Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus)

Journal of Arid Environments xxx (2017) 1e8

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Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus) ~o Carlos Campos a, c, Carola Sanpera b, d, Xavier Santos a, *, Sandra Navarro b, Joa  Carlos Brito a, c Jose ~o em Biodiversidade e Recursos Gen rio de Vaira ~o, 4485-661 Vaira ~o, Portugal CIBIO/InBIO, Centro de Investigaça eticos, Universidade do Porto, Campus Agra Dep. Biologia Evolutiva, Ecologia i Ci encies Ambientals, Universitat de Barcelona, Avgda. Diagonal 643, 08028 Barcelona, Spain c Departamento de Biologia da Faculdade de Ci^ encias da Universidade do Porto, Rua Campo Alegre, 4169-007 Porto, Portugal d Institut de Recerca de la Biodiversitat (irBio), Campus Sud, Universitat de Barcelona, Avgda. Diagonal 643, 08028 Barcelona, Spain a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 August 2016 Received in revised form 8 September 2017 Accepted 13 September 2017 Available online xxx

Stable isotope analysis is a widespread tool in ecological studies of diet composition and habitat use. In deserts, freshwater environments constitute threatened local hotspots of biodiversity. In these environments, stable isotopes may help to describe trophic ecology of top-predators. We examined stable carbon (d13C) and nitrogen (d15N) isotopes from scute keratin samples of 33 Crocodylus suchus and muscle samples from 39 potential prey collected in Southern Mauritania. Isotope ratios were compared among crocodiles according to size (non-adult and adult), and habitat (rock pools and floodplains). There was a significant interaction effect of habitat and size on crocodile d13C values. Whereas d13C was similar for all crocodiles collected in rock pools, adults had lower signatures than non-adults in seasonal floodplains. d15N indicated an ontogenetic dietary shift with adult crocodiles foraging on prey from higher trophic level. Standard ellipse areas showed wider isotopic niches for adult than non-adult crocodiles, and within adults, for those from floodplains than those from rock pools. These environments are small, seasonal, overexploited for livestock watering, and polluted. They support very small and isolated crocodile populations. This study is aimed to provide conservation authorities with baseline information to strictly protect water-bodies where these predators subsist in arid environments. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Isotopes Crocodile trophic ecology Deserts Conservation of freshwater habitats

1. Introduction After the mid-Holocene humid period (around 6.000 years ago), arid conditions developed throughout North Africa and culminated in the formation of the Sahara, the largest warm-climate desert in the world (Schuster et al., 2006). In this arid environment, some species adapted to extreme dryness and developed physiological mechanisms to avoid water-loss. Other species need to be permanently in contact to water bodies (Brito et al., 2014). For aquatic species, water is the most important limiting and temporally variable resource in deserts. Their suitable habitats are naturally patchy and connectivity is important for their populations. Populations of these freshwater species are often irregularly distributed in mountains, where environmental conditions are less harsh and have more available water (Brito et al., 2014). These patches

* Corresponding author. E-mail address: [email protected] (X. Santos).

constitute local hotspots of biodiversity and may contain threatened endemic taxa (Vale et al., 2015). One of the iconic organisms living in the Sahara and completely dependent of water resources is the West African crocodile (Crocodylus suchus). In Mauritania, populations persist mostly in two habitat types: mountain rock pools (locally known as gueltas) and ^moûrts) that are surseasonal floodplains (locally known as ta rounded by inhospitable desert areas (Brito et al., 2011). Most water-bodies have extremely small crocodile populations (Campos et al., 2016). Moreover, water-bodies are affected by overexploitation, faecal contamination and eutrophication (Tellería n et al., 2014; Vale et al., 2015). Mauret al., 2008; Velo-Anto itanian crocodiles have high levels of genetic structure and limited  n et al., 2014). Recent gene flow among populations (Velo-Anto surveys suggest that C. suchus is declining or extirpated throughout much of its distribution, and currently inhabits the western and central-southern Sahara, Sahel and tropical Guinean gulf, as well as the Congo Basin and parts of Uganada (Brito et al., 2011; Hekkala

https://doi.org/10.1016/j.jaridenv.2017.09.008 0140-1963/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

