In the stag beetle family (Lucanidae), males have diverged from females by sexual selection. The males fight each other for mating opportunities with their enlarged mandibles. It is known that owners of larger fighting apparatuses are favoured to win the male-male fights, but it was unclear whether male stag beetles also need to produce high bite forces while grabbing and lifting opponents in fights. We show that male Cyclommatus metallifer stag beetles bite three times as forcefully as females. This is not entirely unexpected given the spectacular nature of the fights, but all the more impressive given the difficulty of achieving this with their long mandibles (long levers). Our results suggest no increase in male intrinsic muscle strength to accomplish this. However, morphological analyses show that the long mandibular output levers in males are compensated by elongated input levers (and thus a wider anterior side of the head). The surplus of male bite force capability is realized by enlargement of the closer muscles of the mandibles, while overall muscle force direction remained optimal. To enable the forceful bites required to ensure male reproductive success, male head size and shape are adapted for long input levers and large muscles. Therefore, the entire head should be regarded as an integral part of male armature.

Two methods are especially suited for tomographic imaging with histological detail of macroscopic samples that consist of multiple tissue types (bone, muscle, nerve or fat): Light sheet (based) fluorescence microscopy (LSFM) and micro-computed tomography (micro-CT). Micro-CT requires staining with heavy chemical elements (and thus fixation and sometimes dehydration) in order to make soft tissue imageable when measured alongside denser structures. LSMF requires fixation, decalcification, dehydration, clearing and staining with a fluorescent dye. The specimen preparation of both imaging methods is prone to shrinkage, which is often not mentioned, let alone quantified. In this paper the presence and degree of shrinkage are quantitatively identified for the selected preparation methods/stains. LSFM delivers a volume shrinkage of 17% for bone, 56% for muscle and 62% for brain tissue. The three most popular micro-CT stains (phosphotungstic acid, iodine with potassium iodide, and iodine in absolute ethanol) deliver a volume shrinkage ranging from 10 to 56% for muscle and 27-66% for brain, while bone does not shrink in micro-CT preparation.

Stag beetle battle behavior and its associated anatomical adaptationsMale stag beetles battle for females with their impressive, oversized mandibles. We describe their fighting behavior, which is essential to understand the evolution and morphology of their weaponry. Our behavioral analysis reveals several anatomical structures that are important for fighting, and our morphological investigations show how these may be adapted for their functions. Stag beetle fightsare much more variable than other armed beetles' battles. They spend considerable time and effort in dislodging their opponent, that clings to the substrate with its tarsal claws. These tarsal claws are also indispensable to maintain balance in the most spectacular battles, when they lift a rival high in the air. The male claws are highly curved and have an increased height for this purpose. The prothoracic muscles are hypertrophied to support the lifting movement. The largest beetle wins in 85% of the fights and the smaller the difference in mandible length is between the rivals, the longer the battles can last. The long mandibles enable males to reach the opponent's legs in order to dislodge it. For this purpose, they bite with all parts of their mandibles, even though the distal part is more vulnerable for failure and transfers less bite force.Blindfolded experiments prove that visual information is not a requisite for a successful battle.Various weapons, evolved by sexual selection and used in male-male combats, are found in a very wide range of taxa: trilobite spines; crustacean chelipeds; stag beetle mandibles; scarab horns; humps, saws and spines of fishes; horns, spines and tusks of frogs; crests, frills, plates and spines of dinosaurs; antlers of deer, elks and moose; horns of bovids and rhinoceros (for a review, see (Emlen 2008)). Knowledge of the nature of fighting tactics and styles is primordial to understand the evolution and morphology of these armaments (Eberhard 1977;Caro et al 2003;Emlen et al 2005;Emlen 2008;Gotoh et al 2012). However, the fighting behavior, as such, has received surprisingly little scientific attention (Emlen 2008). Only for ungulates has the relationship between fighting style,

Locomotion is one of the most important ecological functions in animals. Precocial animals, such as pigs, are capable of independent locomotion shortly after birth. This raises the question whether coordinated movement patterns and the underlying muscular control in these animals is fully innate or whether there still exists a rapid maturation. We addressed this question by studying gait development in neonatal pigs through the analysis of spatio-temporal gait characteristics during locomotion at self-selected speed. To this end, we made video recordings of piglets walking along a corridor at several time points (from 0 h to 96 h). After digitization of the footfalls, we analysed self-selected speed and spatio-temporal characteristics (e.g. stride and step lengths, stride frequency and duty factor) to study dynamic similarity, intralimb coordination and interlimb coordination. To assess the variability of the gait pattern, left-right asymmetry was studied. To distinguish neuromotor maturation from effects caused by growth, both absolute and normalized data (according to the dynamic similarity concept) were included in the analysis. All normalized spatiotemporal variables reached stable values within 4 h of birth, with most of them showing little change after the age of 2 h. Most asymmetry indices showed stable values, hovering around 10%, within 8 h of birth. These results indicate that coordinated movement patterns are not entirely innate, but that a rapid neuromotor maturation, potentially also the result of the rearrangement or recombination of existing motor modules, takes place in these precocial animals.

