Captive Breeding Conditions Decrease Metabolic Rates and Alter Morphological Traits in the Endangered Spanish Toothcarp, Aphanius Iberus

Received: 29 August 2019 | Revised: 8 May 2020 | Accepted: 13 May 2020 DOI: 10.1002/iroh.201902014

RESEARCH PAPER

Captive breeding conditions decrease metabolic rates and alter morphological traits in the endangered Spanish toothcarp, Aphanius iberus

1GRECO, Institute of Aquatic Ecology, University of Girona, Girona, Catalonia, Spain Abstract 2Departamento de Ciencias Médicas Básicas, Physiological features of species can determine the resilience and adaptation of Facultad de Medicina, Universidad San Pablo‐CEU, CEU Universities, Alcorcón, organisms to the environment. Swimming capacity and metabolic traits are key Madrid, Spain factors for fish survival, mating and predator–prey interactions. Individuals of the Correspondence same species can display high phenotypic variation often in response to varying Anna Vila‐Gispert, GRECO, Institute of Aquatic environmental conditions. We investigated the effects of captive breeding Ecology, University of Girona, 17003 Girona, Catalonia, Spain. conditions on swimming capacity, metabolic traits and morphology by comparing a Email: [email protected] captive population with a wild population of the endangered Spanish toothcarp Handling Editor: Patrick Polte (Aphanius iberus). We measured swimming capabilities and oxygen‐uptake rates

Funding information simultaneously, the latter as a proxy for metabolic rate, using a swim tunnel Departament d'Innovació, Universitats i respirometer. Results showed significant differences in standard metabolic rate Empresa, Generalitat de Catalunya, Grant/Award Number: ref. 2017 SGR 548; (SMR), maximum metabolic rate (MMR) and absolute aerobic scope (AAS) between Spanish Ministry of Science, Innovation and populations, as well as differences in morphological features between populations Universities, Grant/Award Number: CGL2013‐ 43822‐R and CGL2016‐80820‐R; Universitat and sexes. In contrast, we did not find significant differences in critical swimming de Girona, Grant/Award Number: IFUdG17 speed between populations or sexes. Differences in SMR between sexes were found in the captive population, and males showed nearly a twofold increase in SMR when compared with females. SMR, MMR and AAS were, on average, twofold lower for the captive population in comparison with the wild population. These differences in metabolic traits likely reflected captivity conditions, which were low food availability and the absence of predators, which in turn, may have influenced morphological traits, such as body and caudal peduncle shape and head size. At the same time, morphological traits also influenced metabolic traits of the populations. The lower SMR and MMR of captive individuals may be related to their deeper body shapes. Taken together, our results suggested that captive breeding conditions caused significant physiological and morphological changes in the endangered Spanish toothcarp. Reduced metabolic traits and changes in morphology may affect fitness‐related traits of the captive populations once reintroduced into the wild, thereby compromising conservation efforts. We therefore recommend to experimentally testing for the effects and consequences of captive breeding conditions

Internat Rev Hydrobiol. 2020;105:119–130. wileyonlinelibrary.com/journal/iroh | 119 120 | LATORRE ET AL.

before fish are released into the wild for successful conservation of them and other endangered species.

KEYWORDS Aphanius iberus, captive breeding conditions, endangered fish, metabolic traits, wild and captive populations

1 | INTRODUCTION areas along the Mediterranean coast, such as salt marshes and lagoons (Doadrio, 2002;Moreno‐Amich, Planella, Fernández‐Delgado, & Swimming capacity and metabolic traits are important physiological García‐Berthou, 1999;Oliva‐Paterna, Torralva, & Fernández‐Delgado, features for fish survival, mating and predator–prey interactions (Huang, 2006). Habitat loss and the presence of invasive species are two key Zhang, Liu, Wang, & Luo, 2013; Killen, Costa, Brown, & Gamperl, 2007; factors that endanger the survival of the remaining populations (Alcaraz, Plaut, 2001). Swimming performance depends on morphological char- Pou‐Rovira, & García, 2008;Doadrio,2002; García‐Berthou & Moreno‐ acteristics of the species (Fisher & Hogan, 2007; Leavy & Bonner, 2009; Amich, 1992;Rincón,2002). Breeding programmes have been developed Rouleau, Glémet, & Magnan, 2010). Specifically, the critical swimming in the Iberian Peninsula to ensure the conservation of the Spanish speed (Ucrit) is a common measurement to determine prolonged swim- toothcarp, as they represent an extended measure in fish conservation ming capacity (Beamish, 1978; Brett, 1964) and it has often been used to programmes (Andrews & Kaufman, 1994; Berejikian, 2000;Flagg,Be- evaluate the relationships between morphological traits and habitat rejikian, Colt, & Dickhoff, 1995) and account for some successful re- features (Hawkins & Quinn, 1996; Oufiero & Whitlow, 2016;Plaut,2001; introductions (Philippart, 1995; Shute, Rakes, & Shute, 2005). However, Rouleau et al., 2010; Ward, Schultz, & Matson, 2003). manystudiesonfishrearingrevealedthat fishes raised in captivity can