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X. Santos et al. / Journal of Arid Environments xxx (2017) 1e8

et al., 2011; Shirley et al., 2015). Data on the biology, ecology, and behaviour of C. suchus is notably unavailable, which currently hinders extensive conservation planning. Stable isotope analysis (SIA) is a widespread tool in studies of diet composition, trophic interactions, and habitat use and migration (Caut, 2013). The most commonly used elements in ecological SIA are carbon (C) and nitrogen (N). Since the carbon isotope ratio (d13C) changes minimally (~1‰) as carbon moves through food webs (Rounick and Winterbourn, 1986), it is commonly used to evaluate the source of carbon at the base of food webs. The nitrogen isotope ratio (d15N) in consumer tissues is typically considered to be enriched by ~3‰ relative to that in the diet (Minagawa and Wada, 1984); it is thus commonly used to estimate trophic position along food chains (Vanderklift and Ponsard, 2003). Thus, d13C can be used to track the original source of a consumer’ nutrients, and d15N can be used to estimate a consumer relative trophic position, i.e. higher d15N indicates higher trophic position (Post, 2002). We quantified carbon and nitrogen stable isotope ratios (d13C and d15N respectively) to infer the trophic ecology of C. suchus in Mauritania with emphasis on ontogenetic (adults and non-adults individuals) and spatial (habitat types) variation. Additionally, we measured stable isotope ratios from potential crocodile prey since identification of baseline isotopic signatures is necessary to infer predator's trophic position and carbon source (Post, 2002). Due to multiple logistic constraints, this technique is an advantageous alternative and a complementary tool to describe crocodile trophic ecology over traditional methods, such as stomach content analysis, scat analysis and feeding observations (Radloff et al., 2012; Wheatley et al., 2012; Caut, 2013). Moreover, stable isotope analysis has been proposed as a surrogate of measuring trophic niche width (Bearhop et al., 2004). This approach provided important advances in food-web ecology in recent years (Layman et al., 2012). We expect that our results could be useful for the management of C. suchus populations and the Mauritanian freshwater habitats in which crocodiles persist. 2. Material and methods 2.1. Study area The study area comprises the mountains of Tagant, Assaba and , in southern Mauritania (Fig. 1). These massifs are the origin Afolle of a number of seasonal rivers organized in six seasonal hydrographic sub-basins that occasionally flow from north to south up to the Senegal River. These seasonal rivers generally are composed of rock pools in mountain slopes and seasonal floodplains at the foothills of the mountains (Campos et al., 2012; Vale et al., 2015). Within the study area, altitude ranges from 9 m a.s.l. on the Senegal River to 625 m a.s.l. in the Tagant. Climate is characterized by three seasons: a cool and dry period from November to February, a hot and dry period from March to June, and a wet season from July to October, with most precipitation in August and September. Annual precipitation has a north-south gradient, and it ranges from 100 mm in the northern desert areas to 900 mm in the extreme southern region of the study area. Variation in average annual temperature is relatively small and tends to follow the altitudinal gradient (Cooper et al., 2006; Brito et al., 2011). 2.2. Water-bodies In Mauritania, C. suchus are mostly found in two freshwater ^moûrts (Fig. 1). Gueltas are located upstream habitats, gueltas and ta of narrow valleys at the base of the mountains. Generally, water is only available during the rainy season (July to September), when torrential waterfalls fill up the pools. The small area of rock pools

(ranging between 0.001 ha and 1.0 ha) restricts their carrying capacity and crocodile populations usually do not exceed eight individuals, and in some cases there are only one to three crocodiles ^moûrts are per pool (Brito et al., 2011; Campos et al., 2016). Ta located on the foothills of the mountains which are larger in size than gueltas and frequently reach more than 1000 ha; water is usually shallow and floodplains are mostly dry during the dry season (October to June), forcing crocodiles to find shelter in nearby rock outcrops during this period. In this habitat type, crocodiles can number up to 30e40 individuals (Brito et al., 2011). 2.3. Fieldwork and sample collection We collected scute keratin samples of C. suchus (n ¼ 33) and muscle of their potential prey (n ¼ 39) from fieldwork expeditions conducted from 2003 to 2015. We opportunistically captured crocodiles by hand or using hand nets (n ¼ 15), and immediately released them at the point of capture after clipping a 5 mm piece of tail tissue for isotopic analysis. We also collected samples from dead crocodiles found near water-bodies (n ¼ 15) or body remains that included scute samples (n ¼ 3). We obtained tissue samples following ethical guidelines for use of live reptiles (Brito et al., 2011). All samples were stored in 70% ethanol. We sampled potential prey (insect, fish, amphibian, and mammal) in and around water bodies. We took whole insects (n ¼ 3 aquatic beetles) or vertebrate muscle tissue. Fish samples were collected from the genera Coptodon/Sarotherodon (n ¼ 8), Clarias (n ¼ 5), Barbus (n ¼ 2), Schilbe (n ¼ 1) and Allestes (n ¼ 1), and amphibian samples from the genera Hoplobatrachus (n ¼ 7) and Amietophrynus (n ¼ 2). We also collected mammal tissues from cow (n ¼ 4), goat (n ¼ 2), sheep (n ¼ 1) and dromedary (n ¼ 2) specimens found dead near water-bodies. Although we found feeders around water bodies, this birds’ keratinized structure is not assimilated by crocodiles. For this reason, we decided to exclude these samples (bird feeders) from lab analyses, and bird muscle was not available. All samples were preserved in 70% ethanol for further lab procedures. Some studies have demonstrated that ethanol did not affect isotope signatures (Hobson et al., 1997; Sarakinos et al., 2002). For example, Barrow et al. (2008) observed in tissues of freshwater and sea turtles subject to lipid extraction, there were no differences between samples dried at 60  C and those preserved in ethanol. We sampled prey items at the same localities as crocodiles (Fig. 1), although some of the prey types had to be collected in some other water-bodies because of fieldwork logistic constraints. We acknowledge that pooling samples from different sites could increase prey isotope variance if sources vary spatially (Layman et al., 2012), thus losing power to detect some small-scale dietary differences. 2.4. Sample preparation and isotope analyses We did not extract lipids from crocodile scales as it mainly corresponded to keratin and because this tissue has low fat content. Crocodile scales have a tough outer keratinized epidermis and a rigid dermal core of extremely dense collagen. Keratin is metabolically inert after formation, and stable isotope values reflect the diet at the time of scale formation (Radloff et al., 2012). Scales were cleaned in 0.1 M NaOH solution for 30 s to remove waste. Afterwards, samples were thoroughly washed in distilled water and dried at 60  C for 24 h. Prey muscle tissue samples were lipid extracted following the Folch method (1957) by rinsing in a 2:1 chloroform: methanol solvent, as tissue lipid content can influence carbon isotope values.

