CAAStools: a toolbox to identify and test Convergent Amino Acid Substitutions.

MOTIVATION: Coincidence of Convergent Amino Acid Substitutions (CAAS) with phenotypic convergences allow pinpointing genes and even individual mutations that are likely to be associated with trait variation within their phylogenetic context. Such findings can provide useful insights into the genetic architecture of complex phenotypes. RESULTS: Here we introduce CAAStools, a set of bioinformatics tools to identify and validate CAAS in orthologous protein alignments for predefined groups of species representing the phenotypic values targeted by the user. AVAILABILITY AND IMPLEMENTATION: CAAStools source code is available at http://github.com/linudz/caastools, along with documentation and examples.

Early tetrapod cranial evolution is characterized by increased complexity, constraint, and an offset from fin-limb evolution.

The developmental underpinnings and functional consequences of modifications to the limbs during the origin of the tetrapod body plan are increasingly well characterized, but less is understood about the evolution of the tetrapod skull. Decrease in skull bone number has been hypothesized to promote morphological and functional diversification in vertebrate clades, but its impact during the initial rise of tetrapods is unknown. Here, we test this by quantifying topological changes to cranial anatomy in fossil and living taxa bracketing the fin-to-limb transition using anatomical network analysis. We find that bone loss across the origin of tetrapods is associated not only with increased complexity of bone-to-bone contacts but also with decreasing topological diversity throughout the late Paleozoic, which may be related to developmental and/or mechanical constraints. We also uncover a 10-Ma offset between fin-limb and cranial morphological evolution, suggesting that different evolutionary drivers affected these features during the origin of tetrapods.

Comparative Analysis of Mammal Genomes Unveils Key Genomic Variability for Human Life Span.

The enormous mammal's lifespan variation is the result of each species' adaptations to their own biological trade-offs and ecological conditions. Comparative genomics have demonstrated that genomic factors underlying both, species lifespans and longevity of individuals, are in part shared across the tree of life. Here, we compared protein-coding regions across the mammalian phylogeny to detect individual amino acid (AA) changes shared by the most long-lived mammals and genes whose rates of protein evolution correlate with longevity. We discovered a total of 2,737 AA in 2,004 genes that distinguish long- and short-lived mammals, significantly more than expected by chance (P = 0.003). These genes belong to pathways involved in regulating lifespan, such as inflammatory response and hemostasis. Among them, a total 1,157 AA showed a significant association with maximum lifespan in a phylogenetic test. Interestingly, most of the detected AA positions do not vary in extant human populations (81.2%) or have allele frequencies below 1% (99.78%). Consequently, almost none of these putatively important variants could have been detected by genome-wide association studies. Additionally, we identified four more genes whose rate of protein evolution correlated with longevity in mammals. Crucially, SNPs located in the detected genes explain a larger fraction of human lifespan heritability than expected, successfully demonstrating for the first time that comparative genomics can be used to enhance interpretation of human genome-wide association studies. Finally, we show that the human longevity-associated proteins are significantly more stable than the orthologous proteins from short-lived mammals, strongly suggesting that general protein stability is linked to increased lifespan.

Anatomical comparison across heads, fore- and hindlimbs in mammals using network models.

Animal body parts evolve with variable degrees of integration that nonetheless yield functional adult phenotypes: but, how? The analysis of modularity with Anatomical Network Analysis (AnNA) is used to quantitatively determine phenotypic modules based on the physical connection among anatomical elements, an approach that is valuable to understand developmental and evolutionary constraints. We created anatomical network models of the head, forelimb, and hindlimb of two taxa considered to represent a 'generalized' eutherian (placental: mouse) and metatherian (marsupial: opossum) anatomical configuration and compared them with our species, which has a derived eutherian configuration. In these models, nodes represent anatomical units and links represent their physical connection. Here, we aimed to identify: (1) the commonalities and differences in modularity between species, (2) whether modules present a potential phylogenetic character, and (3) whether modules preferentially reflect either developmental or functional aspects of anatomy, or a mix of both. We predicted differences between networks of metatherian and eutherian mammals that would best be explained by functional constraints, versus by constraints of development and/or phylogeny. The topology of contacts between bones, muscles, and bones + muscles showed that, among all three species, skeletal networks were more similar than musculoskeletal networks. There was no clear indication that humans and mice are more alike when compared to the opossum overall, even though their musculoskeletal and skeletal networks of fore- and hindlimbs are slightly more similar. Differences were greatest among musculoskeletal networks of heads and next of forelimbs, which showed more variation than hindlimbs, supporting previous anatomical studies indicating that in general the configuration of the hindlimbs changes less across evolutionary history. Most observations regarding the anatomical networks seem to be best explained by function, but an exception is the adult opossum ear ossicles. These ear bones might form an independent module because the incus and malleus are involved in forming a functional primary jaw that enables the neonate to attach to the teat, where this newborn will complete its development. Additionally, the human data show a specialized digit 1 module (thumb/big toe) in both limb types, likely the result of functional and evolutionary pressures, as our ape ancestors had highly movable big toes and thumbs.

