ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah Acta Herpetologica 6(2): 169-174, 2011 Ontogenetic pattern change in amphibians: the case of Salamandra corsica Wouter Beukema ITC, Faculty of Geo-Information Science and Earth Observation, University of Twente, Hengelosestraat 99, Enschede, The Netherlands. E-mail: wouter.beukema@gmail.com Submitted on: 2011, 27th February; revised on: 26th July; Accepted on: 16th September. Abstract. Ontogenetic, post-metamorphic pattern development is a rarely studied topic in amphibian science. As there are indications that the pattern of Salamandra corsica might expand over time, digital image analyses were applied in order to meas- ure several phenotypical variables which were related to the snout vent length. Results show a significant increase of patches which change to irregular shapes while SVL increases. Digital image analysis is identified as a suitable tool to explore pattern shape and change in general, while the documented pattern development in S. corsica might be one of the first quantified cases of post-metamorphic ontogenetic pattern change in amphibians. Keywords. Salamandra corsica, digital image analysis, ontogeny, colour pattern development. Coloration in the amphibian skin is composed of structural different xanthopores, iri- dophores and melanophores, which usually develop during the larval stage (Pederzoli et al., 2003; Vitt and Caldwell, 2009), after which colour patterns typically stabilize shortly after metamorphosis (e.g. Klewen, 1991). However, there are several reports of ontogenet- ic pattern change, i.e. post-metamorphic change in the colour pattern over time in several species of amphibians (e.g. Lantz, 1953; Bogaerts, 2002). While few in number, the major- ity of experimental pattern development research has taken place during the early 20th century and was mostly based on the relation between background colour and amount of dorsal and ventral patches in members of the genera Salamandra and Triturus (Kammer- er, 1913; Herbst, 1924; Lantz, 1953). Likely due to the rarity of such studies and the fact that most were based on anecdotal evidence, ontogenetic pattern change was not men- tioned in several recent reviews on amphibian biology (Duellman and Trueb, 1994; Wells, 2007; Vitt and Caldwell, 2009). Recent developments in digital image analyses of amphibian phenotypes (e.g. Davis and Maerz, 2007; Vörös et al., 2007) could be especially useful in exploring and quantify- ing ontogenetic pattern change. These analyses are increasingly used to measure a vari- 170 W. Beukema ety of morphological and phenotypical features in amphibians such as coloration, pattern and shape (e.g. Relyea, 2004; Davis and Maerz, 2007; Vörös et al., 2007) which can be applied for multiple purposes including evolutionary research and systematics (e.g. Davis and Grayson, 2007; Vörös et al., 2007). Their use permits the quantification of phenotype traits, such as amphibian colour patterns, which have been used widely in taxonomy, but traditionally often have been based on anecdotal evidence only. Members of the genus Salamandra occur over most of Europe and the Mediterranean area, ranging south- and eastwards into North Africa and the Near East (review in Thies- meier, 2004). While a high number of (subspecific) taxa have been described during the last century especially within Salamandra salamandra, the general taxonomy of the genus is unresolved (Steinfartz et al., 2000). Traditionally, the majority of taxa belonging to the ‘Fire salamanders’ (as opposed to the ‘Alpine salamanders’) have been described based on their diagnostic yellow colour pattern composed of xanthopores (Eiselt, 1958). However, data regarding colour patterns is often restricted to the type series of the taxon originat- ing from a single locality, while only limited research has been performed concerning intraspecific pattern colour variation within Salamandra (but see e.g. Bosch and López- Bueis, 1994; Bogaerts, 2002; Donaire Barroso et al., 2009). There are indications of ontoge- netic pattern change in several Salamandra taxa (Eiselt, 1958; Mutz, 1992; Bogaerts, 2002). Contrasting statements exist on the pattern of one of these taxa, Salamandra corsica. Savi (1838) described the species as being characterized by only sparse yellow coloration, while Eiselt (1958) suspected the yellow pattern to expand and merge during development after metamorphosis. Consequently, qualitative analysis of the pattern of S. corsica might both provide an example of rarely studied pattern change, while resolving pending issues on the morphology of the species. In order to gather data on dorsal pattern of S. corsica, fieldwork was conducted in Forêt de Vizzavona (Haute-Corse, 42°06’N, 9°07’E), Corsica at approximately 1200m alti- tude, from the 28th of April until the 2nd of May 2005. The study site is a Fagus sylvatica forest on a granite slope with a thick leaf litter layer and conclusively little undergrowth apart from several Asplenium species and Cyclamen hederifolium. A small brook crosses the area which is used for reproduction by S. corsica. Dorsal photos were made with a Nikon Coolpix E4600 of a total of 35 individuals which were immediately released after measuring their snout vent length in mm (SVL; the distance from the tip of the snout to the posterior margin of the cloaca). Due to undulations of the limbs and tail, images of individual S. corsica were cropped in Adobe Photoshop