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Feb 18, 2005 - (Carcinus maenas L.). The results, based on a virtual grid laid over digital images of crab carapaces, allow for correlations to be made among ...

Ecol Res (2005) 20: 497–501 DOI 10.1007/s11284-004-0034-5


P. A. Todd Æ R. J. Ladle Æ R. A. Briers Æ A. Brunton

Quantifying two-dimensional dichromatic patterns using a photographic technique: case study on the shore crab (Carcinus maenas L.)

Received: 13 September 2004 / Accepted: 29 November 2004 / Published online: 18 February 2005  The Ecological Society of Japan 2005

Abstract Contrasting patterns of pigmentation, such as those associated with crypsis and aposematism, are common in many taxa. In order to determine why patterning varies among individuals or populations, it is important to quantify how these patches of pigment are arranged. Here we present a simple technique for measuring areas of pigmentation as well as their spatial distribution, and demonstrate its application to the study of substrate-associated patterning in shore crabs (Carcinus maenas L.). The results, based on a virtual grid laid over digital images of crab carapaces, allow for correlations to be made among sample populations. The technique, or variations of it, can be applied to any situation where two-dimensional dichromatic patterns need to be quantified. Keywords Dichromatic pattern Æ Shore crab Æ Photographic technique Æ Crypsis Æ Aposematism

Introduction Polymorphisms and polyphenisms are widespread in many taxa and are frequently characterised by differP. A. Todd Æ R. A. Briers Æ A. Brunton School of Life Sciences, Napier University, Merchiston Campus, Edinburgh, EH10 5DT, Scotland, UK R. J. Ladle School of Geography and Environment, University of Oxford, Mansfield Road, Oxford, OX1 3TB, England, UK Present address: P. A. Todd (&) Marine Biology Laboratory, National University of Singapore, 14, Science Drive 4, Blk S1, #02-05, 117543, Singapore E-mail: [email protected] Tel.: +65-68741034 Fax: +65-67792486

ences in colour, dichromatic patterns such as spots and stripes, or a combination of both. These patterns are studied in various contexts, including mimicry, crypsis and aposematism (Mallet and Gilbert 1995; Merilaita 1998; Joron and Mallet 1998; Bedini 2002), selective processes (Jones et al. 1977; Forsman and Appelqvist 1999; Palma and Steneck 2001), genetic differentiation/ phenotypic plasticity (Ekendahl and Johannesson 1997; Wente and Phillips 2003) and habitat selection (Forsman and Shine 1995; Ravigne et al. 2004). To make different morphs statistically amenable they are usually categorised into groups, thus enabling frequencies to be compared and associations to be made (e.g. Ekendahl and Johannesson 1997; Kark et al. 1997; Carretero 2002). In most organisms, however, such grouping is arbitrary as intermediate forms and outliers are common. In addition to the information lost via a classification approach, researchers cannot be certain their definitions exactly match those of other studies. Accurately and simply measuring patterns on threedimensional (3-D) objects is likely to remain difficult for some time (Bythel et al. 2001). But dichromatic variability in a 2-D appendage, for example an insect wing, or a plane that is relatively constant among individuals, such as the profile of a fish, should be quantifiable—especially if an image of the appendage or organism can be captured and examined in detail. The combination of digital photography and image analysis software is a powerful tool that has been used in medicine and dentistry (e.g. Benson et al. 1998; Cochran et al. 2004; Hamza and Reddy 2004; Owen et al. 2004,), as well as for various aspects of plant (Hyder et al. 2003, Jia et al. 2004) and animal (Pech et al. 2004; Heide-Jorgensen 2004) biology and ecology. Todd et al. (2001; 2004) demonstrated that photography offers the study of coral morphometrics various advantages including speed of sampling, non-destructiveness, an instant permanent record, and the opportunity for repeat measurements. These same four advantages should also apply to the study of 2-D dichromatic patterns using digital images.


To successfully discriminate among different pattern morphs a quantitative approach must be able to measure the relative amount of each colour as well at its shape and spatial orientation. For instance, when examining an eye-spot on a butterfly’s wing, the size and shape of the spot, as well as its position, are all important. Furthermore, issues regarding allometry must also be considered, as different sized individuals will tend not to have the same absolute quantities of pigment. Here we demonstrate a photography-based technique for quantifying 2-D dichromatic patterns using variation in shore crab (Carcinus maenas) carapaces as a case study. The shore crab is an interesting study species because it exhibits a large range of carapace patterns (especially when young) of which little is known (Hogarth 1975, 1978). If these patterns represent some form of crypsis, then associations between carapace colouration and substrate is expected (Endler 1988; Ekendahl 1998). To help identify any such associations, we have quantified the carapace patterns of shore crabs from sites where the substrate composition has also been measured.

