A comparative study of predator-induced phenotype in tadpoles across a pond permanency gradient.

A comparative study of predator-induced phenotype in tadpoles across a pond permanency gradient.
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  PRIMARY RESEARCH PAPER A comparative study of predator-induced phenotypein tadpoles across a pond permanency gradient Alex Richter-Boix Æ Gustavo A. Llorente Æ Albert Montori Received: 20 April 2006/Revised: 15 September 2006/Accepted: 14 October 2006/Published online: 26 February 2007 Ó Springer Science+Business Media B.V. 2007 Abstract In a field survey the distribution of pond-breeding anuran species and their potentiallarge predators was investigated along a freshwa-terhabitatgradient,rangingfromephemeralpoolsto permanent ponds. In a laboratory experimentpredator-induced plasticity was examined for alltadpole species to test whether the plasticresponse of ephemeral and temporary pond spe-cies differs from that of permanent pond species.Desiccation and predation pose conflicting de-mands; reduced activity lowers the risk of death bypredation but increases the risk of death bydesiccation. It was expected that species fromtime-constrained habitats would display a mor-photype that would reduce vulnerability to inver-tebrate predators, thus allowing these species tomaintain a high level of activity, whereas speciesfrom permanent ponds would avoid predationboth morphologically and behaviourally. Speciesdistribution and predator composition along thehydroperiod gradient differed. Variations be-tween ephemeral and temporary ponds can beattributed to hydroperiod differences and thepresence of large invertebrate predators in tem-porary ponds, whereas the contrasts betweentemporary and permanent ponds can only beattributed to the hydroperiod, since the presenceand abundance of top predators are similar in bothhabitat types. With the exception of bufonids, allspecies showed predator-induced plasticity inagreement with previous studies. Tadpole speciesdiffered in the integration of the phenotypic traitsmeasured, but differences observed betweenspecies could not be attributed only to habitat.Species from temporary habitats showed anexpected response, with a low reduction of activityincomparisonwith therest ofthe species.Thelackof general patterns in the morphological changessuggests that species within the same habitat typedid not converge on similar phenotypes, perhapsdue to functional constraints on differences inmicrohabitat use in the water column. Keywords Behavioural plasticity Á Mediterranean area Á Morphologicalplasticity Á Phenotypic integration Á Predationrisk gradient Á Spatial distribution Á Temporarypond Á Trade-offs Introduction Models of community structure in lentic systemssuggest that species composition is determined by Handling editor: K. MartensA. Richter-Boix ( & ) Á G. A. Llorente Á A. MontoriDepartament Biologia Animal, Facultat de Biologia,Universitat de Barcelona, Av. Diagonal 645, 08028Barcelona, Spaine-mail:  123 Hydrobiologia (2007) 583:43–56DOI 10.1007/s10750-006-0475-7  a trade-off between pond permanency and pre-dation risk (Schneider & Frost, 1996; Wellbornet al., 1996). Changes in predators are related topond permanency (Woodward, 1983; Werner &McPeek, 1994; Gunzburger & Travis, 2004). Thismodel is particularly applicable to amphibianassemblages. These two antagonistic gradientscreate different selective forces for tadpole spe-cies in ponds from different positions along thegradient (Wellborn et al., 1996; Richardson,2001a, b). Resource acquisition in tadpoles maybe affected by both factors: the need for timelydevelopment in temporary ponds and the need toavoid predators along the hydroperiod gradient.Activity levels and foraging rates are typicallypositively correlated, thus increased activity leadsto an increased predation risk (Skelly, 1994;Anholt & Werner, 1995; Eklo ¨ v & Halvarsson,2000). Activity level is therefore expected toreflect the differences in habitat type along thepond permanency gradient (Woodward, 1983;Skelly, 1996; Wellborn et al., 1996; Richardson,2001b).Several mesocosm and laboratory studies havealso demonstrated that tadpole species haveevolved the ability to asses predation risk andto react in a way that reduces the likelihood of being preyed upon (Skelly & Werner, 1990;Chovanec, 1992; Anholt et al., 1996; McCollum& Leimberger, 1997; Lardner, 2000; Van Buskirk,2002; Relyea, 2004). Typical phenotypic plasticityin the presence of predators includes a reductionin time spent foraging and changes in morphol-ogy which make individuals less vulnerable topredators. All of these changes affect growth anddevelopment, causing individuals to grow anddevelop more slowly when exposed to predatorsthan in the absence of predators (Lardner, 2000;Relyea, 2002; Van Buskirk, 2002). However,species that persist in temporary ponds com-monly face time constraints that limit their abilityto delay growth and development (Altwegg,2002).Due to the behavioural trade-offs that existalong the