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\DOI{10.5802/crgeos.201}
\datereceived{2022-07-12}
\daterevised{2022-11-03}
\datererevised{2022-12-28}
\dateaccepted{2023-01-16}
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\dateposted{2023-08-18}
\begin{document}

\begin{noXML}

%\TopicEF{Research article}{Article de recherche}

\TopicEF{Terrestrial and aquatic ecosystems}{\'Ecosyst\`emes terrestres et aquatiques}

\title{Death and taphonomy of Holocene stromatolites from Lagoa
Vermelha, Brazil}

\author{\firstname{Carolina Neumann} \lastname{Keim}\CDRorcid{0000-0002-3208-4128}\IsCorresp}
\address{Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ,
Brazil}
\curraddr[C. N. Keim]{Instituto de Microbiologia Paulo de G\'{o}es,
CCS, UFRJ, Av. Carlos Chagas Filho, 373, Cidade Universit\'{a}ria,
21941-902, Rio de Janeiro, RJ, Brazil}
\email[C. N. Keim]{cnkeim@micro.ufrj.br}

\author{\firstname{Marcos} \lastname{Farina}}
\addressSameAs{1}{Universidade Federal do Rio de Janeiro, Rio de
Janeiro, RJ, Brazil}
\email[M. Farina]{marcos.farina.souza@gmail.com}

\begin{abstract}
Stromatolites are laminated rocks, comprising both authigenic and
allochthonous materials, which arise under strong influence of both
microorganisms and environmental conditions. Growing stromatolites are
rare nowadays, limiting our understanding on how microbial mats produce
stromatolites. Stromatolites from Lagoa Vermelha in Brazil were claimed
as living, lithifying structures several years ago, but recently have
been reported as dead, bleached skeletons. Here we confirm that they
are currently not forming new laminae. This was confirmed by the
absence of the microbial mat coat, enabling colonization of exposed
surfaces by barnacles, boring cyanobacteria, and burrowing metazoans,
which contributed to erosion and loss of some original features while
adding new, distinct biomarkers. 
\end{abstract}

\thanks{This project has received funding from the Brazilian agencies
CNPq (National Council for Scientific and Technological Development)
and FAPERJ (Carlos Chagas Filho Research Support Foundation).}

\keywords{\kwd{Stromatolite}\kwd{Lagoa
Vermelha}\kwd{Fossilization}\kwd{Hypersaline}\kwd{Fluorescence}}

\maketitle

%\vspace*{-6.3pt plus 1pt minus 1pt}
\vspace*{12pt}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Stromatolites are sedimentary rocks showing domal, conical, columnar or
flat shapes, and laminated facies. Most of them are calcareous,
comprising both authigenic and allochthonous materials in varying
proportions. Stromatolites emerge from \mbox{microbial} mats, which are
benthic microbial ecosystems found in shallow aquatic environments and
tidal flats\break \citep{Riding2000,Duprazetal2009,Riding2011,Bosaketal2013,
Suosaarietal2019}. Microbes in microbial mats arrange themselves in
layers according to their physiological characteristics
\citep{Gemerden1993,DuprazVisscher2005,Duprazetal2009}. They produce
adhesive polymers able to trap solid particles, incorporating them
within the microbial mat as it grows upwards towards sunlight
\citep{Riding2011,Bosaketal2013,Suosaarietal2019}. \mbox{The~interplay}
between microbial physiology and \mbox{environmental} conditions triggers the
precipitation of authigenic minerals, leading to mineral precipitation
within specific microenvironments within the mat, which binds loose
grains together and results in the characteristic laminations
\citep{DuprazVisscher2005,Duprazetal2009,Suosaarietal2019}.  With time,
the precipitation and consolidation of lamina after lamina produces
stromatolites \citep{DuprazVisscher2005,
Duprazetal2009,Riding2011,Bosaketal2013,Suosaarietal2019}.

Stromatolites are very common in sedimentary settings from the
Precambrian Eon, but become scarce towards the Anthropocene 
\citep{Riding2000,Riding2011,Bosaketal2013,Suosaarietal2019}. Today,
living stromatolites are found in a few environments, including
freshwater, brackish, marine, and hypersaline environments 
\citep{Reidetal1995,Reidetal2003,Gischleretal2008,PlanavskyGinsburg2009,
CastroContrerasetal2014,Chagasetal2016,Zeyenetal2021}.  Studies on
living, growing stromatolites have been fundamental for the
interpretation of the ancient stromatolites as fossilized microbial
mats, as well as for mechanistic explanations on how microbial mats
produce consolidated laminations, and how they change with diagenesis
\citep{Riding2011}.\looseness=1 

Several records of living stromatolites in Lagoa Vermelha in Brazil
\citep{Hohnetal1986,Vasconcelosetal2006,Spadaforaetal2010,Vasconcelosetal2014}
contrast with recent works reporting only bleached skeletons from 2004
on \citep{Silvaetal2004,AlvesSilva2011,
Sampaioetal2015,Lautetal2017,Lautetal2019,Keimetal2020}.  The presence
of living stromatolites in Lagoa Vermelha (Rio de Janeiro State,
Brazil) was first described in a scientific paper by
\citet{Hohnetal1986}. Further work described domal stromatolites capped
by 3--5~cm thick, lithifying microbial mats
\citep{Hohnetal1986,Vasconcelosetal2006}. In addition, microbial mats
2--6~cm thick were observed on sediment surfaces between 1982 and 2002
\citep{Hohnetal1986,Lopesetal1986,Moreiraetal1987,
Eliasetal1997,vanLithetal2002,Silvaetal2004,SilvaCarvalhal2005,
Santellietal2006}. These microbial mats contained alternating layers of
whitish carbonate minerals and colored organic layers dominated by
microbial phototrophs, such as cyanobacteria and purple sulfur bacteria
\citep{Vasconcelosetal2006}.  However, a straightforward relationship
between the microbial mats and the lithified laminae in stromatolites
was not established. For example, pure calcite was found in the first
mineral layer of the microbial mat overlying Lagoa Vermelha
\mbox{stromatolites} \citep{Vasconcelosetal2006}, but not within
consolidated laminae
\citep{Spadaforaetal2010,Carvalhoetal2018,Keimetal2020},  which are
largely composed of high Mg-calcite, very high Mg-calcite
(protodolomite), and aragonite \citep{Keimetal2020}. Radiocarbon
analysis suggested that a stromatolite collected in Lagoa Vermelha
stopped growth around 1440 ${\pm}$ 60 AD, but some uncertainty persists
because the reservoir effect was not calculated for Lagoa Vermelha
\citep{Carvalhoetal2018}. Such ages could indicate that the microbial
mats coating Lagoa Vermelha stromatolites in 1982--2004
\citep{Hohnetal1986,Eliasetal1997,
Vasconcelosetal2006,Spadaforaetal2010} were not producing additional,
consolidated laminae to be added to the underlying
stromatolites.\looseness=1 

