phenotypic variability in larvae of two species of mediterranean spadefoot toad: an approach using...

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Phenotypic variability in larvae of twospecies of Mediterranean spadefoottoad: an approach using linear andgeometric morphometricsDaniel Escorizaa & Jihne Ben Hassineba Institute of Aquatic Ecology and Department of EnvironmentalScience, University of Girona, Campus Montilivi, Faculty ofSciences, 17071 Girona, Spainb Faculty of Sciences of Tunis, Department of Biology, University ofTunis-El Manar, 2092 Tunis, TunisiaPublished online: 27 Aug 2014.

To cite this article: Daniel Escoriza & Jihne Ben Hassine (2014) Phenotypic variability inlarvae of two species of Mediterranean spadefoot toad: an approach using linear and geometricmorphometrics, African Journal of Herpetology, 63:2, 152-165, DOI: 10.1080/21564574.2014.948079

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Phenotypic variability in larvae of two speciesof Mediterranean spadefoot toad: an approachusing linear and geometric morphometrics

DANIEL ESCORIZA1* & JIHNE BEN HASSINE2

1Institute of Aquatic Ecology and Department of Environmental Science, University of Girona, CampusMontilivi, Faculty of Sciences, 17071 Girona, Spain; 2Faculty of Sciences of Tunis, Department of Biology,

University of Tunis-El Manar, 2092 Tunis, Tunisia

Abstract.Two species of the genus Pelobates occur in the western Mediterranean region:Pelobates cultripes, in the Iberian Peninsula and southern France, and Pelobates varaldii, endemicin northwestern Morocco. These two species form a monophyletic clade within the EurasianPelobates and are morphologically conservative. However some morphological differences existin their larval stages, which have been used to support the distinction between them as separatespecies. Here we examine the larval morphology at the premetamorphic Gosner stages of bothspecies using linear and geometric morphometrics. The aims were: (i) to investigate the morpho-logical variability of P. varaldii and P. cultripes and (ii) to revise the validity of the taxonomiccriteria used to differentiate the larvae of these species. Our analysis revealed high intraspecificvariability in both species, especially in the dorso-ventral axis, but without a geographicalstructure. Our results suggested the existence of morphological divergence between the larvae ofP. varaldii and those of P. cultripes: P. cultripes showed a more depressed shape than P. varaldii.

Key words.Ibero-Maghrebian region, phenotypic variability, Fourier analysis,taxonomy

INTRODUCTION

The genus Pelobates is widespread through much of the Western Palaearctic ecozone,although only one species appears in northern Africa, Pelobates varaldii Pasteur & Bons,1959 (Garca-Pars et al. 2003). Pelobates varaldii forms a monophyletic clade togetherwith the Iberian Peninsula species Pelobates cultripes (Cuvier). The split between the twospecies is estimated to have occurred after the reopening of the Gibraltar Strait,approximately 5.3 MYA (Veith et al. 2006). These species display a similar naturalhistory; they are semifossorial anurans that occur in loose soils in semiarid to subhumidclimates (Bons & Geniez 1996; Tejedo & Reques 2002). The adult specimens ofP. varaldii and P. cultripes also share similar morphology, although P. varaldii has asmaller size and can display a reddish pigmentation that is absent in P. cultripes (Pasteur &Bons 1959; Beukema et al. 2013).

Pasteur & Bons (1959) described the existence of morphological divergence betweenthe larvae of these two species, particularly in the overall shape of the tail and the posteriorpart of the tail fin, although these assertions were not statistically supported. Furthermore,

*Corresponding author. Email: [email protected]

African Journal of Herpetology,Vol. 63, No. 2, October 2014, 152165

ISSN 2156-4574 print/ISSN 2153-3660 online 2014 Herpetological Association of Africahttp://dx.doi.org/10.1080/21564574.2014.948079http://www.tandfonline.com

