MEMORIA CIENTÍFICO-TÉCNICA DE PROYECTOS INDIVIDUALES

Convocatoria 2021 - «Proyectos de Generación de Conocimiento»

AVISO IMPORTANTE - La memoria no podrá exceder de 20 páginas. Para rellenar correctamente esta memoria,
lea detenidamente las instrucciones disponibles en la web de la convocatoria. Es obligatorio rellenarla en
inglés si se solicita 100.000 € o más (en costes directos).
IMPORTANT – The research proposal cannot exceed 20 pages. Instructions to fill this document are available
in the website. If the project cost is equal or greater than 100.000 €, this document must be filled in English.

1. DATOS DE LA PROPUESTA – PROPOSAL DATA

IP 1 (Nombre y apellidos): David Sánchez-Fernández
TÍTULO DEL PROYECTO (ACRÓNIMO): Predicción del futuro de la
biodiversidad subterránea frente al cambio climático desde una perspectiva
experimental (FRAGGLE)
TITLE OF THE PROJECT (ACRONYM): FoRecAstinG the fate of
subterranean biodiversity to face climate chanGe from an experimentaL
perspectivE (FRAGGLE)

2. ANTECEDENTES, ESTADO ACTUAL Y JUSTIFICACIÓN DE LA

PROPUESTA - BACKGROUND, CURRENT STATUS AND JUSTIFICATION OF THE
PROPOSAL

2.1. Background and state of the art

Amidst interacting climate emergency (Ripple et al., 2020) and biodiversity crises unmatched in human
history (Román-Palacios & Wiens, 2020), scientists face a pressing question: How can we anticipate the
consequences of global change on current ecosystems? Answering this question is fundamental to
achieve most Sustainable Development Goals, which depend upon safeguarding biodiversity in all its
forms and functions. Biodiversity is our insurance to sustain the provisioning of a wealth of nature’s
contribution to people, including services, goods, and life full-filling conditions. Yet, significant
research biases still permeate biodiversity conservation. For example, organisms that live in out-of-sight
ecosystems (e.g. caves, deep-seas) are only alluded to, if not overlooked, in global biodiversity goals.
Meanwhile, aesthetic and charisma bias how the scientific community approaches the study and
protection of flora and fauna (Mammola et al. 2020b; Adamo et al. 2021). Such examples illustrate that
despite recent heartening conservation successes (e.g. Bolam et al., 2021), we are still far from
establishing conservation goals that are comprehensive, ecologically sustainable and cost-effective.
Subterranean ecosystems provide key ecosystem services to humanity, ranging from the supply
of freshwater resources to the sequestration of greenhouse gasses, along with their potential use in
pharmaceutics and engineering (Griebler & Avramov, 2015; Mammola et al., 2019a). To emphasise the
importance and urgency of protecting subterranean biodiversity, the year 2021 was elected the
International Year of Caves and Karst. With subterranean ecosystems in the spotlight, several scientists
published calls for actions in high-rank journals lamenting their lack of protection. In a letter to Nature
Climate Change, we showed that only 6.9% of known subterranean ecosystems overlap with protected
areas globally (Sánchez-Fernández et al., 2021). Importantly, most of these subterranean ecosystems
are protected simply because they occur within protected areas established for surface species/habitats,
thus not designed to account for their vertical dimension. To rectify this omission, along with
representatives from 23 nation-states and 7 indigenous groups, we proposed a roadmap to guide the
conservation of the subterranean biome through 2030 and beyond (Wynne et al., 2021).
On paper, these calls for action emphasize a positive trend. Yet, if scientists’ warnings are not
supported by quantitative evidence on what constitutes critical threats and effective conservation
interventions, all these efforts are merely ‘whistling in the wind’. Subterranean ecosystems are some of
the most understudied ecosystems on Earth and only recently we began to grasp their eco-evolutionary
importance and economic value. So, we have to confront a thorny question: What impediments prevent
the establishment of effective protection of subterranean biota and their inclusion in global conservation

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goals? A main impediment has stalled most attempts so far: we lack a mechanistic understanding of
subterranean species response to climate change.
Thus, in order to make informed decisions, policymakers will need models and predictions as
much accurate as possible (Grimm et al., 2020). In nature, there are only a few habitats in which the
typical uncertainties of climate change vulnerability assessments are minimized, as for example the deep
subterranean environment. Of all natural situations, this is one of the few that can be easily reproduced
under controlled laboratory conditions. In contrast to what happens in surface environments, the range
of variables affecting a species underground is minimal. The humidity in the deep parts of caves is
always near the saturation point and the temperature is highly constant through the day, season and year,
and more interestingly, it is established that it is approximately equal to the mean annual temperature at
the surface (Poulson & White, 1969; Culver & Pipan, 2009; Sánchez-Fernández et al., 2018). In
addition, environmental conditions are practically homogeneous across all microhabitats within a cave
system, so small-scale spatial heterogeneity and the possibility of behavioural adjustments are minimal
compared to surface environments. Also, caves harbour very simple biological communities (Culver &
Pipan, 2009), which minimizes the complexity of accounting for biological interactions. Furthermore,
most highly specialized cave species also have a well-defined distribution, as they show low dispersal
capacity and extremely narrow geographical ranges. Persistence in their current localities is therefore
critical for subterranean species to face climate change, as dispersal to more suitable habitats is not an
option. In sum, unlike superficial environments, here we can have an accurate knowledge about the
environmental conditions that species are currently experiencing. Therefore, these particular natural
laboratories and their inhabitants are ideal model systems to study the effects of global change on
species with poor dispersal capabilities as well as to test biogeographical, ecological and evolutionary
hypotheses (Sánchez-Fernández et al., 2018; Mammola, 2019).
Indeed, it is not surprising that biologists have long been fascinated by the peculiarities of typical
subterranean organisms (Darwin, 1859; Jeannel, 1943), as they show extreme morphological,
physiological, and biological adaptations. For example, many of them have evolved to become blind,
wingless, pigmentless, etc, reflecting severe environmental constraints, which result in an invaluable
evolutionary resource (Juan et al., 2010). In this sense, it is worth noting that subterranean species have
been used for the study of phenotypic and ecological specialization, and the study of the changes
associated with underground colonization have contributed many new insights in evolutionary biology
in recent years (Protas et al., 2011). Other than to exemplify general principles, subterranean fauna is
certainly of interest and value on its own, as they represent an often neglected but substantial portion of
the entire biodiversity. Although there is a general lack of knowledge of most subterranean groups
worldwide, it has been estimated that there are a total of 50,000 to 100,000 obligate subterranean species
with a high level of endemism (Culver & Holsinger, 1992). For example, only in the Pyrenees and in a
tribe of beetles (Leptrodrini) there are more than 150 species and all of them are narrowly distributed
endemics. Such a number of endemic species is much higher than that obtained by adding up the
endemic species of all groups of vertebrates together. Species that have evolved in extremely stable
environments for millions of years must be very susceptible to changes in their habitats, such as those
resulting from climate change and so they could be in high risk of extinction. However, their
vulnerability to an increase of temperature have been poorly explored so far.
An understanding of the specific mechanisms underlying conservation problems is becoming
increasingly important for decision-making, in part because physiological tools and knowledge are
especially useful for developing cause-and-effect relationships, and for identifying the stressor
thresholds for different organisms. We have to be aware that real advances in conservation and resource
management require integration and inter-disciplinarity. When physiological knowledge is incorporated
into ecological models, it can improve predictions of organism responses to environmental change and
provide tools to support management decisions in order to protect biodiversity (Evans et al., 2015;
Rodríguez et al., 2019). Thus, conservation physiology appears as an emerging discipline and its suite
of tools and concepts is a key part of the evidence base needed to address pressing environmental
challenges (Cooke et al., 2013). Data on thermal biology have the potential to contribute greatly to our
understanding of the effects of climate change (Bernardo & Spotila, 2006; Helmuth, 2009), since
warming-induced stress is the most proximate effect of global warming, preceding behavioural or
evolutionary responses to new environmental conditions (Pörtner & Farrell, 2008). Experimental
approaches to obtain such data are well developed for a wide range of taxa, from marine invertebrates
to mammals (Bozinovic et al., 2011). Accordingly, experimental estimates on heat tolerance limits and
individual thermal performance have a great potential for improving predictions on the impact of global

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warming, especially in poor dispersal species in highly stable environments (Somero, 2010) such as
subterranean fauna. In this context, three important considerations in the assessment of how climate
change will impact organisms are (i) how close organisms are to their physiological thermal limits in
nature; (ii) what factors could affect these thermal limits and survival and so, their vulnerability to
climate change; iii) to what extent organisms will be able to adjust such thermal limits through
physiological plasticity and evolutionary change.
While most of the highly interesting research carried out to date on subterranean organisms has
been focused on the process of colonization of subterranean environments and some of the associated
phenotypic changes (loss of eyes, pigments, etc), the related changes in thermal biology of these species
have become to gain interest only during the last years, as this knowledge has been recognised as
essential for estimating the vulnerability of subterranean fauna to climate change (Mammola et al.,
2020a). The current project could shed light on this timely and highly relevant issue.