Two important metabolic traits are estimated during the Ucrit pro- show notable differences in fitness‐related traits (e.g., behaviour, mor- tocol, from which the respiratory capacity of a fish can be described: the phology, growth and metabolic rates) in comparison with their wild standard metabolic rate (SMR) refers to the energy that an animal must conspecifics (Belk, Benson,Rasmussen,&Peck,2008;Einum&Flem- spend on the maintenance of tissues and homoeostatic mechanisms ing, 2001). In some cases, reintroductions of captivity‐reared fish, such as needed to sustain life (Fry, 1971). After satisfying the maintenance salmonids, even compromised the ecological success of the species in the energy requirements, the excess of energy can be allocated to other environment (Einum & Fleming, 2001). By improving the knowledge of functions, but always below the upper limit set by the maximum me- the effects of rearing conditions on physiological and morphological tabolic rate (MMR), which represents the maximal rate at which oxygen traits, we might shed some light on captive breeding programmes to can be supplied to tissues and adenosine triphosphate (ATP) can be enhance the management and conservation of A. iberus. produced (Fry, 1971). The difference between MMR and SMR re- The aim of the present study was to test a wild and a captive‐ presents the absolute aerobic scope (AAS) of an animal and determines reared population of Spanish toothcarp (A. iberus) for differences in the total aerobic capacity to support key life‐history attributes such as swimming capacity, metabolic rates and morphological features. We activity, growth and reproduction (Fry, 1971). Metabolic traits usually hypothesised that captive breeding conditions reduced swimming show allometric scaling with body mass but previous research has capacity, metabolic rates and altered morphological traits in the shown that SMR, MMR and AAS may show a twofold, or even threefold endangered Spanish toothcarp. variation within species (Auer, Salin, Rudolf, Anderso, & Metcalfe, 2015; Burton, Killen, Armstrong, & Metcalfe, 2011; Metcalfe, Leeuwen, & Van Killen, 2015; Norin & Clark, 2016;Norin,Malte,&Clark,2016). Me- 2 | MATERIALS AND METHODS tabolic traits can be influenced by intrinsic (e.g., physiology, behaviour and morphology) and extrinsic factors (e.g., temperature, salinity, prey 2.1 | Study area and experimental fish availability and predation; Burton et al., 2011;Metcalfeetal.,2015; Pettersson & Brönmark, 1999). Morphology is an important determi- A captive and a wild population of A. iberus was compared concerning nant of swimming performance and metabolism in fish (Killen swimming capacity and metabolic traits and tested for the effects of et al., 2016; Pettersson & Hedenström, 2000;Rubio‐Gracia, captive breeding conditions. In June 2017, 32 wild fish were collected García‐Berthou, Guasch, Zamora, & Vila‐Gispert, in press), so that it using a dip net in a hypersaline coastal lagoon (La Rovina) from Em- may reflect differences in environmental conditions as well (Bourke, pordà salt marshes, NE Iberian Peninsula (42°15′38.7″ N, 3°8′38.9″ E). Magnan, & Rodríguez, 1997; Moles, Robinson, Johnston, & Cunjak, 2010). The number of the wild fish sampled was limited by the maximum The Spanish toothcarp Aphanius iberus (Valenciennes, 1846) is number of fish allowed to catch, set by the Regional Government, a sexually dimorphic small fish of the Aphaniidae family (formerly Cy- especially for endangered species. This lagoon is a permanent water- prinodontidae), endemic to the Iberian Peninsula and listed as body with a mean depth of 73 cm, a mean conductivity of 49.3 mS/cm endangered by the IUCN. This fishspeciesisfoundinisolated (range = 8.8–149 mS/cm) and a mean annual temperature of 15.7°C. LATORRE ET AL. | 121

