Sara Usandizaga

Carolina Camus

José Luis Kappes

Marie-Laure Guillemin

Alejandro H Buschmann
email: abuschma@ulagos.cl

EFFECT OF TEMPERATURE VARIATION IN AGAROPHYTON CHILENSIS: CONTRASTING THE RESPONSE OF NATURAL AND FARMED POPULATIONS

Keywords: abiotic factor, domestication, management, general-purpose-genotypes, photosynthesis, origin, seaweed, selection ETRmax, maximal electron transport rate, Fv/Fm, maximal quantum yield, Ek, irradiance of saturation of ETR, NPQmax, maximal non-photochemical quenching, PAR, photosynthetically active radiation, PSII, photosystem II, RLC, rapid light curves abiotic factor, domestication, management, general-purpose-genotypes, photosynthesis, origin, seaweed, selection ETRmax, maximal electron transport rate, Fv/Fm, maximal quantum yield, Ek, irradiance of saturation of ETR, NPQmax, maximal non-photochemical quenching, PAR, photosynthetically active radiation, PSII, photosystem II, RLC, rapid light curves

During the domestication process, farmers influence the reproduction and care of organisms to ensure a predictable supply of the resource of interest, causing changes in phenotypic and genotypic character frequencies. In Chile, as a result of unconscious selection and domestication process, farmed populations of the red alga Agarophyton chilensis have most likely undergone a reduction in genetic diversity and a modification in life-history traits compared to wild populations. In order to understand the implications that these processes may have in A. chilensis, in this study we investigated how temperature variations (10 °C, 15 °C and 20 °C) affect growth and photosynthetic responses of natural and farmed populations from three different localities along the Chilean coast. Natural population's growth decreased at low and high temperature levels while all three farmed populations respond in a very similar way to temperature variation. We propose that a possible outcome of farming, in the A. chilensis vegetatively propagated crops, could have been the selection of general-purpose-genotypes able to perform adequately across the range of temperature tested in our experiment. Furthermore, our results showed that photosynthetic activity was also affected by temperature treatments (e.g., different maximum maximal electron transport rate and quantum yield values depending on the population type and temperature).

In a context of climate change, A. chilensis farmed populations may be better able to cope with impacts of anthropogenic activities than natural populations due to the buffer effect of their general-purpose-genotypes, tolerant to a wide range of conditions.

Introduction

Domestication is considered a long and complex process during which domesticators influence the reproduction and care of domesticated species to guarantee predictable supply of resources presenting selected traits of interest for human use (Zeder, 2015). This selection process generates changes in phenotypic and genotypic character frequencies of cultivated populations (Zohary, 1984). Even if domestication of marine species is much more recent than the one of terrestrial animals and plants (Duarte et al. 2007), strong evidence for domestication has been found in a few cultivated seaweeds (Valero et al. 2017). As for terrestrial plants (Meyer et al. 2012), some domesticated seaweeds are characterized by a shift in their reproductive strategy (e.g., changes from sexual reproduction to vegetative propagation) between natural and farmed populations (Valero et al. 2017). This shift in reproductive strategy has been demonstrated for Agarophyton (referred as Gracilaria in Guillemin et al. 2008) and is probably also present in multiply superior genotypes and maintain desired phenotypes through vegetative propagation (Valero et al. 2017).

In Chile, intensive seaweed farming is limited to the domesticated red alga Agarophyton chilensis used mainly for agar extraction (Buschmann et al. 2017). Even if the domestication process of this species has begun only a few decades ago, the almost complete predominance of diploid individuals in farms demonstrate that farming practices had significantly modified life-history traits as compared to wild populations (Guillemin et al. 2008). Moreover, recent investigations have demonstrated that this red alga colonized the Chilean coast from New Zealand, likely at the end of the Last Glacial Maximum (Guillemin et al. 2014). The lower genetic diversity of the Chilean populations, when compared to the ones from New Zealand, is indeed consistent with a genetic bottleneck resulting from a transpacific range extension and was probably reinforced by the overexploitation of natural Chilean populations during the 90´s (Guillemin et al. 2014). In Chile, active transport and exchange of inoculums of A. chilensis between coastal communities of fishermen for cultivation purposes have contributed to the artificial expansion of the species distribution. Nowadays, natural populations are distributed between 30°S to 45°S while farming extends further north, up to Antofagasta, 17°S (Bird et al. 1986, Guillemin et al. 2008). Human activities have also leaded to a loss of genotypic diversity in farmed populations that could be partially linked to involuntary selection of faster growing thalli, during the first steps of the domestication process (Guillemin et al. 2008, Guillemin et al. 2013, Valero et al. 2017).

Reduced genetic diversity can severely affect the ability of populations to resist pests and pathogens and limit the scope for future genetic improvement in domesticated crops (Robinson et al. 2013). Besides, it has been reported that a reduction in genetic diversity could lead to lower growth and resilience in highly stressful and/or variable environment (Simms 2000). However, to date, few studies have focused on the potential ecophysiological differences between natural and farmed populations of A. chilensis. Gallegos-Sánchez et al. (2018), detected a significant and negative effect of low salinity conditions on thalli sampled from both natural and farmed A. chilensis populations but with farmed population's thalli being less affected than the natural ones. Results suggested that farmed populations might be more tolerant to salt stress than wild ones in this species and the authors proposed that this difference between population types could be due to previous selective process carried out by farmers.

Considering the possible consequences of unconscious selection (as defined in Zohary 2004) and domestication on Agarophyton chilensis, we propose that natural populations will be less sensitive to temperature variations than farmed populations. Genotypic diversity has been shown to be higher in natural than farmed A. chilensis Chilean populations and we propose that the farms will show less effective mechanisms of acclimation (e.g., enhancing of photosynthetic performance) than the one observed in natural populations when confronted with temperature considered as high in their natural environment (i.e., water of the southern coast of Chile do not generally reach 20°C; Westermeier et al. 1993). The aim of the present study was to assess the effect of temperature on growth and photosynthetic responses of both natural and farmed stands of A. chilensis and tetrasporophyte thalli sampled from three sites along the Chilean coast were followed during one month in controlled laboratory conditions at 10°C, 15°C and 20°C.

Materials and methods

Study sites and life cycle phase determination of the sampled thalli

A total of 600 individuals were sampled from 3 natural and 3 farmed populations (i.e., 100 individuals in each population) during a spring season. One farmed and one natural population were sampled from three sites: Concepción, Maullín and Ancud (Fig. 1). The distinction between natural and farmed populations was first based on whether Agarophyton chilensis thalli were actively planted or not by farmers. As previously reported for the species (Guillemin et al. 2008), thalli were attached to small rocks and pebbles by a holdfast in the three natural A. chilensis beds while unattached thalli were found embedded in the sandy bottom in the three farmed stands. In each site, natural and farmed populations were separated by 1 km approximately. In natural beds, individuals sampled correspond to distinct holdfasts (i.e., distinct genotypes produced by sexual reproduction and spore settlement). In farms, to avoid sampling fragments of the same asexually propagated genotype, sampled thalli were separated by at least by 2 m. All the collected thalli were transported in isolated boxes to the CEACIMA hatchery (Centro de Investigación de Acuicultura y Ciencias del Mar, Universidad de Los Lagos) located in the Metri Bay (41°36´S, 72°43´W). Once in the hatchery, each thallus was cleaned with fresh water and all epiphytes were removed by hand. Thalli were individually marked with a numbered tag and maintained in 400 L tanks at 12°C with constant aeration, 12L:12D photoperiod, 20 µmol electron m -2 s -1 photon flux and weekly filtered seawater exchange.

Collected thalli were observed under stereoscope microscope (Stemi DV4, Zeiss, Jena, Germany) to determine phase (i.e., diploid tetrasporophytes or haploid gametophytes) of mature individuals. For vegetative individuals, a 3-cm fragment of tissue was excised from each thallus, placed into plastic bags with silica gel for rapid dehydration. The sex markers available for A. chilensis were amplified following Guillemin et al. (2012) and the amplification products were visualized in 1.5 % agarose gel (w/v) after adding 2 μl of GelRed™ (Biotium, Fremont, USA). Results were used to determine sex and phase of vegetative individuals. In order to prevent experimental bias due to ecophysiological variability between life cycle phases (Guillemin et al. 2013), only diploid tetrasporophytes were selected for our experiments.

Experimental design

The experimental design consisted of 90 2-L Erlenmeyer flasks (i.e., 6 populations of origin of thalli x 3 temperature treatments x 5 replicates per population per temperature treatment) arranged in 15 60-L plastic water tanks fitted with temperature control systems. Three temperature treatments were used: 10°C, 15°C and 20°C. These temperatures were chosen since they roughly represent the temperature range encountered in the field by the study species. Temperature conditions varied widely within sites were A. chilensis is found and between regions populated by the species along the Chilean coasts with values between 9 °C and 16 °C recorded in Maullín (Westermeier et al. 1993) and between 10 °C and 20°C in areas further north such as Concepción, Coquimbo and Antofagasta (Santelices and Ugarte 1990). In each 60-L plastic water tanks, one 50W automatic heater (Whale VK-1000, Regent) and one stainless steel thermometer (Hagen, Phelan) were used to maintain constant temperature. For each population under study, five replicates (i.e., five 2-L flasks) were followed per temperature treatment and eight thalli (5 cm length each), selected randomly from distinct tetrasporophytes, were placed in each 2-L flask. Thalli were selected without replacement from a pool of 40 tetrasporophytes, available for each population of origin (see above). Once a week, thalli were transferred to clean 2-L flasks with fresh Provasoli culture media (McLachlan 1973). After one week of acclimation at 12°C, the laboratory experiment was run during 30 days. All Erlenmeyer flasks were under constant conditions of aeration, photoperiod (12 h light: 12 h dark) and photon flux (20 µmol electron m -2 s -1 ).

Growth

Fresh weight of each thallus was assessed weekly on an analytical balance (Sartorius TE 313 DS, Germany) and the specific growth rate (SGR) was calculated as the percentage of wet weight gain per day according to the formula: SGR= [ln (Wf • Wi -1 )/ (tf-ti)] x100;

where Wi= initial fresh weight, Wf= final fresh weight, and t= time (days). Initial fresh weight in the 2-L flasks was of 0.12 ± 0.06 g, some variation in fresh weight exist between each 2-L flask at the beginning of the experiment since calibration of algal material was based on thallus length (see above).

