Historical Biology

An International Journal of Paleobiology

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Seeking for the best conditions for fish fossil

preservation in Las Hoyas Konservat-Lagerstätte
using microbial mats

Salvador Mollá, Paloma Alcorlo, Angela D. Buscalioni & Ana Isabel López-
Archilla

To cite this article: Salvador Mollá, Paloma Alcorlo, Angela D. Buscalioni & Ana Isabel
López-Archilla (2023): Seeking for the best conditions for fish fossil preservation
in Las Hoyas Konservat-Lagerstätte using microbial mats, Historical Biology, DOI:
10.1080/08912963.2023.2238745

To link to this article: https://doi.org/10.1080/08912963.2023.2238745

Published online: 26 Jul 2023.

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HISTORICAL BIOLOGY
https://doi.org/10.1080/08912963.2023.2238745

Seeking for the best conditions for fish fossil preservation in Las Hoyas Konservat-
Lagerstätte using microbial mats
a a b a
Salvador Mollá , Paloma Alcorlo , Angela D. Buscalioni and Ana Isabel López-Archilla
a
Departamento de Ecología, Ciencias and CIBC-UAM, Madrid, Spain; bDepartamento de Biología, Ciencias and CIPb-UAM, Madrid, Spain

ABSTRACT ARTICLE HISTORY

Actuotaphonomic experiments demonstrate how microbial mats prevent or delay destructive processes. Received 24 March 2023
The rate at which carcasses are covered is a key to their preservation. Because of the growth rate of microbial Accepted 17 July 2023
mats depends on environmental conditions, a set of experiments have been carried out emulating the KEYWORDS
Barremian environmental conditions, analysed for temperatures at 14°C and 26°C (cooler and warmer Experimental taphonomy;
seasons respectively) and atmospheric pCO2 (1000 ppm). For this purpose, the microbial mats were grown microbial mat; Barremian
in mesocosms within an environmental chamber. Variations in primary production were quantified by atmosphere; fish
measuring changes in dissolved O2 concentration in the water. Zebrafish carcasses were laid on the mats, preservation; net community
and their coverage rates were calculated from the daily surface area covered by the mat. The results showed productivity
that the fish was covered twice as fast at 26°C, in coincidence with the highest values for the gross primary
production and community respiration of the microbial mats. Therefore, for these Barremian conditions, the
early stages of carcasses preservation would take place most effectively during the warmer seasons as
decomposing activity would release nutrients that would enhance, together with temperature, the growth
of mats.

