ARTICLE IN PRESS
Deep-Sea Research II 56 (2009) 1700–1712
Contents lists available at ScienceDirect
Deep-Sea Research II
journal homepage: www.elsevier.com/locate/dsr2
Paleodictyon nodosum: A living fossil on the deep-sea floor
Peter A. Rona a,, Adolf Seilacher b, Colomban de Vargas c, Andrew J. Gooday d, Joan M. Bernhard e,
Sam Bowser f, Costantino Vetriani g, Carl O. Wirsen e, Lauren Mullineaux e, Robert Sherrell g,
J. Frederick Grassle g, Stephen Low h, Richard A. Lutz g
a
Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901-8521, USA
Yale University, P.O. Box 208109, New Haven, CT 06520-8109, USA
c
Station Biologique de Roscoff, Bretagne 29682 France
d
National Oceanography Centre, Empress Dock, Southampton SO14 3ZH, UK
e
Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
f
Wadsworth Center, P.O Box 509, Albany, NY 12201-0509, USA
g
Rutgers University, 71 Dudley Road, New Brunswick, NJ 08901-8521, USA
h
The Stephen Low Company, 795 Carson Avenue, Suite 6, Dorval, Quebec, Canada H9S 1L7
b
a r t i c l e in f o
a b s t r a c t
Available online 28 May 2009
We report new in situ observations and laboratory studies of specimens of a small (diameter 2.4–7.5 cm)
strikingly hexagonal form originally described from sedimented steps in a wall of the axial valley of the
Mid-Atlantic Ridge (water depth 3430–3575 m) near 261N, 451W that appears to be identical to the
iconic form Paleodictyon nodosum described as a trace fossil from Eocene flysch deposits at sites in
Europe and Wales. Our findings follow:
Keywords:
Paleodictyon
Living fossil
Hexactinellid sponge
Mid-Atlantic ridge
TAG hydrothermal field
Ediacaran period
The form is apparently agglutinated in sea floor sediment (a veneer of calcareous lutite over red
metalliferous sediment) and consists of three equidistant rows of tiny holes (diameter 1 mm) that
intersect at an angle of 1201 and continuously connect through vertical shafts (length 2–3 mm) with
an underlying horizontal network of tubes or tunnels identical with the fossil form.
The number of rows of holes and spacing of rows increase with overall diameter of the form
indicative of organic growth.
The form is shaped like a shield surrounded by a lip and moat with surface relief (0.5 cm) that is
absent in the fossil form. The surface relief exposes the underlying red sediment and may have been
produced either by excavation (constructional origin) or by infaunal growth (body form).
Protoplasm is absent in recovered specimens, as indicated by negative results of staining techniques,
explained by either initial absence or loss.
Genetic sequencing of material from the form identified different foraminifera that had settled on
the pattern of holes which acts as a baffle to trap organic matter.
Models in flume tanks show that the shield-like form deflects flow of ocean currents into a selfventilating structure capable of aerating and of circulating organic particles through the tubes or
tunnels.
Microbial counting techniques indicated background abundances within and outside the form.
We come to two alternative interpretations of the findings resolvable with further studies:
The modern P. nodosum is a burrow consistent with interpretation of the ancient form as a trace
fossil.
The modern P. nodosum is a compressed form of a hexactinellid sponge adapted to a sedimentary
substrate, which means that the ancient form is a body fossil with possible affinity to the Ediacara
fauna.
& 2009 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +1732 932 6555x241; fax: +1732 932 6557.
E-mail addresses: [email protected] (P.A. Rona), [email protected] (A. Seilacher), [email protected] (C. de Vargas), [email protected] (A.J. Gooday),
[email protected] (J.M. Bernhard), [email protected] (S. Bowser), [email protected] (C. Vetriani), [email protected] (C.O. Wirsen),
[email protected] (L. Mullineaux), [email protected] (R. Sherrell), [email protected] (J. Frederick Grassle), [email protected] (S. Low),
[email protected] (R.A. Lutz).
0967-0645/$ - see front matter & 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dsr2.2009.05.015
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1. Introduction
A field of small (diameter 2.4–7.5 cm) strikingly symmetric
hexagonal patterns was imaged in 1976 by a camera towed over a
sediment-covered area of the lower east wall of the Mid-Atlantic
Ridge (water depth 3415–3585 m) in the course of an investigation of the axial valley at 261090 N, 441480 W for hydrothermal
activity (Fig. 1; Rona and Merrill, 1978). The pattern imaged
comprises three equidistant sets of rows of black dots in the
sediment, inferred to be holes at that time, that intersect at angles
of 1201. Each set of rows parallels two sides of the bounding
equilateral triangle to produce an overall hexagonal outline
encompassing a network of hexagonal cells. Thousands of the
patterns, typically several per m2, were imaged in an area 3 km
along the ridge axis (NE-SW) by 2 km across the axis (NW-SE) at
water depths of 3200–3600 m (Fig. 2). Although the pattern defied
clear classification either as a known benthic organism or its
product, the symmetry and sizes of the pattern were considered
closest to a compressed hexactinellid sponge adapted to an
unconsolidated sediment substrate (Rona and Merrill, 1978).
Subsequently, the pattern has been identified as the inferred
surface expression of the fossil Paleodictyon nodosum found in
flysch sediments of Eocene age near Vienna, Austria and other
areas (Fig. 3; Seilacher 1977, 1978, 2007; Ekdale, 1980; Swinbanks,
1982; Garlick and Miller, 1993), Body fossil Interpretations of the
form, in addition to that of a hexactinellid sponge, are the test of a
large foraminiferan, either an astrorhiziid (Loeblich and Tappan,
1964) or a xenophyophore (Tendal, 1972, 1989; Swinbanks, 1982;
Levin, 1991; Gooday and Tendal, 2002), part of the protistan
supergroup Rhizaria (Adl et al., 2005). Xenophyophores are a
group of large agglutinated protists, confined to deep-sea habitats.
Recent molecular studies have demonstrated that at least two
species are monothalamous foraminifera (Pawlowski et al., 2003a;
Lecroq et al., in press). Both protistan groups reach large body
sizes (0.5 to 410 cm) and construct their tests from sediment
grains. According to the trace fossil interpretation, the hexagonal
form would comprise an open tunnel system, related to other
‘graphoglyptid’ burrows (Seilacher, 1977; Miller, 1991), rather than
the tube of a body fossil. Fossil graphoglyptid burrows are inferred
to have served as farms for growing bacteria or fungi as an
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adaptation to the reduced food supply on the deep-sea floor
(Seilacher, 1977, 2007). In the alternative interpretations as a
sponge or rhizopod, the pattern would reflect growth rather than
behavior. We refer to the ‘‘pattern’’ at the sediment-water
interface and the whole 3-D structure as the ‘‘form’’.
In this paper, we report new observations based on
high-resolution imaging (HDTV ¼ high-definition television;
1920 1080 pixels) of the patterns done in 2001 as part
of the IMAX film Volcanoes of the Deep Sea (Fig. 4; http://
www.volcanoesofthedeepsea.com), examination of specimens
recovered in box cores and push cores using the Deep
Submergence Vehicle (DSV) Alvin on dives to the same site in
four series (1990, 1993, 2001, 2003), and application of
morphologic, genetic sequencing, and microbiological methods
to the core samples. At the same time, preparation of a core
sample cast with epoxy, as well as an in situ erosion method,
confirmed that the surface pattern is indeed connected to a
Fig. 2. Map of TAG hydrothermal field on floor and east wall of the axial valley of
the Mid-Atlantic Ridge showing the spreading axis (dashed line), the active hightemperature sulfide mound, and the distribution of P. nodosum around and to the
north of the Mir inactive sulfide zone (Rona et al., 1993a, b).
