Allometry and phylogenetic divergence: Correspondence or incongruence?

The potential connection between trends of within species variation, such as those of allometric change in morphology, and phylogenetic divergence has been a central topic in evolutionary biology for more than a century, including in the context of human evolution. In this study, I focus on size-related shape change in craniofacial proportions using a sample of more than 3200 adult Old World monkeys belonging to 78 species, of which 2942 specimens of 51 species are selected for the analysis. Using geometric morphometrics, I assess whether the divergence in the direction of static allometries increases in relation to phyletic differences. Because both small samples and taxonomic sampling may bias the results, I explore the sensitivity of the main analyses to the inclusion of more or less taxa depending on the choice of a threshold for the minimum sample size of a species. To better understand the impact of sampling error, I also use randomized subsampling experiments in the largest species samples. The study shows that static allometries vary broadly in directions without any evident phylogenetic signal. This variation is much larger than previously found in ontogenetic trajectories of Old World monkeys, but the conclusion of no congruence with phylogenetic divergence is the same. Yet, the effect of sampling error clearly contributes to inaccuracies and tends to magnify the differences in allometric change. Thus, morphometric research at the boundary between micro- and macro-evolution in primates, and more generally in mammals, critically needs very large and representative samples. Besides sampling error, I suggest other non-mutually exclusive explanations for the lack of correspondence between allometric and phylogenetic divergence in Old World monkeys, and also discuss why directions might be more variable in static compared to ontogenetic trajectories. Even if allometric variation may be a poor source of information in relation to phylogeny, the evolution of allometry is a fascinating subject and the study of size-related shape changes remains a fundamental piece of the puzzle to understand morphological variation within and between species in primates and other animals.

Exploring motion using geometric morphometrics in microscopic aquatic invertebrates: 'modes' and movement patterns during feeding in a bdelloid rotifer model species.

BACKGROUND: Movement is a defining aspect of animals, but it is rarely studied using quantitative methods in microscopic invertebrates. Bdelloid rotifers are a cosmopolitan class of aquatic invertebrates of great scientific interest because of their ability to survive in very harsh environment and also because they represent a rare example of an ancient lineage that only includes asexually reproducing species. In this class, Adineta ricciae has become a model species as it is unusually easy to culture. Yet, relatively little is known of its ethology and almost nothing on how it behaves during feeding. METHODS: To explore feeding behaviour in A. ricciae, as well as to provide an example of application of computational ethology in a microscopic invertebrate, we apply Procrustes motion analysis in combination with ordination and clustering methods to a laboratory bred sample of individuals recorded during feeding. RESULTS: We demonstrate that movement during feeding can be accurately described in a simple two-dimensional shape space with three main 'modes' of motion. Foot telescoping, with the body kept straight, is the most frequent 'mode', but it is accompanied by periodic rotations of the foot together with bending while the foot is mostly retracted. CONCLUSIONS: Procrustes motion analysis is a relatively simple but effective tool for describing motion during feeding in A. ricciae. The application of this method generates quantitative data that could be analysed in relation to genetic and ecological differences in a variety of experimental settings. The study provides an example that is easy to replicate in other invertebrates, including other microscopic animals whose behavioural ecology is often poorly known.

Do brachycephaly and nose size predict the severity of obstructive sleep apnea (OSA)? A sample-based geometric morphometric analysis of craniofacial variation in relation to OSA syndrome and the role of confounding factors.

Obstructive sleep apnea is a common disorder that leads to sleep fragmentation and is potentially bidirectionally related to a variety of comorbidities, including an increased risk of heart failure and stroke. It is often considered a consequence of anatomical abnormalities, especially in the head and neck, but its pathophysiology is likely to be multifactorial in origin. With geometric morphometrics, and a large sample of adults from the Study for Health in Pomerania, we explore the association of craniofacial morphology to the apnea-hypopnea index used as an estimate of obstructive sleep apnea severity. We show that craniofacial size and asymmetry, an aspect of morphological variation seldom analysed in obstructive sleep apnea research, are both uncorrelated to apnea-hypopnea index. In contrast, as in previous analyses, we find evidence that brachycephaly and larger nasal proportions might be associated to obstructive sleep apnea severity. However, this correlational signal is weak and completely disappears when age-related shape variation is statistically controlled for. Our findings suggest that previous work might need to be re-evaluated, and urge researchers to take into account the role of confounders to avoid potentially spurious findings in association studies.