Further information on the sampling area can be found in García‐ conducted between autumn and early spring to avoid the breeding Berthou and Moreno‐Amich (1992) and Trobajo, Quintana, and season of A. iberus. At the end of the experiment, all fish were kept Moreno‐Amich (2002). Fifty‐seven captive fish were randomly col- in quarantine for at least 1 week, and wild and captive individuals lected from a fish stock located at the University of Girona (Girona, were then returned to the capture site. In total, 57 captivity‐reared Spain). Fish stock ancestors were originally captured in 2012 from the and 30 wild individuals successfully completed the respirometry same lagoon than wild fish (La Rovina), then captive individuals re- trials. presented the fifth generation born in captivity. At the University of Girona, fish were kept in freshwater outdoor tanks (1 × 1 m, depth 1.5 m, 1,500 L) to simulate the captivity conditions of governmental 2.2 | Swim tunnel respirometer set up breeding centres for conservation and restocking. The water of the tanks was 30% replaced once a month. These tanks had no substrate Following Rubio‐Gracia, García‐Berthou, Latorre, et al. (2020), re- but they included floating mats of macrophytes. In total, 60 fish (males spirometry trials were conducted using a Blazka‐type swim tunnel and females) were kept in each tank. Fish were exposed to the natural respirometer (Loligo® Systems, Viborg, Denmark). The swim tunnel photoperiod and temperature (10–25°C), and fed zooplankton that respirometer consisted of a 170 ml tubular swimming chamber naturally developed in the tanks. The zooplankton biomass was low (100 mm length × 26.4 mm internal diameter) immersed in an (8.7 μg DW/L) and it was mainly dominated by rotifers, ciliates and external water bath containing 25 L of clean, aerated water. The copepods. Animal research under captive conditions and the fieldwork external water bath was equipped with an automated Eheim pump were authorised by the Autonomous Government of Catalonia that flushed aerated and conditioned water (conductivity (SF/1089/2017), the Commission of Animal Experimentation (Ref.: ~320 µS/cm; pH ~7.6) inside the swim tunnel respirometer between CEA‐OH/9673/1), the Aiguamolls de l'Empordà Natural Park oxygen measurement periods. To ensure stable water quality para- (2017PNATAAEAUT075) and the University of Girona. meters, we additionally connected the external water bath to a Before the respirometry trials, wild and captive fish were held in plastic supply tank containing 300 L of air‐saturated freshwater. An 90 L glass aquariums under experimental conditions for 2 weeks. Pre- automated Eheim pump continuously provided freshwater from the viously, wild fish were acclimated to freshwater for 2 weeks by gradual supply tank to the external water bath, and then the water was changes in salinity conditions (3 ppt/day). Two weeks of acclimatisation recirculated again through a decantation system. The supply tank is sufficient because it has been demonstrated that, in a closely related was equipped with an automated liquid cooler (85 W, 972.46 BTU/h, species (Aphanius dispar, Rüppell, 1829), oxygen consumption rates were J.P. Selecta®) to maintain the temperature at 20°C. similar through a wide gradient of salinities, ranging from freshwater to A propeller connected to the motor outside of the swim tunnel high salt content (i.e., 109.7 mS/cm; Plaut, 2000). Fish were distributed respirometer generated the continuous laminar flow. Flow inside the among aquariums according to population and sex. Sixteen wild males swim tunnel respirometer was made rectilinear by placing a honey- and 16 wild females were placed in two aquariums, respectively. Thirty‐ comb plastic screen at the entrance of the swimming section. We four captive females and 23 captive males were equally distributed in used an optical fibre instrument (Witrox 1; Loligo® Systems, Tjele, four additional aquariums, respectively. Aquariums contained gravel Denmark) for the determination of dissolved oxygen concentration in substrate and conditioned water (with a conductivity ~320 µS/cm and the water. A temperature probe (Pt1000 temperature sensor; pH ~7.6, the last within the range [7.29–8.87] of La Rovina lagoon; Witrox 1) was also used for the automated compensation of oxygen Trobajo et al., 2002). Aquariums were supplied with recirculated, fil- data to changes in temperature and barometric pressure in real time. tered freshwater (particle filtered and ozone sterilised) and vigorous Rates of oxygen consumption were measured using computerised, aeration. Water changes of 30% of the total volume were conducted intermittent‐flow respirometry. During experimentation, the swim twice a week in each aquarium to assist maintaining water quality. tunnel respirometer was periodically flushed with aerated water for During the holding period, water temperature was set to 20±1°C, 2 min (flush phase), followed by a 1 min closed mixing period and which represents the middle range (10–32°C) of the thermal niche of then 20 min of closed respirometry (measurement phase). The oxy- this species (Kottelat & Freyhof, 2007). A natural photoperiod cycle was gen concentration during the measurement phase was never below used during the acclimation period. 80% to avoid any hypoxia‐related effects. For calibration purposes, From the second day of acclimation, fish were fed once a day two‐point calibration with the oxygen sensor was used to record the with frozen bloodworms (Chironomus spp.) with a meal size of ap- highest dissolved oxygen concentration value as 100% air‐saturated proximately 1.5–2% of their wet body mass. This amount of food and the lowest dissolved oxygen concentration value as 0% using a represented a sufficient amount to maintain the fish body condition solution of sodium sulphite (Na2SO3, 0.159 M). throughout the experiment, but it was still below from satiation levels. Fish were fasted 24 hr before the respirometry trials; a postfeeding period that has been shown to be long enough to avoid 2.3 | Swimming performance protocol postprandial effects in several fish species (i.e., cyprinids; Se- cor, 2009). No mortalities occurred during acclimation period and Individual fish were placed into the swim tunnel respirometer and all fish were visually in good health condition. Experiments were allowed to acclimatise for 2 hr to an initial velocity of ca. 0.5 BL/s 122 | LATORRE ET AL.

(body length, taken as the standard length of the fish, per second).

After that, a critical swimming speed (Ucrit) test was performed with stepwise increases in flow speed of approximately 1 BL/s every 20 min until the fish fatigued, that is, when the fish could no longer actively swim against the current and was pulled back against the mesh. The critical swimming speed (Ucrit, cm/s) was calculated following Brett (1964):

Ucrit=+UUTT f i f / i,

where Uf is the highest velocity maintained for a full 20‐min period

(cm/s), Tf is the time swum (min) at the last velocity increment, Ti is the interval time set (20 min in this case) and Ui is the velocity increment FIGURE 1 The exponential function used to describe the

(BL/s). The experiment usually lasted for 4 or 5 hr and finished when relationship between metabolic rate (Ṁ O2,mgO2/h) and swimming fish showed signs of fatigue. Individuals that showed poor rheophilic speed (U, BL/s) for wild and captive populations behaviour were omitted from the analyses (Mateus, Quintella, & Almeida, 2008). Swimming speeds were not corrected by the “solid‐ 2.4 | Morphometric analysis blocking effect” because the cross‐sectional area of the fish never overcame 10% of that of the swim tunnel respirometer (Bell & Following the respirometry trials, the morphology of individual fish was Terhune, 1970). Background microbial respiration inside the swim analysed using geometric morphometrics to determine whether mor- tunnel respirometer was calculated without fish for at least 10 min at phological differences between populations and sex could be related to the end of each trial and was used as “blank”. Estimates of microbial the critical swimming speed and metabolism. Geometric morpho- respiration ranged from 20% (high water flows) to 70% (low water metrics are based on the modifications of the coordinates of "land- flows) of the total oxygen consumption during the respirometry trials. marks" and their covariance (Rohlf & Slice, 1990). As there were no Oxygen consumption by the fish was calculated by fitting a linear studies on geometric morphometrics for this species, landmarks were regression of the oxygen concentration decline over time at each selected based on previously defined morphometric characteristics velocity. The resulting slope was used to calculate oxygen con- identified as having functional significance for locomotion (Blake, 2004; sumption rates (Ṁ O2,inmgO2/h): Higham, 2007; Sagnes, Champagne, & Morel, 2000;Webb,1982). Digital photographs were taken with a Canon Handycam