Introduction
During the Kimmeridgian-Albian time interval, a significant con­
centration of exceptional fossiliferous deposits occurred worldwide.
The concurrence of so many localities in a relatively short geologi­
cal time interval has been related to biochronological stage bound­
aries, mass extinctions, anoxic events, elevated atmospheric carbon
dioxide peaks and transient warm-humid climates (Retallack 2011,
Buscalioni and Poyato-Ariza 2016). One of these fossiliferous local­
ities corresponds to the upper Barremian Konservat Lagerstätte of
Las Hoyas, where fossilisation process has been related to the
expansion of microbial mat communities (Buscalioni and Fregenal-
Martínez 2010; Iniesto et al. 2013). Placed in the Iberia Plate, which
occupied the westernmost area of the Tethys Ocean on a latitude
25–30º N during the Barremian, the Las Hoyas fossil site formed
part of the La Huérguina Formation in the southwestern Iberian
Basin at the Serranía de Cuenca. La Huérguina Formation is mostly
composed of carbonatic sediments deposited in alluvial, palustrine
and lacustrine environments. The fossiliferous site is composed of
finely laminated limestones, and two microfacies associations have
been petrographically distinguished: one made up of massive or
graded millimetric laminae, with detrital rests, carbonate particles
and vegetal debris, and a second one linked to the growth of benthic
microbial mats densely packed with lamination of stromatolites.
This latter has been interpreted as sedimentation during drier and
lower water-level periods, whereas the first one would be deposited
under wetter periods in a persistent shallow lamina of water
(Fregenal-Martínez and Melendez 2016).
Microbial mats are stratified complex communities of metabo­
lically highly varied microorganisms that are mainly composed of
primary producers (photo- and/or chemoautotrophic) and consu­
mers/decomposers (heterotrophic) (Figure 1). They are dynamic
and complex ecosystems exhibiting spatial and temporal heteroge­
neity where physical/chemical gradients and morphological
elements are compressed into a few millimetres of vertically layered
structures (Visscher and Stolz 2005). Their populations are orga­
nised into specific communities interacting with each other and
their environment grouped into specific guilds and assemblages,
based on their metabolic properties. These processes often result in
coupled reactions and biogeochemical cycles and produce impor­
tant end products such as trace gases and mineral precipitates. The
efficiency in cycling is well known in microbial mats, and they
shelter high metabolic rates (e.g. of photosynthesis, aerobic respira­
tion and sulphate reduction) with steep vertical geochemical gra­
dients with extreme diel fluctuations consequence of the combined
functional groups of microbes forming the microbial mats (Decker
et al. 2005). Compared to other benthic ecosystems, photoauto­
trophic microbial mats have extremely high rates of oxygenic
photosynthesis, aerobic respiration, sulphate reduction and sulfide
oxidation (Visscher and Stolz 2005).
The actuotaphonomic experiments have shown the capacity of
microbial mats to embed carcasses of small animals and plants,
forming a crust around that we denominate sarcophagus. The
entombment avoids or delays destructive taphonomic processes,
such as decay, bioerosion, necrokinesis or disarticulation of the
organic remains (Iniesto et al. 2013, 2016, 2017; 2018). Our interest
in actuotaphonomic experiences in mat communities pursues the
understanding of the fossilisation process in the Las Hoyas
Lagerstätte (Barremian, Spain) where the fossil preservation was
bacterially mediated. The taphonomic analyses in Las Hoyas com­
paring the fossil assemblages of the two described microfacies
associations indicate that the layers made up of stromatolite lami­
nation, contain abundant fossil remains. However, the fossil con­
tent of the microfacies that characterise the wetter periods is much
limited in number but shows a high diversity (Buscalioni and
Fregenal-Martínez 2010). For that sake, a set of experiments have

CONTACT Salvador Mollá salvador.molla@uam.es Departamento de Ecología, Darwin 2, Cantoblanco, Madrid 28049, Spain
© 2023 Informa UK Limited, trading as Taylor & Francis Group

