J Paleolimnol
DOI 10.1007/s10933-008-9287-2
ORIGINAL PAPER
Sheathed prokaryotic filaments, major components
of Mid-Cretaceous French amber microcoenoses
Vincent Girard Æ Gérard Breton Æ Luc Brient Æ
Didier Néraudeau
Received: 20 December 2007 / Accepted: 26 November 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Prokaryotes were the first organisms to
colonize Earth, but little evidence of their existence has
been found in the fossil record. Recent studies of
amber, a fossil resin from gymnosperms or angiosperms, have revealed a number of rarely fossilized
microorganisms. Several amber-bearing localities of
Mid-Cretaceous age in southwestern France (Charentes and Aude regions) led to the discovery of a rich and
diverse biota of resin-preserved microorganisms.
These amber microcoenoses are dominated by
sheathed prokaryotic filaments similar to those of the
cyanobacterium Palaeocolteronema cenomanensis
Breton and Tostain (2005) and to those of the
V. Girard (&) G. Breton D. Néraudeau
Geosciences Rennes, Université de Rennes 1,
UMR 6118 CNRS, 263, Avenue du Général Leclerc,
35042 Rennes, France
e-mail: [email protected]
G. Breton
e-mail: [email protected]
D. Néraudeau
e-mail: [email protected]
V. Girard G. Breton D. Néraudeau
IFR CAREN, 263, Avenue du Général Leclerc,
35042 Rennes, France
L. Brient
Université de Rennes 1, UMR 6553 CNRS, 263,
Avenue du Général Leclerc, 35042 Rennes, France
e-mail: [email protected]
bacterium Leptotrichites resinatus Schmidt 2005.
These sheathed filaments appear as peripheral cortexes
around some pieces of amber from the Charentes
outcrops and as peripheral dark areas on amber from
the Aude locality. Macroscopic and microscopic
features, as well as measurements of phycocyanin
concentrations from the filaments, made it possible to
identify two different taxa. The sheathed filaments
from Charentes correspond to P. cenomanensis. They
were growing in freshwater ponds when amber trapped
them. Those of the Aude outcrop represent L. resinatus. The latter were probably trapped in less humid
environments than were P. cenomanensis filaments.
Keywords Sheathed filaments Cyanobacteria Bacteria Amber Phycocyanin Cretaceous
Introduction
Prokaryotic microorganisms have existed on the Earth
since the Archean (Knoll 1990). Today, they are
omnipresent and they have colonized all environments, from the poles to deserts, down to the depths of
the oceans (Schlegel and Jannasch 2006). Despite their
ancient origin and broad environmental distribution,
such microorganisms are rarely fossilized and most
evidence in the fossil record comes from traces of their
activity, in particular bio-sedimentary formations such
as stromatolites (Knoll 1990).
Amber is one of the best media for organism
preservation. It frequently preserves organisms rarely
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found elsewhere in the fossil record, such as insects,
arachnids and delicate plant remains such as flowers.
Microorganisms have also been found in amber since
the nineteenth century. Smith (1896) described filamentous microorganisms from the Carboniferous
amber of Scotland. Thirteen years later, Caspary and
Klebs (1907) published on microorganisms in Eocene
Baltic amber. Despite these pioneer studies, microorganisms in amber were rarely studied until the works of
Waggoner and Poinar (1992), Poinar (1992), Waggoner
(1994a, b), Breton et al. (1999) and Schmidt et al.
(2006), for example. These studies revealed assemblages of microorganisms comprising both prokaryotic
and eukaryotic forms (see for example Poinar et al.
1993; Schönborn et al. 1999; Ascaso et al. 2003; Breton
and Tostain 2005). Prokaryotic filamentous organisms
have been preserved in different ambers (i.e. German,
French, Dominican, Baltic) at different periods (Cenomanian, Eocene, Oligocene). Despite their long period
of study, there is doubt about their systematic position. It
appears that prokaryotic amber microinclusions cannot
be identified with microscopic observations alone.
Some authors tried to extract DNA from such inclusions (Cano and Borucki 1995; Lambert et al. 1998;
Greenblatt et al. 1999, 2004), but they obtained
controversial results, as the DNA signal obtained
seemed to be a contaminant signal, probably from
microorganisms settled in amber cracks (Priest et al.