In this work, we confirm that the stromatolites of Lagoa Vermelha
stopped adding new laminae, analyze how and why stromatolites lose
their microbial mat cap and stopped growth, and how they changed after
they became naked skeletons. Because these are recent stromatolites, it
is easier to unravel original and post-depositional contributions,
which could help in the interpretation of the taphonomy of ancient
counterparts.

\section{Materials and methods}\label{sec2}
\subsection{Field description}\label{ssec21}

Lagoa Vermelha is a coastal, hypersaline lagoon in the municipality of
Saquarema, Rio de Janeiro State, Brazil (Figure~\ref{fig1}). It is
separated from the Atlantic Ocean by a sandbar 200--400~m in width,
from hypersaline Lagoa de Araruama by another sandbar at least 700~m
wide (Figure~\ref{fig1}b), and from freshwater Lagoa de Jacarepi\'{a}
by about 2400~m (not shown). Lagoa Vermelha is small, about 4.3~km in
length and at most 750~m in width \citep{BidegainBizerril2002}, and
shallow, reaching about 1.7~m in depth \citep{Hohnetal1986}. The lagoon
was divided into five parts by artificial sandbars
(Figure~\ref{fig1}b), and the eastern parts are currently used as
\mbox{evaporation} ponds by an artisanal saltern at the Northeast shore.
\mbox{Artificial} channels bring hypersaline water from nearby Araruama
Lagoon, which is eutrophic 
\citep{BidegainBizerril2002,Lautetal2017,Lautetal2019}. Recently, Lagoa
Vermelha was considered a eutrophic environment
\citep{Lautetal2017,Lautetal2019, Pennafirmeetal2019}.\looseness=1 

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig01}
{\vspace*{4pt}}
\caption{\label{fig1}Satellite images showing the localization of Lagoa
Vermelha in Brazil. (a) Part of Rio de Janeiro State in Brazil showing
the location of Lagoa Vermelha along the coast (rectangle). (b) Area
enclosed by the rectangle in (a)  showing Lagoa Vermelha, part of
nearby Lagoa de Araruama, and the Atlantic Ocean. Asterisks show the
places where samples were collected. Images obtained with the software\break
ArcGISEarth.}
{\vspace*{4pt}}
\end{figure*}

\subsection{Sample collection and preparation}\label{ssec22}

Biofilm and stromatolite samples were collected in Lagoa Vermelha at
the south shore (22\textdegree{}55$'$58$''$S,
42\textdegree{}23$'$35$''$W) in January 18, 2013, and at the north-east
shore (22\textdegree{}55$'$35$''$S, 42\textdegree{}22$'$09$''$W) in
October 15, 2015 (Figure~\ref{fig1}b). Several excursions were done in
2013--2015 for additional field work. Field work and analyses focused
on the south shore, and were later confirmed by analysis of samples
collected in the north shore. 

In the laboratory, stromatolites were cut with a saw and sub-sampled.
Small samples were cut perpendicularly to the laminations using a
low-speed diamond wheel saw (South Bay Technology, INC; model 650) and
hand-polished with silicon carbide sandpaper. Some were embedded in
Spurr's resin (Polysciences) before polishing. For transmitted
polarized light microscopy and fluorescence microscopy, samples were
hand-polished with sandpaper until they became sufficiently thin, and
mounted with Entellan$^{\text{\textregistered}}$ and coverslips. Living
biofilm samples were observed in the day after they were collected. 

Reflected light images of both biofilm and stromatolite samples were
obtained with a Nikon AZ1000 stereomicroscope equipped with a Nikon
Digital Sight DS-Ri1 camera. Several high-quality images were used to
construct a high-resolution mosaic of the polished surface using the
Adobe Photoshop CC 2015 software. Transmitted light microscopy
\mbox{images} were obtained in either a Zeiss Axioplan microscope equipped
with an Evolution MP 5.0 color camera (Media Cybernetics) using the
Nomarski interference mode, or in a Zeiss Axioplan 2 light microscope
equipped with a Color View XS digital video camera, using
epi-fluorescence and crossed polarizers. Filters for epi-fluorescence
were from Zeiss: (i) blue fluorescence (365~nm--420~nm), filter set 02
(488002-0000); (ii) yellow-green fluorescence (450/490--515~nm), filter
set 09 (488009-0000); (iii) green fluorescence (470/20--505/530~nm),
filter set 13 (488013-0000-000); and (iv) red fluorescence
(546/12--590~nm), filter set 15 (488015-0000). 

For scanning electron microscopy, we used both freshly broken and
polished samples. Only the broken samples were gold-sputtered using a
Baltec SCD 050 equipment. Scanning electron microscopy using either
secondary or backscattered electrons was done with a Jeol JSM-6490LV
equipped with Noran EDS detector.