Can morphotaxa be assessed with photographs? Estimating the accuracy of two-dimensional cranial geometric morphometrics for the study of threatened populations of African monkeys.

The classification of most mammalian orders and families is under debate and the number of species is likely greater than currently recognized. Improving taxonomic knowledge is crucial, as biodiversity is in rapid decline. Morphology is a source of taxonomic knowledge, and geometric morphometrics applied to two dimensional (2D) photographs of anatomical structures is commonly employed for quantifying differences within and among lineages. Photographs are informative, easy to obtain, and low cost. 2D analyses, however, introduce a large source of measurement error when applied to crania and other highly three dimensional (3D) structures. To explore the potential of 2D analyses for assessing taxonomic diversity, we use patas monkeys (Erythrocebus), a genus of large, semi-terrestrial, African guenons, as a case study. By applying a range of tests to compare ventral views of adult crania measured both in 2D and 3D, we show that, despite inaccuracies accounting for up to one-fourth of individual shape differences, results in 2D almost perfectly mirror those in 3D. This apparent paradox might be explained by the small strength of covariation in the component of shape variance related to measurement error. A rigorous standardization of photographic settings and the choice of almost coplanar landmarks are likely to further improve the correspondence of 2D to 3D shapes. 2D geometric morphometrics is, thus, appropriate for taxonomic comparisons of patas ventral crania. Although it is too early to generalize, our results corroborate similar findings from previous research in mammals, and suggest that 2D shape analyses are an effective heuristic tool for morphological investigation of small differences.

The Physico-Chemical Properties of Glipizide: New Findings.

The present work is a concrete example of how physico-chemical studies, if performed in depth, are crucial to understand the behavior of pharmaceutical solids and constitute a solid basis for the control of the reproducibility of the industrial batches. In particular, a deep study of the thermal behavior of glipizide, a hypoglycemic drug, was carried out with the aim of clarifying whether the recognition of its polymorphic forms can really be done on the basis of the endothermic peak that the literature studies attribute to the melting of the compound. A number of analytical techniques were used: thermal techniques (DSC, TGA), X-ray powder diffraction (XRPD), FT-IR spectroscopy and scanning electron microscopy (SEM). Great attention was paid to the experimental design and to the interpretation of the combined results obtained by all these techniques. We proved that the attribution of the endothermic peak shown by glipizide to its melting was actually wrong. The DSC peak is no doubt triggered by a decomposition process that involves gas evolution (cyclohexanamine and carbon dioxide) and formation of 5-methyl-N-[2-(4-sulphamoylphenyl) ethyl] pyrazine-2-carboxamide, which remains as decomposition residue. Thermal treatments properly designed and the combined use of DSC with FT-IR and XRPD led to identifying a new polymorphic form of 5-methyl-N-[2-(4-sulphamoylphenyl) ethyl] pyrazine-2-carboxamide, which is obtained by crystallization from the melt. Hence, our results put into evidence that the check of the polymorphic form of glipizide cannot be based on the temperature values of the DSC peak, since such a peak is due to a decomposition process whose Tonset value is strongly affected by the particle size. Kinetic studies of the decomposition process show the high stability of solid glipizide at room temperature.

Sexual differences in human cranial morphology: Is one sex more variable or one region more dimorphic?