1995; Austin et al. 1997; Lindhal 2000; Orlando 2005;
Parducci and Bennett 2005; Schmidt and Schäfer 2005).
With the discovery of several amber localities in
SW France since 1999, rich and diverse microfauna
and microflora of Mid-Cretaceous age have been
recognised. These include diverse ciliates, amoebae,
mycelia, and numerous filamentous microorganisms.
Sheathed prokaryotic filaments are the most abundant
microorganisms, comprising 85–90% of the assemblages. We have developed a multi-angle approach to
better characterize and identify Mid-Cretaceous
sheathed prokaryotic filaments. Sheathed prokaryotic
filaments could have two different origins, being
either cyanobacterial or bacterial. Cyanobacterial
filaments are characterized by a high concentration
of phycocyanin (Whitton and Potts 2000), while
bacterial ones do not produce phycocyanin (Bourrelly
1985; Holt et al. 2000). Using only morphological
features, identification of such microorganisms is
problematic, but as demonstrated by Brient et al.
(2008), presence of cyanobacteria can be shown by
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the measurement of phycocyanin concentrations in
samples. Measurements of phycocyanin content were
used here to help determine if the fossil sheathed
filaments were cyanobacteria or bacteria.
Prokaryotic filamentous microorganisms
from amber
Several prokaryotic filamentous organisms have
already been described from amber. Smith (1896)
mentioned small filaments from Carboniferous amber
of Scotland. Caspary and Klebs (1907) described
bacteria and fungi from Eocene Baltic amber. More
recently, Poinar (1992) mentioned ‘‘fungus-like
organisms’’ from Carnian amber of Germany. This
amber has been redated to the Cenomanian (Schmidt
et al. 2001) and the sheathed prokaryotic filaments
reassigned to diverse groups of organisms. Poinar et al.
(1993) reinterpreted the same sheathed filaments
as three different kinds of inclusions: (1) the green
alga Trentepohlia Martius 1817, (2) an unnamed
sheathed bacterium and (3) a sheathed cyanobacterium
resembling extant Scytonema alata [=Scytonema
(Petalomema) alatum Borzi 1879]. Dörfelt and Schäfer
(1998) reinterpreted Poinar et al.’s sheathed bacterium
as a fungal hypha of Palaeodikaryomyces baueri
Dörfelt in Dörfelt and Schäfer (1998), while Schönborn
et al. (1999) interpreted part of an empty sheath from
this German amber as a hypha from a saprophytic
fungus. More recently, Schmidt and Schäfer (2005)
reinvestigated all the filamentous microorganisms from
the Cenomanian German amber and reassigned those
prokaryotic microfilaments to the sheathed bacterium
Leptotrichites resinatus. Waggoner (1994b) described
two different cyanobacteria (belonging to the genera
Plectonema Thuret 1875 and Lyngbya Agardh 1824)
from French Cenomanian amber and two different
actinomycete filaments (one of Paleomonospora
Waggoner 1994 and one of Streptosporangiopsis
Waggoner 1994) from Upper Cretaceous amber of
Mississippi. The same author described sheathed bacteria (related to the living genus Leptothrix Kützing 1843)
from Mid-Cretaceous amber of Kansas (Waggoner
1996). Breton et al. (1999) and Breton (2007) mentioned
the occurrence of actinomycete filaments in Sparnacian
amber from France. Ascaso et al. (2003, 2005) mentioned unidentified mycelia in Lower Cretaceous
Spanish amber. Most recently, the sheathed filamentous
J Paleolimnol
cyanobacterium Palaeocolteronema cenomanensis was
described by Breton and Tostain (2005) and mentioned
by Breton (2007) from the Cenomanian amber of
Ecommoy (W France).
–
Geological setting
The amber samples studied here were collected from
several localities of Mid-Cretaceous age in southwestern France (Fig. 1). Apart from the Middle
Cenomanian amber deposit from Fourtou, located in
the Aude region (S France) and briefly mentioned by
Perrichot et al. (2007a), these localities are located in
the Charentes region (SW France). Their biostratigraphy and sedimentology were the subjects of
detailed previous studies. The deposits were positioned within a regional sequence stratigraphy
framework by Néraudeau et al. (1997):
–
–
The uppermost Albian deposit from ArchingeayLes Nouillers has provided the richest French
Cretaceous amber in arthropod inclusions (Perrichot
2005) and has been dated by dinoflagellate cysts
(Néraudeau et al. 2002; Dejax and Masure 2005).