\section{Results}\label{sec3}

\subsection{Observations in the field}\label{ssec31}

The temperature of the lagoon water remained between 20 and
30~\textdegree C (mean 26~\textdegree C) along 2013, 2014 and 2015,
whereas salinity varied from 51 to 97{$\permil$} (average
78{$\permil$}), and pH values ranged from 7.3 to 8.7 (average 8.2).
Although varied, these values comply with previous observations
\citep{Hohnetal1986,VasconcelosMcKenzie1997,vanLithetal2002,
AlvesSilva2011,Lautetal2017,Pennafirmeetal2019}.

Lagoon water varied from clear and transparent to green and turbid
(Figure~\ref{fig2}a,b). We found several stromatolites removed from
their original places in the lagoon, and deposited at the shore
(Figure~\ref{fig2}a,c,d), whereas those not so easily accessed by
people remained in their original places (Figure~\ref{fig2}e,f). During
field work, we have never observed stromatolites coated by cm-thick
microbial mats as described previously \citep{Hohnetal1986,
Vasconcelosetal2006,Vasconcelosetal2014}. Instead, they alternated
between (i) completely naked, bleached mineral surfaces
(Figure~\ref{fig2}c,e); (ii) a very thin, green slimy coat 
(figure~\ref{fig2}c,d); and (iii) a soft olive-green biofilm, measuring
1--2~mm in thickness (Figure~\ref{fig2}f). The presence of barnacle
shells on most stromatolite skeleton outer surfaces, including the
upper convex surfaces (Figure~\ref{fig2}c), indicate that these mineral
surfaces had been exposed to lagoon water for some time before we began
fieldwork in January 2013. 

\begin{figure*}[t!]
{\vspace*{1pt}}
\includegraphics{fig02}
{\vspace*{1pt}}
\caption{\label{fig2}Photographs of Lagoa Vermelha and stromatolites.
(a,b) Lagoa Vermelha landscapes at the South shore. (a) Picture taken
on January 18, 2013, showing clear water and several stromatolites at
the shore, displaced from their original places. (b) Picture taken on
October 13, 2014, showing green, turbid water due to excess
phytoplankton. (c) Dry stromatolites at the South shore, showing
abundant barnacle shells attached to outer surfaces. For scale, each
individual barnacle shell is about 1~cm wide. (d) Broken stromatolite
found at the shore. Wet surfaces show green color, contrasting with the
almost white color of dry mineral surfaces. (e,f) Stromatolites
observed in their original places. (e) Image taken on October 13, 2014,
at the east border. Partially broken stromatolite heads are seen above
the water level due to an exceptionally dry year. Each stromatolite is
about 40~cm in diameter. (f) Underwater photograph taken close to the
South border on October 15, 2015, showing a thick (1--2~mm)
green-to-brown biofilm coating the lagoon bottom, including the
stromatolite. This individual stromatolite is about 30~cm in diameter.}
{\vspace*{1pt}}
\end{figure*}

The lagoon bottom alternated between light grey exposed sediment
minerals (not shown) and the millimeter-thick biofilm coat, which also
coated the stromatolites (Figure~\ref{fig2}f). This biofilm was soft
and easily displaced from hard surfaces, turning into loose flocs. It
was observed at depths up to $\sim$1~m in Lagoa Vermelha. Microbial
mats 1--3~cm thick were observed only at the north shore of Lagoa
Vermelha, in an area lacking both domal stromatolites and laminated
crusts.

Light microscopy showed that the biofilm coating stromatolites on
October 15, 2015 was composed predominantly by diatoms, fecal pellets,
foraminifera and ostracods (Figure~\ref{fig3}a--c). Several species of
microcrustaceans grazed around (e.g. Figure~\ref{fig3}d).
Figure~\ref{fig3}e shows the main species of foraminifera. In
addition, rare filaments of cyanobacteria (Figure~\ref{fig3}b,f,g) and
gliding colorless sulfur bacteria, probably \textit{Beggiatoa} sp.
(Figure~\ref{fig3}f), were observed. The paucity of filamentous
microbes and their remains could explain the weak cohesiveness observed
in this biofilm [for the roles of filamentous microorganisms in
cohesiveness and textures of biofilms and microbial mats, see for
example the work of \citealp{Gerdesetal2000}]. Local variations in
biofilm composition were common (for example, compare
Figure~\ref{fig3}a,b,f,g).  Thick, laminated microbial mats were
observed only on a tidal flat at the North shore of Lagoa Vermelha and
in the nearby salt evaporation artificial ponds (not shown).{\vspace*{-6pt}}   

\begin{figure*}[t!]
{\vspace*{4pt}}
\includegraphics{fig03}
{\vspace*{4pt}}
\caption{\label{fig3}Light micrographs of the millimeter-thick biofilm
coating stromatolites on October 13, 2014. (a) Sample consisting mainly
of diatoms (olive green dots, D) and foraminifera (white, oblong, F).
(b)~Diatoms (olive green rods, D) and filamentous cyanobacteria (C)
along with abundant fecal pellets (white rods, FP). (c) Fecal pellets
(FP) and remains of an ostracod (O). (d) Example of microcrustacean
that grazed on the biofilms. (e) Remains of a foraminifera. (f) At
least two distinct species of filamentous cyanobacteria (C) along with
colorless sulfur bacteria (B for \textit{Beggiatoa}). (g) High
magnification showing diatoms (D) and filamentous cyanobacteria (C).
(a,b) Stereomicroscopy, reflected light. (c--g) Nomarski interference
contrast, transmitted light.}
{\vspace*{3pt}}
\end{figure*}