2.2. Previous results of the team relevant to the FRAGGLE project

The first attempts to explore both thermal tolerance of subterranean species and their capability to cope
climate change were provided by Rizzo et al., (2015) and Sánchez-Fernández et al., (2016). Estimates
of the thermal niche using information from long-term physiological experiments for only 4 species
living under different thermal conditions (from the Pyreness at 6-7ºC, to the coastal area of Catalonia at
11ºC; see Rizzo et al., 2015), provided unexpected and exciting results. First, physiological data (based
on survival experiments) suggested that these subterranean species actually have a relatively broad
thermal tolerance, allowing them to withstand temperatures never experienced during their evolutionary
history. Surprisingly, no differences were found between the thermal tolerance of these species, and
their viable temperature range was between 0°C and 20-23°C (Rizzo et al., 2015). In sum, their thermal
tolerance range is much narrower than that of surface insects living at the same latitude, but still very
much wider that the temperatures experienced during the evolutionary history of this clade and unrelated
to the current habitat temperature of individual taxa. The experimental approach used in this study
allowed us to get a more realistic estimate of the capability of species to cope with temperatures outside
those of their current environment than a bioclimatic (species distribution models) approach alone would
provide. Then, these results showed that for species with poor dispersal capabilities and even in a system
in which the uncertainties of typical assessments of vulnerability to climate change are minimized,
physiological data on thermal biology are required to get more accurate predictions. The narrow thermal
window of the species studied, apparently conserved/homogeneous across species irrespective of the
climatic conditions of the areas where they live could suggest that i) these species have lost some of the
thermoregulatory mechanisms common in temperate insects, and ii) there is a lack of evolutionary
adjustment to ambient temperature. However, the question that remained was to what extent such
surprising narrow and homogeneous thermal niche was a common feature of subterranean biodiversity
in general, or just a peculiarity of the few species examined by Sánchez-Fernández et al., (2016). These
studies provided the basis for the first project focused on the study of climate change, thermal niche and
conservation of subterranean biodiversity (the CAVEheAT project; PI David Sánchez-Fernández)
funded by the Spanish Ministry of Science and Innovation (former “Excellence Program”; CGL2016-
76995-P).
The main objectives and findings of this project were:
i) To test for differences in the upper thermal tolerances of a number of subterranean species
from different clades, different geographical areas, and living under different environmental conditions.
We provided for the first time data on basal heat tolerance for a subterranean pseudoescorpion (Colado
et al., 2021). We also compared heat tolerance among species of distant arthropod groups (Coleoptera
(Leiodidae and Staphilinidae), Diplopoda, and Collembola) that coexist in a cave located in Jaén, and
therefore represent subterranean taxa with different evolutionary histories but under similar selection
pressures, as they have been exposed to the same constant environmental conditions of the cave for a
long time. We found notable differences in upper lethal limits among these species. Indeed, the diplopod
(Glomeris sp.) showed the highest long‐term heat tolerance detected so far for a troglobiont (i.e., obligate
subterranean) species (median lethal temperature after 7 days exposure: 28°C). These results suggested
that heat tolerance in subterranean species is not determined by environmental conditions, and even
those species living under similar climatic conditions might be differently affected by global warming
(see Pallarés et al., 2019). Using the same experimental approach, we have found differences in heat
tolerance among species of the family Leiodidae from the Baetic System (Pallarés et al., 2019) and the
Pyrenees (Colado et al., in preparation). Bringing together the results of these different studies with

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previous ones (Rizzo et al., 2015; Sánchez- Fernández et al., 2016), a lack of adjustment of heat
tolerance to current habitat temperature could be general for subterranean ectotherms, as a wide gap
between upper thermal limit – cave temperature has been found in different and disparate arthropod
groups. However, the homogeneous (i.e. similar across species) thermal niche suggested before when
explored in a few species (Rizzo et al., 2015) might not be common, as we find differences in upper
thermal limits and acclimation capacity between species (even from the same family). These differences
are likely associated to the evolutionary history or other factors not considered before (e.g. degree of
specialization to subterranean environment).
ii) To explore the acclimation capability of species from subterranean environments. In Pallarés
et al., (2021a), we considered this relevant aspect of thermal tolerance in addition to thermal limits.
Upper lethal limits and heat acclimation capacity were compared among only three related beetles
(Leiodidae: Leptodirini) with differing degrees of specialization to the subterranean environment (deep,
shallow and facultative subterranean, respectively) and therefore exposed to contrasting thermal
variability in nature. Only the facultative subterranean species, exposed in nature to a greater thermal
variability, showed any acclimatory capacity, also having the highest heat tolerance (upper lethal limit)
across the taxa studied (Pallarés et al., 2021a). It is likely that data limitation (shortage of individuals)
in this study (only one acclimation treatment – 20ºC was compared with a control treatment - cave
temperature) provided a simplified vision of a complex process such as plasticity to heat tolerance.
However, the findings suggested that the pathways that determine vulnerability of subterranean species
to climate change (i.e., thermal sensitivity and exposure to climate change) could depend on their degree
of specialization to deep subterranean environments, suggesting that adaptation to cave conditions could
have been selected against the retention of acclimation mechanisms.
iii) To explore the relationship between thermal niche features and the degree of specialization
of species to deep subterranean environments. Using heat tolerance data for 16 species belonging to the
same lineage (Leiodidae: Leptodirini) and with different degree of specialization to subterranean
environments, we demonstrated that heat tolerance is mainly determined by the long-term thermal
stability of the habitat, regardless of the average climatic conditions (Colado et al., 2022). Thus, the
specialization process to live in deep, highly stable subterranean habitats involves a loss of heat
tolerance. More importantly, this extended data set demonstrated that such loss of heat tolerance is not
related the temperature at which species are exposed. Then, there is not a fine-tune adjustment of the
thermal limit to the environment, as seen for example in antarctic organisms that show a strong thermal
specialization (narrow fundamental thermal niche). Our results support the climatic variability
hypothesis (Stevens, 1989), which predicts the evolution of wider thermal tolerance ranges in species
exposed to environments with variable temperatures as well as the evolution of species with narrower
tolerance ranges in thermally stable environments, extending also this interesting relationship to small
spatial scales and narrow gradients of thermal variability within subterranean environments (Colado et
al., 2022).
The CAVEheAT project finished in 2020. We feel that we have greatly advanced in the
understanding of some physiological and ecological aspects on the thermal tolerance of subterranean
biodiversity. However, we also feel that only the surface of these topics with broad interest from
ecological, evolutionary and conservation perspectives have been scratched, and there are still many
open (even more interesting) questions to be solved during the next years.
In parallel, we have just started another highly related, but promising research line, devoted to
assess vulnerability of subterranean fauna to climate change using complementary approaches to typical
survival experiments (Pallarés et al., 2020). By combining experimental measurements of lethal thermal
limits, acclimation capacity and molecular stress biomarkers in the subterranean beetle species
Parvospeonomus canyellesi (Lagar, 1974) (Leiodidae: Leptodirini), we showed that exposure at
temperatures below lethal limits (20ºC) could induced oxidative stress and alteration of the activity of a
key nervous system enzyme (AChE). These results stress the need of exploring performance not only in
terms of survival, but also other thermally sensitive processes at different levels of biological
organization. This approach would allow to obtain more accurate estimates of the capability of poor
dispersal species to cope with temperatures outside those they currently experience (Pallarés et al.,
2020).
Focusing on the vulnerability of species to global warming, we have also evaluated what drives
vulnerability to climate change in other endemic threatened taxa (Arribas et al., 2012). For this purpose,
we employed comparative thermal physiology, species distribution models and estimations of species
dispersal based on genetic data, as a basis to derive conservation strategies for individual species. Other

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aspects of the physiology of insects have also treated by the IP and the members of the research and
working team, e.g. issues on osmorregulation, resistance to desiccation, thermal tolerance, thermal
plasticity and the effects of interactions between multiple stressors on insect performance (e.g. Pallarés
et al., 2016, 2021b; Velasco et al., 2018). We have also explored the congruence of thermal niche
estimations derived from distribution and physiological data (Sánchez-Fernández et al., 2012).