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mailto:[email protected]://dx.doi.org/10.1080/21564574.2014.948079http:///www.tandfonline.com

taxonomic comparisons among anuran larvae need to take into account the high intras-pecific variability in the phenotypic traits that is shown by some anuran species (Relyea2002). This variability occurs because, in a given population, the larval phenotype is theresult of a set of genetically fixed traits (the deterministic phenotype) added to the effect ofenvironmental conditions (the inducible phenotype; van Buskirk 2000). For this reason,the larvae of some species of anuran display marked geographical patterns in their traits(Meril et al. 2000; 2004). This variability can make it difficult to define a set of averagetraits for a species. Moreover, the existence of geographical patterns in phenotypic expres-sion can lead to over-representation of local phenotypes in the definition of the speciesaverage phenotype, and therefore it is desirable to include specimens belonging togeographically distant populations (Gollmann & Gollmann 1995).

The shape of anuran larvae can be described by linear (Altig 2007) or geometricmethods (Arendt 2010; Escoriza & Boix 2012). The geometric approach allows a detailedanalysis of variations in shape, which is useful when comparing relatively complextopologies (Mitteroecker & Gunz 2009). One of these geometric methods is Fouriertransformation (Rohlf & Archie 1984; Lestrel 1997). This method decomposes the shapeof an object into coordinates, and represents this series using simple mathematicalfunctions (Rohlf & Marcus 1993). Thus this transformation provides a series of shapedescriptors (or Fourier coefficients) which can be evaluated by performing a multivariateanalysis (Rohlf 1990). Fourier analysis is applicable to complex two-dimensionalgeometries, provided that a sufficient number of harmonics is present (Kuhl & Giardina1982), and has been successfully applied in studies of morphology in several groups ofbiota (Polihronakis 2006; Hruta 2011; Treinen-Crespo et al. 2012).

This article describes the morphology of the larvae of two sister species, P. cultripesand P. varaldii in order to assess whether there is morphological variability. First weinvestigated the presence of intraspecific variation and whether there is any spatial patternin this variation. Then we compared the morphology of the two Pelobates species, usinglinear and geometric methods in order to evaluate the validity of the taxonomic criteriaproposed by Pasteur and Bons (1959) and to determine whether additional criteria can beproposed on the basis of the external morphology of the larvae.

MATERIALS AND METHODS

Sampling

The study area covers most of the range of the genus Pelobates in the western Mediter-ranean region (Fig. 1), namely the Iberian Peninsula and north-western Morocco (Bons &Geniez 1996; Gasc et al. 1997). This region was surveyed during two periods betweenMarch and May, when it is possible to find larvae in advanced stages of development(Daz-Paniagua et al. 2005; Escoriza 2013). We surveyed typical breeding habitats forboth speciesseasonal ponds, permanent ponds and stream pools (Garca-Pars et al.2004; Escoriza 2013). This study is based on 84 specimens of P. cultripes and 57specimens of P. varaldii captured in the localities shown in Fig. 1. The number ofspecimens and water bodies sampled by site, and the characteristics of these water bodies(i.e. size, larval abundance but no predator occurrence) are detailed in Appendix 1. These

AFRICAN JOURNAL OF HERPETOLOGY 63(2) 2014 153

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variables were related to phenotypic variability in Pelobates larvae (Daz-Paniagua 1992;Lardner 2000; Marangoni & Tejedo 2008).

Morphological Data

The morphological data were obtained from photographs made after anaesthetising thespecimens in a solution containing MS222. Only those specimens with no major injurieson the tail and those that were above Gosners stage 34 and below Gosners stage 40(Gosner 1960) were included in this analysis. The photographs were taken after the larvaecapture, reducing the risk of artifacts depending on whether these specimens could havemore or less filled guts (Relyea & Werner 2000). The photographs were obtained withconstant lighting conditions and at a fixed distance, using a Nikon D-5000 camera,following the methodology described by Touchon and Warkentin (2008), and Escorizaet al. (2014). The images were processed using ImageJ vs 1.47t (Rasband 2012). Thisanalysis yielded several morphological descriptors of size, shape and colour, as follows:total length, body length, maximum tail height, area, tail area, fin area (see also Fig. 2),span ratio (the ratio between the longitudinal and transversal axes), tail roundness(a measure of how a given shape approaches a circle) and colour (mean grey value, ameasure of the mean colour value per pixel, calculated on a grey scale: values close to0 indicate darker colours while values close to 255 indicate lighter colours). Thesemorphological variables evaluated some of the taxonomically distinctive traits proposedby Pasteur & Bons (1959) and included others that can show variability alongenvironmental gradients (McDiarmid & Altig 1999).