Published online 26 Jul 2023

2 S. MOLLÁ ET AL.
Figure 1. Vertical section of the Chiprana saline lake microbial mat. The upper
green layer is dominated by filamentous cyanobacteria (a). In the middle layer are
purple anaerobic bacteria (b), below them, the redox potential is very electrone­
gative and sulphate-reducing bacteria abound (c). Scale bar = 5 mm.
been herein carried out emulating some of the Barremian abiotic
factors such as the atmospheric gas composition and temperature.
The concentration of atmospheric CO2 has varied greatly depend­
ing on the geological period, and even during the Cretaceous
(Retallack 2011; Landwehrs et al. 2021). CO2 may have fluctuated
between 560 and 1680 ppm during the Cretaceous (Barral et al. 2017),
and for the Las Hoyas age, Haworth et al. (2005) showed that the
pCO2 could have varied between 560 and 960 ppm. These values are
higher than the atmospheric CO2 concentration of 416 ppm recorded
in 2021 (NOAA 2022). Recent studies confirm that the Cretaceous
was a period of warm to hot greenhouse conditions with reduced
latitudinal gradient and with an enhanced hydrologic cycle (Burgener
et al. 2023). According to the classification of the Köppen climate
zones the Iberian Plate was placed at a temperate, humid subtropical
zone during the interval Hauterivian-Barremian; a regime, otherwise,
dominant for all the Cretaceous periods. This climate zone would
record more than 21º C during the warmest mean monthly tempera­
ture (Burgener et al. 2023). The palaeoclimatic model of Haywood
et al. (2004) evidence a seasonal surface temperature regime for the
southwestern Eurasian landmasses including the Iberian Plate during
the Barremian. In the Haywood model, the palaeoclimate tempera­
tures range from 12°C−28°C in the moderate season, whereas 8°C
−12°C for the coldest, and 32°C−36°C for the warmer. In addition,
the average sea surface temperature (SST) during the Barremian has
been estimated in Barral et al. (2017) to be approximately 22°C (the
20th century average SST was 13.9°C according to NOAA’s 2022
annual global climate report).
Due to both the higher concentration of atmospheric CO2 and
temperatures for the Barremian for the Iberian area than today’s atmo­
spheric conditions, it is expected that the microbial communities would
have had a greater rate of coverage. As the temperature increases so
does the metabolic rate (Noll et al. 2020), and second, a higher partial
pressure of CO2 stimulates the growth of autotrophic organisms (De
Luca et al. 1999; De Kluijver et al. 2013; Russell et al. 2013). The main
objective of this work is to value the coverage of zebrafish carcasses
(Danio rerio) by microbial mats that were laid on a microcosmos in
controlled conditions at temperature and CO2 similar to the ones
prevailing during the Barremian. The rate of body coverage depends
on the growth of the mats, and therefore on their net production (i.e.
net productivity of the community since the mats are made up of both
primary producers and consumers). This data has been evaluated based
on the photosynthetic oxygen production. Finally, we discuss the
implications of the obtained results in the fossilisation.
Material and methods
Culture of microbial mats in mesocosms and design of the
experiments
In order to assess the effect of the Barremian environmental con­
ditions in the Iberian area on the growth of microbial mats, a set of
experiments were carried out during 6 months. To perform the
experiments, we used mesocosms with microbial mats (Figure 2)
placed inside an environmental chamber where they were subjected
to two different temperatures (14°C and 26°C) at a constant pCO2
of 1000 ± 100 ppm which was the maximum CO2 concentration
estimated during the Barremian (Haworth et al. 2005).
Four experiments were developed using two levels of temperature
and the presence or absence of zebrafish carcasses (Danio rerio). In
the fish treatments, three individuals were placed in each mesocosm
on the microbial mats with 2 cm gap between specimens. Previous
experiments demonstrated that this separation is sufficient to avoid
the interaction between individuals of the phenomena that develop
after their placement on the mats (the initial decomposition and
subsequent growth of the microbial mat) (Iniesto et al. 2013).
The treatments were: (1) 14°C, with three carcasses per meso­
cosm laid on the microbial mat; (2) 26°C, with three carcasses laid
on the microbial mat per mesocosm; (3) 14°C, no carcasses; (4)
26°C, no carcasses.
The experimental design allows us to make the following com­
parisons, evaluated by linear mixed effect models:
● Metabolic rates at 14°C vs 26°C with carcasses laid on the
microbial mat.
● Metabolic rates at 14°C vs 26°C without carcasses laid on the
microbial mat.
● Metabolic rates at 14°C without carcasses vs carcasses lied on
the microbial mat.
● Metabolic rates at 26°C without carcasses vs carcasses lied on
the microbial mat.
The mats used in the experiment were collected near the shore in
the Salada de Chiprana saline lake (Zaragoza) at a depth of 10–20
cm. This shallow lake has a salinity ranging from 30 to 70 g/L
depending on the season of year, mainly MgSO4 (0.5 M) and
ClNa (0.35 M) (Guerrero 1991). The microbial community is domi­
nated by the cyanobacteria Coleofasciculus chthonoplastes, which
forms bundles in a common sheath, although there are also other
oxygenic phototrophs in the most superficial zone, mainly from the
diatom genera Nitzchia sp. and Navicula sp (De Wit 2016). In
deeper areas where there is still light, various types of anoxygenic
photosynthetic bacteria are observed, especially purple sulphur
bacteria and Chlorothrix halophila, of the Chloroflexaceae family.
Chiprana mats were crushed and prepared as described in pre­
vious experiments (Iniesto et al. 2013; Iniesto et al. 2015a) and
placed in three 30 × 15 × 17 cm mesocosms containing a 2–3 cm
base of limestone, overlain by a 3–4 cm layer of sediment from the
lake (Figure 2). Tanks were filled with water from the lake up to
approximately 4 cm over the surface of the crushed mat or sedi­
ment. Thereafter, microbial mats need to be grown in the labora­
tory for 3 months until maturation, i.e. when the mats exhibit the
characteristic multilayer organisation (Figure 1). Mesocosms were
illuminated with a cold beam (OSRAM Decostar 51 Titan) with
a photoperiod adjusted to 12 h of daylight and 12 h of darkness. To