2.3. Justification of the proposal and theoretical framework

The FRAGGLE is the natural continuation of the CAVEheAT project, but now expanding both the
conceptual and applied dimensions on the vulnerability of species to climate change. Thus, the results
and experience acquired during the CAVEheAT project (database, network, facilities, previous
knowledge and analytical tools) will now be used as the starting point.
With this project, taking advantage of the unique natural laboratory offered by caves for
ecological, biogeographical and conservation research (Sánchez-Fernández et al., 2018; Mammola,
2019), we expect to develop the field of conservation physiology for subterranean biodiversity in three
directions: i) improving the understanding of the mechanisms underlying tolerance of insects to heat
stress and their evolution; ii) establishing direct links between species' physiology and vulnerability to
climate change and iii) providing one of the most accurate predictions of species response to climate
change estimated so far for a complete clade of species. Whether subterranean organisms have lost the
ability to adapt to a changing environment has been stressed as one of the fundamental research
questions in subterranean biology (Mammola et al., 2020a), being the degree of specialization to deep
subterranean environments a key driver for the pathways that determine vulnerability to climate change
(Pallarés et al., 2021a; see Figure 1).
The climatic variability hypothesis (CVA) provides the theoretical framework to shed light into
the relationship between thermal tolerance, subterranean specialization and environmental conditions,
something that is pivotal for the conservation of subterranean fauna in a climate change perspective (see
Figure 1). One of the main limitations of mechanistic approaches to predict species vulnerability to
climate change is that obtaining physiological
information for species is costly, and then the main
insights provided in this fields are derived from a very
reduced set of species. The challenge now is to get a
compromise between the need to accurately predict
vulnerability to climate change for as many species as
possible, or complete lineages, and our capacity to
obtain physiological data, in a context of ongoing
global warming at unprecedented rates. The loss of heat
tolerance in the process of specialization to the deep
subterranean environment (Colado et al., 2022) could
represent another example of convergent evolution in
caves, as other troglomorphies (e.g. eye reduction or
elongation of appendages). Thus, if morphological
adaptations to subterranean environments parallel with
the reduction of heat tolerance, such correlation makes
it possible to predict the heat tolerance values for a
complete clade just using morphological traits as
predictive variables, which are by far easier to obtain
than physiological data.
Despite the valuable information provided by
thermal tolerance experiments based on typical survival
assays by themselves, predictions of species
vulnerability to climate change based on physiological
data could be refined at a deeper level by considering
the multiple factors that could affect species thermal
tolerance.
Figure 1. Conceptual framework

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 To explore this issue, subterranean habitats are at an advantage with respect to other habitats, as
such factors are in principle minimal. Indeed, the most important feature that corroborates the idea that
caves are ideal laboratories for the study of climate change in natural environment is their unique
climatic stability. However, apart from temperature, it is worth noting that air moisture content (i.e.
relative humidity) is one of the most important limiting factors for terrestrial cave obligate species
(Mammola et al., 2019b). Accordingly, the maintenance of high humidity levels appears to be essential
for the survival of different troglobionts. This is generally explained by the high cuticular permeability
of many species, associated with an extremely low resistance to desiccation. In a context of changing
rainfall patterns in many parts of the world, humidity levels may decrease in Mediterranean subterranean
habitats. Effects of increasing temperature on survival and fitness of subterranean species have been
recently explored at length whereas, surprisingly and as far as we known, there are no studies focused
on the effects of the predicted decrease of relative humidity on the heat tolerance of subterranean species
(nor in isolation nor combined with the increase of temperature). In the context of the climatic variability
hypothesis and according to our previous results on heat tolerance in subterranean beetles (Colado et al.,
2022), the negative effect of decreasing humidity on heat tolerance could be also different among species
depending on their degree of degree of specialization to deep subterranean environments being higher
for highly specialized species exposed to lower climatic variability.
In the same way, acclimation capacity will be fundamental in determining the sensitivity of
subterranean species to climate change (Figure 1). Acclimation responses can provide a measure of
species thermal plasticity, and therefore their capacity to adapt to changing conditions (Somero, 2010).
However, despite the first advances provided by Pallarés et al., (2021a), we still do not know how
acclimation capacity performs in subterranean species. Using thermal response curves (TRC) obtained
from thermal tolerance values at several temperature treatments (from below the cave temperature to
stressful sublethal temperatures), we could show how, when and for which species heat tolerance
plasticity is displayed (Figure 2). Besides, we could formally test the hypothesis raised by Pallarés et al.
(2021a), that is, that the pathways that determine vulnerability of subterranean species to climate change
depend on their degree of subterranean specialization, suggesting that adaptation to cave conditions
could have been selected against the retention of acclimation mechanisms. Such hypothesis is consistent
with CVH, according to which both heat tolerance and acclimation capacity are expected to decrease
with increasing subterranean
specialisation. However, if trade-
offs between thermal limits and
plasticity have shaped the
evolution of thermal tolerance in
these taxa, as found in other
invertebrates (Stillman, 2003),
such a relationship with
subterranean specialisation might
not be so evident for acclimation
capacity (i.e., species living in the
more variable habitats could
tolerate higher temperatures but
show little thermal plasticity).
Figure 2. Theoretical acclimation response curves expected in subterranean
species depending on their degree of subterranean specialization

Besides, in a context of global warming, another interesting and generally overlooked approach
is to assess the potential importance of evolutionary adaptations that occurs quickly enough to prevent
species or populations extinctions, i.e., the so called evolutionary rescue (Carlson et al., 2014). This is
especially relevant for species with low dispersal capacity (which have to face of these changes in situ),
and also with little capacity for behavioral adjustments. By reconstructing the heat tolerance (as ancestral
character) in the phylogeny would allow us to explore how far the evolutionary rescue could be an option
to face climate change for these species with low thermal tolerance and insufficient phenotypic
plasticity.
The approaches exposed above would allow us to obtain more accurate estimates of the capability
of subterranean species to cope with temperatures outside those they currently experience, and
consequently, better estimates of their capacity to face climate change.

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3. OBJETIVOS, METODOLOGÍA Y PLAN DE TRABAJO - OBJECTIVES,
METHODOLOGY AND WORK PLAN

3.1. Specific aims and hypothesis

The main aim of this project is to predict the capacity of subterranean biodiversity to face climate change
from an experimental perspective. The specific aims are:

O1. To infer heat tolerance values for a whole lineage of cave beetles from their morphological
traits. As the selective pressures during the process to specialization to live in deep subterranean
environments appear to have generated convergent syndromes spanning both morphological and
physiological traits, we expect to be able to predict the heat tolerance values for the complete clade (ca.
160 species) just using morphological traits as predictive variables (much easier to obtain than
physiological data). This prediction will be validated with new heat tolerance experiments for a set of
species not considered during the model training.

O2. To assess the impact of a decrease in relative humidity on the thermal physiology of beetles
with differing degrees of specialization to the subterranean environment. It is expected that the
predicted decrease of relative humidity has an effect (reduction) on the heat tolerance of subterranean
species. Following the climatic variability hypothesis (Stevens, 1989), we expect that the decrease in
heat tolerance caused by desiccation stress (loss of relative humidity - RH) will depend on the degree of
specialization to deep subterranean environment, being higher for highly specialized species exposed to
lower climatic variability.

O3. To assess plasticity in heat tolerance in subterranean beetles and its relationship with heat
tolerance and subterranean specialization. We will test if there are differences in the thermal
plasticity of species in function of their degree of subterranean specialization when they are previously
exposed to different acclimation temperatures in laboratory conditions. For this, we will generate
thermal response curves (TRC) to assess how acclimation at different temperatures changes heat
tolerance in species with different degrees of subterranean specialization. We will focus on the response
of the species regarding two critical points (acclimation temperatures above the temperature of their
caves and close to their physiological limits obtained in O1). The climatic variability hypothesis (CVH,
Stevens, 1989) predicts that animals from more thermally variable habitats will have greater plasticity,
while the trade-off hypothesis (Stillman, 2003) predicts that individuals with the lowest heat tolerance
will have the greatest plasticity. In our case, it is expected that highly specialized subterranean species
have lost the ability to acclimate to different ambient temperatures, as the acclimatization mechanisms
are costly in terms of energy economy (Pallarés et al., (2021b), and the specialization process to
subterranean environment would also imply fewer available resources.