The quantification of cranial sexual dimorphism (CSD) among modern humans is relevant in evolutionary studies of morphological variation and in a forensic context. Despite the abundance of quantitative studies of CSD, few have specifically examined intra-sex variability. Here we quantify CSD in a geographically homogeneous sample of adult crania, which includes Italian individuals from the 19th and 20th centuries. Cranial morphology is described with 92 3D landmarks analyzed using Procrustean geometric morphometrics (PGMM). Size and shape variables are used to compare morphological variance between sexes in the whole cranium and four individual regions. The same variables, plus Procrustes form, are used to quantify average sex differences and explore classification accuracy. Our results indicate that: (a) as predicted by Wainer's rule, males present overall more variance in size and shape, albeit this is statistically significant only for total cranial size; (b) differences between sexes are dominated by size and to a lesser extent by Procrustes form; (c) shape only accounts for a minor proportion of variance; (d) the cranial base shows almost no dimorphism for shape; and (e) facial Procrustes form is the most accurate predictor of skeletal sex. Overall, this study suggests developmental factors underlying differences in CSD among cranial regions; stresses the need for population-specific models that describe craniofacial variation as the basis for models that facilitate the estimation of sex in unidentified skeletal remains; and provides one of the first confirmations of "Wainer's rule" in relation to sexual dimorphism in mammals specific to the human cranium.

Modern morphometrics and the study of population differences: Good data behind clever analyses and cool pictures?

The study of phenotypic variation in time and space is central to evolutionary biology. Modern geometric morphometrics is the leading family of methods for the quantitative analysis of biological forms. This set of techniques relies heavily on technological innovation for data acquisition, often in the form of 2D or 3D digital images, and on powerful multivariate statistical tools for their analysis. However, neither the most sophisticated device for computerized imaging nor the best statistical test can produce accurate, robust and reproducible results, if it is not based on really good samples and an appropriate use of the 'measurements' extracted from the data. Using examples mostly from my own work on mammal craniofacial variation and museum specimens, I will show how easy it is to forget these most basic assumptions, while focusing heavily on analytical and visualization methods, and much less on the data that generate potentially powerful analyses and visually appealing diagrams.

How flat can a horse be? Exploring 2D approximations of 3D crania in equids.

Quantitative analyses of morphological variation using geometric morphometrics are often performed on 2D photos of 3D structures. It is generally assumed that the error due to the flattening of the third dimension is negligible. However, despite hundreds of 2D studies, few have actually tested this assumption and none has done it on large animals, such as those typically classified as megafauna. We explore this issue in living equids, focusing on ventral cranial variation at both micro- and macro-evolutionary levels. By comparing 2D and 3D data, we found that size is well approximated, whereas shape is more strongly impacted by 2D inaccuracies, as it is especially evident in intra-specific analyses. The 2D approximation improves when shape differences are larger, as in macroevolution, but even at this level precise inter-individual similarity relationships are altered. Despite this, main patterns of sex, species and allometric variation in 2D were the same as in 3D, thus suggesting that 2D may be a source of 'noise' that does not mask the main signal in the data. However, the picture that emerges from this and other recent studies on 2D approximation of 3D structures is complex and any generalization premature. Morphometricians should therefore test the appropriateness of 2D using preliminary investigations in relation to the specific study questions in their own samples. We discuss whether this might be feasible using a reduced landmark configuration and smaller samples, which would save time and money. In an exploratory analysis, we found that in equids results seem robust to sampling, but become less precise and, with fewer landmarks, may slightly overestimate 2D inaccuracies.

Geometric morphometrics on juvenile crania: Exploring age and sex variation in an Australian population.

For medico-legal forensic practitioners the identification of unknown remains is an important part of any investigation, often predicated on having accurate estimations of age and sex. In considering the specific skeletal elements available to facilitate such biological information, the cranium is frequently targeted for analysis, as it exhibits marked traits of sexual dimorphism, and also has a predictable pattern of growth. There are, however, instances where it may not be possible to estimate skeletal sex, especially in the juvenile skeleton. There is still considerable uncertainty surrounding the age at which the human cranium is quantifiably dimorphic. The aim of the present study is to explore age and sex variation in three-dimensionally reconstructed MDCT scans of the juvenile cranium. The study sample comprises 152 juvenile crania from a Western Australian population; a total of 52 three-dimensional landmarks are acquired and analyzed using Procrustean geometric morphometrics. Group discrimination is assessed between sexes and across age classes. Results demonstrate that sexual dimorphism and age variation is discernible through geometric morphometric analysis of form, size and shape. Relative to sex and age, size is found to be generally equivalent to, or even more accurate than, shape data. There is little quantifiable sexual dimorphism in individuals younger than 12years of age with most variation related to age; discrimination improves with increasing age, with average hit rate (HR) values increasing from just over 50% (52-58%) to more than 90% (93-94%) accuracy at 18years. In contrast, differences between contiguous age classes follow the opposite trend and tend to be larger in prepubertal groups, while becoming progressively smaller in older age classes. This study demonstrates that simple linear interlandmark distances describing overall cranial size may provide a simple option for preliminary classifications of age and sex in skeletal remains of forensic interest. However, although recombining size and shape to perform analyses using form generally does not appreciably improve predictive accuracy, it potentially contributes to increased confidence in group assessment (especially for sex) and thus offers a promising, albeit complex, type of information to discriminate groups based on cranial size and/or shape.

