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2 Abstract: We report on a reticulated filament found in modern and fossil cave samples that cannot be correlated to any known microorganism or organism part. These filaments were found in moist environments in five limestone caves (four in New Mexico, U.S.A., one in Tabasco, Mexico), and a basalt lava tube in the Cape Verde Islands. Most of the filaments are fossils revealed by etching into calcitic speleothems but two are on the surface of samples. One hundred eighty individual reticulated filaments were imaged from 16 different samples using scanning electron microscopy. The filaments are up to 75 mm (average 12 mm) long, but all filaments appear broken. These reticulated filaments are elongate, commonly hollow, tubes with an open mesh reminiscent of a fish net or honeycomb. Two different cross-hatched patterns occur; 77% of filaments have hexagonal chambers aligned parallel to the filament and 23% of filaments have diamond-shaped chambers that spiral along the filament. The filaments range from 300 nm to 1000 nm in diameter, but there are two somewhat overlapping populations; one 200-400 nm in size and the other 500-700 nm. Individual chambers range from 40 to 100 nm with 30-40 nm thick walls. Similar morphologies to the cave reticulated filaments do exist in the microbial world, but all can be ruled out due to the absence of silica (diatoms), different size (diatoms, S-layers), or the presence of iron (Leptothrix sp.). Given the wide range of locations that contain reticulated filaments, we speculate that they are a significant cave microorganism albeit with unknown living habits.
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Abstract: We report on a reticulated filament found in modern and fossil cave samples
that cannot be correlated to any known microorganism or organism part. These
filaments were found in moist environments in five limestone caves (four in New Mexico,
U.S.A., one in Tabasco, Mexico), and a basalt lava tube in the Cape Verde Islands. Most
of the filaments are fossils revealed by etching into calcitic speleothems but two are on
the surface of samples. One hundred eighty individual reticulated filaments were imaged
from 16 different samples using scanning electron microscopy. The filaments are up to
75 mm (average 12 mm) long, but all filaments appear broken. These reticulated filaments
are elongate, commonly hollow, tubes with an open mesh reminiscent of a fish net or
honeycomb. Two different cross-hatched patterns occur; 77% of filaments have
hexagonal chambers aligned parallel to the filament and 23% of filaments have
diamond-shaped chambers that spiral along the filament. The filaments range from
300 nm to 1000 nm in diameter, but there are two somewhat overlapping populations;
one 200–400 nm in size and the other 500–700 nm. Individual chambers range from 40 to
100 nm with 30–40 nm thick walls. Similar morphologies to the cave reticulated
filaments do exist in the microbial world, but all can be ruled out due to the absence of
silica (diatoms), different size (diatoms, S-layers), or the presence of iron (Leptothrix sp.).
Given the wide range of locations that contain reticulated filaments, we speculate that
they are a significant cave microorganism albeit with unknown living habits.
Microbes are well known from cave systems (Danielli
and Edington, 1983; Northup and Lavoie, 2001; Barton et
al., 2001; Barton and Northup, 2007). Scanning electron
microscopy of the surface of speleothems commonly
reveals a variety of spheroid or filamentous features,
interpreted as either microorganisms or biofilms (mucous/
EPS) (Jones and Motyka, 1987; Jones and Kahle, 1986;
Jones, 2001; Vlasceanu et al., 2000; Baskar et al., 2006).
Etching calcitic samples with weak acid sometimes reveals
fossil microorganisms and/or biofilms that were entombed
in the calcite (Melim et al., 2001; Boston et al., 2001). We
report herein on an unusual reticulated filament found in
modern and fossil cave samples that cannot be correlated
to any known microorganism or organism part. Since all of
our samples are from caves, we cannot comment on their
possible wider distribution. We document here the
morphology, distribution and context of these reticulated
The samples for this study (Table 1) came from caves in
the Guadalupe Mountains of southeastern New Mexico,
from a lava tube in the Cape Verde Islands, and a cave in
Tabasco, Mexico (Fig. 1). All locations are from the
aphotic zone of the cave; all but one are from speleothems
that formed underwater (pool fingers) or in wet areas (cave
The Guadalupe Mountains include over 300 known
caves (DuChene and Martinez, 2000) administered vari-
ously by Carlsbad Caverns National Park, the National
Forest Service, and the Bureau of Land Management.
Hidden Cave and Cottonwood Cave are located in the
Guadalupe Ranger District of the Lincoln National Forest
in southeastern New Mexico (Fig. 2). The samples from
Hidden Cave are giant pool fingers (Fig. 3a), pendant
features that form underwater (Davis et al., 1990; Melim et
al., 2001). In Cottonwood Cave, the samples are thin pool
fingers with abundant u-loops; curved connections between
fingers (Fig. 3b; Davis et al., 1990). Both areas are
currently dry but the features formed when the pools were
full (unpublished data; Hill, 1987 Melim et al., 2001).
Endless Cave is located on Bureau of Land Management
property in the McKittrick Hill area of the Guadalupe
Mountains (Fig. 2). The sample is a warclub (Hill and
Forti, 1997) from the Warclub Room, a currently dry
room. A warclub forms when the end of a stalactite is
Dept. of Geology, Western Illinois University, Macomb, IL 61455, USA
Biology Dept., University of New Mexico, Albuquerque, NM 87131, USA
Institute of Meteoritics, University of New Mexico, Albuquerque, NM 87131, USA
Dept. of Earth and Atmospheric Sciences, University of Alberta, Edmonton, AB,
T6G 2E3, Canada
Dept. of Earth and Environmental Science, New Mexico Institute of Mining and
Technology, Socorro, NM 87801, USA
L.A. Melim, D.E. Northup, M.N. Spilde, B. Jones, P.J. Boston, and R.J. Bixby – Reticulated filaments in cave pool speleothems:
microbe or mineral? Journal of Cave and Karst Studies, v. 70, no. 3, p. 135–141.