Figure 2. Diagram of the mesocosm showing the arrangement of the microbial mats, fish carcasses, and the position of the dissolved oxygen probe, 1 cm above the surface
of the mat, centred and 11 cm from one of the short sides of the mesocosm. (Draw by one of the authors PA).
prevent light penetration and the subsequent growth of the mats on
the walls of the tanks, they were lined with cardboard in such a way
that the mats could only grow upwards, as occurs in their natural
environment. One additional mesocosm is used as control, pre­
pared with the same protocol but covered with a lid to keep in
darkness and to prevent the development of the microbial mat.
Once grown the microbial mats, the mesocosms were placed inside
an IBERCEX MOD E-600-BV CO2 environmental chamber with
LED tubes (Systion: SE-EGA-045-8W-4000K-CRI80-CL) to con­
tinue illuminating them for 12 hours to start the experiment.
Photosynthetically Active Radiation (PAR) was measured on the
water surface of the aquariums with a Quantum-Photo-Radiometer
(Delta OHM HD9021) ranged from 50 to 60 µE m−1 s−1, depending
on the aquarium.
Electric conductivity (EC) and pH (measured with 340i/SET
WTW conductivimeter and a 3210 SET2 WTW pH-metre, respec­
tively), and total alkalinity (using HACH-titration method 8203)
were periodically measured (4–7 days) to monitor physicochemical
changes.
Estimation of metabolism in mesocosms
We estimate the response of the mats by measuring the Net
Community Productivity (NCP) = Gross Primary Productivity
(GPP) – Community Respiration (CR) from diel changes in dissolved
oxygen concentration (DO). DO and temperature were continuously
measured using oxygen probes (AQUALABO. PONSEL OPTOD)
connected to a datalogger (CR300-Series), which recorded data every
10 minutes. Periodically (4–7 days) data recorded (pCO2, DO, air and
water temperature) were downloaded to a computer. Water in the
mesocosms were refilled when needed. The average depth of the three
mesocosms ranged from 3.1 to 4.7 above the microbial mat.
Gross primary productivity (GPP) and community respiration
(CR) were determined using a modification of the one-station diel
oxygen change method (Odum 1956), based on direct measure­
ments of changes in DO concentrations. During darkness, the
HISTORICAL BIOLOGY 3
change in oxygen concentration was assumed to be due to CR
and diffusion (D, exchange of oxygen across the air-water bound­
ary). During daylight, changes in oxygen concentration were
assumed to be due to CR, D and GPP. Diffusion was determined
from the rate of change of dissolved concentration in night-time
data. The re-aeration coefficients for diffusion (K2) were estimated
from a sequence of 10-min measurements of DO and temperature
during the night. Linear regression of the rate of change against the
saturation deficit gives K2 as the slope and community respiration
as the intercept (Thyssen et al. 1987). Markager and Sand-Jensen
(1989) observed sharp changes in night-time respiration just before
dawn and after dusk. Therefore, measurements up to 2 hours after
turning off the lights and 1 hour before turning on the lights were
excluded when calculating K2. Since re-aeration coefficients depend
on temperature, the following modified Arrhenius equation was
used to obtain the standardised K2 at 20°C and its value for diel
temperatures:
K2 ðT� CÞ¼K2 ð20� Þ�ΩðT 20Þ
where T is the actual temperature in Celsius degrees, and Ω is the
temperature coefficient, which remains constant (1.0241) for a wide
range of environmental characteristics (Gromiec 1989). Curves for
the rate of change of DO corrected for diffusion, based on data for
a 10-min interval, were plotted to calculate the metabolic rates.
Daytime community respiration was estimated by drawing a line
from the lowest pre-dawn value to the minimum post-sunset value
on the rate of oxygen change curve (Kelly et al. 1974; Hall and Moll
1975). Areas under the curves were integrated and used to calculate
the NCP, GPP at ecosystem level, and CR (Hall and Moll 1975). The
rates of change were converted to surface units (square metres) by
multiplying by mean depth. Curves of oxygen saturation concen­
tration were experimentally calculated for the water in the meso­
cosms. Water was cooled to 4°C and air saturated in a 1 L beaker.
Then, temperature was slowly increased with a heater and dissolved
oxygen concentration was continuously measured with optical oxi­
meter probe.
4 S. MOLLÁ ET AL.