Figure 1. Map showing the study region. The area covered with diagonal lines shows the range ofP. cultripes and the area covered with horizontal lines shows the range of P. varaldii. Black circles:sampling sites of P. cultripes, black triangles: sampling sites of P. varaldii.

154 ESCORIZA & BEN HASSINEPhenotypic variability in larvae of spadefoot toads

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Morphological Analysis

First we assessed the existence of autocorrelated patterns in the external morphologywithin the Pelobates species studied. The existence of these spatial patterns would justifythe exclusion of some populations from the analysis, because they could lead to an over-representation of local phenotypes in the description of the average species shape. To dothis we calculated the median values of the variables for each population and subsequentlycomputed the spatial correlation by performing Mantels test (after 9 999 permutations;Koenig 1999). We were also interested to assess whether the observed phenotypicvariability could be explained by some habitat descriptors (water body size and tadpoleabundance). The possible correlation between tadpole morphology, water body surfacearea, water body average depth and surface area average depth (as proxy for thehydroperiod; Brooks & Hayashi 2002), and tadpole abundance, measured as catch per unitof effort (CPUE; Shulse et al. 2010) were assessed by Spearman rank-order correlationtests, after 9 999 random replicates.

Figure 2. Tadpoles of P. cultripes (A) and P. varaldii (B); description of the morphological variablesmeasured (C).


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After examining the existence geographical patterns and the effect of the habitatconditions in the phenotypic traits, we tested for the existence of morphologicaldivergence between the two species. The variability in the morphological variables wasvisualised using a Kernel density function (Baylac et al. 2003) and their statisticaldifferences were assessed by means of permutation t-tests (after 9 999 permutations).In the case of size-dependent variables, their variability was visualised using linearregression (morphological variable against size) and their statistical differences weredetermined by permutational MANOVA (PERMANOVA, one fixed factor: species). Thelatter analysis was based on a resemblance matrix of Euclidean distances, including thelarval size (total length or total area) as a covariate (Anderson 2005). These analyses werecarried out using the PRIMER-E package (PRIMER-E Ltd, Plymouth), vegan (Oksanenet al. 2013) and sm (Bowman & Azzalini 2014) for R (R Development Core Team 2012).

We also performed an outline shape analysis based on Fourier transformation in orderto test the validity of some of the criteria proposed by Pasteur & Bons (1959). The imageswere processed using SHAPE vs. 1.3 (Iwata & Ukai 2002a) which generated 80 EllipticFourier Coefficients (EFCs) per image, after setting the number of harmonics to 20 (Iwata& Ukai 2002b). The efficiency of the EFC in describing the shape of the Pelobates larvaewas evaluated after reconstructing the specimens outlines from the EFC and comparingthem with the actual images, using the routine NEF-view (Iwata & Ukai 2002b).Subsequently, we examined whether the Fourier descriptors allowed the larvae to beassigned correctly to the two Pelobates species. To do this, the EFC were included in apartial least squares discriminant analysis (PLSDA), a particularly robust method fordiscriminating between groups using high dimensionality datasets such the EFC series(Costa et al. 2009). We were also interested in assessing whether the outline divergencewas statistically significant. We reduced the number of variables by performing a principalcomponent analysis (PCA) on the EFC series (Nikolakakis et al. 2014). The scores ofthe significant axes (those with eigenvalues greater than one) were included in aPERMANOVA test, following the procedure described previously. The significance ofthis test was obtained after 999 unrestricted permutations (Anderson 2005). Finally, tovisualise the possible variability in the outlines of both species, we superimposed thespecies average shapes. These shapes were generated by inverse Fourier transformation ofthe effective PCA factors (Iwata & Ukai 2002b). The outline shape analyses wereperformed using PRIMER-E (PRIMER-E Ltd, Plymouth) and DiscriMiner (Sanchez &Determan 2013) for R (R Development Core Team 2012).

