Figure 3-1: Preparation of the medium for the aquatic species Proasellus lusitanicus (Frade, 1938), collected from the Olho de Mira Cave, Estremenho Karst Massif, Portugal.
Paper in preparation for submission:
Medina M.J, Reboleira A.S.P.S. Lethal upper-limit temperatures in cave-adapted species from Western Europe.
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3.1 Abstract
Climate change predictions impose growing importance in understanding how species will be affected by rising temperatures. Still, subterranean ecosystems, such as caves, are still neglected even though they are dependent on the surface climate and as such, are expected to be affected by climate change.
Moreover, little is known about cave species’ response to climate change, partly due to the difficulty in accessing caves. However, these habitats are responsible for multiple ecosystem services that are of high importance to humans, namely groundwater storage and purification through the feeding habits of aquatic species. Therefore, assessing cave species’ vulnerability to climate change is crucial. We tested the upper thermal limits (UTL) of six cave-adapted species, belonging to different trophic levels, from Western Europe and compared it with the predicted scenarios of temperature increase for those areas.
Our results show that the UTL50 range between 27 and 29°C and UTL100 ranged between 28 and 31°C.
This shows that terrestrial cave species are more endangered by the predicted scenarios of global warming. These results show that temperature increase poses a risk to the ecological integrity of subterranean ecosystems in Western Europe, emphasizing the need to assess sub-lethal effects of temperature in cave species, as physiological changes resulting from the increase in temperature may affect these ecosystems even before mortality occurs.
Keywords: thermal tolerance, caves, stygobiont, troglobiont, climate change.
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3.2 Introduction
Caves are natural underground isolated habitats that provide a microclimate characterized by very particular conditions such as darkness, high humidity near the saturation point, and thermal stability with surface influence decreasing with depth (Castaño-Sánchez et al., 2020a; Colado et al., 2021). Due to the environmental stability that characterizes subterranean environments, reproducing these conditions in a laboratory setting is relatively straightforward (Colado et al., 2021; Mammola et al., 2019b). Caves' stable temperature corresponds to the mean annual temperature for the surface at its vertical (Domínguez-Villar et al., 2015; Mammola et al., 2019b). However, this dependency on the surface results in these habitats being particularly vulnerable to climate change, as it is expected that as the Earth's mean annual temperature increases due to climate change, so will the temperature inside the caves.
Caves have extreme environmental conditions, such as absence of light, low food availability and stable conditions. Animals that have adapted to these conditions have specific morphological, physiological, and behavioural adaptations (Delić et al., 2016; Mammola et al., 2019a; White et al., 2019). These traits include low genetic variability, poor dispersal capabilities, slow metabolisms, long life cycles and limited population growth (Castaño-Sánchez et al., 2020; Colado et al., 2021; Mammola et al., 2019a;
Pallarés et al., 2019). The environmental conditions provided by caves are also expected to influence the organisms' thermal capacity and possible behaviour adjustments, given the thermal stability throughout the caves (Pallarés et al., 2020a; Raschmanová et al., 2018). It is expected that cave dwellers may have altered their physiological limits to account for the short range of temperatures experienced inside caves (Pallarés et al., 2019).
How climate change will affect species is a fundamental question in subterranean biology as subterranean ecosystems are often disregarded (Mammola et al., 2019a; Mammola et al., 2020; Sánchez-Fernández et al., 2021). Climate change is affecting all ecosystems, with predictions for the increase in the average annual temperature for the surface until 2100 being very extreme, while occurring other problems such as climate extremes, droughts (IPCC, 2014). However, the study of subterranean ecosystems and how climate change will affect them is still lacking even though the Intergovernmental Panel on Climate Change (IPCC) predictions are as drastic as ever (Mammola et al., 2019a). The Intergovernmental Panel on Climate Change (IPCC) report from 2014 (IPCC, 2014) suggests a sub-regional classification of the world according to climate change's impact in those regions. This panel situates Portugal in the Southern and Atlantic regions, accordingly, climate change in Europe will be most dramatic in the Southern sub-region in the Summer and in Northern Europe in the winter.
Species vulnerability to climate change depends on three main aspects: exposure impact, thermal sensitivity (determined by its physiological tolerance limits), and its potential for adaptation (Dawson et al., 2011; Williams et al., 2008). Species’ upper thermal limit, i.e. the maximum survival temperature, is expected to be the most direct determinant of climate change effects, because heat-induced stress is the most immediate consequence of global warming's extremely high rate of temperature rise (Colado et al., 2021). Considering the characteristics that provoke a higher vulnerability to climate change, cave fauna is particularly at risk due to its constraints in developing adaptations and low dispersal capabilities (Culver & Pipan, 2019). We predict that the level of exposure impact on caves is region-dependent, and the most at-risk caves will be situated in the most at-risk surface locations.