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

X. Santos et al. / Journal of Arid Environments xxx (2017) 1e8

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Fig. 1. Geographic distribution of samples from Crocodylus suchus and their prey in southern Mauritania (top), and two representative water-bodies inhabited by crocodiles in ^moûrt Bouga ^ri (bottom right). Mauritania: Guelta Metraoucha (bottom left) and Ta

Carbon stable isotope values decrease when lipid concentration increases in tissues (Post et al., 2007), and a lipid extraction is recommended when C:N ratio > 3.5 for aquatic animals and >4 for terrestrial animals (Post et al., 2007). Ruiz-Cooley et al. (2011) studied the effect of ethanol on C and N isotopic signatures as well as on C:N ratios of muscle from aquatic organisms over time (400 days of alcohol preservation) and observed that ethanol increased d13C and d15N by 0.8‰, but lipid extraction removed the effect of ethanol. Thus, lipid extraction from tissue samples ensures the lack of isotopic bias due to collection of samples during long periods and its preservation in ethanol. Once dried, we individually grounded the hardest samples to a fine powder with liquid nitrogen. We weighed 0.25e0.5 mg of each sample and placed individually into tin capsules for d13C and d15N analyses. Analyses were carried out at the Centres Científicocnics from the University of Barcelona, with a Thermo-Finnigan Te Flash 1112 (CE Elantech, Lakewood, NJ, USA) elemental analyzer coupled to a Delta-C isotope ratio mass spectrometer via a CONFLOIII interface (Thermo Finnigan MAT, Breman, Germany). We expressed stable isotope ratios in conventional delta notation as parts per thousand (‰). Reference materials from the International Atomic Energy Agency (IAEA CH6, IAEA CH7 and USGS 24 for C, and IAEA N1, IAEA N2 and IAEA NO3 for N) were inserted every 12 samples to calibrate the system and compensate for any drift over time. d 15N values are expressed relative to atmospheric nitrogen (VAIR) and d 13C values are expressed relative to Pee Dee Belemnite (VPDB). Replicate assays of standards indicated analytical

measurement errors of ±0.1‰ and ±0.2‰ for d13C and d15N, respectively. 2.5. Data analysis We examined d15N and d13C from crocodile scutes by general linear models (GLM) using body-size class (i.e. adult and nonadult), habitat (rock pool and seasonal floodplains), and its interaction as predictor variables. Crocodile body-size was considered due to the general ontogenetic dietary variation in crocodiles (Hutton, 1987; Tucker et al., 1996; Platt et al., 2006, 2013; Rosenblatt et al., 2015). For our C. suchus samples, adults were >200 mm of head length, and non-adults < 200 mm which roughly corresponded to 1.5 m body length (authors, personal observation). This body size does only constitute an approxiamte measure of sexual maturity due to the lack of feasible size/age/maturity relation in the Mauritanian populations. In the study area there is a rainfall gradient with latitude as the northernmost localities at Tagant Mountains are more arid than the southernmost ones at the Senegal River (Hijmans et al., 2005). Given that d15N and d13C signatures may be affected by this latitudinal gradient (Farquhar et al., 1982), we assessed putative latitudinal effects on isotopic signatures by including the latitude at which samples were collected in the GLMs. To meet the assumption for ANOVA, we examined residuals of the dependent variable on the grouping factors. Similarly, we examined differences in d13C and d15N values from fish, amphibian and mammals samples by GLM according to water-body