Fig. 1. One of original 1976 towed camera photos of Paleodictyon nodosum patterns in sediment on the east wall of the axial valley of the Mid-Atlantic Ridge (lower half;
Rona and Merrill, 1978) and ink drawings of the top three forms in the photo (upper half; Seilacher, 2007, plate 55). The patterns range in diameter from 2.4 to 7.5 cm.
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Fig. 3. Cast of fossil P. nodosum on the sole of an Eocene turbidite near Vienna
Austria. Note partial erosion on right side, showing nodose pattern of vertical
shafts.
Fig. 4. High-definition TV image of P. nodosum at the discovery site on the MidAtlantic Ridge (Fig. 2) with laser beams for scale (10 cm separation). Note the
shield-shaped elevation, marginal elevated rim and mote, and color (pale pink) of
the area of the pattern compared with the surrounding veneer of gray calcareous
lutite (image courtesy The Stephen Low Company). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
this article.)
horizontal layer of hexagonal tunnels or tubes a few millimeters
below, as had been postulated in the definition of the trace fossil
P. nodosum (Seilacher, 1977). Thus the modern form can be safely
called P. nodosum. The only question is whether the earlier
interpretation of this taxon as a trace fossil has been correct.
(Fig. 2; Rona et al., 1993a, b; Rona, 2004) and is absent on the
sulfide substrate within the Mir zone. In this area the average
radiometrically determined accumulation rate of lutite is
1.8 cm 103 y (Scott et al., 1978). The veneer of light gray lutite is
up to 1 cm thick, consistent with the radiometrically dated
cessation (650 y ago) of black smoker-type venting that
deposited massive sulfides and metalliferous sediments in the
Mir zone (Lalou et al., 1995). Patchy low-temperature venting and
seepage still continues, and conductive heat flow remains above
background level, in the Mir zone (Rona et al., 1996). Sparse
occurrences of P. nodosum extend about 4 km to the north end of
the presently inactive portion of the TAG field. P. nodosum has not
been observed, however, in calcareous lutite that is mottled by
fallout from black smoker plumes of fresh dark metalliferous
particles surrounding an active high-temperature sulfide mound
2 km west of the Mir zone (Fig. 2).
Where P. nodosum occurs the sediment surface undulates
gently on a scale of meters and exhibits patches of current ripples
with centimetric wavelengths and heights. Millimetric east-west
trending striations evidence winnowing by currents. Actual
near-bottom currents up to several cm s1 have been observed
from displacement of the submersible and drift of suspended
particulate matter. Three current meters moored 150 m above
bottom on the floor and lower east wall of the TAG field recorded
predominantly east-west flow with velocities up to 8 cm s1 that
reversed with semidiurnal tidal cycles (2 weeks August 1988;
Rudnicki, 1996). These currents are too weak to account for the
observed bedforms, which may be related to past or episodic flow
events.
Sparse worm trails are present on the sediment surface and
have been observed to intersect the edge of the P. nodosum pattern
and to turn away. Video recorded a tiny isopod crustacean
(3 mm long) crossing a P. nodosum pattern (C. Allen, pers.
comm.). A polychaete worm was observed next to another one,
but neither organism entered the holes. Dead pteropod tests and
light-colored fluffy (apparently organic) particles were also
observed on top of some of the patterns.
Adjacent P. nodosum patterns on the sea floor vary. Sharp
shield-like mounds up to 7.5 cm in diameter and with relief up to
0.5 cm generally expose red metalliferous sediment. The mounds
are surrounded by a marginal lip that bounds an outer shallow
moat-like depression (Fig. 4). Flat forms are also present with
rounded edges covered by light gray lutite (Fig. 5). These
variations are inferred to reflect the progressive degradation and
burial of inactive forms. The shield-like relief and reddish
sediment in the fresh patterns indicates that the form has either
been excavated by shoveling-out of the underlying red
metalliferous sediment or uplifted by infaunal growth to expose
2. Setting and distribution
P. nodosum was first imaged in 1976 (Rona and Merrill, 1978)
within an inactive portion of the TAG hydrothermal field prior to
the discovery of the adjacent active high-temperature portion of
this field in 1985 (Rona et al., 1986). The TAG field is a 5 5 km
area of the floor and lower wall of the Mid-Atlantic Ridge at water
depths of 2400–3700 m situated 2.4–8 km east of the spreading
axis near 261N, 451W (Fig. 2). The field encompasses an
assemblage of large active (‘‘black smokers’’) and inactive massive
sulfide mounds up to hundreds of meters in diameter and tens of
meters high (Rona et al., 1986; Rona, 2008). P. nodosum is most
abundant (o45 patterns m2) on a thin layer of light gray
hemipelagic calcareous lutite that veneers fine-grained red
metalliferous sediment on the margins of the Mir relict hydrothermal zone in the TAG field at water depths of 3430–3575 m
Fig. 5. Degraded P. nodosum pattern at the discovery site on the Mid-Atlantic Ridge
(Fig. 2; Rona and Merrill, 1978). Original relief is smoothed and partially covered by
recent sediment.
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Fig. 6. Top section of a sediment core (6.65 cm diameter; 5 cm long) showing
oblique view of P. nodosum pattern in pink sediment surrounded by a veneer of
gray calcareous lutite at the sediment-water interface and an underlying layer of
red metalliferous sediment. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
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Fig. 7. A regression of predicted diameter fit to a plot of the sum of the number of
whole (full) rows counted in each of three sets of parallel rows that intersect at
1201 versus the measured diameter of the P. nodosum pattern (based on laserscaled video images of 30 patterns). The plot shows that the number of rows
increases linearly with diameter of a pattern, in addition to the increase in distance
noted between rows with increase in diameter of a pattern.
the red sediment through the gray lutite veneer (Fig. 6). Coverage
of the more degraded forms by several millimeters of the light
gray lutite suggests that the forms may last for tens to hundreds of
years under the prevailing conditions. Smoothly rounded mounds
of similar size and rose color without holes, as well as lightcolored gray spots of similar dimensions, are interspersed with
the intact patterns. They may either represent degrading stages of
P. nodosum or be produced by other organisms.
3. Architecture
P. nodosum ranges from 2.4 to 7.5 cm in diameter, with a mean
of 5 cm based on measurements from sea floor images scaled by
two laser beams 10 cm apart (Fig. 4). The high-resolution imagery
confirms that the dark dots on the original photos are actually
small holes in the sediment each about 1 mm wide arranged in
three sets of parallel rows that intersect at 1201 in an overall
hexagonal shape encompassing a hexagonal network. The distance between parallel rows remains constant in all three
directions within a given pattern and gradually increases as the
diameter of the pattern increases based on observation of still
images of 25 forms. The network remains uniform within a
pattern (e.g., Fig. 1). The holes are regularly spaced within each
row. Alternate rows exhibit half the number of holes and twice the
spacing between holes as adjacent rows.
