Morphology can be adaptive through its effect on performance of an organism. The effect of performance may, however, be modulated by behavior; an organism may choose a behavioral option that does not fully utilize its maximum performance. Behavior may therefore be decoupled from morphology and performance. To gain insight into the relationships between these levels of organization, we combined morphological data on defensive structures with measures of defensive performance, and their utilization in defensive behavior. Scorpion species show significant variation in the morphology and performance of their main defensive structures; their chelae (pincers) and the metasoma (“tail”) carrying the stinger. Our data show that size-corrected pinch force varies to almost two orders of magnitude among species, and is correlated with chela morphology. Chela and metasoma morphology are also correlated to the LD50 of the venom, corroborating the anecdotal rule that dangerously venomous scorpions can be recognized by their chelae and metasoma. Analyses of phylogenetic independent contrasts show that correlations between several aspects of chela and metasoma morphology, performance and behavior are present. These correlations suggest co-evolution of behavior with morphology and performance. Path analysis found a performance variable (pinch force) to partially mediate the relationship between morphology (chela aspect ratio) and behavior (defensive stinger usage). We also found a correlation between two aspects of morphology: pincer finger length correlates with the relative “thickness” (aspect ratio) of the metasoma. This suggests scorpions show a trade-off between their two main weapon complexes: the metasoma carrying the stinger, and the pedipalps carrying the chelae.

In other groups important evolutionary connections have been observed in the relationships between venom compounds and the evolution of the venom gland. For example, in squamates [28] the structure of the delivery system [29] and its functional performance [30] are intimately associated with the evolution of the venoms themselves [31] . The evolution of the venom, and its mechanical delivery system, are therefore intimately related and must be studied together. We here present comparative data on the association between the morphology and performance of the defensive structures of different species of scorpions, and their defensive behavior. Our data show a large variety in defensive behaviors, and an evolutionary association with both morphology and performance.

Occurring worldwide in terrestrial habitats ranging from temperate forest to deserts and tropical forests, scorpions have ecologically diversified considerably, with nearly 2000 described species, and many more cryptic species are awaiting discovery [14] , [15] . Several ecomorphotypes based on relative sizes of specific body parts have been qualitatively described [16] , [17] . A large part of the morphological variation of scorpions resides in their most emblematic body parts; the pincers or chelae [18] , and the tail-like metasoma carrying the venomous stinger. These structures are used in defense [19] , as well as in prey capture and incapacitation. Scorpions can form a large part of the animal biomass in some habitats [17] , and are therefore an important food resource for some predators. Scorpions possess defensive responses that elicit fear in mammals [20] . Some predators, however, such as Hemprich’s long-eared bat (Otonycteris hemprichii), have developed insensitivity to scorpion defenses in order to utilize this important resource [21] . Most scorpions will avoid contact with predators by retreating to a burrow or other hiding place. When cornered or apprehended by a predator, a scorpion can choose to use either its chelae or its venomous telson (stinger), or both. The distribution of the defensive capacities of scorpions between the chelae and the telson may results in an evolutionary trade-off in the investment in these two systems; some species have developed powerful chelae and others have a well-developed metasoma carrying the venomous stinger. In interspecific interactions, scorpion species with larger chelae are known to use them more, whereas the Buthidae, having more slender chelae, use their metasoma more in defense [22] . In fact, the relative size of chelae and metasoma is often used as a rule of thumb to assess whether an unknown scorpion may be dangerously venomous or not [23] – [25] . The species with more robust chelae produce a much higher pinch force [26] , and finite element analyses show that their low-aspect ratio shapes allow cuticular stresses to remain lower during application of maximum forces, making them better suited for use in defense [18] . The “bite” force of the chelae of scorpions relative to their body mass is highly variable, and spans a range of almost three orders of magnitude [27] , suggesting this performance variable may be subject to differential selection for its different functions (such as prey prehension, mating, sensing, defense etc.), although neutral variation cannot be excluded.

