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Testate Amoebae in the Neoproterozoic Era: Evidence from Vase-shaped Microfossils in the Chuar Group, Grand Canyon


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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 tests, ~20-300 µm long and ~10-200 µm wide, that are circular in transverse section, expand aborally 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 restate 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 eukaryotes in marine ecosystems. Organismic and Evolutionary Biology Version of Record
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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:
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
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.
Geological Setting
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
area in
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
Breed 1973).
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
200–340-m-thick Awa-
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
250 m
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,
1.5 m
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-
lain by
69 m of black organic-rich shales
which, at Nankoweap Butte, are directly over-
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.
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-
nication 1999).
Depositional Environment
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
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
Raiswell 1991).
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
of 742
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-
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
bar, 25
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
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
m. Bloeser
(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)
noted numerous
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
is 1.0–3.0
m (mean
m; SD
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
m (mean
m; n
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-
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):
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
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).
Biostratigraphic Range
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
850-Ma Mi-
royedikha Formation or the
1000-Ma Lak-
handa and Turukhansk Groups, in Siberia
(German 1990; Sergeev et al. 1997). Nor have
VSMs been discovered in rocks that postdate
2. Characters of VSM populations from eleven localities; ‘‘yes’’ means at least some members of the population have the character, ‘‘no’’ means none do.
Size range;
l/w ratios
vesicles Wall Honeycomb
Mode of
preservation References
Chuar Group,
Grand Canyon,
w: 17–202
l: 25–286
l/w: 1.0/1 to
yes hexagonal, trian-
gular, and cir-
cular aper-
tures; opercu-
lum reported
but not con-
but not
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-
w: 25–62
l: 60–130
no aperture shape
not noted; no
no no siliceous, calcareous,
and phosphatic
casts and molds
Ewetz 1933; Knoll and Vi-
dal 1980
Eleonore Bay
Group, East
l: 50–265
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-
di Arabia
w: 25–105
l: 35–160
no aperture shape
not noted;
noted but not
no not noted no noted but
not con-
dolomite internal
molds with iron ox-
ide coats
Binda and Bokhari 1980
Rysso¨ Formation,
w: 16–119
l: 34–257
no hexagonal aper-
tures; no oper-
no not noted no no calcareous and sili-
ceous casts
Knoll and Calder 1983;
this study
Chatkaragai Suite,
Tien Shan,
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-
munication 1999
Togari Group, Tas-
w: 30–80
l: 40–120
no aperture shape
not noted;
noted but not
no not noted no noted but
not con-
siliceous casts Saito et al. 1988
Formation, Spits-
not noted no hexagonal aper-
ture; no oper-
no not noted no no mineralized internal
Knoll et al. 1989
Draken Conglomer-
ate Formation,
w: 30–105
l: 64–230
no aperture shape
not noted; no
no not noted no no siliceous internal
Knoll et al. 1991
Pahrump Group,
southeast Califor-
w: 50–150
l: 40–140
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
l: 25–286
l/w: 1.0–5.0/1
yes hexagonal, trian-
gular, circular,
and crenulate
yes 1–3
yes no mineralized casts
and/or molds,
coated with organic
debris or iron com-
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
15 Ma
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
C excursion
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).
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 (
4 permil)
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, Schopfs (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-
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 (
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
m. Cour-
tesy of R. Meisterfeld.
ameter in Cyphoderia ampulla [Bovee 1985a;
D. Patterson personal communication
1999]; to
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-
erally 20–50
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-
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
none do.
Length commonly 25–
commonly 60–
m 20–50
m commonly
200 to
Sac- or tear-shaped
1 2 2112 11
Nonresistant wall
Rigid wall
Curved neck
1 2 2222 11
Aperture diameter
mostly 5–40
m pores are
m 20–100
m 50–125
m 10–50
Apertural shapes
1 2 2222 22
1 2 1221 11
Honeycomb: non-
organic scales
1 1 2122 22
Other features that
contradict an affini-
ty with VSMs
with a stalk; no
often with
warts or
spines on
usually with
long spines
collar often
with a stalk
Faure´ -Fremiet1936;
Hamilton 1952;
Lee et al. 1985;
Loeblich and Tappan 1988;
Tappan 1993;
S. Bamforth,
R. Meisterfeld,
D. Patterson,
P. Siver, all personal communications 1999;
Graham and Wilcox 2000.