Journal of Cave and Karst Studies, December 2008 N135
submerged by a rising pool level and is then coated in pool
spar. Our sample is a surface piece of this pool spar. Two
more samples came from Carlsbad Cavern in Carlsbad
Caverns National Park (Fig. 2). One sample is a thin pool
finger from an active pool, whereas the other is webulite
from a dry pool in Lower Cave, a portion of the cave
generally closed to the public. Webulite is a thin draping
calcite web that connects adjacent pool fingers (Davis et
al., 1990, Queen and Melim, 2006).
The Cape Verde sample is from a small, unnamed lava
tube on Fogo Island. The sample came from a purple and
white crust on the wall of the basalt lava tube (Peter Roe,
personal communication). The Mexican sample is a cave
pearl collected from Cueva de Las Canicas, a cave in
Tabasco famous for containing millions of cave pearls
(Pisarowicz and Snow, 2003).
All samples were collected under permit from the
appropriate agency (U.S. National Park Service, U.S.
Forest Service, and Bureau of Land Management) or
landowner permission (Sr. Arturo Cano, Cueva de Las
Can˜icas) and were selected from those broken by past
visitors to avoid disturbance of intact speleothems. Thin
sections were cut from all samples from Hidden Cave,
Cottonwood Cave, and Carlsbad Cavern and examined for
possible microbial fabrics in a standard petrographic
microscope. The cave pearl was cut in half, ground flat,
and then etched. For SEM analysis, samples were either
etched with 5% hydrochloric acid for 10–15 seconds before
rinsing in distilled water, drying and mounting on SEM
stubs (Hidden Cave, Cottonwood Cave, Carlsbad Cavern
Table 1. List of locations containing reticulated filaments.
Cave Location Speleothem Collector
Number of
Imaged Filaments
Hidden Cave Guadalupe Mountains, New
Mexico, USA
Pool fingers Authors 92
Cottonwood Cave Guadalupe Mountains, New
Mexico, USA
Pool fingers and U-loops Authors 65
Endless Cave Guadalupe Mountains, New
Mexico, USA
Warclub Authors 9
Carlsbad Cavern Guadalupe Mountains, New
Mexico, USA
Webulite Authors 5
Cueva de Las Canicas Tabasco, Mexico Cave pearls Arturo C. Conde 2
Fogo Island, Basalt
Lava Tube
Cape Verde Islands Coating on basalt lava
Peter Roe 11
Figure 1. World map of locations where reticulated filaments have been found by this study: 1. Guadalupe Mountains, New
Mexico, U.S., 2. Tabasco, Mexico, 3. Cape Verde Islands. Also shown are locations for two examples found by other workers:
4. Poland, Gradzinski (2003); 5. Frasassi Caves, Italy, Macalady (pers. comm.).
136 NJournal of Cave and Karst Studies, December 2008
and Cueva de Las Canicas) or mounted without etching
(Cape Verde lava tube and Endless Cave).
All samples were examined with either a JEOL 5800LV
scanning electron microscope at the University of New
Mexico (UNM) or a JSM 6301FXV field emission
scanning electron microscope at the University of Alberta.
The UNM samples were coated with approximately 200
angstroms of gold-palladium alloy in an evaporative
coater. The thin film of gold-palladium provides a
conductive layer that is relatively free of artifacts and
allows light element x-rays of carbon to pass with only
moderate attenuation. The University of Alberta samples
were coated in gold alone, which resulted in minor artifacts
at higher magnification. The UNM SEM is equipped with
an Oxford Isis 300 Energy Dispersive X-ray (EDX)
analyzer. This modern EDX system utilizes a thin
polymer-film window, which allows the analysis of low-
energy x-rays of light elements such as boron and carbon
(Z .5). The SEM was operated at 15 kV accelerating
voltage, the beam current was 10 picoamps as measured in
a Faraday cup. This gives a beam diameter of less than
50 nm. However, the beam samples a greater volume. With
these operating conditions, the EDX provides a qualitative
estimate of elements present in the upper 2–3 mmofa
calcitic sample.
Three samples were analyzed for total organic carbon
using a Carlo Erba elemental analyzer from samples
dissolved in 6 N hydrochloric acid to remove carbonate.
Smooth, commonly hollow, filaments are locally
abundant in our cave samples and have been interpreted
as fossil microbes (Melim et al., 2001; Boston et al., 2001).
We have also found a less common reticulated filament
that is similar in size to the smooth filaments but has a
reticulated form (Fig. 4). Over 180 individual examples of
these unusual reticulated filaments were found in 16
different cave samples (out of 22 samples examined in this
study). Most (87%) of these filaments are in pool fingers
from Hidden and Cottonwood Caves (Figs. 2 and 3), but
this at least partly reflects the extended time we have spent
with these samples. In addition, Gradzinski (2000, 2003)
imaged an example from Polish cave pearls and J.
Macalady has found them in the Frasassi caves in Italy
(Macalady, personal communication).
Filaments are preferentially found in dense micritic
calcite, but can also occur in clear spar (Melim et al., 2001).
All but two of our samples come from cave pool biothems,
Figure 2. Locations of major caves in the Guadalupe
Mountains. We have found reticulated filaments in samples
from Hidden Cave, Carlsbad Cavern, Cottonwood Cave, and
Endless Cave. (Map, after Palmer and Palmer, 2000.)
Figure 3. (a) Giant pool fingers in Hidden Cave, NM. (b) Small pool fingers coating an earlier stalactite in Cottonwood
Cave, NM (Spanish moss of Hill, 1987).