Materials and methods Due to their pelagic larval phase, shore crabs have high levels of gene flow (Brian 2002); nevertheless, different morphs have been associated with different substrates (Hogarth 1978). To test whether carapace patterns vary among sites with differing substrates in this study, crabs were sampled from three intertidal rocky shores along the Firth of Forth, Scotland, i.e. Ferny Ness (5559.215¢N, 00254.091¢W), Milsey Bay (5603.674¢N, 00243.016¢W) and Long Craigs (5600.317¢N, 00232.518¢W). At each site a sampling area of 50 · 120 m perpendicular to the shore was delimited. This large area was then subdivided into 15 smaller (10 · 40 m) quadrats, arranged so that there were five adjacent replicates at each of three shore positions: high, middle, and low. From each shore position, two of the five replicate quadrats were randomly selected (using

Substrate cover (%)


random number tables) and all crabs found therein were collected (45 min searching time). The substrate composition of these three sites, as determined using line intercept transect methodology (English et al. 1997), is provided in Fig. 1. All patterned crabs were photographed in the laboratory with a high-quality digital camera at 640·480 pixel resolution before being released at a location >20 km from the sampling sites. RGB colour images, as oppose to black and white ones, were used as they were slightly easier to interpret. A millimetre scale (an engineer’s ruler) and a code for sex and quadrat of origin (written on paper) were physically included in each photograph; when each image was downloaded onto computer this information was added as a label. Using a raster-based graphics software [Paint Shop Pro version 7.04 (Jasc Software 2000)] the images were straightened and cropped so that all four carapace extremities (anterior and posterior edge, left and right edge) were just touching the four sides of the frame (Fig. 2) with the rostrum to the top. In Microsoft PowerPoint a 20·20 cell, font size 10, table was created and placed over each imported image. Both table and image were then adjusted so that the two matched exactly. To quantify patterns zeros were allocated to areas of dull green carapace as well as the non-crab regions of the image, whereas ones were used to identify cells that (visually and subjectively) appeared to contain >50% white pigment (Fig. 2). After all 400 cells were filled, the curser was dragged over the entire table and the data copied and pasted into Microsoft Excel (in most versions of Excel the 400 data points will automatically form into one column) and appropriately labelled. All the patterned crabs from any one quadrat were treated in the same way, thus one spreadsheet contained columns representing individual crabs and 400 rows that represented presence and absence of white pigment. After all







cobble rock


mussel - bed

0 Ferny Ness

Milsey Bay

Long Craigs

Fig. 1 Substrate composition of the three sampling sites. At each site a total of 1,200 m of line intercept transect data were collected

Fig. 2 The technique is based upon a 20 · 20 virtual grid laid over the image. Depending on the level of resolution required, grids with more, or fewer, cells can also be used. Here, for clarity, the noncarapace regions of the image have been blocked out and different colours used for ones and zeros

the crab data from one quadrat had been entered, the mean value for each row was calculated. Thus, one column of 400 variables effectively encapsulated the mean quantity, shape, and spatial orientation of white pigment for all patterned crabs from that quadrat. After all crabs from the 18 quadrats had been treated in the same way, a similarity matrix was formed using the correlation statistic in Excel (Pearson correlation coefficient) applied to the 18 summary columns. Due to the high number of zeros and similar spatial arrangement of patterns, these correlation figures were very high and could not be interpreted in the usual manner. However, the required information was contained within the relative correlations among quadrats. This was analysed by ordinating the correlation matrix using non-metric multidimensional scaling (MDS). We chose to do this with Primer version 5 (Clarke and Gornley 2001) as this programme allows the user to input their own distance (in this case correlation) matrix. The significance of differences in patterning among shores was assessed using the one-way analysis of similarity (ANOSIM; Clarke and Warwick 1994) procedure in Primer. To visualise the actual differences in carapace pattern among sites, the mean row values for each site were reassembled into 20·20 tables and used to create 3-D graphs.

Results and discussion A total of 1,772 crabs were collected of which 789 were patterned: 373 from Ferny Ness, 155 from Milsey Bay and 261 from Long Craigs. All patterned crabs were successfully photographed, and the resultant images analysed. For each crab, the entire process from downloading the image to entering the data into the spreadsheet took 2.5––3 min. Thus approximately 40 h of processing was needed for this, relatively large, data set. Multidimensional scaling ordinates our correlation matrix in two dimensions with a low stress value of 0.07. There was a significant among-site difference in patterning (ANOSIM, global R=0.302, P=0.002). Pairwise comparisons between the sites indicated that there was a significant difference between Ferny Ness and Milsey Bay (R=0.428, P=0.004) and Ferny Ness and Long Craigs (R=0.481, P=0.002), but not between Milsey Bay and Long Craigs (R=0.019, P=0.372). This separation is reflected in the MDS plot where Ferny Ness forms a distinct group, but there is a strong overlap between Milsey Bay and Long Craigs (Fig. 3). The 3-D representations of the data for each site indicate that Ferny Ness hosts crabs with more widely distributed white areas, especially in the central and anterior part of the carapace (Fig. 4). Using predator-exclusion experiments, Palma and Steneck (2001) demonstrated that polymorphic rock crabs (Cancer irroratus) possessing non-adult carapace colours had a higher survival rate in polychromatic habitats than monochromatic ones. As the substrate at Ferny Ness is characterised by mussel-