RESULTS

Our analysis did not detect any spatial structure in the morphological traits of P. cultripesand P. varaldii (Table 1). Therefore we did not remove any of the sampled populationsfrom the analysis. The results of the correlation matrix between the characteristics of theaquatic habitats and tadpole morphology are shown in Appendix 2. This analysis yielded asignificant correlation between the tadpole body area and the water body average depth,but only in the case of P. cultripes. The statistical tests revealed that the two speciesdiffered in size in the premetamorphic Gosner stages: P. varaldii showed higher totallength (Table 2 and Fig. 3). Pelobates cultripes and P. varaldii also diverged in theiraverage span ratios (higher in P. cultripes, Table 2 and Fig. 3), and in the relativemaximum tail height (greater in P. varaldii; Table 2 and Fig. 4). The PLSDA supported


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the hypothesis of differences in the shape of the two species, with a misclassification errorrate of 0.04 (Table 3). The PCA ordination revealed that P. varaldii and P. cultripesoccupied distinct positions in the first factorial plane, although with some overlap (Fig. 5).The first 16 PCA axes had eigenvalues greater than 1, and explained 81.8% of the total

Table 1. Spatial patterns in the phenotypic expression of P. cultripes and P. varaldii. N: number oflocalities; LAT: latitudinal range of localities; AD: average distance between localities (in km). Thestatistics refer to the results of the Mantel test, assessing the spatial correlation of the variables. TL:total length.

P. cultripes P. varaldii

Localities N 14 4LAT 42.436.5 35.233.7AD 437.4 75.3

TL r 0.03 0.06p 0.49 0.41

Area r 0.11 0.03p 0.72 0.50

Span ratio r 0.17 0.66p 0.87 0.17

Tail roundness r 0.12 0.83p 0.81 0.06

Colour r 0.27 0.34p 0.07 0.30

Table 2. Interspecific variability. Results of t-test and PERMANOVA. The dispersion of thevariables is displayed in Figs 3 and 4. Significant results are marked in bold. TL: total length; BL:body length; TMH: tail maximum height. The variables BL, TMH, tail area and fin area wereanalysed holding the size constant (TL, area or tail area).

Variable Statistic Results

TL T 4.91P 0.0001

BL TL Pseudo-F 0.76P 0.45

TMH TL Pseudo-F 7.15P 0.001

Area t 6.71P 0.0001

Tail area area Pseudo-F 0.71P 0.46

Fin area tail area Pseudo-F 1.95P 0.06

Tail roundness t 7.66P 0.0001

Span ratio t 8.92P 0.0001

Colour t 0.15P 0.14


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400

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500 1000 1500 2000 2500 3000 60 80 100 120 140

Figure 3. Kernel density estimation describing the larval morphological variability. A: TL (totallength), in mm; B: area, total body area in mm2; C: span ratio (ratio between the longitudinal andtransversal axes); D: tail roundness (a measure of how the tail shape approaches a circle); E: meangrey value (mean colour value per pixel, calculated on a grey scale).

40 50 70 90

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Figure 4. Variability in the relationship between morphological variables and size, established bylinear regression. A: BL TL: body length vs total length, in mm; B: TMH TL: tail maximumheight vs total length, in mm; C: tail area area, in mm2; D: fin area area, in mm2. Squares:P. varaldii; black dots: P. cultripes.


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Table 3. Classification matrix obtained by performing a partial least squares discriminant analysison 80 elliptic Fourier coefficients (EFCs).

Original group Correct Incorrect Correct classification (%)

P. varaldii 54 3 94.7P. cultripes 81 3 96.3

Figure 5. Principal component analysis (PCA) ordination showing the variability of the shape basedon 20 harmonics in the first factorial plane. The plot shows the convex hull and standard error ellipsegenerated for P. cultripes (PC) and P. varaldii (PV).