Journal of Cave and Karst Studies, December 2008 N137
features with external morphology suggestive of microbial
involvement (Cunningham et al., 1995; Queen and Melim,
2006). This, however, could be an artifact of our study, as
we have not closely examined many other cave pool
precipitates, other speleothems or similar features in non-
cave environments. Other speleothems or other environ-
ments may contain similar filaments, but etching of
samples and meticulous, very high magnification SEM
work is required to find them.
Reticulated filaments have an open cross-hatched
pattern reminiscent of a fish net or a honeycomb (Fig. 4).
Preservation of filaments varies substantially between
individuals. The best preserved are hollow tubes that are
partly collapsed (Fig. 4a). Others are torn open (Fig. 4b) or
completely filled with calcite (Fig. 4c). Recently living
filaments are hollow tubes (Fig. 4d). The filaments are up
to 75 mm (average 12 mm) long. The measured length,
however, is largely an artifact of preservation as virtually
all of the specimens are torn, broken or have their ends
buried in the matrix. Filament diameter is 300–1000 nm
(average 590 nm) but this overall range encompasses two
overlapping populations; one with an average diameter of
200–400 nm and the other with an average diameter of
500–700 nm. Both populations are found in the same
samples (Fig. 5).
The filaments are characterized by two different styles
of cross-hatched patterns, both with individual chambers
40 to 100 nm long and walls between chambers that are 30–
40 nm wide. The larger diameter filaments typically have
larger chambers, but not always. The more common form
(77% of imaged filaments) has approximately hexagonal
chambers that align in rows parallel to the length of the
filament (Figs. 4b, 5). The less common form (23%) has
more diamond-shaped chambers that spiral along the
filament (Fig. 4c). The cross-hatch pattern does not
correlate to filament size.
In etched samples, the reticulated filaments occur within
the sample and are partially revealed by the removal of the
surrounding calcite (Fig. 4). Preliminary data show 1–2%
organic carbon in the sample containing the most filaments
(from Cottonwood Cave). EDX analysis of individual
Figure 4. Scanning electron micrographs of reticulated filaments. (a) Hollow reticulated filament with diamond-shaped
chambers that form a spiral. Hidden Cave pool finger, etched sample. (b) Reticulated filament that is hollow and torn open,
with chambers that align along length of filament. Hidden Cave pool finger, etched sample. (c) Reticulated filament that is solid
with diamond-shaped chambers that spiral. Hidden Cave pool finger, etched sample. (d) Reticulated filament from recently
living sample (not etched). Note similarity to etched samples, particularly (a). Cape Verde lava tube.
138 NJournal of Cave and Karst Studies, December 2008
filaments commonly shows calcium, oxygen, and more
carbon than the surrounding calcite (Fig. 6). Since the
depth of analysis (2–3 mm) is greater than the thickness of
the filament, the results are a mixture of the surrounding
calcite and the filament. We speculate that the extra carbon
found is either a coating on the filament, protecting it from
the acid etch (Melim et al., 2001), or the filaments are
simply composed of a carbon-rich (hence organic) material
and the etch has removed the surrounding calcite. Since the
filaments from surface samples (not etched, Fig. 4d) and
the embedded samples (etched out, Fig. 4a–c) have
identical textures, it seems more likely that the filaments
are preserved organic material and not carbon-coated
calcitized filaments.
The morphology and high carbon content of these
filaments suggests that they are biogenic. An extensive
survey of known microorganisms and associated struc-
tures, and consultation with colleagues who work with a
number of different microbial forms, however, shows
nothing similar to the reticulated filaments in the same
size range. Although the general filament shape is a
common morphology, reticulated chambers are not. We
are uncertain as to the origin of the reticulated chambers,
as known microbes lack any structure of comparable size.
Larger chambers are common; for example, some diatoms
(Bacillariophyceae) have similar complex structures in their
cell walls (areolae), but they are far larger in scale and very
different in aspect ratio. Filamentous diatoms such as those
in the genus Aulacoseira have similar cell morphology with
regular patterns of pores termed areolae, but these
filaments are significantly larger in diameter (3–30 mm
Figure 5. Scanning electron micrographs showing the two different sizes of reticulated filaments with parallel hexagonal
chambers. Note the scale is identical in these images. (a) Larger filament. (b) Smaller filament. Hidden Cave pool finger.
Figure 6. Scanning electron micrograph of reticulated
filament and two EDX analyses; one centered on the filament
and one centered on the calcite next to the filament. The
analyses extend 2–3 mm into the sample so both plots show
calcite (Ca +C+O), trace amounts of clays (Mg +Si +O)
and the gold-palladium coating medium (Au +Pd). Note that
the filament contains more carbon than the calcite sample
indicating that the filament itself is carbon-rich.
Journal of Cave and Karst Studies, December 2008 N139
diameter, 2.5–24 mm mantle height; Krammer and Lange-
Bertalot, 1991). In general, diatoms in valve view range
from eight or ten microns to a millimeter in length and are
commonly 10–20 microns in diameter. At the extreme size
ranges, diatoms can be as small as one micron in diameter
(rarely) and as wide as 30–40 microns in diameter (Round
et al., 2000). Some diatoms, including Aulacoseira, produce
resting cells of similar sizes to the normal cells (Round et
al., 2000). Thus, there is only the most marginal overlap in
size with our observed structures. More importantly, all
diatoms contain biogenic silica as a major component of
their cell walls (Round et al., 2000). The reticulated
filaments found in the cave samples completely lack silica.
Fungi, especially the filamentous, microscopic forms,
were eliminated from consideration due to their larger size,
generally two microns in diameter or larger. Thus, while
they are often tens of microns in length, their diameter is
insufficiently small to qualify as a bona fide candidate. The
fungal morphologies we examined, or with which we are
experienced, lack this form of reticulated patterning.