pond permanence gradient, it can beexpected that levels of phenotypic plasticity inresponse to predation pressure––like activitylevels––will differ among species evolving underdifferent selective regimes, with varying integra-tion of the different traits (behavioural andmorphological) to fine-tune the plastic phenotyperesponse depending on species ecology and his-tory (Relyea, 2004). Species from ephemeral andtemporary ponds can be expected to exhibit apredominantly morphological, predator-inducedplasticity in order to allow the species to maintainhigh levels of activity and to develop more rapidlywhilst also reducing predation risk (Relyea &Werner, 1999; Anholt et al., 2000). In mostpermanent ponds, morphological changes in spe-cies can be accompanied by behavioural changesin order to reduce encounters with and detectionby predators (Chovanec, 1992; Anholt et al.,2000).To date, only three studies have tested thedistribution and phenotypic plasticity of morethan two closely related species (Relyea &Werner, 2000; Richardson, 2001b; Van Buskirk,2002). The study made by Relyea & Werner(2000) focuses only on the morphological changesof four species along a predation risk gradient anddoes not incorporate measures of behaviouralactivity. In contrast, Richardson (2001b) studiesonly activity levels and their plasticity withoutconsidering morphological plasticity, whereasVan Buskirk (2002) studies both plastic traitsbut from an adaptive point of view of phenotypicplasticity that gives no consideration to the trade-offs that species face along the pond permanencygradient. None of these studies address howbehavioural and morphological traits are inte-grated along the hydroperiod gradient. The pres-ent study evaluates the links between activitybehaviour, predator phenotypic plasticity andhabitat use of species along the pond permanencygradient and, ultimately, explains distributionpatterns. Consequently, the distributions of sixtadpole species are compared in a field surveywith regard to what are commonly considered thetwo principle sources of mortality: pond perma-nency and predation risk. This is followed by alaboratory experimental procedure to assesspredator-induced plasticity (morphology andactivity level) and its effects on development. 44 Hydrobiologia (2007) 583:43–56  123  Materials and methods SiteThe field study was carried out in protectednatural areas near Barcelona (NE Iberian Penin-sula), including the Natural Park of Garraf andthe Metropolitan Park of Collserola. The climatein this zone is Mediterranean, with hot, drysummers, mild winters and two rainy periods,one in spring and the other in autumn. Theamount of precipitation varies considerably fromyear to year in this region (see Richter-Boix et al.,2006b for a well description and localization of the areas of study). The amphibian community inthis zone comprises seven anuran species (  Alytesobstetricans (Laurenti 1768), Pelobates cultripes (Cuvier 1829), Pelodytes punctatus (Daudin1802), Bufo calamita (Laurenti 1768), Bufo bufo (Linne´ 1758), Hyla meridionalis (Boettger 1874)and Rana perezi (Seoane 1885)) and Salamandra salamandra (Linne´ 1758  ) . We focused our studyon the anuran community but excluded Pelobates because it is a rare species in the area and fewdata are available.Field sampling methodsWe evaluated amphibian larvae and their poten-tial invertebrate predators in four sampling peri-ods during the spring and summer, from March toAugust of 2002 in a total of 193 isolate ponds.These localities span the range of aquatic habitatsin which the species studied breed in the area of study, including ephemeral pools, temporary andpermanent ponds. Sampling time periods weredictated by preliminary sampling and accountedfor temporal differences in breeding activityamong species (Richter-Boix et al., 2006b) andensured that all species breeding were captured.Due to variation in hydroperiod not all sites couldbe surveyed in all sampling periods, thus samplesizes of ponds were not uniform. Both, amphibianlarvae and predaceous invertebrates were sam-pled with dip-net sweeps (30 cm · 40 cm) toobtain relative species densities. This is a stan-dardized technique used to sample both groups(e.g. Schaffer et al., 1994; Babbitt et al., 2003). Aminimum of 5–10 dip-net sweeps were taken ineach possible tadpole microhabitat followingstandard techniques determined by pond size(Schaffer et al., 1994). All tadpoles were identi-fied in the field and photographed with a gridbackground; the number of individuals of eachspecies were counted and finally returned towater. Predaceous invertebrates shown in previ-ous studies (e.g. Woodward, 1983; Travis et al.,1985; Cronin & Travis, 1986) to prey on tadpoleswere identified, counted and photographed with agrid background. Three types of insects wereconsidered as potential predators: Odonata larvae(considering as predators aeshnid and libellulidnaiads), Heteroptera (notonectids and Nepa spp.)and Coleoptera (larvae and adults diving beetles).Fish presence was determined through