Microbial mats coverage rate Table 2. Results of linear mixed effects models for comparison of metabolic rates
with and without fish carcasses laid on microbial mats (GPP: Gross primary pro­
For the calculation of the coverage rates, nine zebrafish (Danio ductivity, CR: Community respiration, NCP: Net community productivity, df 1:
rerio) (three per mesocosm) were used for each of the treatments. numerator degrees of freedom, df 2: denominator degrees of freedom. GPP, CR
Fish were euthanised with tricaine mesylate (MS-222, 0.06%) and NCP in gO2 m−2 d−1).
diluted in TRIS-phosphate (0.29%), following standard animal Average±SD Average±SD
care protocol used at the Universidad Autónoma de Madrid. 14°C N No fish laid on N Fish laid on df 1 df 2 F p-value
Dead bodies were laid on the surface of microbial mats with a 2 GPP 22 1.448 ± 0.818 20 0.659 ± 0.154 3 36 23.446 <0.001
cm gap between the specimens. Individuals were photographed CR 22 1.029 ± 0.343 20 0.327 ± 0.117 3 36 36.164 <0.001
together with a millimetre scale every 24 h until they were comple­ NCP 22 0.419 ± 0.868 20 0.332±.0.232 3 36 11.276 <0.001
tely covered. The size of the coated area of each fish is measured 26°C
using the ImageJ software (Abramoff et al. 2004). Coverage rate has GPP 18 0.569 ± 0.308 14 3.212 ± 2.641 3 26 9.183 0.001
CR 18 0.604 ± 0.372 14 6.369 ± 6.415 3 26 10.132 <0.001
been calculated as the total surface area of the fish (in mm2) divided NCP 18 −0.037 ± 0.352 14 −3.157 ± 4.489 3 26 7.937 0.001
by the number of days it took for the fish to be covered.

Data analysis
Differences between metabolic and coverage rates for each treat­ without fish, the GPP and CR were higher at 14°C than at 26°C,
ment are assessed by linear mixed effect models (LMEM, i.e. nested but the NCP was positive (Table 1).
ANOVAs with both fixed and random effects) for each variable. The second contrast explores independently the effect that car­
The LMEMs used in this paper accounted for the hierarchical casses had on GPP and CR at 14°C and 26°C (Table 2). At 14°C, the
nature of the experimental design, with ‘aquarium’ nested within GPP and CR were significantly higher without fish than with fish,
each fixed factor (temperature, fish/no fish) as a random factor. The resulting in high and positive NCP, as GPP exceeds CR. At 26°C,
Shapiro–Wilk test is used to assess normality of the analysed vari­ the GPP and CR were higher with fish than without fish, and CR
ables, and transformations when necessary. All statistical analyses exceeded GPP, resulting in negative NCP.
described above were performed with IBM SPSS Statistics v. 22 During the experiments, the average and standard deviation of
(Laas et al. 2012). electric conductivity, pH and alkalinity were 34.7 ± 9.1 mS cm−1,
8.2 ± 0.2 and 255.6 ± 84 mg L−1 CaCO3, respectively, and no sig­
nificant differences were found between treatments in any of these
Results variables.
Effects of temperature on the metabolism of microbial mats
Assessment microbial mat coverage rates over zebra fish
To evaluate the effect of temperature on metabolic rates, we per­ carcasses
formed two contrast analyses comparing the temperature in meso­
cosms with and without zebrafish on microbial mats (Table 1). In The rate of coverage of zebrafish carcasses under Barremian pCO2
mesocosms with fish, the GPP and CR were significantly higher at conditions (1000 ppm CO2 atm), although variable among meso­
26°C than at 14°C, but the NCP was negative. In mesocosms cosms, was rapid in all of them. The number of days required to
cover the fish was more variable, and considerably longer (about 16
days) at 14°C than at 26°C (Table 1, Figure 3a). At 26°C the cover­
Table 1. Results of linear mixed effects models for comparison of zebra fish coverage age was more constant, and 7% of the fish became covered in 7 days
and metabolic rates at 14°C and 26°C (GPP: Gross primary productivity, CR:
Community respiration, NCP: Net community productivity, df 1: numerator degrees
(in two of three mesocosms), whereas in the other took 2 days
of freedom, df 2: denominator degrees of freedom. GPP, CR and NCP in gO2 m−2 d−1). longer (Figure 3b). The applied statistical analysis (LMEM) shows
Average ± SD Averag ± SD significant differences of coverage between 14°C and 26°C, as well
as in the coverage rate (mm2/days to cover) (Table 1 and Figure 4).
df df The average area covered by the mat in time follows an ascend­
N 14°C N 26°C 1 2 F p-value ing line. At 26°C the maximum slope occurs between second and
Coverage 9 16.774 ± 9 24.789 ± 3 12 7.766 0.004 third days (Figure 5), then the slope softened but increased again in
rate 3.015 5.453
(mm2
the seven to nine, when the zebra-fish are completely covered. At
day−1) 14°C, the microbial mats barely grew over the fish during the first 2
Coverage 9 16.00 ± 3.391 9 7.44 ± 0.726 3 12 49.478 <0.001 days, and the maximum slope occurs days three-four (Figure 5).
time The percentage of coverage allows us to estimate the mat growth in
(day) surface over the fish daily, with respect to its size. The percentage is
Fish laid on
GPP 20 0.659 ± 0.155 14 3.212 ± 3 28 8.651 <0.001 a complementary calculation to the surface area covered per day,
2.640 providing similar results (Figure 6).
CR 20 0.327 ± 0.117 14 6.369 ± 3 28 22.850 <0.001 Interestingly, as in previous experiments (Iniesto et al. 2013), the
6.415 fish covered by the microbial mats broke less during handling and
NCP 20 0.332 ± 0.232 14 −3.157 ± 3 28 24.494 <0.001
4.489
preserved its colour and skin integrity (Figure 7).
No fish laid
on
GPP 22 1.448 ± 18 0.569 ± 3 34 26.212 <0.001 Discussion
0.818 0.308 The experiments took place under controlled conditions and have
CR 22 1.029 ± 18 0.604 ± 3 34 5.327 0.004
0.343 0.372 reproduced the environmental general features of the Chiprana
NCP 22 0.419 ± 0.868 18 −0.037 ± 3 34 6.266 0.002 saline lake in the laboratory, because the hydro-chemical data of
0.352 the mesocosms (electric conductivity, pH and alkalinity) kept
 HISTORICAL BIOLOGY 5