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

X. Santos et al. / Journal of Arid Environments xxx (2017) 1e8 Table 1 General linear model results of d13C and d15N values as dependent variables for crocodiles Crocodylus suchus and their prey. For each analysis, degrees of freedom (df), F values and probability are included for each factor and interaction. **P < 0.01; * 0.05 < p < 0.01;. 0.1 < p 0.05. Factors

df

d13C

d15N

F

P

F

P

Crocodiles

Latitude Body size (BS) Habitat (H) BS * H

1 1 1 1

0.382 5.04 3.532 7.002

0.5 0.03* 0.07 0.01*

12.384 9.204 0.18 0.552

0.002** 0.005** 0.7 0.5

Prey

Latitude Prey type (PT) Habitat (H) PT * H

1 2 1 2

1.027 4.974 0.783 2.564

0.3 0.01* 0.4 0.09

0.03 2.609 0.302 0.696

0.8 0.09 0.6 0.5

floodplains was smaller than in rock pools (Fig. 2a). The GLM showed higher d15N in adult than in non-adult crocodiles both in rock pools and seasonal floodplains (Fig. 2b), and a significant effect with latitude (higher values with latitude, Appendix Fig. A2). In contrast, there were no differences in d15N according to habitat or its interaction with crocodile body-size (Table 1). Layman's metrics (NR, CR, CD, and MNND) showed higher values for crocodile samples collected in floodplains than in rock

-14

a

δ13C

-16

-18

-20

-22

Adult

Non-adult

15

b 14

13

15

(rock pool and seasonal floodplains) where samples were collected and the prey category/taxa e habitat interaction. We did not include aquatic insects in the GLM as these were only collected in rock pools. For this reason, we also performed an ANOVA analysis including only prey collected in rock pools. We conducted the statistical analyses with Statistica 10.0 (Statsoft, 2010). We inferred trophic niche width for adult and non-adult crocodiles from carbon and nitrogen signatures using several metrics (Layman et al., 2007). Layman metrics represent community-wide measures of: 1) trophic diversity, e. g., 15N and d13C ranges (NR and CR respectively), the convex hull area (TA) occupied by all individuals of each group in the d15N - d13C biplot, and the mean distance to the d15N and d13C centroid (CD); and 2) trophic redundancy, such as the mean nearest neighbour distance (MNND) that characterizes communities where individuals have similar trophic ecologies. These metrics can be biased by low sample size and outliers; for this reason, Jackson et al. (2011) proposed the calculation of the standard ellipse areas (SEA) with a correction for small sample sizes (SEAc). We calculated SEAc following Jackson et al. (2011) implemented in the R package SIBER (Parnell and Jackson, 2013). SIBER applies a Bayesian approach that takes into account uncertainty of the data, computes SEA.B (Bayesian estimation of SEA), and compares isotopic community metrics between groups (Jackson et al., 2011). We inferred diet reconstruction of C. suchus using prey isotopic values with the R package SIAR (Parnell and Jackson, 2013). Two major parameters are the basis for stable isotope interpretation (Caut, 2013): the turnover rate (i.e. the time an isotope takes to be assimilated into the consumer tissue), and the discrimination factor (i.e. D, the difference between the stable isotope composition of a given tissue and that of the diet). It is well known that discrimination values and turnover rates can vary considerably among species and tissue types because of variable metabolic rates and pathways (Caut et al., 2009). So far, only three studies have used stable isotopes in crocodilians: the Nile crocodile Crocodylus niloticus (Radloff et al., 2012), the American alligator Alligator mississippiensis (Rosenblatt and Heithaus, 2013), and the caiman Caiman latirostris (Caut, 2013). Rosenblatt and Heithaus (2013) and Caut (2013) used stable isotopes to determinate diet of crocodilians, but only the former study was performed on scutes. Therefore, we selected the discrimination factor and turnover rates from Rosenblatt and Heithaus (2013) to reduce bias related to values in other tissues (Steinitz et al., 2016). The estimated d13C and d15N complete turnover time for scales was 590 and 414 days respectively (Rosenblatt and Heithaus, 2013). The mean Dd13C value for scales was 0.61‰ ± 0.12‰ SE, whereas the mean Dd15N was 1.22‰ ± 0.08‰ SE, a value lower than assumed for this isotope in all tissues (Rosenblatt and Heithaus, 2013). Within each prey category, we identified species belonged to the same trophic guild: fish genera were mostly omnivorous (Yatuha et al., 2013; Dadebo et al., 2014), amphibians were mostly arthro€ del, 2011) and pophagous (Akani et al., 2011; Hirschfeld and Ro mammals herbivorous. Thus, although each sample was analyzed individually, within each prey category the data were pooled for statistical analysis given the small sample size.