Summary1. In many animal species, male armature has evolved through sexual selection. This male weaponry can increase reproductive success, but only if the owner overcomes the associated costs. 2. Male stag beetles bear one of the most extreme examples of male weaponry: their mandibles can be almost as long as their own body. We question whether the armature of male Cyclommatus metallifer negatively affects terrestrial locomotion (stability and cost). If so, we investigate whether these effects are potentially compensated by morphological and/or behavioural features, as seen in other specialized insect species. Conspecific females are used to represent the non-dimorphed condition. 3. The presence of the huge male mandibular apparatus shifts the body centre of mass (bCOM) anteriorly. Concomitantly, the male fore limbs are 28% longer and are systematically positioned in a more anterior angular sector than in females. Thus, the rostral border of the support area of the leg tripod also moves forward. This suggests a stability enhancing mechanism. However, in contrast to load-carrying ants, the anteriorly placed bCOM still creates two pronounced statically instable periods each locomotor cycle. 4. Due to the static instability, males must adjust their locomotor behaviour: they walk at higher cycle frequencies when compared to females of the same size, to ensure they proceed to the next stance before bumping to the ground with their heavy heads. 5. Contrary to other specialized load-carrying insect species, the (muscle) mass specific mechanical cost of transport of males exceeds that of females by 40%. 6. Since neither stability nor cost of transport benefit from the male forelimb size and positioning, their role in guaranteeing adequate terrestrial locomotion while carrying an enlarged mandibular apparatus seems doubtful. Instead, the long limbs are themselves functional in fights, by pitching the body upwards in order to throw opponents backwards. 7. The oversized male stag beetle armature comes at high ecological costs: locomotion economics as well as stability clearly suffer from the large mandibles. The observed limb length dimorphism does not prevent this, but should probably be considered part of sexual selection, rather than a compensation for its consequences.

Male stag beetles have evolved extremely large mandibles in a wide range of extraordinary shapes. These mandibles function as weaponry in pugnacious fights for females. The robust mandibles of Cyclommatus metallifer are as long as their own body and their enlarged head houses massive, hypertrophied musculature. Owing to this disproportional weaponry, trade-offs exist with terrestrial locomotion: running is unstable and approximately 40% more costly. Therefore, flying is most probably essential to cover larger distances towards females and nesting sites. We hypothesized that weight, size and shape of the weaponry will affect flight performance. Our computational fluid dynamics simulations of steady-state models (without membrane wings) reveal that male stag beetles must deliver 26% more mechanical work to fly with their heavy weaponry. This extra work is almost entirely required to carry the additional weight of the massive armature. The size and shape of the mandibles have only negligible influence on flight performance (less than 0.1%). This indicates that the evolution of stag beetle weaponry is constrained by its excessive weight, not by the size or shape of the mandibles and head as such. This most probably paved the way for the wide diversity of extraordinary mandible morphologies that characterize the stag beetle family.

Male stag beetles carry large and heavy mandibles that arose through sexual selection over mating rights. Although the mandibles of Cyclommatus metallifer males are used in pugnacious fights, they are surprisingly slender. Our bite force measurements show a muscle force reduction of 18% for tip biting when compared with bites with the teeth located halfway along the mandibles. This suggests a behavioural adaptation to prevent failure. We confirmed this by constructing finite-element (FE) models that mimic both natural bite situations as well as the hypothetical situation of tip biting without muscle force modulation. These models, based on micro-CT images, investigate the material stresses in the mandibles for different combinations of bite location and muscle force. Young's modulus of the cuticle was experimentally determined to be 5.1 GPa with the double indentation method, and the model was validated by digital image correlation on living beetles. FE analysis proves to be a valuable tool in the investigation of the trade-offs of (animal) weapon morphology and usage. Furthermore, the demonstrated bite force modulation in male stag beetles suggests the presence of mechanosensors inside the armature.

Foraging mode plays a pivotal role in traditional reconstructions of squamate evolution. Transitions between modes are said to spark concerted changes in the morphology, physiology, behaviour, and life history of lizards. With respect to their sensory systems, species that adopt a sit-and-wait strategy are thought to rely on visual cues primarily, while actively hunting species would predominantly use chemical information. The morphology of the tongue and the vomeronasal-organs is believed to mirror this dichotomy. Still, support for this idea of concerted evolution of the morphology of the lizard sensory system merely originates from studies comparing only a few, distantly related taxa that differ in many aspects of their biology besides foraging mode. Hence, we compared vomeronasal-lingual morphology among closely related lizard species (Lacertidae). Our findings show considerable interspecific variation indicating that the chemosensory system of lacertids has undergone substantial change over a short evolutionary time. Although our results imply independent evolution of tongue and vomeronasal-organ form, we find evidence for co-variation between sampler and sensor, hinting towards an ‘optimization’ for efficient chemoreception. Furthermore, our findings suggest species’ degree of investment in chemical signalling, and not foraging behaviour, as a leading factor driving the diversity in vomeronasal-lingual morphology among lacertid species.