Actinomycetes, a filamentous group of bacteria, com-
monly occur in caves, are tens of microns in length at times
and have similar diameters, but all known strains that we
have investigated in the literature lack this kind of
reticulated patterning. The authors have examined many
cultures and environmental samples of actinomycetes using
scanning electron microscopy and have never observed this
morphology nor seen it in the literature
There is the possibility that the reticulate structure is a
form of S-layer, a symmetrical arrangement of hexagonal
protein units in the outer cell surface layer that has been
observed in some Gram-negative bacteria (e.g. Schultze-
Lam et al., 1992) and in Archaea (Messner et al., 1986).
Interestingly, S-layers have a cross-hatched pattern when
imaged using TEM (Phoenix et al., 2005) that is akin to the
chamber patterns found in the reticulated filaments. The
hexagonal units of the S-layer, however, are generally
,50 nm wide (Messner et al., 1986; Schultze-Lam et al.,
1992; Phoenix et al., 2005) and are therefore smaller than
70–100 nm long hexagonal units found in the reticulated
filament. In the reticulate filaments, the center-to-center
distance between adjacent chambers is generally two to
three times larger than found in S-layer units. In addition,
images of S-layers give the impression of a solid lattice
layer rather than the open chambers of the reticulated
filaments that we have observed in our cave samples.
In reviewing bacteria described in Bergey’s Manual of
Systematic Bacteriology (Holt, 1984–1989; Garrity, 2001–),
only three bacteria were found with a similar morphology.
The first of these is Nitrosomonas, which is pictured with
‘‘an additional cell wall layer’’ with units that are ,20 nm
(see Holt 1984–1989, p. 1824). The units in this extra cell
wall layer in Nitrosomonas are three to four times smaller
than the hexagonal units found in the cave reticulated
filaments and also give the impression of a solid layer.
Nitrosomonas’ additional layer may represent an S-layer, as
it is similar in morphology. The image of Prosthecomicro-
bium polyspheroidum (Garrity, 2001–) depicts cells with
numerous short prosthecae that give the appearance of a
corn cob, superficially resembling the reticulate structures,
but the comparison breaks down on closer inspection. The
third possibility is an image of Helicobacter bilis (Garrity,
2001–) with ‘‘tightly wound periplasmic fibers and multi-
ple sheathed flagella.’’ Although this organism is similar
in diameter, it is only three microns in length.
In a study of putative Leptothrix sp. from a pool
enriched in iron oxides in Carlsbad Cavern, Caldwell and
Caldwell (1980) described filaments with ‘‘a hexagonal
matrix over the surface of the cells.’’ These hexagonal
subunits, 0.1 microns in diameter, are more irregular in
shape and are composed of iron, thus ruling out Leptothrix
as a candidate for our filaments.
Thus, similar morphologies to the cave reticulated
filaments do exist in the microbial world, but all can be
ruled out due to the presence of silica (diatoms), size
(diatoms, S-layers), or the presence of iron (Leptothrix sp.),
leading us to conclude that this is a heretofore unreported
morphology probably bacterial in nature.
Reticulated filaments are common in speleothems from
moist or wet environments. These reticulated filaments are
tubes of cross-hatched mesh with either hexagonal or
diamond-shaped chambers. They are up to 75 mm in length
(avg. 12 mm) but this size range is partly an artifact of
preservation. Two overlapping populations occur, one
200–400 nm in diameter and the other 500–700 nm. EDX
indicates that the filaments are composed of predominantly
carbon, hence they are not mineral. We speculate that
reticulated filaments are from an unknown, but possibly
common, subsurface type of microorganism or group of
microorganisms that prefer moist cave environments. We
continue to look for more examples, particularly living
examples, in the hope of culturing and/or obtaining DNA
in order to more precisely identify their phylogenetic
position and to understand their role in the cave ecosystem.
This material is based upon work supported by the
National Science Foundation under Grants No. 0719710,
0719507 and 0719669. We thank the U.S. Forest Service
for permission to sample in Hidden and Cottonwood
Caves, the U.S. National Park Service for permission to
sample in Carlsbad Caverns National Park, and the U.S.
Bureau of Land Management for permission to sample in
Endless Cave. In addition, special thanks are owed to
USFS employees Jerry Trout, Ransom Turner, Mike Baca,
Deanna Younger, and Kevin Glover; USNPS employees
Dale Pate, Paul Burger, and Jason Richards; and BLM
140 NJournal of Cave and Karst Studies, December 2008
employee James Goodbar. J. Michael Queen, Ginny Rust,
Neil Shannon and Andy Brehm all assisted in the field
work. Special thanks to Susan Herpin, who managed to
train L. Melim in vertical work for entering Hidden Cave
despite the odds and who assisted with field work and
other logistics. Western Illinois students Kristen Shinglman
Woody, Neil Shannon and Randi Liescheidt assisted in the
UNM SEM work. Peter Roe collected the sample from the
Cape Verde Islands. We extend our gratitude to Sen˜or
Arturo Cano Conde, owner of Cueva de Las Can˜ icas, in
Teapa, Mexico, for cave pearl samples. Many thanks to
George Braybrook who took the SEM images at the
University of Alberta. Special thanks to Kenneth Ingham
who took many great photographs of our sampling sites
and pool precipitates in the field. Figure 3 images courtesy
of K. Ingham.
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ogy Journal, v. 17, p. 125–139.