Figure 6. Mean shapes 2 SD obtained from Elliptic Fourier Coefficients (EFCs), correspondingto the first PCA component (which explained 45.5% of the variance for P. varaldii and 41.7% forP. cultripes). The outline obtained for P. cultripes was superimposed on that of P. varaldii. In darkgrey: P. varaldii; light grey: P. cultripes + P. varaldii; white: P. cultripes.


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variance. The scores of these axes were included in a PERMANOVA test in order todetermine whether there were significant divergences in the species outlines, and thisyielded significant results (Pseudo-F = 6.21, P = 0.001). Finally, the visualisation of theover-imposed outlines of the average shapes (mean 2 SD) supported the statisticalresults, but also revealed that most of the shape variation between the two speciesappeared along the dorso-ventral axis (Fig. 6). Additionally, most of the intraspecificvariation also appeared in the same body axis (Fig. 6).

DISCUSSION

Our results provide evidence that this method of outline analysis, based on Fouriertransformation, provides a detailed quantitative description of relatively complex bi-dimensional geometries, which can be used as a complement to other measures commonlyused in traditional morphometry (Baltans & Danielopol 2011; Carlo et al. 2011).Particularly Fourier transformation could be an alternative to the landmark-basedmorphometrics, which in the case of the analysis of the shape of anuran larvae requiresthe use of a high number of semilandmarks (Arendt 2010). In our example, the EFCscaptured quite well the regular and lanceolate contours of Pelobates tadpoles using a lownumber of harmonics. These 80 EFCs were sufficient to classify 96% of the specimensproperly in the discriminant analysis, suggesting that this method may be useful in theanalysis of phenotypic variation in anuran larvae.

Our analysis also revealed the existence of high intraspecific variability in bothspecies, although we did not detect any geographical structure. This may be because thisvariability depends mainly on local habitat conditions and so appears within a population.Both species show long periods of larval development, usually greater than 34 months,and even extending up to 6 months (Daz-Paniagua 1982; Escoriza 2013). However, theselarvae can be exposed to irregular hydroperiods, and variations in the volume of water cantrigger an early metamorphosis, causing significant variability in the sizes of premeta-morphic tadpoles (lvarez et al. 1990). Moreover our results also suggested that the larvalsize can depend on the depth of the water body, which could account for part of theobserved variation. The existence of high intraspecific variability is a commonphenomenon in anuran larvae, and is usually an adaptive response to erratic changes inthe conditions of their aquatic habitats (Ghioca-Robrecht et al. 2009). This fact should betaken into account when producing taxonomic descriptions of anuran larvae, particularlythose of generalists or species that breed regularly in temporary ponds with highly variablehydroperiods (Newman 1989; Pfennig 1992).

We believe that these results also have important taxonomic implications. Pasteur &Bons (1959) suggested that P. varaldii can be distinguished from P. cultripes bydifferences in the shape of the tail. Specifically, these authors indicated that P. varaldii hasa more elongated tail (i.e. a higher span ratio) that is proportionally longer than that ofP. cultripes. Pasteur & Bons (1959) also considered that P. varaldii has concave edges tothe posterior caudal fin (convex or rectilinear in P. cultripes) and a more rounded tip of thetail. However, our analysis of the morphology of Pelobates tadpoles clearly showed thatthe tail outline is a highly variable trait and that it should not be used as a taxonomiccriterion. Our analysis also did not confirm in an unequivocal way the differences in theedges of the tail fin and in the contour of the tip of the tail. The shape of the tail was verysimilar in the mean and 2 SD shapes, and only when the +2 SD shape was taken into


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account did P. cultripes display a slightly more rectilinear contour. These discrepanciesbetween our results and those described by Pasteur & Bons (1959) can be explained by thehigh inter-population variability of the species studied. Pasteur & Bons were unable toexamine specimens of P. cultripes, and their comparisons were based solely ondescriptions provided in the literature, such as those of Boulenger (1897). Subsequently,no other authors have revised or proposed other criteria to distinguish the larvae of thesespecies (see Busack et al. 1985; Salvador 1996; Schleich et al. 1996; Beukemaet al. 2013).

