Climate change goes underground: temperature in caves and its effects on subterranean organisms

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2023

UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE BIOLOGIA ANIMAL

Climate change goes underground: temperature in caves and its effects on subterranean organisms

Maria João Alves Lopes de Oliveira Medina

Mestrado em Ecologia e Gestão Ambiental

Dissertação orientada por:

Professora Doutora Ana Sofia P. S. Reboleira

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Acknowledgements

To summarize an entire year in a page is no easy task, much less a year such as this one. I could not have been happier with the dissertation topic I chose, and to be able to do it with such an amazing team was an opportunity I will never forget.

First of all, I want to thank my incredible supervisor, Professor Ana Sofia Reboleira for this opportunity and for believing in me. I could not have had a better support to my work, and it was an amazing privilege to learn from her and it is something I hope I get to continue doing. This work could not have been possible without her constant support, kindness, and encouragement during difficult times and celebrating all my achievements with me. I will be forever grateful for her amazing introduction to a world that was unknown to me in caves, a world I will continue to explore.

I want to thank my dear friends and co-workers Cláudia Duarte, Rita Eusébio and Maria Miguel Gomes.

This work could have been done without them, but it would not have been as fun. I am glad I got to share a workspace with such an amazing group and I am very happy for the friendship we grew along the way. You will do great in life, and I hope I get to keep celebrating each of your achievements with you all.

I want to thank Marta Palma for her availability to help with all things lab related.

To my friends Filipa Bernardo, Diogo Teixeira and Rui Saraiva that I can always count on for a kind word or just laughing our way through self-loathing, a HUGE thank you.

A very special thanks to my boyfriend, Duarte, who was an extraordinary emotional support. To have someone to lean on to through happy times or challenging times and to be able to do it with someone as kind and caring as him is not to be taken for granted. Thank you.

Finally, I want to thank my family, and especially my mom and my brother, for their patience, their kindness and love they showed me during all my life but that was especially important during this year.

Thank you for celebrating my achievements with me, lifting me up when I was down and being my biggest fans. Your support is crucial to be able to do and be my best.

During this year I discovered a new world in caves, learned a lot, experienced some amazing things, and had a lot of fun. I was challenged like never before and I loved every bit of the way.

This work was supported by the project “Sustainability of subterranean ecosystems” financed by the Cooperation protocol with the Municipality of Alcanena and by Portuguese National Funds through

“Fundação para a Ciência e a Tecnologia” (FCT) within the cE3c Unit funding UIDB/00329/2020.

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List of abbreviations

MDT Mean Daily Temperature

RCP Representative Concentration Pathways

UTL50 Upper Thermal Limit for 50% of the population

UTL100 Upper Thermal Limit for 100% of the population

D Thermal amplitude between the average habitat temperature and the UTL50/LT50

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Abstract

Climate change is one of the most significant threats to all ecosystems. Yet, there are fundamental knowledge gaps in understanding its effects on caves, mainly due to the difficulty in accessing and studying these ecosystems. Caves provide incredibly stable conditions, such as constant temperature, often corresponding with the average annual temperature for the surface. This dissertation studies the temperature variation in caves and at their respective surface in several climatic regions, and tested the effect of temperature in cave-adapted species and compared it with previously published studies on cave-adapted species thermal tolerance. Temperature was measured in 12 different locations across different climatic zones during one year. Results showed that cave temperature corresponds to the average annual temperature of their correspondent surface, independently on the type of cave or location.

Three types of thermal regimes were found with caves thermally similar to the surface, caves with a slight thermal delay, and caves with an extreme thermal delay from the surface. Daily thermal cycles were found in some caves, with implications for the fauna’s circadian rhythms. Thermal tolerance was assessed for six cave-adapted species from Western Portugal karst areas. Some species started dying at temperatures very close to the highest predicted temperature. It is crucial to consider sub-lethal effects that may be occurring prior to mortality. However, caves provide vital ecosystem services. Subterranean ecosystems store 97% of freshwater used in multiple human activities, in which groundwater species play the crucial role of maintaining its ecological equilibrium by filtering and cleaning the water.

Furthermore, some terrestrial species are also key decomposers of organic matter, encouraging nutrient cycling. Considering the cave dependence on the surface to regulate its temperature, temperature increases at the surface will be reflected underground, impacting caves, their fauna and ecosystem services.

Keywords: global warming, karst, subterranean ecosystem, cave fauna, thermal tolerance.

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Resumo

As alterações climáticas e os seus efeitos nos ecossistemas e na humanidade são um dos maiores problemas da atualidade, e como tal, entender como é que os ecossistemas vão ser afetados por este fenómeno é imprescindível para poder desenvolver medidas compensatórias e de mitigação assim como compreender a gravidade da situação atual. No entanto, os ecossistemas subterrâneos, e nomeadamente as grutas, têm vindo a ser negligenciados apesar da sua importância e possível suscetibilidade.

As grutas são ecossistemas subterrâneos compostos por cavidades naturais verticais ou horizontais, ou ambas, que podem conter ar, água ou até hidrocarbonetos. Estes ecossistemas são caracterizados pela ausência de luz, temperatura constante e humidade muito elevada próxima dos 100%. Apesar das dificuldades de estudar os ambientes subterrâneos, tendo em conta a estabilidade de fatores abióticos que caracteriza as grutas (humidade, temperatura, ausência de luz), estes sistemas são relativamente simples de reproduzir em laboratório, o que é uma mais-valia para o estudo das grutas. Para além disto, é ainda importante superar a lacuna científica existente nos ecossistemas subterrâneos, melhorando assim a sua proteção. A temperatura no interior da gruta tem sido comummente relacionada com a temperatura média anual à superfície, pelo que existe uma óbvia ligação entre as grutas e a superfície por meio de vários fatores, nomeadamente a circulação do ar, a condutividade térmica da rocha ou até a infiltração de água. As grutas podem ter géneses diferentes, sendo a maioria formadas em ambiente cársico pela dissolução de rochas como o calcário ou a dolomite. No entanto, ocorrem também grutas em tubos de lava em ambientes vulcânicos.

Nas grutas vivem diversas espécies com adaptações específicas (troglomorfismos) e adequadas ao ambiente em que se encontram. Os animais troglóbios, animais que permanecem na gruta durante todo o seu ciclo de vida e que apresentam troglomorfismos, são caracterizados por despigmentação, cegueira ou ausência dos órgãos oculares, apêndices longos e órgãos sensoriais desenvolvidos. Tendo em conta a ausência de luz nestes habitats, a disponibilidade de nutrientes por produção fotossintética é muito escassa, pelo que a maior parte das espécies cavernícolas são decompositores, predadores ou parasitas.