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
communication 1999).
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
small (
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-
Evolutionary Implications
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 [1993]), 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.
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-
munication 1999).
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
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-
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
m) sphero-
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-
proterozoic times.
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
800-Ma Bitter
Springs Formation, and the
570-Ma Perta-
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
(Tappan 1980).
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).
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-
phic eukaryotes.
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
(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-
lowship to Porter, and by the Conoco Corpo-
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... van Hengstrum et al., 2007); they have also been found in marine settings in the Paleozoic and Precambrian (e.g. Porter and Knoll, 2000;Kumar et al., 2011) and possibly coinhabited both fresh and marine water environments in the Mesozoic (see Singh et al., 2015, for full discussion). However, these documented marine occurrences are few in number (<4) and there is uncertainty in many cases about the marine context (e.g. ...
... However, these documented marine occurrences are few in number (<4) and there is uncertainty in many cases about the marine context (e.g. Porter and Knoll, 2000) with questions on how they could concurrently exist in both fresh and marine settings (Singh et al., 2015). However, many of these studies examine samples over a limited stratigraphic range and none have examined testate amoebae in pre-Cenozoic fluvio-deltaic successions ranging from fresh to brackish estuarine and the marine basin, or considered that testate amoebae maybe transport into marine basins despite several Holocene examples (e.g. ...
... As discussed, there are several Holocene studies that have documented the transported of testate amoebae into deep marine basins by river flood events (Babalola, 2009;Riveiros and Patterson, 2009;Laut et al., 2017). The oldest record of testate amoebae is from Neoproterozoic marine sediments of the Chuar Group, Grand Canyon, Arizona, where testate amoebae are described as vase-shaped microfossils (Porter and Knoll, 2000). Well-preserved testate amoebae (e.g. ...
Morphogroup and biofacies analyses were conducted on strata of the Upper Cretaceous (Turonian) Ferron-Notom delta, Central Utah, USA. This study investigates associations between ancient deltaic environments and foraminiferal and testate amoebae assemblages with an assessment of their ecological zonation based on their extant Holocene counterparts. Sixty-six hand samples of mudstone were collected from the three outcrops (Caineville North, Blue Hills, and Steamboat) along strike and dip profiles. Morphological analysis showed four morphogroups with eleven morphotypes of foraminifera and testate amoebae reflecting relationships between test morphology and depositional environment. Morphotypes were used to define the main biofacies of the study sections using cluster analysis. Due to a possible fossilization biases with the calcareous foraminiferal fraction, the biodiversity index is only used as relative measure in defining biofacies. Despite this possible taphonomic bias, the agglutinated foraminifera and testate amoebae morphogroups show plausible trends of salinity both among the outcrops and stratigraphically within each outcrop. The Blue Hills outcrop represents the most landward and thus the lowest salinity environment (tidally-influenced backwater), Steamboat is more coast proximal with a slightly higher salinity found in the delta front and the fluvial/estuarine environment, while the Caineville North outcrop represents the most coast proximal with the highest salinity of the prodelta/delta front and fluvial/estuarine environment. It appears that landward and coastward transport of tests led to a taphonomic bias. Nonetheless, the assemblages provide useful information that correlates with previously documented, lithofacies data. A salinity index, which is a ratio of trochospiral taxa versus testate amoebae taxa, provides a useful measure of coastal proximity that matches lithofacies trends. In addition, an Organic Matter (OM) index, which is the ratio of bolivinid-type taxa versus trochospiral taxa shows relationships with original probable OM content of the sediment inferred from the lithofacies and the plant macrofossils.