Behavior, i.e. the response of an animal when faced with behavioral options [1] , is often viewed as an important driver of evolutionary diversification. Some authors have argued that behavioral flexibility may also constrain phenotypic evolution [2] . The fact that animals can use different behaviors depending on the context may blur the relationships between morphology, performance and ecology [1] , [3] , [4] . Consequently, selection on performance capacity may be decoupled from ecology [4] , and behavioral variation can result in a many-to-one mapping of performance on ecology [5] . On the other hand, links between behavior, performance, and morphology have been demonstrated at the inter- and intra-specific level [6] – [10] . Moreover, behavioral traits can have a genetic basis and as such can be under direct selection [11] , [12] . Yet, which aspects of morphology and performance are related to behavior and whether morphology and performance variation constrains or enhances the evolution of different behaviors, or vice versa, remains poorly understood. Given that behavioral traits typically show less phylogenetic signal [13] , the evolution of behavior may indeed be decoupled from the evolution of morphology and performance for many functions. In active defensive behavior against predators; however, the prey’s fitness is maximized by successfully deterring the predator. The arms race between the prey’s deterrence capacity and the predator’s ability to withstand or circumvent these defenses requires the prey to select its maximum performance in the context of its defensive behavior. It is therefore likely that defensive behavior against predators, which we study here, is more closely correlated to maximum performance than behaviors for which non-maximum performance also has a fitness benefit [4] .

Materials and Methods

Ethics Statement Buthid scorpions were kept under ICNB license 05/2010/CAPT. No additional permits were required for the described experimental manipulations. When necessary, subjects were anesthetized using Isoflurane. All efforts were made to minimize suffering.

Taxon Selection and Animal Maintenance A total of 26 scorpion species were selected to represent a broad range of chela and telson morphologies based on their availability (Table 1). All specimens were procured from the pet trade, and kept in captivity for at least several weeks before experiments commenced. Species were identified using specific keys [17], [32]–[40]. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Species and numbers of specimens used for each aspect of the study. https://doi.org/10.1371/journal.pone.0078955.t001 All animals were kept under species-specific circumstances [26] and appeared in good health throughout the test period and beyond. Desert species (Androctonus, Buthacus, Buthus, Hadogenes, Hadrurus, Hottentotta, Leiurus, Orthochirus, Parabuthus, and Scorpio) were kept in plastic boxes (123×190×80 mm for small species; 200×230×130 mm for larger species) on a layer of ground cork substratum. Species requiring more humid circumstances (Caraboctonus, Euscorpius, Grosphus Hetrometrus, Iomachus, Opisthacanthus, Opistophthalmus and Pandinus) were kept in plastic boxes with humid substrate and sprayed with water regularly. All animals were fed with crickets (Acheta sp.) and cockroaches (Blaptica sp.) once every 1–2 weeks before and during the experiment. All specimens were provided with a piece of polyethylene tubing as a hiding place, and kept at 24°−26°C. Although optimum temperatures for the species in this study are not known, the used maintenance conditions were chosen as all species have been kept under these conditions in good health for several years by one of us (AvdM). Data from specimens that died, gave birth or molted during the study were excluded. From a small subsample a haemolymph smear was inspected for the presence of parasites after the test period, and none were discovered.

Behavioral Trials Behavioral trials were executed to estimate qualitative differences in the defensive response of scorpions. Before the trial proper was started, the scorpion was aroused by gently tapping the pedipalps and/or prosoma until an alert posture was assumed (chelae extended and metasoma erect). Each trial consisted of first restraining each of the chelae in arbitrary order for five seconds using large rubber-tipped tweezers, followed by a similar restriction of the prosoma (figure 1). Each trial therefore resulted in three behavioral responses. Two responses from restraining each of the two chelae, and one from restraining the prosoma. Only actual gripping motion on the tweezers and directed stinging were scored as defensive behaviors. All behavioral trials were performed in the enclosure of the scorpion. Each specimen was subjected to these behavioral trials five times, spaced by at least one day. To allow a comparative study, and as optimum temperatures are unknown for most of the included species, we chose to make all behavioral and performance observations at standardized environmental conditions. All behavior trials were performed by a single person (AvdM) in a climate controlled room at 23-24°C. This temperature range was chosen as both tropical and desert species have been observed to be active at these temperatures. This temperature range is on the low end of the temperature range to which all specimens were acclimated for several weeks before the experiments started, thus mimicking the nighttime conditions during which scorpions are normally active. Behavioral responses were scored in the following categories: (0) none; (1) chelae only; (2) telson only; (3) Both chelae and telson. For further analysis, except where stated otherwise, the responses from the chela restrictions and the prosoma restrictions were pooled. We also calculated the proportions of the active responses without the non-responses (0), in order to quantify what a scorpion uses in response if it chooses to respond at all (designated if1, if2 and if3). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Defensive behavior trials, shown on a specimen of Hadogenes cf. paucidens. First each chela is pinned to the ground using rubber-tipped forceps for 5 seconds (a.). Subsequently, the prosoma is pinned down for 5 seconds (b.), and the defensive response categorized as using one or both chelae (1), the telson (2), chelae and telson (3) or neither (0). https://doi.org/10.1371/journal.pone.0078955.g001 For visualization of the data, hierarchical cluster analysis was performed using Euclidian distances. We also clustered the species based on active responses only (if1, if2, if3), and on the proportion of the usage of chelae (category 1+3; TotC) and telson (category 2+3; TotT) in defense. We performed non-parametric Fisher's exact tests on the behavioral data in order to identify significant differences in behavior among species (Table 2). In addition, a Fisher’s exact test was used to test for differences in behavioral responses to chela restriction versus prosoma restriction. These statistical analyses were performed in R [41]. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Differences in defensive responses between species as tested with a two-sided Fisher's exact test. https://doi.org/10.1371/journal.pone.0078955.t002