Journal of Cave and Karst Studies, December 2008 N141
... The study of the samples by field emission scanning electron microscopy (FESEM) revealed the presence of filamentous microorganisms in the vermiculations of Morgana Cave ( Figure 3). Most of the microbial features observed in samples M1, M3, M4, M5, and M6 resemble the enigmatic reticulated filaments reported in caves worldwide (Melim et al., 2008;Miller et al., 2012), which are long tubular filaments with peculiar surface ornamentation (Miller et al., 2012;Melim et al., 2015). In general, these long filaments (<100 µm) were found embedded in a slimy matrix of extracellular polymeric substances ( Figures 3A,D,F). ...
... In both types of vermiculations, EPS and reticulated filaments were evident. These enigmatic filaments have been found in microbial mats from caves worldwide, such as limestone caves and basaltic lava tubes (Melim et al., 2008;Jones, 2009Jones, , 2010Jones, , 2011Miller et al., 2014;Riquelme et al., 2015a), as well as in a granite spring water tunnel in Portugal (Miller et al., 2012). They were characterized as long microbial filaments with hexagonal and diamond-shaped chambers resembling honeycombed structures, but attempts to decipher their taxonomic affiliation have failed thus far. ...
... December 2020 | Volume 8 | Article 586248 Melim et al. (2008) reported long reticulated filaments (up to 75 μm long and 0.5 μm in diameter) in calcite speleothems from limestone caves in New Mexico, and in lava tubes from Cape Verde Islands. (Jones 2009(Jones , 2010(Jones , 2011 described the occurrence of reticulated filaments in caves from the British West Indies, and suggested they are calcified filaments. ...
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Morgana Cave is located in Val di Scerscen, Central Italian Alps. The cave opens at an altitude of 2,600 m a.s.l. close to the retreating glacier Vedretto di Scerscen, and its entrance was discovered 30 years ago hidden underneath the glacier. A characteristic of this cave is the occurrence of vermiculation deposits on the walls and ceiling. In general, the composition of the microbial communities in cave vermiculations is relatively unknown and rarely investigated. Here we present the data of a geomicrobiological study of vermiculations from an Alpine cave subjected to extreme climate conditions. The microbial communities were dominated by 13 main phyla of Bacteria, and contained a negligible percentage (<1%) of Archaea. The two major bacterial classes were Gammaproteobacteria and Betaproteobacteria, whose metabolic traits were mainly associated with the nitrogen cycle. In addition, psychrophilic and methanotrophic bacterial groups were identified. The occurrence of a large number of uncultured members, at the lowest taxonomic ranks, indicated the presence of still unexplored microbial taxa in the vermiculations.
... These bioforms were found embedded in a slimy matrix of EPS, which can provide nucleation sites for the precipitation of minerals, such as NFC (Cañ averas et al., 2006;Cuezva et al., 2012;Miller et al., 2012;Riquelme et al., 2015). iScience Article long filamentous forms with 0.5 mm in diameter and >50 mm in length, similar to the reticulated filaments reported by Melim et al. (2008) and Miller et al. (2012) These filaments were widely observed on the surface of the coralloid speleothems in association with EPS ( Figure 5F, arrows). Reticulated filaments have been frequently reported in caves worldwide and have been associated with biomineralization of opal-A in lava tubes (Miller et al., 2014), calcium in limestone caves (Jones, 2009;Melim et al., 2008), and manganese oxides in a granite spring water tunnel (Miller et al., 2012). ...
... iScience Article long filamentous forms with 0.5 mm in diameter and >50 mm in length, similar to the reticulated filaments reported by Melim et al. (2008) and Miller et al. (2012) These filaments were widely observed on the surface of the coralloid speleothems in association with EPS ( Figure 5F, arrows). Reticulated filaments have been frequently reported in caves worldwide and have been associated with biomineralization of opal-A in lava tubes (Miller et al., 2014), calcium in limestone caves (Jones, 2009;Melim et al., 2008), and manganese oxides in a granite spring water tunnel (Miller et al., 2012). Yet, they could not be affiliated to any known microorganism, as most of these filaments are found as hollow mineralized sheaths (Miller et al., 2012). ...
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The network of lava tubes is one of the most unexploited natural wonders of the Galapagos Islands. Here, we provide the first morphological, mineralogical, and biogeochemical assessment of speleothems from volcanic caves of the Galapagos to understand their structure, composition, and origin, as well as to identify organic molecules preserved in speleothems. Mineralogical analyses revealed that moonmilk and coralloid speleothems from Bellavista and Royal Palm Caves were composed of calcite, opal-A and minor amounts of clay minerals. Extracellular polymeric substances, fossilized bacteria, silica microspheres and cell imprints on siliceous minerals evidenced microbe-mineral interactions and biologically-mediated silica precipitation. Alternating depositional layers between siliceous and carbonate minerals and the detection of biomarkers of surface vegetation and anthropogenic stressors indicated environmental and anthropogenic changes (agriculture, human waste, cave visits) on these unique underground resources. Stable isotope analysis and Py-GC/MS were key to robustly identify biomarkers, allowing for implementation of future protection policies.
... Análisis moleculares de las comunidades microbianas que colonizan estos ambientes subterráneos sugieren la participación de distintas clases de microorganismos, incluidos los microorganismos fototróficos, como cianobacterias y algas microscópicas en zonas donde incide la luz solar o artificial (Albertano y Urzì, 1999;Hernández-Mariné et al., 2001), una variedad de bacterias (Schabereiter-Gurtner et al., 2002, 2004 y hongos (Martin-Sanchez et al., 2012). Además, un cierto número de estudios de geomicrobiología de cuevas han revelado la presencia de morfologías microbianas que no se relacionan con ninguna forma de vida o estructura de algún organismo conocido (Melim et al., 2008;Northup et al., 2011), o representan nuevas especies de microorganismos no descritas anteriormente (Jurado et al., 2006. Al mismo tiempo, la creciente información sobre la ocurrencia y distribución de microorganismos en ambientes subterráneos está cambiando la actual percepción sobre estos ecosistemas (Frierdich et al., 2011;Northup et al., 2011). ...