Para além da sua morfologia, também fisiologicamente estes animais sofreram adaptações para sobreviverem num ambiente como as grutas. Estes animais têm ciclos de vida longos, metabolismos lentos, tendo em conta a escassez de alimento; e baixas taxas de reprodução. Sendo as grutas sistemas semifechados, tal como as ilhas, a dispersão das espécies cavernícolas, muitas vezes endémicas, encontra-se limitada, o que contribui para a sua suscetibilidade.

A maioria dos animais cavernícolas são invertebrados ectotérmicos. Como tal, tendo em conta que estes animais dependem de condições externas para regular a sua temperatura corporal, o aumento da temperatura da gruta vai afetar estes animais mais do que a um animais endotérmicos. Para além disto, tendo em conta a estabilidade da temperatura nas grutas, é expectável que a tolerância térmica dos animais cavernícolas seja menor que a de animais de superfície que estão expostos a maiores variações no clima. No entanto, tendo em conta que o aumento da temperatura à superfície se deverá sentir nos ecossistemas subterrâneos e que é expectável que os animais cavernícolas sejam mais sensíveis à variação da temperatura, conhecer os efeitos do aumento das temperaturas nas grutas é indispensável.

As grutas são responsáveis por múltiplos serviços de ecossistema imprescindíveis para o ser humano, para além do seu valor natural intrínseco, nomeadamente serviços de regulação, de provisão ou culturais.

Cerca de 97% da água doce em estado líquido encontra-se armazenada em ecossistemas subterrâneos.

Esta água é limpa e filtrada por meio de várias espécies cavernícolas aquáticas que contribuem para o equilíbrio ecológico da água subterrânea. Espécies cavernícolas terrestres detritívoras contribuem para

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a decomposição de matéria orgânica e ciclo de nutrientes. Um outro benefício do estudo destas espécies é a possibilidade de novos conhecimentos que possam ser aplicados à medicina ou até à engenharia.

Para além disto, culturalmente as grutas são também um património importante contendo vários vestígios arqueológicos, sendo assim uma importante janela para civilizações passadas.

O IPCC (Painel Intergovernamental para as Alterações Climáticas) divide Portugal em duas sub-regiões, a Atlântica e a do Sul. A sub-região Sul será a região mais afetada pelo aquecimento global e é nesta região que se situam as grutas portuguesas estudadas nesta dissertação. Nesta dissertação foram utilizados três cenários para o aumento da temperatura média anual até 2100: RCP2.6, o melhor cenário possível em que é possível diminuir as emissões de gases de efeito de estufa a um nível substancial;

RCP4.5, um cenário intermédio e o RCP8.5, como um cenário mais conservador em que se assume a continuação e eventual aumento da emissão de gases de efeito de estufa sem medidas compensatórias adicionais. De acordo com o pior cenário (RCP8.5), a temperatura média anual à superfície pode aumentar quase 6°C até 2100.

O principal objetivo desta dissertação é avaliar a suscetibilidade dos ecossistemas subterrâneos ao aquecimento global e prever se espécies cavernícolas do centro de Portugal se encontram ameaçadas pelo aumento de temperatura previsto para Portugal pelo IPCC. Para tal foram utilizadas duas abordagens:

1. Para tal a temperatura de 12 grutas e respetivas superfícies foi medida ao longo de um ano em diferentes zonas climáticas. Foi possível verificar a similaridade entre a temperatura média anual no interior das grutas e a mesma à superfície independentemente da zona climática. Para além disto, ciclos diários de temperatura foram encontrados em algumas grutas, apesar de com menor intensidade que à superfície, o que poderá ter implicações ao nível dos ritmos circadianos da fauna cavernícola. No entanto, para compreender quão dependente o microclima cavernícola é da superfície, é essencial o estudo de outros fatores como a circulação do ar no interior das cavidades, assim como compreender quanto tempo este ecossistema demorará a ajustar a sua temperatura à temperatura média anual da superfície.

2. Foram efetuados estudos de tolerância térmica em seis espécies cavernícolas do centro de Portugal nas regiões de carso Estremenho, de Sicó e de Sintra-Cascais. Em todas as espécies foi observada 100% de sobrevivência até acima da temperatura mais alta estimada pelo IPCC para o RCP8.5. No entanto, foi possível verificar uma maior suscetibilidade das espécies terrestres à temperatura, com mortalidade a ocorrer imediatamente após o valor de temperatura correspondente à previsão mais grave do IPCC em duas espécies de isópodes. Assim, é importante compreender que a experienciação em animais recolhidos em diferentes alturas do ano ou em estados de vida diferentes pode dar origem a resultados diferentes, pelo que é necessária a continuação do estudo desta temática. Por fim, apesar de estes animais só atingirem a mortalidade a uma temperatura mais elevada que as previsões do IPCC, podem estar a ocorrer alterações fisiológicas que podem afetar a performance dos indivíduos, por exemplo ao nível da reprodução, da muda, ou mesmo da respiração. Todos os indivíduos morreram com o aumento da temperatura.

Os resultados desta dissertação permitem afirmar que as temperaturas nas grutas refletem a temperatura média anual à superfície. Tendo em conta as atuais previsões para as alterações climáticas à superfície, é importante considerar que as grutas e as suas espécies serão também afetadas. Nas grutas esconde-se um vasto mundo essencial para o funcionamento dos restantes ecossistemas e para a humanidade que pode vir a ser deteriorado devido às alterações climáticas por meio de extinção de espécies ou pela

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perturbação dos ambientes aquáticos subterrâneos devido à seca. É essencial que seja dada continuidade ao estudo destes habitats e das suas espécies dando ênfase não só ao estudo da temperatura letal máxima capaz de extinguir 50% das populações mas também a testes com temperaturas sub-letais, como por exemplo com a utilização de biomarcadores, que poderão mais eficazmente determinar os efeitos do aumento da temperatura nos animais.

Palavras-chave: aquecimento global, carso, ecossistemas subterrâneos, fauna cavernícola, tolerância térmica.

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List of Figures

FIGURE 1-1: SAMPLING IN ASSAFORA CAVE (SINTRA-CASCAIS) KARST AREA. CREDITS: A.S.P.S.

