
gave eukaryotes an ecological foothold in bac-
terial/archeal ecosystems; cell-ingesting preda-
tors added new dimensions to microbial eco-
systems (Knoll and Bambach in press).
381NEOPROTEROZOIC TESTATE AMOEBAE
If eukaryotic heterotrophs existed more
than 1200 million years ago (as inferred from
the presence of red algae in rocks of this age),
why are their earliest fossil representatives
not found until ca. 800 Ma? Older hetero-
trophs may not have had preservable struc-
tures that were sufﬁciently diagnostic for us to
recognize them—many heterotrophic clades
have no fossil record, and, conceivably, some
Mesoproterozoic or earliest Neoproterozoic
acritarchs could be the remains of heterotro-
Alternatively, older heterotrophic protists
may not have been diverse. A strong case can
be made that global primary productivity was
limited in Mesoproterozoic oceans and in-
creased thereafter (Brasier and Lindsay 1998;
Anbar and Knoll 1999). The ﬁrst appearance
of recognizable heterotrophs during the early
to mid-Neoproterozoic Era coincides with an
increase in acritarch diversity (Knoll 1994) as
well as a major shift toward increased C-iso-
topic variability in the marine carbon cycle
(Kaufman and Knoll 1995; documented local-
ly in the Grand Canyon by Dehler et al. 1999).
Thus, while VSMs are not a proxy for the or-
igin of heterotrophy in eukaryotes, they may
reﬂect a diversiﬁcation of eukaryotic preda-
tors facilitated by increasing primary produc-
tivity—an event that may also have included
other groups such as the microscopic ances-
tors of fungi and animals.
New morphological observations and taph-
onomic inferences derived from exceptionally
preserved VSM populations in the upper
Chuar Group, Grand Canyon, support the hy-
pothesis that testate amoebae were wide-
spread and relatively diverse constituents of
mid- to late Neoproterozoic marine ecosys-
tems. The fossils appear to represent a multi-
species assemblage of ﬁlose and lobose testate
amoebae; VSMs with a distinct honeycomb-
patterned wall are nearly identical to scale-
bearing testate amoebae, such as Euglypha.
The presence of testate amoebae in marine
rocks of this age more than doubles their
stratigraphic range, which until now extended
from the Recent to the Triassic (Cushman
1930; Bradley 1931; Frenguelli 1933; as dis-
cussed in Medioli et al. 1990a; Medioli et al.
1990b; Poinar et al. 1993; Waggoner 1996;
Boeuf and Gilbert 1997), with questionable
representatives found in Carboniferous rocks
(Vasicek and Ruzicka 1957; as discussed in
Medioli et al. 1990a; Wolf 1995).
The presence of testate amoebae in Neo-
proterozoic rocks supports the inference,
drawn from molecular phylogenies in combi-
nation with the fossil record, that the major
clades of eukaryotic organisms diverged from
one another and began to diversify during late
Mesoproterozoic/early Neoproterozoic time.
It also conﬁrms the existence of additional eu-
karyote clades in Neoproterozoic oceans.
Most signiﬁcantly, it provides the earliest
morphological evidence for heterotrophic eu-
karyotes in marine ecosystems, thereby indi-
cating that complex (multi-tiered) ecosystems
were in place by Neoproterozoic time.
We thank the Grand Canyon research team,
and in particular K. Karlstrom, C. Dehler, M.
Timmons (all from the University of New
Mexico), and A. Weil (University of Michigan)
for many useful discussions about Chuar
Group geology. C. Dehler is also thanked for
sharing unpublished data and for many help-
ful comments on the manuscript. S. Bamforth
(Tulane University), W. Foissner (University of
Salzburg), V. Golemansky (Bulgarian Acade-
my of Sciences), J. Lipps (University of Cali-
fornia at Berkeley), R. Meisterfeld (Institute for
Biology II, Aachen), F. Medioli and D. B. Scott
(Dalhousie University), D. Patterson (Univer-
sity of Sydney), L. Rothschild (NASA/Ames
Research Center), P. Siver (Connecticut Col-
lege), M. Sogin and colleagues (Marine Bio-
logical Laboratories), and Anna Wasik (Nen-
cki Institute of Experimental Biology, Warsaw)
all provided useful advice and/or informa-
tion on protistan biology. We also thank S.
Awramik (University of California at Santa
Barbara), T. Fairchild (University of Sa˜ o Pau-
lo), and B. Runnegar (University of California
at Los Angeles) for providing fossil material
or photographs for comparative study, L. N.
Kraskov (VSEGEI, Russia) for providing pho-
tographs for publication, D. Lange (Harvard
University) for technical help, and S. Xiao
382 SUSANNAH M. PORTER AND ANDREW H. KNOLL
(Harvard University) for beneﬁcial discussion.
N. Butterﬁeld (University of Cambridge) and
R. Meisterfeld provided many valuable sug-
gestions for improvement of the manuscript.
Finally, we thank the National Park Service for
permission to conduct research and collect
samples within the Grand Canyon National
Park. This work was supported by the Nation-
al Science Foundation through grant EAR-
9706496 to Knoll and a graduate student fel-
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