O4. To assess the capability of different species and populations to face climate change. It is
expected that species with higher heat tolerance are more able to respond to the new conditions imposed
by climate change and so their vulnerability should be lower to such environmental change. Such
vulnerability will be estimated by obtaining thermal safety margins (TSM), as the difference between
the estimated temperature of the locality for both current and future scenarios and the estimated
physiological thermal limits gathered for the previous objectives. Besides this information, the rates of
evolutionary change of heat tolerance estimated from a molecular phylogeny will be also considered as
a potential way to explore the evolutionary rescue. The general theory for surface organisms predicts
that high altitude species (exposed to colder temperatures) will be at high extinction risk under future
climatic conditions because they are less tolerant to heat. In the case of subterranean species, a lack of
adjustment of thermal tolerance to habitat temperature would mean that those species living in warmer
areas will be the most vulnerable to an increase of temperature. Furthermore, for those species living
already at temperatures close to the upper limit of their fundamental thermal niches, the possibilities of
survival are severely limited. For example, some of the species living in warmer lowland areas (e.g.
coastal populations of Troglocharinus) could be close to their physiological limits in the near future,
which combined with their reduced dispersal capacity and habitat confinement increases concerns for
their long term survival. Therefore, physiological information combined with evaluation of the exposure
to climatic changes at their current locations is fundamental in order to identify species or populations
at greatest risk.

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3.2. Methods
The study species belong to the Speonomus and Bathysciola clades of a lineage of the beetle tribe
Leptodirini (Coleoptera, Leiodidae, Cholevinae) distributed in the Pyrenees, Basque Country and
coastal mountain systems in Catalonia (Fresneda & Salgado, 2017). This lineage encompasses around
160 species and subesespecies showing different specificity for the subterranean environment
(facultative subterranean species and obligate subterranean species specialized either in deep or shallow
subterranean habitats) and different morphological traits related with such degree of specialization.
Besides, there is a recent published species-level phylogeny for this group, that includes 116 species
(Cieslak et al., 2014).

O1. To infer heat tolerance values for the whole clade of subterranean beetles from their
morphological traits.
Responsible: David Sánchez-Fernández
Participants: Postdoc, Javier Fresneda, Susana Pallarés, Raquel Colado, Stefano Mammola.
Collaborators: Pau Balart, Aitor Montes, Valeria Rizzo (sampling).
Task 1a) Getting predictive variables (measurements of morphological traits):
For all the species of the clade, different biological and ecological traits commonly associated with
adaptation to the deep subterranean environment in arthropods will be measured (Trajano, 2005; Ribera
et al., 2018). There are not apparent differences in the degree of depigmentation and eye reduction (all
species are blind and depigmented), but larger body size and elongated body appendages are typically
found in highly specialized subterranean species. Thus, we will measure total body length (LT), the
length of the antennae (LA) and the tibias (LL) relative to the total body length (LA/LT and LL/LT)
using a stereoscopic microscope (Olympus SZX16) and a camera lucida (Olympus C5060WZ camera),
already available at the University of Murcia. We have already gathered morphological data of 16
species, and we expect to get these data for the whole set of species in the phylogeny. Javier Fresneda
(member of the working team) has an outstanding entomological personal collection in Llesp (Lleida)
with well-preserved specimens of most of these species (Fresneda & Salgado, 2017).
Task 1b) Selection of species to validate the model: We will select a minimum of 10 extra species from
the lineage to extend/validate/refine the model that relates heat tolerance and the degree of subterranean
specialization (Colado et al., 2022). We will select species with different degree of specialization and
living under different environmental conditions (see Table 1).
Task 1c) Field sampling program and material transport: Individuals of the selected species will be
collected using baits and food traps set 24 h in advance. Specimens will be handled and stored in
conditions as similar as possible to those in their caves. Adult beetles will be collected as close as
possible to the central point of their latitudinal ranges. Unlike surface environments, the possible
confounding effects of i) local adaptation in range edge populations; and ii) latitudinal differences
between collection sites is minimal for subterranean species. Collected specimens will be placed in a
portable fridge, with substratum from the cave and moss from the entrance to retain humidity, and kept
and transported in the same conditions to the laboratory where the experiments will be conducted
(University of Murcia). Once in the laboratory, specimens will be kept with moss, substratum from the
caves at the same temperature where they were collected (i.e., considered to be closer to the natural
conditions in the cave), without being disturbed until used for experiments. Specimens will be fed ad
libitum with freshly frozen Drosophila melanogaster. In previous experiments, we have shown that
individuals can be successfully kept in these conditions for more than nine months, with almost no
mortality.
Task 1d) Physiological experiments: We will conduct long term thermal tolerance experiments with the
selected species following the same methodology as in the former project (CAVEheAT) to make the
data comparable (information for 16 species is already available). For this we will use the climatic
chambers at the University of Murcia that will allow us to control temperature, humidity and light. The
experimental boxes containing test individuals (10 individuals per treatment) will be placed in a climatic
test cabinet (Sanyo S.A., temperature range +4°C/+40°C). We will explore survival at different
temperatures, placing test individuals directly from the stock into a test cabinet at one of three set
temperatures (20, 23 or 25°C, plus controls), and survival will be recorded over a period of 7 days.
Experiments will include a control kept in the same conditions at the approximate temperature of the
cave and with RH close to saturation. Thus, our work will require min. 40 individuals of each species.
Specimens will be checked every 24 h, the stones and the tissue wetted and fresh food added whenever

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necessary. Specimens will be recorded as alive if capable of some movement, or dead if there is no
movement when prodded with a seeker. As a measure of the upper lethal temperature, we will calculate
the LT50 (median lethal temperature, i.e., the temperature at which 50% of the exposed individuals have
died) at 7 days exposure. To assess LT50, we will fit survival data to a logistic regression model (i.e.,
binomial GLM) using the R package brglm v. 0.6.2, and estimated LT50 values with the dose.p function
in the R package MASS v. 7.3-51.5.
Task 1e) Predicting heat tolerance for the whole lineage: We will predict heat tolerance (using LT50, as
the response variable: see Task 1d) from the morphological traits obtained in the point above using
classical statistics (Random Forest and/or Phylogenetic Generalized Least Squares (PGLS) in the
package caper v.1.0.1. The choice of PGLS allows us to account for the non-phylogenetic independence
among the species. We will incorporate species relationships in the models using the most recent
published species-level phylogeny of the group (Cieslak et al., 2014). The predictions of this model,
constructed using physiological data for 16 species, will be validated using independent data, i.e., with
the physiological data obtained for the new set of species (tasks 1b-d).
Task 1f) Manuscript writing: We will write a scientific paper, which would be published in a journal
included in the JCR. We expect to demonstrate that easily measurable morphological traits can predict
thermal physiology, showing an evolutionary convergence in the process to deep subterranean
specialization in morphology and thermal tolerance. Irrespective of our precise findings these data are
highly publishable, and test clear hypotheses.
Milestone M1.1: Completion of fieldwork for this objective.
M1.2: Completion of the experiments of thermal tolerance.
M1.3: Predictive modelling and validation.
Deliverable D1.1: Results of the experiments on thermal tolerance.
D1.2: Results of the predictive modelling and validation.
D1.3: Manuscript(s) with the results of O1.

O2. To assess the impact of a decrease in relative humidity on the thermal physiology of beetles
with differing degrees of specialization to the subterranean environment.
Responsible: Josefa Velasco
Participants: David Sánchez-Fernández, Postdoc, Susana Pallarés, Raquel Colado.
Collaborator: Pau Balart, Aitor Montes, Valeria Rizzo (sampling).
Task 2a) Selection of species to be tested: We already have heat tolerance values for 16 species at near
saturation humidity conditions RH >90%, (LT5090%RH). We will select at least 10-12 of these 16 species
(with different degree of specialization to subterranean environments, see Colado et al.,2022) to test the
effect of decreasing humidity on heat tolerance.
Task 2b) Field sampling program and material transport: (same as Task 1d).
Task 2c) Physiological experiments. We will calculate heat tolerance values (LT50) within a
hypothetical case of reduction in RH to 70-75% (LT5075%RH) following the same approach than that for
obtaining LT5090%RH (i.e. survival during a week at 20, 23 and 25ºC, plus control; see Task 1e) but
varying humidity conditions. We plan to buy two chambers with control for temperature and humidity
MLR-352H-PE (temperature range +0°C/+50°C; humidity range 55-90%), that together with the
facilities that already exist in the University of Murcia (see point 1.8. Infrastructures, equipment and
facilities), would allow to conduct these experiments.
Task 2d) Data Analyses: We will calculate for each species a value that represent the magnitude of the
reduction in heat tolerance by desiccation stress (decrease in RH), as LT50 dif = (LT5090%RH -–
LT5075%RH). Then, LT50dif will be related with the degree of subterranean specialization (as estimated
by morphological traits, see Colado et al., 2022 and task 1a) by a Phylogenetic Generalized Least
Squares (PGLS) in the package caper v.1.0.1. to account for phylogenetic relationship among species.
We expect that LT50dif will be higher in the case of highly specialized species to deep subterranean
environment.
In the case that LT5075%RH could be calculated for a sufficient number of species (10-15) we will
try to predict heat tolerance values considering a scenario of decrease in RH for the whole lineage using
the same morphological variables (task 1a). Otherwise, we will perform a cluster analysis to group
species from the whole lineage into homogeneous classes of subterranean specialization, using the
morphological traits measured. Using standardized values of traits, we will estimate clusters via
Euclidean distances and the Ward2’s hierarchical clustering method (Murtagh & Legendre, 2014), with