The uppermost Albian-lowermost Cenomanian
amber from Cadeuil quarry, at Sainte-Gemme, is
the second richest fossiliferous amber and has been
dated both by sequence stratigraphy correlations
–
–
–
–
with the Archingeay-Les Nouillers quarry and by
ostracods (Néraudeau et al. 2008).
The uppermost Albian deposit from Puy-Puy
quarry, at Tonnay-Charente, has been dated
mainly by sequence stratigraphy correlations with
the previous quarries (Néraudeau et al. 2005). It is
relatively poor in amber, but has rich fossil plant
accumulations (Gomez et al. 2004).
The mid Lower Cenomanian of Fouras yields
amber from the tidal flat. The rocks here are
paralic deposits interbedded with limestones
containing the lower Cenomanian foraminifer
Orbitolina conica (Néraudeau et al. 2003).
The mid Lower Cenomanian amber from the tidal
flat of Aix Island corresponds to the same
stratigraphic level as that of Fouras and constitutes its continuation to the west (Néraudeau et al.
2009).
The mid Lower Cenomanian deposit from La
Buzinie corresponds to the same stratigraphic
level as the two previous localities, but is a
continuation of the outcrop several dozen kilometres to the east (Perrichot et al. 2007b).
The Middle Cenomanian deposits from Fourtou
are not well known. They are only accessible by
argillaceous mine debris rich in plant remains
(cuticles, wood). Fourtou stratigraphy is not
constrained.
Materials and methods
Fig. 1 Map of France showing the Cretaceous amber localities
studied
Most of the studied material comes from the collections of the Geosciences Rennes laboratory
(University of Rennes 1). This comprises a total of
about 50 kg of amber from the outcrops of Charentes
and Fourtou. These pieces were collected by the
authors or donated by amateur palaeontologists.
The pieces of amber range from tiny (a few
millimetres long) to big (15–20 cm in diameter) and
comprise intact or broken nodules of amber of
different colours. The amber from Charentes can be
sorted into four different categories: translucent and
yellow, more or less translucent and honey, opaque
and milky, translucent and red with a peripheral
brown to grey cortex of filamentous microorganisms.
Most amber from Fourtou is translucent and red with
rare opaque and brown samples.
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Sample preparation
Amber pieces are subjected to great surface contamination by recent microorganisms. Thus, samples
were treated to eliminate contamination using the
protocol described by Girard et al. (2009). They were
cleaned by ultrasound and then washed in 9–10%
H2O2 for 5 h to eliminate contaminant organic matter
(such as recent bacteria or mycelia), and finally
washed in 5% HF for 5 min to eliminate contaminant
inorganic matter (such as diatom frustules). Very thin
fragments of such treated samples were mounted on
slides with Canada balsam and then observed under a
Leica DLMP microscope. When necessary, immersion oil was used to observe more details. In some
cases, bigger fragments were observed directly under
the Leica DMLP microscope, without any embedding
in Canada balsam.
Measurement of pigment concentration
To identify the groups of microorganisms present, a
TriOs microFlu-blue probe was used to detect the
presence of preserved phycocyanin in the filaments.
This probe was placed 5 cm from the samples, and
the concentrations of phycocyanin (measured in lg/l)
were obtained for the peripheral cortex of prokaryotic
Fig. 2 Macroscopic
aspects of the peripheral
cortexes of sheathed
filamentous prokaryotes in
French Cretaceous ambers.
Arrows indicate the
presence of the peripheral
cortexes
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filamentous microorganisms and for the amber core.
The probe was calibrated on modern cyanobacterial
specimens. Concentrations between 0 and 100 lg/l
were measured, with the probe saturating when the
phycocyanin concentration was more than 100 lg/l
(Ahn et al. 2007; Gregor and Marsalek 2005; Brient
et al. 2008).
Results
Features of Mid-Cretaceous prokaryotic filaments
of French ambers
Macroscopic features
Although amber microinclusions are in general hard
to detect, the prokaryotic filaments within the MidCretaceous French amber are not. They generally
appear as a peripheral cortex surrounding a heart of
more or less translucent amber, although the overall
appearance varies in amber from different sites (Fig. 2).