visualsurveys in addition to dip-net captures. Indepen-dently, for tadpole surveys, we visited pondsevery month throughout the year to establish thedata of drying and determine the position of thepond across the hydroperiod gradient. In thismanner, we were able to evaluate the number of days (in 30 · 30 days) that ponds retain water.We defined hydroperiods in terms of threecategories: (1) ephemeral pools, (2) temporaryponds and (3) permanent ponds. Ephemeral pools(ponds containing surface water for a maximumof two months) refilled after each rainfall period.Temporary ponds were flooded by spring andautumn rainfall. The shallowest temporary pondsoften dried out in winter, whereas the deepestponds held water until the end of spring or earlysummer. It is important to consider that a pond’sposition on the gradient of the hydroperiod couldbe dynamic, and especially that some temporaryponds change position along this gradient annu-ally as a result of climatic conditions. We did notinclude a category for permanent ponds with orwithout fish as in previous studies (Babbitt et al.,2003; Stocks & McPeek, 2003; Van Buskirk,2003), because in the study area only 6 of the193 ponds contained fish, and these were notincluded in the study, leaving 187 localities foranalysis.Total counts for each amphibian species andpredaceous invertebrate captured in each pondwere divided by the number of dip-net sweepstaken in each pond (Babbitt et al., 2003). Thisyielded an abundance based on catch per unit Hydrobiologia (2007) 583:43–56 45  123  effort which could be compared across the threedifferent habitats considered. Body lengths of predaceous invertebrates were measured frompictures on a computer and divided into twogroups: (1) small invertebrates from 5 to 15 mmlong, and (2) large invertebrate predators with abody length of over 15 mm. We conductedanalyses of variance ANOVAs to determine if amphibian species and predator group abun-dances and relative densities varied among hy-droperiod categories. Ponds were considered asthe units in all analyses and variables log-trans-formed before analyses. Predation risk at whicheach amphibian species was exposed was com-pared with analysis of variance. Also, larvalamphibian species abundance was correlated withinvertebrate predator abundance, and a partialset correlation analysis was performed to removethe effect of hydroperiod.Experimental procedure: effect of predationrisk on larval morphology and activityMorphological and behavioural anti-predatorresponses of the six species to a common predatorwere tested in a short-term laboratory experi-ment. Clutches of the six species were collectedfrom natural ponds. In all cases egg masses weretaken during species amplexus period to reducethe time of exposition of embryos to predators toavoid phenotypic plasticity induction on hatch-ings. For Pelodytes egg samples were taken from 5egg masses and hatched separately. For Hyla werecollected 15 egg masses from three differentponds. In the case of bufonids, fragments of threedifferent egg strings were taken for each species( B. bufo and B. calamita ). In the case of  Rana ,tadpoles were obtained from 5 clutches from twodifferent ponds. The Alytes larvae had recentlyhatched and just been deposited in the pond bymales when we recollected it. As males of   Alytes have parental care of eggs, negligiblepredation on hatchings should take place duringthe egg period and no defences should have beendeveloped.The experiment was conducted in laboratorytanks (30 l) filled with dechlorinated water underapproximately the same conditions as the naturalphotoperiod and at a temperature of around22 ° C, during the spring of 2001. Thirty tadpoleswere randomly drawn from a mixture of allclutches and transferred from hatching aquariumsto experimental tanks when they reached Gosner25 life stage. During the entire experiment tad-poles were fed rabbit pellets ad libitum . Twotransparent cylindrical predator cages wereplaced on both sides of each tank to preventpredators capturing tadpoles but permitting theflow of chemical signals. Both treatments (pred-ator presence and absence of predator) werereplicated several times (6 for Alytes , 10 for Pelodytes , 10 for Hyla , 7 for B. calamita , 6 for B. bufo and 6 for Rana ). In the predator presencetreatment aeshnid odonate naiads larvae wereused as predators. Aeshnid larvae are known tofeed on tadpoles and trigger defences. Eachpredator was fed every day with tadpoles rearedin separate containers.After 2 weeks from the start of the experimenttadpole activity was measured. We sampledactivity behaviour by counting the number of tadpoles moving in each tub the instant the tubwas first viewed (Skelly, 1995). Each tub wasobserved every half hour for a total of 30replicates during 2 weeks. This protocol wasrepeated for all six species.In order to obtain data on tadpole morphology,4 weeks after the start of the experiment all thetadpoles were individually photographed with agrid background. We noted Gosner developmen-tal stage and measured