δ N

4

12

3. Results 3.1. Stable isotope results for crocodile according to body-size and habitat Based on crocodile d13C values (Appendix Table A.1), the GLM showed a significant interaction between body-size and habitat (Table 1). Whereas non-adult crocodiles had similar d13C signatures in both habitat types, for adults, the d13C signature in seasonal

11 Adult

Non-adult

Fig. 2. Boxplot of the mean and standard error d13C (a) d15N (b) values of adult and non-adult crocodiles C. suchus in Mauritanian rock pools (squares) and seasonal floodplains (triangles).

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

X. Santos et al. / Journal of Arid Environments xxx (2017) 1e8

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3.2. Stable isotope values of crocodile prey and diet reconstruction

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For d13C, the GLM showed differences among prey types and also in the interaction between prey types and habitats (Table 1). Fish collected on seasonal floodplains had lower d13C signatures, whereas all prey samples collected on rock pools had similar d13C values (Fig. 4a). For d15N, we found marginal variation among prey types, but we did not find differences related to habitat (Table 1). In both habitats, mammals have lower d15N values (i. e. lower trophic levels) than fish and amphibians (Fig. 4b). We have not included aquatic insects in the GLM as we only collected this prey type in

-26

TA SEA SEAc

Mammal

Pisces

b

14 13 12 11 10 9

Seasonal floodplains

Rock pools NR CR CD MNND

Amphibia

15

δ 15 N

Table 2 Layman's metrics for crocodiles collected in rock pool and seasonal floodplain habitats, and convex hull and standard ellipse areas for adult and non-adult crocodiles collected in both habitat types. 15N range (NR), d13C range (CR), mean distance to the d15N and d13C centroid (CD), mean nearest neighbour distance (MNND), convex hull area (TA), standard ellipse area (SEA), and standard ellipse area corrected for small sample sizes (SEAc).

a

-14

δ 13 C

pools (Table 2), this indicates higher trophic diversity and lower redundancy in the former habitat. Similarly, the total convex hull areas (TA) and standard ellipse areas (SEA and SEAc) for adult and non-adult crocodiles in rock pools and seasonal floodplains indicated differences between both crocodile-size groups (Table 2 and Fig. 3). The Bayesian approach confirmed differences between groups of crocodiles as SEA.B was higher for adult than for nonadult crocodiles in rock pools (probability 0.83) and floodplains (0.89 respectively). Likewise, SEA.B was higher for floodplain than for rock pool adult crocodiles (probability 0.76), and no differences in SEA.B were detected for non-adult crocodiles collected in both habitat types.

5

0.949 0.366 0.542 1.084

1.620 3.351 1.873 3.746

Adults

Non-adults

Adults

Non-adults

24.455 9.633 10.596

12.555 6.273 7.057

18.404 11.707 14.049

3.306 4.210 6.315

8

Amphibia

Mammal

Pisces

Fig. 4. Boxplot of the mean and standard error of d13C (a) d15N (b) values for three prey crocodile types collected in Mauritanian rock pools (squares) and seasonal floodplains (triangles).

rock pools. An ANOVA analysis including this prey type showed that insects had the lowest d13C and the highest d15N values compared to other prey types collected in rock pools (Appendix Fig. A3). The d13C - d15N biplot derived from SIAR with average-prey and individual-crocodile values showed a high overlap of isotope signatures (Appendix Fig. A4). Overlap between crocodiles and prey suggests that these prey items apparently cover the dietary resources of the predator. However, the overlap of isotope signatures among prey suggests low discriminatory power to infer the diet reconstruction of Mauritanian crocodiles, i.e. which prey type could be preferred for non-adult and adult crocodiles. 4. Discussion

Fig. 3. Standard ellipse areas based on the d13C - d15N biplot of adult (circles) and nonadult crocodiles (triangles) Crocodylus suchus collected in Mauritanian rock pools and seasonal floodplains. Ellipse colours: rock-pool adults (green); rock-pool non-adults (blue); floodplain adults (black); floodplain non-adults (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Despite limitations concerning our sampling design, i.e. multiple years and sites, our study shows consistent dietary patterns throughout crocodile body sizes and habitats. Indeed, stable isotope analysis of C. suchus and its prey living in isolated waterbodies of Mauritanian arid regions revealed some basic traits of this top predator. 4.1. Ontogenetic dietary shift Our results of d15N signatures indicated an ontogenetic dietary