Physiological variables

Thallus pieces were collected at the end of the experiment to measure rapid light curves (RLC). As an indicator of quantum efficiency and photoinhibition, we used, Fv/Fm, which was determined after incubation of 20 min of the thalli in darkness (Schreiber et al. 1995) with a Junior PAM (Walz GmbH, Effeltrich, Germany). The electron transport rate (ETR, µmol electrons • m -2 • s -1 ) was determined after 20 s exposure in 12 increasing intensities of PAR (up to 1500 µmol photon • m -2 • s -1 ) provided by a blue light of the Junior PAM device (Schreiber et al. 1995). ETR was calculated according to Schreiber et al. (1995) as follows:

ETR = ∆F/F´m • E • A • FII (µmol electrons • m -2 • s -1 ) (1)
Where ∆F/F´m is the effective quantum yield, E is the incident PAR (photosynthetically active radiation) irradiance expressed in µmol photons m -2 s -1 , A is the thallus absorptance and FII is the fraction of chlorophyll related to photosystem II (PSII, 400-700m), being 0.15 in red seaweed (Grzymski et al. 1997). As an estimator of photosynthetic efficiency, the initial slope of ETR (αETR) and maximum ETR (ETRmax) were obtained from the tangential function reported by Eilers and Peeters (1988) and the irradiance saturation (EkETR) was calculated from the intercept between these two parameters. Representing potential thalli photoprotective mechanism, non-photochemical quenching (NPQ) was measured according to Schreiber et al. (1995). The maximal non-photochemical quenching (NPQmax) was calculated from the tangential function of NPQ versus irradiance function according to Eilers and Peeters (1988).

Statistical treatment

All analyses were performed in R (3.2.4 version) (Cayuela 2011). Assumptions of homogeneity of variances and normal distribution were tested using Levene`s test and Shapiro-Wilk, respectively. When non-normal residuals and heteroscedasticity were detected, the data were transformed using logarithm (for Ek and SGR) or Box-cox (for Fv/Fm). The experimental design fitted a three-way ANOVA with treatment (temperature:

10°C, 15°C and 20°C), site of origin (Concepción, Maullín and Ancud) and population type (natural or farmed) considered as fixed factors. Statistical differences between groups were analyzed using comparisons of means (Tukey's HSD). Significances were set at p < 0.05.

Results

Significant differences were detected in Agarophyton chilensis specific growth rate (SGR) after 30 days of experimentation between temperature treatments (F(2, 72) = 15.46; P < 0.0001), between population types (F(1, 72) = 4.93; P= 0.03) and a significant interaction was also detected between population type and site of origin (F(2, 72) = 5.00; P= 0.009). In Maullín and Ancud, no significant differences between temperature treatments were detected for farmed thalli (Fig. 2C andE), while farmed thalli from Concepción showed a slightly but significantly lower SGR at 20°C than at 15°C (7.26 ± 1.48 and 9.45 ± 1.92 g • day -1 , respectively; Fig. 2A). In addition, differences among Concepción farmed and natural populations were observed (Tukey test; P<0.05, see Figure 2A andB). In contrast, SGR was significantly higher at 15°C for thalli sampled in natural A. chilensis stands, whatever the site under study (SGR at 15°C: 7.49 ± 2.07 g • day -1 in Concepción, 8.81 ± 2.60 g • day -

1 in Maullín and 8.67 ± 0.84 g • day -1 in Ancud; Fig. 2B, D andF). Finally, the growth rate did not decrease significantly (Tukey test; P<0.05) at the lower (10 o C) and the higher (20 o C) temperature tested for Maullin and Ancud populations (Fig. 2 C, D, E andF).

Regarding the photosynthetic responses, significant effect of temperature treatments were detected for all parameters measured: the optimal quantum yield of fluorescence (Fv/Fm,), the maximal electron transport rate (ETRmax), the saturation irradiance (Ek)and the maximal non-photochemical quenching (NPQmax) (see Table 1). Interactions between temperature treatment and population type were also observed for ETRmax and Ek (Table 1). Values of ETRmax and Ek were significantly higher for thalli sampled in the farmed population of Concepción and grown at 20°C than for thalli sampled in the farmed population of Ancud and grown at 10°C (18.63 ± 12.76 and 1.44 ± 0.37 µmol electrons • m -2 • s -1 , 442.97 ± 306.60 and 31.86 ± 4.78 µmol photons • m -2 • s -1 , for ETRmax and Ek, respectively; Tukey's HSD tests; p < 0.05; Table 2). Significant interactions between temperature treatment, population type and site of origin were observed for Fv/Fm and NPQmax (Table 1). In Maullín at 15°C, values of NPQmax were significantly higher for thalli sampled in the farmed bed than the natural population (Tukey's HSD tests; p < 0.05; Table 2).

Discussion

The present study confirms that natural and farmed Agarophyton chilensis populations respond differentially to temperature variations. Indeed, contrarily to our expectations, our results indicate that farmed populations are less sensitive to temperature variations than natural ones (i.e., thalli growth rate are mostly similar at 10°C, 15°C and 20°C for farms while thalli from natural populations have a higher growth rate at 15°C). In Chile, farmed population has been under unconscious human selection pressure for, at least, three decades and we propose that a possible outcome could have been the selection of general-purposegenotypes (as in Baker 1974) in these vegetatively propagated crops. Our results also showed that photosynthetic activity was affected by temperature treatments (e.g., different maximum maximal electron transport rate and quantum yield values depending on the population type and temperature).

Regardless of the origin of the natural population under study, our experiment shows that thalli specific growth rate (SGR) was higher at 15°C than at lower (i.e., 10°C) or higher (i.e., 20°C) temperatures. Even if SGR tended to be much more homogeneous for farmed thalli at all temperatures, one slight difference was observed for the farmed thalli from Concepción that show significantly less growth at 20°C. Supporting our results, a study realized by Santelices and Ugarte (1990) on A. chilensis (as Gracilaria chilensis) natural populations from Maullín, also revealed better growth rates at 15°C than at 10°C or 20°C.

Most temperate species of the genus Agarophyton has been shown to grow faster in temperature ranging between 15° and 20°C (McLachlan and Bird 1984). A study performed in Gracilaria gracilis (as G. verrucosa) reported a slow growth rate at 10°C and high mortality after a 14 days' heat-wave in Saldanha Bay, South Africa (Engledow and Bolton 1992). A. chilensis, is a temperate-water species with a distribution limited to southern part of the Pacific (Bird et al. 1986, Guillemin et al. 2008) and it is possible that the species present a metabolism with limited temperature tolerance.

Although farming has begun only a few decades ago (i.e., during the 80´s) in Chile, the predominant mechanism for stock propagation by cuttings used in Agarophyton chilensis farms has already significantly decreased their genotypic diversity when compared with wild populations (Guillemin et al. 2008). There is an overall agreement that genetically more variable populations may be associated with higher resilience, increased productivity and population growth rate as compared with less variable populations (Forsman 2014).

However, contrasting with our expectations, farmed thalli in our experiment clearly show a strong ability to grow in contrasting environmental conditions, including the quite "extreme" temperature of 20°C tested. In the same way, Gallegos-Sanchez et al. (2018) concluded that A. chilensis farmed populations may be less sensitive to salt stress and able to grow in a greater range of salinity than natural populations. One possible explanation for these results is that farmed populations of A. chilensis are composed mostly of general-purpose genotypes, able to grow in highly stressful and/or variable environments. Generalpurpose-genotypes are sometime also referred as 'Jack-of-all-trades, master of none' since they are described as versatile genotypes that are able to perform adequately across a range of environments but are not superior in any of them. These general-purpose-genotypes can confer a species or population a broad tolerance to environmental changes and are often associated with species invasion (Baker 1974, Richards et al. 2006). In Chile, farms have been developed using material growing embedded in muddy estuaries and sandy bays.

These habitats are typically highly heterogeneous and present strong seasonal variations in temperature and salinity (Westermeier et al. 1993, Buschmann et al. 1995) In benthic algae, temperature variations affect photosynthetic metabolism (Davison 1991) determining, for example, seasonal distribution (De Nicola 1996). However, habitats characterized by strong spatial and temporal variations of abiotic factors, request constant adjustment of photosynthetic processes in species populating them (Ensminger et al. 2001).

In our study, the highest values of ETRmax and Ek were observed in the A. chilensis farmed population from Concepción grown at 20°C. These results suggest that the effect of high temperatures on photosynthetic metabolism of this farmed population could be mild. excess light energy that may cause photoinhibition due to damage to the PSII apparatus (Moll and Steinback 1986). However, short-term response of photosynthesis to temperature cannot easily be used to infer the long-term response of algal growth (Wienke and Dieck 1989). Indeed, despite the possible signal of photoinhibition detected at 15°C, no limited growth could be observed in these Agarophyton chilensis thalli.

Populations of Agarophyton chilensis from different sites in Chile have been reported to present ecological differences, potentially linked to local adaptation in response to specific abiotic and biotic environmental characteristics (Santelices and Ugarte 1990, Usandizaga et al. 2018). The success of Agarophyton farming depends in part on the origin of the initial inoculum since differences in thallus morphology, agar yield and gel strength and susceptibility to epiphytes exist among regions and populations. Indeed, it have been suggested that random transplantation between distinct habitats could lead to cultivation failure (Santelices and Ugarte 1990). However, in our experiment, even if we included sampling sites located more than 700 km apart, no major effect of the site of origin were detected on growth or photosynthesis. A possible explanation for this discrepancy is that continuous transplantations and exchanges during the last decades have leaded to the homogenization of the genetic diversity among the whole Chilean coast. However, this hypothesis is not in accordance with population genetic studies showing the presence of clear genetic divergence between regions in Chile (Guillemin et al. 2008 and2014). Studies focused on the effect of other stressors (e.g., nutrient supply, salinity, irradiance) and the cumulative effects of various of these stressors on the physiological responses of distinct genotypes is now needed in order to better explore the resistance of A. chilensis populations to stress.