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“Las cuevas turísticas como activos económicos: conservación e innovación” es un libro donde se recogen los resultados de trabajos de investigación básica, de innovación, gestión, conservación y planificación que se han realizado o se están llevando a cabo en cavidades turísticas de España, Francia y Portugal. Está constituido por las contribuciones presentadas en el IV Congreso español sobre cuevas turísticas (CUEVATUR), celebrado en Aguilar de Campoo (Palencia) del 18 al 20 de octubre de 2012. Es la cuarta edición de CUEVATUR, en la que se recogen 36 trabajos. Los trabajos presentados y que recopila esta obra abarcan estudios en todo el ámbito de la geografía nacional, incluidos el archipiélago canario y balear, con sendas portaciones. La distribución geográfica de los trabajos es amplia, con 13 comunidades autónomas representadas. Algunas regiones, como Andalucía con 10 aportaciones, Cantabria (3), Murcia (3) o la anfitriona, Castilla León (3), con especial énfasis en la provincia de Palencia, donde se sitúa la cueva de los Franceses, suponen más de la mitad del total de la obra. Pero además, esta monografía cuenta también con un número importante de contribuciones que abordan el conocimiento y estudio de las cuevas en general, sin una ubicación geográfica determinada, con 6 trabajos recogidos. Esta libro es, por tanto, un compendio actualizado de los diferentes aspectos abordados en el ámbito de las cuevas turísticas por técnicos e investigadores de numerosos organismos públicos de investigación, universidades, administraciones públicas y empresas públicas y privadas. Todos ellos aunando esfuerzos en ampliar el conocimiento, la la conservación y la innovación en el ámbito de las cuevas turísticas y de su territorio más próximo.
... Interestingly, sample from "Grotta Lunga" (GL_1) showed the presence of reticulated filaments embedded in a matrix of EPS (Fig. 5A, B). The presence of reticulated filaments has been frequently reported in caves worldwide, in both lava and limestone caves [49][50][51][52] but their nature still results enigmatic for microbiologists [54]. FESEM observations of the sample GL_3 revealed a variety of actinobacteria-like filamentous forms spread all over the sample (Fig. 5C, D). ...
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While microbial communities in limestone caves across the world are relatively understood, knowledge of the microbial composition in lava tubes is lagging behind. These caves are found in volcanic regions worldwide and are typically lined with multicolored microbial mats on their walls and ceilings. The Mount Etna (Sicily, S-Italy) represents one of the most active volcanos in the world. Due to its outstanding biodiversity and geological features, it was declared Natural Heritage of Humanity by the UNESCO in 2013. Despite the presence of more than 200 basaltic lava tubes, the microbial diversity of these hypogean systems has never been investigated so far. Here, we investigated bacterial communities in four lava tubes of Mount Etna volcano. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features. We documented an abundant presence of microbial cells with different morphotypes including rod-shaped, filamentous, and coccoidal cells with surface appendages, resembling actinobacteria reported in other lava tubes across the world. Based on 16S rRNA gene analysis, the colored microbial mats collected were mostly composed of bacteria belonging to the phyla Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria. At the genus level, the analysis revealed a dominance of the genus Crossiella, which is actively involved in biomineralization processes, followed by Pseudomonas, Bacillus, Chujaibacter, and Sphingomonas. The presence of these taxa is associated with the carbon, nitrogen, and ammonia cycles, and some are possibly related to the anthropic disturbance of these caves. This study provides the first insight into the microbial diversity of the Etna volcano lava tubes, and expands on previous research on microbiology of volcanic caves across the world.
... Other biofilm structures resemble sheets that have had holes poked in them, some creating geometric patterns. Some of the shapes observed overlap with those seen from carbonate caves and include the reticulated filaments [74,75] and the beads-on-a-string [76]. Lava cave microbial mat SEM photomicrographs provide great insights into microbial mat structure, but also provide an opportunity for interpretive materials for cave visitors as the images reveal that the walls truly are alive. ...
... In this case, thin section fabrics were closely tied to the SEM, differing only by material lost to the saw blade kerf (ca 1 mm). Most samples were etched with 5% HCl, using the method of Melim et al. (2008Melim et al. ( , 2016. A few samples were examined on freshly broken surfaces or polished thin sections to control for etching artifacts. ...
Speleothems form layered archives of the climate and local cave conditions during their formation. The origin of layering in cave pearls, however, is not well‐understood. Cave pearls grown in two adjacent mine sites between 2006 and 2014 elucidate the complexity of speleothem growth and recrystallization. Site A cave pearls grew under an active drip, while Site B cave pearls grew in small rimstone‐dam pools that filled with reverse‐graded fitted pearls between about 2009 and 2014. Despite the variation in pool setting, all samples are layered in grey and/or brown laminations and dendrites. The order and number of these layers varies widely, even between pearls growing millimetres apart in the same pool. However, stable isotope values reflect homogenized local precipitation. The variability between adjacent samples supports control by very local factors within each pool, likely related to CO2 degassing at the water–air interface and water flow within the confined space of each pool. Recrystallization of calcite to calcite occurs resulting in triangular microspar patches and much less obvious bladed calcite. Laminations of brown or grey 1 to 5 µm calcite crystals recrystallize to bladed calcite up 100 µm long, all the while retaining a memory of the original layers in the form of ‘ghost’ layers, as revealed by gentle acid etching. Pearls at the top of rimstone‐dam pools grew faster than those just a few millimetres deeper, resulting in reverse grading. This model is applicable to reverse grading in marine and lacustrine pisolites. This study suggests cave pearls in active flow regimes (drips or currents) are similar and largely abiogenic, in contrast to other locations with less flow, where more biological input is common. Recrystallization of calcite to calcite proceeds not only to equant spar (classic Ostwald ripening), but also to bladed calcite. Thus, bladed calcite in speleothems needs to be carefully evaluated for recrystallization even when aragonite is absent.