\subsection{Stromatolite structure and taphonomy}\label{ssec32}

Figure~\ref{fig4} shows the surface of the sagittal plane of a Lagoa
Vermelha stromatolite. Laminated facies predominate at the core,
whereas clotted facies appear at the top, the bottom, and the
periphery. Some cm-wide voids are also present. Burrows 0.7--1.0~mm
wide are widespread on peripheral regions, but not in the laminated
core. The occurrence of a well-laminated core and thrombolitic portions
at the base and the top of Lagoa Vermelha stromatolites suggests a
changing environment in the timescale of stromatolite growth, as
proposed by \citet{Carvalhoetal2018} based on radiocarbon age
determination.

\begin{figure}
{\vspace*{3pt}}
\includegraphics{fig04}
{\vspace*{3pt}}
\caption{\label{fig4}Stromatolite from Lagoa Vermelha (reflected light
microscopy, mosaic). Note the laminated facies in the core, and clotted
facies on both top and bottom. Circular burrows, about 1~mm in
diameter, occur at peripheral regions, specially at the top right
corner.}
\end{figure}

The stromatolite core showed millimeter-thick, convex, parallel
laminae, showing shade variations, sometimes separated by empty voids
(Figure~\ref{fig5}a). Color variations could be observed in both
laminated and clotted regions (Figure~\ref{fig5}a--d) and indicate
differences in composition and/or texture as observed previously
\citep{Keimetal2020}.  Extensive regions of both laminated and clotted
regions consisted of rather \mbox{irregular} peloids cemented together
(Figure~\ref{fig5}b), contrasting with the regular size and shape of
fecal pellets (Figure~\ref{fig5}d). 

\begin{figure*}
{\vspace*{1pt}}
\includegraphics{fig05}
{\vspace*{1pt}}
\caption{\label{fig5}Reflected light stereomicroscopic images of
polished stromatolite surfaces. (a) Laminated facies in the
stromatolite core. (b) Clotted region showing abundant peloids
(asterisks) cemented together by a thin mineral layer. (c) Holes
consisting of metazoan burrows (asterisks). (d) Cluster of fecal
pellets cemented to the stromatolite (white rods). (e) Space between
two laminae containing a quartz grain (asterisk) and a foraminifera
shell (arrow). (f) Cluster of foraminifera shells with both inner and
outer surfaces coated by a thin mineral layer, which cements them to
the stromatolite surface (arrows).}
{\vspace*{1pt}}
\end{figure*}

Fecal pellets were found mostly in the outer regions, where they were
cemented within metazoan burrows and other surfaces
(Figure~\ref{fig5}d) as observed before \citep{Keimetal2020}. The
peripheral regions contained metazoan burrows, 0.7--1.0 mm wide,
reaching up to 1~cm from the outer surface (Figure~\ref{fig5}c).
Gastropod, bivalve, ostracod and foraminifera shells were found both on
the surfaces and embedded in the stromatolite fabric, but were more
common in the voids between laminae (Figure~\ref{fig5}e,f), indicating
that most of them arrived after laminae consolidation. Previous work
found the same types of shells within Lagoa Vermelha stromatolites
\citep{Silvaetal2004,Spadaforaetal2010,Keimetal2020}.
\citet{Silvaetal2004} identified most gastropods as \textit{Hydrobia},
most ostracods as \textit{Cyprideis}, and the main bivalve species as
\textit{Anomalocadia brasiliana}. Inorganic clastic materials were rare
and consisted mainly of quartz (Figure~\ref{fig5}e). A thin mineral
layer coated many surfaces, including laminae, peloids, fecal pellets,
shells and metazoan burrows (Figure~\ref{fig5}b--f). This mineral coat
seems to be responsible for cementing most peloids, fecal pellets and
shells to the stromatolite fabric as proposed before
\citep{Keimetal2020}.

Figure~\ref{fig6} shows the minerals coating a small gastropod shell
found in a stromatolite. The texture of the coat was similar within and
outside the shell (Figure~\ref{fig6}a), as well as in the surrounding
areas. In either transmitted or reflected light microscopy images, or
in scanning electron microscopy, this mineral coat was
5--100~${\upmu}$m wide. In both foraminifera and gastropods, the coat
was slightly thinner on the inner shell surface as compared to the
outer surface (Figure~\ref{fig6}a). Figure~\ref{fig6}b shows minerals
arranged in bunches on the mineral fringe, as well as filamentous
microorganisms, diatom frustules, and EPS remains in what appears to be
additional layers deposited onto the mineral fringe coating the shell.
Higher magnification showed the mineral bunch at the top to be composed
of long, thin minerals with trigonal symmetry, sometimes presenting a
hole in the center suggesting a hollow head (Figure~\ref{fig6}c). The
proximity with an EPS mass could suggest a role of EPS in nucleation,
although the underlying carbonates are probable nucleating sites as
well. The mineral bunch at the bottom in Figure~\ref{fig6}b consisted
of thin rhombohedral plates with the acute angles pointing outwards
(Figure~\ref{fig6}d). Both morphologies have been observed before in
Lagoa Vermelha stromatolites \citep{Spadaforaetal2010}, and very
similar shapes have been found in freshwater stromatolites 
\citep{CastroContrerasetal2014}, low Mg-calcite minerals from a coastal
hypersaline microbial mat \citep{Perrietal2018}, in an ancient reef 
\citep{Cabiochetal1999}, and also in soil extracts
\citep{Parragaetal2004}. Thus, these seem to be features common in
authigenic Ca--Mg carbonates.