... One was the apatitic scale microfossil (ASM) from the Fifteenmile Group chert nodules (Fig. 8a-d) (~0.81 Ga, Canada) , which was interpreted as a biomineralizing eukaryote. The other novel class was the vase-shaped microfossil (VSM) preserved in the shales, carbonates and cherts that were deposited in the shallow marine (Mus and Moczydowska, 2000;Porter and Knoll, 2000). Exceptionally preserved VSMs were short-lived and globally distributed in the Tonian and Cryogenian successions (Fig. 2, Table 1), including the Kwagunt Formation of the Chuar Group (~0.74 Ga, Arizona) (Porter and Knoll, 2000) and the near-contemporaneous Callison Lake dolostone of the Mount Harper Group (~0.74 Ga, Canada) (Strauss et al., 2014), the Visingsö Formation (~0.80-0.70 ...
... The other novel class was the vase-shaped microfossil (VSM) preserved in the shales, carbonates and cherts that were deposited in the shallow marine (Mus and Moczydowska, 2000;Porter and Knoll, 2000). Exceptionally preserved VSMs were short-lived and globally distributed in the Tonian and Cryogenian successions (Fig. 2, Table 1), including the Kwagunt Formation of the Chuar Group (~0.74 Ga, Arizona) (Porter and Knoll, 2000) and the near-contemporaneous Callison Lake dolostone of the Mount Harper Group (~0.74 Ga, Canada) (Strauss et al., 2014), the Visingsö Formation (~0.80-0.70 Ga, Sweden) (Mus and Moczydowska, 2000), as well as the Urucum Formation (<749 ± 3 Ma, may be deposited at the Cryogenian, Brazil) (Morais et al., 2021). ...
... Ga, Sweden) (Mus and Moczydowska, 2000), as well as the Urucum Formation (<749 ± 3 Ma, may be deposited at the Cryogenian, Brazil) (Morais et al., 2021). Therefore, VSMs were widely suggested as ideal index fossils for the late Tonian sedimentary successions (Mus and Moczydowska, 2000;Porter and Knoll, 2000;Strauss et al., 2014). Based on the morphological and taphonomic features, VSMs were commonly interpreted as testate amoebae (Porter and Knoll, 2000;Porter et al., 2003). ...
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The emergence and diversity of eukaryotes that occurred in the Proterozoic Eon (2.5–0.541 Ga) are the most important parts of life’s evolution on the Earth. However, the critical time and key environmental factor(s) are still controversial. Herein, we systematically compiled the reported fossils and convinced biomarkers of Proterozoic eukaryotes. The Proterozoic eukaryotic evolutionary timeline was proposed to be divided into four stages: (1) ambiguous origin (2.3–1.8 Ga), (2) slow evolution (1.8–0.8 Ga), (3) diversity fluctuation (0.8–0.635 Ga), and (4) first radiation (0.635–0.541 Ga). As aerobic organisms, the emergence and evolution of eukaryotes have been proposed to be hindered by low oxygen levels during the Proterozoic Eon. However, increasing evidence suggests that the Proterozoic oxygen content might have been higher than 1% PAL (present atmospheric level) since 2.3 Ga, allowing eukaryotes, including early animals, to survive. The Great Oxygenation Event (GOE) did not wake up eukaryotes. The Mesoproterozoic Oxygenation Event (MOE) did not accelerate the evolution of eukaryotes. The first and rapid radiation of eukaryotes after the Neoproterozoic Oxygenation Event (NOE) and global glaciations acted like the opening of Pandora's box. Hence atmospheric oxygen alone cannot be invoked as the only control on the evolution of eukaryotes. Instead, we suggest that oceanic temperature may have been an important environmental factor that limited eukaryotic physiology until the end-Proterozoic.
... Putative metazoan body-fossil candidates of unknown affinity include Otavia antiqua (Brain et al. 2012), a 0.3 to 5 mm organism from the Otavi and Nama Group in the Okavuvu Formation in Namibia dated at 760 Ma; Eocyathispongia qiania (Yin et al. 2015), from the Doushantou Formation in central Guizhou, China dated at 600 Ma; and Thectardis avalonensis (Clapham et al. 2004), from the Mistaken Point and Drook Formations dated at 575 Ma. All of these have a proposed poriferan affinity, or alternatively are considered possible amoebae (Otavia), or are thought to represent a state (stage) of other known vendobionts (Thectardis) (Porter and Knoll 2000;Sperling et al. 2011;Antcliffe et al. 2014). ...