Diameters of P. nodosum patterns were measured on 30 laserscaled video images and plotted against the sum of the number of
rows with the whole rather than half number of holes in each of
three directions (Fig. 7). A regression of predicted diameter fit to
the plot shows that the number of rows increases linearly with
diameter of a pattern, in addition to the increase in distance noted
between rows with diameter of a pattern. These observations are
indicative of growth of an organism.
In few patterns (about 1% of those observed) the outline is
elongate with unequal numbers of rows along the three axes (e.g.,
17 21 25; 18 21 24; 20 25 28; 20 29 29; shown in
Rona and Merrill, 1978, Fig. 5). The continuity of rows in these
variants evidences twinning of a body form or an error in
executing a construction program, rather than the overlapping
of two patterns.
Fig. 8. Photo of plasticine reconstruction (3-D) of the modern P. nodosum pattern
based on observation of the hexagonal pattern of holes at the sediment–water
interface and vertical shafts connecting with an underlying horizontal hexagonal
network of tunnels or tubes (model and photo by Hans Luginsland).
The 3-D architecture of the modern P. nodosum has been
confirmed using three methods:
(1) an Alvin push core (6.87 cm diameter by 50 cm long; D.S.V.
Alvin dive 2592, core 3, 1993; 26108.60 N, 44148.80 W; water
depth 3583 m) with the pattern of holes exposed on the
surface was dried and impregnated with epoxy at sea. Careful
preparation of the impregnated core from below revealed a
hexagonal network of open tunnels or tubes in a single plane
2–3 mm below the sediment surface. Light projected through
the epoxy from above showed that the vertical shafts connect
with the horizontal tunnels or tubes in between network
nodes, as had been inferred from fossil specimens (Fig. 14h in
Seilacher, 1977). These observations guided reconstruction of
a 3-D model of the whole tunnel or tube system, which is
continuous (Fig. 8).
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Fig. 9. X-ray picture made orthogonal to top of sediment core containing an intact
P. nodosum form (minimal core deformation assumed). Note the convex-up lensshaped cross-section in upper 0.5 cm (darker gray layer). The field of view is 5 cm
wide.
(2) Two Alvin push cores (dive 2195; cores 4 and 5; 1990;
26108.70 N, 44148.60 W; water depth 3535 m) and a third push
core recovered in a box core (dive 2195–7) all contain
P. nodosum at the sediment–water interface. X-ray pictures
made at sea perpendicular to the long axes of the cores are
clear along the lengths of the cores except for a darker zone
within the upper 0.5 cm. In the X-ray picture of one of the
cores shown in Fig. 9, a P. nodosum with a horizontal diameter
of 5 cm corresponds to a darker zone with a slightly convex
surface and a flat base 0.5 cm beneath the surface. The darker
shade apparently results from increased radiation
transmission due to the vertical shafts and horizontal
tunnels or tubes relative to underlying solid sediment. The
image likely shows the cross-section through a complete
individual. When formalin (10% in seawater) was poured onto
the cores at room temperature, the surface layer initially
persisted and then the vertical shafts gradually disintegrated,
starting to reveal a coherent underlying layer of tunnels or
tubes (Fig. 10). This step-wise disintegration suggests gradual
dissolution of a cement that agglutinates the P. nodosum
structure. If the cement were organic, it may not have
dissolved in formalin. Sieving of the upper 4 cm of each core
(100 mm mesh), as well as the sediment remaining in the cores
and box core (300 mm mesh), yielded only sediment.
(3) A water pump normally used for collecting organisms by
suction was inverted, so that it issued a jet of water (flow rate
25 cm s1) through a tube attached to the manipulator arm
of D.S.V. Alvin (dive 3900, 2003; 26108.830 N, 44148.240 W;
water depth 3415 m). The pilot directed the jet at the shieldlike mound of a distinct P. nodosum pattern on the sea floor.
Instead of suspending a cloud of fine sediment particles, the
sediment between the holes peeled off in the form of small
tablets, indicating the presence of a cementing material.
Within seconds the flowing water removed the top millimeters of fine-grained sediment and a planar hexagonal
network identical to that in the fossil form appeared
(Fig. 11A–C). The size, shape and relation between the
surface holes and underlying hexagonal network with six
vertical shafts located in the center of each side of a hexagon
confirmed the identity of the 3-D architecture of P. nodosum
on the sea floor (Fig. 4) and in fossils (Fig. 3).
Fig. 10. Modern P. nodosum pattern in a sediment core (inner diameter 6.87 cm)
showing cohesion of structure indicating presence of agglutinating cement as the
form differentially dissolves in 10% formalin-seawater solution.
4. Hydraulics
In order to test the hydraulic effect of the shield-like relief on
the tunnel or tube system, simplified models with a horizontal
base tunnel and multiple vertical exits were drilled in plexiglas
and placed in a flume channel. In the flat-topped model, inkstained water passed without entering the tunnels. In contrast, a
shield-shaped model became automatically flushed by water
sucked out of the more central shafts and replaced from the
margins, as predicted from an airfoil analogy (Fig. 12). The
elevated rim enhances this effect by reducing water flow in the
marginal zone of the shield.
5. Sediment composition
The carbonate lutite and underlying red metalliferous sediment hosting a P. nodosum form were analyzed to search for a
possible chemical energy source that could sustain microbes and
to test for increased barium concentrations indicative of xenophyophores associated with the pattern. Multi-element analysis
was carried out using ICP optical emission spectroscopy to
determine a suite of elements associated with sulfides, carbonates, organic matter, terrigenous phases, and sea salt including
Al, P, Ca, Mg, Ti, Fe, Mn, Co, Cu, Zn, Sr, Na, K, and Rb.
Sediment samples (c. 100 mg) were extracted from locations
within and laterally distal to the P. nodosum pattern (Alvin dive
3900, cores 3 and 7) on the surface of the core (upper 5 mm) and
from a depth of about 1–2 cm in the same core. Duplicate
subsamples of 10–20 mg were digested in acid (nitric and
hydrofluoric) prior to multi-element analysis. While significant
variations in elemental concentrations were observed among the
samples, no consistent differences were observed as a function of
either location (within or outside the pattern), or depth in the
sediment. Barium concentrations were 60 ppm in most samples
(average crustal Ba 585 ppm). We conclude from these findings
that there is no evidence for an obvious chemical depth gradient
that could provide an energy source, nor evidence for elevated
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Fig. 11. Application of the induced erosion method showing three successive stages (A–C) in removal of upper 2–3 mm of sediment by jet of water directed at P. nodosum
pattern on sea floor progressively exposing horizontal hexagonal network beneath vertical shafts (video frame grabs, D.S.V. Alvin dive 3900). Compare (B,C) with Fig. 3.
6.2. Microscopic examination
Fig. 12. Passive ventilation in plexiglas model of shield-shaped P. nodosum. Test
made by H. Luginsland with ink injected in a flume tank (modified from Seilacher,
2007, Plate 55).
barium within the pattern that would suggest local barite
enrichments.
