# Environmental organic carbon availability determines metabolic and consumption rates in freshwater isopods

Florian Mermillod-Blondin, Christophe J Douady, Clémentine M François, Frédéric Hervant, Laurent Simon

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## To cite this version:

Florian Mermillod-Blondin, Christophe J Douady, Clémentine M François, Frédéric Hervant, Laurent Simon. Environmental organic carbon availability determines metabolic and consumption rates in freshwater isopods. Oikos, In press, 10.1002/oik.11149. hal-05095703

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Distributed under a Creative Commons Attribution 4.0 International License
Environmental organic carbon availability determines metabolic and consumption rates in freshwater isopods

Florian Mermillod-Blondin<fnref n="1" />,*, Christophe J. Douady<fnref n="1" />,<fnref n="2" />, Clémentine M. François<fnref n="1" />, Frédéric Hervant<fnref n="1" /> and Laurent Simon<fnref n="1" />

Acknowledgements – We are grateful to Margot Jacquy and Martin Lawniczak for their invaluable help during the monitoring of the experiment.

Funding – Funding was provided by the Agence Nationale de la Recherche (ANR-22-CE02-0019 DIET) and the Centre National de la Recherche Scientifique (EC2CO “CoCoNutS” project). This work was performed in the framework of the ZABR – Rhone Basin LTSER, and of the EUR H₂O’Lyon (ANR-17-EURE-0018) of Université de Lyon (UdL), within the program "Investissements d'Avenir" operated by the French National Research Agency (ANR).

Conflict of interest – None of the authors have any conflict of interest.

Author contributions – **Florian Mermillod-Blondin**: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Investigation (equal); Methodology (equal); Visualization (equal); Writing – original draft; Writing – review and editing (equal). **Christophe J. Douady**: Conceptualization (equal); Investigation (equal); Methodology (equal); Writing – review and editing (equal). **Clémentine M. François**: Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Writing – review and editing (equal). **Frédéric Hervant**: Investigation (equal); Writing – review and editing (equal). **Laurent Simon**: Conceptualization (equal); Funding acquisition (equal); Investigation (equal); Writing – review and editing (equal).

Data availability statement – Data are available from the Zenodo Repository: 10.5281/zenodo.15066238

Supporting information – The Supporting Information associated with this article is available at the end of the document.

<footnote n="1">¹ Université Claude Bernard Lyon 1, LEHNA UMR 5023, CNRS, ENTPE, F-69622, Villeurbanne, France</footnote>

<footnote n="2">² Institut Universitaire de France, Paris, France</footnote>

<footnote>* corresponding author</footnote>

<footnote>email: [florian.mermillod-blondin@univ-lyon1.fr](mailto:florian.mermillod-blondin@univ-lyon1.fr)</footnote>
Abstract

In forested freshwater ecosystems, benthic detritivorous invertebrates have been recognized as important agents of organic carbon (OC) cycling. In comparison with these detrital-rich ecosystems, OC processing by invertebrates in heterotrophic groundwater ecosystems characterized by low OC availability has been poorly documented. The availability of OC should significantly influence the basal metabolism and the feeding rates of aquatic invertebrates, thus affecting how quickly these organisms are able to process OC sources. Therefore, obligate-groundwater species should process OC sources at lower rates than surface-dwelling species to satisfy their energy requirements. Despite their implications for groundwater ecosystem functioning, conclusive tests of these predictions are lacking. We tested these two predictions by evaluating how the environmental availability of three main OC sources (coarse and fine particulate organic matter, and sedimentary biofilm) influences the basal metabolic and OC consumption rates by four species of freshwater asellid isopods. The four isopod taxa used consist of two surface-dwelling and two obligate-groundwater species inhabiting environments characterized by profoundly asymmetric OC availabilities. As expected, obligate-groundwater asellids exhibited reduced basal metabolic rates in comparison with surface-dwelling asellids. This result was associated with a positive correlation between basal metabolic rates of isopods and the availability of OC in their environments. The processing rate of each OC source was significantly and positively correlated with the basal metabolic rate of isopod species. Finally, obligate-groundwater asellids exhibited reduced metabolic and associated OC processing rates to cope with resource scarcity in groundwater ecosystems. Nevertheless, when OC consumption rates were expressed relative to the quantity of OC in the environment of each species, obligate-groundwater asellids processed at least the same percentages of OC available in their environments as surface-dwelling asellids. This finding suggests that obligate-groundwater isopods could contribute significantly to carbon cycling in their ecosystems.
Keywords: metabolic theory of ecology, obligate-groundwater isopods, particulate organic matter, resource availability, sedimentary biofilm, surface-dwelling isopods
Introduction

Many ecosystems exchange nutrients and organic matter to adjacent ecosystems (e.g., Pace et al. 2004, Marczak et al. 2007, Marcarelli et al. 2011, Wallace et al. 2015), often influencing community and ecosystem processes in recipient habitats (Polis et al. 1997, 2004). Studies demonstrating the significance of these fluxes and subsidies include terrestrial inputs to lakes (e.g., Pace et al. 2004, Carpenter et al. 2005, Bartels et al. 2012) and streams (e.g., Wallace et al. 1997), subsidies from lake to terrestrial systems (Hoekman et al. 2011), surface inputs to caves (Schneider et al. 2011) and cave streams (Venarsky et al. 2012). In forested freshwater ecosystems, the inputs of terrestrial leaves have long been recognized as a major food resource for lentic- and lotic-dwelling invertebrate animals, supporting aquatic food webs (e.g., Webster & Meyer 1997, Hall et al. 2000). Due to the dependence of these ecosystems to terrestrial inputs, linkages between allochthonous inputs, detrital processing, and detritivores have been well documented (e.g., Fisher and Likens 1973, Cummins 1974, Anderson and Sedell 1979, Vannote et al. 1980, Wallace and Webster 1996, Hieber and Gessner 2002, Kominoski et al. 2011). These linkages have been used to evaluate the contribution of benthic invertebrates to carbon cycling in streams and rivers. For example, several studies (e.g., Dangles et al. 2004, Piscart et al. 2011) demonstrated that the processing rate of leaf litter (coarse particulate organic matter) in streams was positively linked with the density of the aquatic invertebrates acting as shredders. However, the role of invertebrates on organic carbon (OC) processing has been predominantly studied in freshwater ecosystems receiving large amount of leaf litter while few studies tackled this role of invertebrates in the extreme oligotrophic end of the detritus-supply spectrum that can be found in groundwater ecosystems (Venarsky et al. 2014). Indeed, invertebrates living in groundwater ecosystems have received little interest despite the crucial services played by these ecosystems and their biota for human populations (Boulton et al. 2008, Griebler et al. 2014, Hose et al. 2022, Mermillod-Blondin et al. 2023).