Morphology All external measurements were made using digital calipers on preserved or isoflurane anesthetized specimens. These specimens were either the specimens used in other aspects of this study, or specimens of similar size from the same source. Measurements of the distance between the fulcrum of the movable finger of the chela and the manus, and the muscle insertion point furthest away from it were either made by hand using digital calipers on the disjointed movable finger of preserved specimens, or taken from high-resolution CT or synchrotron scans [18]. These internal measurements to determine the force inlever of the movable finger were made on a subsample of the specimens available for each species, or only once when anatomical scan data were available. Because scorpions can vary considerably in length, girth and weight depending on their feeding state, we used the length of the prosoma, which does not vary between molts, as an indicator of overall size [32]. Regression of morphological variables on prosoma length as a proxy for size was not significant, as there are large differences in the relative sizes of the chelae and metasoma between species independent of overall size variation. The calculation of regression residuals was therefore not appropriate. Several linear measurements were combined in order to give functionally relevant ratios. Chela aspect ratio (AR) is the height of the chela manus divided by the total length of the chela. This ratio has been shown to be highly correlated with pinch force [26]. Similarly, metasoma AR was calculated by dividing the metasoma length by the product of the average height of the 1st, 3rd and 5th metasomal segment and the average of the width of those segments to provide a single value for metasoma girth. The ratio of the movable finger to the chela length was obtained by dividing the total length of the chela by the length of the movable finger, and captures the relative length of the chela fingers. Longer-fingered chelae will thus result in a smaller value. Since relatively longer-fingered chelae will have a longer outlever, and a reduced space for muscles, we expect that long fingers will correlate to reduced pinch performance. Mechanical advantage was calculated by dividing the average of the distance from the muscle insertion to the axis of rotation for the left and right chela with the average of the length of the movable finger for both chelae. This measure is therefore the displacement advantage, and inverse to the force advantage, and expected to be lower in species with stronger chelae. Some scorpion species have reduced metasoma lengths and relative metasoma length was obtained by dividing metasoma length by the prosomal length, the latter being a good estimate of overall size (see above). A logistic regression was carried out to identify correlations between morphological characters and behavioral variables, with the behavioral classes as the dependent variable.

Performance Measurement In vivo pinch forces were measured using either a Kistler force transducer (type 9203, Kistler Inc., Switzerland) mounted on a purpose-built holder [42], or using a similar setup using a Sauter FH20 external force sensor (Sauter ltd., Germany). Measurements were made in a climate-controlled room set at 23–24°C. During pinch-force measurements, scorpions were restrained between sponge pads in which a cutout was made to accommodate the body, or by placing a padded clamp over the last segments of the metasoma to allow safe handling. Five trials were performed, separated by at least one day. Only the maximum force per individual was retained for further analyses. In order to obtain pinch forces corrected for body size, we attempted to use a linear regression of pinch force on prosoma length across all species. As these variables did not show any linear relationship (R2<0.02), presumably due to the effects of chela design obscuring the effects of body size, we chose to correct for size by dividing pinch force by the square of the prosoma length. Pinch force must be scaled by prosoma length squared, as force scales with the physiological cross section of the muscle, which in turn scales with length squared [43]. The LD50 of 14 species of scorpions were included as a second defensive performance variable. Where no LD50 was available, the value of a closely related species was used given that this variable is thought to be conserved within genera (see table 3). PPT PowerPoint slide

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larger image TIFF original image Download: Table 3. All behavioral responses by category, proportions of each response type, number of specimens, LD50 and measured variables. https://doi.org/10.1371/journal.pone.0078955.t003