6. Morphology
6.1. Dissection
Several methods were applied to determine morphology of
P. nodosum. A D.S.V. Alvin core (dive 2592) containing an intact
pattern was preserved (10% buffered gluteraldehyde) at sea,
refrigerated, and examined at room temperature on land. Dissection followed by sieving (177 mm mesh) of the sediment along and
beneath barely visible remaining traces of P. nodosum failed to
reveal tubes, mucus, protoplasm, or tests except for small
foraminiferal shells characteristic of calcareous deep-sea lutite.
Only the core cited above from the same dive that was
impregnated with epoxy resin revealed a horizontal network of
tunnels or tubes.
Another Alvin core (dive 3900) containing the P. nodosum
pattern was frozen, stored at –80 1C and defrosted for examination. The surface and subsurface structures were examined under
a binocular microscope and recorded photographically. The upper
layer of the sediment was later sieved on a 300-mm screen,
stained with rose Bengal and examined under a binocular
microscope. The inner part of the core surface was raised and
bounded on one side by an arcuate depression, on the other side
by a lower, flat area with redeposited sediment. Apart from a few
irregularly shaped holes, no subsurface structures were encountered within the top 1 cm of sediment. The 4300-mm fraction of
the upper 1 cm of sediment consisted largely of planktonic
foraminifera (including abundant Orbiculina universa) and fragments of pteropod shells. Benthic foraminifera were fairly
uncommon and dominated by komokiacians, a group with diffuse
protoplasm that does not stain with rose Bengal. The only stained
foraminifera were four specimens of Reophax sp. and of Cibicicoides wuellerstorfi. The metazoan macrofauna consisted of a
single isopod crustacean. The residue yielded four small, nondescript agglutinated fragments, possibly derived from a psamminid xenophyophore. They were not alive but did contain the
decayed remains of stercomata. The residue did not yield any
trace of a fragmented naked (atestate) xenophyophore, e.g.
protoplasmic strands (granellae) or stercomata masses (stercomare). The only curious feature of the 4300-mm fraction was the
presence of numerous small, irregularly shaped metallic fragments, each with a patch of stained material attached. There was
no evidence, either from a careful dissection of the core or an
examination of the sieved sediment (top 1 cm), that a xenophyophore or other large protist was associated with the structure. The
four small possible xenophyophore fragments are not unexpected
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in a deep-sea sample. The metallic fragments are likely sulfides
from the substrate of red metalliferous sediments and could
potentially serve as energy sources for chemosynthesis of
microbes (Edwards et al., 2005).
6.3. Core-embedding
Core-embedding methods (e.g., Watling, 1988) were used to
study the sediment micromorphology associated with freshappearing P. nodosum patterns in Alvin cores (dive 3900, 2003)
preserved at sea (10% buffered gluteraldehyde), refrigerated, and
examined at room temperature on land (D.S.V. Alvin dive 3900,
2003). Two sub-cores were taken, dehydrated in serial ascending
alcohol and acetone solutions for extended periods (weeks), and
finally embedded in Spurr’s epoxy resin (Polysciences Inc.). Fully
polymerized cores were cut into 2-mm-thick slices using a
diamond saw. The slices were glued to glass slides, polished
manually with 240-, 320, 400-, and 600-grit Carbimet papers,
covered with number 1.5 cover slips mounted with glycerol, and
finally imaged using reflection optics. Excess sediment recovered
during the sub-coring step was carefully sieved over a 63-mm
mesh and then picked by hand with the aid of a stereomicroscope.
Slices through one core did not show any surface texture; the
other core surface appeared hummocky, as expected for the
P. nodosum pattern. The sediment of this latter core was of
particular interest, revealing a structure that occurred in the
trough between hummocks which might be interpreted as an
organic matrix of some sort. However, for the most part, the
sediment fabric appeared as a heterogeneous composite of fine
particles typical of deep-sea mud. Numerous foraminiferal tests
were seen scattered throughout both slices; these were mostly
empty planktonic species mixed with an assortment of agglutinated benthic species. Based on their microscopic appearances,
the benthic assemblage probably represented a mix of dead and
live specimens, but this observation requires confirmation using a
proper live/dead assay.
An attempt to use glutaraldehyde-induced autofluorescence
(from the sediment fixation step) to image the infauna and
unmask microorganisms responsible for generating the hexagonal
pattern produced negative results. Additionally, the slices were
stained with fluorescent probes for DNA (DAPI) and protein (i.e.,
the fluorgenic amine—derivatization reaction of fluorescamine).
Appropriate epifluorescence and confocal optics were not useful
in imaging the contained biota. Finally, a classic histochemical
stain (Alcian Blue) was used to find mucosubstances using
transmitted light. However, low pH incubations destroyed the
slices.
The core-embedding methods employed revealed interesting
sediment fabric in certain areas, but none of the structural
approaches revealed an organism that might produce the
patterns.
7. Genetic sequencing
Four Alvin cores containing distinct P. nodosum patterns were
decanted and stored at –801C immediately after recovery of the
submersible (Alvin dive 3900), and kept frozen. Three of the cores
(3, 7 and 9) were used for genetic analyses for identification of
eukaryotic and prokaryotic biota that may build or inhabit the
forms (a fourth core was saved). The methods and their
application are presented in Appendix I.
In our search for a genetic signature to identify the taxonomic
status of P. nodosum, we tried to PCR amplify SSU rDNA fragments
directly from the surface sediments of the core. In the three cores
preserved for DNA analyses, the areas of sediment displaying the
hexagonal pattern were clearly distinguishable from the surrounding flat sediments. In order to minimize the risk of
contamination and validate the genetic identification, each core
was analyzed independently. Several subsamples of sediment,
both within and outside the hexagonal pattern, were DNA –
extracted separately within each core. The PCRs performed using
universal-eukaryotic primers did not produce any positive
amplification from the sediment containing and surrounding the
pattern.
Recent molecular studies suggest that two species belonging to
the order Psamminida are monothalamous foraminifers (Pawlowski et al., 2003a; Lecroq et al., in press). To test whether the
P. nodosum is a species related to the xenophyophores, primers
targeting the full diversity of living foraminifers were used.
Positive strong PCR products were obtained in the three cores and
in three of nine DNA extractions of sediments displaying the
P. nodosum pattern. Negative results were obtained from the six
extractions of surrounding sediment. The foraminiferal PCR
products were sequenced and aligned to an extensive dataset of
more than 400 foraminiferal SSU rDNA, representing the modern
diversity of foraminifers, including xenophyophores (J. Pawlowski
et al., personal communication).
Phylogenetic analyses revealed that the three foramininferal
DNA sequences were different and did not come from a single
species. All three sequences cluster within the mega-group of the
agglutinated monothalamous foraminifers (Pawlowski et al.,
2003b). The two DNA sequences (cores 3 and 7) branch together
and form a sister group to representatives of the genus
Hippocrepinella. The third sequence is closely related to a species
of Vanhoeffenella and thus clearly belongs to this genus (Loeblich
and Tappan, 1964).