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

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X. Santos et al. / Journal of Arid Environments xxx (2017) 1e8

shift that was consistent throughout the two habitat types inhabited by C. suchus. Higher d15N signatures from adult C. suchus demonstrate that larger individuals forage on prey of higher trophic position (Vanderklift and Ponsard, 2003). In addition, we detected that adult individuals in both habitats had larger standard ellipse areas than non-adult crocodiles, suggesting wider trophic niches (Bearhop et al., 2004). Ontogenetic dietary shift linked to morphological growth is a general pattern in the Animal Kingdom, especially in ectotherm species in which individuals of different ages greatly differ in body size (e.g. sharks, Lowe et al., 1996; snakes, Vincent et al., 2007). In crocodiles, the average increase in body size over the lifespan is three to five orders of magnitude (Radloff et al., 2012). Specifically, skull growth and cranium allometric growth (Hall and Portier, 1994) would facilitate adult crocodiles including Mauritanian C. suchus to handle a wider variety of prey compared to non-adult individuals (Tucker et al., 1996). This trend coincides with a general pattern observed in many crocodile studies both based on diet (Hutton, 1987; Tucker et al., 1996; Platt et al., 2006, 2013; Rosenblatt et al., 2015) and stable isotope (Radloff et al., 2012; Rosenblatt and Heithaus, 2013) analysis. Several studies reported an ontogenetic shift from aquatic small invertebrates foraged on by young crocodiles to predominantly vertebrate prey among larger crocodiles (Cott, 1961; Tucker et al., 1996). However, this prey-size ontogenetic shift is not the exclusive trend in crocodiles. For example, adult C. suchus in Uganda continued foraging on aquatic beatles (Thorbjarnarson and Shirley, 2009), adult C. acutus in Belize subsisted primarily on marine crustaceans (Platt et al., 2013), and adult C. moreletti consumed gastropod, fish and crustaceans (Platt et al., 2006). Thus, dietary patterns described for crocodile species are rather opportunistic and eclectic, and mostly dependent on habitat type (Platt et al., 2006). This fact can be especially relevant in habitats where low productivity, limited water abundance, and seasonal climatic variability can drastically reduce the available resources (Thorbjarnarson and Shirley, 2009). We suspect that this is the case of C. suchus in arid environments of Mauritania, where our isotope results detected an effect of habitat on crocodile diet. 4.2. Habitat effect on crocodile diet

d13C signature changes minimally through food webs (Rounick and Winterbourn, 1986), and shows original carbon source of a consumer’ nutrients, e.g. type of habitat (Post, 2002). Our GLM detected significant d13C differences in the interaction between crocodile size and habitat; adults collected in seasonal floodplains showed lower d13C signatures than adults collected in rock pools and all non-adult crocodiles. This result suggests that adult crocodiles from floodplains forage on a higher proportion of terrestrial prey than crocodiles from rock pools. Interpopulation differences in the proportion of terrestrial prey have been reported in C. niloticus (Hutton, 1987; Woodborne et al., 2012). For example, Hutton (1987) indicated that the diet of C. niloticus in Zimbabwe changed from insects to fish and birds in parallel to changes in habitat. Woodborne et al. (2012) also detected differences among five C. niloticus populations in South Africa and Mozambique, confirming the presence of terrestrial food remains in pellets from populations with lower d13C signatures. Similar to these two studies, Thorbjarnarson and Shirley (2009) showed dietary differences in C. suchus from Uganda according to the habitat and the season. The most plausible explanation for why adult C. suchus forage on a higher proportion of terrestrial prey in floodplain than in rock pools is prey availability. Floodplains are temporary water-bodies usually located in more accessible sites and with larger water surface areas compared to rock pools, attracting a greater number of