Author Correction: Larger mammals have longer faces because of size-related constraints on skull form.

In Table 1 of this article, the descriptions of landmarks 14, 15, and 36 are incorrect. Landmarks 14 and 36 should read "Posterior extremity of occipital condyle along margin of foramen magnum" and landmark 15 should read "Opisthion". A correct version of Table 2 appears in the Author Correction associated with this article; the error has not been fixed in the original article.

Procrustes-based geometric morphometrics on MRI images: An example of inter-operator bias in 3D landmarks and its impact on big datasets.

Using 3D anatomical landmarks from adult human head MRIs, we assessed the magnitude of inter-operator differences in Procrustes-based geometric morphometric analyses. An in depth analysis of both absolute and relative error was performed in a subsample of individuals with replicated digitization by three different operators. The effect of inter-operator differences was also explored in a large sample of more than 900 individuals. Although absolute error was not unusual for MRI measurements, including bone landmarks, shape was particularly affected by differences among operators, with up to more than 30% of sample variation accounted for by this type of error. The magnitude of the bias was such that it dominated the main pattern of bone and total (all landmarks included) shape variation, largely surpassing the effect of sex differences between hundreds of men and women. In contrast, however, we found higher reproducibility in soft-tissue nasal landmarks, despite relatively larger errors in estimates of nasal size. Our study exemplifies the assessment of measurement error using geometric morphometrics on landmarks from MRIs and stresses the importance of relating it to total sample variance within the specific methodological framework being used. In summary, precise landmarks may not necessarily imply negligible errors, especially in shape data; indeed, size and shape may be differentially impacted by measurement error and different types of landmarks may have relatively larger or smaller errors. Importantly, and consistently with other recent studies using geometric morphometrics on digital images (which, however, were not specific to MRI data), this study showed that inter-operator biases can be a major source of error in the analysis of large samples, as those that are becoming increasingly common in the 'era of big data'.

Estimating the magnitude of morphoscapes: how to measure the morphological component of biodiversity in relation to habitats using geometric morphometrics.

Ecological indicators are currently developed to account for the different facets of loss of biological diversity due to direct or indirect effects of human activities. Most ecological indicators include species richness as a metric. Others, such as functional traits and phylogenetic diversity, account for differences in species, even when species richness is the same. Here, we describe and apply a different indicator, called morphoscape dimension, accounting for morphological variability across habitats in a geographical region. We use the case of ground beetles (Coleoptera: Carabidae) in four different habitats in the Po Plain in Northern Italy to exemplify how to quantify the magnitude of the morphological space (i.e. the dimension of the morphoscape) occupied by the species in each habitat using geometric morphometrics. To this aim, we employed a variety of metrics of morphological disparity related to univariate size, and more complex multivariate shape and form. Our 'proof of concept' suggests that metrics assessing size and form might largely tend to simply mirror the information provided by species richness, whereas shape morphoscape disparity may be able to account for non-trivial differences in species traits amongst habitats. This is indicated by the woodland morphoscape being on average bigger than that of crops, the most species-rich habitat, despite having almost 20% less species. We conclude suggesting that the analysis of morphoscape dimension has the potential to become a new additional and complimentary tool in the hands of conservation biologists and ecologists to explore and quantify habitat complexity and inform decisions on management and conservation based on a wide set of ecological indicators.