REBOLEIRA. ... 1 FIGURE 1-2:MIKTONISCUS LONGISPINA COLLECTED FROM THE CERÂMICA CAVE (SICÓ KARST AREA). 2 FIGURE 2-1:CAVE EXPLORATION.CREDITS:MARIA MIGUEL GOMES. ... 6 FIGURE 2-2:LOCATION OF THE STUDIED CAVES ACROSS CLIMATIC ZONES. ... 9 FIGURE 2-3:THERMAL AMPLITUDE OF DEEP ZONES IN CAVES AND THEIR RESPECTIVE SURFACE. ... 11 FIGURE 2-4:AVERAGE MONTHLY THERMAL VARIATION FOR THE 12 STUDIED LOCATIONS ALONG ONE YEAR. ... 14 FIGURE 2-5:SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR DEEP ZONES OF ALL STUDIED CAVES.

... 15 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. ... 19 FIGURE 3-2:LOCATION OF THE SAMPLING LOCATIONS IN WESTERN PORTUGAL. ... 22 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. ... 25 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. ... 26 SUPPLEMENTARY FIGURE 2-1:SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR DEEP ZONES OF ALL STUDIED CAVES RESPECTIVE SURFACES. ... 40 SUPPLEMENTARY FIGURE 2-2:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE BALCÕES CAVE (AZORES). ... 41 SUPPLEMENTARY FIGURE 2-3:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE JAZINKA CAVE (CROATIA). ... 42 SUPPLEMENTARY FIGURE 2-4:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE LAZAREVA CAVE (SERBIA). ... 43 SUPPLEMENTARY FIGURE 2-5:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE TALOFOFO CAVE (GUAM). ... 44 SUPPLEMENTARY FIGURE 2-6:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE VAMPIRJEVA CAVE (SLOVENIA). ... 45 SUPPLEMENTARY FIGURE 2-7:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE SETERGROTTA CAVE (NORWAY). ... 46 SUPPLEMENTARY FIGURE 2-8:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE SANT JOSEP CAVE (SPAIN). ... 47 SUPPLEMENTARY FIGURE 2-9:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE HONDA DE GUIMAR CAVE (CANARY ISLANDS). ... 48 SUPPLEMENTARY FIGURE 2-10:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE VALE TELHEIRO CAVE (SOUTHERN PORTUGAL). ... 49 SUPPLEMENTARY FIGURE 2-11:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE CERÂMICA CAVE (CENTRAL PORTUGAL). ... 50

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SUPPLEMENTARY FIGURE 2-12:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE PLANINSKA CAVE (SLOVENIA). ... 51 SUPPLEMENTARY FIGURE 2-13:SEASONAL SPECTRAL DENSITY ANALYSIS OF TEMPERATURE FOR THE DEEP ZONE OF THE VIENTO CAVE (CANARY ISLANDS). ... 52

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List of Tables

TABLE 2-1:STATISTICS AND COMPARISON BETWEEN DEEP ZONES OF CAVES (C) AND THEIR RESPECTIVE SURFACES (S).MIN T–MINIMUM TEMPERATURE,MDT– MEAN DAILY TEMPERATURE,AAT– AVERAGE ANNUAL TEMPERATURE, MAX T – MAXIMUM TEMPERATURE, TA – THERMAL AMPLITUDE, WILCOXON – WILCOXON SIGNED RANK TEST, CORRELATION – CORRELATION COEFFICIENT (P–PEARSON,SP –SPEARMAN) FOR THE COMPLETE DATA,CSD–CAVE SPECTRAL DENSITY,SSD–SURFACE SPECTRAL DENSITY,NDC–NO DAILY CYCLE. ... 12 TABLE 3-1:SAMPLED CAVES LOCATIONS AND AVERAGE ANNUAL TEMPERATURE. ... 23 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. ... 24 SUPPLEMENTARY TABLE 2-1:LOCATION AND COORDINATES OF THE 12 STUDIED CAVES. ... 39 SUPPLEMENTARY TABLE 3-1:LITERATURE REVIEW OF THERMAL TOLERANCE STUDIES IN CAVE FAUNA ACROSS REGIONS. ... 53

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1. Introduction

1.1 Characteristics of caves and their inhabiting fauna

Caves are natural subterranean environments composed of vertical and/or horizontal spaces that can be large, small, or maze-like interconnected crevices filled by air, water, or hydrocarbons (Culver & Pipan, 2019). The extent of their connectivity depends on the permeability of the rock which yields limited dispersal for cave fauna (Culver & Pipan, 2019; Howarth & Moldovan, 2018; Lauritzen, 2018).

Subterranean ecosystems encompass geological, biological, archaeological and climatological significance (White et al., 2019). Caves may be formed through different geneses. They can be formed either in karst from the dissolution of soluble rocks (e.g., limestone, dolomite, gypsum) by percolating waters and other dynamic interactions, or in volcanic landscapes that can create lava tubes (Canedoli et al., 2021; Howarth & Moldovan, 2018; Lauritzen, 2018). Caves embody unique environmental characteristics, such as total darkness, stable temperature, and high relative humidity (Culver & Pipan, 2019; Howarth & Moldovan, 2018; Lauritzen, 2018) (Fig. 1-1). These conditions become more homogeneous with increasing distance from the surface (Culver & Pipan, 2019; Howarth & Moldovan, 2018; Lauritzen, 2018). Because caves lack solar radiation, food and nutrients provided by photosynthetic producers are very scarce (Castaño-Sánchez et al., 2020a). As a result of being semi- closed environments, caves behave as isolated habitats, with an island-like behaviour, hosting specialized, endemic species, mostly invertebrates, with very distinct evolutionary traits (Castaño- Sánchez et al., 2020; Hose et al., 2022; Howarth & Moldovan, 2018; Lauritzen, 2018; Mammola et al., 2019a). Given the difficulty in accessing these environments, caves are among some of the less known and less studied environments and may be susceptible to multiple threats (Mammola et al., 2019a).

However, due to the stability of their environment, caves are exceptional systems for conducting biological research (Culver & Pipan, 2019; Howarth & Moldovan, 2018; Mammola et al., 2019b) although this is still very much an under-explored territory (Canedoli et al., 2021).

Figure 1-1: Sampling in Assafora Cave (Sintra-Cascais) karst area. Credits: A.S.P.S. Reboleira.