As most groundwater ecosystems are OC-limited due to the absence of photosynthesis and thus rely
on surface ecosystems for carbon supply (Malard & Hervant 1999, Hüppop 2005, Hose et al. 2022),
the availability and diversity of OC sources have been recognized as major determinants of the trophic strategies of invertebrates living in these ecosystems (Simon et al. 2003, Venarsky et al. 2014, 2018, Saccò et al. 2019a, 2021, François et al. 2020). It suggests that the availability of OC sources in the groundwater environment could shape the ability of a species to process these OC sources. For example, Navel et al. (2011) showed that the subterranean amphipod *Niphargus rhenorhodanensis* living in the hyporheic zone of streams fed preferentially on fine particulate organic matter (FPOM) rather than on coarse particulate organic matter (CPOM) under laboratory conditions, suggesting a better ability of *N. rhenorhodanensis* to feed on FPOM which is the dominant source in the hyporheic habitat inhabited by the tested population of *N. rhenorhodanensis*. However, this conclusion may not apply to OC-limited groundwater environments because the hyporheic zone often receives much larger amounts of organic matter from the surface (Cornut et al. 2012, Pacioglu et al. 2021). Thus, there is a gap of knowledge on OC processing in groundwater ecosystems having low exchanges with the surface (e.g., aquifers characterized by a low permeability of rocks and sediments, Robertson et al. 2023) while these ecosystems constitute a major source of drinking water worldwide (Griebler & Avramov 2015). In these systems, the scarcity of OC is expected to significantly influence the processing rates of carbon sources by obligate-groundwater invertebrates because energy availability (e.g. quantity of food sources and nutrients) should have a key influence on the basal metabolic rate (e.g., Hervant et al., 1999, 2001, Di Lorenzo et al. 2023) and the feeding activities (e.g., Foulquier et al., 2010) of aquatic invertebrates.

The energy availability constitutes a key factor in the evolution of physiological and life history strategies of organisms. The basal metabolic rates of fauna living in groundwater environments are typically low, suggesting that reduced metabolic rate can be a selective trait to survive chronically low and/or discontinuous food supplies (Hervant 2012, Bishop et al. 2014, Mezek et al., 2010; Wilhelm et al., 2006, Di Lorenzo et al. 2015, Di Lorenzo et al. 2023). Although most comparisons between surface and groundwater invertebrates were not designed to disentangle the effect of environmental selective pressures from random changes linked to neutral drift (e.g., Hervant et al.
1997, Spicer 1998, Mezek et al. 2010, Di Lorenzo et al. 2015), a recent study including a comparative phylogenetic approach (Lefebure et al. 2017) demonstrated that the metabolic rates (determined by the RNA/protein ratios) of groundwater species of crustacean isopods (asellids) were significantly lower than those of their surface-related counterparts. According to the Metabolic Theory of Ecology (MTE), the basal metabolic rate would control ecological processes, such as the rate of resource uptake, from the individual to the ecosystem scale (Brown et al. 2004). Following this theoretical framework, groundwater species having a reduced metabolism would process a lower quantity of OC than surface species to satisfy their energy requirements. Nevertheless, the predictions concerning the positive influences of the environmental availability of carbon sources on the metabolism and the processing rates of carbon sources by invertebrates has never been tested (as stated by Pacioglu et al. 2021).

To fill this gap, we developed an experimental approach in the laboratory to measure separately the processing rate of the three main OC sources found in freshwater environments (CPOM, FPOM and sedimentary biofilm) by four species of freshwater asellids. These four species constitute two phylogenetically independent pairs (sensus Felsenstein, 1988) of surface-dwelling and obligate-groundwater species as replicates of the ecological transition from energy-rich surface water to energy-poor groundwater environments (Lefebure et al., 2017). In contrast with most previous trophic experiments performed on groundwater species which focused on a single carbon source (Foulquier et al. 2010, Navel et al. 2011, Cooney & Simon 2009, Kinsey et al. 2007), we considered the three dominant carbon sources contributing to the diet of surface-dwelling and groundwater asellids (François et al. 2016, 2020). We studied species collected in ecosystems characterized by contrasting quantities of carbon sources with deep cave systems (among the most energy-limited environments on Earth; Poulson & Lavoie, 2000; Venarsky et al. 2014) and highly productive surface streams. The availability of the three carbon sources tested in laboratory were quantified in the field to evaluate the potential link between this environmental resource availability and the metabolism of each species (estimated through respiration rate) and the consequences on the processing rates of each OC source
measured in the laboratory.

# Materials and Methods

## Biological material

The present study focused on four freshwater asellid isopods, the two obligate-groundwater species *Proasellus valdensis* (Chappuis, 1948) and *Proasellus cantabricus* (Henry & Magniez, 1968) and the two surface species *Proasellus meridianus* (Racovitza, 1919) and *Proasellus banyulensis* (Racovitza, 1919). Based on the Aselloidea timetree, these four species were selected as they constitute two independent pairs of surface and subterranean asellid species as independent replicates of the ecological transition from energy-rich surface water to energy-poor groundwater environments (Saclier et al. 2023). Independence among the two pairs was ensured as the tree paths from one species to the other within the first pair did not contain any branches in common with the other pair (Felsenstein, 1988). *P. cantabricus* and *P. meridianus* are members of the well supported ibero-aquitanian clade, while *P. valdensis* and *P. banyulensis* are member of a putative alpine-anophtalmus-coxalis clade (Saclier et al., 2023). Noteworthy, whatever the support for the alpine-anophtalmus-coxalis clade, both comparisons will remain phylogenetically independent *sensu* Felsenstein (1988). Subterranean species were collected in caves from Cantabrian and Jura mountains for *P. valdensis* and *P. cantabricus* respectively (Table 1). Surface species were sampled from a stream near Geneva and a dead arm of the Rhône River near Lyon for *P. banyulensis* and *P. meridianus*, respectively (Table 1). For each species, around 100 individuals were carefully collected from stones using a paintbrush and brought back to the laboratory in aerated and refrigerated plastic containers filled with water from the sites. *Proasellus* species can feed on a wide variety of food sources including detritus, microbial biofilms, algae and plant roots (Basset & Rossi 1987, Zimmer et al. 2005, Mondy et al. 2014, François et al. 2020). However, based on stable isotope analyses of the diets of 14 *Proasellus* species collected from cave and surface environments, three food sources were predominant in
*Proasellus* diets (François et al. 2016, 2020): coarse particulate organic matter (CPOM; 1 mm < particle size < 6 mm), fine particulate organic matter (FPOM; particle size < 1mm), and microbial biofilm associated to the sediment, to which we referred as sedimentary biofilm. These three resources were thus selected to estimate OC resources availability in the field and to quantify the processing of organic matter by asellid species in the laboratory.