Conclusion

Agarophyton chilensis tolerance of a wide range of abiotic conditions has been proposed as one of the main reasons of the species successful expansion in the Pacific and establishment in a wide array of habitats (Santelices andUgarte 1990, Chow et al. 2001). These successful extension waves have probably also been facilitated by intrinsic characteristics of the species, such as its capacity to shift between sexual and asexual reproduction (Guillemin et al. 2014). We suggest that the possible selection for general-purposegenotypes in the asexually reproducing farmed populations may help modulating the impact of environmental variation on population dynamics (Reed et al. 2010) authors are grateful for the help in the field of R. Altamirano and S. V. Pereda for the revision of this manuscript. We also wish to thank two anonymous referees who greatly improved the manuscript with constructive suggestions. The authors declare no conflict of interest.

Introduction

Domestication is considered a long and complex process during which domesticators influence the reproduction and care of domesticated species to guarantee predictable supply of resources presenting selected traits of interest for human use (Zeder, 2015). This selection process generates changes in phenotypic and genotypic character frequencies of cultivated populations (Zohary, 1984). Even if domestication of marine species is much more recent than the one of terrestrial animals and plants (Duarte et al. 2007), strong evidence for domestication has been found in a few cultivated seaweeds (Valero et al. 2017). As for terrestrial plants (Meyer et al. 2012), some domesticated seaweeds are characterized by a shift in their reproductive strategy (e.g., changes from sexual reproduction to vegetative propagation) between natural and farmed populations (Valero et al. 2017). This shift in reproductive strategy has been demonstrated for Agarophyton (referred as Gracilaria in Guillemin et al. 2008) and is probably also present in multiply superior genotypes and maintain desired phenotypes through vegetative propagation (Valero et al. 2017).

In Chile, intensive seaweed farming is limited to the domesticated red alga Agarophyton chilensis used mainly for agar extraction (Buschmann et al. 2017). Even if the domestication process of this species has begun only a few decades ago, the almost complete predominance of diploid individuals in farms demonstrate that farming practices had significantly modified life-history traits as compared to wild populations (Guillemin et al. 2008). Moreover, recent investigations have demonstrated that this red alga colonized the Chilean coast from New Zealand, likely at the end of the Last Glacial Maximum (Guillemin et al. 2014). The lower genetic diversity of the Chilean populations, when compared to the ones from New Zealand, is indeed consistent with a genetic bottleneck resulting from a transpacific range extension and was probably reinforced by the overexploitation of natural Chilean populations during the 90´s (Guillemin et al. 2014). In Chile, active transport and exchange of inoculums of A. chilensis between coastal communities of fishermen for cultivation purposes have contributed to the artificial expansion of the species distribution. Nowadays, natural populations are distributed between 30°S to 45°S while farming extends further north, up to Antofagasta, 17°S (Bird et al. 1986, Guillemin et al. 2008). Human activities have also leaded to a loss of genotypic diversity in farmed populations that could be partially linked to involuntary selection of faster growing thalli, during the first steps of the domestication process (Guillemin et al. 2008, Guillemin et al. 2013, Valero et al. 2017).

were followed during one month in controlled laboratory conditions at 10°C, 15°C and 20°C.

Materials and methods

Study sites and life cycle phase determination of the sampled thalli

A total of 600 individuals were sampled from 3 natural and 3 farmed populations (i.e., 100 individuals in each population) during a spring season. One farmed and one natural population were sampled from three sites: Concepción, Maullín and Ancud (Fig. 1). The distinction between natural and farmed populations was first based on whether

Agarophyton. chilensis thalli were actively planted or not by farmers. As previously reported for the species (Guillemin et al. 2008), thalli were attached to small rocks and pebbles by a holdfast in the three natural A. chilensis beds while unattached thalli were found embedded in the sandy bottom in the three farmed stands. In each site, natural and farmed populations were separated by 1 km approximately. In natural beds, individuals sampled correspond to distinct holdfasts (i.e., distinct genotypes produced by sexual reproduction and spore settlement). In farms, to avoid sampling fragments of the same asexually propagated genotype, sampled thalli were separated by at least by 2 m. All the collected thalli were transported in isolated boxes to the CEACIMA hatchery (Centro de Investigación de Acuicultura y Ciencias del Mar, Universidad de Los Lagos) located in the Metri Bay (41°36´S, 72°43´W). Once in the hatchery, each thallus was cleaned with fresh water and all epiphytes were removed by hand. Thalli were individually marked with a numbered tag and maintained in 400 L tanks at 12°C with constant aeration, 12L:12D photoperiod, 20 µmol electron m -2 s -1 photon flux and weekly filtered seawater exchange.

Collected thalli were observed under stereoscope microscope (Stemi DV4, Zeiss, Jena, Germany) to determine phase (i.e., diploid tetrasporophytes or haploid gametophytes) of mature individuals. For vegetative individuals, a 3-cm fragment of tissue was excised from each thallus, placed into plastic bags with silica gel for rapid dehydration. The sex markers available for A. chilensis were amplified following Guillemin et al. (2012) and the amplification products were visualized in 1.5 % agarose gel (w/v) after adding 2 μl of GelRed™ (Biotium, Fremont, USA). Results were used to determine sex and phase of vegetative individuals. In order to prevent experimental bias due to ecophysiological variability between life cycle phases (Guillemin et al. 2013), only diploid tetrasporophytes were selected for our experiments.

Experimental design

The experimental design consisted of 90 2-L Erlenmeyer flasks (i.e., 6 populations of origin of thalli x 3 temperature treatments x 5 replicates per population per temperature treatment) arranged in 15 60-L plastic water tanks fitted with temperature control systems. Three temperature treatments were used: 10°C, 15°C and 20°C. These temperatures were chosen since they roughly represent the temperature range encountered in the field by the study species. Temperature conditions varied widely within sites were A. chilensis is found and between regions populated by the species along the Chilean coasts with values between 9

°C and 16 °C recorded in Maullín (Westermeier et al. 1993) and between 10 °C and 20°C in areas further north such as Concepción, Coquimbo and Antofagasta (Santelices and Ugarte 1990). In each 60-L plastic water tanks, one 50W automatic heater (Whale VK-1000, Regent) and one stainless steel thermometer (Hagen, Phelan) were used to maintain constant temperature. For each population under study, five replicates (i.e., five 2-L flasks)

were followed per temperature treatment and eight thalli (5 cm length each), selected randomly from distinct tetrasporophytes, were placed in each 2-L flask. Thalli were selected without replacement from a pool of 40 tetrasporophytes, available for each population of origin (see above). Once a week, thalli were transferred to clean 2-L flasks with fresh Provasoli culture media (McLachlan 1973). After one week of acclimation at 12°C, the laboratory experiment was run during 30 days. All Erlenmeyer flasks were under constant conditions of aeration, photoperiod (12 h light: 12 h dark) and photon flux (20 µmol electron m -2 s -1 ).

Growth

Fresh weight of each thallus was assessed weekly on an analytical balance (Sartorius TE 313 DS, Germany) and the specific growth rate (SGR) was calculated as the percentage of wet weight gain per day according to the formula: SGR= [ln (Wf • Wi -1 )/ (tf-ti)] x100;

where Wi= initial fresh weight, Wf= final fresh weight, and t= time (days). Initial fresh weight in the 2-L flasks was of 0.12 ± 0.06 g, some variation in fresh weight exist between each 2-L flask at the beginning of the experiment since calibration of algal material was based on thallus length (see above).

Physiological variables

Thallus pieces were collected at the end of the experiment to measure rapid light curves (RLC). As an indicator of quantum efficiency and photoinhibition, we used, Fv/Fm, which was determined after incubation of 20 min of the thalli in darkness (Schreiber et al. 1995) with a Junior PAM (Walz GmbH, Effeltrich, Germany). The electron transport rate (ETR, µmol electrons • m -2 • s -1 ) was determined after 20 s exposure in 12 increasing intensities of PAR (up to 1500 µmol photon • m -2 • s -1 ) provided by a blue light of the Junior PAM device (Schreiber et al. 1995). ETR was calculated according to Schreiber et al. (1995) as follows:

ETR = ∆F/F´m • E • A • FII (µmol electrons • m -2 • s -1 ) (1)
Where ∆F/F´m is the effective quantum yield, E is the incident PAR (photosynthetically active radiation) irradiance expressed in µmol photons m -2 s -1 , A is the thallus absorptance and FII is the fraction of chlorophyll related to photosystem II (PSII, 400-700m), being 0.15 in red seaweed (Grzymski et al. 1997). As an estimator of photosynthetic efficiency, the initial slope of ETR (αETR) and maximum ETR (ETRmax) were obtained from the tangential function reported by Eilers and Peeters (1988) and the irradiance saturation (EkETR) was calculated from the intercept between these two parameters. Representing potential thalli photoprotective mechanism, non-photochemical quenching (NPQ) was measured according to Schreiber et al. (1995). The maximal non-photochemical quenching (NPQmax) was calculated from the tangential function of NPQ versus irradiance function according to Eilers and Peeters (1988).

Statistical treatment

All analyses were performed in R (3.2.4 version) (Cayuela 2011). Assumptions of homogeneity of variances and normal distribution were tested using Levene`s test and Shapiro-Wilk, respectively. When non-normal residuals and heteroscedasticity were detected, the data were transformed using logarithm (for Ek and SGR) or Box-cox (for Fv/Fm). The experimental design fitted a three-way ANOVA with treatment (temperature:

10°C, 15°C and 20°C), site of origin (Concepción, Maullín and Ancud) and population type (natural or farmed) considered as fixed factors. Statistical differences between groups were analyzed using comparisons of means (Tukey's HSD). Significances were set at p < 0.05.

Results

Significant differences were detected in Agarophyton. chilensis specific growth rate (SGR) after 30 days of experimentation between temperature treatments (F(2, 72) = 15.46; P < 0.0001), between population types (F(1, 72) = 4.93; P= 0.03) and a significant interaction was also detected between population type and site of origin (F(2, 72) = 5.00; P= 0.009). In Maullín and Ancud, no significant differences between temperature treatments were detected for farmed thalli (Fig. 2C andE), while farmed thalli from Concepción showed a slightly but significantly lower SGR at 20°C than at 15°C (7.26 ± 1.48 and 9.45 ± 1.92 g • day -1 , respectively; Fig. 2A). In addition, differences among Concepción farmed and natural populations were observed (Tukey test; P<0.05, see Figure 2A andB). In contrast, SGR was significantly higher at 15°C for thalli sampled in natural A. chilensis stands, whatever the site under study (SGR at 15°C: 7.49 ± 2.07 g • day -1 in Concepción, 8.81 ± 2.60 g • day - 1 in Maullín and 8.67 ± 0.84 g • day -1 in Ancud; Fig. 2B, D andF). Finally, the growth rate did not decrease significantly (Tukey test; P<0.05) at the lower (10 o C) and the higher (20 o C) temperature tested for Maullin and Ancud populations (Fig. 2 C, D, E andF).