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Subterranean habitats harbour highly specialized life-forms, many endemic with limited dispersal and geographic range (Sánchez-Fernández et al., 2018). For animals to succeed in such a constrained environment, they developed important behavioural and morpho-physiological traits, given that caves lack the cues (e.g. daily cycles) used by organisms to regulate their cycles at the surface (Hose et al., 2022). Caves are divided into four areas: entrance, twilight, transition, and deep zone, each with different abiotic characteristics (Howarth & Moldovan, 2018; White et al., 2019), which will affect the biodiversity in each location. Given the absence of light in most of these environments, caves often lack primary producers and herbivores, although decomposers, predators, or parasites are regularly well represented (Jensen et al., 2019; Saccò et al., 2019). Subterranean fauna is often biologically simple and of a very small body size (Moldovan et al., 2018; Sánchez-Fernández et al., 2016).

Subterranean fauna is classified according to how populations use caves and their dependence and permanence on these habitats (Castaño-Sánchez et al., 2020a; Culver & Pipan, 2019; White et al., 2019).

There are two prefixes to ecologically classify cave biota: (i) troglo-, for terrestrial organisms and (ii) stygo-, for aquatic organisms (Castaño-Sánchez et al., 2020a; Moldovan et al., 2018; White et al., 2019).

Troglobionts/stygobionts are considered "true cave organisms" and share specific morphological adaptations to help them navigate their environment called troglomorphisms (Castaño-Sánchez et al., 2020a; Moldovan et al., 2018). Species with these adaptations are often blind, lack pigment, and have elongated appendages and increased sensory organs to help them circulate around their habitat and find food in their nutrient depleted environment (Castaño-Sánchez et al., 2020a; Culver & Pipan, 2019) (Fig.

1-2). These animals complete their entire life cycle in underground ecosystems. Cave species also developed other special physiological traits such as slow metabolism, given the scarcity of nutrients available in their habitat (Carpenter, 2021; Castaño-Sánchez et al., 2020a), long life cycles, and low reproductive rates (Pallarés, Colado, et al., 2020). When considering stygobionts, crustaceans are the most diverse group (Moldovan et al., 2018). Species frequently found in the subterranean environment but may not complete their whole life cycle inside a cave are classified as troglophiles/stygophyles.

Lastly, the animals classified with the suffix -xene occasionally occur in caves but cannot establish a subterranean population (Castaño-Sánchez et al., 2020a; Moldovan et al., 2018).

Figure 1-2: Miktoniscus longispina collected from the Cerâmica Cave (Sicó karst area).

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Due to the stable conditions inside the caves, subterranean ectothermic biota is expected to present a shorter thermal tolerance, as opposed to most surface animals acclimated to a larger temperature spectrum (Mammola et al., 2019a; Pallarés et al., 2020). For this reason, cave fauna may present a strict set of temperature constraints that may make them vulnerable to rapid changes in their environment (Domínguez-Villar et al., 2015; Pallarés et al., 2019; Sánchez-Fernández et al., 2018). However, taking into account the conditions inside the subterranean environment and due to the characteristics of cave species’, their dispersal capability is limited to the vertical and lateral void connectivity inside the rock and the effects of anthropogenic changes will be much more pronounced (Castaño-Sánchez et al., 2020;

Mammola et al., 2019a; Sánchez-Fernández et al., 2016), which also results in these organisms being more vulnerable to threats to their environment than surface species, as they have no possibility to migrate to another habitat (Sánchez-Fernández et al., 2016).

1.2 Ecosystem services provided by caves and cave fauna

Humanity is highly dependent on multiple ecosystem services provided by different environments (Griebler & Avramov, 2015), and caves are no exception (Mammola et al., 2019a). Yet, the protection level provided to these ecosystems inadequately reflects their importance. In fact, subterranean ecosystems provide a variety of essential services, from water purification, groundwater storage, refuge to several species, to tourism and educational services (Griebler et al., 2014). Karst caves are the main reservoirs of groundwater, an essential human resource, with over 94% of the available freshwater underground and about 25% of the world's drinking water being from aquifers (Culver & Pipan, 2019;

Griebler & Avramov, 2015; Mammola et al., 2019a). However these benefits highly depend on the well-being of these ecosystems (Griebler et al., 2014).

Cave species are responsible for providing important ecosystem services of provision and regulation (Mammola et al., 2019a), such as maintaining the ecologic equilibrium of groundwater by cleaning and filtering groundwaters through their feeding habits or nutrient cycling (Canedoli et al., 2021; Castaño- Sánchez et al., 2020a; Ravn et al., 2020). The study of cave species may also provide significant knowledge that could be applied to medicine, or even engineering. The study of cave species could help us understand survivability strategies in extreme environments but also resistance to infections, leading to the discovery of new medicines or treatments for human diseases (Zada et al., 2022). Engineering studies in caves could also promote the development of energy-efficient lighting systems. Caves are also important models for the study of evolution and genetic studies (Juan et al., 2010).

Caves represent a vast natural heritage with important geo and biodiversity with countless unknown species and even archaeological artifacts that helps archaeologists and anthropologists better understand past civilizations (Gillieson et al., 2022). Considering this, the opportunity for nature tourism and recreation is immense while spreading conservation knowledge about these environments to visitors.

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1.3 An overview on climate change on subterranean ecosystems

Climate change is a growing threat to biodiversity and all ecosystems, and is now well-documented across many terrestrial and aquatic ecosystems (IPCC, 2014). Impacts of climate change have been observed in terrestrial, freshwater and oceanic environments (IPCC, 2014), however, this phenomenon is still poorly understood in caves and by extension in all subterranean ecosystems (Mammola et al., 2019a) affecting their ecosystem services, increasing the propagation of invasive species, and impacting humans (IPCC, 2014).

Representative Concentration Pathways (RCP) are different trajectories of greenhouse gas (GHG) emissions that result in different scenarios which consider different mitigation measures that may be put into action. An RCP of 2.6 corresponds to the best-case scenario achieved by keeping global warming below 2°C when considering pre-industrial temperatures as the baseline. The 4.5 RCP is an intermediate scenario and the RCP of 8.5 is a “business as usual” scenario with no additional mitigation measures and with very high emissions of GHG (IPCC, 2014). According to the IPCC synthesis report published in 2014, along with the European supplementary material, Portugal’s Southern subregion will likely suffer more with increasing mean surface temperatures than the northern region of Portugal, the Atlantic subregion. In fact, following the “business as usual” scenario, mean annual temperature in Portugal may be increased by almost 6°C until 2100 (IPCC, 2014).