Charentes In samples from these outcrops,
sheathed prokaryotic filaments appear in several
different forms. Commonly they appear as a cracked,
yellowish to greyish peripheral cortex, 2–10 mm
thick, around flattened red translucent amber pieces.
J Paleolimnol
In Archingeay-Les Nouillers, such amber pieces are
relatively abundant (10%), while they are rarer in the
other localities of Charentes. In Cadeuil, this type of
amber represents only 5%, and just 2% in Fouras. In
Aix Island, it is present, but in \1% of the amber
pieces. It has never been found in La Buzinie, nor in
the quarry of Puy-Puy. Sheathed prokaryotic filaments can occasionally be found as a thin (1–2 mm
thick) peripheral cortex around honey-coloured
amber at most sites, except in La Buzinie and in
the quarry of Puy-Puy. In La Buzinie, sheathed
prokaryotic filaments are very common, with 41% of
La Buzinie amber pieces having preserved sheathed
prokaryotic filaments. These always look like an
uncracked, very thick (1–1.5 cm) white cortex around
brown or milky amber pieces. Few pieces of amber
have been found in the quarry of Puy-Puy and most of
them contained sheathed prokaryotic filaments, macroscopically looking like those of La Buzinie outcrop.
Fourtou Sheathed prokaryotic filaments in this
locality appear either as discontinuous dark areas at
the periphery of red translucent amber pieces or in
brown and opaque milky amber pieces. In the latter
case, sheathed prokaryotic filaments have entirely
colonized the amber pieces. Table 1 recapitulates, for
each locality the different kinds amber in which
prokaryotic filaments have been preserved.
Table 1 Ambers that have provided sheathed filamentous
prokaryotes
Period
Upper Albian
Locality
Amber with
prokaryotes
filaments
Archingeay-les (1) flattened and red
Nouillers
(2) honey
Cadeuil
(1) flattened and red
Microscopic features
The growth of filaments is directional, with individual filaments initiating on the surface of the amber
and extending into the amber pieces. These sheathed
filaments are 4–12 lm in diameter. Small differences
exist among the different localities. Sheathed filaments from Fouras have a diameter between 8.5 and
12 lm, while in the other localities they are only
between 4 and 8.5 lm. These filaments divide
regularly by dichotomy. Near the branching points,
filament diameter increases a little. Branch angle
varies between 50° and 100° and no significant
variation of this character has been observed between
localities. The filaments are generally composed of
two parts, with a sheath surrounding a uniseriate cell
chain. The cell chain is frequently not preserved and
is replaced by a lumen of the same diameter. The
sheath appears more or less hyaline, often having a
dusty appearance. In amber from Charentes, the
sheath is divided into two layers, the outer one being
slimmer than the inner one. The cell diameter varies
from 0.9 to 1.8 lm and the cells are 1.2–2.3 lm long.
Rare examples of longer cells (2.5–3.5 lm diameter)
have been observed. Significant variations in the cell
diameter have been observed in Fouras, where cell
diameter is 1.4–1.8 lm while in the other localities it
is only 0.9–1.2 lm. No variation in cell length has
been observed from one locality to another. The
filament extremities are often marked by the presence
of a cell chain protruding from the sheath for a few
micrometers. Cell chains without a sheath occur very
often in the central part of the peripheral cortex. On
the other hand, the sheaths are more common in the
inner part of the cortex, at the boundary with hyaline
amber. No preparation shows heterocysts associated
with the sheathed filaments (Fig. 3).
(2) honey
Lower Cenomanian
Puy-Puy
(1) brown
(2) red
Aix Island
(1) flattened and red
(2) honey
Fouras
(1) flattened and red
(2) honey
La Buzinie
(1) brown
(2) red
Middle Cenomanian
Fourtou
(1) red
(2) opaque and brown
Pigment concentrations
Phycocyanin concentrations have been obtained for
diverse amber pieces containing sheathed prokaryotic
filaments. The results are summarized in Table 2. In
the absence of filamentous fossils, the amber shows a
background level of phycocyanin. This signal comes
from autofluorescence, and this may bias a possible
primary signal. Despite this, differences in phycocyanin concentration between the amber itself and the
part with filaments demonstrate that a primary signal
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Fig. 3 Microscopic aspects
of the sheathed filamentous
prokaryotes in French
Cretaceous ambers. a–c
Sheathed filamentous
prokaryotes from Charentes
ambers. d–e ‘‘Budding
filament’’ from Charentes
ambers. f–g Sheathed
filamentous prokaryotes
from Fourtou amber
has been preserved. In amber from Charentes,
phycocyanin concentration decreases from the filamentous cortexes to the amber itself. This shows that
filaments from these cortexes contain a substantial
concentration of fossil phycocyanin. In Fourtou
amber, phycocyanin concentration increases from
the dark areas full of filaments to the amber core.