six traits which had beentaken as indications of plasticity in previousstudies (Van Buskirk & Relyea, 1998; VanBuskirk, 2002): body length, body depth, tail finlength, tail fin depth, tail muscle length and tailmuscle depth.Experiment statistical analysesTo measure the behavioural response of tadpolesin the presence of a predator the mean proportionof active tadpoles per tub was calculated. Theseproportional data were arc-sine square roottransformed previously to test the hypothesis thatthe mean number of active tadpoles would differbetween treatments (presence or absence of predator) using ANOVA analyses. Developmen-tal stage data were log transformed, and differ- 46 Hydrobiologia (2007) 583:43–56  123  ences between treatments within species wereanalysed with ANOVA analyses.Before morphological plasticity response anal-yses, tadpole measurements were corrected forvariation in body size. To generate size-correctedmeasures, we used the residuals of the morpho-logical measurements of log-transformed traitsafter regressions against body size. We usedcentroid size, obtained from landmarks, as ameasure of body size (Loy et al., 1993). Coordi-nates of these landmarks were collected using theTPSDIG computer program, version 1.30 (Rohlf,2001). The centroid size, the square root of thesum of squared distances of a set of landmarksfrom their centroid (Bookstein, 1991), was calcu-lated for each specimen and used to representsize. After performing this correction, tadpolemorphology for each species in the two treat-ments was tested, first with multivariate analysisfor all traits together and second with a univariateanalysis for each variable.We measured differences in plasticity amongspecies (both activity and morphological traits)and their effect on development by examiningchanges in traits that occurred between treat-ments divided by the mean value of the trait inthe absence of predator treatment ([presence of predator – absence of predator]/absence of pred-ator) (Van Buskirk, 2002). In the presence of predator, positive values of plasticity reflect anincrease in the value of the trait, whereas negativevalues show a decrease in this trait. The magni-tude of plasticity in traits can be compared amongspecies because they are all represented in unit-less measures of proportional change. We calcu-lated a overall proportional change inmorphology as the mean of the absolute valuesof proportional changes in the six size-correctedtraits considered (Van Buskirk, 2002). If the traitsmeasured affect species performance within ahabitat type (temporality and predation risk),then species from distinct habitats should differ intrait values. Therefore, we first tested for differ-ences among species regardless of the kind of habitat their occupied. We used univariate andmultivariate analyses on activity, developmentalstage and on morphological traits to determinewhether phenotypes of the species were affectedby the presence of predator. To consider theeffects of habitat we ran analyses of varianceusing habitat as a fixed factor and species nestedwithin habitat as a random factor. Species werenested within habitats following field data. As weexpected that the plasticity response would be infunction of habitat temporality, we consideredtwo groups of species: temporary ponds breeders(species which preferably occupied ephemeraland temporary ponds) and permanent pondbreeders (species from more predictable season-ally and permanent water).As species cannot be considered independentdata points because trait values could be influ-enced by shared common ancestry (Felsenstein,1985; Richardson, 2001a), we tested whether thedistribution of a particular species in terms of phenotypic plasticity is correlated with its phy-logeny. We compare proportional change inactivity for each species and the overall propor-tional change in morphology depending on thephylogenetic topology of the six species studied.The phylogenetic relationships between the sixspecies were reconstructed using the combineddata set of three genes: 12S, 16S and cyt b .Sequences were obtained from specimens in apersonal collection (collected and sequenced byS. Carranza) and from the GenBank database. Allsequences were compiled, aligned and refinedmanually using Sequence Navigator. Observeddistances in pairwise comparison were obtainedusing PAUP software. We tested phylogeneticindependence of larval traits with the computerprogram ‘‘Phylogenetic Independence 2.0’’ (Re-eve and Abouheif, 2003). Tests For Serial Inde-pendence (TFSI) on continuous data wereperformed using the phylogenetic topology andnode distances obtained from molecular recon-struction. Topology was randomly rotated10,000 times to build the null hypothesis. Results Quantification of species abundances andpredation risk along the gradientWe surveyed 49 ephemeral pools, 85 temporaryponds and 53 permanent ponds, with a total of 24,938 tadpoles counted during field sampling. Hydrobiologia (2007) 583:43–56 47  123
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