... Otavia is a 0.3 to 5 mm organism with openings of highly irregular morphology that are dispersed throughout the body. It has no clear anchoring point as would be expected for a sessile animal, and so its morphology most readily resembles a testate amoeba or even abiogenic calciphosphate grains (Porter and Knoll 2000;Antcliffe et al. 2014). Eocyathispongia is even smaller than Otavia, reaching only 1.2 mm across. ...
... Yet the folded shape is unlike sponges, as it unnecessarily minimizes the area available for feeding, and although the cavities are proposed as chambers (Yin et al. 2015), it lacks any obvious openings that might be true ostia as opposed to gaps in a testate amoeba (Botting and Muir 2018). Moreover, such "honeycomb" structures have been reported in a testate amoeba cast resulting from mineral precipitation (Porter and Knoll 2000). From the morphometric standpoint, there is only one specimen, which makes any further analysis of dimensions impossible. ...
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The timing of early animal evolution remains one of the biggest conundrums in biology. Molecular data suggest Porifera diverged from the metazoan lineage some 800 Ma to 650 Ma, which contrasts with the earliest widely accepted fossils of sponges at 535 Ma. However, the lack of criteria by which to recognize the earliest animals in the fossil record presents a challenge. The sponge body plan is unchanged since the early Cambrian, which makes a sponge-type animal a good candidate for the earliest fossils. Here we propose a method for identifying an organism as sponge grade by translating the sponge pump character into a quantifiable morphological trait. We show that the ratio between the two major components of the aquiferous system, the cross-sectional area of the osculum (OSA) and the surface area of the whole sponge (SA), is an effective metric of the pump character of extant sponges and that the slope of this ratio is distinct for three classes of Porifera: Demospongiae, Calcarea, and Hexactinellida. Furthermore, this metric is effective at distinguishing as sponges both extant taxa and fossils from two extremes of the Phanerozoic, the Cambrian and Paleogene. We tested this metric on the putative Ediacaran sponge Thectardis avalonensis from Mistaken Point, Newfoundland, and found Thectardis fits both with Cambrian sponges and with modern demosponges. These analyses show that the OSA/SA ratio is a reliable character by which to identify fossils as sponge grade, opening up exciting possibilities for classifying new fossils as sponges.
... Several fossil and extant organisms and structures are similar to Ostiosphaera from the Weng'an Biota in overall morphology and internal structures, for example the early Ediacaran unicellular Sporosphaera (Landon et al. 2019), certain vase-shaped microfossils and testate amoebae ( Fig. 3c) (Porter & Knoll 2000;Porter et al. 2003;Nikolaev et al. 2005;Lahr & Lopes 2009;Lahr et al. 2019), reproductive cysts of certain green algae (Fig. 3d), spore masses of cellular slime mold Fonticulida (Brown et al. 2009), co-occurring MCH embryo-like fossils (Yin & Zhu 2012;Xiao et al. 2014a;Cunningham et al. 2017b;Yin et al. 2020) or even sponge gemmules (Manconi & Pronzato 2015). To figure out the affinity of Ostiosphaera, we compare it with potential analogues as below. ...
... in general the maximum length of most testate amoebae, both extant and fossil, is less than 200 μm (Porter & Knoll 2000;Beyens & Meisterfeld 2001;Porter et al. 2003) and the test of amoebae is normally single-layered and only 2-5 μm in thickness (Porter & Knoll 2000;Beyens & Meisterfeld 2001;Porter et al. 2003). Hence, when compared with most testate amoebae, Ostiosphaera are larger in size and more complex in terms of envelope microstructure. ...
... in general the maximum length of most testate amoebae, both extant and fossil, is less than 200 μm (Porter & Knoll 2000;Beyens & Meisterfeld 2001;Porter et al. 2003) and the test of amoebae is normally single-layered and only 2-5 μm in thickness (Porter & Knoll 2000;Beyens & Meisterfeld 2001;Porter et al. 2003). Hence, when compared with most testate amoebae, Ostiosphaera are larger in size and more complex in terms of envelope microstructure. ...