This signal is due to a decrease of the autofluorescence of the amber because of the presence of
filaments.
Discussion
Affinities of the sheathed prokaryotic filaments
of SW French ambers
Microscopic appearances of the sheathed filaments
indicate that these fossils can be (1) sheathed
cyanobacteria (as seen in some Stigonematales) or
(2) sheathed bacteria (as in Clonothrix Roze 1896,
Crenothrix Cohn 1870, Lieskeella Perfiliev 1926).
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In 2005, Schmidt and Schäfer, followed by Breton
and Tostain, described two different fossil sheathed
filaments. Breton and Tostain (2005) described the
cyanobacterium P. cenomanensis from Ecommoy
amber (Western France, Cenomanian) and assigned it
to the Stigonematales due to its morphological
features and the fact that they observed wellpreserved blue cells typical of the cyanobacteria.
Schmidt and Schäfer (2005) described other sheathed
filaments in amber from Schliersee (southern
Germany, Cenomanian) as the sheathed bacterium
L. resinatus. They considered this fossil close to the
recent genera Leptothrix and Sphaerotilus, by its
morphology. Breton (2007) mentioned that these two
fossils are morphologically similar, with both presenting the same cell diameter. Sheathed filaments
from SW France are morphologically similar to these
two fossils and show similar diameter ranges for both
cells and sheath as P. cenomanensis and L. resinatus.
They also exhibit the same sheath characteristics as
these two fossils, with a granulated surface giving
a dusty appearance to the sheath, and regular
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dichotomic ramifications. It is difficult to assign the
sheathed prokaryotic filaments of SW French ambers
to one of these two fossils. Some preparations,
especially from Archingeay and Puy-Puy amber,
showed few cells that appear blue. These peculiar
cases indicate that the sheathed filaments from these
localities may represent P. cenomanensis rather than
L. resinatus. However these cases are too rare to be
diagnostic.
Phycocyanin concentrations are different in material from the Charentes localities and Fourtou.
Filamentous cortexes of amber from Charentes show
higher phycocyanin concentrations than the amber
(Table 2), which may reflect the fact that those
cortexes contain cyanobacteria. This material can
therefore be assigned to the genus Palaeocolteronema. The macroscopic appearance of those
filaments corroborates this conclusion. Both Ecommoy amber (Breton and Tostain 2005; Breton 2007)
and material from Charentes exhibit these sheathed
filaments as a more or less thick cortex around the
amber piece, the latter being more or less cracked
from one locality to another (Fig. 1). Graphing ‘‘cell
diameter versus sheath diameter’’ permits one to
differentiate two groups of filaments within the
Charentes sheathed filaments (Fig. 4). The first group
is composed of relatively small filaments (group A of
Fig. 4) and the second group is made up of bigger
filaments (group B of Fig. 4). These variations in the
cell and sheath diameters are not significant and do
not prove the existence of two different species of
Palaeocolteronema in Charentes localities, but may
reflect the fact that two different populations of
Palaeocolteronema co-inhabited these localities
during the Mid-Cretaceous.
In Fourtou amber, phycocyanin concentrations
decrease from the amber itself to the filamentous
cortex. It may reflect the fact that the sheathed
filaments of Fourtou amber are not related to
cyanobacteria, but to sheathed bacteria. Their morphology is similar to the Cenomanian sheathed
filaments of Schliersee amber (Schmidt and Schäfer
2005) and thus they can be assigned to the genus
Leptotrichites. Sheathed filaments of Fourtou mainly
appear as peripheral dark areas on red amber pieces.
Their macroscopic appearance is similar to Leptotrichites filaments of Schliersee amber, which appear
as peripheral white areas on tiny amber pieces
(Fig. 1-1 of Schmidt and Schäfer 2005).
Table 2 Pigment concentrations in the sheathed filamentous
prokaryotes from French Cretaceous ambers
Locality
Kind of
amber
Aix Island
?