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the R package ‘fastcluster’, which will allow us to have an estimate of the LT50 dif for species of the
same classes of subterranean specialization, assuming a similar performance within groups.
Task 2e) Manuscript writing: We will write a scientific paper, which would be published in a journal
included in the JCR. We expect to show variation in thermal niche related to the process of colonization
of deep subterranean environments. Irrespective of our precise findings these data are highly
publishable, and test clear hypotheses.
Milestone M2.1: Completion of field work.
M2.2: Completion of the experiments of thermal tolerance.
M2.3. Compilation of the results of previous experiments.
Deliverable D2.1: Results of the experiments on thermal tolerance.
D2.2: Table with the results of all experiment.
D2.3: Manuscript(s) with the results of O2.

O3. To assess plasticity in heat tolerance in subterranean beetles and its relationship with heat
tolerance and subterranean specialization.
Responsible: Josefa Velasco
Participants: David Sánchez-Fernández, Postdoc, Susana Pallarés, Raquel Colado.
Collaborator: Pau Balart and Aitor Montes (sampling), David T. Bilton (theoretical framework).
Task 3a) Selection of species to be tested: We will select at least 10 out of the 16 species for which we
already known their LT50 and degree of specialization, trying to maximize the representation of species
within this axis of specialization (see task 1b).
Task 3b) Field sampling program and material transport: (see task 1c).
Task 3c) Experimental set-up for assessing acclimation capability: Specimens will be maintained for 2
days under constant conditions to minimize the effects of recent thermal history on measures of
temperature tolerance. After this, specimens of each species will be divided into different equal-sized
groups (eight individuals in each one) and exposed to six different acclimation temperatures (5, 10, 12.5,
17.5, 20 and 22.5 °C) with all other conditions (humidity, food etc.) as described above (task 1d). Beetles
will be maintained under their corresponding thermal acclimation treatments for 5 days. After this
period, individuals from each treatment will be used to measure heat tolerance using short term
experiments (task 3d). These acclimation temperatures cover the range of temperatures in which all
subterranean leiodids studied to date can survive in relatively long-term exposures (i.e., between 5 and
20°C), but also include temperatures close to their upper lethal limits (Rizzo et al., 2015; Pallarés et al.,
2019; Colado et al., 2022).
Task 3d) Physiological experiments: After acclimation, we will estimate heat coma temperature (HCT)
for each individual. This is defined as the temperature at paralysis prior to death, preceded by spasmodic
movements of legs and antennae, and is a typical response used to assess critical thermal maximum in
insects (see Chown & Nicolson, 2004). HCT will be measured using a dynamic, ramping method
(Terblanche et al., 2007), with a heating rate of 1°C min−1 and infrared thermography. CTmax values
measured with such a fast ramping rate tend to be overestimated (Terblanche et al., 2007) and cannot
be used to directly extrapolate actual thermal limits, but this method provides a robust comparative
estimate of the relative thermal tolerance amongst related species and is the most commonly used in
thermal tolerance assays on ectotherms (Bennett et al., 2018). Furthermore, infrared thermography has
proven to be a highly sensitive technique to measure HCT in insects (e.g., Gallego et al., 2016).
Specimens will be placed in a programmable BINDER MK53 incubator (BINDER GmbH, Tuttlingen,
Germany) in which the temperature will be increased 1°C min−1, starting at the corresponding
acclimation temperature. The incubator is coupled with an infrared thermographic camera (FLIR
SC305) to record body temperature of the specimens, from which HCT will be obtained. High quality
images will be also recorded with a video camera (Sony DCR-DVD110E, Sony Co., Tokyo, Japan) to
determine the moment of paralysis (cessation of movement of legs and antennae) of each individual.
Thermal images will be analysed with the software ThermaCAM Researcher Professional 2.10 (FLIR
Advanced Thermal Solutions; ATS; Croissy-Beaubourg, France).
Task 3e) Data Analyses: We will get thermal response curves (TRC) for each species. We will perform
different analysis to fit acclimation response curves to a model (lineal, bimodal, logistic), and relate the
species response (e.g. slopes of lineal functions, and/or other parameters of the models) with the degree
of subterranean specialization of each species (see Fig. 2). Acclimation effects might differ under
stressful (sublethal) and non-stressful temperatures, and species might differ in the temperature range
over which acclimatory adjustments could be made (Pallarés et al., 2021b). Then, we will focus in the

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different performance of the species heat tolerance at acclimation temperatures below the cave
temperature, between the cave temperature and the LT50 value and above LT50. All statistical analyses
will be conducted in R, version 3.6.2 (R Core Team 2019), and/or SPSS for Windows, Version 15.0.1.
2006 (SPSS Inc., Chicago, IL, USA).
Task 3f) Manuscript writing: We will write a scientific paper, which would be published in a journal
included in the JCR. We could demonstrate the relationship between the thermal plasticity (acclimation
capability) and both the degree of specialization of species to deep subterranean environments and heat
tolerance.
Milestone M3.1: Completion of field work (=Milestone 1.1).
M3.2. Completion of the acclimation treatments.
M3.3: Completion of the experiments of thermal tolerance (Milestone 1.2).
Deliverable D3.1: Results of the experiments.
D3.2: Manuscript(s) with the results of O3.

O4. To assess the capability of different species and populations to face climate change.
Responsible: David Sánchez-Fernández
Participants: Postdoc, Josefa Velasco, Javier Fresneda, Susana Pallarés, Stefano Mammola.
Collaborator: Pedro Abellán (University of Seville)
Task 4a) Selection of species to be tested: We will use data for all the species considered in the previous
objectives.
Task 4b) Compiling distributional data: We will use the distributional database published in Sánchez-
Fernández et al., (2016), with additions of the georeferenced information on the occurrences of the
remaining species, including both a bibliographical compilation and the data from our own sampling
from the CAVEheAT project.
Task 4c) Evolutionary change of thermal tolerance: For the estimation of the minimum accumulated
change in temperature experienced through the evolutionary history of the lineage, we will use an
updated version of the phylogeny published for this clade (Sánchez-Fernández et al., 2016) and we will
perform an ancestral trait reconstruction in BEAST, using as a quantitative trait, not the temperature of
the cave in which each specimen was found as in Sánchez-Fernández et al (2016) but the values of
physiological heat tolerance (LT50) estimated in the objective 1 and a multivariate diffusion model of
evolution. To estimate the minimum rates of change in heat tolerance that the species must have
experienced we will use: i) the average rate of thermal niche change, calculated as the average rate of
all branches connecting all the specimens of each species with the root of the tree. For each branch, rate
evolution will be calculated as the absolute difference between the reconstructed initial and final
temperatures divided by the length of the branch. ii) Maximum per-branch rate of thermal niche change,
or the highest per-branch reconstructed rate among all the branches of the tree. This obtained rates of
thermal tolerance changes will be added to the LT50 values.
Task 4d) Assessing vulnerability of species and populations under climate change: The concept of a
thermal safety margin (TSM) has been previously used to assess vulnerability to climate change, as it
approximates the average amount of environmental warming an ectotherm could tolerate before
performance drops to fatal levels (Deutsch et al., 2008). TSM will be estimated for each species and
locality as the difference between species heat tolerance obtained from the Objective 1 also considering
both the outputs from the results obtained for the Objective 2 and the rates of evolutionary change in
thermal tolerance obtained in the task above (task 4c) and the future mean annual temperature of the of
each locality (i.e., BIO1 from WORLDCLIM, variables derived from WorldClim v. 2.1
(www.worldclim.org; Fick & Hijmans, 2017) for both present and future scenarios. We will use two
widely used global climate models (BCC-CSM2-MR and CNRM-ESM2-1) and two Shared Socio-
economic Pathways (SSPs): 245 and 585 for the periods 2041-2060, and 2081-2100. Thus, for each
locality of each species we will calculate TSMcurrent and TSMfuture. To calculate TSMfuture we will
consider a TSM range obtained for each species considering the minimum and maximum thermal
tolerance (depending on its variation under desiccation stress and its capacity for evolutionary change).
Task 4e) Manuscript writing: We will write at least one scientific paper, which would be published in a
journal included in the JCR and another one in a popular-science journal (in Spanish). We could
accurately estimate what will be the fate of a complete clade of subterranean species under different
climate change scenarios. Besides, we could demonstrate that the species living at high altitude will not
be those at higher extinction risk under warming conditions, but those highly specialized species to
subterranean environment currently living in warm areas.