Given the impact of climate change on the Earth’s surface and the future projections provided by entities such as the IPCC, it is crucial to understand how it impacts all ecosystems, including subterranean ones,
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like caves. We tested the upper limit temperature (UTL) of six cave-adapted species from the Westernmost part of continental Europe (Portugal), from different trophic levels and underground compartments (aquatic and terrestrial). We compare those values with three scenarios of the maximum average temperature predicted to the area by the IPCC, to understand the vulnerability of subterranean ecosystems to predicted temperature rise.
3.3 Material and methods
3.3.1 Sampling, species and acclimation
All specimens were collected in caves of mainland Portugal, in karst areas Sicó (Cerâmica Cave), Estremenho (Olho de Mira Cave and Alviela Spring) and Sintra-Cascais (Assafora Cave) (Fig. 3-2 and Table 3-1). In situ, temperature was measured in water using a multiparameter probe (Aquaread AP-2000) for the aquatic species and soil temperature was measured using a temperature datalogger (HOBO TidbiT v2, during two years) for terrestrial species. Aquatic species were sampled using large pipettes, and terrestrial species were sampled using soft bristle brushes from 2018 to 2022 and were transported to the lab in portable cooler boxes with local water (aquatic) or sediment (terrestrial species).
Figure 3-2: Location of the sampling locations in Western Portugal.
Aquatic species tested: 1) Proasellus lusitanicus (Frade, 1938), a groundwater-adapted asellid crustacean (Crustacea: Isopoda) endemic to the Estremenho Karst Massif (A. S. P. S. Reboleira et al., 2013), a detritivorous species collected from the Olho de Mira Cave, Mira de Aire and Alviela Spring, Alcanena (Table 3-1). The species is known to feed on organic matter at the sediment level (A. S. P. S.
Reboleira et al., 2013); and 2) Pseudoniphargus n. sp., a groundwater adapted amphipod crustacean,
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endemic to Assafora Cave (Table 3-1) in Sintra-Cascais karst area, typically a predatory species seen feeding on Proasellus assaforensis Afonso, 1988 (Reboleira pers. obs.).
Terrestrial species tested: 1) Scutogona minor Enghoff & Reboleira, 2013, a cave-adapted millipede endemic to the Sicó karst area, and a detritivorous species (Enghoff & Reboleira, 2013); 2) Miktoniscus longispina Reboleira & Taiti, 2015, a cave-adapted terrestrial crustacean isopode with a disjunct distribution in Sicó karst and Cesaredas karst plateau (A. S. P. S. Reboleira et al., 2015); 3) Trichoniscoides sicoensis Reboleira & Taiti, 2015, a cave-adapted terrestrial crustacean isopod endemic to the Sicó karst area (A. S. P. S. Reboleira et al., 2015); and 4) Podocampa cf. fragiloides (Silvestri, 1932), a detritivore cave dipluran (Sendra & Reboleira, 2020). All terrestrial species were sampled in the Cerâmica cave (Table 3-1) in the Sicó karst area.
Table 3-1: Sampled caves locations and average annual temperature.
Cave Coordinates Annual average temperature (°C) Olho de Mira 39°32'28.4"N 8°43'20.0"W 17
Alviela 39°26'44.4N 8°42'43.6"W 17
Assafora 38°54'31.67"N 9°25'18.98"W 17 Cerâmica 37°10'12.5"N 8°2'5.21"W 15
Animals were acclimated in the lab within a temperature-controlled chamber (Memmert ICP 400) to 17°C prior to the experiment to ensure minimal stress during the assay, using the same setup as the below described test conditions.
3.3.2 Test conditions and maintenance
Aquatic species were individually placed in glass vials with sand, small rocks, and water from their habitat. Since the glass vials with were open for this experiment, for medium oxygenation. Terrestrial species we placed in transparent plastic boxes with sediment from the Cerâmica Cave sterilized in an autoclave (120°C for 45 minutes). Humidity in the soil of the terrestrial species was maintained using a pipette to drop water and trays with water were placed inside the incubator. Species were monitored and tended to every 24h.
Following the same protocol used in Castaño-Sánchez et al. (2020b), species were tested in a temperature-controlled chamber at 17°C (VWR INCU-Line 68R, accuracy ±0.1 at 37°C). Temperature was increased by 1°C every three days, until reaching 100% mortality in the test group. Control groups were kept at the annual average temperature of their habitat (Table 3-1) from the start of the acclimation process until the end of the test. All animals were adult, and gender was undetermined to avoid specimen’s excessive manipulation, except for ovigerous females of crustaceans, which were excluded.