\begin{figure*}
{\vspace*{4pt}}
\includegraphics{fig06}
{\vspace*{4pt}}
\caption{\label{fig6}Scanning electron micrographs of freshly broken
stromatolite surfaces, showing minerals grown onto a gastropod shell.
(a) Broken gastropod shell showing mineral fringes growing both inside
(bottom arrow) and outside (top arrow) of the shell. Note that the
fringe is wider on the outer shell surface (top) than on the inner
surface (bottom). (b) Larger magnification of the top of the shell
showing the mineral fringe in detail, along with a filamentous
prokaryote (F), a diatom frustule (D), and EPS (E). Boxed areas are
enlarged in (c, d). (c) Larger magnification of the region
enclosed in the upper rectangle in (b) showing thin crystals growing
from the EPS underneath (E). Side view of the crystals show they are
long and thin, with enlarged tops (arrowheads). Top view shows
triangular tops with small holes in the center. (d) Larger
magnification of the region enclosed in the bottom rectangle in (b)
showing rhombohedral plates growing radially, with the acute angles
pointing upwards (arrow). Secondary electrons, 25~kV.}
{\vspace*{4pt}}
\end{figure*}

Polarized light microscopy showed that the minerals in stromatolites
were largely birefringent (Figure~\ref{fig7}a,c,e), as described before
\citep{Spadaforaetal2010,Keimetal2020}. Fluorescence microscopy put on
evidence otherwise hidden features, which may be interpreted as organic
matter and remains of microorganisms (Figure~\ref{fig7}b,d,f).  As a
general finding, bright areas in the polarized light image showed the
weakest fluorescence, indicating that these areas are poor in organics
as compared to surrounding areas. Conversely, weakly birefringent areas
showed the strongest fluorescence (Figure~\ref{fig7}).  Observation of
several samples using polarized light and SEM suggests that the weak
birefringence would be due to reduced sizes of Ca--Mg carbonate
crystals, which is the \mbox{predominant} minerals in Lagoa Vermelha
stromatolites \citep{Keimetal2020}.

\begin{figure*}
\includegraphics{fig07}
\caption{\label{fig7}Polished slices of stromatolite observed by
polarized and fluorescence light microscopy. Note the inverse
brightness of polarized light (a,c,e) and fluorescence images (b,d,f).
(a,b) Border area showing heterogeneous birefringence and fluorescence,
as well as weakly birefringent, highly fluorescent, well-delimited
areas probably consisting of fecal pellets (FP). Note the bright
fluorescent spots within small voids (arrows). Highly birefringent,
weakly fluorescent mineral fringes coat all surfaces, cementing the
putative fecal pellets to the stromatolite. (c,d) Void in a laminated
region filled by clastic materials. Highly fluorescent, weakly
birefringent regions are intercalated by weakly fluorescent, highly
birefringent areas. The mineral fringe coats all surfaces, showing a
single layer lining the clasts, including foraminifera shells (F), and
a double layer coating stromatolite mineral surfaces. Fluorescence is
stronger in small spots, corresponding to dark areas in the polarized
light image. (e,f) Fossilized filamentous microorganisms (asterisks)
and microscopic tubules (arrows).}
\end{figure*}

Most features fluoresced green and yellow
(Figures~\ref{fig7},~\ref{fig8}), in contrast to weaker and more
specific blue (not shown) and red fluorescence (Figure~\ref{fig8}). The
strongest birefringence and lowest fluorescence were observed in the
radial mineral fringe coating several surfaces (Figure~\ref{fig7}a--d).
Considering birefringence as a proxy for Ca--Mg carbonates and
fluorescence as a proxy for organic materials, these images indicate
that the fringe consists largely of minerals growing radially from
available mineral surfaces, with little (if any) contribution of
organic materials. Both light \mbox{microscopy} techniques show that in
some places the fringe coating inner stromatolite surfaces is composed
by at least two layers (Figure~\ref{fig7}a--d), which indicate that the
fringe precipitated intermittently.

\begin{figure}
\includegraphics{fig08}
{\vspace*{-3pt}}
\caption{\label{fig8}\fontsize{9.7}{11}\selectfont Polarized light microscopy (a) and fluorescence
microscopy (b,c) of a thin, polished sample of a peripheral
stromatolite region. (a) Border area showing abundant microscopic
tubules. (b) Highly fluorescent spots juxtaposed to tubule walls and
filaments running on the bottom left are probably due to remains of
microorganisms that thrived within the tubules. A weak, heterogeneous
and widespread green fluorescence is also observed. (c) The strong red
fluorescence co-localized with the green fluorescence within tubules
and in the filaments at the left is compatible with that of the
photosynthesis pigment chlorophyll.}
\end{figure}

Figure~\ref{fig7}a,b show highly fluorescent, poorly birefringent
circular profiles cemented to stromatolite surfaces by the mineral
fringe. Their rather regular shape and size indicate that they consist
of fecal pellets. In addition, highly fluorescent spots occurred within
small voids (Figure~\ref{fig7}a,b). Shapes include rods, cocci, and
filaments, indicating that these could represent fresh remains of
microbial cells. Figure~\ref{fig7}c,d show bioclasts displaying
fluorescence levels and patterns similar to the laminae, with no
evidence of organic remains from the original organisms inside most of
them. Bright spots showing size and shape compatible with microbial
cells are abundant in poorly birefringent areas (Figure~\ref{fig7}c,d).
\mbox{Comparison} of fluorescence levels of Figures~\ref{fig7}b 
and~d indicate that these could represent aged cell
remains.

In Figure~\ref{fig7}e,f, the remains of several well-preserved,
mineralized, filamentous microorganisms can be recognized by their
conspicuous morphology. They show birefringence and fluorescence levels
similar to the surrounding minerals, indicating that carbonate minerals
largely substituted the original cell materials, whereas fluorescent
organic remains were lost and/or distributed around. Circular holes are
observed in both polarized and fluorescent light images
(Figure~\ref{fig7}e,f), but presumably empty tubular holes parallel to
the plane of the sample are particularly evident in the fluorescent
light image, where they appear darker than their surroundings
(Figure~\ref{fig7}f).