In summary, this study provides a detailed analysis of the larval morphology of twoclosely related species of anuran, suggesting that, although they are similar, the larvae ofthese species show sufficient morphological divergence to be correctly classified asdistinct species. More studies are needed to determine whether these differences alsoextend to other aspects of their natural history.

ACKNOWLEDGEMENTS

The authors wish to express their gratitude to two anonymous referees for their usefulremarks.

Permits for field work were provided by the Haut Commissariat aux Eaux et Forts et la Lutte Contre la Dsertification in Morocco and the Departament de Medi Ambient deCatalunya, Spain (ref. SF/574).

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Appendix 1. Sampling Effort per Site, Water Body Size and TadpoleAbundance

PC: Pelobates cultripes; PV: Pelobates varaldii. Coord: geographical coordinates; N:number of water bodies surveyed; Spec: number of larvae specimens sampled; surfacearea: surface area of the water body (m2); average depth: mean depth of the water body(cm), after five random measurements. CPUE: catch per unit of effort (specimens/dipnet).

PC Coord N Spec Surface area Average depth CPUE

1 38 N 1 3 735 76 0.36.7 W

2 36.5 N 1 7 592 33 0.76.1 W

3 37.3 N 1 13 685 94 1.38.7 W

4 41.8 N 1 7 102 45 1.42.9 E

5 40.9 N 1 3 150 81 0.60.6 E

6 41.8 N 2 7 522 34 0.12.8 E 148 26 1.2

7 42.3 N 1 3 10337 88 0.23 E

8 42.3 N 3 9 7011 43 0.12.9 E 8715 23 0.1

5017 17 0.39 41.4 N 2 4 944 81 0.2

0.7 E 1113 71 0.110 41.8 N 1 7 1017 9 0.4

3.1 E11 42.3 N 1 1 250 81 0.2

3.1 E12 40.6 N 2 5 682 73 0.3

3.9 W 724 74 0.213 40.3 N 1 1 2290 77 0.3

4.3 W14 40.2 N 2 14 367 21 0.8

4.5 W 116 14 2.0


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Appendix 2. Environmental Factors and Phenotypic Variability

Spearman rank-order correlations between the morphological variables, water body sizeand tadpole abundance (CPUE). Significant values at p < 0.05 are marked in bold.PC: Pelobates cultripes; PV: Pelobates varaldii; TL: total length; CPUE: catch per unit ofeffort (specimens/dipnet).

PV Coord N Spec Surface area Average depth CPUE

1 34 N 7 22 6313 19 0.26.6 W 1170 42 0.5

805 35 0.2421 32 1.81000 27 0.14725 71 0.1

2 34.2 N 7 25 40828 94 0.26.5 W 1118 26 0.5

3666 45 0.11400 50 0.12306 24 0.11340 38 0.12774 23 0.21827 41 0.1

3 35.1 N 2 9 12344 39 0.36.1 W 2122 21 0.2

4 33.6 N 1 1 2530 26 0.17.1 W

Surface area Average depth Surface areaaverage depth CPUE

PC TL 0.17 0.48 0.29 0.35Area 0.09 0.54 0.34 0.26span ratio 0.23 0.45 0.08 0.24

PV TL 0.10 0.28 0.12 0.42area 0.18 0.12 0.10 0.26span ratio 0.15 0.30 0.17 0.15


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AbstractIntroductionMaterials and MethodsSamplingMorphological DataMorphological Analysis

ResultsDiscussionAcknowledgementsReferencesAppendix 1. Sampling Effort per Site, Water Body Size and Tadpole AbundanceAppendix 2. Environmental Factors and Phenotypic Variability

phenotypic variability in larvae of two species of mediterranean spadefoot toad: an approach using...

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