CS2 to include only the dorsum and head defined as the Region Of Interest (ROI). The Noise Filters ‘Despeckle’ (detects the edges in an image and blurs all of the selection except those edges) and ‘Median’ (within the radius of a pixel those pixels with similar brightness are selected while discarding pixels that differ too much from adjacent pixels; the centre pixel is replaced with the median brightness value of the searched pixels) were used to facilitate subsequent selection of the ROI against heteroge- neous backgrounds. Using the ‘Magic Wand Tool’ with a tolerance of 50, yellow patches (defined as areas of xanthopores) on the dorsum and head, as well as lateral patches con- tinuing onto the dorsum were selected and saved as a separate image (Fig. 1). Pixel sur- faces of the ROI, area of yellow patches, perimeter of yellow patches and circularity (CI) were calculated with the freeware ImageJ (Ferreira and Rasband, 2011). The area of yellow patches was converted to a percentage of the ROI area. Patch circularity was defined as 171Ontogenetic pattern change in amphibians CI = 4π(area/perimeter2) in which a CI of 1 represents a perfect circle while 0 is an infinitely elongated polygon. Furthermore, the number of yellow patches was visually summed. Statistics were computed in SPSS16. A Shapiro-Wilk test was used to investi- gate normality. Subsequently, Spearman’s Rank Correlation Test was used to examine the strength of relations between SVL, CI, number of patches and area of patches. Significant relations between variables were tested with a P < 0.05. Descriptive statistics on the measured variables (N = 35) are given in Table 1. SVL displayed a strong correlation with circularity (Spearman’s rs = -0.889, P < 0.0001) and the number of patches (rs = 0.783, P < 0.0001). Additionally, circularity was strongly cor- related with the number of patches (rs = -0.864, P < 0.0001). Although a weak correlation was recovered between SVL and the area of patches (rs = 0.254) the relation proved to be insignificant (P = 0.141). Table 1. Descriptive statistics on the phenotypical variables derived from digital image analyses and the snout vent length of thirty-five Salamandra corsica. Variable Mean Minimum Maximum SD Circularity (index) 0.0000046 0.0000023 0.0000086 0.0000017 Area of patches (%) 29.73 15.68 49.78 10.04 Number of patches 19.54 11 35 7.04 SVL (mm) 74.71 32.43 100.01 21.83 The current results show a significant positive relationship between SVL and number of yellow patches, and a negative relationship between SVL and patch circularity. While Fig. 1. Example of unprocessed images (A: juvenile Salamandra corsica, C: adult Salamandra corsica) to processed images showing the selected patch area on the Region Of Interest of the same individuals (B,D). 172 W. Beukema the area of yellow patches does show a weak relation with SVL, this relationship is not sig- nificant. This could be an effect of low sample size. On the other hand, the relative area of yellow patches might increase only slightly or remain approximately equal when patches ontogenetically change to a more irregular shape. Visual inspection of the photographed individuals indeed showed several cases of merged patches on the head (see also Fig. 1) as described by Eiselt (1958), but the general pattern seems to be highly inconsistent with considerable variation in patch number and area of yellow patches in adults (Fig. 2). Therefore, as displayed by the highly significant relationships ontogenetic pattern develop- ment is present in S. corsica, but is characterised by substantial intraspecific variation. Bogaerts (2002) provided photographical evidence on the ontogenetic pattern develop- ment in a small sample of south-Iberian S. s. crespoi and S. s. gallaica over the course of a Fig. 2. Correlations between snout vent length and number of patches (A) and circularity (B). 173Ontogenetic pattern change in amphibians few years, while noting that Moroccan S. algira tingitana seems to show ontogenetic reduc- tion of xanthopores. A similar tendency of iridophore decrease was suspected by Beukema et al. (2009) for a population of Anatolian Lyciasalamandra luschani finikensis. In addition to its relevance for general morphology and systematics, ontogenetic pattern change can be also an important factor within long term population studies based on individual recogni- tion by means of colour pattern. Such studies have been applied on different subspecies of S. salamandra (e.g. Kopp-Hamberger, 1998; Carafa and Biondi, 2004), without taking sig- nificant changes in pattern into account. Errors could potentially arise when recognition of prior identified individuals fails due to post-metamorphic development, similar to a recent case described for the viperid Vipera ursinii (Tomović et al., 2008). Conclusively, the currently documented pattern development in S. corsica might be one of the first quantified cases of post-metamorphic pattern change in amphibians. While according to literature ontogenetic pattern change appears to occur rarely, it is at least pre- sent in several taxa within the Salamandridae, and presents an interesting topic for future research. ACKNOWLEDGEMENTS Jan Beukema and Mark Bakkers are thanked for assistance in the field. Sergé Bogaerts pro- vided valuable discussion on pattern development among Salamandra taxa. REFERENCES Beukema, W., de Pous, P., Brakels, P. 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