terrestrial vertebrates (Cooper et al., 2006; Brito et al., 2011). Thus, availability of resources at each habitat type and functional constraints according to crocodile size would interact to understand d13C results of the GLM model. In rock pools, food availability is presumably limited and dietary segregation between non-adult and adult crocodiles would be related to a shift from small to large prey items as crocodiles increase in size. In contrast, higher prey availability in floodplains could cause diet diversification, especially in a generalist forager like C. suchus. Highly mobile predators such as crocodiles may travel long distances in the face of seasonal peaks of food availability (Rosenblatt and Heithaus, 2011). Oueds (rivers) are apparently crucial for crocodile dispersal in the Sahara desert between gueltas ^moûrts (Campos et al., 2016). This is, however, unlikely in and ta Mauritania as the arid conditions between water points limit dispersal to rainfall periods and attempted dispersal outside of rainy periods is a significant source of mortality in C. suchus (Brito et al., 2011). Despite the increase of trophic opportunities due to increase in size, wider trophic diversity in adults (a conclusion supported by Layman metrics and SEA.B estimations of adult and non-adult C. suchus) is not a general pattern in crocodiles (e.g. Tucker et al., 1996; Platt et al., 2006; Wallace and Leslie, 2008). In tropical and marine environments for example, unlimited food resources allowed food partitioning among sympatric crocodile species (Magnusson et al., 1987) or among crocodile groups within species (Platt et al., 2006, 2013). We acknowledge that there are still several gaps in our ecological study. For example, there is limited evidence that floodplains have higher resources availability than rock pools (see also Cooper et al., 2006), we lack a complete list of potential prey in both habitat types, and we do not have isotopic signatures of birds, a potential important food source for crocodiles in floodplains (Wallace and Leslie, 2008; Thorbjarnarson and Shirley, 2009). Despite these limitations, our results support that multiple factors, such as prey availability, habitat, and body size, are driving the diet of these top (generalist) predators (Hutton, 1987; Magnusson et al., 1987; Wallace and Leslie, 2008; Radloff et al., 2012; Rosenblatt et al., 2015). In Mauritania, these factors are most likely constrained by the local harsh conditions, which may greatly influence the trophic ecology and demography of C. suchus populations (Brito et al., 2011). 4.3. Isotopic signature of prey and diet reconstruction Isotopic signatures of crocodile prey (fish, amphibians and mammals) partially matched their dietary traits. For example, d15N signatures were smaller for mammals, in accordance with the herbivorous diet of the species sampled. In contrast, we did not identify differences in d15N signatures between fish and amphibians. Although we do not know the diet of fish and amphibian species in the study area, the available literature in the region suggests that fish species are mostly omnivorous and amphibian €del, species insectivorous (Akani et al., 2011; Hirschfeld and Ro 2011; Yatuha et al., 2013; Dadebo et al., 2014), causing similar d15N signatures and similar trophic level between both groups. The analysis of d13C signatures identified differences between rock pools and floodplains. d13C values were similar for all rock pool prey, suggesting lower habitat and resource heterogeneity in these small water bodies. In contrast, floodplain fish showed lower values than most terrestrial amphibians and herbivorous mammals. This result might be related to the origin of fish that are essentially dispersers that colonise floodplains after a flooding event as these seasonal water bodies remain dry a significant part of the year (Brito et al., 2011). Our opportunistic sampling design precludes experimental confirmation of this hypothesis.

Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008

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Inference from our dietary reconstruction for C. suchus was limited, likely for two predominant reasons. First, C. suchus might have an eclectic and opportunistic diet, foraging on all available (and scarce) resources. The large standard ellipse areas of adults would support this statement. Second, isotopic signatures of prey mostly overlap, and predator diet reconstruction can only be achieved when prey d values do not overlap (Layman et al., 2012). We suspect several additional factors limiting our capacity to full reconstruct C. suchus diet in Mauritanian water bodies: 1) our sample size was small both for crocodiles and prey; 2) our sampling design was incomplete and prey were captured opportunistically, i.e. all prey types were not available for this isotopic study from all the localities with crocodile samples; 3) some prey types consumed by crocodiles were under-represented notably birds (a prey type consumed by C. suchus in other populations like Kidepo Valley, Uganda; Thorbjarnarson and Shirley, 2009), and some reptiles with aquatic habits such as Varanus sp.; 4) temporal and spatial variation in prey resources (and their isotope signatures) might add additional among-individual variability to the crocodile isotopic values (Willson et al., 2010). Future analysis of crocodile scats can contribute to describe C. suchus diet. For crocodiles, scat analysis can have two limitations: underrepresentation of particular prey types due to their quick rate of digestion, e.g., amphibians (Thorbjarnarson and Shirley, 2009), and pseudoreplication, i.e. many scats from a single individual, a problem especially in small water bodies with extremely low population size as in Mauritania (Brito et al., 2011). 4.4. Conservation remarks To successfully conserve C. suchus populations and their aquatic ecosystems in Mauritanian mountains, it is helpful to know their basic life-history traits. We identified critical dietary patterns that can help to elucidate the West African crocodile trophic ecology. Specifically, we identified variation in dietary patterns according to water-bodies inhabited by crocodiles. Both habitats have many threats related to the extent of water-bodies, crocodile population densities, and water seasonality. This is especially critical in gueltas which are often overexploited resulting in contaminated water quality (Tellería et al., 2008; Brito et al., 2014). Water pollution may jeopardize the presence of crocodile prey and at the same time may affect crocodile survival. The extremely small populations in some rock pools (Brito et al., 2011, 2014), low genetic diversity at a regional level, and isolation between neighbouring hydrological n et al., 2014), suggests low chance for crocodile basins (Velo-Anto re-colonization after local extinction. During the dry season, crocodiles are forced to aestivate (Trutnau and Sommerlad, 2006; Brito et al., 2011). In rock pools, they shelter between boulders of the rocky slopes, while in floodplains, they bury themselves below the mud surface or migrate to nearby rock outcrops (Shine et al., 2001). Feeding and reproductive periods of C. suchus seem to be restricted to the rainy season, 10 weeks or less per year (Brito et al., 2011), which may increase vulnerability. Despite this reduced period of suitable conditions, water-bodies provide enough prey, both aquatic and terrestrial since these freshwater habitats constitute local biodiversity hotspots (Vale et al., 2015). Our study has demonstrated that C. suchus in Mauritania has a generalist diet related to limited feeding opportunities over long periods of time. Overexploitation is reducing water quality and consequently may compromise the presence of (small) aquatic prey, the main food resource for small crocodiles. For this reason, complete protection of the habitat including crocodiles, their prey, and water quality is needed. Loss of top predators can have serious consequences for ecosystem stability (Rosenblatt et al., 2013). This can be more