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The embryo-like fossils from the early Ediacaran Weng'an Biota (Southwest China, 609 million years ago), widely interpreted as members of holozoans, potentially provide insights to understanding the early evolution of development of metazoans and the rise of the animal kingdom. However, the biodiversity of the embryo-like fossil assemblage is largely underestimated, and its more precise phylogenetic affinities within the holozoan tree are still under debate. Here we describe a new species of embryo-like fossil Ostiosphaera rara n. gen. n. sp. from the Ediacaran Weng'an Biota. These three-dimensional, phosphatized specimens exhibit a spherical morphology, an ornamented thick envelope with a circular opening and a membrane-bounded, multicellular inner body. In terms of biological characteristics, Ostiosphaera rara show similarities to a number of extant and fossil analogues including testate amoebae, unicellular green algae, cellular slime mold Fonticuida and co-occurring Weng'an embryo-like fossils. Although the phylogenetic affinity of Ostiosphaera rara is difficult to constrain very precisely based on the available evidence, it is reasonable to follow the holozoan interpretation for them, since that they share the same grade complexity with the co-occurring embryo-like fossils such as Megasphaera and Helicoforamina in terms of the combination of biological features. It is worth mentioning that the new holozoans resemble asexual reproductive gemmules of fossil and living demosponges in size, morphology, circular opening, and cellular anatomy. If the similarity between them reflects biological affinity rather than convergent evolution, this discovery would force us to rethink the evolutionary history of Precambrian sponges. Thematic collection: This article is part of the Advances in the Cambrian Explosion collection available at:
... Wörndle et al. 2019) and ending before the Cryogenian low-latitude glaciations (Cohen and Macdonald 2015). The increase in diversity at this time also includes the emergence of novel microfossil groups in addition to OWM, notably microorganisms capable of biomineralization, i.e. producing biomineralized components like tests and scales, that first appeared in the Tonian, starting around 810 Ma (Porter and Knoll 2000;Cohen et al. 2017). ...
... (16) Chuar Group, Arizona, USA . (17) Fifteenmile Formation, Yukon, Canada ). (18) Chuar Group, Arizona, USA (Porter and Knoll 2000). (19) See compilation of biomarker data from various Neoproterozoic units in Brocks et al. 2017;Hoshino et al. 2017. ...
The emergence of a complex, eukaryotic cell is one of the major steps in the evolution of life on Earth. Eukaryotic organisms include a range of macroscopic life such as animals, plants, fungi, as well as a plethora of microscopic single-celled or colonial protists. The first evidence for eukaryotic life appears in the geologic record around 1650 million years ago (Ma), as organic-walled microfossils—cellular vesicles often preserved as carbonaceous compressions in siliciclastic rocks. Early eukaryotes were predominantly single-celled and minute for about a billion years, until the onset of macroscopic multicellularity in algae and animals in the Ediacaran Period (635–538 Ma). Here I review the earliest evidence of eukaryotic life, including a range of Proterozoic organic-walled microfossils and problematica. These fossils contain a suite of morphological and geochemical characters that offer clues about their palaeobiology. Complex microfossil morphology like spines can be considered a proxy for the appearance of a cytoskeleton, very early in eukaryote history, in the late Paleoproterozoic. Multidisciplinary studies on fossil features such as cell morphology, cell wall ultrastructure and its chemical composition, ancient forms of multicellularity, as well as understanding the environments these microorganisms inhabited, enable the use of the fossil record to inform the timing and mode of eukaryogenesis.
... 古生 物学家在美国大峡谷距今约7.5亿年前的丘尔群(Chuar Group)中发现一类瓶状 微体化石,它与现今具骨骼的变形虫非常类似 [99] . 这就表明,单细胞的原生动物 至少在距今7.5亿年前已经发生了分异. ...