Survival was monitored every 24h by observing the specimen’s motion or carefully touching the individuals with a soft bristle brush in both the test and the control group. Test was considered valid with <20% mortality in the control group. For aquatic species, we followed additional test validation guidelines for ecotoxicity testing for stygobitic crustaceans by Di Lorenzo et al. (2019), regarding also pH and oxygen levels of the medium.
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Upper Limit Temperatures for 50% of the populations (UTL50) is defined as the temperature at which 50% of the population died when exposed to a defined temperature (Castaño-Sánchez et al., 2020a). All analyses were made in R and R Studio (v1.3.1073) (R Core Team, 2020) and visualized with the ggplot2 package (Wickham, 2016).
3.3.4 Literature review and predicted climate change scenarios
A literature review was condensed into a table (Supplementary table 3-1) to consider different species’
UTL50 and UTL100 from other regions to assess the vulnerability of subterranean fauna to climate change.
Searches were made using Web of Science, as well as Google Scholar using the following keywords:
"UTL50", "UTL100", "cave", “groundwater” and "thermal tolerance".
To predict the susceptibility of cave fauna to climate change we used the upper-bound of three scenarios projected for the increase in average annual temperature in the IPCC report of 2014 for the area of the caves: a “best case” scenario (RCP2.6), an intermediate scenario (RCP4.5) and a “business as usual”
scenario (RCP8.6).
3.4 Results
3.4.1 UTL experiments
Tested species revealed close UTL50 and UTL100, from 1 to 4ºC apart. The UTL50 were between 27 and 29ºC, and the UTL100 were between 28 and 31ºC (Table 3-2, Fig 3-3 and 3-4). The lowest UTL50 was 27ºC obtained for the terrestrial millipede Scutogona minor and the terrestrial isopods Miktoniscus longispina and Trichoniscoides sicoensis. The highest UTL50 (29ºC) was obtained for the aquatic isopod Proasellus lusitanicus. The lowest UTL100 was found for the terrestrial isopod M. longispina (28ºC), while the highest UTL100 was obtained for the aquatic isopod P. lusitanicus (31ºC). Two species also have particularly close UTL50 and UTL100, with just a 1ºC difference (Pseudoniphargus n. sp. and M.
longispina). In general, test groups revealed close UTL50 and UTL100, from 1 to 4ºC apart. The UTL50
were between 27 and 29ºC, and the UTL100 were between 28 and 31ºC (Table 3-2, Fig 3-2).
Table 3-2: Thermal tolerance obtained for the cave-adapted studied species. From left to right, N - number of individuals in the control and test groups, UTL50 - upper limit temperature for 50% of the population and ULT100 - upper limit temperature for 100% of the population, D - difference between the UTL50 and the average annual temperature of the species’ habitat.
Taxa N UTL50
(°C)
UTL100
(°C) Lifestyle D (°C)
Scutogona minor 9 27 30 Terrestrial 12
Pseudoniphargus n.
sp. 10 28 29 Aquatic 11
Proasellus
lusitanicus 10 29 31 Aquatic 12
Miktoniscus
longispina 10 27 28 Terrestrial 12
Trichoniscoides
sicoensis 10 27 29 Terrestrial 12
Podocampa cf.
fragiloides 7 28 31 Terrestrial 13
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Figure 3-3: Survival percentage in function of temperature increment for all studied species and comparison to IPCC scenarios for each species’ habitat. UTL - upper thermal limit.
Western Portugal is situated in the Southern sub-region, and is expected to suffer an increase in 1.7, 3.2 or 5.7ºC according to the upper-bound of three different IPCC scenarios – RCP 2.6, 4.5 and 8.5 (IPCC, 2014). These temperatures were added to the average temperature of each of the studied caves and added as vertical lines in Fig. 3-3. All specimens died with temperature, yet all above the predicted temperature to affect the region by 2100 according to the worst RCP scenario.
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Figure 3-4: Photos of the species used in the experiment. a) Scutogona minor, b) Pseudoniphargus n. sp. (Photo credits:
Cláudia Duarte), c) Proasellus lusitanicus, d) Miktoniscus longispina, e) Trichoniscoides sicoensis (Photo credits: Rita Eusébio), f) Podocampa cf. fragiloides.