Regarding the photosynthetic responses, significant effect of temperature treatments were detected for all parameters measured: the optimal quantum yield of fluorescence (Fv/Fm, F(2, 35)= 13.20; P< 0.00001), the maximal electron transport rate (ETRmax; F(2, 35)= 6.17; P= 0.02), the saturation irradiance (Ek; F(2, 35)= 6.97; P= 0.04) and the maximal nonphotochemical quenching (NPQmax; F(2, 35)= 6.02; P= 0.01) (see 1). In Maullín at 15°C, values of NPQmax were significantly higher for thalli sampled in the farmed bed than the natural population (Tukey's HSD tests; p < 0.05; Table 2).

Discussion

The present study confirms that natural and farmed Agarophyton chilensis populations respond differentially to temperature variations. Indeed, contrarily to our expectations, our results indicate that farmed populations are less sensitive to temperature variations than natural ones (i.e., thalli growth rate are mostly similar at 10°C, 15°C and 20°C for farms while thalli from natural populations have a higher growth rate at 15°C). In Chile, farmed population has been under unconscious human selection pressure for, at least, three decades and we propose that a possible outcome could have been the selection of general-purposegenotypes (as in Baker 1974) in these vegetatively propagated crops. Our results also showed that photosynthetic activity was affected by temperature treatments (e.g., different maximum maximal electron transport rate and quantum yield values depending on the population type and temperature).

Regardless of the origin of the natural population under study, our experiment shows that thalli specific growth rate (SGR) was higher at 15°C than at lower (i.e., 10°C) or higher (i.e., 20°C) temperatures. Even if SGR tended to be much more homogeneous for farmed thalli at all temperatures, one slight difference was observed for the farmed thalli from Concepción that show significantly less growth at 20°C. Supporting our results, a study realized by Santelices and Ugarte (1990) on A. chilensis (as Gracilaria chilensis) natural populations from Maullín, also revealed better growth rates at 15°C than at 10°C or 20°C.

Most temperate species of the genus Agarophyton has been shown to grow faster in temperature ranging between 15° and 20°C (McLachlan and Bird 1984). A study performed in Gracilaria gracilis (as G. verrucosa) reported a slow growth rate at 10°C and high mortality after a 14 days' heat-wave in Saldanha Bay, South Africa (Engledow and Bolton 1992). A. chilensis, is a temperate-water species with a distribution limited to southern part of the Pacific (Bird et al. 1986, Guillemin et al. 2008) and it is possible that the species present a metabolism with limited temperature tolerance.

Although farming has begun only a few decades ago (i.e., during the 80´s) in Chile, the predominant mechanism for stock propagation by cuttings used in Agarophyton. chilensis farms has already significantly decreased their genotypic diversity when compared with wild populations (Guillemin et al. 2008). There is an overall agreement that genetically more variable populations may be associated with higher resilience, increased productivity and population growth rate as compared with less variable populations (Forsman 2014).

However, contrasting with our expectations, farmed thalli in our experiment clearly show a strong ability to grow in contrasting environmental conditions, including the quite "extreme" temperature of 20°C tested. In the same way, Gallegos-Sanchez et al. (2018) concluded that A. chilensis farmed populations may be less sensitive to salt stress and able to grow in a greater range of salinity than natural populations. One possible explanation for these results is that farmed populations of A. chilensis are composed mostly of generalpurpose genotypes, able to grow in highly stressful and/or variable environments. Generalpurpose-genotypes are sometime also referred as 'Jack-of-all-trades, master of none' since they are described as versatile genotypes that are able to perform adequately across a range of environments but are not superior in any of them. These general-purpose-genotypes can confer a species or population a broad tolerance to environmental changes and are often associated with species invasion (Baker 1974, Richards et al. 2006). In Chile, farms have been developed using material growing embedded in muddy estuaries and sandy bays.

These habitats are typically highly heterogeneous and present strong seasonal variations in temperature and salinity (Westermeier et al. 1993, Buschmann et al. 1995) In benthic algae, temperature variations affect photosynthetic metabolism (Davison 1991) determining, for example, seasonal distribution (De Nicola 1996). However, habitats characterized by strong spatial and temporal variations of abiotic factors, request constant adjustment of photosynthetic processes in species populating them (Ensminger et al. 2001).

In our study, the highest values of ETRmax and Ek were observed in the A. chilensis farmed population from Concepción grown at 20°C. These results suggest that the effect of high temperatures on photosynthetic metabolism of this farmed population could be mild.

Driven by seasonal changes in river discharge, precipitation, and coastal upwelling, high variability in abiotic conditions (e.g., temperature, salinity and turbidity; (Moll and Steinback 1986). However, short-term response of photosynthesis to temperature cannot easily be used to infer the long-term response of algal growth (Wienke and Dieck 1989). Indeed, despite the possible signal of photoinhibition detected at 15°C, no limited growth could be observed in these Agarophyton chilensis thalli.

Populations of Agarophyton. chilensis from different sites in Chile have been reported to present ecological differences, potentially linked to local adaptation in response to specific abiotic and biotic environmental characteristics (Santelices and Ugarte 1990, Usandizaga et al. 2018). The success of Agarophyton farming depends in part on the origin of the initial inoculum since differences in thallus morphology, agar yield and gel strength and susceptibility to epiphytes exist among regions and populations. Indeed, it have been suggested that random transplantation between distinct habitats could lead to cultivation failure (Santelices and Ugarte 1990). However, in our experiment, even if we included sampling sites located more than 700 km apart, no major effect of the site of origin were detected on growth or photosynthesis. A possible explanation for this discrepancy is that continuous transplantations and exchanges during the last decades have leaded to the homogenization of the genetic diversity among the whole Chilean coast. However, this hypothesis is not in accordance with population genetic studies showing the presence of clear genetic divergence between regions in Chile (Guillemin et al. 2008 and2014). Studies focused on the effect of other stressors (e.g., nutrient supply, salinity, irradiance) and the cumulative effects of various of these stressors on the physiological responses of distinct genotypes is now needed in order to better explore the resistance of A. chilensis populations to stress.

Conclusion

Agarophyton chilensis tolerance of a wide range of abiotic conditions has been proposed as one of the main reasons of the species successful expansion in the Pacific and establishment in a wide array of habitats (Santelices andUgarte 1990, Chow et al. 2001). These successful extension waves have probably also been facilitated by intrinsic characteristics of the species, such as its capacity to shift between sexual and asexual reproduction (Guillemin et al. 2014). We suggest that the possible selection for general-purposegenotypes in the asexually reproducing farmed populations may help modulating the impact of environmental variation on population dynamics (Reed et al. 2010)

  . It have been demonstrated that intraspecific competition for resources utilization in clonal individuals living in habitats characterized by fluctuating environmental conditions could lead to positive selection of general-purpose-genotypes (Arnaud-Haond et al. 2012).

  and Chilean Agarophyton crop to better cope with impacts of climate change and direct anthropogenic activities. Nevertheless, implementation of breeding strategy and cultivar selection for mariculture systems improvement has not yet begun in Chile and development of long-term management plans for the sustainable exploitation of A. chilensis populations is dearly needed.

Fig 1 .

 1 Fig 1. Location of the three sites sampled along the Chilean coast. Photographs of farmed

Fig 2 .

 2 Fig 2. Mean (±SE) of the specific growth rate (SGR) of Agarophyton chilensis thalli

  . It have been demonstrated that intraspecific competition for resources utilization in clonal individuals living in habitats characterized by fluctuating environmental conditions could lead to positive selection of general-purpose-genotypes (Arnaud-Haond et al. 2012).

  and Chilean Agarophyton crop to better cope with impacts of climate change and direct anthropogenic activities. Nevertheless, implementation of breeding strategy and cultivar selection for mariculture systems improvement has not yet begun in Chile and development of long-term management plans for the sustainable exploitation of A. chilensis populations is dearly needed.

Figure legends Fig 1 .

 legends1 Figure legends

Fig 2 .

 2 Fig 2. Mean (±SE) of the specific growth rate (SGR) of Agarophyton chilensis thalli

Table 2 .

 2 Photosynthetic parameters measured in Agarophyton chilensis natural and farmed thalli submitted to three temperature treatments. Thalli from one natural and one farmed populations were sampled in three sites (i.e. Concepción, Maullín and Ancud). Data are given as mean ± S.E.D (n = 5). Distinct uppercase letters denote significant differences after Tukey test.

  In our experiment, the response of the maximum quantum yield (Fv/Fm) to temperature was quite variable. The lowest value of Fv/Fm (indicating photoinhibition) was observed at intermediate temperature (15°C) in the farm of Maullín. In plants, it is well known that limitation of electron transport that reduces the ability of plants to use light result in an

	Driven by seasonal changes in river discharge, precipitation, and coastal upwelling, high
	variability in abiotic conditions (e.g., temperature, salinity and turbidity; Saldías et al.

2016) has been observed in the river mouths were A. chilensis thalli are planted in Concepción. These characteristics could be associated to distinctive heat susceptibility of the photosynthetic metabolism of the A. chilensis thalli growing in Concepción farms.