MOfObV̇ O2 =–(Δ –Δ )× , DS126491 (Japan; image resolution of 18 Mp) of the right side of each specimen, and nine landmarks were recorded as two‐ where ΔOf and ΔOb are the rates of oxygen consumption in mg dimensional (x and y) coordinates (Figure 2) using the software −1 −1 O2 ·L ·min due to fish respiration and microbial respiration, ImageJ 1.50i (Schneider, Rasband, & Eliceiri, 2012). Landmark respectively, and V is the volume of the swim tunnel respirometer coordinates were adjusted with a Generalised Procrustes Analysis

(after subtracting the fish volume). For individual fish, Ṁ O2 was used (GPA; Rohlf & Slice, 1990). This procedure centres each specimen as a measure of metabolic rate (MR). MMR was defined as the highest onto a common centroid, scales all specimens to a common unit size MR during the swimming trial, which was usually close to the highest by dividing each total configuration by centroid size and lastly rotates velocity (c.f. Rubio‐Gracia, García‐Berthou, Latorre, et al., 2020,Srean, each specimen to a common orientation that minimises the differ- Almeida, Rubio‐Gracia, Luo, & García‐Berthou, 2017). The exponential ences between corresponding landmarks. We then estimated a function was used to describe the relationship between MR and “consensus” form composed of the mean coordinates for each land- swimming speed (U; Beamish, 1978;Brett,1964; Tudorache, Viaene, mark averaged across all specimens. For each specimen, we esti- Blust, Vereecken, & De Boeck, 2008;Webb,1975), as it fitted the data mated 18 partial warps plus the corresponding centroid size using better than the power function (Figure 1): the MorphoJ 1.06d programme (Klingenberg, 2011). To help visua- lising and interpreting shape differences, we used deformation grid cU MR=× SMR e , plots, which reflect the degree and type of shape change between the consensus form, and the form of wild males, wild females, captive where SMR is the estimated standard metabolic rate at zero swim- males and captive females. ming speed, and c is the speed exponent. The AAS was calculated as the difference between MMR and SMR. At the end of each experiment, individuals were measured (standard body length, SL) to the 2.5 | Data analysis nearest 1 mm and weighed (wet mass, M) to the nearest 0.1 mg. For comparison purposes, SMR, MMR and AAS were mass‐corrected To test for significant differences in swimming capacity and meta- −1 −1 as O2 ·h ·g . bolism between the two populations of Spanish toothcarp, we LATORRE ET AL. | 123

FIGURE 2 Location of the nine landmarks used in the geometric morphometric analysis to characterise the body shape variation between the two populations of Iberian toothcarp. (1) Tip of the snout; (2) dorsal head end; (3) anterior dorsal fin insertion; (4) posterior dorsal fin insertion; (5) dorsal caudal fin insertion; (6) ventral caudal fin insertion; (7) posterior anal fin insertion; (8) anterior anal fin insertion; (9) ventral end of the operculum

conducted an analysis of covariance (ANCOVA) using population morphological traits and fish swimming performance and metabo- (wild vs. captive) and sex as fixed effects and SL or M as covariates. lism. A Tukey's multiple comparison test (Bonferroni corrected) was

Critical swimming speed (Ucrit), SMR, MMR and AAS were used as applied to elucidate differences of covariate (Ucrit)adjustedmeans dependent variables. Critical swimming speed was more related to SL of MMR between wild males, wild females, captive males and cap- than to M and the opposite was true for SMR and MMR; therefore, SL tive females. For all analyses, the statistical significance level was ® was used as a covariate for Ucrit and M for metabolic traits (SMR, set at p < .05. Statistical analyses were conducted with the IBM MMR). The assumption of homogeneity of slopes of ANCOVA was SPSS Statistics v.25. tested by analysing the interactions between the covariate (SL or M) and the categorical factors (population and sex). If such interactions were not significant, the assumption of homogeneity of slopes was 3 | RESULTS satisfied and the interactions were removed from the model to im- prove the statistical power of the ANCOVA (García‐Berthou & 3.1 | Critical swimming speed and metabolism Moreno‐Amich, 1993). AAS was not significantly correlated with SL nor M, and therefore an analysis of variance (ANOVA) was performed Values of critical swimming speed and metabolic traits for the wild to test for differences between populations and sexes. To test for and captive populations of Spanish toothcarp are summarised in differences in body condition (weight‐length relationship) between Table 1. ANCOVA results showed that the interactions between SL populations, we also applied an ANCOVA with population and sex as or M and the categorical factors (population and sex) were not sig- fixed effects, SL as a covariate and M as a dependent variable. All nificant (p > .05) and, hence, homogeneous slopes among wild males, variables were log10 transformed to linearise the data and residual wild females, captive males and captive females were assumed. plots of dependent variables were used to confirm the homo- Critical swimming speed was positively correlated with SL and M, scedasticity and normality of residuals. Pearson's product‐moment and was not significantly different between populations and sexes correlations (r) and linear regression analyses were also conducted to (Table 2). SMR and MMR were also positively correlated with SL analyse the relationship between swimming performance and meta- and M, and MMR was significantly different between populations bolic traits. (Table 2). Irrespective of sex, MMR was almost twice as high in wild Partial warps are the minimal shape parameters needed to fish when compared with captive fish (Table 1). For SMR, the inter- deform the “consensus” configuration to each one of the analysed action between population and sex was significant after accounting specimens and they contain shape information, which was analysed for SL or M (Table 2), thereby showing a different relationship be- using multivariate statistics (multivariate analysis of variance tween populations and sexes. SMR of captive males was almost twice