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Milestone M4.1: Compilation of physiological information.
M4.2. Completion of species distributional data.
M4.3. Completion of data on temperature (current and future) of each locality.
M4.4. Reconstruction of heat tolerance values in the phylogeny.
M4.5. TSM for each species (current and future).
Deliverable D4.1: Detailed distribution maps for the target species.
D4.2. Table with the average rate of evolutionary change of heat tolerance for each species.
D4.3. Table with current and future TSM estimated for each population.
D4.4. Ranking of species (and populations) regarding their vulnerability to climate change.
D4.5. Manuscript(s) with the results of O4.

Figure 3. Project outline.

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3.3. Relationship with other national and international research groups
At international scale, David T. Bilton and John Spicer (University of Plymouth, UK) have studied the
relationship between thermal tolerance and acclimation and the size of geographical ranges in some
aquatic species (both marine and freshwater organisms). We have a fluent communication with them.
Indeed, David T. Bilton was member of the working team in the CAVEheAT project. On the other hand,
Steven L. Chown (Monash University, Australia) is one of the most prestigious researchers on insects’
physiology. Regarding thermal tolerance in subterranean insect, Ana Sofia Reboleira (University of
Lisboa) has worked on different aspects of subterranean biodiversity (mainly taxonomy and systematic).
Although there is no established formal collaboration in this project with these researchers yet, the PI of
the project is in contact with all of them.
There are only a few groups in Spain that are directly related with the scope of this project. Jorge
M. Lobo (MNCN, CSIC) and José Ramón Verdú (University of Alicante) have explored thermal biology
of dung beetles using similar approaches than those proposed in this project, Miguel Tejedo (Doñana
Biological Station, CSIC) have studied different aspects on physiology of amphibians (including thermal
limits). We have regular collaborations with all of them (some of them have been supervisors of different
postdoctoral positions of the IP), and they will be willing to collaborate punctually in some objectives
of the project. Other national specialists in subterranean environments that would be interesting to
collaborate are Vicente Ortuño, Alberto Sendra and Alberto Jiménez-Valverde (University of Alcalá),
Carles Ribera and Miquel Arnedo (University of Barcelona) and Pedro Oromí (University of La
Laguna). Ignacio Ribera (Institute of Evolutionary Biology, CSIC-UPF) have developed an extensive
research line focused on ecology and evolution of subterranean biodiversity. Unfortunately, Ignacio,
one of the most inspiring and productive researchers in this field, passed away in 2020.
During the last years, other European researchers have started to work on thermal physiology
of subterranean fauna. Indeed, we have created an international network of young European researchers
working on subterranean biology (e.g., Stefano Mammola (Italian National Research Council (INRC,
Italy, also included in the working team), Ana Sofia Reboleira (University of Lisboa, Portugal), Pedro
Cardoso (University of Helsinki, Finland), Arnaud Faille and (Stuttgart State Museum of Natural
History, Germany), etc.) to develop several exciting projects to improve our understanding on how
subterranean organisms will respond to potentially rapid environmental change.

3.4. Feasibility, potential difficulties and contingency plan

The project is ambitious, but not overly so. It is feasible to complete within three years given the
capabilities of the PI and members of the research and working team, and the facilities and expertise
available at the institutions involved. The collaboration between the people integrated in the proposal
unites all the elements necessary for addressing the challenge of the proposed questions with confidence.
We have demonstrable knowledge of the model system, results of previous studies (specially within the
CAVEheAT project) that allow the formulation of precise hypotheses to be tested, and expertise in all
the required methodologies, as demonstrated by our publication record. Our previous history of
collaboration also makes it easier for us to undertake an integrative, complex project like this and a
guarantee of success.
David Sánchez-Fernández (IP) is starting a Ramón y Cajal contract at the University of Murcia
(from 1st of January 2021). He has a wide theoretical background and skills in a variety of ecological
topics, including conservation biology, beetle taxonomy, biogeography, physiology and evolutionary
biology, all integral to the proposed research. Josefa Velasco has a demonstrated experience in
ecophysiology of insects, mainly on the tolerance of natural stressors (thermal, salinity and desiccation)
and their interactions. The members of the working team show complementary profiles with a broad
experience in conducting all planned tasks, from sampling and identification of species to experiments
on thermal tolerance proposed here. Dr. Stefano Mammola (National Research Council, Italy &
University of Helsinky, Finland) has become one of the most renowned experts in subterranean biology
internationally. Javier Fresneda is a leading speleologist likely the best specialist on taxonomy,
systematics and biogeography of the species of this lineage. In spite of his youth, Dr. Susana Pallarés
(postdoc at the University of Seville) has a strong background in ecophysiology of insects, especially
on thermal tolerance of subterranean insects and resistance to desiccation in aquatic beetles. Raquel
Colado is an enthusiastic FPI student at the University of Murcia. Both Susana and Raquel have been
working on the previous CAVEheAT project acquiring key skills and theoretical background on thermal
physology and subterranean biodiversity conservation.

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 We have also the support of other prestigious researchers (see support letters), as Jorge M. Lobo
(National Museum of Natural Sciences, CSIC) expert in biogeography and conservation of insects, Juan
Carlos Sánchez-Hernández (Institute of Environmental Sciences, UCLM, Toledo (Spain),
ecotoxicologist expert in biomarker assays and oxidative stress, Pedro Abellán (University of Sevilla,
Spain) expert in modelling species distribution and physiology and Valeria Rizzo (Università degli
Study di Roma La Sapienza, Italy), a speleologist and expert in the evolution and ecology of cave
beetles, that will be willing to collaborate in some of the objectives of this project (see methodology).
Besides, in case we have a problem with our own infrastructure to conduct physiological experiments
(breakage, etc.), they will allow us to use the infrastructure need to carry out both short physiological
experiments (specially the climatic chambers at the UCLM).
We are aware that sampling in these particular habitats could be one of the critical stages of this
project. For this we have the support and previous experiencing collaborating with of a network of
acknowledge speleologist such as Pau Balart (Barcelona) and Aitor Montes (Segura, Basque Country)
and speleological associations such as the “Grupo de Bioespeleología de Villacarrillo” ensuring both
identification in the field and, at the same time, overcoming the technical difficulties of sampling in
these habitats. If some of them are temporarily out (for personal reasons or other incidentals) or have
problems to carry out the sampling, other members of the speleological associations will help us to carry
out these tasks.
In the same way, one of the most notable difficulties of this project is the possible death of
species during transport from the cave to the laboratory. To minimize this setback, we will use a portable
electric cooler connected to the car battery which allows species to be in the conditions of the cave
during transport. This transport system was used before (CAVEheAT) with success. If we have any
problem with this device, we plan to send the material via specialized companies (e.g. SeurFrío or
PolarExpress) that offer services at controlled temperatures.
We plan to include ca. 10 species with different degrees of specialization to subterranean
environments (using morphology as a surrogate) to objetives 1 and 3 + other 10 species for objective 2
(species for wich we already have data on LT50 calculated witout humidity restrictions. However, if we
have problems with the sampling and we can obtain only 7-8 species, it would still be sufficient to
robustly test all the exciting hypotheses proposed. At this time it is impossible to state exactly which
new species we will be able to get sufficient individuals (this depends on the cave and other
unpredictable factors). However we have constructed a ‘long list’ from which species will be selected
(Table 1). As can be seen from this table, our plans have sufficient contingency to allow for difficulties
which may be encountered with collecting particular species.