  Table1). Interactions between temperature treatment and population type were also observed for ETRmax (F(2, 35)= 9.57; P= 0.0005) and Ek (F(2, 35)= 5.34; P= 0.01) (Table1). Values of ETRmax and Ek were significantly higher for thalli sampled in the farmed population of Concepción and grown at

	20°C than for thalli sampled in the farmed population of Ancud and grown at 10°C (18.63
	± 12.76 and 1.44 ± 0.37 µmol electrons • m -2 • s -1 , 442.97 ± 306.60 and 31.86 ± 4.78 µmol
	photons • m -2 • s -1 , for ETRmax and Ek, respectively; Tukey's HSD tests; p < 0.05; Table 2).
	Significant interactions between temperature treatment, population type and site of origin
	were observed for Fv/Fm (F(4, 35)= 2.86; P= 0.04) and NPQmax (F(4, 34)= 3.31; P= 0.02) (Table

  Saldías et al. 2016) has been observed in the river mouths were A. chilensis thalli are planted in Concepción. These characteristics could be associated to distinctive heat susceptibility of the photosynthetic metabolism of the A. chilensis thalli growing in Concepción farms. In our experiment, the response of the maximum quantum yield (Fv/Fm) to temperature was quite variable. The lowest value of Fv/Fm (indicating photoinhibition) was observed at intermediate temperature (15°C) in the farm of Maullín. In plants, it is well known that limitation of electron transport that reduces the ability of plants to use light result in an excess light energy that may cause photoinhibition due to damage to the PSII apparatus

Table 2

 2 Click here to access/download;table;Table 2.docx

						Ek
	20°C	15°C	10°C Farmed	20°C	15°C	10°C Natural	20°C	15°C
	442.97±306.60 a	93.56±21.17 ab	94.85±87.88 ab	200.16±196.5 ab	185.94±70.68 ab	143.97±53.30 ab	18.63±12.76 a	3.97±0.68 ab
	20°C	15°C	10°C Farmed	20°C	15°C	10°C Natural	20°C	15°C
	233.80±108.15 ab 20°C	173.11±32.20 ab 15°C	66.76±32.14 ab 10°C Farmed	111.39±48.50 ab 20°C	111.63±58.52 ab 15°C	129.40±88.27 ab 10°C Natural	16.11±8.19 ab 20°C	8.25±3.32 ab 15°C
	184.44±134.12 ab	110.38±70.53 ab	31.86±4.78 b	124.41±91.02 ab	229.16±92.04 ab	73.94±45.65 ab	10.03±8.65 ab	9.21±5.67 ab

Acknowledgements

This research was supported by a doctoral fellow from Universidad de Los Lagos (Chile) to SU, CeBiB (CONICYT, FB-0001) to AHB and CC and Fondecyt N°1150987 and N°1170541 (CONICYT) to AHB and MLG, respectively. Additional support was provided by the grant "Término de Tesis" awarded by Universidad de Los Lagos. The

Acknowledgements

This research was supported by a doctoral fellow from Universidad de Los Lagos

Reduced genetic diversity can severely affect the ability of populations to resist pests and pathogens and limit the scope for future genetic improvement in domesticated crops (Robinson et al. 2013). Besides, it has been reported that a reduction in genetic diversity could lead to lower growth and resilience in highly stressful and/or variable environment (Simms 2000). However, to date, few studies have focused on the potential ecophysiological differences between natural and farmed populations of A. chilensis. Gallegos-Sánchez et al. (2018), detected a significant and negative effect of low salinity conditions on thalli sampled from both natural and farmed A. chilensis populations but with farmed population's thalli being less affected than the natural ones. Results suggested that farmed populations might be more tolerant to salt stress than wild ones in this species and the authors proposed that this difference between population types could be due to previous selective process carried out by farmers.

Considering the possible consequences of unconscious selection (as defined in Zohary 2004) and domestication on GAgarophyton. chilensis, we propose that natural populations will be less sensitive to temperature variations than farmed populations. Genotypic diversity has been shown to be higher in natural than farmed A. chilensis Chilean populations and we propose that the farms will show less effective mechanisms of acclimation (e.g., enhancing of photosynthetic performance) than the one observed in natural populations when confronted with temperature considered as high in their natural environment (i.e., water of the southern coast of Chile do not generally reach 20°C; Westermeier et al. 1993). The aim of the present study was to assess the effect of temperature on growth and photosynthetic responses of both natural and farmed stands of A. chilensis and tetrasporophyte thalli sampled from three sites along the Chilean coast (Chile) to SU, CeBiB (CONICYT, FB-0001) to AHB and CC and Fondecyt N°1150987 and N°1170541 (CONICYT) to AHB and MLG, respectively. Additional support was provided by the grant "Término de Tesis" awarded by Universidad de Los Lagos. The authors are grateful for the help in the field of R. Altamirano and S. V. Pereda for the revision of this manuscript. We also wish to thank two anonymous referees who greatly improved the manuscript with constructive suggestions. The authors declare no conflict of interest.

growth results

Click here to access/download;line figure;Fig. 2A,B,C,D,E,F.TIF


Marion Chatelain
email: marion.chatelain@cent.uw.edu.pl

J Mathieu

How good are epigeic earthworms at dispersing? An investigation to compare epigeic to endogeic and anecic groups

Keywords: density-dependent dispersal, departure, diffusion behaviour, ecosystem engineers, functional groups, habitat quality

Dispersal capacities can strongly determine an individual's ability to respond to changing environmental conditions, which would consequently influence the structure of natural communities. Nonetheless, we know little about the dispersal behaviour of soil organisms, despite some of these organisms, such as earthworms, have key roles in ecosystem functioning (e.g. organic matter decomposition). We expect that species exposed to frequent environmental changes would benefit from the capacity to escape from adverse environmental conditions and to disperse to settle in a more suitable habitat. In earthworms, we expect the epigeic group, which lives at or close to the soil surface, to have evolved higher dispersal capacities than the two other functional groupsanecic and endogeic, which live deeper in the soil. In this study, we investigated dispersal and diffusion behaviour of three species of epigeic earthworms (i.e. Eisenia fetida, Eisenia andrei and Lumbricus rubellus) and compared these behaviours with those of anecic and endogeic earthworms, whose behaviour has been previously measured through similar experiments. In accordance with our hypothesis, our study shows that dispersal behaviour of epigeic earthworms depends on habitat quality and population density, but that those responses vary among species and that it differs only to a limited extent from behaviour of anecic and endogeic earthworms.

Introduction

Dispersal is a central ecological process that allows colonization of new habitats and exploitation of spatially and temporally variable resources [START_REF] Ronce | How does it feel to be like a rolling stone? Ten questions about dispersal evolution[END_REF]. Active dispersal of animals (as opposed to passive dispersal, where individuals are transported by an external agent, and has not necessarily a cost for the disperser) is the result of three successive behavioural stages (following the definition given by [START_REF] Clobert | Informed dispersal, heterogeneity in animal dispersal syndromes and the dynamics of spatially structured populations[END_REF][START_REF] Clobert | Dispersal[END_REF]. It involves the departure from a breeding site, crossing to a new place, and settlement [START_REF] Clobert | Informed dispersal, heterogeneity in animal dispersal syndromes and the dynamics of spatially structured populations[END_REF]. It is thought to depend on the balance between the costs and benefits of dispersal [START_REF] Bonte | Costs of dispersal[END_REF][START_REF] Bowler | Causes and consequences of animal dispersal strategies: relating individual behaviour to spatial dynamics[END_REF], which are strongly determined by both environmental conditions (e.g. habitat quality, habitat fragmentation, patch size, density, predation; [START_REF] Bonte | Geographical variation in wolf spider dispersal behaviour is related to landscape structure[END_REF][START_REF] Schtickzelle | Didpersal depression with habitat fragmentation in the bog fritillary butterfly[END_REF] and individual life traits (e.g. age, hormonal levels, movement abilities). Therefore, dispersal capacities are expected to strongly determine an individual's ability to respond to changing environmental conditions, which would consequently influence the dynamics and persistence of populations, the distribution and abundance of species, the structure of natural communities but also the functioning of ecosystems [START_REF] Cuddington | Invasive engineers[END_REF]. Nonetheless, we know little about the dispersal behaviour of soil organisms, even if some of these organisms, such as earthworms, play key roles in ecosystem functioning [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services: Earthworm impact on ecosystem services[END_REF].

Earthworms species are often classified into three functional groups based on their morphology, and their foraging behaviour [START_REF] Bouché | Stratégies lombriciennes[END_REF][START_REF] Bouché | Lombriciens de France, écologie et systématiques[END_REF]: endogeic earthworms live and feed in the soil, epigeic earthworms mainly live and feed on the leaf litter at the soil surface, anecic earthworms make vertical burrows in soil and feed on leaf litter which they drag into their burrows. Earthworms are of primary importance for ecosystem functioning because they modify the availability of resources for other organisms through physical and chemical changes in their surrounding soil environment [START_REF] Jones | A framework for understanding physical ecosystem engineering by organisms[END_REF][START_REF] Jones | Organisms as ecosystem engineers[END_REF][START_REF] Rillig | Soil microbes and community coalescence[END_REF]. As a consequence, they fulfil numerous soil-based ecosystem services [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services: Earthworm impact on ecosystem services[END_REF]. While anecic earthworms, and to a lower extent endogeic and epigeic earthworms, are of particular importance for cropping systems [START_REF] Bertrand | Earthworm services for cropping systems. A review[END_REF][START_REF] Van Groenigen | Earthworms increase plant production: a meta-analysis[END_REF], epigeic earthworms play a key role in organic matter decomposition in deciduous forests because of the ingestion of poorly decomposed litter [START_REF] Manna | Comparative efficacy of three epigeic earthworms under different deciduous forest litters decomposition[END_REF] and the interactions they established with decomposer microorganisms [START_REF] Gómez-Brandón | Species-specific effects of epigeic earthworms on microbial community structure during first stages of decomposition of organic matter[END_REF][START_REF] Monroy | Changes in density of nematodes, protozoa and total coliforms after transit through the gut of four epigeic earthworms (Oligochaeta)[END_REF], which explains their use in vermicomposting (e.g. [START_REF] Suthar | Earthworms as bioindicator of metals (Zn, Fe, Mn, Cu, Pb and Cd) in soils: Is metal bioaccumulation affected by their ecological category?[END_REF].