Cortex
Milky
Fossil resin
70–80
Cortex
30
Cortex
[100
?
Archingeay-Les Red 1
Nouillers
Red 2
Red 3
Honey
La Buzinie
Brown
Red
Cadeuil
Red
Milky
Fourtou
Fouras
55–55
Fossil resin
50
Cortex
Fossil resin
[100
40–50
Cortex
70–80
Fossil resin
50–60
Cortex
90–100
Fossil resin
55–60
Cortex
35–40
Fossil resin
18–25
Cortex
60–70
Fossil resin
[100
Cortex
[100
Fossil resin
40–50
Cortex
[100
Fossil resin
[100
Cortex
[100
Red
Fossil resin
40–50
Brown
Cortex
?
20–40
20–30
Milky 1
Cortex
90–100
Milky 2
Milky 3
Puy-Puy
Pigment
concentration
(lg/l)
Fossil resin
50–60
Cortex
[100
Fossil resin
70
Cortex
[100
Fossil resin
[100
[100
Honey
Cortex
Honey
Fossil resin
75–80
?
Cortex
20–25
?
Cortex
20–25
Palaeoecology
Palaeocolteronema is the most common microorganism in amber of the Charentes localities. It is present
in more than 10% of the collected amber pieces from
Archingeay-Les Nouillers. More than 40% of La
Buzinie amber pieces have preserved filaments of
Palaeocolteronema, this locality being one of the
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Fig. 4 Comparison
between cell diameter and
sheath diameter of the
sheathed filamentous
prokaryotes from French
Cretaceous ambers
richest in this fossil cyanobacterium. It is less
common in the other localities: 5% in Cadeuil, 2%
in Fouras and less than 1% of the collected amber
pieces in Aix Island have preserved Palaeocolteronema filaments. In Puy-Puy quarry, few amber pieces
have been collected, but 80% of them contain
filaments of Palaeocolteronema, making this perhaps
the richest Palaeocolteronema locality known.
A diverse microbiota is associated with the Palaeocolteronema filaments in amber from Charentes.
Diverse freshwater testate amoebae have been found in
Archingeay-Les Nouillers, Aix Island, Cadeuil and
Fouras ambers (including several specimens of Centropyxis Stein 1857, one specimen of Leptochlamys
West 1901 and other still unidentified specimens).
Freshwater green algae of the genus Enallax appear to
always be associated with P. cenomanensis (Girard
2009). A ciliate of the genus Brachonella Jankowski
1964 was identified in Archingeay-Les Nouillers
amber and one related to the genus Pattersoniella
Foissner 1987 was discovered in Cadeuil amber.
Diverse freshwater actinomycetes and mycelia have
been preserved in amber from Charentes. Leptotrichites is the most common microorganism found in
Fourtou amber. More than 5% of the collected amber
pieces have preserved Leptotrichites filaments. Few
microorganisms have been found associated with these
Leptotrichites filaments. They correspond to a possible
testate amoeba (related to the genus Porosia?), an
unidentified naked amoeba, and a bacterial structure
that looks like bacterial mats found in stromatolites.
The Fourtou microassemblage is limited, but clearly
indicates a freshwater environment with the presence
of freshwater amoebae.
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Microorganism assemblages associated with both
Palaeocolteronema and Leptotrichites filaments indicate that they lived in wet forest microhabitats
(cf. Peyrot et al. 2005), with Palaeocolteronema
and Leptotrichites probably growing in aquatic
environments such as little ponds on the forest floor,
or in humid environments such as the forest litter or
in very wet bark.