Complex multicellular eukaryotic organisms (or “complex organisms”), including animals, land plants, fungi, and macroalgae, have dominated the Earth’s biosphere since the Precambrian-Cambrian transition. Although fossil evidence and molecular clock estimates indicate that the early adaptive radiation of complex organisms occurred no later than the Ediacaran Period (~635−541 Ma) or even the Cryogenian Period (~720−635 Ma), the timing and dynamics of the evolutionary transitions from unicellular ancestors to different complex organisms remain largely elusive. This is mainly due to the deficiency of ancestral protists and transitional multicellular forms of complex organisms in the middle Proterozoic (~1800−800 Ma), a period commonly known as the “Boring Billion” for a billion years of apparent environmental quiescence and evolutionary stasis in Earth history. However, recent paleontological and molecular clock advances have suggested that complex organisms have a deep root in the “Boring Billion”. In this review paper, we systematically scrutinized available fossil records, molecular clock estimates, and phylogenetic data regarding to the evolutionary mechanisms from unicellular ancestors to complex organisms. We propose that eukaryotic multicellularity may have originated through incomplete reproduction, where reproductive cells failed to liberate but retained in mother cells for ecological benefits. In addition, symbiosis, such as parasitism and mutualism, and horizontal gene transfer may have occurred multiple times among prokaryotes and protists during the “Boring Billion”, leading to the advent of differentiated cells, such as reproductive cells. Furthermore, the persistently stratified waters and stable environment during the “Boring Billion” have provided an unprecedented avenue for the trials of these evolutionary innovations. Although it is difficult to directly test all these hypotheses, integrated paleontological, molecular clock, and phylogenetic data suggest that single-celled ancestors may have diverged into major groups of complex organisms in the “Boring Billion”, setting up the basis for their adaptive radiations in the Ediacaran and ecological dominance at the Precambrian-Cambrian transition. Therefore, the “Boring Billion” is not “boring” at all in the perspective of early life evolution, it fostered the earliest divergence of complex life. 复杂生物,包括动物、陆生植物、真菌和宏体藻类等,它们是寒武纪至现今地球生物圈的主体. 根据化石记录,地球生命自38亿年前起源以来,有近30亿年是单细胞的微体生物世界,直到距今10-8亿年的新元古代早期,复杂生命才开始出现. 成冰纪可靠的化石记录极少,我们对这近一亿年的生命史的认识非常有限. 在埃迪卡拉纪,丰富的化石记录和分子钟的估算结果均指示,复杂生物已经发生了适应辐射,也进一步诠释了达尔文的自然选择学说. 现如今,如果还以宏体复杂生物化石的大量出现来定义“显生宙”,它应该包括埃迪卡拉纪更为合适. 距今18-8亿年的元古宙中期是地质历史上持续时间最长、环境最为稳定的时期,生物演化也似乎处于停滞状态,被地质学家称为“枯燥的十亿年”. 本文根据现代生物学的研究进展,结合以往报道的化石资料,特别是对近年来中国一系列新化石的发现进行综合分析,认为:正是在这长期稳定的环境中,原生生物和原核生物悄然地发生了寄生、共生和基因转移等一系列相互作用,演化出了复杂生物各大类型的单细胞祖先,并进一步实现了多细胞化和细胞分化,“枯燥的十亿年”并不“枯燥”,它建造了复杂生命的根基.
... To trace the evolutionary patterns of Arcellinida, their general shell shape was compared with the Neoproterozoic vase shaped microfossils , which are considered as the oldest fully reliable eukaryotic fossils (Porter and Knoll, 2000). These comparisons seemed to confirm the hypothesis of phenotypic continuity between Precambrian fossils and extant forms, which was formulated based on the striking resemblance between fossils and extant species (Lahr, 2021;Porter et al., 2003). ...