Therefore, we were not able to target a single eukaryotic
genetic signature systematically associated with the P. nodosum
pattern. The new foraminiferal SSU rDNA sequences that we
amplified from the form probably belong to microscopic monothalamous foraminifers using the form as a support. Interestingly,
no such ‘‘squatters’’ were detected from the surrounding sediments, which may indicate that the foraminifers associated with
the P. nodosum pattern are feeding on biogenic particles physically
trapped when water flows over the network of ridges and
concavities. Alternatively, they may be using the baffle structure
as a habitat (Levin, 1991). The absence of PCR amplification (using
both broad eukaryotic and more specific foraminiferal primers)
from six out of nine total DNA extractions from the three cores
may be explained as either that the P. nodosum pattern does not
enclose a living organism, or that the specimens that we analyzed
were simply dead, like most of the shells, tests, and other skeletal
material residing in deep-sea sediments. However, as noted, one
of the DNA sequences we obtained corresponds to a Vanhoeffenella
species. Interestingly, Vanhoeffenella sp. builds a flat test that
includes an agglutinated ‘‘hemitubular margin’’ (Loeblich and
Tappan, 1964). This is sometimes approximately hexagonal in
shape and is somewhat reminiscent of an isolated P. nodosum
hexagon.
8. Microbial distribution
Two experiments were performed to determine the microbial
population within the P. nodosum pattern and in the surrounding
sediments.
8.1. Microbial experiment 1
Sediment samples were extracted to 1 cm depth from the form
within the P. nodosum pattern and 3 cm away from it in two cores
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Table 1
Bacterial counts from sediment samples within and outside of P. nodosum pattern.
Box core
Within pattern ¼ 1.1 109 cells/cm3 sediment
Outside pattern ¼ 9.9 108 cells/cm3 sediment
Tube core
Within pattern ¼ 9.8 109 cells/cm3 sediment
Outside pattern ¼ 1.2 109 cells/cm3 sediment
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changes in the composition of the two microbial communities
were detectable in the two microenvironments.
9. Discussion
9.1. Fossil Paleodictyon nodosum form
(Alvin dive 2195, cores 4 and 7) and preserved in buffered
glutaraldehyde (10%). Direct bacterial counts were done on
diluted samples, using acridine orange staining and epifluorescence microscopy. The counts were fairly typical of oceanic
sediments and exhibited no significant difference between
samples taken inside and outside the pattern (Table 1).
8.2. Microbial experiment 2
8.2.1. DNA extraction
Genomic DNA was extracted from sediment samples from
three cores (Alvin dive 3900, cores 3, 7 and 9; same cores as used
for genetic sequencing) within the P. nodosum pattern, using the
Ultra Clean Soil DNA kit (Mo Bio, Solana Beach, CA). In brief, about
1.0 g of sediment was subjected to bead beating to lyse the cells,
and DNA was subsequently purified using spin columns. Using an
identical procedure, genomic DNA was also extracted from
reference sediments in the immediate surroundings of the
hexagonal form.
8.3. Denaturing gradient gel electrophoresis (DGGE)
A nested PCR approach was used to amplify the variable region
3 (V3) of the bacterial 16S rRNA gene. The full length bacterial 16S
rDNA was amplified from the genomic DNA using primers Bact8F
(50 -AGAGTTTGATCCTGGCTCAG-30 ) and Univ1517R (50 -ACGGCTACCTTGTTACGACTT-30 ), as previously described (Vetriani et al.,
2003). The PCR products were gel-purified and used as a template
to amplify the V3 region using the GC-clamp primer 338F(GC
50 -CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCGCCCTCCTACGGGAGGCAGCAG-30 ) and either primer Univ518R (50 ATTACCGCGCGGCTGCTGG-30 ) or primer Univ907R (50 -CCGTCAATTCMTTT
RAGTTT-30 ), yielding fragments of about 200 and 600 bp, respectively. DGGE was performed with a D Gene system (Bio-Rad
Laboratories, Hercules, CA). Template DNA was incubated in a
thermal cycler in the presence of Taq DNA polymerase for 30
cycles under the following conditions: 941C, 30 s; 55C, 30 s; 72C,
30 s. PCR products (20.0_I) were applied directly onto 6% (wt/vol)
polyacrylamide gels in 1X TAE (40 mM Tris, 20 mM acetate, 1 mM
EDTA), with denaturant gradient from 20% to 60% (where 100%
denaturant contains 7 M ureas and 40% formamide). Electrophoresis was performed at a constant voltage of 200 V and a
temperature of 60 1C for 4 h. After electrophoresis, the gels were
incubated for 15 min in ethidium bromide (0.5 mg/liter), rinsed for
10 min in distilled water, and photographed with a UV Foto
Analyst system (Fotodyne Inc., Hartland, WI).
In order to establish if there were significant changes in the
composition of the microbial communities associated with both
the P. nodosum pattern and with reference surrounding sediments,
we used denaturing gradient gel electrophoresis (DGGE) analysis
to obtain a 16S rDNA-based fingerprint of the microbial communities of these two microhabitats. Bacterial 16S rDNA fragments
were amplified from the same sediment subsamples from which
the foraminifera-related sequences were obtained. The DGGE
analysis revealed identical profiles both from the hexagonal form
and from reference sediments, indicating that no significant
The term ‘‘living fossil’’ is usually applied to animals or plants
that had a long geological history and survive today only in
restricted or inaccessible refugia. Usually, they were first described from the fossil record. Such refugia may be deep or
shallow water environments depending where a particular type of
organism finds shelter from competition, predator pressure, or
environmental change. So each deep dive with a research
submersible is a journey into an ancient world. Siliceous sponges,
stalked crinoids, the slit-shell gastropod Pleurotomaria and the
monoplacophoran Neopilina are familiar examples of deep-sea
survivors. The situation for trace fossils is different from body
forms, because they leave the maker unknown, as is the case in
complex backfill burrows with a long geologic history (e.g.,
Chondrites; Zoophycos).
Because trace fossils found in the geologic record have passed
though a taphonomic filter, they are rarely identical to structures
observed on modern sediment surfaces. Surface traces generally
have a low fossilization potential, because they are wiped out
during the erosive phase of the same sedimentation event that
would have served as a casting agent. Therefore, traces observed
on fossil bedding planes are likely to be either undertracks
pressed through a covering veneer, or burrows made within the
sediment.
The spectacular preservation of P. nodosum and other fossils on
the soles of sandy turbidites is inferred to have resulted from an
unusual kind of erosion and deposition that uncovered the
delicate tunnel or tube systems and immediately replicated the
exposed reliefs with sand without destroying them (Seilacher,
1977). These preservational conditions may occur near the distal
reaches of a turbidity current, where the event is inferred to start
with a shock wave that brings the unconsolidated top sediment
into suspension without tractional forces (‘‘suction erosion’’). This
phase must have been immediately followed by the vertical
settling of suspended sand. In more proximal areas the exposed
structure did not survive due to the tractional erosion by
suspended particles expressed as tool marks and flute casts.
Consequently, the fossils are interpreted to show the bottom
sides of the P. nodosum tunnel or tube systems (Fig. 3; Seilacher,
2007) as opposed to the top views seen on the sea floor (Figs. 1
and 4). The link between the two aspects is provided by
specimens, in which part of the horizontal tunnel or tube system
was incompletely exposed when deposition began, so that the cast
replicated the vertical outlets rather than the horizontal tunnels.
The original reconstruction of the P. nodosum system interpreted
as tunnels was based on such variants of preservation, which
explain the lack of shield-like relief of the fossil forms (Fig. 14h in
Seilacher, 1977).
9.2. Modern P. nodosum form
Sedimentation at the sea floor site on the Mid-Atlantic Ridge is
hemipelagic by particles settling from the water column rather
than by turbidity flows as assumed for the fossil preservation.