Figure 3. Coating process of zebrafish laid on the microbial mat at an atmospheric pCO2 of 1000 ppm and (a) 14°C of temperature and (b) at 26°C. Scale bar, 5 mm.

Figure 4. Comparison of mean coverage rate ± SD (mm2/day) between both temperatures tested in each of the mesocosms.

values similar to that of the original ecosystem (Guerrero 1991;
Jonkers et al. 2005). Further, the experience aims approaching the
taphonomic processes occurred in microbial mats measuring its
metabolic and growth rates below pCO2 values and average tem­
peratures that emulate the Barremian atmosphere (Haworth et al.
2005; Barral et al. 2017; Burgener et al. 2023) to explore the effects
on fish corps preservation.
The fish laid on the mat at the Barremian atmospheric condi­
tions remained articulated along the experiment, with skin and
scales almost intact (preserving their original pigmentation), as in
previous taphonomic experiments, which were executed at current
conditions of pCO2 (~416 ppm) and ambient temperature (25°C)
(Iniesto et al. 2013; Alfonso et al. 2015; Buscalioni and Poyato-Ariza
2016). Therefore, no significant differences on early decay were
detected and neither related to the changes expressed in the abiotic
variables. However, the results of the present experiments provide
new clues about the mat cover rate and the environmental condi­
tions of the mat community subjected to these changes; but bio­
precipitation still needs of longer termed data collection.
The rate with which carcasses are covered by the mat is signifi­
cantly different depending on the experimental conditions; 8 days
at 26°C and 14 days at 14°C (at a pCO2 = 1000). In fact, the increase
6 S. MOLLÁ ET AL.

Figure 5. Mean area covered ± SD every 24 hours for fish located in each of the mesocosms for both temperatures (14°C and 26°C). In the black line, the last points belong
to data from a single fish that took six days longer than the rest to be covered.

Figure 6. Mean percentage ± SD of fish covered every 24 hours in each of the mesocosms for both temperatures (14°C and 26°C). In the black line, the last points belong to
data from a single fish that took six days longer than the rest to be covered.