For these reasons, it is essential to identify the environmental factors that may influence earthworm prevalence and abundance in ecosystems [START_REF] Curry | Factors affecting earthworm abundance in soil[END_REF][START_REF] Palm | Modelling distribution patterns of anecic, epigeic and endogeic earthworms at catchment-scale in agro-ecosystems[END_REF], especially in the context of global changes. Indeed, anthropogenic activities, including urbanisation and agriculture, are responsible for considerable modifications of the natural environment through e.g. light, noise and chemical pollutions, temperature modifications and habitat fragmentation. These changes may have considerable impact at the individual level and in terms of population dynamics and functioning (e.g. [START_REF] Dupont | Dispersal constraints and fine-scale spatial genetic structure in two earthworm species: Spatial genetic structure of earthworms[END_REF][START_REF] Johnston | Effects of agricultural management practices on earthworm populations and crop yield: validation and application of a mechanistic modelling approach[END_REF][START_REF] Orwin | Effects of climate change on the delivery of soil-mediated ecosystem services within the primary sector in temperate ecosystems: a review and New Zealand case study[END_REF]. To cope with these natural (e.g. soil heterogeneity, daily and seasonal cycles, etc.) or human-induced environmental constraints, high sensory capacities associated with acclimatization or dispersal abilities may have been naturally selected in earthworm species (e.g. [START_REF] Fisker | Genetic adaptation of earthworms to copper pollution: is adaptation associated with fitness costs in Dendrobaena octaedra?[END_REF][START_REF] Spurgeon | The development of genetically inherited resistance to zinc in laboratory-selected generations of the earthworm Eisenia fetida[END_REF]. Amongst other things, we expect species exposed to frequent environmental changes to benefit from a large tolerance range, meaning from high flexibilities (e.g. earthworms exposed to changing concentrations of pollutants should benefit from high flexibility in the synthesis of detoxification proteins; [START_REF] Lukkari | Biomarker responses of the earthworm Aporrectodea tuberculata to copper and zinc exposure: differences between populations with and without earlier metal exposure[END_REF] or from the capacity to escape from these detrimental environmental conditions and to disperse to settle in a more suitable habitat.

The three functional groups of earthworms inhabit three different niches whose exposure to aboveground conditions increases from the endogeic group to the epigeic group. The aboveground environment is characterized by a high temporal and spatial heterogeneity.

Therefore, epigeic earthworms have to face highly fluctuating environments (e.g. temperature and humidity changes) and are more directly exposed to soil inputs (e.g. pesticides, hydrocarbons, fertilizer, etc.), soil surface state (e.g. subsidence exerted by vehicles, ploughing or bioturbations) and predators. Consequently, we may expect epigeic earthworms to have evolved higher sensibility to surface conditions (i.e. quicker responses) and higher dispersal abilities (i.e. lower dispersal costs, associated with physiological and anatomical adaptations for low latency and high speed movements) than endogeic and anecic groups.

Habitat (i.e. soil and litter) structure (e.g. particle size distribution), composition (e.g. amount of organic carbon) and pH, both linked to bioavailability of chemicals in soils and earthworms' ability to move in the habitat, are expected to influence earthworm habitat preference [START_REF] Lanno | The bioavailability of chemicals in soil for earthworms[END_REF] and as a consequence dispersal behaviour. Moreover, the amount of food in the environment is often limited; therefore, food availability per individual is negatively correlated with population density [START_REF] Curry | Factors affecting earthworm abundance in soil[END_REF]. Alternatively, population density may affect soil physical and chemical properties [START_REF] Jones | A framework for understanding physical ecosystem engineering by organisms[END_REF][START_REF] Jones | Organisms as ecosystem engineers[END_REF][START_REF] Rillig | Soil microbes and community coalescence[END_REF], which may lead to facilitating mechanisms [START_REF] Caro | Is earthworms' dispersal facilitated by the ecosystem engineering activities of conspecifics?[END_REF][START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF].

Nonetheless, to our knowledge, few studies have investigated the environmental factors that influence dispersal behaviour in earthworms. [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF][START_REF] Caro | Is earthworms' dispersal facilitated by the ecosystem engineering activities of conspecifics?[END_REF] and [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF] showed that low soil quality increased dispersal rate of Aporrectodea icterica, Allolobophora chlorotica, Aporrectodea caliginosa (endogeic species) and of Aporrectodea longa, Lumbricus terrestris and Aporrectodea giardi (anecic species) [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF][START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF]. Moreover, the absence of litter increased dispersal rate in Dendrobaena venata, an epigeic species [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF]. High intraspecific density also increased dispersal rate in the three anecic species and in A. icterica but not in A. chlorotica and A. caliginosa. Finally, while dispersal speed was increased by conspecifics through the use of existing galleries in Aporrectodea giardi [START_REF] Caro | Is earthworms' dispersal facilitated by the ecosystem engineering activities of conspecifics?[END_REF], dispersal rate was reduced by the pre-use of soil in A. icterica [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF]. Previous studies on earthworm dispersal were mostly carried out on endogeic and on anecic groups [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF][START_REF] Caro | Is earthworms' dispersal facilitated by the ecosystem engineering activities of conspecifics?[END_REF] or on a single epigeic species [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF]. Therefore, we still know little about the dispersal behaviour of the epigeic group and whether it is different from the two other groups.

To investigate diffusion and dispersal behaviour of epigeic earthworms, we performed three different experiments on three epigeic species (i.e. Eisenia fetida, Eisenia andrei and Lumbricus rubellus), following the same experimental protocol as a previous experiment [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]. We investigated diffusion behaviour in a homogeneous environment (experiment 1), to measure earthworm propensity to explore, their distance of exploration and their tendency to follow their conspecifics. We measured dispersal rate in response to habitat quality (experiment 2) or in response to population density (experiment 3). Then, we compared diffusion and dispersal behaviours of epigeic earthworms with that of the two other functional groups: anecics and endogeics, whose behaviours have been previously measured through similar experiments [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]. We expected high specificity in epigeic dispersal behaviours because of their specific relative selective pressures.

Methods

Subjects and housing

Free-living adult earthworms from three epigeic species -Eisenia fetida, Eisenia andrei and Lumbricus rubellus -were collected in November 2015 from several rural locations in Ile-de France, near Paris, France (between 48°69'N, 2°60'E and 48°74'N, 2°68'E). Earthworms were kept in acclimatizing mesocosms (12 cm x 10 cm x 8 cm) filled with suitable soil (see 2.2 Diffusion and dispersal mesocosms set-up) with a density of 10 earthworms per mesocosm, at a constant 17°C. Before the start of the trials (section 2.3) earthworms were acclimatized for a period of at least 3 weeks, depending on their capture date and the trial dates, to remove potential stress effects of capture. Acclimatizing mesocosms were humidified and enriched with homogenized horse dropping twice a month. All individuals were kept for at least three weeks before onset of experiments. The species names used herein conformed to the Fauna Europaea web site.

Diffusion and dispersal mesocosms set-up

First, we investigated diffusion in a homogeneous environment (experiment 1). Then, two different environmental factors were tested on epigeic earthworm dispersal: population density (experiment 2) and habitat quality (experiment 3). The experiments' setting followed the protocol used by [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]. Diffusion behaviour in a homogeneous environment was tested in mesocosms (300 cm x 20 cm x 20 cm) that consisted of a suitable habitat. The suitable habitat consisted of grassland soil collected from a brunisol at the IRD research centre (48°54'N, 2°29'E), which hosts large earthworm populations. Because epigeic earthworms mainly inhabit litter, lime leaves (Tilia vulgaris) were added on the surface of the suitable soil. This litter is generally well consumed by earthworms [START_REF] Hendriksen | Leaf litter selection by detritivore and geophagus earthworms[END_REF]. This first experiment tested the natural spread of individuals while removing the effect of heterogeneity. The mesocosms were divided in 13 regular sections (each 23 cm long) which were named according to their distance from the central section (see Fig. 1).

The influence of population density and habitat quality were investigated in dispersal mesocosms (100 cm x 20 cm x 20 cm; [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF], which were divided into three equal sections (see Fig. 1): the "inoculation" section (1) was a suitable habitat in population density trials and either a suitable or an unsuitable habitat in habitat quality trials; the "crossing" section ( 2) was always an unsuitable habitat; finally, the "arrival" section (3) was always a suitable habitat. The suitable habitat was similar to the one previously described (i.e. grassland soil + leaf litter). The unsuitable habitat consisted of a sandy soil collected from a luvisol in an area deprived of earthworms and litter in the forest of Fontainebleau (48°24'N, 2°44'E). Because of their niche, epigeic earthworms were expected to be strongly affected by litter quality. Therefore, the unsuitable soil was not topped with litter. We checked in a preliminary experience that the three species did prefer the suitable soil to the unsuitable one.

Both soils were air dried, sieved to 2 mm and rewetted manually at 25% of humidity. Lime leaves were washed, air dried and soaked in tap water for 15 minutes. Soil (suitable or unsuitable) was filled to 5 cm high and 2 cm high of lime leaves were spread on the top of appropriate sections. This setup allowed the reproduction of the three stages of dispersal: departure, crossing and settlement in a suitable site [START_REF] Clobert | Informed dispersal, heterogeneity in animal dispersal syndromes and the dynamics of spatially structured populations[END_REF]. The unsuitable habitat in the crossing section represented a physical barrier generating dispersal costs. It allowed us distinguishing between mechanisms of diffusion (random movements with potentially returns in the starting point) from active dispersal [START_REF] Clobert | Informed dispersal, heterogeneity in animal dispersal syndromes and the dynamics of spatially structured populations[END_REF]. As expected, diffusion rate in a homogeneous high quality habitat was higher than dispersal rate from a high-quality habitat (see Table 1 and Fig. 2), verifying that our dispersal mesocosm set-up successfully induced a dispersal cost, and suggesting that dispersal rate rather than diffusion rate was measured in dispersal experiments.

Diffusion and dispersal trials

When investigating diffusion behaviour in a homogeneous environment (experiment 1), we simultaneously inoculated 10 earthworms of the same species in the central section of a threemeter long mesocosm (i.e. section 0). To avoid earthworms from escaping during inoculation, earthworms were placed in the centre of a rectangular plastic gate (12 cm x 10 cm x 6 cm) previously buried at 1.5 cm deep in the centre of the inoculation section, which was ever in the middle of the mesocosm (experiment 1) or at the left (experiments 2 and 3). We checked that all earthworms entered the soil or remained motionless before removing the gate and closure of the mesocosms. Each trial of the first experiment (experiment 1) lasted 24h; this time was short enough to prevent individuals from reaching the end of the mesocosms and going back towards the inoculation section. At the end of each trial, the 13 sections were separated and the number of earthworms per section was counted. We calculated diffusion rate as the proportion of individuals that left the central section. Despite the mesocosms being carefully closed, some earthworms escaped; the escape rate was low during the diffusion trials (mean ± se: 2 ± 7%, 0 ± 6% and 0 ± 0% in E. fetida, E. andrei and L. rubellus, respectively).