Comparison of polarized and fluorescence light images shows strong
birefringence and weak autofluorescence in the minerals around such
tubules (Figure~\ref{fig8}a,b). Fluorescence microscopy showed many of
them partially filled with strongly autofluorescent materials, which
would probably correspond to remains from the microorganisms that
inhabited the tubules (Figure~\ref{fig8}b,c). Their autofluorescence is
strong in yellow, green and red. In particular, the red
autofluorescence (Figure~\ref{fig8}c) is compatible with the
autofluorescence of the photosynthesis pigment chlorophyll, indicating
that these could be remains of cyanobacteria. \looseness=-1

Accordingly, scanning electron microscopy showed the remains of
filamentous microorganisms within microscopic tubules
(Figure~\ref{fig9}a,b). Figure~\ref{fig9}a shows several of them at the
border of a stromatolite, some still containing the organic remains of
filamentous microorganisms. Their organic nature could be identified
due to the translucent character at the scanning electron microscope
when using backscattered electrons (Figure~\ref{fig9}a). In other
samples, filaments remaining within the tubules were brittle,
indicating that their organic remains were largely substituted for
minerals (Figure~\ref{fig9}b), as observed by light microscopy in
Figure~\ref{fig7}e,f. Their shapes suggest that they were filamentous
microorganisms (Figure~\ref{fig9}b). In samples embedded in resin, the
outlines of stromatolite laminae are more evident. Figure~\ref{fig9}c
shows a site at the outer surface dominated by microscopic tubules.
They are more concentrated towards the surface, as observed in
Figure~\ref{fig9}a. Some entombed remains of microorganisms could be
observed, but they did not seem related to the nearby tubules
(Figure~\ref{fig9}c) and may represent microbial remains from the time
minerals in this lamina were precipitated, as previously described in
similar environments \citep[e.g.][]{Gischleretal2008,Couradeauetal2013,
CastroContrerasetal2014,Perrietal2018,Shiraishietal2020, Debrieetal2022}. 

\begin{figure}
{\vspace*{2pt}}
\includegraphics{fig09}
{\vspace*{2pt}}
\bcaption{\label{fig9}Scanning electron micrographs of polished
stromatolite samples showing peripheral regions containing abundant
microscopic tubules, some still harboring filamentous microorganisms.
(a) Image showing both polished (right) and the original stromatolite
surface, which consists largely of openings of the tubules (left). Note
abundant organic remains of filamentous microorganisms, some still
filling the tubules (arrowheads). Their translucent appearance is due
to the organic nature. (b) Abundant tubules in a peripheral area, some
harboring fossilized, brittle,~filamentous microorganisms (arrowhead).}{8}{
(c) Resin-embedded sample. The contrast between minerals and the resin
filling the tubules shows clearly that most of the perimeter of the
border consists of tubules. The circle encloses some remains of
entombed microbial cells. (a,c) Gradients of tubules across
stromatolite laminae indicate that microorganisms living in these
tubules prefer areas close to the outer surfaces. Backscattered
electron images, 10~kV (a,b) or 30~kV (c).}
\end{figure}

Figures~\ref{fig7}e,f, \ref{fig8} and \ref{fig9}a,b show both fresh and
mineralized filamentous microorganism remains within tubules. Their
size, shape, probable presence of chlorophyll, habitat, and proximity
with outer surfaces (Figures~\ref{fig8} and~\ref{fig9}a,c) indicate
that they consisted of remains of filamentous cyanobacteria which lived
within the stromatolite minerals. The green, thin slime observed on
some wet stromatolite surfaces in the field (Figure~\ref{fig2}d), the
strong autofluorescence observed within the tubules
(Figure~\ref{fig8}), and the organic character observed by SEM
(Figure~\ref{fig9}a) indicate that some of them were still alive at the
time the samples were collected. Even though red fluorescence was not
observed in the brittle, mineralized boring microfossils shown in
Figures~\ref{fig7}e,f and~\ref{fig9}b, their size, shape and habitat
are similar to the fresh ones observed in Figures~\ref{fig8}
and~\ref{fig9}a, suggesting that they were cyanobacteria as well. 

Figure~S1 (Supplementary Material) presents several reflected light and
scanning electron micrographs taken from a stromatolite slab, ranging
from the millimeter to the micrometer scale. It shows that the tubules
observed in Figures~\ref{fig7}e,f, \ref{fig8} and~\ref{fig9} occur
only close to stromatolite borders, usually not exceeding
500~${\upmu}$m from the outer surface, and are completely absent from
the inner laminae (Figure~S1a--c). At the mm-scale, the outer borders
enriched in tubules seem smoother as compared to inner surfaces, which
showed more kinks (Figure~S1a--c). At the micrometer scale, several
stretches of outer borders are dominated by the tubules
(Figure~\ref{fig9}a,c; Figure~S1), which could explain the smoother
appearance at low magnification. Such differences between inner and
outer surfaces indicate that the tubules would be associated to
corrosion of stromatolite outer surfaces, which would not occur in
inner surfaces.

The activities of cyanobacteria can have strong influence on the
carbonate minerals in stromatolites, leading to either precipitation or
dissolution of minerals. Those cyanobacteria involved in mineral
precipitation usually increase the pH in the nearby microenvironment
during photosynthesis leading to increased supersaturation, and/or
produce abundant EPS where minerals are nucleated and grow 
\citep{DuprazVisscher2005,Duprazetal2009}. Accordingly, mineral
precipitation around cyanobacteria it is relatively common, specially
onto secreted EPS, generating characteristic textures and/or mineral
composition \citep{Gischleretal2008,Couradeauetal2013,
CastroContrerasetal2014,Perrietal2018,Shiraishietal2020,
Debrieetal2022}. On the other hand, some cyanobacteria burrow into
carbonate minerals, pumping Ca\tsup{2+} ions through their cells to
decrease saturation in the area of contact of the apical cell with the
mineral. Localized undersaturation induce mineral dissolution in the
microborings inhabited by these cyanobacteria 
\citep{GarciaPicheletal2010,GuidaGarciaPichel2016}. Microboring
cyanobacteria are widespread in carbonate minerals from marine
habitats, including microbialites \citep{Golubic1969,
CampionAlsumardetal1996,Perry1998,Macintyreetal2000,
ReidMacintyre2000,Arpetal2003,Reidetal2003,Pantazidouetal2006,
PlanavskyGinsburg2009,Duguidetal2010,RadtkeGolubic2011,Wynessetal2022}. 