Availability of OC sources at sampled sites

The trophic resources available for asellids were sampled in each site following the approach of
Huntsman et al. (2011) and François et al. (2020). The sampling consisted in collecting on a depth of
1 cm all available resources occurring on a standardized area (256 cm²). Three samples were
performed and mixed per site to obtain a composite sample taking into account the spatial variability
of OC sources in each site. Then, samples of trophic resources were flash-frozen with liquid nitrogen
in the field for conservation during the journey back to the laboratory.

At the laboratory, all collected trophic resources were unfrozen and sorted according to the food
categories described above by using sieves of different mesh sizes (200 µm, 1 mm and 6 mm). Food
categories were then frozen, freeze-dried and weighed to obtain a dry mass of each trophic resource
per square meter (g of dry weight [DW]/m²) for each site. All obtained resource samples were then
ground using a ball mill grinder (Retsch MM-200) for homogenization before the determination of
the OC content of each resource, as % of dry mass. For sedimentary biofilm, samples were treated
with 1 N HCl before OC analysis to remove carbonates using the ‘capsule method’, as described in
Brodie et al. (2011). The quantity of each resource (g of DW/m²) and its elemental composition
(%OC) enabled the calculation of the OC content for each resource per site (g of OC sequestered in
a given resource/m²).

Dry mass and N content of asellids to normalize metabolic and processing rates
The mean dry masses of individuals that were used to measure the basal metabolism and the processing rates of OC sources (see below) were 0.18 ± 0.06 mg (mean ± standard deviation) for *P. cantabricus*, 0.38 ± 0.13 mg for *P. meridianus*, 0.41 ± 0.13 mg for *P. valdensis* and 0.36 ± 0.09 mg for *P. banyulensis*. However, we did not use dry masses to normalize metabolic and OC processing rates because differences in dry mass among asellid individuals and among species from different environments can be partly associated with differences in mass of isopod components contributing marginally to oxygen respiration rate like exoskeleton (Vittori et al. 2017) and body reserves (especially lipids and glycogen, Hervant & Renault 2002). Following this point, analyses of the percentages of N per dry mass of individual isopods (see method below) showed marked differences among species with mean percentages of 9.09%, 7.58%, 7.96% and 6.52% in *P. cantabricus*, *P. meridianus*, *P. valdensis* and *P. banyulensis*, respectively (see Supporting Information 1). As the percentage of N in animals is mainly associated with proteins (Moore et al. 2010) which are main constituents of tissue structure and main contributors to organism functions, these contrasting percentages of N among species indicated that the four species did not contain comparable masses of body tissues per individual dry mass. Consequently, we chose to express basal metabolic rates and carbon processing rates per mg of asellid N, to normalize to asellid protein content. For this, nitrogen but also carbon contents of each asellid individual were determined by high-temperature combustion of dried asellid individuals and subsequent measurement of N₂ and CO₂ by thermal conductometry using an elemental analyzer (FlashEA, Thermo Electron Corporation). The carbon content of isopods was measured in addition with nitrogen content to measure the molar ratio between organic carbon and nitrogen (hereafter indicated C:N ratio) for all species. Following the stoichiometry theory (Sterner & Elser 2002, Cross et al. 2005), the mismatch between the C:N ratios of a consumer and its food source is expected to be a main determinant of the processing rate of this food source by a consumer: the higher the mismatch is, the higher the quantity of food is needed for the consumer to fill its nutritional requirements. Thus, mismatches in C:N ratio between asellids and the three studied OC sources was evaluated to determine its potential effect on OC source processing rates.
## Basal metabolism (respiration rate)

To determine the basal metabolic rates of the four isopod species, individual respiration rates of asellids were measured using a Micro-Respiration System (Unisense, Aarhus, Denmark), according to Navel et al. (2010) and Eme et al. (2014). After 3 weeks of acclimation to laboratory conditions, asellids were individually introduced into beakers of 250 mL filled with aerated chemically-controlled water (96 mg L⁻¹ NaHCO₃, 39.4 mg L⁻¹ CaSO₄·2H₂O, 60 mg L⁻¹ MgSO₄·7H₂O and 4 mg L⁻¹ KCl, US EPA) during 48 hours to clear their gut contents and therefore to ensure that an overshoot in O₂ consumption due to digestive metabolism did not affect the results. After this step, each individual was introduced into a micro-respiration chambers (4.5 mL) filled with chemically-controlled water which was continuously stirred to prevent any vertical oxygen gradient in the chambers. Oxygen uptake rates were determined from changes over time in concentration of O₂ measured with micro-sensor inserted into respiration chambers (records every 30 min during 6 h). After measurements, each asellid individual was recovered, dried at 60°C (for 48 h), weighed and its nitrogen and carbon contents were determined (see above). Then, oxygen uptake rates were expressed as µg of O₂ h⁻¹ mg⁻¹ of asellid N content. A total of 10 individuals were used per asellid species.