Despite these differences, the identity of the Paleodictyon form on
the sea floor and the fossil P. nodosum is confirmed by the 3-D
architecture observed in the push core impregnated with epoxy
resin (not shown) and by the horizontal tunnel or tube system
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exposed millimeters below the hexagonal pattern of holes by the
induced sediment erosion experiment (Fig. 11a–c; Rona et al.,
2003). These observations are consistent with the X-ray crosssection (Fig. 9), and with high-resolution images of the pattern on
the sea floor (Fig. 4). The lines of evidence together show that in
the modern as well as the fossil form (Figs. 3 and 4): (1) the
hexagonal pattern of surface holes connects through shafts with
the mid-points of an underlying horizontal tunnel or tube system
(Fig. 8); and (2) this tunnel or tube system forms a single-level
hexagonal network that is only 2–3 mm below the sediment
surface. The top of the modern system exhibits a shield-like
elevation (c. 0.5 cm) relative to the surrounding sediment surface
(Figs. 4 and 9), which is not preserved in the fossil form.
9.3. Origin of P. nodosum form
Worms and other invertebrates including crustaceans construct a large variety of burrows at all water depths (e.g., website
www.annelida.net). On the other hand, agglutinating species of
protists belonging to astrorhiziids and xenophyophorean Forami-
nifera may form networks of comparable size, but less symmetric
than those considered here. They have been reported from
sediments in all the major ocean basins (Tendal, 1972; Ekdale,
1980; Swinbanks, 1982; Tendal et al., 1982; Gooday, 1990; Levin,
1994). Xenophyophores are unique among agglutinated Foraminifera in having (1) fecal pellets (stercomata; typical diameter
10–20 mm) packaged in strings or masses (stercomare) encased by
an organic sheath within the test; (2) barite crystals (BaSO4;
granellae) within the plasma; and (3) an agglutinated organic
tube system enclosing the branched protoplasmic strands (granellare system; Tendal, 1972; Gooday and Nott, 1982).
Xenophyophores exhibit features that merit their consideration as possible makers of P. nodosum. These features include their
present restriction to deep-sea environments and the reticulated
test structure of several endobenthic species immersed in the
upper millimeters of sediment (Swinbanks, 1982; Tendal et al.,
1982; Levin, 1994). One such species, Occultammina profunda, lives
from o1 to 6 cm beneath the surface of deep-sea turbidite
sediments in trenches of the western Pacific (Ogasawara Trench;
Tendal et al., 1982). Structures assigned to O. profunda were
described as ‘‘large networks (diameter 5–10 cm) 2–5 cm beneath
Fig. 13. Examples of fossil graphoglyptids. Only the ones with multiple exits would be spotted on the modern sea floor, but all can potentially documented by the induced
erosion method (modified from Seilacher, 1977).
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openings in the sediment surface; 81% polygons hexagonal; others
pentagons or quadrilateral’’ (S. Ohta, personal communication in
Levin, 1994). Small fragments of similar networks are reported
from the Japan Trench (Swinbanks, 1982) and the abyssal NE
Atlantic (Gooday, 1991, unpublished data). Nevertheless, a
xenophyophore origin of P. nodosum appears unlikely, because:
(1) vertical branches or reticulate mounds protruding above the
sediment for effective baffling are rare in modern xenophyophores
with reticulated test structure (Gooday, 1996); (2) the modern P.
nodosum appears to be an open tunnel or tube system, rather than
a test filled with accumulations of stercomata (stercomare) and
branched protoplasmic strands; and (3) the barium concentration
caused by the barite crystals in the xenophyophoran protoplasm,
which have low solubility so would remain in dead constructs for
long periods of time, is apparently absent in the modern P.
nodosum.
Some similarities exist between the surface expression of the
P. nodosum form and sediment structures created by Toxisacron alba, a
giant naked foraminiferan that is common at sublittoral depths
(10–30 m) off the west coast of Scotland (Wilding, 2002). This species
lacks a test but creates a ‘‘distinctive, slightly raised, approximately
circular mound of 18–40 mm diametery through which there are
many perforationsy’’ (Wilding, 2002, p. 359). However, the cell body
that underlies this mound usually has a highly irregular branched
form, or less commonly a ‘diffuse form’ that comprises very fine
filaments. A large and morphologically extremely variable cell of this
sort is unlikely to produce the very regular P. nodosum form.
The shield-like relief of the modern P. nodosum partially
surrounded by a marginal ridge and a moat-like depression (Fig. 4)
may be explained as excavated sediment brought to the surface or,
alternatively, by uplift produced by the growth of an infaunal
organism, as in certain xenophyophores (Fig. 4; Levin, 1994; Gooday,
1996). The presence of agglutinating cement inferred from the
coherent openings and the network can be explained by either
hypothesis. The failure of staining techniques to detect protoplasm in
the P. nodosum may indicate the form is constructed of empty
excavated tunnels. Alternatively, if it is a body form with tubes, then
either protoplasm was removed by decomposition and consumption
or the tubes never contained protoplasm. In either case, the
accumulation of foraminiferal tests and flocculent material, and the
passive ventilation model (Fig. 12) support its function as a baffle that
concentrates and circulates particulate organic material (Levin and
Thomas, 1988; Levin and Gooday, 1992; Levin, 1994; Seilacher, 2007).
The most intriguing aspect of P. nodosum is its complex
architecture. As Seilacher (2007) notes, ‘‘Though similar hexagonal patterns result automatically from close packing of soap
bubbles, eggs (Abel, 1935), corals and honeycomb cells, it turns
out that ‘‘weaving’’ them is a more difficult task (compare the
fabrication of hexagonal chicken wire!)’’. Computer simulations
suggest that, if P. nodosum is a burrow, it is the product of ‘‘spiral
excavation by an organism of outstanding navigational skills’’
(Garlick and Miller III, 1993). The maker executes turns within 21
of the nominal 601 and regularly uses its pre-existing burrow in
order to re-adjust position and orientation. Application of graph
theory and geometric analysis to the 3-D form (Plotnick, 2003;
Honeycutt and Plotnick, 2005) suggests that a P. nodosum-forming
burrowing animal would have to travel an unreasonable multiple
of its own body length (103–104) to move within such a structure.
10. Summary and conclusions
10.1. Summary
The hexagonal forms observed in sediment on the present MidAtlantic Ridge at water depths between 3200 and 3600 m are
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identical with the fossil P. nodosum described from Eocene deepsea turbidites. Its intricate morphology has been documented by
cast and by an in situ erosion method in combination with highdefinition images of the hexagonal pattern of holes at the
sediment–water interface and the underlying hexagonal network.
Staining techniques failed to detect an organic morphology.
Genetic sequencing identified various small foraminifera apparently associated with the form rather than constituting the form
itself. The hexagonal pattern of holes on the shield-like form of
the modern P. nodosum connect continuously through vertical
shafts to an underlying horizontal hexagonal tunnel or tube
system and constitutes a baffle that traps organic particles
including plankton. As shown by models in flume tanks, the
shield-like form deflects flow of ocean currents into a selfventilating structure that apparently aerates and circulates
organic particles through the system. The equal background
numbers of bacteria measured inside and outside the P. nodosum
form is inconsistent with the hypothesis that the tunnel or tube
system is a farm for bacteria. While P. nodosum shares some
characteristics with certain xenophyophore protists (net-like
form, size range, habit), differences outweigh similarities. Increase
in number of rows of holes and expansion of network size in direct
proportion to the outer diameter of forms and the presence of
whole rather than partial forms indicates growth of an organism.
