The Shape of Weaver: Investigating Shape Disparity in Orb-Weaving Spiders (Araneae, Araneidae) Using Geometric Morphometrics Abstract Sexual size dimorphism in orb-weaving spiders is a relatively well-studied phenomenon, and numerous works have documented evolutionary variation in interspecific size and degree of dimorphism. To date, these studies have been largely limited to assessing the evolution of a single or few linear measurements correlated with body size. While the descriptive and comparative literature is rich with qualitative and linear comparisons that distinguish the sexes and characterize species, the extent to which interspecific or dimorphic variation in size correlates with morphological shape remains relatively unexplored. The carapace of spiders is generally conserved in shape, especially among members of the same family, but is neither well-characterized as a potential facet of spider sexual dimorphism nor as a variable structure overall. Here, we use geometric morphometric techniques to quantify differences in carapace shape among members of the family Araneidae and test for allometric influences on interspecific and dimorphic shape differences across orb-weavers. We show that females and males differ in shape, occupying overlapping but distinct areas of morphospace, with males having more piriform carapaces than females. Araneid spider subfamilies overlap substantially in morphospace, though interspecific differences in shape are generally greater than those distinguishing males and females of a species. Furthermore, we show that female carapace shape shows phylogenetic signal and is more conserved than is male shape. Carapace shape differences made evident from canonical variates analysis are congruent with the more mobile lifestyle adopted by males, as a broader carapace may support more robust leg musculature.

Seeing Distinct Groups Where There are None: Spurious Patterns from Between-Group PCA Abstract Using sampling experiments, we found that, when there are fewer groups than variables, between-groups PCA (bgPCA) may suggest surprisingly distinct differences among groups for data in which none exist. While apparently not noticed before, the reasons for this problem are easy to understand. A bgPCA captures the g − 1 dimensions of variation among the g group means, but only a fraction of the \(\sum {n_{i} } - g\) dimensions of within-group variation ( \(n_{i}\) are the sample sizes), when the number of variables, p, is greater than g − 1. This introduces a distortion in the appearance of the bgPCA plots because the within-group variation will be underrepresented, unless the variables are sufficiently correlated so that the total variation can be accounted for with just g − 1 dimensions. The effect is most obvious when sample sizes are small relative to the number of variables, because smaller samples spread out less, but the distortion is present even for large samples. Strong covariance among variables largely reduces the magnitude of the problem, because it effectively reduces the dimensionality of the data and thus enables a larger proportion of the within-group variation to be accounted for within the g − 1-dimensional space of a bgPCA. The distortion will still be relevant though its strength will vary from case to case depending on the structure of the data (p, g, covariances etc.). These are important problems for a method mainly designed for the analysis of variation among groups when there are very large numbers of variables and relatively small samples. In such cases, users are likely to conclude that the groups they are comparing are much more distinct than they really are. Having many variables but just small sample sizes is a common problem in fields ranging from morphometrics (as in our examples) to molecular analyses.

Macroevolution of Toothed Whales Exceptional Relative Brain Size Abstract Toothed whales (Odontoceti, Cetacea) are well-known for their ability to produce complex vocalizations, to use tools, to possess self-recognition, and for their extreme behavioural plasticity. The toothed whale intelligence is said to compete with that of primates, so does their extremely large brain to body size ratio. Common explanations for the acquisition of such large brains over the evolutionary time (encephalization) in toothed whales range from their demanding, complex social lives, to their feeding habits, to echolocation. Yet, several studies found no macroevolutionary trend in Odontoceti encephalization, which casts doubts on its selective advantage. We applied a recently developed phylogenetic comparative method to study macroevolutionary trends in relative brain size (RBS) and brain size evolutionary rates in cetaceans, comparing toothed whales to the other cetaceans and contrasting groups of species as ascribed to different feeding categories. We found that cetaceans as a whole followed a trend for increased encephalization over time, starting from small-brained archaeocete ancestors. Toothed whales do not show this same trend in RBS but have possessed larger RBS than any other cetacean ever since the beginning of their existence. The rate of RBS evolution in Odontoceti is significantly slower than in other Cetacea and slower than the rate of Odontoceti body size evolution. These results suggest that toothed whales’ history is characterized by high and conservative relative encephalization. Feeding lifestyle does not explain these patterns, while the appearance of echolocation within stem group Odontoceti remains a viable candidate for them.