As previously stated, caves are stable environments where constant temperature is a primary aspect (Badino, 2004). Temperature in caves corresponds typically to the average annual temperature for the surface (Domínguez-Villar et al., 2015; Mammola et al., 2019b). Given its dependency on surface temperature, it is expected that cave temperatures will also be affected by climate change (Mammola et al., 2019a). Yet, the extent to which caves will be affected and how subterranean biota will respond to these environmental changes is mostly unknown. Therefore, understanding how cave temperatures are dependent on the surface and its impacts on cave species is crucial to predict the effects on their ecosystem services that we all depend on.

1.4 Structure and Goals of this dissertation

Main objective

The primary objective of this dissertation is to understand the thermal conditions inside caves, how these conditions affect cave fauna and how climate change will impact these ecosystems, particularly in Portugal. Additionally, a goal of this study is to provide better evidence of the interconnectivity between cave and surface, scratch the surface of the fundamental research question “How does climate change affect subterranean adapted organisms” (Mammola et al., 2020), as well as improve the assessments to species vulnerability, while bringing attention towards an often forgotten ecosystem.

Specific objectives

1- Identify thermal patterns in caves, by studying temperature in caves and at their surface across different climatic regions, to understand how the surface interferes and regulates caves;

2- Test lethal responses to temperature increase on cave species from Portugal, using laboratory- controlled conditions, in order to assess the impact of climate change on cave-adapted species.

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This dissertation is divided into introduction, two chapters and final remarks. The introduction provides a general state of the art of the research subject, the two chapters correspond to original data analysis, field and laboratory work, and the final remarks conclude the most relevant findings of this dissertation.

The two chapters correspond to:

I

–

Temperature variation in caves and its significance for subterranean ecosystems

Analysis of continuously measurements of temperature inside 12 caves across different climatic zones from Tropical to Continental regions and comparison their correspondent surfaces.

Differentiate and categorize the different caves according to different thermal patterns, understand the importance of external factors in regulating the caves temperature and evaluate the possible impact of climate change in these environments.

II – Lethal upper-limit temperatures in cave-adapted species from Western Europe

Determine the upper limit temperatures of different cave animal species from Central Portugal and understand how these species respond to incremental temperatures. Review literary results already published from similar studies in cave species and compare them to our obtained results.

Additionally, to estimate how the temperatures expected for Portugal will affect those cave species.

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2. Chapter I – Temperature variation in caves and its significance for subterranean ecosystems

Figure 2-1: Cave exploration. Credits: Maria Miguel Gomes.

Paper in preparation for submission:

Medina M.J., Borko Š., Oromí P., Martín J.L., Prevorčnik S., Lauritzen S.E., Puliafico K., Pavlek M., Antić D., Sendra A., Borges P., Reboleira A.S.P.S.. Temperature in caves.

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2.1 Abstract

Climate change affects all ecosystems, but subterranean ecosystems are often neglected from political and public agendas. Temperature in subterranean ecosystems is known to be highly dependent on the surface’s climate. We studied the annual variation of cave temperatures vis-à-vis surface temperature in different climatic areas, from Tropical to Continental regions, during one year. We hypothesize that cave temperatures follow the average temperature pattern at the surface for each location with a slight delay in the signal. Our results show that caves have had always a lower thermal amplitude than the surface, whose average corresponds approximately to the annual average temperature at the surface.

We found three different thermal patterns occurring in cave: 1) caves with a high positive correlation and a similar thermal pattern to the surface, 2) caves with low correlation and a slight thermal delay of the signal from the surface, and 3) caves with high negative correlation with an extreme delay from the surface. Cave’s thermal patterns are related to the cave’s traits, such as the rock type, the morphology, altitude and latitude, and air and water circulation. This shows that even exhibiting similar average year temperatures, caves can respond with different patterns expressing the signal from surface’s temperature. Despite the high thermal stability and very little temperature amplitude throughout the year, some caves show daily thermal cycles, i.e. 24h-day cycle. This may potentially control circadian rhythms in the underground. These finding are also important in the context of paleoclimatic and archaeological reconstructions.

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2.2 Introduction

Climate change is a main driver of biodiversity loss, affecting species’ geographical distribution, ecosystem functioning and services (human benefits provided by the ecosystems) (Hooper et al., 2005;

IPCC, 2014; Mammola et al., 2019a; Pires et al., 2018). These include support, provisioning, regulation and cultural services (Millennium Ecosystem Assessment, 2005). Thus, understanding how global warming affects all ecosystems is crucial (Pires et al., 2018).

Our capability of making predictions regarding climate change at surface increased significantly over the last decades (IPCC, 2014). Nevertheless, below the ground hides a vast subterranean ecosystem, whose response to climate change remains largely unknown (Castaño-Sánchez et al., 2020a). Caves are the most accessible underground ecosystems and constitute a window to study the vast dimension of the underground habitat (Canedoli et al., 2021; Lauritzen, 2018). Caves lack light and are known to have stable environmental parameters, with surface influence decreasing with depth (Canedoli et al., 2021; Castaño-Sánchez et al., 2020a; Lauritzen, 2018; Mejía-Ortíz et al., 2020; Pallarés, Colado, et al., 2020; Sánchez-Fernández et al., 2018; Smithson, 1991). Temperature in caves show lower amplitude than at surface (Domínguez-Villar et al., 2015; Lauritzen, 2018) though caves’ stable temperature has been directly associated with the average annual temperature at the surface for the same location (Domínguez-Villar et al., 2015; Mammola et al., 2019a; Mejía-Ortíz et al., 2020; Smithson, 1991).

Because caves are isolated habitats with stable environmental conditions, they provide good models to predict ecological responses to multiple environmental stressors, including climate change (Mammola et al., 2019; Pallarés et al., 2020).

Caves have unique and adapted organisms, including many short-range endemics, representing independent colonisations of surface ancestors, many ancient lineages and countless species yet to be discovered (Castaño-Sánchez et al., 2020a; Juan et al., 2010). These ecosystems provide multiple benefits to humans (Castaño-Sánchez et al., 2020a; Griebler et al., 2014; Griebler & Avramov, 2015).

They include the largest spaces for groundwater storage, but also water purification, in which subterranean biota plays the crucial role in the degradation of organic matter and pollutants (Castaño- Sánchez et al., 2020a; Griebler & Avramov, 2015). Considering the importance of these ecosystems, understanding the thermal behaviour in caves is crucial to predict the fate of these ecosystems (Castaño- Sánchez et al., 2020; Mammola et al., 2019a).