[MANOVA]). Next, we conducted a discriminant function analysis as high as for captive females (F1, 50 = 20.9, p < .001), whereas sexes (DFA, Wilks's method) on the partial warps matrix to highlight of the wild population did not differ significantly from each other shape differences between populations and sexes of the Spanish in their SMR (F1, 22 = 0.94, p > .05), when comparing individuals of toothcarp. This analysis included a cross‐validation procedure to similar size. For AAS, we found significant differences between identify the percentage of correct classifications by comparing the populations (F1, 69 = 21.2, p < .001) but not between sexes morphology‐related classification of each specimen made by (F1, 69 = 0.20, p > .05). Irrespective of sex, AAS was almost twice as the DFA with our a priori classification of each specimen into one of high for the wild population (Table 1). The interaction between po- the following groups: wild males, wild females, captive males and pulation and sex was not significant (F1, 69 = 0.03, p > .05). We also captive females. Individual scores associated with the two first ca- found significant positive associations between Ucrit and MMR nonical axes of the DFA were correlated (Pearson's correlation) (r = .418, p < .001) or AAS (r = .333, p < .01), whereas Ucrit and SMR with Ucrit, SMR, MMR and AAS to evaluate the relationship between were not significantly correlated. 124 | LATORRE ET AL.

TABLE 1 Critical swimming speed and metabolic traits for wild and captive populations of the Spanish toothcarp

Wild Captive

Males (N = 16) Females (N = 16) Males (N = 23) Females (N = 34)

Mean SE Mean SE Mean SE Mean SE

SL (cm) 2.06 0.376 2.36 0.551 2.30 0.256 2.45 0.427

M (g) 0.30 0.183 0.39 0.348 0.29 0.089 0.38 0.210

Ucrit (cm/s) 8.01 0.856 8.80 0.838 10.66 0.806 9.92 0.648

*Ucrit (BL/s) 3.88 0.381 3.70 0.242 4.61 0.311 4.08 0.256

SMR (mg O2/h) 0.21 0.048 0.31 0.074 0.13 0.014 0.07 0.011

−1 −1 *SMR (mg O2 ·h ·g ) 0.83 0.218 1.25 0.383 0.46 0.054 0.19 0.021

MMR (mg O2/h) 0.39 0.053 0.52 0.072 0.27 0.029 0.21 0.020

−1 −1 *MMR (mg O2 ·h ·g ) 1.67 0.276 2.03 0.557 1.00 0.103 0.72 0.109

AAS (mg O2/h) 0.26 0.041 0.24 0.032 0.12 0.119 0.14 0.018

−1 −1 *AAS (mg O2 ·h ·g ) 1.07 0.212 0.98 0.255 0.49 0.061 0.46 0.067 Note: Means and SE are shown. Abbreviations: AAS, absolute aerobic scope; *AAS, absolute aerobic scope per unit weight; MMR, maximum metabolic rate; *MMR, maximum metabolic rate per unit weight; SE, standard errors; SL, standard body length; SMR, standard metabolic rate; *SMR, standard metabolic rate per unit weight; Ucrit, critical swimming speed; *Ucrit, size‐corrected critical swimming speed.

ANCOVA of body condition showed that the interactions between 3.2 | Body shape, critical swimming speed and SL and the categorical factors (population and sex) were not significant metabolism (p > .05) and, hence, there was no evidence of heterogeneity of slopes among populations and sexes. Weight was positively correlated with MANOVA performed on the matrix of 18 partial warps showed

SL, and was significantly different between populations (F1, 83 =6.30, differences in body shape between populations and sexes (Table 3). p <.05)butnotbetweensexes(F1, 83 =2.35,p > .05), and there was no DFA showed significant differences among groups (wild males, interaction between population and sex. The mean weight of wild wild females, captive males and captive females) for the first (Wilks's individuals measuring 2.30 cm SL was 0.316 g (±1.04 SD) and for λ = 0.14, χ2 = 117.8, p < .001) and second axis (Wilks's λ = 0.38, captive individuals measuring the same SL it was 0.275 g (±1.03 SD). χ2 = 58.7, p < .001). The first axis of DFA explained 57.7% of the

TABLE 2 Analyses of covariance of swimming performance and metabolic traits for wild and captive populations of the Spanish toothcarp