10.2. Conclusions
We come to alternative interpretations of the nature of P.
nodosum based on available observations. According to one
hypothesis the modern P. nodosum is constructional and the fossil
form is a trace fossil (Seilacher, 1977, 2007). According to an
alternative hypothesis, the modern form is the compressed body
of a hexactinellid sponge adapted to an unconsolidated sedimentary substrate, as surmised in the original discovery paper (Rona
and Merrill, 1978), so that the fossil form is a body fossil.
10.2.1. Constructional hypothesis
Any paleoichnologist would have recognized the strange
photos from the modern deep-sea bottom as P. nodosum, because
they correspond exactly to the surface expression reconstructed
from only partially eroded specimens of that trace fossil
(Seilacher, 1977, 2007). P. nodosum is best known from Eocene
turbidites near Vienna (Austria) as a particularly complex member
of a large group of pre-turbidite trace fossils called ‘‘grapholyptids’’. They are characterized by ornamental patterns in the form
of hexagonal meshworks, guided meanders, spirals, and dendritic
arrays. Despite the unusual diversity of patterns, they all share a
tendency to subdivide a given surface into units of subequal
diameters, just as human-made drainage systems do. This, as well
as signs of re-visiting, led to the interpretation of graphoglyptids
as a kind of subsurface mushroom garden, in which foods were
cultivated by unknown (but related) animals. An additional
baffling function was only possible in graphoglyptids with multiple exits (Fig. 13). The many graphoglyptids lacking multiple exits
cannot be explained by the sponge hypothesis ((Seilacher, 2007).
In this view, the unusual diversity of patterns also made sense:
just as house keys, they were not differentiated by fitness, but
served to keep other species from usurping the gardens (and/or
traps), while the owner was attending other gardens. Interestingly, an unfamiliar tunnel course was sufficient as a barrier
against other species.
Graphoglyptid tunnel systems also required very specific
sedimentary conditions to leave a fossil record. In the first place,
the horizontal galleries had to be laid open without the
destructive effect of the sand grains carried by a turbidity current.
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This probably happened in the shock wave preceding the arrival of
the sediment-laden water mass itself. Sudden acceleration of the
still clear bottom water delaminated the top sediment by a kind of
suction erosion. In this process, the horizontal gallery systems
presumably had a similar effect as the perforations in a sheet of
postage stamps: their cover was removed, leaving the lower
halves of the tunnels unaffected.
The second step, casting of the exposed relief by sand, could
follow only after the turbid cloud had arrived. But its kinetic
energy must have been so low that sand grains rained vertically
down from suspension. Only in some tunnel casts (mostly in
stretches running transverse to the current direction) incipient
tractional erosion can be noted in the form of microflutings on the
down-current side. If this kind of erosion had continued and
carved-out veritable flute casts, all pre-turbiditic burrows would
have become erased. Neither could open tunnels leave a trace
without a turbidite; they simply collapsed when the mud became
compacted.
The present TAG field is way out of the reach of turbidity
currents. Yet our induced erosion experiment mimicked the
postulated preservational process and yielded the expected result:
it laid open the hexagonal meshwork of galleries below the
present sea floor. Videos also reveal why our experiment did not
produce a cloud of mud obstructing the view: the microbially
bound surface sediment became removed in the form of small
tablets rather than suspendable particles.
More importantly, our hasty experiment also uncovered other
types of graphoglyptid burrows (e.g. Cosmorhaphe) that lack the
systematic exits and can therefore not be spotted at the surface
(Fig. 13). So the method exists to map this hidden biotope on
modern sea bottoms. If the survey would be done with a robotic
vehicle instead of a human occupied vehicle, one could even
afford to let it sit for hours next to a fresh P. nodosum and wait for
the unknown trace maker to visit its farm. Or would it be deterred
by the light in its eternally dark environment?
10.2.2. Body hypothesis
Alternatively, the modern form is the compressed body of a
hexactinellid sponge adapted to an unconsolidated sedimentary
substrate (Rona and Merrill, 1978). If this interpretation is correct,
then the fossil form is a body rather than trace fossil.
In making this attribution, Rona and Merrill (1978) state, ‘‘The
discoidal (hexagonal) object corresponds either to the sieve
(water inflow) or body wall (water outflow) of the sponge raising
questions as to the mode of water circulation.’’ The characteristics
that support interpretation of P. nodosum as a hexactinellid
sponge of agglutinated sediment (Reiswig and Mackie, 1983)
are: (1) shield shape (2.4–7.5 cm diameter) projecting above the
sediment surface (0.5 cm); (2) an outer wall penetrated by a
hexagonal array of narrow tubes (1 mm diameter; pores);
connection of the tubes through short (1–2 mm long) vertical
shafts to a compressed body cavity consisting of a continuous
horizontal network of hexagonal tubes; (3) passive ventilation of
the body cavity (Fig. 13; sieve inflow at margins enhanced by
marginal ridges; body wall outflow at center).
The whole form apparently functions as a feeding strategy of
trapping of organic matter (particles including plankton) by its
surface structure as a baffle and circulation through vertical shafts
and the horizontal hexagonal network of the body cavity by
passive ventilation. The passive ventilation mechanism (Fig. 12;
Seilacher, 2007) answers the original question as to the mode of
water circulation. A number of related studies have been made of
induced flow in fossil and living sponges (Boyajian and Labarbera,
1987; Labarbera and Boyajian, 1991; Labarbera, 1993; Savarese,
1992; Vogel, 1977). The hexactinellid sponge interpretation is
consistent with the presence of agglutinating material, the
trapping of particulate matter, the presence of only complete
modern forms with internally regular networks, apparent organic
growth by concurrent expansion of distance between rows of
holes (pores) and addition of number of rows with increasing
diameter (Fig. 7), and the passive ventilation mode of circulation
(Fig. 12). If the interpretation of the modern P. nodosum form as a
sponge is correct, then the identity of the modern with the fossil
form implies that P. nodosum is a body fossil. The Porifera are
among the earliest body fossils with the first probable hexactinellid sponge Palaeophragmodictya (Gehling and Rigby, 1996) from
the Neoproterozoic Ediacaran Period (635–541 Ma) fauna of South
Australia (Knoll et al., 2006; Xiao and Laflamme, 2009). Palaeophragmodictya reticulata (Gehling and Rigby, 1996, Fig. 4-1,2,3)
exhibits similar size, shape, and hexagonal mesh to P. nodosum.
These similarities would support P. nodosum as one of the oldest
and, possibly, the oldest known living fossil.
Additional diagnostic studies of the form on the sea floor are
needed to resolve remaining questions. These studies include
examination of the sediment texture of the pattern to determine if
it is excavated (constructional) or exhumed (body growth) and
whether sponge spicules (calcareous or siliceous), protoplasm and
sponge biomarkers (e.g., Love et al., 2009) are present. Now that
P. nodosum has been identified at a site on the Mid-Atlantic Ridge, it
is likely to be found at other sites in the world ocean. The induced
erosion experiment that we used to expose the 3-D architecture of
P. nodosum opens a window to a world of ancient and modern
infaunal forms in sea floor sediment and to their evolution.