We studied the temperature variation of deep zones in caves and on their respective surface, at sediment level during one year. Our hypothesis is that temperature in the deep zones of caves follows the pattern of average temperature at surface for each location, expressing a slight delay and lower amplitude. We focused this study on cave deep zones as these are the less surface-influenced areas. We selected 12 caves located in different climatic zones, from tropical to cold continental climates, to study the relationship between surface and cave temperatures. We analysed the overall amplitudes, thermal patterns among seasons and months, and unveiled cyclicity patterns. Understanding how temperature in caves is related to surface temperatures in different climatic areas is crucial to predict how climate change will affect subterranean ecosystems and consequently affect the ecological quality of groundwater.

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2.3 Methodology

2.3.1 Data collecting

We selected twelve caves in different climate zones: Tropical (Am Tropical - Monsoon), Arid (BWk – Arid – Desert – Cold, BSk – Arid – Steppe – Cold), Temperate (Cfa – Temperate – No dry season – Hot summer, Cfb – Temperate – No dry season – Warm summer, Csa – Temperate – Dry summer – Hot summer, Csb – Temperate – Dry summer – Warm summer), and Continental (Dfb – Continental – No dry season – Warm summer, Dfc – Continental – No dry season – Cold summer) (Fig. 2-2, Supplementary Table 1). For each location, temperature was recorded inside the cave at the deep zone to ensure minimal influence from outside sources, and vertically above the cave at surface. Temperature data loggers (HOBO TidbiT v2) recorded the temperature every two hours during a 12-month period, with an accuracy of ± 0.21°C and a resolution of 0.02°C. These were installed 2 cm below the soil at the surface and below the sediment inside the caves. These caves vary in lithology, genesis, size, climatic zones, number of entrances, depth, and altitude. The map of localities was produced in ArcGIS (v10.7.1), over a Köppen-Geiger climate classification layer adapted from Peel et al. (2007).

Figure 2-2: Location of the studied caves across climatic zones.

2.3.2 Data analysis

The data was analysed with basic statistics in R and R Studio (v1.3.1073) (R Core Team, 2020) and visualized with the package ggplot2 (Wickham, 2016). Mean daily temperature (MDT), average annual temperature (AAT) and maximum and minimum temperatures were calculated for both cave and surface for every location. Correlation between cave and surface environments for the complete and monthly data was analysed using a Pearson coefficient for variables with a linear relationship, while a Spearman coefficient was used for non-linear. To check if the difference in MDT between cave and surface was statistically significant, initially, a Shapiro-Wilk test was performed to check the normality

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of the variables. If the variables were parametric, we would perform a t-test. If not, a Wilcoxon test would be used.

Thermal cyclicity was studied using spectral density analysis in JMP software (v16.0.0) to investigate the existence of 24h cycles in caves. Spectral density analysis is used to study a signal’s periodicity linked to a cyclic behaviour (Stoica & Moses, 2005). Thermal cycles were analysed for the complete data of cave and surface for each location during a 60h period, and for the seasonal data, i.e. analysing independently the data from each season. Because the temperature was recorded every 2h, the 24h cycles are detectable as 12 time intervals, and the 12h cycles are detectable as 6 time intervals.

2.4 Results

2.4.1 Temperature variation in caves vs surface

We found temperature in caves to be consistently more stable than at surface, with the lowest amplitudes occurring in the caves (Fig. 2-3, Table 2-1). The highest temperature was recorded at surface in Southern Portugal (Vale Telheiro – 39.1°C) and the highest cave temperature in Guam (Talofofo – 26.7°C). In comparison, the lowest temperatures were recorded in Northern Norway (-0.5°C at surface, and 2°C in the Setergrotta Cave) (Table 2-1). We found a statistically significant difference between the cave and surface MDT for seven of the studied locations (Balcões, Honda de Güímar, Jazinka, Cerâmica, Lazareva, Planinska and Sant Josep) (Table 2-1).

Thermal amplitude in caves ranged from 0.1°C in Planinska Cave in Slovenia, to 8.8°C in Balcões Cave in the Atlantic Island Terceira of the Azores archipelago (Fig. 2-3, Table 2-1). The lowest thermal amplitudes at surface were recorded in a Am (Tropical – Monsoon) climate zone in Guam (2.5°C in the cave and 4.9°C at surface), and in a Csb (Temperate – Dry summer – Warm summer) in Azores (8.8°C in the cave and 12.4°C at surface), with Guam having the most similar values between cave and surface thermal amplitudes. The highest thermal amplitude was recorded at surface at the vertical of Vale Telheiro Cave, Southern Portugal (32.2°C).

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Figure 2-3: Thermal amplitude of deep zones in caves and their respective surface.

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Table 2-1: Statistics and comparison between deep zones of caves (C) and their respective surfaces (S). Min T – Minimum temperature, MDT – Mean daily temperature, AAT – Average annual temperature, Max T – Maximum temperature, TA – Thermal amplitude, Wilcoxon – Wilcoxon Signed Rank Test, Correlation – Correlation coefficient (P – Pearson, Sp – Spearman) for the

complete data, CSD – Cave spectral density, SSD – Surface spectral density, NDC – No daily cycle.

Location Zone Min T (°C) MDT (°C) AAT (°C) Max T (°C) TA (°C) Wilcoxon

(C and S) Correlation CSD (24h)

SSD (24h)

C S C S C S C S C S

Balcões (Azores) Csb 9.9 8.6 13.4 14.1 13.4 14.1 18.6 21.0 8.8 12.4 𝑝 = 0.0040 𝑟(𝑃) = 0.93

𝑝 = 5.48𝑒^!"#$ 0.022 4.50 Honda de Güímar

(Canary Islands) BWk 18.5 13.2 20.1 23.5 20.1 23.5 21.5 37.1 3.0 23.9 𝑝 = 2.2𝑒^!"# 𝑟 (𝑃) = 0.53

𝑝 = 1.41𝑒^!%& 0.301 801.06 Jazinka (Croatia) Csa 8.1 1.7 11.4 13.1 11.4 13.1 13.6 27.4 5.6 25.7 𝑝 = 0.0002 𝑟 (𝑃) = 0.85

𝑝 = 1.46𝑒^!"'$ 0.263 195.22 Lazareva (Serbia) Cfa 8.6 3.1 9.3 12.0 9.3 12.0 9.6 21.6 1.0 18.4 𝑝 = 1.75𝑒^!( 𝑟(𝑆𝑝) = 0.85