Overall Bone Structure as Assessed by Slice-by-Slice Profile Abstract Quantifying the inner structure of bones is central to various analyses dealing with the phenotypic evolution of animals with an ossified skeleton. Computed tomography allows to assess the repartition of bone tissue within an entire skeletal element. Two parameters of importance for such analyses are the global compactness (Cg) and total cross-sectional area (Tt.Ar). However, no open-source, time-efficient methods are available to acquire these parameters for whole bones. A methodology to assess the variation of these parameters along a profile following one of the studied bone’s anatomical axes is also wanting. Here I present an ImageJ macro and associated R script to automatically acquire Cg and Tt.Ar along an axis of the skeletal element of interest using a slice-by-slice approach. No manual segmentation is required and several bones can be present on the analysed scan, as long as the bone of interest is isolated and the largest element on each slice. While some bias might be involved by the automatic acquisition, semi-automatic slice exclusion and correction procedures can be used to efficiently account for it. As a test case, µCT-data was gathered for the mid-lumbar vertebra of over 70 mammals. The two evaluated correction procedures proved to perform equally well, with a slight advantage for the one relying on the exclusion of local outliers. The presented macro allows to efficiently build a dataset concerned with the quantification of bone inner structure. The code being readily available, further improvement of the methodology and adjustment to particular needs can be easily performed.

Pathologies of Between-Groups Principal Components Analysis in Geometric Morphometrics Abstract Good empirical applications of geometric morphometrics (GMM) typically involve several times more variables than specimens, a situation the statistician refers to as “high p/n,” where p is the count of variables and n the count of specimens. This note calls your attention to two predictable catastrophic failures of one particular multivariate statistical technique, between-groups principal components analysis (bgPCA), in this high-p/n setting. The more obvious pathology is this: when applied to the patternless (null) model of p identically distributed Gaussians over groups of the same size, both bgPCA and its algebraic equivalent, partial least squares (PLS) analysis against group, necessarily generate the appearance of huge equilateral group separations that are fictitious (absent from the statistical model). When specimen counts by group vary greatly or when any group includes fewer than about ten specimens, an even worse failure of the technique obtains: the smaller the group, the more likely a bgPCA is to fictitiously identify that group as the end-member of one of its derived axes. For these two reasons, when used in GMM and other high-p/n settings the bgPCA method very often leads to invalid or insecure biological inferences. This paper demonstrates and quantifies these and other pathological outcomes both for patternless models and for models with one or two valid factors, then offers suggestions for how GMM practitioners should protect themselves against the consequences for inference of these lamentably predictable misrepresentations. The bgPCA method should never be used unskeptically—it is always untrustworthy, never authoritative—and whenever it appears in partial support of any biological inference it must be accompanied by a wide range of diagnostic plots and other challenges, many of which are presented here for the first time.

The Skull Integration Pattern and Internal Constraints in Myotis myotis – Myotis blythii Species Group (Vespertilionidae, Chiroptera) Might be Shaped by Natural Selection During Evolution Along the Genetic Line of Least Resistance Abstract Evolutionary dynamics of covariation patterns in craniometric traits was studied for bats from the Myotis myotis–Myotis blythii species group (Mammalia, Chiroptera, Vespertilionidae) namely M.m.myotis, M.b.oxygnathus, M.b.omari, M.b.blythii, and M.b.altaicus. These species evolved towards increasing the size. One more species, M.dasycneme, which is phylogenetically, morphologically, and ecologically rather distant from these OTUs, also was studied for comparison with them. A set of 30 craniometric traits was studied for each of the OTUs using the quantitative genetics approaches. Phenotypic covariance matrices were used as proxies for additive genetic covariance matrices. The analysis has shown that multivariate divergence of the studied Myotis OTUs was governed by selection rather than by random drift and that these OTUs evolved in the direction close to the line of least evolutionary resistance. As overall size increases, the skull integration, respondability, and evolvability increase, while flexibility slightly decreases. Such a pattern can possibly be explained as a consequence of adaptation of M.blythii and especially M.myotis to prey on considerably large hard-shelled insects. A high cohesion of skull structures is needed for the effective functioning of their jaw apparatus. For Myotis bats with other foraging strategies (such as M. dasycneme that catches insects mainly using the claws of hind feet) this cohesion seems to be of lesser importance.