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critical in small ecosystems such as Saharan water bodies. For this reason, Crocodylus suchus in Mauritania has been proposed as an umbrella species, since its conservation is tied to other aquatic or semi-aquatic organisms, namely fishes (e.g. Barbus spp., Coptodon/ Sarotherodon spp. and Clarias anguillaris), amphibians (e.g. Amietophrynus xeros and Hoplobatrachus spp.) and reptiles (e.g. Varanus niloticus and Python sebae), which also live in isolated spots across  n et al., 2014). Our study is a seminal the Sahara-Sahel (Velo-Anto contribution to better understanding the ecology of this iconic Saharan species. Future studies including the analysis of scats and stomach pumping will increase our knowledge of the functioning of this top predator on these vulnerable water-bodies. Author contributions XS originally formulated the idea; JCC and JCB conducted fieldwork; SN generated isotope data; XS, SN and CS performed statistical analyses; XS and SN wrote the manuscript; and all the authors provided ideas and improvements to early versions of the manuscript. Acknowledgments Fieldwork was funded by National Geographic Society (CRE 8412-08), Mohammed bin Zayed Species Conservation Fund ~o para a Cie ^ncia e a Tecnologia (FCT: PTDC/BIA(11052707), Fundaça BEC/099934/2008 and PTDC/BIA-BIC/2903/2012), and by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE (FCOMP-01-0124-FEDER-008917/028276). Individual support was given to JCB, XS and JCC by FCT (IF/459/2013, SFRH/BPD/73176/2010, and SFRH/BD/87885/2012). Logistic support for fieldwork was given by P.N. Banc d’Arguin, Fondation Interre de l’Environnement et du nationale du Banc d'Arguin, Ministe veloppement Durable of Mauritania, and Faculty of Sciences and De Technology (USTM; Mauritania). Authors thanks very constructive  ski, DV comments of two anonymous reviewers, and Z Boratyn Gonçalves, R Guerreiro, P Sierra, N Sillero, TL Silva, P Tarroso, CG  n, and AS Sow for their assistance during Vale, G Velo-Anto field work. Appendix A. Supplementary data Supplementary data related to this chapter can be found at https://doi.org/10.1016/j.jaridenv.2017.09.008. References Akani, G.C., Luiselli, L., Amuzie, C.C., Wokem, G.N., 2011. Helminth community structure and diet of three Afrotropical anuran species: a test of the interactiveversus-isolationist parasite communities hypothesis. Web Ecol. 11, 11e19. Barrow, L.M., Bjorndal, K.A., Reich, K.J., 2008. Effects of preservation method on stable carbon and nitrogen isotope values. Physiological Biochem. Zool. 81, 688e693. Bearhop, S., Adams, C.E., Waldron, S., Fuller, R.A., Macleod, H., 2004. Determining trophic niche width: a novel approach using stable isotope analysis. J. Animal Ecol. 73, 1007e1012. Brito, J.C., Martínez-Freiría, F., Sierra, P., Sillero, N., Tarroso, P., 2011. Crocodiles in the Sahara desert: an update of distribution, habitats and population status for conservation planning in Mauritania. PLoS One 6 (2), e14734. Brito, J.C., Godinho, R., Martínez-Freiría, F., Pleguezuelos, J.M., Rebelo, H., Santos, X., n, G., Boratyn  ski, Z., Carvalho, S.B., Ferreira, S., Vale, C.G., Velo-Anto Gonçalves, D.V., Silva, T.L., Tarroso, P., Campos, J.C., Leite, J.V., Nogueira, J.,  Alvares, F., Sillero, N., Sow, A.S., Fahd, S., Crochet, P.-A., Carranza, S., 2014. Unravelling biodiversity, evolution and threats to conservation in the SaharaSahel. Biol. Rev. 89, 215e231. Campos, J.C., Sillero, N., Brito, J.C., 2012. Normalized difference water indexes have dissimilar performances in detecting seasonal and permanent water in the Sahara-Sahel transition zone. J. Hydrol. 464e465, 438e446. m, F., Brito, J.C., 2016. Update of Campos, J.C., Martínez-Freiría, F., Sousa, F.V., Santare distribution, habitats, population size, and threat factors for the West African

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Please cite this article in press as: Santos, X., et al., Stable isotopes uncover trophic ecology of the West African crocodile (Crocodylus suchus), Journal of Arid Environments (2017), https://doi.org/10.1016/j.jaridenv.2017.09.008