Ucrit SMR MMR 2 2 2 R adj = 0.224 R adj = 0.364 R adj = 0.414

SS df p SS df p SS df p

M 0.502 1 <.001 2.196 1 <.001 0.420 1 <.01

Population 0.102 1 >.05 2.498 1 <.001 1.485 1 <.001

Sex 0.008 1 >.05 0.200 1 >.05 0.007 1 >.05

Population × Sex 0.033 1 >.05 1.130 1 <.01 0.186 1 <.05

Residuals 1.903 84 9.259 73 2.909 72

2 2 2 R adj = 0.216 R adj = 0.350 R adj = 0.442

SL 0.482 1 <.001 1.986 1 <.001 0.562 1 <.001

Population 0.044 1 >.05 3.161 1 <.001 1.707 1 <.001

Sex 0.025 1 >.05 0.310 1 >.05 0.022 1 >.05

Population × Sex 0.012 1 >.05 0.893 1 <.05 0.147 1 >.05

Residuals 1.924 84 9.469 73 2.767 72

Note: Models tested differences between populations and sexes with fish mass (M) or standard length (SL) as covariates. Variables were log10 transformed. See Table 1 for abbreviations of response variables. LATORRE ET AL. | 125

TABLE 3 Summary of the multivariate analysis of variance on the To interpret the first two DFA axes, we only considered landmark effects of population (i.e., wild vs. captive), sex and their interaction coordinates with significant loadings (p < .05). on morphological variation AscanbeseeninFigure3, the first axis of the DFA separated Source of variation Wilks's λ Fpcaptive females with short heads, short and deep caudal peduncles

Population (C or W) 0.475 4.191 <.001 and deep bodies from wild males with long heads, long and narrow caudal peduncles and narrow bodies. Captive males displayed an Sex (M or F) 0.476 4.170 <.001 intermediate morphology along this axis. The second axis of the Population × Sex 0.651 2.029 >.05 DFA separated wild females with narrow bodies and intermediate Note: Morphological variation was measured in 18 partial warps for each size of heads and caudal peduncles from wild males and captive individual. For each test, df = 14 (effect) and 54 (error). males and females.

The correlation of individual DFA scores with Ucrit revealed that variation in morphology among groups and the second one explained there was not a significant relationship between morphology and this 32.9% (Figure 3). Cross‐validation matrices generated from the DFA trait, whereas MMR, SMR and AAS showed significant correlations with indicated that 65.5% of captive females (n = 19), 73.7% of captive morphology (Table 4). Specifically, MMR and AAS were more correlated males (n = 14), 50.0% of wild females (n = 7) and 66.7% of wild males with DF1, whereas SMR was more correlated with DF2 (Table 4).

(n = 6), were correctly classified. The first DFA axis (DF1) primarily We also found a significant linear relationship between Ucrit revealed differences in the x coordinate of the posterior anal fin and MMR (F1, 72 = 22.8, p < .001), which was significantly different insertion (DF loading = −0.468), the x coordinate of the ventral end of (F3, 72 = 20.1, p < .001) when comparing the morphological groups the operculum (DF loading = 0.338), the y coordinate of the posterior previously described (captive females, captive males, wild males dorsal fin insertion (DF loading = 0.302) and the y coordinate of the and wild females). However, Tukey‐test comparisons of the dorsal head end (DF loading = 0.157). The second DFA axis (DF2) was covariate‐adjusted means between groups showed no significant mainly associated with differences in the x coordinate of the anterior differences between captive males and females nor between wild anal fin insertion (DF loading = ‐0.572), the x coordinate of the dorsal males and females (Figure 4). The same pattern was observed for head end (DF loading = 0.462), the x coordinate of the ventral caudal AAS. In contrast, SMR did not show any relationship with Ucrit. fin insertion (DF loading = 0.453), the x coordinate of the dorsal caudal fin insertion (DF loading = 0.369), the x coordinate of the posterior dorsal fin insertion (DF loading = −0.205) and the y co- 4 | DISCUSSION ordinate of the ventral end of the operculum (DF loading = 0.171). 4.1 | Critical swimming speed and metabolism

To the best of our knowledge, this is the first study that compares the critical swimming speed, along with metabolic and morphological traits among wild and captive populations of the Spanish toothcarp. We found differences in metabolic traits such as SMR, MMR and AAS between populations; differences in morphology between popula-

tions and sexes, but no differences in Ucrit. Regarding the swimming capacity, wild and captive populations of the Spanish toothcarp showed on average low relative (size‐corrected) critical swimming

speeds, that is, 3.75 BL/s (Ucrit mean = 8.40 cm/s) for the wild

population and 4.35 BL/s (Ucrit mean = 10.29 cm/s) for the captive population. These swimming capacities were lower when compared

with similar small‐bodied species (Ucrit mean = 14.11 cm/s for Gam- busia holbrooki (Girard, 1859; Srean et al. 2017) and similar to those

TABLE 4 Pearson's linear correlations between critical swimming

speed (Ucrit), maximum metabolic rate (MMR), standard metabolic rate (SMR) and absolute aerobic scope (AAS) and the two first axes (DF1 and DF2) of the discriminant function analysis FIGURE 3 Ordination of all Spanish toothcarp specimens along U the first two axes of the discriminant function analysis on crit MMR SMR AAS morphological partial warps data. White circle (CF, captive females), DF1 0.094 (>.05) −0.366 (<.01) −0.290 (<.05) −0.272 (<.05) black circle (CM, captive males), black square (WF, wild females), DF2 −0.147 (>.05) 0.327 (<.01) 0.350 (<.01) 0.065 (>.05) white square (WM, wild males). Black triangles represent population centroids Note: Variables were log10 transformed. p Values are in parenthesis. 126 | LATORRE ET AL.