## Organic matter processing rate

In the laboratory, we performed a 28-day long experiment to measure separately the processing rate of the three food sources (FPOM, CPOM and sedimentary biofilm) by the four species. Sedimentary biofilm was grown in the laboratory using cave sediments (100µm < particle size < 1000 µm) incubated in the dark at 10 ± 0.5°C during 1 month in a chemically-controlled water (96 mg L⁻¹ NaHCO₃, 39.4 mg L⁻¹ CaSO₄·2H₂O, 60 mg L⁻¹ MgSO₄·7H₂O and 4 mg L⁻¹ KCl) enriched with dissolved nitrogen (10 mg L⁻¹ KNO₃) and organic carbon (25 mg L⁻¹ CH₃CO₂Na). The CPOM and FPOM were derived from alder (*Alnus glutinosa*) leaves. Abscised leaves were collected from alders and dried at 30°C during 1 week in an oven. Thirty dry leaves were ground using a mortar and a
pestle, sieved (< 1 mm) and homogenized to obtain samples of leaf fragments, which were used as FPOM food source. Twelve dry leaves were re-wetted and then cut into 18 mm diameter discs, which were used as CPOM food source. During disc preparation, main leaf veins were removed to avoid biases in processing rates due to heterogeneity in leaf toughness. Afterwards, CPOM and FPOM food sources were incubated in river water at 10°C during 1 month to allow their colonization by hyphomycetes which increases the palatability of POM for shredders (Arsuffi & Suberkropp 1984, Suberkropp & Chauvet 1995). We used the same CPOM and FPOM food sources for surface-dwelling and obligate-groundwater species while differences in organic matter quality between surface and groundwater environments can be expected (Pacioglu et al. 2023). We made this choice for comparison purposes but also because data obtained by François et al. (2020, in Table S2) showed that C:N ratios of CPOM and FPOM did not differ significantly between surface and groundwater environments (Supporting Information 2).

Experiments were performed in 1-L glass beakers (diameter: 10 cm, height: 15 cm) previously filled with chemically-controlled water (96 mg L⁻¹ NaHCO₃, 39.4 mg L⁻¹ CaSO₄·2H₂O, 60 mg L⁻¹ MgSO₄·7H₂O and 4 mg L⁻¹ KCl; pH = 7.5; US EPA 1991). A total of 108 beakers were prepared with 36 beakers per carbon source (CPOM, FPOM and sedimentary biofilm). For CPOM, 1 leaf disc corresponding to a dry mass of 27.9 ± 3.2 mg was introduced per beaker. A mass of 34.4 ± 0.9 mg of FPOM (in dry mass) was introduced per beaker. For sedimentary biofilm, 7.6 g (dry mass) of sediment previously enriched in biofilm were introduced per beaker. For the three sources, carbon quantity per beaker was not limiting for asellids during the experiment. As measured for isopods, the C:N ratios of the three sources were determined at the start of the experiment to evaluate the mismatches in C:N between asellids and their OC sources and their potential consequences on the processing rates of the three sources.

Before starting the experiment, 90 individuals per species were starved for 3 days to clear their gut content. For each species and carbon source, 6 groups of 5 individuals were randomly placed in beakers. Thus, asellids were introduced in 72 beakers (3 carbon sources × 4 species × 6 groups). The
remaining 36 beakers without isopods were used to evaluate the mean quantity of OC introduced in the beakers at the start of the experiment for each carbon source (n=18, 3 carbon sources × 6 replicates) and the OC loss from each carbon source at the end of the experiment without isopods (n=18, 3 carbon sources × 6 replicates). All beakers were covered with aluminum foil to prevent contamination and continuously aerated to maintain concentration of dissolved oxygen between 9.5 and 10.5 mg L⁻¹. The experiment was conducted under constant darkness and water temperature was set at 10°C ± 0.5 (close to the cave temperatures of the two groundwater species, Mermillod-Blondin et al. 2013, Eme et al. 2014) in a thermo-regulated room. These experimental conditions were also suitable for the two surface-dwelling species, which commonly live under rocks and litter, and experience large variations of light and temperature in their environments. The experiment was run for 28 days, with sampling at the start of the experiment (18 beakers used to evaluate the quantity of OC supplied at the start of the experiment) and at the end of the experiment (18 beakers used to evaluate OC loss without isopods and 72 beakers with isopods).

The tested density of isopods (5 individuals per beaker) corresponded to 641 individuals per m² for all species. This density was selected as a mean value because the densities of these species in the field varied from 0 to several thousands of individuals per m² due to their gregarious behavior (Supporting Information 3). For example, Chambers (1977) reported densities in *Proasellus meridianus* (previously named *Asellus meridianus*) varying from 468 to 3768 individuals per m² in reed beds. Žganec et al. (2024) reported densities varying from 0 to 1285 individuals per m² in surface water channels for the species *Proasellus coxalis*. In groundwater environments, Mathieu et al. (1992) reported a comparable variability in density for the species *P. valdensis* in cave sediments: 0 to 30 individuals were collected in sediment buckets having a surface of 78 cm², corresponding to densities varying from 0 to 3846 individuals per m². Published data on *P. cantabricus* are lacking but, as for *P. valdensis* (Supporting Information 3), individuals of this species show a gregarious behavior (personal observation in the field).

During the experiment, survival was monitored daily to remove dead individuals and to calculate the
average number of living asellids processing organic matter during the whole course of the experiment for each beaker with isopods. When more than 1 individual per beaker died, the beaker was removed from the analyses. For all beakers collected at the start and at the end of the experiment, sediment, CPOM and FPOM sources were frozen, freeze-dried, weighted and their OC and N contents were determined by high-temperature combustion of pre-acidified (with HCl 2N to remove inorganic carbon) dry samples and subsequent measurement of CO2 and N2 by thermal conductometry using an elemental analyzer (FlashEA, Thermo Electron Corporation). For each carbon source, differences in OC mass between the start and the end of the experiment were used to evaluate the processing rate of OC with and without isopods. Then, for each carbon source, the average processing rate obtained from beakers without asellids was subtracted from the processing rates measured with asellids to obtain the carbon processing rates due to asellids. As for the measurements of asellid basal metabolism, the dry mass and nitrogen content of asellids collected at the end of the experiment were determined for each beaker to express the processing rate of organic matter in mg of OC per day and per mg of asellid N content.