Based on the distribution of tubules only close to outer surfaces of
the stromatolites (Figure~S1), on the presence of organic,
autofluorescent remains or fossilized cells inside some of the tubules
(Figures~\ref{fig7}e,f, \ref{fig8}, \ref{fig9}a,b), on the difference
in texture and relief between inner and outer stromatolite surfaces
(Figure~S1), and on the absence of any structural or compositional
relation between tubules, microorganisms inhabiting them, and the
surrounding minerals  (Figures~\ref{fig9} and S1), we interpreted these
micrometer-wide tubes as microborings, and the cyanobacteria inhabiting
them as microboring cyanobacteria.

This implies that under some environmental conditions, boring
cyanobacteria grow and bore into illuminated areas of Lagoa Vermelha
stromatolites, leading to mineral dissolution. When environmental
conditions change, their dead, soft bodies can be \mbox{replaced} by
minerals, leading to fossilization. Thus, they could act both in
dissolution and precipitation of minerals close to the stromatolite
surfaces, depending if they are alive or dead, as well as on the
environmental conditions. 

{\vspace*{2pt}}

\section{Discussion}\label{sec4}
\subsection{Life and death of stromatolites from Lagoa
Vermelha}\label{ssec41}

Field work confirmed that the cm-thick microbial mats coating
stromatolites were lost. Instead, Lagoa Vermelha stromatolites were
intermittently coated by a thin microbial coat, 1--2~mm in thickness,
containing diatoms as main phototrophs (Figure~\ref{fig3}). In
contrast, the microbial mats that once coated Lagoa Vermelha
stromatolites included two green layers dominated by cyanobacteria 
(\textit{Gloeocapsa}, \textit{Spirulina} and \textit{Microcoleus}
species), along with a purple layer dominated by purple sulfur bacteria
(e.g. \textit{Thiocystis} sp.) and a layer enriched in Ca--Mg
carbonates \citep{Vasconcelosetal2006}. In the biofilm, the
birefringence characteristic of carbonates is concentrated in
cylindrical fecal pellets and in the shells of foraminifera and
ostracods, whereas in microbial mats collected at the north border of
Lagoa Vermelha, carbonates are observed mainly in irregular to rounded
``microoncoids'' about 0.2--1.0~mm in diameter \citep{Guedesetal2022}.
Cylindrical fecal pellets were observed on stromatolite outer surfaces
and burrows (Figure~\ref{fig5}d) but not in the laminated core, whereas
the peloids observed in the stromatolite fabric present irregular
shapes [Figure~\ref{fig5}(b); \citealp{Keimetal2020}] rather similar to
the ``microoncoids'' observed by \citet{Guedesetal2022}. Indeed, a
previous work proposed that these peloids within Lagoa Vermelha
stromatolites originated from sequential precipitation of authigenic
minerals, with bioclasts observed mainly close to voids
\citep{Keimetal2020}. Thus, we consider that this biofilm did not
participate in the genesis of new stromatolite laminae. Perhaps, under
the appropriate conditions, this biofilm may develop into a lithifying
microbial mat able to produce additional stromatolitic laminae.  

In the stromatolites of Lagoa Vermelha, we observed barnacle shells
only at the outer surfaces, whereas cyanobacteria microborings and
metazoan burrows reached at most 500~${\upmu}$m and 10~mm into the
stromatolite, respectively. Bioerosion and \mbox{barnacle} shells were never
observed deep into the \mbox{stromatolite} core. It seems that the
stromatolite's mineral surfaces were protected from those animals and
\mbox{microorganisms} during growth by the microbial mat coat, consisting of a
thick web of microorganisms, exopolysacharides and dead cell materials.
Once the stromatolites lost their microbial mats, colonization by
barnacles, burrowing metazoans, and boring microorganisms could occur
on the exposed mineral surfaces, but this biota could not reach the
stromatolite core because they thrive close to the outer surface. The
absence of the thick microbial mat coating the stromatolites described
before \citep{Hohnetal1986,Vasconcelosetal2006,Vasconcelosetal2014},
the inconsistency of the thin biofilms with the stromatolite cores, the
presence of barnacle shells only at the outer surfaces, and the finding
of both metazoan and microorganism borings only close to outer surfaces
indicate that these stromatolites are not adding new mineral laminae to
their tops, and have been ``dead'' for a while before they were
collected in January~2013.

Thin mineral fringes, consisting of a palisade of coarse crystals, were
observed coating several surfaces, including laminae, shells, peloids,
and also the walls of some metazoan burrows. \citet{Spadaforaetal2010}
and \citet{Keimetal2020} also observed such fringes in Lagoa Vermelha
stromatolites and considered that they precipitated directly from water
onto available carbonate mineral surfaces. Observation of banding in
some of these mineral coats indicate stepwise mineral deposition, which
could be related to climate cycles in Lagoa Vermelha. Because metazoan
burrows occurred after the microbial mat coat has been lost, and some
presented these mineral fringes, precipitation of such fringes probably
continued after loss of the microbial mat coat, resulting from abiotic
processes as proposed previously
\citep{Spadaforaetal2010,Keimetal2020}. The olive-green biofilms that
grew intermittently onto outer stromatolite surfaces would not
participate in precipitation of these mineral fringes, since the
fringes were equivalent in inner and outer surfaces, whereas the
biofilm grew only onto external surfaces.\looseness=1