FIGURE 4 Linear regressions between

log10 transformed MMR (mg O2/h) and Ucrit (cm/s) for morphological groups (captive females [CF], captive males [CM], wild males [WM] and wild females [WF])

1 found in other cyprinidontiforms (range of relative Ucrit =7–11 BL/s (zooplankton biomass 107.2 µg DW/L; Badosa, Boix, Brucet, & Quinta- for Aphanius dispar, Plaut, 2000; relative Ucrit mean = 5.5 BL/s for na, 2007). Therefore, the reduced SMR of the captive population may be Fundulus notatus (Rafinesque, 1820; Leavy & Bonner, 2009). Our attributed to differences in their recent energy intake history (Rosenfeld, results, along with findings from other studies that examined Van Leeuwen, Richards, & Allen, 2015), which agrees with the low body swimming capabilities in the Spanish toothcarp (Rubio‐Gracia, condition showed by the captive individuals. However, we cannot rule out García‐Berthou, Latorre, et al., 2020), are consistent with the idea the possibility that the longer acclimatisation of wild individuals (4 weeks) that this species has a poor swimming performance, corresponding to as compared with captive ones (2 weeks) could have also contributed to its preference for slow‐flowing habitats (Alcaraz et al., 2008). differences in body condition among populations and, therefore, to a The captive population showed lower SMR and MMR (mean = 0.325 reduced SMR in the captive population. Furthermore, it was observed −1 −1 and 0.860 mg O2 ·h ·g , respectively) than the wild population that the presence of predators in a given habitat can induce prey fish to −1 −1 (mean = 1.035 and 1.850 mg O2 ·h ·g , respectively). Compared with show higher SMR than those that live in environments without predators other species, SMR and MMRs of the captive population were far below (Auer, Dick, Metcalfe, & Reznick, 2018; Fu, Yuan, Cao, & Fu, 2015). For −1 −1 the average for Gambusia holbrooki (1.114 and 1.637 mg O2 ·h ·g , instance, Auer et al. (2018) found consistent differences in SMR between respectively; Srean et al. 2017), whereas the wild population had similar low‐ and high‐predation risk populations of Trinidadian guppy Poecilia values to those of Gambusia holbrooki. Metabolic traits of the Spanish reticulata Peters (1859) under common garden conditions. Thus, the toothcarp were low if compared with other cyprinidontiforms absence of predators in captivity conditions could have also caused a (Plaut, 2000) and cyprinids, but high if compared with limnophilic species reduction in SMR in the captive population. Although some studies of larger size (Rubio‐Gracia, García‐Berthou, Guasch, et al., in press). The reported differences in metabolic rates when comparing fish populations differences in metabolic traits between populations may have important experiencing different osmoregulatory demands (Dalziel, Vines, & implications for the success of captive breeding populations reintroduced Schulte, 2012,Norinetal.,2016, Prakoso & Chang, 2018), it has been into the wild, because it has been suggested that changes in metabolic found in a closely related species (Aphanius dispar) that critical swimming traits can affect the behaviour and fitness‐correlated traits, such as speed and oxygen consumption rates were similar from freshwater to growth, reproduction and survival (Biró & Stamps, 2010;Burton high salinities (i.e., 109.7 mS/cm; Plaut, 2000). Therefore, it seems that et al., 2011). Several studies have documented that SMR and MMR can differences in salinity acclimatisation between wild and captive popula- decrease when fish are subject to a period of food restriction (Auer tions did not affect the measured swimming capacity and metabolic rates. et al., 2015;Biró&Stamps,2010;DuPreez,1987;Wieser, However, the influence of unexplored, both environmental and genetic Krumschnabel, & Ojwang‐Okwor, 1992) and can increase when food is (i.e., founder effects and genetic drift) factors on metabolic rates cannot supplied above reference levels (Auer et al., 2015; O'Connor, Taylor, & be ruled out.

Metcalfe, 2000; Van Leeuwen, Rosenfeld, & Richards, 2011;Van We found positive associations between Ucrit and MMR or AAS