## Statistical analyses

As data obtained in the field and in the laboratory experiment did not satisfy the assumptions of normality and homoscedasticity, they were ln (x+1) transformed to perform parametric tests (analyses of variance and linear models). Analyses of variance (ANOVAs) were used to test for the influence of species ecological status (i.e., surface-dwelling asellid versus groundwater asellid) on respiration rate and carbon processing rates of CPOM, FPOM and sedimentary biofilm. A nested design with isopod phylogenetic clade and species ecological status nested in clade as main effects was used in order to avoid confounding between-clade variation (linked to shared evolution within each clade) with variation induced by species ecological status (i.e., surface-dwelling *versus* groundwater species). Post hoc Tukey's tests were performed to determine whether significant pairwise differences occurred between species.
Despite the statistical limitation linked to the low number of species studies (n = 4), regression analyses were performed to assess (1) the potential influence of OC availability in the field on basal metabolic rates of asellid species and (2) the potential influences of metabolic rates on processing rates of each OC source by the asellid species. The significance of explanatory variables on basal metabolic rates of asellid species and the processing rates of each OC source by the asellid was tested using type II ANOVAs using the “car” R package.

Statistical analyses (nested ANOVA tests, linear regression models, codes in Supporting Information 4) as well as graphics were performed with R version 4.1.2 (R Core team 2021). Significance for all statistical tests was accepted at α<0.05.

# Results

## Availabilities of carbon sources in the field

The four environments covered a wide range of OC content varying from 0.72 to 315.7 g per m² (Table 1). As expected, groundwater environments presented OC availabilities that were at least 10-fold lower than values reported from surface environments. The differences between surface and groundwater environments were observed for the three carbon sources, although less marked for the sedimentary biofilm. It is also worth noting that the surface environment of *P. meridianus* presented 15-fold higher concentration of OC per m² than the environment of the other surface-dwelling species (*P. banyulensis*, Table 1).

Table 1: Quantity of organic carbon (OC) in g per m² for each of the three available OC sources in the four sampled sites. ‘Total’ corresponds to the total amount of OC (g / m²) available in these trophic resources in each site. Between the brackets is indicated the proportion in percent of OC in the resource relative to the total OC quantity. CPOM = coarse particulate organic matter. FPOM = fine particulate organic matter. X and Y stand for the latitude/longitude (in decimal degrees) of the
sampling site location.

<table><thead><tr><th>Site</th><th>Y</th><th>X</th><th>Species</th><th>Clade</th><th>Habitat</th><th>Sedimentary biofilm in g per m² (% of total)</th><th>CPOM in g per m² (% of total)</th><th>FPOM in g per m² (% of total)</th><th>Total OC in g per m²</th></tr></thead><tbody><tr><td>Geneva (Switzerland)</td><td>46.19</td><td>6.18</td><td>Proasellus banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>Surface stream</td><td>3.76 (18%)</td><td>9.48 (45%)</td><td>7.66 (37%)</td><td>20.89</td></tr><tr><td>Grottes de la Balme (France)</td><td>45.85</td><td>5.34</td><td>Proasellus valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Cave stream</td><td>0.70 (50%)</td><td>0.12 (9%)</td><td>0.57 (41%)</td><td>1.39</td></tr><tr><td>Cubio (Spain)</td><td>43.28</td><td>-3.62</td><td>Proasellus cantabricus</td><td>Ibero-aquitanian</td><td>Cave stream</td><td>0.70 (23%)</td><td>0.54 (74%)</td><td>0.02 (3%)</td><td>0.73</td></tr><tr><td>Balan (France)</td><td>45.82</td><td>5.09</td><td>Proasellus meridianus</td><td>Ibero-aquitanian</td><td>Surface stream</td><td>0.80 (&lt; 1%)</td><td>239.5 (76%)</td><td>75.44 (24%)</td><td>315.7</td></tr></tbody></table>

## Stoichiometric ratios (molar C:N ratios)

Measurements performed on aquatic isopods indicated that their mean C:N ratios were comparable with 4.71, 4.35, 4.18 and 4.13 for *P. meridianus*, *P. valdensis*, *P. banyulensis* and *P. cantabricus*, respectively (data in Supporting Information 1). The mean C:N ratios of the sedimentary biofilm was close to the C:N ratio of isopods with a value of 4.3 ± 0.5 (mean ± standard deviation). The mean C:N ratios of CPOM (14.9 ± 1.9) and FPOM (12.4 ± 1.2) were comparable but they were markedly higher than those measured on sedimentary biofilm and isopods.

## Respiration rates of the four asellid species

The consumptions of O₂ per hour and mg of asellid N content were significantly different among surface and groundwater species (nested ANOVA, F₁,₃₇=21.3, p < 0.0001, Figure 1a, Supporting Information 1) while no significant difference between clades was detected (nested ANOVA, F₁,₃₆=3.58, p > 0.05). The two groundwater species (*P. valdensis* and *P. cantabricus*) had comparable basal metabolic rates (Figure 1a). In comparison, the mean respiration rates of the two surface-dwelling species were significantly different (Figure 1a). The mean respiration rate of *P. banyulensis* was 20% higher than those of its relative in groundwater (*P. valdensis*) while *P. meridianus* had the
highest respiration rate with a mean value more than 2-fold higher than those of its groundwater relative (*P. cantabricus*). Despite a low number of points (n= 4 species), linear regression model showed that the availability of OC in the environment was a significant predictor of the basal metabolic rate of asellids (R²=0.985, p < 0.05). More precisely, the basal respiration rate of the asellid species was significantly and positively correlated with the total availability of OC in the environment (Supporting Information 5).

<figure><img src="image_3.png" /><figcaption>Figure 1: Oxygen uptake rate of the four asellid species (n=10 per species). Different letters display significant differences in respiration rate among species. *Pc* = *Proasellus cantabricus*, *Pm* = *Proasellus meridianus*, *Pv* = *Proasellus valdensis* and *Pb* = *Proasellus banyulensis*.</figcaption></figure>