... FESEM observations of Royal 2 sample showed abundant microbial structures and extracellular polymeric substances (EPS) associated with the coralloid stalactites ( Figure 3). Long reticulated filaments, resembling those reported by Melim et al. [25] and Miller et al. [16,26], were widely observed on the surface of the coralloid speleothems ( Figure 3A-C). These enigmatic filaments have been frequently reported in caves worldwide, but their origin is still unknown. ...
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The Galapagos Islands (Ecuador) have a unique ecosystem on Earth due to their outstanding biodiversity and geological features. This also extends to their subterranean heritage, such as volcanic caves, with plenty of secondary mineral deposits, including coralloid-type speleothems and moonmilk deposits. In this study, the bacterial communities associated with speleothems from two lava tubes of Santa Cruz Island were investigated. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features associated with moonmilk and coralloid speleothems from Bellavista and Royal Palm Caves. Microbial cells, especially filamentous bacteria in close association with extracellular polymeric substances (EPS), were abundant in both types of speleothems. Furthermore, reticulated filaments and Actinobacteria-like cells were observed by FESEM. The analysis of 16S rDNA revealed the presence of different bacterial phylotypes, many of them associated with the carbon, nitrogen, iron and sulfur cycles, and some others with pollutants. This study gives insights into subsurface microbial diversity of the Galapagos Islands and further shows the interest of the conservation of these subterranean geoheritage sites used as show caves.
... Also SEM images of the microstructures in moonmilk from the Proschalnaya Cave were similar to calcitic nanofibres, needle fibre calcite, tubular-and filament-like structures in other scientific literature (Shankar and Achyuthan, 2007;Maciejewska et al., 2015). However, the tubular nanostructures found by us are not similar to the reticulated filaments described earlier (Melim et al., 2008). ...
... Accordingly, clusters of cells, spores and organic filaments, mostly associated with clay deposits, were commonly observed in all the analyzed samples (Fig. 7). Interestingly, some of the filaments (Fig. 7B) can be classified as "enigmatic reticulated filaments" (Melim et al., 2008(Melim et al., , 2015Northup et al., 2011;Miller et al., 2012Miller et al., , 2014: honeycomb hollow tubular structures thought to be microbial sheets belonging to still unidentified microorganisms (Melim et al., 2015). The traces of microbial activity embodied in the dissolution morphologies and the secondary minerals, with evidence of diverse microbial communities in the vermiculations from the Pertosa-Auletta Cave, support the hypothesis of an interaction between biogenic and abiogenic processes in the development of vermiculations (Dong et al., 2009;Cuadros, 2017), paving the way to the understanding of how the environment influences these processes. ...
Unlike the spectacular speleothems that can often be found in numerous caves, vermiculations are rather un-assuming formations, whose origin and evolution still subject of several heated debates. In order to provide a quantitative basis for the understanding of the nature and evolution of vermiculations in karst environments, the geochemical properties of one of the most important karst systems of southern Italy, the Pertosa-Auletta Cave, were studied through a comprehensive approach which included elemental, mineralogical and microscopyanalyses. The chemical element abundances, mineral composition and microstructure of the vermiculations covering the entire range of morphologies and colours observed in the case-study cave were investigated, thus providing the first quantitative record of these traits.The vermiculations presented exceptional diversity in their morphology, colour, chemical and mineral composition,with it being due to exogenous determinants such as the deposition of stream sediments ororganic matter as well as the development of photoautotrophic communities. They were invariably composed of calcite, associated to quartz as well as clays and other secondary minerals, the formation of which may be biologically mediated. This occurrence, and the evidences of microbial activity observed through dissolution traces,support the possible involvement of biogenic processes in vermiculation development.
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Many helictites in Lechuguilla Cave, New Mexico, show conclusive evidence for a subaqueous origin. Some are apparently still growing, or remain in the environment in which they grew. Helictites have previously been interpreted as exclusively subaerial in origin. Growth of the subaqueous helictites is triggered by the common-ion effect, where calcite-saturated water comes in contact with gypsum blocks. Dissolution of gypsum rapidly drives the water to supersaturation with calcite, forming helictites if the water enters a pool as discrete strands. Another previously undescribed speleothem type, tentatively named "pool fingers' is also subaqueous in origin. They are elongate growths of calcite that appear to coat organic filaments. Many are connected by curved bridges ("U-loops'). A third variety of speleothem, rarely described in the literature, consists of iron-oxide stalacites and columns lined with calcite. At least part of their origin was subaqueous. The iron oxide coats organic (bacterial?) filaments associated with oxidation reactions. -Authors
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The Guadalupe Mountains of New Mexico and west Texas are a northeast-tilted fault block cut by canyons that increase in frequency and topographic relief from east to west. The processes of erosion and mass wasting have exposed more than 300 known caves, which range from systems like Lechuguilla Cave (>170 km) and Carlsbad Cavern (>49 km) in the east, to caves with less than 10 m of passage in the west. Erosion of the Capitan, Yates and Seven Rivers formations progressively removed more cave-bearing strata and destroyed more caves from east to west. It is likely that modern-day canyons in the central and western Guadalupe Mountains were once sites of long cave systems that have been truncated or destroyed by erosion and mass wasting.