Leeuwen, Rosenfeld, & Richards, 2012). Since the captive population was whereas Ucrit and SMR were not correlated, which may be explained by maintained in outdoor tanks (semi‐controlled mesocosms) and was not the considerably high interindividual variations in SMR. This finding artificially fed during their captivity, food availability was lower (zoo- could likely be related to the short acclimation period (2 hr) used before plankton biomass 8.7 µg DW/L) than that from the wild population starting the Ucrit protocol, since several hours are needed to obtain the LATORRE ET AL. | 127 best accuracy of SMR estimates (Norin & Clark, 2016). Consequently, (Figure 4) and AAS, varied almost in accordance with the morpholo- we could have overestimated SMR and could have measured values gical groups previously described. This is not surprising because it is reflective of resting and routine metabolic rates (Clark, Sandblom, & generally assumed that fish with different morphological traits can Jutfelt, 2013). However, these elevated estimates of SMR may not display variations in swimming performance and metabolism (Killen substantially affect the general outcomes of this study because the et al., 2016; Pettersson & Hedenström, 2000). For instance, Pettersson same protocol was applied for all individuals. In addition, we found that and Brönmark (1999) and Rubio‐Gracia, García‐Berthou, Guasch, et al. individuals with higher MMR had also higher SMR (r = .696, p < .001) (in press) found that deep‐bodied fish showed lower SMR than suggesting that an elevated MMR entails important energy require- shallow‐bodied individuals, at intra‐ and interspecific levels, respec- ments to maintain baseline metabolism (Biró & Stamps, 2010;Rubio‐ tively. Therefore, we can conclude that the lower SMR and MMR of Gracia, García‐Berthou, Guasch, et al., in press). We also observed that the captive individuals may be related to their deeper body shapes. fish with higher MMR showed higher AAS (r = .746, p < .001) too, but Intraspecific morphological variation is common in fish, and is, there was no correlation between SMR and AAS. This indicated that the among other factors, expected to reflect differences in habitat use, variation in AAS was mainly driven by the variation in MMR, and that food preferences, and predatory pressure (Bourke et al., 1997; Moles the impact of variation in SMR on variation in AAS was negligible. This et al., 2010; Rouleau et al., 2010). Although the Spanish toothcarp is a finding agrees with what was found by Killen et al. (2016)whenex- species that can live in a wide variety of brackish and freshwater amining interrelationships between metabolic traits in teleost fish. habitats (García‐Berthou & Moreno‐Amich, 1999), it prefers complex Thus, the positive associations between AAS and MMR on the one habitats with abundant submerged vegetation and slow current flow hand, and MMR and SMR on the other, together with the weak effect (Alcaraz & García‐Berthou, 2007; Magellan & García‐Berthou, 2016; of SMR variations on AAS variations, indicated that wild individuals Vargas Pera, 1993). Moreover, benthic invertebrates are preferred with a high AAS exhibited elevated MMRs and SMRs as well. In food items (Alcaraz & García‐Berthou, 2007) and the search for contrast, individuals of the captive population with low AAS showed benthic prey in structurally complex habitats requires manoeuvring low MMR and SMR as well. Although we did not assess the fitness and intermittent swimming bouts (Walker, 1997). Species that live in consequences of metabolic differences between wild and captive po- these types of habitats usually show morphological adaptations, such pulations, it would be important to bear in mind that the fitness con- as deep bodies through the caudal region, medium fins and large sequences of a given metabolic phenotype may be context‐dependent caudal fins with low aspect ratios (broad surface area for powerful (Auer et al., 2015, Burton et al., 2011). For instance, individuals with thrust, but high frictional drag; Blake, 1983; Domenici, Turesson, high SMR can enhance their fitness when environmental conditions are Brodersen, & Brönmark, 2008; Walker, 1997). However, in our study, favourable (i.e., there is no restriction of food), thereby allowing to these morphological features were characteristic of individuals from offset the higher cost of maintenance. In contrast, the fitness of the captive population, which lived in a structurally simpler en- individuals with low SMR will be favoured in poor environmental vironment than the wild population. Furthermore, the lack of pre- conditions (i.e., low food availability) because of their lower main- dators in captivity conditions can also induce morphological changes. tenance requirements (Burton et al., 2011). In short, it may be con- For instance, Langerhans, Layman, Shokrollahi, and DeWitt (2004) cluded that the success of the captive population, which was found morphological differences in Western mosquitofish Gambusia characterised by lower metabolic traits, could be threatened if the re- affinis (Baird & Girard, 1853) related to the presence of predators. introduction does not occur under adequate environmental conditions. Specifically, they found that fish in environments with predators had larger caudal peduncles, smaller heads and more elongated bodies. In contrast, Domenici et al. (2008) observed that the presence of pre- 4.2 | Body shape, critical swimming speed and dators induced deeper bodies in the crucian carp (Carassius carassius L.). metabolism Some of these results are not in accordance with ours, as the captive individuals, which were not exposed to predators, showed deeper We found that morphological attributes influenced the metabolic bodies, shorter heads and shorter and deeper caudal peduncles. It traits of the populations studied. That is, captive females, which were seems that captivity conditions could promote differences in morpho- characterised by shorter and deeper caudal peduncles, deeper body logical traits of captive and wild fish populations when environmental shapes and shorter heads, showed lower MMR than wild males and conditions do not adequately mimic the original habitat (Belk females. On the contrary, wild males and females, which had more et al., 2008; Näslund & Johnsson, 2016;Saraiva&Pompeu,2016). For fusiform body shapes and intermediate to longer sizes of heads and instance, the restriction of swimming opportunities in captivity could caudal peduncles, showed higher SMR than captive males and females. cause the development of specific shapes in fish, such as reduced In agreement with this, Rubio‐Gracia, García‐Berthou, Guasch, et al. streamlining (Fabre, Vila‐Gispert, Galobart, & Vinyoles, in press). (in press) found that species that were more streamlined displayed Therefore, it appears that differences in morphological traits maximum metabolic rates than deep‐bodied species. Although we did between wild and captive populations could be closely related not find differences in metabolic traits among the morphological to captivity conditions (i.e., structurally simple habitat and/or the ab- groups corresponding to captive males, captive females, wild males sence of predators), although contemporary selection might not be and wild females, the relationship between Ucrit and both MMR excluded considering that five generations have passed. The observed 128 | LATORRE ET AL. differences in morphology could also have a genetic basis due to the Bell, W. H., & Terhune, L. D. B. (1970). Water tunnel design for fisheries particular source of individuals originally sampled from the wild popu- research (Fisheries Research Board of Canada Technical report no. 195). 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