## Processing rates of the three OC sources by the four asellid species

The processing rates of CPOM and FPOM by the asellids presented the same trend among species (Figures 1bc, Supporting Information 6) with higher rates for surface-dwelling than groundwater species (Figure 2ab, nested ANOVAs, F₁,₁₄=9.2 and p < 0.01 for CPOM, F₁,₁₄=8.0 and p < 0.05 for FPOM). Despite significant differences in processing rates of CPOM between clades (nested
ANOVAs, F₁,₁₄=6.5 and p < 0.05), the two groundwater species (*P. valdensis* and *P. cantabricus*) exhibited comparable CPOM decomposition rates (Figure 2ab). The clade effect was due to the high processing rates performed by *P. meridianus* which were at least 5-fold higher than those measured with the three other asellid species. The contributions of the surface species *P. banyulensis* to decomposition rates of CPOM and FPOM were higher than the contributions of its relative groundwater species (*P. valdensis*, Figures 2ab). The processing rates of sedimentary biofilm by asellid species were also significantly higher for surface-dwelling than for groundwater species (nested ANOVAs, F₁,₁₄=9.5 and p < 0.01) but no significance of clade affiliation was measured on this processing rate (nested ANOVAs, F₁,₁₄=4.2 and p > 0.05). Nevertheless, it is worth noting that differences in processing rates of sedimentary biofilm were not significant between surface-dwelling and groundwater species within each pair (Figure 2c). The results also showed that the processing rates of OC by each species varied strongly between sedimentary biofilm source and POM (CPOM and FPOM) sources, with lower processing rate for sedimentary biofilm (Figure 2). For example, *P. meridianus* processed less than 1 mg of OC per day and mg of asellid N content when feeding on sedimentary biofilm, while it processed more than 2 mg of OC per day and mg of asellid N content (figure 2c) when feeding on POM sources (Figure 2a and 2b).

Statistical analyses showed that the basal metabolic rate (respiration rate) was a significant predictor of the processing rates of CPOM and FPOM by asellids despite the low number of points (n=4 species, Supporting Information 5). More precisely, the processing rates of CPOM and FPOM by asellid species was significantly and positively correlated with the basal metabolic rates of the isopods (Supporting Information 5, linear regression models, R² =0.861 and p < 0.05 for CPOM, R² =0.925 and p < 0.05 for FPOM). In contrast with CPOM and FPOM processing rates, basal metabolic rate was not detected as a significant predictor of the processing rate of sedimentary biofilm by asellids (Table 3c). This lack of significance was due to the limited number of studied species (n=4) as we observed a positive trend between the basal metabolic rate of species and their processing rate of sedimentary biofilm (Supporting Information 5, linear regression model, R² =0.745 and p = 0.089).
<figure><img src="image_4.png" /><figcaption>Figure 2: Processing rates of (a) CPOM, (b) FPOM and (c) sedimentary biofilm by the four asellid species (n=3-5 replicates per species). Different letters display significant differences in respiration rate among species. Pc = *Proasellus cantabricus*, Pm = *Proasellus meridianus*, Pv = *Proasellus valdensis* and Pb = *Proasellus banyulensis*. Note the large differences in Y-axis among carbon sources.</figcaption></figure>

Discussion

Using two pairs of surface-groundwater species associated with two independent events of
groundwater colonization by asellids, we confirmed that obligate-groundwater species had depressed
metabolisms in comparison with surface-dwelling species. By estimating growth rate in 16 species
of asellid isopods using the RNA/protein ratio, Lefebure et al. (2017) also showed that subterranean
asellid species display lower growth rates than surface asellid species. These results are in line with
the commonly accepted idea that groundwater species can present specific energy saving strategies
(behavioral and metabolic strategies, Hervant et al. 1997, Hervant 2012) to cope with the low food
availability encountered in groundwater ecosystems. However, the link between basal metabolism of
groundwater invertebrates and the availability of OC sources in the field has never been demonstrated
before. Thus, the present study goes beyond previous works by highlighting a positive link between
the respiration rates of asellids and the availability of OC in the environment. This positive
relationship supports the energy-limitation hypothesis stating that species evolving in groundwater
ecosystems placed in the extreme oligotrophic end of the detritus-supply spectrum display reduced
metabolic and growth rates (Spicer, 1998; Hervant et al., 2001; Hervant and Renault, 2002; Venarsky
et al. 2014, 2018). In agreement with the metabolic theory of ecology (MTE, Brown et al. 2004),
groundwater asellids with depressed metabolisms processed OC sources at lower rates than surface
asellids. More precisely, MTE predicts how metabolic rate, by setting the rates of resource uptake
from the environment and resource allocation to survival, growth, and reproduction, controls
ecological processes at all levels of organization from the individuals to the biosphere (Brown et al.
2004). Nevertheless, MTE also constitutes a synthetic framework that characterizes the effects of
body size and temperature on the metabolism of individual organisms (Brown et al. 2004). This facet
of the MTE was not supported by the present study because differences in basal metabolism among
isopod species were not driven by temperature (metabolic analyses were done at the same temperature
for the four species) nor by differences in body size. For this last point, the lower basal metabolisms
of obligate-groundwater species compared with surface-dwelling species were not associated with
strong differences in individual dry masses (as a proxy of size) between groundwater (mean individual
dry mass of groundwater species: 0.335 mg) and surface-dwelling (mean individual dry mass of
surface-dwelling species: 0.385 mg) isopod species. As observed at the population level in the subterranean isopod *Proasellus lusitanicus* (Di Lorenzo & Reboleira 2022), the reduced basal metabolic rate measured in groundwater isopods is more likely due to a metabolic optimization in groundwater habitats, which are often limited by the availability of OC and nutrients. Therefore, we concur with several authors (Allen & Gillooly 2009, Brown & Sibly 2012, Vanni & McIntyre 2016, Pacioglu et al. 2021) who aim to combine MTE and the availability of nutrients through the ecological stoichiometry theory into a synthetic metabolic theory. As already done in previous studies (Schneider et al. 2010, François et al. 2016, 2020, Pacioglu et al. 2023), integrating the concepts of ecological stoichiometry in subterranean environments is a pertinent way to include the availability of nutrients (C, N and P) as a main determinant of the metabolism of animals and their contribution to organic matter recycling. In this direction, observed differences in OC processing rates between the sedimentary biofilm and the POM sources (CPOM and FPOM) could have been linked to differences in C:N ratios among C sources leading to contrasting feeding constraints for aquatic isopods. More precisely, the C:N ratios of asellid species (C:N ratios comprised between 4.1 and 4.7) were close to the C:N ratio of sedimentary biofilm (C:N ratio ~ 4.3), while they were markedly lower than the FPOM and CPOM ratios (C:N ratios ~ 12.4 and 14.9 for FPOM and CPOM, respectively). Under these conditions, aquatic isopods had to process more CPOM and FPOM than sedimentary biofilm to complete their requirements in N, explaining the higher OC processing rates for CPOM and FPOM than for sedimentary biofilm. Although these results were obtained on a limited number of species (n=4), they support the ecological stoichiometry theory stating that organic matter recycling by animals depends on stoichiometric mismatches between the consumers and their foods (Elser & Urabe 1999). Finally, the present study clearly validated the hypothesis that OC availability (energy-limitation hypothesis, Venarsky et al. 2018) in the environment was a main driver of the metabolic and feeding rates of freshwater isopods and it pledges for a full integration of ecological stoichiometry concepts to understand groundwater ecosystem functioning.