3D Photogrammetry of Bat Skulls: Perspectives for Macro-evolutionary Analyses Abstract Photogrammetry (PH) is relatively cheap, easy to use, flexible and portable but its power and limitations have not been fully explored for studies of small animals. Here we assessed the accuracy of PH for the reconstruction of 3D digital models of bat skulls by evaluating its potential for evolutionary morphology studies at interspecific (19 species) level. Its reliability was assessed against the performance of micro CT scan (µCT) and laser scan techniques (LS). We used 3D geometric morphometrics and comparative methods to quantify the amount of size and shape variation due to the scanning technique and assess the strength of the biological signal in relation to both the technique error and phylogenetic uncertainty. We found only minor variation among techniques. Levels of random error (repeatability and procrustes variance) were similar in all techniques and no systematic error was observed (as evidenced from principal component analysis). Similar levels of phylogenetic signal, allometries and correlations with ecological variables (frequency of maximum energy and bite force) were detected among techniques. Phylogenetic uncertainty interacted with technique error but without affecting the biological conclusions driven by the evolutionary analyses. Our study confirms the accuracy of PH for the reconstruction of challenging specimens. These results encourage the use of PH as a reliable and highly accessible tool for the study of macro evolutionary processes of small mammals.

The Inhibitory Cascade Model is Not a Good Predictor of Molar Size Covariation Abstract The inhibitory cascade (IC) model is a widely used evolutionary developmental explanation of among-species differences in relative molar tooth size. The IC model posits that, as molars develop from front to back, the relative strength of activating and inhibiting influences establishes a “ratcheting” mechanism leading to predictable relative molar sizes. Such a constraint on molar covariation would lead to strong variational biases on the evolutionary paths that the molar row can traverse through phenotypic space. These constraints manifest themselves in characteristic patterns of variation among species that loosely match observed macroevolutionary patterns. In this paper, we write out the predictions of the IC model for within-species covariation in molar size in a framework that unifies evolutionary developmental biological and quantitative genetic perspectives on the evolution of complex traits. We then evaluate these predictions about aspects of molar covariation in eight anthropoid primate species. We find that the IC model tends to over-predict aspects of within-species covariation by substantial margins. Only macaques exhibit covariation in and among individual teeth consistent with the IC model, but they do not show signs of the strong evolutionary constraint predicted by the model. Gorillas meet none of the predictions. While we cannot rule out an IC-like process as a contributor, causes of molar size covariation other than those described in the IC model must be major contributors to covariation in molar teeth within populations.

Description and Analysis of Spatial Patterns in Geometric Morphometric Data Abstract The development of techniques for the acquisition of high-resolution 3D images, such as computed tomography and magnetic resonance imaging, has opened new avenues to the study of complex morphologies. Detailed descriptions of internal and external traits can be now obtained, allowing the intensive sampling of surface points. In this paper, we introduce a morphometric and statistical framework, grounded on Procrustes and Procrustes-like techniques as well as standard spatial statistics, to explicitly describe and incorporate the spatial pattern of these surface points into the analyses. We exemplified this approach by analyzing ontogenetic changes in a sample of human brain endocasts and inter-specific differences between primate skulls. An intensive sampling of points on 3D surfaces was performed by automatic techniques and the morphometric variation among specimens was measured by the residuals obtained after the alignment of points. Our results showed that shape changes in both examples are spatially structured. Different results were attained by using methods that incorporate or not the spatial structure in the evaluation of the effect of specific biological factors on shape variation. Particularly, these analyses indicated that the effect of biological factors acting at local scales can be confounded with more systemic factors (by example, the effect of the diet on the facial skeleton) if the spatial structure is not taken into account. Overall, our results suggest that the intensive description of shape differences among structures using densely sampled points on 3D surfaces combined with spatial statistical methods can be used to explore problems not widely addressed in morphological studies.

The Apex Set-Up for the Major Transitions in Individuality Abstract Morphological and functional hierarchies occurring in contemporary biological entities are amalgamated via a small number of progressive key-steps termed as Major Transition in Evolution (MTE) that encompass steps of Major Transition in Individuality (MTI). Literature views MTE/MTI in nature as a sequential increase in complexity, and has contributed insights into the emergence of genuine MTI candidates that actually build higher order individuals from simpler entities and into their specific properties. The theory- By considering a novel MTI trajectory termed the ‘MTI continuum’ (independent of the tree of life that contemplates taxonomic correlations), I found no literature consensus for this continuum’s apex. Next, I consider the properties of biological entities termed as ‘superorganism’ (eusocial insects, humans), also considered as highly-developed MTIs. I classify ‘superorganism’ as being on the level of ‘miscellaneous transitions’ that have not yet developed into real MTIs and that do not meet the ‘individual’ physiognomy. Then I assign the emergence of three new MTI diachronic-classes, the colonial-organisms, chimerism and multi-chimerism, suggesting that they represent highly complex MTIs that belong at the apex of the MTI continuum. These novel MTIs are neither fraternal, nor egalitarian, deprived of ‘kinship’ and ‘fairness’ considerations, yet still generate genuine and distinct libertarian entities. I posit that these MTIs embody the qualities of real units of selection.