Table 1. Initial selection of species which could be used in the FRAGGLE project, including the locality where the
species has been found to be relatively abundant in previous sampling ( SP: Spain; FR: France).
Species
Bathysciola meridionalis
Aranzadiella leizaolai
Parvospeonomus delarouzei
Speonomidius crotchi mazarredoi
Speonomidius c. oberthuri
Speonomus bourgoini
Speonomus orgibetensis
Antrocharis querilhaci s.str.
Paraspeonomus vandeli
Speonomus piochardi
Speonomus longicornis
Speonomus normandi hydrophilus
Speonomus pireneus
Speonomus abeillei
Speomomus diecki
Speonomus colluvii
Speonomus zophosinus
Speonomus carrerei
Trapezodirus cerberus
Locality
F-31, Mérigon, Grotte de la Quère (Ariege, FR)
Sima de Kobeta, Olatz (Guipuzkoa, SP)
Gr. de Can Pey -Arles·sur·Tech (P. Orientales, SP)
Cv. de San Valerio -Mondragon (Guipuzcoa, SP)
Cv. de San Adrián -Zegama (Guipuzcoa, SP)
Gr. de la Cascade -Sarracolin (Hautes P., FR)
MSS massif d’Orgibet (Ariège, FR)
Grs. de Tarascon (Ariège, FR)
Gr. las Souleillos -Seix (Ariège, FR)
Gr. de la Bastide- La Bastide·de·Sérou (Ariège, FR)
Gr. Lavelanet -Freychenet/Lavelanet (Ariège, FR)
MSS /Gr. d’Olote -Saint Girons/m. d’Arize (Ariège, FR)
Gr. de Ker -Massat/Biert (Ariège, FR)
Gr. du Mas d’Azil -Mas d’Azil/Plantaurel (Ariège, FR)
Gr. d’Aubert -Aubert /massif Sourroque (Ariège, FR)
MSS Bois de Candail, NO. pic d’Estibat (Ariège, FR)
MSS /Grs. environs de Massat /Vallés Salat (Ariège, FR)
MSS /Gr. de Payssa -Salseiu (SP)
Espluga des Tosses, Bonansa (Huesca, SP)

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3.5. Infrastructures, equipment and facilities
The project will be developed mainly in the University of Murcia. The 'Aquatic Ecology' group of the
University of Murcia, to which D. Sánchez-Fernández (PI) and Josefa Velasco belongs, has a fully
equipped laboratory for the management and identification of entomological samples (collection and
storage equipment, transmission and stereoscopic microscopes, photographic equipment, etc.). It also
has the necessary GIS software and infrastructure for the cabinet analysis to be developed during the
project, all the necessary infrastructure for physiology experiments, including four climatic chambers,
one of them (Sanyo) with control of temperature and humidity, for the acclimation of individuals, which
will be completed with the purchase of another one chamber (PHC_ L6MLR-352H-PE) with control of
temperature and humidity. An environmental simulation chamber (Binder) for testing extreme
temperatures (between - 70°C and 180ºC), an infrared video camera (FLIR A325SC) for analysis of
thermal tolerance, a digital video camera (JVC), temperature data-logger (ECOLOG) and other
laboratory basic equipment required to conduct both short (ramping) and long term thermal experiments.
Anyway, we will also have access to common research facilities at the service University of Murcia
(CAID), including one climate controlled room and one chamber, with control of temperature, light and
humidity.

3.6. Chronogram
The following chronogram shows a schedule (quarters for each year) of the different tasks for each
specific goal as indicated in the Methodology section, indicating the duration of the task (in blue) and
when the milestones (MX.X) and deliverables (DX.X) for each goal are expected to be meet.

1st year 2nd year 3rd year

Task 1 2 3 4 1 2 3 4 1 2 3 4
Task 1a
Task 1b
Task 1c M1.1
Task 1d M1.2; D1.1
Task 1e M1.3; D1.2
Task 1f D1.3
Task 2a
Task 2b M2.1
Task 2c M2.2; D2.1
Task 2d M2.3; D2.2
Task 2e D2.3
Task 3a
Task 3b M3.1
Task 3c M3.2
Task 3d M3.3; D3.1
Task 3e
Task 3f D3.2
Task 4a M4.1
Task 4b M4.2; D4.1
Task 4c M4.3; M4.4; D4.2
Task 4d D4.3 D4.4
Task 4e D4.5
Outreach

3.7. Justification of the need of requested personal

Due to the large amount of expected field, laboratory and analytical work it is requested a dedicated
postdoctoral with a full-time two-year contract. This request is primarily due to the need for a skilled
researcher with a background in insect thermal biology in order to develop highly specialized tasks
aimed at achieving the milestones during the last two years of the project (see methods and chronogram).
Obtaining experimental measurements of physiological thermal limits for subterranean species is not
trivial, given the logistical difficulties for sampling in caves and conducting experiments with
subterranean animals. Then, an expert researcher is needed, as we could not afford problems during the
experimental and/or analytical steps. Thus, his/her tasks will be: i) maintenance and tuning of the
infrastructure necessary to carry out the experiments (mainly climate chambers and thermographic
devices); ii) participation in the last samplings (e.g. sampling in the cave, transporting the samples from
the cave to the lab) iii) organization and participation in physiology experiments; iv) general
management of the laboratory, including basic training of students and visitors; v) molecular and

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phylogenetic analyses; vi) participation in the analytical (statistical) phase for the four objectives. To
hire a postdoc is very important to address the goals presented in the proposal. It should be noted that
due to the nature of these experiments (animal maintenance and monitoring survival has to be made
daily at least during 8 days in each experiment), at least two people are required to conduct this task.
Besides, an overlapping between experiments on heat tolerance and acclimation capacity is expected
during the end of the second year and the first months of the last year. Lastly, beyond the specific tasks
of this project, the postdoc would contribute to the creation of a research team at the University of
Murcia devoted to the study of climate change effects on biodiversity (a novel research line for the
University of Murcia) associated with the Ramón y Cajal contract to the IP.

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D (2021a). Loss of heat acclimation capacity could leave subterranean specialists highly vulnerable
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4. IMPACTO CIENTÍFICO-TÉCNICO - SCIENTIFIC-TECHNICAL IMPACT

The results of this project could provide important insights to improve our ability to understand and
predict changes in biological communities that are exposed to global warming effects. Indeed, contrary
to the general theory that high altitude species in surface environments will be at high extinction risk
under future climatic conditions, we could demonstrate that subterranean species living in the warmer
areas (usually at lower altitude) will be the most vulnerable to an increase in temperature. The
importance of i) the predicted changes in humidity within caves, and ii) the plasticity of heat tolerance
could be a key factor for those species already close to the upper limit of their fundamental thermal
niches (i.e. with the lower Thermal Safety Margins), limiting their possibilities of survival in the future.
Besides testing the climactic variability hypotheses, these refinements to get accurate values of heat
tolerance could provide relevant insights to rethink the lack of adjustment of heat tolerance to ambient
temperatures. The studied subterranean specialists will be poorly able to cope physiologically with
temperature increase, but in contrast will be also exposed to lower magnitude and rate of warming in
the deep subterranean environment (which is buffered from external changes to some extent). Thus, we
could launch some relevant scientific perspectives on this paradox of climate change vulnerability and
trying to explore how far this paradox is a rule in other distant biological groups exposed to different
environmental stability gradients.
The information generated by this project will be of great interest for the recently created IUCN-
Cave Invertebrate Specialist Group and for the Spanish Office for Climate Change from the Spanish
Ministry of Agriculture, Food and Environment. An indirect benefit from the project will be a deeper
knowledge of the ecology of some of the most endangered habitats in Spain and Europe and the biology
of the species living in them: the deep subterranean environments, likely, represent the most widespread
nonmarine environments on Earth. The global importance and the conservation challenges associated
with subterranean ecosystems was recently highlighted by Mammola et al. (2020; Bioscence) in their
“Scientists' Warning on the Conservation of Subterranean Ecosystems”. The expected results would add
significantly to our understanding of the relationships between global change and the loss of
biodiversity, and will be of general interest to ecologists. Our results could show that only an
experimental approach can provide evidence of the capability of species to cope with temperatures
outside those they currently experience. This should be a strong cautionary warning for studies trying
to infer fundamental niches (and predictions of responses to future scenarios) from characteristics of the
environment only, especially considering that in most cases organisms have many different ways to use
the temporal and spatial heterogeneity of the habitat to accommodate their needs. Despite the general
neglect, these habitats harbour a wealth of exclusive biodiversity of the highest interest, but also the
highest vulnerability to both anthropogenic effects and global change, so it is expected a great impact in
the scientific community.
Thus, depending on the outcome, we will raise further collaborations with the application of
other more ambitious projects, as new exciting issues would be opened up (especially once accurate
values of heat tolerance can be predicted for the whole lineage). Transcriptome approaches (RNA-seq)
would be further used to explore changes in gene expression between congeneric species with different
subterranean adaptations leading to open new and exciting evolutionary questions regarding rates of

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evolutionary changes in thermal tolerance. For example, why and how thermal regulation is lost? What
are the costs of maintaining a given thermal tolerance? What are the mechanisms to cope with
temperature? When was the temperature control system established? Is the ability to acclimatize a
reversible trait? Besides, this project will allow reinforcing links between these researchers opening new
avenues for future collaborations, and what is more important allow consolidating an emerging research
line for the IP, as new “Ramón y Cajal” researcher at the University of Murcia.