Based on the present study, one would argue that the contribution of obligate-groundwater isopods to
carbon cycling is extremely low compared to surface-dwelling isopods in an ecosystem functioning perspective. Similarly, Foulquier et al. (2010) showed in a ¹³C tracing experiment that the obligate-groundwater amphipod *Niphargus rhenorhodanensis* did not impact the sedimentary microbial biofilms because of its low grazing activity. However, this experiment was performed under organic matter-rich conditions (initial conditions with 25 mg/L of ¹³C-acetate in Foulquier et al. 2010) favoring a rapid renewal of the grazed biofilm while groundwater ecosystems often exhibited low organic matter availability (dissolved OC concentrations often lower than 2 mg/L, Chapelle 2022). This raises the question about the contribution of obligate-groundwater invertebrates to C cycle under realistic field conditions. The present study allowed us to make this evaluation because it is possible to report the C processing rates induced by isopods measured in the laboratory to the availability of OC in the environment for each asellid species. Assuming a comparable biomass of 1 mg of asellid N content per m² (corresponding to a carbon biomass of 4-5 mg of asellid per m²) for the four species in the field, mean processing rates of OC per m², day and mg of asellid N content were estimated as the average of processing rates obtained for the three carbon sources during the laboratory experiment for each species: 0.12 (mg of C processed per m², day and mg of asellid N content) for *P. cantabricus* (groundwater species), 3.52 for *P. meridianus* (surface species), 0.05 for *P. valdensis* (groundwater species) and 0.50 for *P. banyulensis* (surface species). Reported to the total quantity of OC in mg per m² in the field (Table 1), we estimated that 16.4%, 1.1%, 3.3% and 2.4% of available resources in the field would be processed per day by *P. cantabricus*, *P. meridianus*, *P. valdensis* and *P. banyulensis*, respectively. We are aware that these estimations of the contributions of asellids to OC cycling in their respective environments are rough and need to be improved by considering, for example, the isopod biomass in the field and the OC fluxes rather than the OC stocks in the studied environments. Nevertheless, these estimations suggest that obligate-groundwater asellids living in energy-poor environments could significantly contribute to C cycling although they process the available organic matter sources at low rates (compared with asellids living in high-energy environments). As observed for *Asellus aquaticus* in surface freshwater ecosystems (e.g., Bergfur et al. 2007, Cook and Hoellein
2016), obligate-groundwater asellids could also significantly contribute to C processing in their environment despite biological traits permitting them to cope with low energy availability in cave streams like reduced metabolism, long fasting abilities (Hervant and Renault 2002) and increased assimilation rates for a given carbon source (François et al. 2016).

## Conclusion

The present study suggests that the availability of resources is a main determinant of basal metabolic rates and trophic activity of freshwater isopods. While further studies with other species are needed to determine if they are widespread patterns, reduced basal metabolic rates associated with reduced OC processing rates of all available resources may represent an efficient strategy to cope with resource scarcity in groundwater ecosystems (Gibert and Deharveng 2002). Despite the lower processing rates of OC sources by obligate-groundwater asellids in comparison with surface-dwelling ones, obligate-groundwater isopods could contribute significantly to C cycle in their OC-poor groundwater ecosystems. Following these promising results and the recent advances made in groundwater ecology (Venarsky et al. 2018, Saccò et al. 2019b, 2022, Malard et al. 2023), multidisciplinary field approaches based on carbon and nutrient flux measurements using stable isotopes and assessments of invertebrate diversity and densities are greatly needed to evaluate the significance of the role of invertebrates in groundwater ecosystem functioning.

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# Supporting Information

Supporting Information 1: Dry masses, contents of N, contents of C, C:N ratios and respiration rates measured in the laboratory for the individuals of the 4 species used to estimate basal metabolic rates.