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Protists, the micro-eukaryotes that are neither plants, animals nor fungi build up the greatest part of eukaryotic diversity on Earth. Yet, their evolutionary histories and patterns are still mostly ignored, and their complexity overlooked. Protists are often assumed to keep stable morphologies for long periods of time (morphological stasis). In this work, we test this paradigm taking Arcellinida testate amoebae as a model. We build a taxon-rich phylogeny based on two mitochondrial (COI and NADH) and one nuclear (SSU) gene, and reconstruct morphological evolution among clades. In addition, we prove the existence of mitochondrial mRNA editing for the COI gene. The trees show a lack of conservatism of shell outlines within the main clades, as well as a widespread occurrence of morphological convergences between far-related taxa. Our results refute, therefore, a widespread morphological stasis, which may be an artefact resulting from low taxon coverage. As a corollary, we also revise the groups systematics, notably by emending the large and highly polyphyletic genus Difflugia. These results lead, amongst others, to the erection of a new infraorder Cylindrothecina, as well as two new genera Cylindrifflugia and Golemanskia.
... This stands in contrast with the fact that most species described in Cyphoderia (14) are from freshwater environments, while only three species (Cyphoderia littoralis, Cyphoderia compressa, and Cyphoderia truncata [Schulze, 1875]) have been described from the marine environment. This knowledge gap demands more research on testate amoebae from marine ecosystems, a surprising fact given that current hypotheses based on fossils and phylogenomic reconstructions support a marine origin for the group (Porter et al., 2003;Porter & Knoll, 2000). ...
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The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the Mid Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.
... More recent works regard thecamoebians as an extremely resilient and conservative group with a long stratigraphic range and apparently very little morphological variation through time (e.g. Porter and Knoll 2000;Singh et al. 2015). ...
The Venta del Moro site (Valencia, Spain) is especially known for its rich content in Late Miocene fossil mammals but it also holds other fossil vertebrates, invertebrates and plants. A micropalaeontological study of a 70-cm long sedimentary section carried out in the lacustrine beds of the site has yielded 2 foraminifer, 7 thecamoebian and 19 ostracod taxa (two of these last probably new species). A constrained cluster analysis allowed the recognition of three consecutive zones based on their micropalaeontological content. The variation of the microfossil assemblages closely matches lithology and reflects environmental changes in a palaeolake that was subject to a gradual drying process. Anoxic bottom conditions at the base of the sequence (Zone 1) prevented the occurrence of aerobic benthic microorganisms. Later, the appearance of ostracods and thecamoebians (Zone 2) documents the increasing oxygenation of the lake and suggests a permanent freshwater to slightly brackish lake with low temperature and stream influence. The dominance of foraminifers and the changes in the thecamoebian and ostracod assemblages in the upper part of the sequence (Zone 3) reflect more stable and oxygenised conditions and probably increasing temperature and evaporation in oligohaline temporary waters, during the final drying phase of the lake
... Shells are also abundant in 1600 Mya sediments, however lack of ornamental diagnostic structures. Silica and calk tests of putative amoeba and simple foraminifera are found in the mid Neoproterozoic (Porter & Knoll, 2000). Nodules and vessels comparable to extant chrisophytes are dated 800Mya (Allison & Hilgert, 1986), before Ediacarian radiation. ...
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The origin and early evolution of eukaryotes are one of the major transitions in the evolution of life on earth. One of its most interesting aspects is the emergence of cellular organelles, their dynamics, their functions, and their divergence. Cell compartmentalization and architecture in prokaryotes is a less understood complex property. In eukaryotes it is related to cell size, specific genomic architecture, evolution of cell cycles, biogenesis of membranes and endosymbiotic processes. Explaining cell evolution through form and function demands an interdisciplinary approach focused on microbial diversity, phylogenetic and functional cell biology. Two centuries of views on eukaryotic origin have completed the disciplinary tools necessarily to answer these questions. We have moved from Haeckel SCALA NATURAE to the un-rooted tree of life. However, the major relations among cell domains are still elusive and keep the nature of eukaryotic ancestor enigmatic. Here we present a review on state of art views of eukaryogenesis; the background and perspectives of different disciplines involved in this topic
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The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid), and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. Ribosomal RNA trees show the myxogastrid and dictyostelid slime molds as unrelated early branching lineages, but actin and ß-tubulin trees place them together as a single coherent (monophyletic) group, closely related to the animal–fungal clade. We have sequenced the elongation factor-1a genes from one member of each division of the Mycetozoa, including Dictyostelium discoideum, for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, with the myxogastrid and dictyostelid slime molds most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals + fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four that place these taxa within the ‘‘crown’’ of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and we emphasize the importance of developing multiple sequence data sets.