Figure 7. Zebrafish after 8 days being laid on the sediment (no microbial mat) in
the control mesocosm (left). Zebrafish after 30 days laid on microbial mat in
a mesocosm (right). Both fish were subjected to 1000 ppm pCO2 and 26°C
temperature at the same time. Scale bar = 5 mm.
in the coverage rate is due to the accelerated metabolic rates that
exert the microbial mat mostly with the temperature when fish
carcasses were present, with much higher gross primary productiv­
ity (GPP) at 26°C than at 14°C (3.212 and 0.659 gO2 m−2d−1
respectively, Table 1). This result agrees with the increase in cell
growth and primary production with the temperature (Lepock
2005; Zoboli et al. 2018).
The effect of temperature on the GPP and CR has been well
studied in different primary photosynthetic producers, although
there are few studies concerned on microbial mats (Epping and
Kúhl 2000; Abed et al. 2006, 2006; Jodloswska and Latala 2013;
Zhang et al. 2020). Recent studies in currently coastal microbial
mats, tested the effect of the changes in temperature (Berlanga et al.
2022), finding that temperature strongly regulates the physico­
chemical processes involved in mass transfer mat within the mat
and across the water interface, such as solute diffusion and solute
solubility, and the metabolic rates under high summer tempera­
tures. In general, an increase in temperature also conveys the
increase in GPP and the CR. However, interestingly, at 26º the
(CR) exceeded the GPP resulting in a negative NCP value, unlike
at 14°C, where the GPP and the RC were lower, and the GPP
exceeded the CR and the NCP was positive. It is interesting to
note that the rapid growth of the mats in days 2 and 3 at 26°C
(Figures 5 and 6) related to the decomposition of the fish. The lysis
of the fish cells (autolysis) by catabolically active enzymes generates
an acidic, anaerobic and nutrient-rich environment (Mukundan
et al. 1986), and many of these nutrients are organic molecules
that the heterotrophic microorganisms in the mat would rapidly
consume releasing inorganic nutrients that would be used by cya­
nobacteria and other autotrophic microorganisms for rapid growth.
The effect of the carcasses on the microbial mats and the further
increase of the CR can be analogised with what occurred in ecosys­
tems with and without an external energy input as organic matter
(Sand-Jensen and Staehr 2009; Laas et al. 2012; Brett et al. 2017;
Adamczuk et al. 2019; Scharfenberger et al. 2019). Usually, the CR
and the GPP are balanced since all the resources that can be
HISTORICAL BIOLOGY 7
consumed come from the primary productivity (GPP). However,
those ecosystems that receive external energy inputs (subsidised
according to Odum 1971; Babcock et al. 2006), the CR usually
exceeds the GPP since it consumes the extra contribution of
resources, resulting in a heterotrophic ecosystem. The community
respiration increases are linked to the carcass decomposition pro­
voking the consumption of oxygen.
Notwithstanding, the result was not as expected for the meta­
bolic rates with fish and without fish at 26°C. At that temperature,
the CR exceeded the GPP, and the NCP was negative even without
fish on the mat (Table 2). Because the only reason for the negative
value of NCP was the consumption of the accumulated organic
matter, we suppose that a deterioration of the microbial mat should
be the cause. Although it has not been quantified, this deterioration
has been observed, with a progressive weakening and deteriorating
over time. Nonetheless, at 14°C without fish (and also with fish) laid
on microbial mats, the GPP did exceed the CR, with a positive NCP.
Supposing a parallelism with our experiment between the
changes in temperature and seasonality in the aquatic ecosystems
(without and with mat communities), such as the Mediterranean,
arid and semi-arid areas, it can be noticed that an alternation in the
trophic state occurred when inputs of terrestrial energy resources
are very limited (Florín and Montes 1998; López-Archilla et al.
2004; Alfonso et al. 2015; Iniesto et al. 2018). In such areas, the
ecosystems alternate yearly from autotrophic (GPP>CR and posi­
tive NCP) being the resources of organic matter (OM) accumulated
in the substratum, to heterotrophic in which the previously accu­
mulated resources (OM) are consumed, and its trophic state
becomes resulting in a negative net community productivity. In
the monitored wetland ecosystems with temperature fluctuation
and external organic matter input (e.g. urban, agricultural or live­
stock discharges) would have produced an alternance, in the energy
budget of the ecosystem from heterotrophic (consuming organic
matter) to autotrophic (accumulating organic matter) (i.e. López-
Archilla et al. 2004).
Inferences on the fossilisation process
There are no differences in the carcasses preservation mediated by
microbial mats at the Barremian atmospheric conditions in com­
parison with the current ones. In that regard, our results are in
consonance with other experiments with microbial mats, and sup­