Diffusion, internationalisation and data management plan

We plan to publish at least four/five SCI papers directly related to this project. Potential target journals
for the first and third objective could be “Proceedings of the Royal Society Series B”, “Ecology” or
“Ecography”, for the second one “Biology Letters”, “Scientific Reports” or “Physiological
Entomology”, and for the last objective “Global Change Biology” or “Conservation Letters”. We also
plan to publish one article on popular science (“The Conversation“ or “Quercus”), and to present these
results in at least three communications in both national and international congress and workshops (e.g.
the Bienal Conference of the International Biogeography Society; the meeting, the SIBECOL meeting
or the Annual Meeting of the Society for Experimental Biology) and two national/Iberian conferences
during the three years (e.g. annual congress of the Spanish Association of Terrestrial Ecology). We will
disseminate results in the frame of academic courses, counseling activities, and seminars. Further
visibility will be ensured by creating a web page dedicated to the project connected with main social
networks (Twitter, ResearchGate). We will propose key results to magazines or internet portals intended
for the general public (e.g. NewScientist, Nature research highlights, Le Scienze). We will take
advantage of the communication tools currently available by the Unit of Scientific Culture of the
University of Murcia (training and informative "pills", Pint of Science, MedNight, the TV program
LaboratoriUM, etc..) and the knowledge transfer group “Advances in Biodiversity” (UMU) to
disseminate the results to a public especially of interest. In the same way, we will also take the advantage
of the name of the project, a famous children's puppet television series (Fraggle Rock) about
subterranean creatures, created by Jim Henson, to draw the attention of children (and not so children) to
the importance of the conservation of the underground environment. All members of the team and their
institutions are well aware of reproducibility issues that permeate today’s scientific landscape. We fully
support open science, transparent science, and FAIRness (sensu Wilkinson et al., 2016 Scientific Data)
in data management. More informally, the dissemination of the results will be carried out through
participation in seminars, summer and master courses, discussion groups and personal contacts. We plan
to create an outreach video of the project with a specialized audiovisual company (e.g. linknature.es;
which is included in the budgets). In addition, preliminary results, raw data and PDF files of some
scientific articles will be published on the website of the groups involved. Other results will be available
through public repositories (for example, GBIF, Dryad, Zoobank, etc.).

5. IMPACTO SOCIAL Y ECONÓMICO - SOCIAL AND ECONOMIC IMPACT

The Habitats Directive (Directive 92/43/EEC) on the conservation of natural habitats and of wild fauna
and flora provides a legal basis for Community action to report upon and conserve European
biodiversity, being pivotal to the establishment of the European Natura 2000 network. However, a 2011
Terrestrial Invertebrates Platform Meeting within the EC’s LIFE programme has highlighted
inconsistent and insufficient data on invertebrates as one of the main challenges for implementing
conservation actions within the Habitats Directive. New approaches are required to provide the
necessary improvements in invertebrate conservation, and this has been argued for by the head of the
LIFE Nature Unit of the Directorate-General for the Environment within the EC. The objectives of this
project fall within the EU invertebrate conservation policy framework by addressing data limitations to
policy implementation. Specifically, these limitations will be addressed by integrating experimental
thermal physiology approaches to better understand climate change consequences for the arthropod
fraction of subterranean diversity. Thus, the results will also be relevant for conservation agencies
(administration, conservation agencies, NGOs). Thus, the project will contribute to the development of
proactive strategies to reduce climate change impacts on biodiversity in subterranean ecosystems.
Furthermore, the project could also allow knowing the performance of Natura 2000 network of protected
areas (on the surface) to promote the persistence of subterranean species.
The present project also has a great potential for applied conservation, in the form of engaging
key stakeholders in the protection of the subterranean biome. Proactive interactions with stakeholders
(e.g. managing authorities of caves and karst areas, NGOs) could be organized in the forms of Living

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Labs on the field inside touristic caves to increase their interest from a scientific and biological (natural
patrimony) perspective, raising awareness about subterranean life and promote the implementation of
Nature-Based Solutions. We will prepare reports focused on main conservation issues to be sent to the
key actors (Departament de la Vicepresidència i de Polítiques Digitals i Territori de la Generalitat de
Catalunya; Departamento de Agricultura, Ganadería y Medio Ambiente del Gobierno de Aragón;
Departamento de Desarrollo Económico, Sostenibilidad y Medio Ambiente del País Vasco;
Departamento de Desarrollo Rural y Medio Ambiente de la Comunidad Foral de Navarra; and the
Ministry for the Ecological Transition of the Gouvernement of France). Also, we will closely cooperate
with the different working groups at IUCN (e.g. Cave Invertebrate Specialist Group) to take our message
to relevant international actors. We will promote meetings and working groups with these entities to
transmit the results obtained and facilitate the implementation of conservation and adaptation strategies.
The outreach activities organized in the point above along with some post that will be submitted
to the “Agencia SINC”, “The Conversation” or the Ecomandanga blog (the IP is regular collator) will
allow the main results of the project to reach many more (not scientific) people. Finally, as a result of
the project we expect to substantially contribute to the common pool of primary data available to the
scientific community. This will include a large volume of physiological data on species belonging to
different lineages of cave beetles, and associated data on their ecology and distribution. We will also
deposit in public institutions a large number of curated and well preserved specimens for future research.

6. CAPACIDAD FORMATIVA - TRAINING CAPACITY

The FRAGGLE project offers an excellent opportunity for research for a PhD student at the University
of Murcia, developing an integrated approach that combines physiological and ecological skills with
clear purposes related to subterranean biodiversity conservation. The student would develop her/his
formation in the International School of Doctorate at the University of Murcia (EIDUM) and would be
able to attend the seminars of the PhD program “Biodiversidad y Gestión Ambiental”, as well as courses
at the university or other academic opportunities. The stay of the PhD student in the research centres of
other members of the team will be promoted (specially the Molecular Ecology Group, Water Research
Institute (IRSA), National Research Council of Italy (CNR), Verbania Pallanza, Italy). In addition, as
part of the FPI grant, the student will take full advantage of the program for short-stages in foreign
institutions. So it is expected that the student will spend between 2-4 months per year at least during the
first two years of the PhD visiting foreign research centres. Our group considers that short-stages in
foreign institutions to work with leading researchers in the field are of crucial concern, and all our
previous students have done them. Other potential labs to conduct research stages are: Steven L. Chown
Lab (School of Biological Sciences at Monash University, Australia); David T, Bilton and John Spicer
at the University of Plymouth (UK) and Michael Angilletta Jr. (Arizona State University, USA).
The IP of the project have co-supervised successfully five Master students (University Pablo
Olavide (Sevilla), University of Murcia (2008, 2019 and 2021), Rey Juan Carlos University (Madrid)
and Complutense University of Madrid; and three PhD students (+2 in course), all of them with top
marks and with international (European) mention. All of them are currently working at different
institutions as postdoc researchers.
Besides, Dr. Velasco has supervised ten PhDs, eight of them in the last ten years, and three
others are currently in different phases of development. One of them was also co-supervised by the IP.
Of special value is that all of these PhDs have obtained the European Doctorate, the awards obtained by
some of them (from the Faculty of Biology and the International Scholl of Doctorate of the University
of Murcia, Asociación Ibérica de Limnología and the European Freshwater Sciences) and the
postdoctoral positions they have gained in other international and national research centres. In addition,
Dr. Velasco has supervised 8 Degree theses and 15 Master theses within the two Masters Programs of
the University of Murcia in which she lectures.

7. CONDICIONES ESPECÍFICAS PARA LA EJECUCIÓN DE DETERMINADOS

PROYECTOS – SPECIFIC CONDITIONS FOR THE EXECUTION OF CERTAIN PROJECTS

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