In order to take into account these escapes, diffusion rates were calculated as the number of individuals that left the central section (i.e. section 0) compared to the number of individuals that were found in the inoculation section at the end of a trial. We also investigated the mean of the absolute distance crossed and the tendency to follow conspecifics as the absolute value of the difference in the diffusion rates to the right and to the left of a starting section; thus the tendency to follow their conspecifics varies from 0 (individuals equally distributed in each side) to 1 (all individuals in only one side).

When investigating the effect of habitat quality on dispersal rate (experiment 2), we simultaneously inoculated 10 earthworms of the same species in a one meter long mesocosm.

When testing the effect of population density on dispersal rate (experiment 3), we inoculated earthworms in the mesocosm, at one of these four densities: 1, 10, 20 or 30 individuals of the same species. Each trial of experiments 2 and 3 lasted 7 days. At the end of each trial, the 3 sections were separated in order to prevent earthworms from switching between sections. Soil and lime leaves were carefully searched and earthworms were removed. Earthworms were then replaced in their resting mesocosms. The number of earthworms per section was counted; earthworms found in the arrival section were considered as disperser, while earthworms found in the inoculation and crossing section were considered as non-dispersers. Some of the earthworms also escaped these dispersal devices (mean ± se: 31 ± 5%, 27 ± 5% and 4 ± 5% in E. fetida, E. andrei and L. rubellus, respectively). The escape rate did not depend on the initial density (χ²ddl=1=0.01, P=0.909). Nonetheless, dispersal rates were calculated as the number of individuals in the arrival section compared to the number of individuals found in the two other sections at the end of the trials, which allowed measuring dispersal behaviour rather than departure behaviour. Each experimental design (i.e. homogeneous environment, high quality, low quality, density 1, density 10, density 20 and density 30) was replicated 5 times per species under the same conditions of light (a daily photoperiod of 10h) with temperature fluctuating between 15°C and 17°C.

Comparison of diffusion and dispersal behaviour between functional groups

We compiled the data obtained in these experiments with those obtained in a previous similar study on anecic and endogeic earthworms [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF] in order to compare dispersal behaviour between functional groups. Note that in order to take into account for the specific ecology of epigeics, the suitable and unsuitable habitats slightly differed in our study from the ones in [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]: we added a layer of leaf litter on top of the suitable soil, and soil depth was reduced to 5 cm high instead of 15 cm in the previous study. Both studies aimed at mimicking a suitable and an unsuitable habitat, which necessarily implied some minor habitat differences to adjust to the focus group.

Statistical analyses

Diffusion and dispersal behaviour in epigeic earthworms -To investigate whether

the diffusion rate, the mean distance crossed and the tendency to follow conspecifics in a homogeneous environment varied between species (experiment 1), we performed a Generalized Linear Model (GLM) with a binomial distribution (model 1) and Linear Models (model 2 and 3), respectively: diffusion rate, diffusion direction or the mean distance crossed was the dependent variable and species identity was the explanatory variable. In order to test the effects of habitat quality (model 4) and population density (model 5) on dispersal rate, we used GLM with binomial distributions: dispersal rate was the dependent variable and habitat quality (i.e. high vs. low), species identity (i.e. Eisenia fetida, Eisenia andrei, Lumbricus rubellus) and their interactive effects (model 4) or population density (i.e. 1, 10, 20, 30), species identity and their interactive effect (model 5) were the explanatory variables. Because some of the earthworms escaped during the experiment and we have no means to know when it happened, we also tested the effect of final density on dispersal rate (model 5) by considering the number of earthworms that have been found at the end of the experiment as the explanatory variable instead of the number of earthworms inoculated.

Comparison of diffusion and dispersal behaviour between functional group -We

compared the results from our three experiments on epigeic species to the ones obtained in a previous study with a similar experimental setup carried on three species of anecic (A. longa, L. terrestris and A. giardi) and three species of endogeic earthworms (A. chlorotica, A. icterica and A. caliginosa; [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]. We re-run our five previous models aggregating our results with the results from the previous study. In model 1 (Generalized Linear Mixed Models), 2 and 3 (Mixed-Effects Models), functional group was the explanatory variable and species was added as a random factor. In models 4 and 5 (Generalized Linear Mixed Models), andrei (F1,8=8.20, P=0.022, Table 1). The tendency to follow conspecifics was not significantly different between species (F2,12=1.22, P=0.329; Table 1).

Habitat quality (experiment 2)

-There was a significant interaction between habitat quality and species identity on the dispersal rate (χ²df=2=10.49, P=0.005; Fig. 2): in all three species, dispersal rate was higher when earthworms were inoculated into a low quality habitat than into a high quality habitat (χ²df=1=5.15, P=0.023; χ²df=1=17.40, P<0.001 and χ²df=1=45.09, P<0.001 for E. fetida, E. andrei and L.rubellus, respectively). However, dispersal rate from high quality habitat significantly differed between species (χ²df=2=24.89, P<0.001): L. rubellus dispersed less from high habitat quality than the two other species (χ²df=1=16.58, P<0.001 and χ²df=1=21.48, P<0.001 for E. fetida and E. andrei, respectively); dispersal rate from low quality habitat did not differ between species (χ²df=2=5.24, P=0.073).

Population density (experiment 3) -

There was a significant interaction between population density (i.e. 1, 10, 20, 30) and species identity on the dispersal rate (χ²df=6=27.38, P<0.001; Fig. 3): dispersal rate increased with increasing population density in E. andrei (χ²df=1=7.85, P=0.005), while it decreased with increasing population density in L. rubellus (χ²df=1=15.81, P=0.001); the correlation between dispersal rate and population density was not significant in E. fetida (χ²df=1=3.00, P=0.083). The results were quite similar when considering the number of earthworms found at the end of the experiment as the explanatory variable; there was a significant interaction between the final density and species identity on the dispersal rate (χ²df=2=24.44, P<0.001): dispersal rate increased with increasing the final density in E. andrei (χ²df=1=12.95, P<0.001) while it decreased with increasing population density in L. rubellus (χ²df=1=5.36, P=0.021) and E. fetida (χ²df=1=5.19, P=0.023).

Comparison of diffusion and dispersal behaviour between functional groups

Homogeneous environment -

There was no significant effect of functional group on diffusion rate in an homogenous soil (χ²df=2=1.90, P=0.387; Table 2), on the mean distance crossed (χ²df=2=0.91, P=0.634; Table 2) and on the tendency to follow conspecifics (χ²df=2=0.23, P=0.890; Table 2).

Habitat quality -

There was a significant interaction between habitat quality and functional group on the dispersal rate (χ²ddl=2=16.59, P<0.001; Fig. 4): in all three functional groups, dispersal rate was higher when earthworms had been inoculated into a low quality habitat than into a high quality habitat (χ²ddl=1=46.62, P<0.001; χ²ddl=1=112.51, P<0.001 and χ²ddl=1=67.05, P<0.001 for epigeic, anecic and endogeic earthworms respectively). However, dispersal rate from low quality habitat significantly differed between functional groups (χ²ddl=2=7.34, P<0.026): it was lower in endogeic earthworms than in the two other functional groups (χ²ddl=1=5.29, P=0.021 and χ²ddl=1=4.43, P=0.035 when compared to anecic and epigeic earthworms, respectively); on the contrary, dispersal rate from high quality habitat did not significantly differed between functional groups (χ²ddl=2=3.06, P=0.216).

Population density -

There was no significant effect of population density (χ²df=1=1.83, P=0.176), functional group (χ²df=1=3.64, P=0.162) and their interactions (χ²df=2=3.03, P=0.220) on dispersal rate (Fig. 5). The results were quite similar when considering the number of earthworms found at the end of the experiment as the explanatory variable: there was a significant interaction between the final density and species on the dispersal rate (χ²ddl=2=15.66, P<0.001) but final density tended to influence dispersal rate in anecic earthworms only (χ²ddl=1=0.30, P=0.584, χ²ddl=1=2.81, P=0.094 and χ²ddl=1=0.23, P=0.635 in epigeic, anecic and endogeic earthworms).

PLS-DA components analysis -

Two components were retained in the PLS-DA: the first and the second component explained 67.20% and 12.58% of the among group variation respectively. The sensitivity to habitat quality, the sensitivity to population density and the dispersal rate in homogeneous environment were the most important variables in the projection (i.e. VIP index greater than 1). The first component was correlated with the sensitivity to habitat quality (t=-4.11, df=7, P=0.005), while the second component was correlated with the sensitivity to population density (t=-10.87, df=7, P<0.001), the dispersal rate in homogeneous environment (t=3.26, df=7, P=0.014) and the mean distance crossed in homogeneous environment (t=2.77, df=7, P=0.028).

Discussion

Diffusion and dispersal behaviour in epigeic earthworms

Our study demonstrated that dispersal behaviour of epigeic earthworms depends on habitat quality and population density, and that this response varies among epigeic species.

Firstly, results showed a higher dispersal rate of Eisenia fetida and Eisenia andrei than of Lumbricus rubellus from high quality habitat. This elevated basal dispersal rate may be related to the narrower ecological niche of E. fetida and E. andrei, characterized by a high proportion of organic matter (i.e. they inhabit compost and manure; [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. Therefore, the so-called "high quality" habitat, composed of grassland soil topped with lime leaves may actually be moderately favourable for those species, consequently inducing high dispersal rates. Alternatively, both species might have high basal dispersal behaviour associated with high exploratory behaviour. This interpretation is supported by the results of our first experiment in a homogeneous environment. Indeed, diffusion rates in homogeneous environment were higher in E. fetida and E. andrei than in L. rubellus, and the mean distance crossed was also higher in E. andrei than in L. rubellus. It might also explain the higher escape rate measured in these two species than in L. rubellus. Moreover, our study clearly demonstrated an effect of habitat quality on dispersal rate, with earthworms exhibiting a higher dispersal rate in a low than in a high quality habitat. Therefore, as for anecic and endogeic earthworms [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF], habitat quality should be a strong driver of epigeic earthworm spatial distribution in ecosystems. Future studies should identify more thoroughly which habitat characteristics count for epigeic earthworms' decision to disperse. In particular, we need to disentangle the role of leaf litter presence and characteristics (e.g. plant variety, decomposition state) from the role of soil characteristics (e.g. composition, humidity). E. fetida response to habitat quality was less pronounced comparing to E. andrei and L. rubellus but also more variable between individuals, suggesting that this species would either be less sensitive to habitat quality, or suffer high dispersal cost, which is doubtful regarding their high dispersal rate in high quality habitat.