Evolutionary and ontogenetic changes of the anatomical organization and modularity in the skull of archosaurs.

Comparative anatomy studies of the skull of archosaurs provide insights on the mechanisms of evolution for the morphologically and functionally diverse species of crocodiles and birds. One of the key attributes of skull evolution is the anatomical changes associated with the physical arrangement of cranial bones. Here, we compare the changes in anatomical organization and modularity of the skull of extinct and extant archosaurs using an Anatomical Network Analysis approach. We show that the number of bones, their topological arrangement, and modular organization can discriminate birds from non-avian dinosaurs, and crurotarsans. We could also discriminate extant taxa from extinct species when adult birds were included. By comparing within the same framework, juveniles and adults for crown birds and alligator (Alligator mississippiensis), we find that adult and juvenile alligator skulls are topologically similar, whereas juvenile bird skulls have a morphological complexity and anisomerism more similar to those of non-avian dinosaurs and crurotarsans than of their own adult forms. Clade-specific ontogenetic differences in skull organization, such as extensive postnatal fusion of cranial bones in crown birds, can explain this pattern. The fact that juvenile and adult skulls in birds do share a similar anatomical integration suggests the presence of a specific constraint to their ontogenetic growth.

A node-based informed modularity strategy to identify organizational modules in anatomical networks.

The study of morphological modularity using anatomical networks is growing in recent years. A common strategy to find the best network partition uses community detection algorithms that optimize the modularity Q function. Because anatomical networks and their modules tend to be small, this strategy often produces two problems. One is that some algorithms find inexplicable different modules when one inputs slightly different networks. The other is that algorithms find asymmetric modules in otherwise symmetric networks. These problems have discouraged researchers to use anatomical network analysis and boost criticisms to this methodology. Here, I propose a node-based informed modularity strategy (NIMS) to identify modules in anatomical networks that bypass resolution and sensitivity limitations by using a bottom-up approach. Starting with the local modularity around every individual node, NIMS returns the modular organization of the network by merging non-redundant modules and assessing their intersection statistically using combinatorial theory. Instead of acting as a black box, NIMS allows researchers to make informed decisions about whether to merge non-redundant modules. NIMS returns network modules that are robust to minor variation and does not require optimization of a global modularity function. NIMS may prove useful to identify modules also in small ecological and social networks.

A network approach to brain form, cortical topology and human evolution.

Network analysis provides a quantitative tool to investigate the topological properties of a system. In anatomy, it can be employed to investigate the spatial organization of body parts according to their contiguity and patterns of physical contact. In this study, we build a model representing the spatial adjacency of the major regions of the human brain often considered in evolutionary neuroanatomy, to analyse its topological features. Results suggest that the frontal lobe is topologically independent of the posterior regions of the brain, which in turn are more integrated and influenced by reciprocal constraints. The precentral gyrus represents a hinge between the anterior and posterior blocks. The lateral temporal cortex is particularly influenced by the neighbouring regions, while the parietal cortex is minimally constrained by the overall brain organization. Beyond the reciprocal spatial influences among cortical areas, brain form is further constrained by spatial and mechanical influence of the braincase, including bone and connective elements. The anterior fossa and the parietal bones are the elements more sensitive to the brain-braincase spatial organization. These topological properties must be properly considered when making inferences on evolutionary variations and macroscopic differences of the human brain morphology.

Evolutionary parallelisms of pectoral and pelvic network-anatomy from fins to limbs.