As discussed above, Lagoa Vermelha stromatolites lost their thick
microbial mat coats some time ago. The high turbidity and green color
of the water \mbox{observed} in the field provide a clue that
eutrophication could be the cause. Indeed, previous work suggested that
Lagoa Vermelha is currently eutrophic
\citep{Lautetal2017,Lautetal2019,Pennafirmeetal2019}.  Recurrent
episodes of high turbidity due to excess phytoplankton, which is a
characteristic of eutrophic environments, could have led phototrophs in
the microbial mats on the stromatolites to death due to insufficient
light. Since phototrophs are the base of the food web in microbial
mats, their death could lead to the collapse of microbial mats. Thus,
eutrophication could explain the death of the microbial mats that once
coated Lagoa Vermelha stromatolites.\looseness=1

In nearby Araruama Lagoon, which is hypersaline and eutrophic but much
larger than Lagoa Vermelha, eutrophication changed the ecosystem
metabolism from net heterotrophic, benthic-dominated, and limited by
phosphorus in 1993--1995, to net autotrophic, planktonic-dominated in
2017 \citep{Knoppersetal1996,Cotoviczetal2021}. In Araruama Lagoon,
increased photosynthesis in phytoplankton driven by eutrophication
consumes most of the dissolved inorganic carbon (DIC) and modulates
alkalinity, decreasing the saturation state and the amount of
precipitated CaCO\tsub{3} \citep{Cotoviczetal2021}. Similarly,
increased photosynthesis in the water column due to eutrophication
could interfere with CaCO\tsub{3} precipitation in Lagoa Vermelha,
including the stromatolites.

Figure~\ref{fig10} summarizes the findings and interpretations of the
present work relative to living beings. In addition to the
biologically-driven changes, climate-driven episodes of low water level
could lead cyanobacteria, barnacles and burrowing metazoans to death by
increased salinity or desiccation. Such conditions could also drive
increased Ca--Mg carbonate precipitation, which could entomb boring
cyanobacteria, add new layers to the mineral fringe, and/or cement
fecal pellets and other bioclasts to some stromatolite surfaces,
particularly within metazoan burrows. 

\begin{figure*}
\includegraphics{fig10}
\caption{\label{fig10}Model of Lagoa Vermelha stromatolites and their
post-deposition changes driven by eutrophication and the biota. (a)
Scheme of a growing stromatolite coated by a cm-thick, multilayered
microbial mat. (b) Close-up of the top of the stromatolite, showing two
green layers and a purple layer, along with an incipient mineral lamina
in between. Water is clear, enabling photosynthesis up to several mm
deep in the microbial mat. (c) Eutrophication drives phytoplankton
blooms, which makes the water turbid. The microbial mat collapses due
to the insufficient light, exposing the naked skeletons. (d) The
exposed mineral surfaces become colonized by barnacles, burrowing
metazoans, and boring cyanobacteria. (e) High turbidity due to
phytoplankton blooms, desiccation, virus infection etc. could lead
boring cyanobacteria to death, resulting in entombed filaments or empty
microborings. (f) Eventually the boring metazoans die or move away,
leaving fecal pellets, which can be cemented to stromatolite surfaces,
particularly within the burrows. (g) During periods of clear water, a
thin, olive-green, loose biofilm grows on illuminated surfaces. This
biofilm is enriched in diatoms, foraminifera and fecal pellets and
could contribute with additional bioclasts to the stromatolite. (d--g,
arrows) Recurrence of phytoplankton blooms lead to collapse of the
olive-green biofilm, leaving naked stromatolite surfaces available to
be colonized by barnacles, burrowing metazoans, and cyanobacteria,
which can die or move away. These steps can repeat several times, in
different orders.}
\end{figure*}

\section{Conclusions}\label{sec5}

Lagoa Vermelha stromatolites are currently not capped by microbial
mats, nor adding new laminae to the top. Eutrophication leading to
periods of excess phytoplankton and high turbidity of the water seems
to be the probable cause of death of the thick microbial mats
previously reported on the top of the stromatolites.

The thin, olive-green biofilm coat that sometimes coats the
stromatolites shows no incipient mineral laminae and is enriched in
diatoms, fecal pellets, foraminifera and ostracods, whereas the core of
Lagoa Vermelha stromatolites is largely formed by peloids, with little
contributions of foraminifera and ostracod shells \citep{Keimetal2020},
and no fecal pellets. Thus, the olive-green biofilm probably does not
contribute with new laminae to the stromatolites.

The presence of barnacle shells, borings and microborings only close to
outer surfaces suggest that barnacles, boring metazoans, and boring
cyanobacteria colonized the stromatolite mineral surfaces only after
they were devoid of mats and exposed to lagoon water. Thus, they are
regarded here as post-depositional changes. In addition, the presence
of a mineral fringe lining some borings suggest that the stromatolite
skeletons remained naked for some time before we began field work on
January 2013. 

The precipitation of the mineral fringe lining stromatolite surfaces
and cementing bioclasts seems to be independent of the presence of a
microbial mat, but result largely from supersaturation of lagoon water
with respect to Ca--Mg carbonates.

\section*{Conflicts of interest}

Authors have no conflict of interest to declare.

\section*{Acknowledgements} 

We thank M.Sc. Mair M. M. Oliveira for technical assistance, and
Beatriz Ramos Rabello, Em\'{i}lio Telles de S\'{a} Moreira, Felipe
Pitzer de Souza, J\'{e}ssica Alves de Paiva, Lukas Bolini and Mirian
Crapez for help in field work. We thank also LABNANO for electron
microscopy facilities, and the Brazilian agencies CNPq and FAPERJ for
financial support.

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crgeos-201-suppl.pdf}}

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