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The phylogenetic relationships of the "golden algae", like all algae, were rarely addressed before the advent of electron microscopy because, based upon light microscopy, each group was so distinct that shared characters were not apparent. Electron microscopy has provided many new characters that have initiated phylogenetic discussions about the relationships among the "golden algae". Consequently, new taxa have been described or old ones revised, many of which now include non-algal protists and fungi. The haptophytes were first placed in the class Chrysophyceae but ultrastructural data have provided evidence to classify them separately. Molecular studies have greatly enhanced phylogenetic analyses based on morphology and have led to the description of additional new taxa. We took available nucleotide sequence data for the nuclear-encoded SSU rRNA, fucoxanthin/chlorophyll photosystem I/II, and actin genes and the plastid-encoded SSU rRNA, tufA, and rbcL genes and analysed these to evaluate phylogenetic relationships among the "golden algae", viz., the Haptophyceae (= Prymnesiophyceae) and the heterokont chromophytes (also known as chromophytes, heterokont algae, autotrophic stramenopiles). Using molecular clock calculations, we estimated the average and earliest probable time of origin of these two groups and their plastids. The origin of the haptophyte host-cell lineages appears to be more ancient than the origin of its plastid, suggesting that an endosymbiotic origin of plastids occurred late in the evolutionary history of this group. The pigmented heterokonts (heterokont chromophytes) also arose later, following an endosymbiotic event that led to the transfer of photosynthetic capacity to their heterotrophic ancestors. Photosynthetic haptophytes and heterokont chromophytes both appear to have arisen at or shortly before the Permian-Triassic boundary. Our data support the hypothesis that the haptophyte and heterokont chromophyte plastids have independent origins (i.e., two separate secondary endosymbioses) even though their plastids are similar in structure and pigmentation. Present evidence is insufficient to evaluate conclusively the possible monophyletic relationship of the haptophyte and heterokont protist host cells, even though haptophytes lack tripartite flagellar hairs. The molecular data, albeit weak, consistently fail to present the heterokont chromophytes and haptophytes as monophyletic. Phylogenetic resolution among all classes of heterokont chromophytes remains elusive even though molecular evidence has established the phylogenetic alliance of some classes (e.g., Phaeophyceae and Xanthophyceae).
The upper half of the younger Precambrian strata of the Grand Canyon presents a sequence of some 6800 feet (2100 m) of rocks not seen anywhere else in the southwestern US. They have not been metamorphosed, and the sedimentary rocks include an unparalleled assemblage of late Precambrian fossils. For these reasons, the Nankoweap and Chuar deserve a chapter to themselves. Furthermore, in the Sixtymile Formation they provide unique evidence of sedimentation during tectonic activity, with examples of massive block falls from a fault scarp. The late Precambrian paleontological record from the Chuar Group consists of a variety of microfossils from filamentous algal sheaths, coccoid algae, acritarchs (including the megascopic Chuaria), vase-shaped microfossils of unknown affinity, and stromatolites. The lobate markings from the Nankoweap Formation can be regarded at best as a dubio-fossil and may not be of organic origin at all. -from Author
Using scanning electron micrographs, this book illustrates most of the common species of testate amoebae found in freshwater habitats. Information on the biology, ecology, geographical distribution, and classification is followed by descriptions of 95 species, each illustrated by several views of the shell. The text serves both as an identification guide and as an introduction to the biology and taxonomy of these freshwater protozoa. Readership: protozoologists, ecologists, limnologists, water tyreatment specialists, and micropaleontologists interested in recent sediments.
Microscopic organic objects extracted from the Cretaceous freshwater deposits of Ruby Creek (Alberta) are discussed and illustrated. The authors reach the conclusion that these objects are fossilized thecamoebian tests which, on the basis of the evidence available, comprise ten species and seven genera all new to Science, but contained in Families and Superfamilies already known.