<table><thead><tr><th>Species</th><th>Clade affiliation</th><th>Individual dry mass (mg)</th><th>Individual biomass (µg of N content)</th><th>Individual biomass (µg of C content)</th><th>C:N ratio</th><th>Respiration rate (µg of O<sub>2</sub> consumed per hour and mg of asellid N content)</th></tr></thead><tbody><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.281</td><td>25.4</td><td>104.9</td><td>4.13</td><td>14.1369</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.147</td><td>13.4</td><td>52</td><td>3.88</td><td>10.3418</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.146</td><td>13.2</td><td>53.5</td><td>4.05</td><td>11.0096</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.243</td><td>22.9</td><td>99.6</td><td>4.35</td><td>7.8194</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.107</td><td>10.1</td><td>42.2</td><td>4.18</td><td>6.8543</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.146</td><td>13.6</td><td>61.2</td><td>4.50</td><td>13.5101</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.165</td><td>15.1</td><td>58.9</td><td>3.90</td><td>4.9373</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.194</td><td>17</td><td>64.3</td><td>3.78</td><td>10.7057</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.223</td><td>19.4</td><td>81.5</td><td>4.20</td><td>7.9720</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>0.126</td><td>11.2</td><td>47.26</td><td>4.22</td><td>9.0949</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.611</td><td>46.3</td><td>218.5</td><td>4.72</td><td>19.5914</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.475</td><td>36.7</td><td>165.2</td><td>4.50</td><td>21.5997</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.252</td><td>19.4</td><td>93.3</td><td>4.81</td><td>23.1726</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.407</td><td>31.9</td><td>153.1</td><td>4.80</td><td>24.4531</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.320</td><td>25.7</td><td>119.8</td><td>4.66</td><td>22.1865</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.223</td><td>16.7</td><td>80.3</td><td>4.81</td><td>26.9148</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.349</td><td>25.9</td><td>115.2</td><td>4.45</td><td>25.3619</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.543</td><td>40</td><td>191.2</td><td>4.78</td><td>15.4935</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.301</td><td>22.3</td><td>109.3</td><td>4.90</td><td>16.1143</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>0.340</td><td>24.7</td><td>115.1</td><td>4.66</td><td>27.1240</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.534</td><td>42.5</td><td>184.0</td><td>4.33</td><td>11.6090</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.349</td><td>28.3</td><td>121.7</td><td>4.30</td><td>12.5141</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.291</td><td>23.8</td><td>100.0</td><td>4.20</td><td>11.9777</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.369</td><td>30.8</td><td>136.0</td><td>4.41</td><td>11.7624</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.281</td><td>23.4</td><td>105.3</td><td>4.50</td><td>11.3362</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.223</td><td>17.7</td><td>74.5</td><td>4.21</td><td>10.8692</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.398</td><td>31.4</td><td>137.5</td><td>4.38</td><td>9.1589</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.466</td><td>35.9</td><td>152.6</td><td>4.25</td><td>9.7452</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.563</td><td>42.8</td><td>175.5</td><td>4.10</td><td>9.8964</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.601</td><td>45.5</td><td>218.4</td><td>4.80</td><td>10.5171</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.359</td><td>23.5</td><td>98.7</td><td>4.2</td><td>12.1174</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.330</td><td>21.6</td><td>84.24</td><td>3.9</td><td>15.4151</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.320</td><td>21.7</td><td>93.31</td><td>4.3</td><td>13.3479</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.359</td><td>24.5</td><td>102.165</td><td>4.17</td><td>16.8153</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.446</td><td>31</td><td>122.45</td><td>3.95</td><td>16.8291</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.243</td><td>16</td><td>68.48</td><td>4.28</td><td>13.1528</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.242</td><td>15.6</td><td>68.64</td><td>4.4</td><td>14.2908</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.339</td><td>21.3</td><td>93.294</td><td>4.38</td><td>13.5670</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.524</td><td>32.3</td><td>129.523</td><td>4.01</td><td>17.0892</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>0.398</td><td>24.1</td><td>102.425</td><td>4.25</td><td>13.2217</td></tr></tbody></table>
Supporting Information 2: C:N ratios of FPOM (fine particulate organic matter) and CPOM (coarse particulate organic matter) measured in groundwater (GW) and surface water (SW) environments. These data were obtained from eight habitats of obligate-groundwater isopod species and seven habitats of surface-dwelling isopod species (François et al. 2020 in Table S2).

<figure><img src="image_5.png" /></figure>
Supporting Information 3: (a) Photo highlighting the gregarious behavior observed for the obligate-groundwater species *P. valdensis* in caves and (b) Photos showing individuals and the habitat of the surface-dwelling species *P. meridianus* with estimated densities

<figure><img src="image_6.png" /><figcaption>(a)</figcaption></figure>

*Proasellus valdensis* (Chappuis, 1948). Grotte des Foules (Jura, France).

Source: http://www.aricia.fr/jura-patrimoine/faune_cavernicole.htm

<figure><img src="image_7.png" /><figcaption>(b)</figcaption></figure>

*Proasellus meridianus* (Racovitza, 1919). Etang rompu (Indre, France).

A total of 78 individuals (left panel) was collected by sorting 1.2 L of decaying leaf litter collected in the ditch (foreground of right panel).

Source: C. J. Douady. Density = 750 individuals per m²
Supporting Information 4: R codes used for statistical analyses

Nested ANOVAs to take into account between-clade variation (linked to shared evolution within each clade) with variation induced by species status (i.e., surface-dwelling versus groundwater species).

Code:

model<-aov(Response variable ~ Couple/Status, data=data)

Response variable = Ln(metabolic rate + 1) OR Ln(CPOM processing rate + 1) OR Ln(FPOM processing rate + 1) OR Ln(Sedimentary biofilm processing rate + 1)

Linear models to test the influence of organic carbon (OC) quantity on metabolic rates OR the influence of metabolic rates on processing rates of coarse particulate organic matter (CPOM), fines particulate organic matter (FPOM) or Sedimentary biofilms

Code:

library("car")

model <- lm(Response variable ~ Explanatory Variable, data=data)

Anova(model)

Response variable = Ln(metabolic rate +1) OR Ln(CPOM processing rate + 1) OR Ln(FPOM processing rate + 1) OR Ln(Sedimentary biofilm processing rate + 1)

Explanatory Variable = Ln(OC quantity +1) OR Ln(metabolic rate +1)
Supporting Information 5: Linear regressions between the (a) organic carbon availability (total organic carbon) and respiration rates of asellid species, and between mean respiration rates of each asellid species and the processing rates of (b) coarse particulate organic matter (CPOM), (c) fine particulate organic matter (FPOM) and (d) sedimentary biofilm. Confidence intervals for respiration rates and processing rates are displayed for each species to give information about the estimation of response and predictor variables.

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>
<table><caption>Supporting Information 6: Organic carbon processing rates measured in the laboratory for the 4 species and for the 4 organic carbon sources</caption><thead><tr><th>Species</th><th>Clade affiliation</th><th>Organic carbon source</th><th>Processing rate (mg of OC per day and mg of asellid N content)</th></tr></thead><tbody><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>0.00000</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>0.40546</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>0.17467</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>0.25135</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>8.14496</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>5.02890</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>CPOM</td><td>5.73850</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.17892</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.00000</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.00000</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.00000</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.07594</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>1.10942</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.52042</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>1.09040</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.19082</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>CPOM</td><td>0.55338</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>0.00000</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>0.02677</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>0.05377</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>0.14053</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>1.93928</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>4.81594</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>FPOM</td><td>5.08643</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.31978</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.00000</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.08931</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.00000</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.00000</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.95410</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.43729</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.95410</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.43616</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>FPOM</td><td>0.28270</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.11415</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.06803</td></tr><tr><td>P. cantabricus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.10570</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.15543</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.34211</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.71201</td></tr><tr><td>P. meridianus</td><td>Ibero-aquitanian</td><td>Sedimentary biofilm</td><td>0.10487</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.00525</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.04012</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.00725</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.00264</td></tr><tr><td>P. valdensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.01306</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.05291</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.16628</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.20004</td></tr><tr><td>P. banyulensis</td><td>Alpine-anophtalmus-coxalis</td><td>Sedimentary biofilm</td><td>0.36396</td></tr></tbody></table>