Lost in the Other Half: Improving Accuracy in Geometric Morphometric Analyses of One Side of Bilaterally Symmetric Structures.

Systematists and evolutionary biologists have widely adopted Procrustes-based geometric morphometrics for measuring size and shape in biology. Many structures, and in fact most animals, are bilaterally symmetric with an internal plane of symmetry (also called object symmetry). Often, when quantifying asymmetric variation is not an aim, only one or the other side is measured and analyzed. This approach has been used in hundreds of studies. Its implicit assumption is that the information on the other side is redundant and a single side will, therefore, produce results mirroring those one would have obtained from the analysis of the entire structure with all its left and right landmarks. However, the extent to which this assumption is met has, to my knowledge, never been explored. Using two example data sets, I will show that congruence may be high in analyses at a macroevolutionary level but much lower at a microevolutionary one, and inaccuracies might especially affect shape. I will discuss some of the other factors that may influence results and will suggest a simple expedient that can improve both the visualization and accuracy of shape analyses in one-side-only studies.

The 'temporal effect' in hominids: Reinvestigating the nature of support for a chimp-human clade in bone morphology.

In 2004, an analysis by Lockwood and colleagues of hard-tissue morphology, using geometric morphometrics on the temporal bone, succeeded in recovering the correct phylogeny of living hominids without resorting to potentially problematic methods for transforming continuous shape variables into meristic characters. That work has increased hope that by using modern analytical methods and phylogenetically informative anatomical data we might one day be able to accurately infer the relationships of hominins, including the closest extinct relatives of modern humans. In the present study, using 3D virtually generated models of the hominid temporal bone and a larger suite of geometric morphometric and comparative techniques, we have re-examined the evidence for a Pan-Homo clade. Despite differences in samples, as well as the type of raw data, the effect of measurement error (and especially landmark digitization by a different operator), but also a broader perspective brought in by our diverse set of approaches, our reanalysis largely supports Lockwood and colleagues' original results. However, by focusing not only mainly on shape (as in the original 2004 analysis) but also on size and 'size-corrected' (non-allometric) shape, we demonstrate that the strong phylogenetic signal in the temporal bone is largely related to similarities in size. Thus, with this study, we are not suggesting the use of a single 'character', such as size, for phylogenetic inference, but we do challenge the common view that shape, with its highly complex and multivariate nature, is necessarily more phylogenetically informative than size and that actually size and size-related shape variation (i.e., allometry) confound phylogenetic inference based on morphology. This perspective may in fact be less generalizable than often believed. Thus, while we confirm the original findings by Lockwood et al., we provide a deep reinterpretation of their nature and potential implications for hominid phylogenetics and we show how crucial it is not to overlook size in geometric morphometric analyses.

Morphometric analysis of pelvic sexual dimorphism in a contemporary Western Australian population.

Requisite to routine casework involving unidentified skeletal remains is the formulation of an accurate biological profile, including sex estimation. Choice of method(s) is invariably related to preservation and by association, available bones. It is vital that the method applied affords statistical quantification of accuracy rates and predictive confidence so that evidentiary requirements for legal submission are satisfied. Achieving the latter necessitates the application of contemporary population-specific standards. This study examines skeletal pelvic dimorphism in contemporary Western Australian individuals to quantify the accuracy of using pelvic measurements to estimate sex and to formulate a series of morphometric standards. The sample comprises pelvic multi-slice computer tomography (MSCT) scans from 200 male and 200 female adults. Following 3D rendering, the 3D coordinates of 24 landmarks are acquired using OsiriX® (v.4.1.1) with 12 inter-landmark linear measurements and two angles acquired using MorphDb. Measurements are analysed using basic descriptive statistics and discriminant functions analyses employing jackknife validation of classification results. All except two linear measurements are dimorphic with sex differences explaining up to 65 % of sample variance. Transverse pelvic outlet and subpubic angle contribute most significantly to sex discrimination with accuracy rates between 100 % (complete pelvis-10 variables) and 81.2 % (ischial length). This study represents the initial forensic research into pelvic sexual dimorphism in a Western Australian population. Given these methods, we conclude that this highly dimorphic bone can be used to classify sex with a high degree of expected accuracy.