𝑝 = 1.97𝑒^!"') 0.0004 10.33 Talofofo (Guam) Am 24.2 23.0 26.1 26.0 26.1 26 26.7 27.9 2.5 4.9 𝑝 = 0.476 𝑟(𝑃) = 0.90

𝑝 = 2.17𝑒^!")' 0.065 12.81 Setergrotta (Norway) Dfc 2.0 -0.5 2.3 4.4 2.3 4.4 2.6 14.3 0.6 14.7 𝑝 = 0.801 𝑟(𝑆𝑝) = 0.77

𝑝 = 3.33𝑒^!&% NDC 6.46 Vale Telheiro

(Portugal) Csa 17.1 6.9 17.2 18.0 17.2 18 17.4 39.1 0.4 32.2 𝑝 = 0.0874 𝑟(𝑃) = −0.62

𝑝 = 1.705𝑒^!$' NDC 410.91 Cerâmica (Portugal) Csb 14.7 2.8 14.9 15.1 14.9 15 15.3 30.2 0.6 27.3 𝑝 = 0.0181 𝑟(𝑆𝑝) = −0.87

𝑝 = 1.38𝑒!"%" NDC 369.29 Planinska (Slovenia) Cfb 9.2 1.2 9.3 10.3 9.3 10.3 9.3 20.2 0.1 19.0 𝑝 = 0.0011 𝑟(𝑃) = −0.79

𝑝 = 8.735𝑒^!*% NDC 39.05 Vampirjeva (Slovenia) Dfb 9.0 1.3 10.4 10.9 10.4 10.9 11.4 22.6 2.4 21.3 𝑝 = 0.801 𝑟(𝑆𝑝) = 0.32

𝑝 = 3.315𝑒^!"' 0.0024 43.44 Sant Josep (Spain) BSk 18.8 6.6 19.1 17.7 19.1 17.7 19.4 26.7 0.6 20.1 𝑝 = 3.63𝑒^!( 𝑟(𝑆𝑝) = −0.21

𝑝 = 2.052e^!+ NDC 40.29 Viento (Canary

Islands) BWk 14.6 7.3 14.6 14.9 14.6 14.9 14.7 26.5 0.2 19.2 𝑝 = 0.257 𝑟(𝑆𝑝) = −0.75

𝑝 = 9.7241e^!&' 0.00003 95.79

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2.4.2 Annual temperature variation patterns in caves compared to surface

We found three main correlation patterns between annual cave and surface temperature variation: 1) caves with high positive correlation values to temperature at the surface, that have the same thermal signal (0-1 months difference) as the surface but with a smaller amplitude (Balcões, Jazinka, Lazareva, Talofofo), 2) caves with low correlation to surface temperature, and with a slight thermal delay of the signal from the surface (1-4 months) (Vampirjeva, Setergrotta, Sant Josep, Honda de Güímar), and 3) caves with high negative correlation to surface temperature, that exhibit an extreme delay from the surface (5-6 months) (Vale Telheiro, Cerâmica, Planinska, Viento) (Table 2-1).

The caves with the lowest correlation with the surface (Vale Telheiro, Cerâmica, Planinska and Viento) show the most extreme delay in the coldest and hottest surface peaks (Fig. 2-4, Table 2-1). In these cases, the coldest temperatures inside the caves correspond to the hottest temperatures at surface and vice-versa. Contrarily, the caves that had the highest correlation with the surface show similar behaviour to the surface, with little to absent delay.

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Figure 2-4: Average monthly thermal variation for the 12 studied locations along one year.

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We found daily thermal cycles in seven caves (Balcões in Azores, Jazinka in Croatia, Lazareva in Serbia, Talofofo in Guam, Vampirjeva in Slovenia, Viento and Honda de Güímar in the Canary Islands) (Fig.

2-5), with values always lower than those obtained for their surface (Table 2-1, Supplementary figure 1). All other caves show no daily peaks, even at low spectral densities (Fig. 2-5). Caves with daily thermal cycles, show daily thermal cyclicity for individual seasons, but not necessarily for the four of them, while caves lacking daily thermal cyclicity, also lack cyclicity for seasonal data (Supplementary figures 1-2 to 1-13).

Figure 2-5: Spectral density analysis of temperature for deep zones of all studied caves.

Balcões Cave in Azores Jazinka Cave in Croatia Lazareva Cave in Serbia

Talofofo Cave in Guam Vampirjeva Cave in Slovenia Setergrotta Cave in Norway

Sant Josep Caves in Spain Honda de Güímar Cave in the Canary Islands Vale Telheiro Cave in Southern Portugal

Cerâmica Cave in Central Portugal Planinska Cave in Slovenia Viento Cave in the Canary Islands

Spectral Density

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2.5 Discussion

Temperature in caves was always more stable than at the surface. We found this across climate zones, lithologies, altitudes and latitudes and cave morphologies. The annual average temperature in caves is highly correlated to the average surface temperature. This agrees with other comparative studies that measured, simultaneously, temperature in caves and at their surface in tropical caves (Mejía-Ortíz et al., 2020), in cave air temperature in Slovenia (Pipan et al., 2019), and in cave-floor temperature in the Czech Republic (Zacharda et al., 2007). This implies that temperature variations – such as those imposed by climate change – at surface will be reflected underground. Thermal stability in caves exercises strong selective pressure in all life in the underground (Mammola et al. 2019a), and the temperature increase due to climate change is known to threaten the ecological sustainability of subterranean ecosystems (Castaño-Sánchez et al., 2020a; Di Lorenzo & Galassi, 2017).

Cave temperatures are known to react “to long term temperature drifts with some delay” (Badino, 2018), due to the inertia of the rock and fluids infiltration. Therefore, cave temperature is directly affected by external atmosphere temperature, and the surface heat transmission through the Earth’s upper crust mainly occurs through conductivity (Badino, 2004; Domínguez-Villar et al., 2015; Mammola et al., 2019b). The signal delay has putatively been related to the depth of the cave zone (Mammola et al., 2019b), i.e., in deep zones of caves forced ventilation seems to be the primary influence on temperature (Domínguez-Villar et al., 2013).