Macroscopic and microscopic appearances of
Palaeocolteronema filaments (peripheral filamentous
cortexes around amber pieces) indicate that the resin
may have ended up in more or less deep ponds. The
resin stayed trapped in the cyanobacterial mat that
had developed on the pond surface. Presence of
bubbles on the sheath and the cells of Palaeocolteronema, similar to the ‘‘budding filaments’’ described
by Waggoner (1994a) and Breton and Tostain (2005)
in some preparations, may reflect the filament
trapping in ponds. Cyanobacteria continued to
develop and grow into the fresh resin, encrusting all
the surface of the resin flow and creating the
peripheral filamentous cortexes. Rikkinen and Poinar
(2000) inferred that such growth in resin had to be
possible for some fungi. Schmidt and Schäfer (2005)
cultivated Leptothrix filaments on conifer resins and
showed that filaments were able to grow in the resin
to a depth of 200–300 lm when resin dried slowly, as
it can in ponds. Similar phenomena are inferred to
have occurred within these ambers; some Palaeocolteronema filaments were in contact with the resin
when it fell, and filaments developed on the surface
before growing into the resin. Compound amber
fragments proved that sheathed filaments were
trapped in situ (on the forest floor) and not a long
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Fig. 5 Compound amber pieces from la Buzinie (a) and from
Archingeay-Les Nouillers (b) showing a filamentous cortex
composed of the cyanobacteria P. cenomanensis (arrows)
covered by a younger resin flow (F2; F1 being the first resin
flow). This exceptional amber piece proves that the peripheral
cortexes are not due to modern amber degradation
time after resin secretion, some of these filamentous
cortexes being recovered by another amber flow
(Fig. 5). Such kinds of cortexes must be produced in
the same place as the amber.
Macroscopic and microscopic appearance of
Leptotrichites filaments can indicate that the Leptotrichites filaments did not develop in ponds, but just
in humid environments such as forest litter or on wet
bark. No ‘‘budding filament’’ was observed in
Fourtou amber. The resin may have been secreted
or may have fallen down close to the place where
Leptotrichites filaments were present and so preserved a part of the filament mat as peripheral dark
areas on amber pieces. Schmidt and Schäfer (2005)
noticed that filaments of such bacteria are very fragile
and resin-engulfed filaments would be expected to
have been broken. Their integrity in Fourtou amber
may indicate that they grew into resin during an
undetermined time after resin secretion; the fact that
some Fourtou amber pieces have been completely
colonized by Leptotrichites filaments indicates that
this would have taken place over a long time period.
Schmidt and Schäfer (2005) showed that Leptothrix
filaments were able to grow for 200–300 lm into
conifer resin in 2 or 3 days. Leptotrichites filaments
are known to have colonized pluricentimetric amber
pieces and it is supposed that Leptotrichites filaments
grew in resin over a few weeks.
Conclusions
Prokaryotic filaments have been recorded from the
Precambrian (Knoll 1990). Unfortunately, they are
rarely fossilized and, despite their long history, few are
known as fossils. Amber offers a unique opportunity to
find new well-preserved fossils of prokaryotic filaments. Mid-Cretaceous amber of SW France is rich in
such microorganisms. The sheathed cyanobacterium
Palaeocolteronema was one of the main components
of the forest microcoenosis during the Mid-Cretaceous, preserved in Charentes amber (western France).
This cyanobacterium developed in freshwater forest
ponds when fresh resin flowed into and trapped some
filaments. These used the resin as a substratum and
developed all around the resin, creating the peculiar
cortexes observed on the Charentes amber. In comparison, Palaeocolteronema is not found in Fourtou
amber (southern France). In this locality, the main
component of this Middle Cenomanian microcoenosis
was the bacterium Leptotrichites. It appears that
Leptotrichites did not grow in ponds, but probably in
forest litter or on wet barks.
Schmidt and Schäfer (2005) postulate that Leptotrichites filaments found in Schliersee amber
(southern Germany, Cenomanian) were not trapped
in the amber, but grew into the fresh resin. Amongst
the sheathed filaments of the SW French ambers, it
appears that both phenomena co-existed. First, fresh
liquid resin may have trapped a part of the filament
mat in ponds with Palaeocolteronema and on forest
litter or on wet bark with Leptotrichites. Filaments
then grew on and in the resin, directing their growth
centripetally, before the resin solidified.
Acknowledgments This article is a contribution to the projects
Global Change IFB ‘Interactions biodiversité végétalechangements globaux à la transition Crétacé inférieursupérieur d’Europe occidentale’ and ANR AMBRACE (No.
BLAN07-1-184190) of the French Research Agency. We thank
Malvina Lak, Florentin Paris, Bernard Gomez (Univerity
of Rennes 1), Vincent Perrichot (Paleontological Institute,
Lawrence) and Alexander R. Schmidt (Museum für
Naturkunde, Berlin), Eric Depre (INRA, Surgères) for their
help and advice, and Charlie Underwood (Birkbeck College,
London) for improving the English of the original manuscript.
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