Extreme climate, rather than population history, explains mid-facial morphology of Northern Asians.

Previous studies have examined mid-facial cold adaptation among either widely dispersed and genetically very diverse groups of humans isolated for tens of thousands of years, or among very closely related groups spread over climatically different regions. Here we present a study of one East Asian and seven North Asian populations in which we examine the evidence for convergent adaptations of the mid-face to a very cold climate. Our findings indicate that mid-facial morphology is strongly associated with climatic variables that contrast the temperate climate of East Asians and the very cold and dry climate of North Asians. This is also the case when either maxillary or nasal cavity measurements are considered alone. The association remains significant when mtDNA distances among populations are taken into account. The morphological contrasts between populations are consistent with physiological predictions and prior studies of mid-facial cold adaptation in more temperate regions, but among North Asians there appear to be some previously undescribed morphological features that might be considered as adaptive to extreme cold. To investigate this further, analyses of the seven North Asian populations alone suggest that mid-facial morphology remains strongly associated with climate, particularly winter precipitation, contrasting coastal Arctic and continental climates. However, the residual covariation among North Asian mid-facial morphology and climate when genetic distances are considered, is not significant. These findings point to modern adaptations to extreme climate that might be relevant to our understanding of the mid-facial morphology of fossil hominins that lived during glaciations.

Larger mammals have longer faces because of size-related constraints on skull form.

Facial length is one of the best known examples of heterochrony. Changes in the timing of facial growth have been invoked as a mechanism for the origin of our short human face from our long-faced extinct relatives. Such heterochronic changes arguably permit great evolutionary flexibility, allowing the mammalian face to be remodelled simply by modifying postnatal growth. Here we present new data that show that this mechanism is significantly constrained by adult size. Small mammals are more brachycephalic (short faced) than large ones, despite the putative independence between adult size and facial length. This pattern holds across four phenotypic lineages: antelopes, fruit bats, tree squirrels and mongooses. Despite the apparent flexibility of facial heterochrony, growth of the face is linked to absolute size and introduces what seems to be a loose but clade-wide mammalian constraint on head shape.

Biogeographic variation in the baboon: dissecting the cline.

All species demonstrate intraspecific anatomical variation. While generalisations such as Bergman's and Allen's rules have attempted to explain the geographic structuring of variation with some success, recent work has demonstrated limited support for these in certain Old World monkeys. This study extends this research to the baboon: a species that is widely distributed across sub-Saharan Africa and exhibits clinal variation across an environmentally disparate range. This study uses trend surface analysis to map the pattern of skull variation in size and shape in order to visualise the main axes of morphological variation. Patterns of shape and size-controlled shape are compared to highlight morphological variation that is underpinned by allometry alone. Partial regression is used to dissociate the effects of environmental terms, such as rainfall, temperature and spatial position. The diminutive Kinda baboon is outlying in size, so analyses were carried out with and without this taxon. Skull size variation demonstrates an east-west pattern, with small animals at the two extremes and large animals in Central and Southern Africa. Shape variation demonstrates the same geographical pattern as skull size, with small-sized animals exhibiting classic paedomorphic morphology. However, an additional north-south axis of variation emerges. After controlling for skull size, the diminutive Kinda baboon is no longer an outlier for size and shape. Also, the east-west component is no longer evident and discriminant function analysis shows an increased misclassification of adjacent taxa previously differentiated by size. This demonstrates the east-west component of shape variation is underpinned by skull size, while the north-south axis is not. The latter axis is explicable in phylogenetic terms: baboons arose in Southern Africa and colonised East and West Africa to the north, diverging in the process, aided by climate-mediated isolating mechanisms. Environmental terms appear poorly correlated with shape variation compared with geography. This might indicate that there is no simple environment-morphology association, but certainly demonstrates that phylogenetic history is an overbearing factor in baboon morphological variation.