Measuring temperature at soil level in the deep zone of each cave, we found three main correlation patterns in the annual temperature variation of caves compared to the surface: 1) caves with a high positive correlation to surface temperature, with identical thermal signal (0-1 month difference) to the surface but with a smaller amplitude (Balcões, Jazinka, Lazareva, Talofofo), 2) caves with low correlation to surface temperature, and a slight thermal delay of the signal from the surface (1-4 months) (Vampirjeva, Setergrotta, Sant Josep, Honda de Güímar), and 3) caves with high negative correlation to surface temperature, exhibiting an extreme delay from the surface (5-6 months) (Vale Telheiro, Cerâmica, Planinska, Viento). This points out that cave thermal regimes are influenced by the surface but also by the individual traits of each cave: 1) rock properties, in which igneous rocks have higher thermal conductivity than sedimentary rocks (Badino, 2004; Labus & Labus, 2018; Mammola et al., 2019a), result in a smaller thermal delay in volcanic caves that may explain the patterns found in Talofofo Cave (Guam) and Balcões Cave (Azores), but the opposite was found for Viento Cave (Tenerife), which was the most thermally stable of all caves measured; 2) cave morphology, in which deep parts of large caves tend to be more stable (Smithson, 1991; Sendra & Reboleira, 2012), observed for Planinska and Viento caves, however Vale Telheiro (Portugal) is a very shallow cave (<20m depth) and was also very stable; 3) latitude and altitude, which controls the temperature at surface and consequently in depth (White et al., 2019); and 4) air and water circulation are also significant contributors to regulating cave temperatures (Badino, 2010, 2018; Středa et al., 2012).

Caves are semi-closed complex systems and therefore must be understood as dynamic environments in which the interaction of previous factors plays a role in controlling the caves’ temperature. Air circulation is particularly relevant for caves with large and multiple entrances, when air renewal can rapidly occur as in Guam, Azores and Honda de Güímar Cave in Tenerife. Surface air during winter is denser than during summer, resulting in cooler air entering the cave towards its lower points. The cold air pushes the warm air deeper into the most stable parts of the cave, forming an atmospheric “cul-de- sac”, where air renewal is more limited (Covington & Perne, 2016), which might explain the pattern observed for caves with negative correlation to surface temperature (Audra & Nobécourt, 2012). These

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include some of the most biodiverse caves in the world (Oromí & Socorro, 2021; A. S. Reboleira, 2012;

A. S. Reboleira et al., 2011; Zagmajster et al., 2021).

At higher latitudes and altitudes, seasonal ice-layers formed during the cold season, act as a thermal buffer, resulting in a lower thermal amplitude inside caves, higher than the temperatures expected during winter, due to the lack of percolating water during that season (Badino, 2018), as happens in Setergrotta cave in Northern Norway where we see a plateau during winter and start of spring.

Other factors may be influencing the cave’s temperatures such as surface vegetation, by providing shadow (Mammola et al., 2019a), humidity (Forbes, 1998), and potentially the geothermal gradient where temperature increases with depth (Audra & Nobécourt, 2012; Pérez-López et al., 2017) although in karst regions this effect seems to be buffered by the advection of groundwater (Badino, 2010).

Despite the general thermal stability in caves compared to the surface, caves with temperatures highly correlated to the surface expressed daily thermal cycles. We expected this cycle to occur in the caves most influenced by their respective surface, such as the caves with multiple entrances, where airflow plays a significant role in controlling the cave microclimate. This pattern had been observed in tropical caves, but related to non-deepest parts of caves (Mejía-Ortíz et al., 2020). We found daily cycles in deep zones of the caves located in different climatic areas, pointing out that the daily temperature cycles in deep parts of caves may be more frequent than previously thought.

Circadian rhythms are intimately related to environmental cues such as light, and regulate different processes in the organisms (Yerushalmi & Green, 2009). It was previously believed that caves had no daily variations that could exert control over the organisms (Yerushalmi & Green, 2009). However, the observed daily thermal cycles may play an important role to mark the circadian rhythms in cave-adapted organisms. Cave-adapted biodiversity is controlled by ecological, climatological, temporal and geological conditions (Sendra & Reboleira, 2014), but interestingly, we can observe that some of the most biodiverse caves (Planinska, Vale Telheiro and Cerâmica) lack daily cycles, emphasizing the importance of thermal stability for below-ground high richness.

Thermal stratification may also occur in caves (Edenborn et al., 2012; Forbes, 1998; Sendra & Reboleira, 2012; Smithson, 1991; Středa et al., 2012). We studied the variation at soil level in the deepest parts of different caves located in many parts of the world. The variation in temperature across the cave zones and between the floor and the roof of a gallery may have a large impact on speleogenesis, on the formation and maintenance of ice inside the caves and on the creation of distinct ecological niches (Badino, 2018).

Apart from global climate change, other human activities are also known to increase the temperature in subterranean ecosystems, such as the proximity to cities (Becher et al., 2022).

While at surface species have the ability to disperse to other altitudes and latitudes, isolated in caves and with no survival capacity at surface, cave-adapted communities are fatally conducted to perish with no dispersal possibility (Culver & Pipan, 2019). Below the ground, we have 95% of the global resources of freshwater available for direct human consumption and the largest water reservoir for plants and agriculture (Becher et al., 2022; Mammola et al., 2019b). Considering this, it is extremely relevant to comprehend the factors affecting cave temperatures and how they may affect cave species and ecosystem services.

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These findings are particularly relevant for studying the impact of climate change in subterranean ecosystems and niche partitioning, but also for speleothems genesis (Hellstrom et al., 2020), and paleoclimatic reconstructions (Li et al., 2021), with implications at our capacity to interpret historical data from cave records. Further studies are needed to disentangle the role of the different drivers influencing cave microclimates.

2.6 Future perspectives

The International Panel on Climate Change synthesis report from 2014 confirms that climate change has and will continue to impact ecosystems and geographical species’ distribution at surface, which will be reflected underground, by mean annual surface temperatures increase but also by the disruption of precipitation levels and extreme climatic events (IPCC, 2014). We observed that the average temperature of deep zones of caves reflects the average surface temperature for each cave, therefore, we expect the rise of temperature at the surface to be reflected underground. Moreover, in caves where temperature is highly dependent on the surface, such as Balcões in Azores and Talofofo in Guam, climate extremes may even be detected inside the cave. However, this work also shows the difficulty in explaining the factors controlling the cave’s climate, as this is still a very understudied area and should be further explored to better comprehend the depth to which caves are dependent on the surface and how susceptible they are to anthropogenic threats like climate change.

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3. Chapter II – Lethal upper-limit temperatures in cave-adapted species from Western Europe

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.

Climate change goes underground: temperature in caves and its effects on subterranean organisms

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