axis 2


axis 1 = Ferny Ness

= Milsey Bay

= Long Craigs

Fig. 3 This multidimensional scaling plot, based on the carapace pattern correlation matrix, illustrates how the crabs from the Ferny Ness quadrats form a distinct group whereas those from Milsey Bay and Long Craigs overlap

bed and is thus highly polychromatic, it is possible the relationship between this site and strong carapace patterns is not simply stochastic (Ekendahl 1998). Exactly where on the carapace the white spots appear most frequently can be identified from the 3-D graphs, i.e. the hepatic and facial regions, plus some marking along the anterior–posterior axis (Fig. 4). This provides clues as to whether the colouration is some form of crypsis or mimicry. The high frequency of white spots touching the edge of the carapace is suggestive of disruptive colouration as this type of marking helps break up the outline of an organism (Merilaita 1998). The fact that most of the white pigmentation appears towards the anterior, rather than the posterior, of the carapace may be associated with shore crab behaviour, i.e. their tendency to peer out from under rocks or among mussel shells, thus only revealing their anterior region (P. Todd, personal observation). Finally, the hepatic patches could possibly be interpreted as eye-spots, and thus aposematic. The advantages of photographic techniques discussed in Todd et al. (2001), and outlined in the Introduction of this paper, also apply to the present study. Sampling is fast and non-destructive, only requiring a photograph of the carapace that includes a scale bar and some indicator of sex and site. Downloading images onto a computer or some other form of electronic memory creates a permanent record; and the technique would certainly be suitable for studies of pattern change with growth. The ensuing analysis is also straightforward, and while it may be time-consuming once specimen numbers become high, compared to manual scoring methods the time saving is substantial. The majority of the process can be conducted on a personal computer loaded with standard software (Excel, PowerPoint, Paint Shop Pro); the only specialist programme is the one selected to ordinate the similarity matrix. A virtual grid placed over a crab image means that direct comparisons of patterns can be made among individuals of varying size. If absolute measurements of



b) anterior

c) non-crab areas

non-crab areas


Fig. 4 Three-dimensional graphs illustrating the relative mean frequency of white pigment on the carapaces of crabs from a Ferny Ness, b Milsey Bay, and c Long Craigs. The crabs from Milsey Bay and Long Craigs tend to have less white pigment in the central region of their carapaces, as indicated by the depressed areas to either side of the anterior–posterior axis, than those from Ferny Ness

pigment patch size were used, they would have to be adjusted to overall organism/appendage size before comparisons among individuals could be made. Using a grid means that every cell has a constant, isometric (sensu Bookstein 1989), relationship with its neighbouring cells, regardless of size. This constant relationship also means that, when the grid and image are re-sized to match each other, the information extracted from the grid is still an accurate reproduction of the pattern being studied. Issues regarding correlations between pattern and sex, or between pattern and organism size, need to be addressed just as they would if the patterns had been categorised into groups (Hoffman and Blouin 2000; Hull and Rollinson 2000). In cases of sexual dimorphism, treating sexes separately is appropriate. Where patterns change with size, organisms/appendages can be pooled by some representative mensuration and a semi-continuous gradient of pattern created. Alternative approaches to statistically explore polymorphic populations grouped by morph type include ANOVA (Palma and Steneck 2001), diversity indices and v2-test (Ekendahl and Johannesson 1997). All these analyses can be adversely affected by low sample size and/or ‘‘empty cells’’. As the technique presented here does not split the populations into smaller components, it is not compromised by low or zero counts of particular morphs. Naturally, there remains a positive relationship between sample size and the information content of the data. Furthermore, a balanced design is preferable and, ideally, each replicate should comprise an equal number of specimens. The primary drawback of the technique is that it is only applicable to dichromatic patterns. For polychromatic patterns each colour has to either be treated separately or weighted. Treating the colours separately means different distance matrices for every colour combination of interest, although the matrices themselves can be amalgamated to produce a single result. Alternatively, if some colours are considered more important

than others, they can be assigned a number higher than 0 or 1. This produces a distance matrix that accurately reflects the weighting, but the 3-D graphs become redundant as they can only represent two colours. The technique has successfully identified differences in shore crab carapace pattern among three sites—information that helps to address questions regarding crypsis, mimicry, and habitat selection. The combination of ordination plots and 3-D graphs provides an easily interpretable summary of large data sets — in the present case, >800,000 data points. Only requiring readily available hardware and software, the process is straightforward. This novel approach could be applied to the profiles of higher animals, but is probably best suited to the study of patterns on the appendages and bodies of invertebrates, or on plant leaves and petals. Acknowledgements This study was supported by Napier University, Edinburgh, UK. We would like to thank the numerous students who have assisted with fieldwork and laboratory analysis.

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