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The Karst Waters Institute Breakthroughs in Karst Geomicrobiology and Redox Geochemistry conference in 1994 was a watershed event in the history of cave geomicrobiology studies within the US. Since that time, studies of cave geomicrobiology have accelerated in number, complexity of techniques used, and depth of the results obtained. The field has moved from being sparse and largely descriptive in nature, to rich in experimental studies yielding fresh insights into the nature of microbe-mineral interactions in caves. To provide insight into the changing nature of cave geomicrobiology we have divided our review into research occurring before and after the Breakthroughs conference, and concentrated on secondary cave deposits: sulfur (sulfidic systems), iron and manganese (ferromanganese, a.k.a. corrosion residue deposits), nitrate (a.k.a. saltpeter), and carbonate compounds (speleothems and moonmilk deposits). The debate concerning the origin of saltpeter remains unresolved; progress has been made on identifying the roles of bacteria in sulfur cave ecosystems, including cavern enlargement through biogenic sulfuric acid; new evidence provides a model for the action of bacteria in forming some moonmilk deposits; combined geochemical and molecular phylogenetic studies suggest that some ferromanganese deposits are biogenic, the result of redox reactions; and evidence is accumulating that points to an active role for microorganisms in carbonate precipitation in speleothems.
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Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms are isotopically light, suggesting that in situ chemoautotrophic activity plays an important role in this subsurface ecosystem.
Sulfur isotope data, whole rock analyses, and pH-dependence of the clay mineral endellite support the hypothesis that the large cave passages in the Guadalupe Mountains were dissolved primarily by sulfuric acid rather than carbonic acid. As the Guadalupe Mountains uplifted and tilted to the northeast during the Pleistocene, hydrogen sulfide liberated at the base of the Castile Formation in the Gypsum Plain migrated updip into the Capitan reef and there combined with downward-moving oxygenated ground water to form sulfuric acid. -from Author
Txtbook on cave minerals and speleothems
Cyanobacteria belonging to the Synechococcus group are ubiquitous inhabitants of diverse marine and freshwater environments. Through interactions with the soluble constituents of their aqueous habitats, they inevitably affect the chemistry of the waters they inhabit. Synechococcus strain GL24 was isolated from Fayetteville Green Lake, New York, where it has a demonstrated role in the formation of calcitic minerals. In order to understand the detailed interactions which lead to mineral formation by this organism, we have undertaken detailed ultrastructural studies of its cell surface and the initial events in mineral growth using a variety of electron microscopic and computer image enhancement techniques. Synechococcus strain GL24 has a hexagonally symmetrical S layer as its outermost cell surface component. The constituent protein(s) of this structure appears as a double band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r)s of 104,000 and 109,000. We demonstrate that the S layer acts as a template for fine-grain gypsum and calcite formation by providing discrete, regularly arranged nucleation sites for the critical initial events in the mineralization process. To our knowledge, this is the first time that a bacterial S layer has been shown to have a role in mineral formation in a natural environment, and this report provides conclusive evidence for the specific involvement of bacterial surfaces in natural mineral formation processes.
Uncalcified and calcified algae grow on and color walls of cavities that occur in karst breccia, which fills pockets in the Tertiary Bluff Formation of Grand Cayman Island. The calcified algae are important because the calcification has been achieved through the formation of dendritic calcite crystals. Although common in metals, such branching crystals have not been widely recognized in naturally occurring calcite. The dendritic calcite crystals are exactly like most dendritic crystals in that they have a primary needle or stem with secondary and tertiary branches. Most dendritic calcite crystals have an open, treelike form and are easy to recognize. As calcification proceeds, the dendritic crystals become progressively filled in and are increasingly difficult to recognize as dendritic crystals. Filling of the dendritic crystals can result in two-dimensional sheets or three-dimensional columns. Assuming the three-dimensional columnar dendritic crystals developed planar and/or curving smooth surfaces, then, lacking other evidence, they would be indistinguishable from normal columnar calcite crystals, and their dendritic origin would be hidden. Variables thought to control dendrite crystal formation include a) creation of localized, highly supersaturated solutions leading to rapid rates of nucleation and crystal growth, and b) "impurities" in the form of organic molecules and other substances. Algae are excellent candidates for controlling a via their metabolism and contributing to b via their decay. Combining these ideas with the results of this study and with the known common occurrence of algae in many speleothemic environments provides an impetus for a reexamination of the origin of subaerially formed calcite crystals.
Although speleothems are usually considered inorganic precipitates, recent work has demonstrated hitherto unsuspected biogenic influence in some twilight areas. We have expanded this notion to the dark zone, examining pool fingers from Hidden Cave, New Mexico, to test for possible bacterial involvement. The pool fingers in Hidden Cave are pendant speleothems that formed subaqueously in paleo-pools. They are 1 to 4 cm in diameter and 5 to 50 cm long. A knobby, irregular external shape is underlaid by a layered interior on two scales, a 0.5 to 1.0 cm alternation between dense and porous layers and a mm-scale alternation between dark micritic calcite and clear dogtooth spar. The micrite is similar to microbialites identified in modern and ancient carbonates. Fossil bacteria were found in all layers. These include (1) calcified filaments 1m in diameter and 5–50m long and (2) micro-rods 0.1 m by 1–2 m. Most filaments are curved rods with a smooth surface but rare examples display a diamond crosshatch surface. The micro-rods occur as isolated crystals to dense meshes. We interpret the micro-rods as calcified bacilliform bacteria and the filaments as calcified filamentous bacteria. Carbon isotopic data are slightly more negative (by - 0.5 to - 1.0% in micritic layers than in dogtooth spar layers, suggesting a greater microbial influence in the micritic layers. Based on these similarities to known microbialites (e.g., petrographic fabrics, the presence of fossil bacteria, and the suggestive carbon isotopic data), we conclude that microbial activity was an intimate part of pool finger formation in Hidden Cave. The significance of such involvement goes beyond speleological contexts to wider questions of identification of biosignatures in rocks on earth and beyond.