3.5 Discussion
The UTL50 varied from 27 (S. minor, M. longispina, T. sicoensis) to 29ºC (P. lusitanicus) while the UTL100 ranged from 28 (M. longispina) to 31ºC (P. lusitanicus). The difference between UTL50 and the measured annual average habitat temperature (D) ranged from 11 (Pseudoniphargus n. sp.) to 13ºC (P.
cf. fragiloides). Therefore, the temperature increase due to climate change, that will likely be reflected underground is expected to have pernicious effects in subterranean ecosystems across the globe (Castaño-Sánchez et al., 2020a).
Several UTL studies in cave species are available (Supplementary Table 3-1). However, studies lack standardization and varied in methodology, from measuring only at specific temperatures during seven days (Colado et al., 2021; Pallarés et al., 2019; Pallarés et al., 2020b); studying the critical thermal maxima, the highest temperature from which animals can still recover when returned to suitable conditions (Jones et al., 2021); or using a slow ramping of 1ºC increase every 24h but only measuring species physiological responses at certain temperatures (Mermillod-Blondin et al., 2013). From the selected studies, only one followed the same methodology as this one (Castaño-Sánchez et al., 2020b).
Cave fauna often showed high thermal tolerance when compared to the temperatures these animals experience in their habitats (Supplementary Table 3-1). The studied species D (11 to 13ºC) fits in the range of D found in the literature review, while they may be difficult to compare due to the use of different methodologies (Table 3-2 and Supplementary Table 3-1).
The lowest D (thermal amplitude between the average habitat temperature and the UTL50/LT50) seems to occur in Atlantic areas species, while species collected from Australasia or the Southern sub-region are more tolerant. The highest D was obtained for the terrestrial millipede Glomeris sp. (Pallarés et al., 2019) (14.59ºC) and the lowest D was obtained for an aquatic isopod, the Proasellus n. sp. 2 in Mermillod-Blondin et al. (2013) (3ºC).
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Although the tested species show no mortality at the highest temperature from the RCP8.5 scenario, they will still be at risk due to climate change. Both terrestrial isopods (M. longispina and T. sicoensis) reached their UTL50 very close to the RCP8.5 temperature upper-bound (20.7ºC), therefore, longer exposure to sublethal temperatures may lead to mortality at lower temperatures. Also we should consider that UTL’s may change across different populations of the same species, and within the same population depending on organisms’ health and nutrient availability at a certain time in those caves (Castaño-Sánchez et al., 2020a). Moreover, different life-stages, especially juveniles of cave animals tend to show differential responses to stress (Castaño-Sánchez et al., 2021; Pallarés et al., 2020b), and therefore some individuals may be affected slightly earlier or later than others. It is essential to understand that cave animals experience physiological stress at sublethal temperatures that limit their performance and fitness (Pallarés et al., 2020b). Individually oxygen consumption of P. lusitanicus, with a fast-ramping thermal increment shows their metabolic vulnerability to climate change (Di Lorenzo & Reboleira, 2022), despite of showing a UTL values above the highest predicted scenario.
Subterranean ecosystems provide a wide variety of ecosystem services, from provision and regulation to cultural services (Gillieson et al., 2022; Mammola et al., 2019a). Aquatic cave fauna, for example, is responsible for the ecological equilibrium in groundwater as they clean and filter the water for humans and ecosystems (Canedoli et al., 2021; Castaño-Sánchez et al., 2020a; Ravn et al., 2020). Detritivorous cave fauna are also essential for the degradation of organic matter in caves playing a vital role in nutrient cycling (Ravn et al., 2020; A. S. P. S. Reboleira et al., 2015). Caves also harbour many endemic species that have low dispersal capabilities and may be at risk of going extinct (Sánchez-Fernández et al., 2021).
Considering this and given that cave temperature reflects the average annual temperature at the surface, climate change will affect cave temperature along with cave species and ecosystem services.
Due to the difficulty in accessing underground environments (Castaño-Sánchez et al., 2021), including caves, sampling enough individuals to produce an unbiased assay in the laboratory is challenging.
However, to develop effective conservation measures for these habitats, increasing the knowledge of caves and cave fauna is crucial (Castaño-Sánchez et al., 2021). The studying caves and cave-adapted species is of growing importance to improve conservation measures covering these environments, especially as climate change poses other issues such as drought, a problem that affects both terrestrial and aquatic species (Sánchez-Fernández et al., 2021). Additionally, as different species respond differently to temperature variation, therefore predicting UTL values for taxa across trophic levels will lead to more accurate predictions to the effect of climate change in subterranean ecosystems.
We show that cave-adapted species across geographic regions respond differently to temperature variation, therefore predicting UTL values for taxa across trophic levels will lead to more accurate predictions to the effect of climate change in subterranean ecosystems.
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