port the evidence that photosynthetic organisms have a high capa­
city to regulate the pH and mat communities are able to adapt to
conditions, such as acidification and warming of surface water
(Mazière et al. 2022). Thereby, biostratinomic alterations to reach
exceptional preservation does not depend upon atmospheric con­
ditions where the carcasses were deposited.
It is recognised that microbe-sediment interaction occurs in
most of the terrestrial sediments, although in some sediments
microbial growth is more favoured than in others, and it is also
recognised the microbenthos responds differently to the sediment
dynamics (Noffke 2021). In addition, environmental conditions,
such as anoxia, cold temperatures and high salinity, modulate the
activity of the microbial mat (Janssen et al. 2022). Both the inter­
action of the benthic microbial community with the sediment and
the environmental conditions noted above can promote the pre­
servation and fossilisation. Being these certain, what we in addition
highlight is that the role played by the organism entombed in the
mat community is also important, because it stimulates the mat
activity, changing the mat conditions (e.g. it may contribute to
favour the anoxia nearby and inside the carcass, see Iniesto et al.
2015b, 2018), and consequently favouring the early diagenesis and
mineralisation. Zebrafish carcasses in mesocosms subjected to the
8 S. MOLLÁ ET AL.
Figure 8. Flowchart of the effects promoted by an animal corpse in the MM (microbial mat) community. In red, is represented atmosphere variables (temperature and
carbon dioxide).
atmospheric conditions of pCO2 and temperature estimated for the
Barremian were covered twice as fast at the higher temperature.
After this first phase, the release of nutrients would be less, which
would explain why the growth rate was slower afterwards at both
temperatures. At 26°C the GPP also showed the highest values, as
well as those of the CR, although the effect of the high temperature
would promote preservation over decomposition. According to the
above, the results suggest that the capture of organism would occur
more efficiently during warmer periods, while in cooler ones the
carcasses would be covered much more slowly by microbial mats,
decreasing the probability of preservation and fossilisation of the
remains. Therefore, the results of the experiment would support the
argument that the microfacies with stromatolite lamination and
significantly higher abundance of fossils would have produced
during periods of higher temperature.
Further experiments
Future actualistic experiments should be focused on assessing
the changes in organic and inorganic carbon (i.e. organic matter
and calcium carbonate) in the sediment to understand biomi­
neralization, concretely by calcium carbonate. To address this
objective, we assume that in the experiment the microbial mat
is stimulated by the presence of the animal corpses, especially at
high temperature (Figure 8). During decaying, the action of the
heterotrophic microorganisms, which are an active part of the
community, releases inorganic nutrients by processing the labile
organic compounds from the decayed corpse (initially by
autolysis). These inorganic nutrients, in turn, stimulate photo­
synthesis, the pH, and increase the amount of carbonates
(Ludwig et al. 2005), and further, more organic matter is gen­
erated by the autotrophic organisms. Some of this organic
matter is integrated into the autotroph cells, but some is
released into the environment. Studies in the currently micro­
bial mats from the Salada of Chiprana showed that 50% of fixed
organic is exported daily (Jonkers et al. 2003). In the microbial
mats a fraction of the released matter constitutes EPS, which
play an important role in bioprecipitation (Dupraz et al. 2009;
Noffke 2021). However, the organic matter integrated into the
cells of the autotrophs together with the EPS themselves will
become buried detrital matter as the community grows. They
may also play a role in calcification by stimulating anaerobic
decomposition, e.g. through the activity of heterotrophs such as
sulphate-reducers by maintaining high concentrations of dis­
solved inorganic carbon in that zone, which stimulates biopre­
cipitation (Ludwig et al. 2005). Accordingly, our next
experiments aim to relate the processes occurred above the
microbial mat, the ones within the mat, and in the sediment,
taking into account that the type of organism may play a role in
metabolic response by mats.
Acknowledgments
We thank Taphos organisers for gathering the meeting contributions. This study
was funded by the Spanish Ministry of Science, Innovation and Universities
[project PID2019-105546GB-I00].

Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Salvador Mollá http://orcid.org/0000-0001-5769-139X
Paloma Alcorlo http://orcid.org/0000-0002-0048-0038
Angela D. Buscalioni http://orcid.org/0000-0003-1598-7963
Ana Isabel López-Archilla http://orcid.org/0000-0002-3673-6189
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