Secondly, our study highlighted no clear pattern of response to population density in epigeic earthworms: dispersal rate increased with increasing population density in E. andrei but not in the two other species. Insufficient information is available regarding the biotic environmental factors that influence habitat choice in earthworms (but see [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF][START_REF] Curry | Factors affecting earthworm abundance in soil[END_REF][START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF][START_REF] Palm | Modelling distribution patterns of anecic, epigeic and endogeic earthworms at catchment-scale in agro-ecosystems[END_REF]. For instance, the presence of conspecifics could be used as a clue of a high quality habitat [START_REF] Lister | Information, behaviour and population dynamics[END_REF]. For instance, E. fetida lives at high density, arguably because they chose patches of abundant food. In accordance, attraction mechanisms were observed in this species [START_REF] Zirbes | Self-assemblage and quorum in the earthworm Eisenia fetida (Oligochaete, Lumbricidae)[END_REF](Zirbes et al., , 2010)). In contradiction, dispersal rate of E. fetida was not negatively correlated with population density in our study.

Moreover, earthworms' motions and secretions (i.e. earthworm cast and mucus) induce strong modifications of the physical and chemical parameters of the soil [START_REF] Jones | A framework for understanding physical ecosystem engineering by organisms[END_REF][START_REF] Rillig | Soil microbes and community coalescence[END_REF]. Therefore, earthworm presence modifies the habitat in a way that may increase earthworm propensity to establish [START_REF] Caro | Impact of soil engineering by two contrasting species of earthworms on their dispersal rates[END_REF]. In accordance with this hypothesis, dispersal rates of Aporrectodea icterica (endogeic) were lower when the soil has been preused by conspecifics in a former experiment [START_REF] Mathieu | Habitat quality, conspecific density, and habitat pre-use affect the dispersal behaviour of two earthworm species, Aporrectodea icterica and Dendrobaena veneta, in a mesocosm experiment[END_REF]. On the contrary, in the species E. andrei, population density would trigger dispersal. Positive density dependence in dispersal behaviour due to resource depletion is quite widespread in animals in general [START_REF] Murray | Dispersal in Vertebrates[END_REF][START_REF] Waser | Does competition drive dispersal?[END_REF]. [START_REF] Uvarov | Inter-and intraspecific interactions in lumbricid earthworms: Their role for earthworm performance and ecosystem functioning[END_REF] showed that more than 85% of the studies found negative effects of population density on earthworm demographic parameters, whatever their functional group. For instance, negative density dependence was found for growth rates and maturation in E. fetida, E. andrei and L. rubellus. The lack of a clear relationship between dispersal rate and population density in E. fetida and L. rubellus did not verify that density dependent dispersal is a key mechanism regulating population dynamics in the rapidly reproducing species such as E. fetida and most epigeic earthworms [START_REF] Kammenga | Explaining densitydependent regulation in earthworm populations using life-history analysis[END_REF].

It would suggest that, in these two species, either dispersal response to population density would not covary with fitness improvement, or dispersal costs were higher than its benefits [START_REF] Ronce | How does it feel to be like a rolling stone? Ten questions about dispersal evolution[END_REF]. We may also hypothesize that the density tested in our experiment (i.e. 1, 10, 20, 30) were under the threshold needed to induce the costs associated with crowding.

Comparison of dispersal and diffusion behaviour between functional groups

The comparison of dispersal behaviour of epigeic earthworms to the ones of anecics and endogeics, investigated in a previous study [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF], suggests that dispersal rate of endogeic earthworms from low habitat quality was lower than the one of the epigeic and anecic earthworms (see Fig. 4). This would suggest that either endogeic earthworm have a broader ecological niche, a reduced sensitivity to habitat quality or poorer dispersal capacities (i.e. higher dispersal costs) compared to the two other functional groups. This last explanation would verify our hypothesis that earthworms experiencing high interactions with the aboveground environment would be more exposed to environmental stochasticity and therefore likely evolved higher dispersal capacities. Future studies should disentangle these hypotheses by investigating the effects of dispersal choice on fitness (i.e. survival and reproductive success) or health parameters (e.g. weight variation over time).

On the contrary, none of the three functional groups showed a clear response to population density (see Fig. 6) and the three groups exhibited similar behaviour in dispersal rate, in mean distance crossed and in the tendency to follow conspecifics in an homogeneous environment (see Table 2). Our study showed that there was significant variability in the dispersal behaviour of epigeic earthworm. Such variability had also been measured in anecic and endogeic groups [START_REF] Caro | Are dispersal behaviours of earthworms related to their functional group?[END_REF]. The high variability of dispersal behaviour among species of the same functional group may explain the lack of clear dispersal difference between earthworms' functional group. It suggests that functional group would not be the main factor explaining dispersal behaviour variability between species. It would be interesting to perform similar experiments on a greater number of species per functional group to lower the intragroup (i.e. within functional group) variation but also to extend the time and spatial scale of the diffusion experiment which may help measuring variations in diffusion behaviour dynamics along time in the longer term. Alternatively, functional groups may have to be redefined in view of species diffusion and dispersal behaviours; although these behaviours directly influence burrowing activities, which has known impact on soil processes, soil invertebrate species are mainly categorised according to their foraging behaviour and as a consequence to their position in foodwebs [START_REF] Lavelle | Diversity of soil fauna and ecosystem function[END_REF][START_REF] Mariani | Régime alimentaire d'un ver de terre anécique des savanes colombiennes: une remise en question des types écologiques[END_REF]. Indeed, several species, although belonging to a same functional group, inhabit different habitat. For instance, E. fetida and E. andrei habitat (i.e. mainly composts and manures) are very different from L. rubellus one (i.e. mainly leaf litter but also soil), despite they are all classified as epigeics. This may explain the low difference of dispersal behaviour between functional groups. Note also that the two comparative studies had slightly different experimental designs (i.e. presence of leaves and amount of soil). Because epigeic earthworms mainly live on the leaf litter at the soil surface, we assume that their dispersal behaviour only slightly depends on the amount of soil. However, the presence of leaf litter, by influencing food and shelter availability, likely affect dispersal behaviour of anecics, and to a lower extent of endogeic earthworms. Moreover, a deeper soil (> 15 cm depth) may have been more relevant in the previous study to mimic a high quality habitat for anecic earthworm. Consequently, we may think that in previous experiments, dispersal rate in high quality habitat might have been lower in these groups if the soil had been topped with leaf litter or had been thicker. However, dispersal rate of anecic and endogeic earthworms was already very low in high quality habitat (lower than 20%), suggesting that the amount of food and soil was sufficient for the experimental time and would not have significantly modulated dispersal behaviour.

In addition to identifying important factors influencing earthworms' dispersal choice, our study underlines the high variability in the responses to these factors among species and functional groups of earthworms. These complex patterns in dispersal behaviours contribute to earthworm community composition and as a consequence to ecosystem functioning. Efforts in maintaining ecosystem functioning and high ecosystem services, that often relies on species and functional diversity [START_REF] Balvanera | Quantifying the evidence for biodiversity effects on ecosystem functioning and services: Biodiversity and ecosystem functioning/services[END_REF][START_REF] Mace | Biodiversity and ecosystem services: a multilayered relationship[END_REF], should be optimized by taking into account such diversity in environmental factors sensitivity and consequently in dispersal behaviours.

Acknowledgments

Fig. 1

 1 Fig. 1 Schematic representation of the experimental designs. The soil disposition in the mesocosms is symbolized by rectangles whose colour

Fig. 2 .

 2 Fig. 2. Dispersal rate (mean ± se) of three epigeic earthworm species inoculated in low quality

Fig. 3

 3 Fig. 3 Relationship between dispersal rate and population density (i.e. 1, 10, 20 or 30

Fig. 4

 4 Fig. 4 Dispersal rate (mean ± se) of epigeic (circles), anecic (triangles) and endogeic (squares)

Fig. 5

 5 Fig. 5 Relationship between dispersal rate (calculated as the number of earthworms counted

Fig. 6

 6 Fig. 6 Partial Least Squares -Discriminant Analysis (PLSDA function in R) on dispersal

Fig. 1

 1 Fig. 1

Fig. 2

 2 Fig. 2

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

Fig. 6

 6 Fig. 6

the interactive effect between habitat quality or population density and functional group (i.e. epigeic, anecic, endogeic) was the explanatory variable and species identity was added as a random factor. Finally, we performed a Partial Least Squares -Discriminant Analysis (PLS-DA; mixOmics and RVAideMemoire packages in R) to compare dispersal behaviour between functional groups as a combination of the diffusion rate in homogeneous environment, the mean distance crossed in homogeneous environment, the tendency to follow conspecifics, the mean sensitivity to habitat quality (calculated as the difference between the mean dispersal rate from low quality habitat and from high quality habitat within a species), the sensitivity to population density (calculated as the slope of the regression between dispersal rate and population density), averaged for each species. PLS-DA is a supervised multivariate analysis that classifies groups of observations (i.e. functional groups) based on multivariate predictors (results from the different experiments; i.e. diffusion rate, mean distance crossed, tendency to follow conspecifics, sensitivity to habitat quality and sensitivity to population density), and which helps to identify which variables best discriminate the groups.

Statistical analyses were performed using R software (version 3.1.2). Full models included all factors and covariates and all their interactions. We retained final models based on their AIC.

Results

Diffusion and dispersal behaviour in epigeic earthworms

Homogeneous environment (experiment 1) -Diffusion behaviour in an homogenous

environment was significantly different between species (χ²df=2=14.97, P=0.001): diffusion rate was lower in L. rubellus than in E. fetida (F1,8=6.57, P=0.010) and E. andrei (F1,8=14.18, P<0.001; Table 1). The mean distance crossed was also significantly different between species (F2,12=5.61, P=0.019): the mean distance crossed was shorter in L. rubellus than in E. Zirbes, L., Deneubourg, J.-L., Brostaux, Y., Haubruge, E., 2010