Acknowledgements
The Stephen Low Company imaged Paleodictyon nodosum as
part of the award-winning Imax film of hydrothermal vents and
their ecosystems, Volcanoes of the Deep Sea, and supported a D.S.V.
Alvin dive dedicated to experiments and sampling reported in this
paper after the film was wrapped. E. ‘‘Dolly’’ Dieter of the National
Science Foundation instigated the dedicated dive support. E.
Kristof of the National Geographic Society and W. Reeve did the
HDTV photography on the 2001 dives, as part of the filming by the
Stephen Low Company. A. Low, a producer of the film, generously
provided the images. D. Seilacher praises E. Seilacher as a partner
in paleontological research and H. Luginsland, University of
Tuebingen, for masterful laboratory contributions including
plasticine 3-D reconstruction of the P. nodosum pattern and the
plexiglas model for the hydraulics experiment. The cores containing P. nodosum were skillfully recovered on various dives by D.S.V.
Alvin pilots D. Foster, P. Hickey, T. Tengdin, and B. Williams. Chief
Pilot and 2003 Expedition Leader P. Hickey rigged the induced
erosion method. S. Humphris of the Woods Hole Oceanographic
Institution and R. Petrecca of Rutgers University performed
shipboard X-rays and sieving of cores containing the P. nodosum
pattern. J. E. Sanders formerly of Barnard College of Columbia
University advised on the epoxy impregnation technique of the
cored specimen that first revealed the 3-D structure of the
modern P. nodosum. A.A. Ekdale of the University of Utah first
published the connection between the modern and ancient
P. nodosum forms. We thank G. Hollis, M. Lomas and B. Williams
of the Bermuda Biological Station for Research for provision of the
reagents needed for preserving the P. nodosum specimens at sea.
We thank R.E. Plotnick of the University of Illinois at Chicago for a
constructive review generously providing citations of related
sponge circulation and biomarker publications and corroborative
findings from his own flow circulation studies. A second
anonymous reviewer provided helpful comments. We are grateful
to R. Reeves-Sohn of the Woods Hole Oceanographic Institution
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and S. Humphris for accommodating one of our D.S.V. Alvin
Paleodictyon dives (3900) on their 2003 cruise to the TAG
hydrothermal field on the Mid-Atlantic Ridge. C. de Vargas thanks
J. Pawlowski, University of Geneva, for constructive discussion. P.
Rona thanks K. Ruetzler, Smithsonian Institution, and honors the
memory of G. Voss (formerly the University of Miami) and F. Bayer
(formerly the Smithsonian Institution) for enlightening discussion
of attribution when he and G. Merrill found the hexagonal pattern
on the sea floor in 1976.
Appendix I. Genetic sequencing (Section 7)
(1) DNA extraction: Total genomic DNA was extracted from the
first 3 mm of surface sediments at different locations inside
and outside each pattern. About 1 g of sediment was subjected
to bead beating to lyse the potential biological material and
cells. DNA was subsequently purified using spin columns from
the Ultra Clean Soil DNA kit (Mo Bio, Solana Beach, CA). In
total, nine independent extractions were made from the three
P. nodosum patterns, and six extractions were made as
negative controls from surrounding sediment.
(2) Genetic analyses of eukaryotic life within the P. nodosum
pattern: In the absence of information about the biological
identity of the form, the most common phylogenetic marker,
the gene coding for the small sub-unit of the ribosomal DNA
(SSU rDNA), was targeted. Standard PCR (94 1C/300 –50 1C/
300 –721/20 for 40 cycles) were performed using the couples of
general eukaryotic primers S14 50 -ACTTAAAG(AG)AATTGACGG-30 and SB 50 -TGATCCTTCTGCAGGTTCACCTAC-30 , S18
50 -TAACAGGTCTGTGATGCC-30 and S20r 50 -GACGGGCGGTGTG
TGTACAA-30 , and S12.2 50 -GATYAGATACCVTCSTAGTC-30 and
SB, as well as a couple of primers amplifying specifically
all sorts of foraminifers (S15rf-gtgcatggccgttcttagttc and
S19f0cccgtacraggcattcctag-). Amplification products were
either ligated into the pGEM-T Vector System (Promega),
cloned in XL-1 ultra-competent home-made cells, and
sequenced
on both strands using the ABI Big Dye Cycle Sequencing kit
(Perkin-Elmer) and an ABI 3100-Avant automatic sequencer,
or purified using shrimp alkaline phosphatase and exonuclease I and directly sequenced. The new sequences reported
herein have been deposited in GenBank. The SSU rDNA
fragments were compared with the Genbank database using
BLASTn, and manually aligned to related species. For the
foraminiferal SSU rDNA, sequences integrated into a dataset of
more than 400 orthologous sequences representing the entire
diversity of living foraminifers (J. Pawlowski, personal communication). Phylogenetic trees were inferred to analyze the
taxonomic position of the foraminiferal SSU rDNA with
Bayesian and Neighbor Joining statistics using the software
MrBayes (Huelsenbeck and Ronquist, 2001) and PAUP*
(Wilgenbusch and Swofford, 2003).
(3) Genetic analyses of the prokaryotic life within the P. nodosum
pattern: for analysis of the bacterial diversity within the
pattern, we chose a first-order approach using denaturing
gradient gel electrophoresis (DGGE) to test if the prokaryotic
communities were different within and outside the pattern. A
nested PCR approach was used to amplify the variable region
3 (V#) of the bacterial 16S rRNA gene (16S rDNA). The fulllength bacterial rDNA was first amplified from the genomic
DNA using primers Bact8F (50 -AGAGTTTGATCCTGGCTCAG-30 )
and Univ 1517R (50 -ACGGCTACCTTGTTACGACTT-30 ), as previously described (Vetriani et al., 2003). The PCR products
were gel-purified and used as a template to re-amplify the V#
region using thee GC-clamp primer 338F(GC) (50 -cgcccgccg
1711
cgccccgcgcccgtcccgccgcccccgccctcctacgggaggcagcag-30 )
and
either primer Univ518R (50 -ATTACCGCGGCTGCTGG-30 ) or
primer Univ907R (50 -CCGTCAATTCMTTTRAGTTT-30 ), yielding
fragments of about 200 and 600 bp, respectively. DGGE was
performed with a D Gene system (Bio-Rad Laboratories,
Hercules, CA). Template DNA was incubated in a thermal
cycler in the presence of Taq DNA polymerase for 30 cycles
under the following conditions: 94 1C, 30 s; 55 1C, 30 s;72 1C.
30 s. PCR products (20.0 ml) were applied directly onto 6% (wt/
vol) polyacrylamide gels in 1X TAE (40 mM Tris, 20 mM
acetate, 1 mM EDTA), with denaturant gradient from 20% to
60% (where 100% denaturant contains 7 M urea and 40%
formamide). Electrophoresis was performed at a constant
voltage of 200 V and a temperature of 60 1C for 4 h. After
electrophoresis, the gels were incubated for 15 min in
ethidium bromide (0.5 mg/liter), rinsed for 10 min in distilled
water, and photographed with a UV Foto Analyst system
(Fototdyne Inc., Hartland, WI).
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