Lobe-fins transformed into limbs during the Devonian period, facilitating the water-to-land transition in tetrapods. We traced the evolution of well-articulated skeletons across the fins-to-limbs transition, using a network-based approach to quantify and compare topological features of fins and limbs. We show that the topological arrangement of bones in pectoral and pelvic appendages evolved in parallel during the fins-to-limbs transition, occupying overlapping regions of the morphospace, following a directional trend, and decreasing their disparity over time. We identify the presence of digits as the morphological novelty triggering topological changes that discriminated limbs from fins. The origin of digits caused an evolutionary shift toward appendages that were less densely and heterogeneously connected, but more assortative and modular. Disparity likewise decreased for both appendages, more markedly until a time concomitant with the earliest-known tetrapod tracks. Last, we rejected the presence of a pectoral-pelvic similarity bottleneck at the origin of tetrapods.

Disconnecting bones within the jaw-otic network modules underlies mammalian middle ear evolution.

The origin of the mammalian middle ear ossicles from the craniomandibular articulation of their synapsid ancestors is a key event in the evolution of vertebrates. The richness of the fossil record and the multitude of developmental studies have provided a stepwise reconstruction of this evolutionary innovation, highlighting the homology between the quadrate, articular, pre-articular and angular bones of early synapsids with the incus, malleus, gonial and ectotympanic bones of derived mammals, respectively. There are several aspects involved in this functional exaptation: (i) an increase of the masticatory musculature; (ii) the separation of the quadrate bone from the cranium; and (iii) the disconnection of the post-dentary bones from the dentary. Here, we compared the jaw-otic complex for 43 synapsid taxa using anatomical network analysis, showing that the disconnection of mandibular bones was a key step in the mammalian middle ear evolution, changing the skull anatomical modularity concomitant to the acquisition of new functions. Furthermore, our analysis allows the identification of three types of anatomical modules evolving through five evolutionary stages during the anatomical transformation of the jawbones into middle ear bones, with the ossification and degradation of Meckel's cartilage in mammals as the key ontogenetic event leading the change of anatomical modularity.

Unique skull network complexity of Tyrannosaurus rex among land vertebrates.

Like other diapsids, Tyrannosaurus rex has two openings in the temporal skull region. In addition, like in other dinosaurs, its snout and lower jaw show large cranial fenestrae. In T. rex, they are thought to decrease skull weight, because, unlike most other amniotes, the skull proportion is immense compared to the body. Understanding morphofunctional complexity of this impressive skull architecture requires a broad scale phylogenetic comparison with skull types different to that of dinosaurs with fundamentally diverging cranial regionalization. Extant fully terrestrial vertebrates (amniotes) provide the best opportunities in that regard, as their skull performance is known from life. We apply for the first time anatomical network analysis to study skull bone integration and modular constructions in tyrannosaur and compare it with five representatives of the major amniote groups in order to get an understanding of the general patterns of amniote skull modularity. Our results reveal that the tyrannosaur has the most modular skull organization among the amniotes included in our study, with an unexpected separation of the snout in upper and lower sub-modules and the presence of a lower adductor chamber module. Independent pathways of bone reduction in opossum and chicken resulted in different degrees of cranial complexity with chicken having a typical sauropsidian pattern. The akinetic skull of opossum, alligator, and leatherback turtle evolved in independent ways mirrored in different patterns of skull modularity. Kinetic forms also show great diversity in modularity. The complex tyrannosaur skull modularity likely represents a refined mosaic of phylogenetic and ecological factors with food processing being probably most important for shaping its skull architecture. Mode of food processing primarily shaped skull integration among amniotes, however, phylogenetic patterns of skull integration are low in our sampling. Our general conclusions on amniote skull integrity are obviously preliminary and should be tested in subsequent studies. As such, this study provides a framework for future research focusing on the evolution of modularity on lower taxonomic levels.

First anatomical network analysis of fore- and hindlimb musculoskeletal modularity in bonobos, common chimpanzees, and humans.

Studies of morphological integration and modularity, and of anatomical complexity in human evolution typically focus on skeletal tissues. Here we provide the first network analysis of the musculoskeletal anatomy of both the fore- and hindlimbs of the two species of chimpanzee and humans. Contra long-accepted ideas, network analysis reveals that the hindlimb displays a pattern opposite to that of the forelimb: Pan big toe is typically seen as more independently mobile, but humans are actually the ones that have a separate module exclusively related to its movements. Different fore- vs hindlimb patterns are also seen for anatomical network complexity (i.e., complexity in the arrangement of bones and muscles). For instance, the human hindlimb is as complex as that of chimpanzees but the human forelimb is less complex than in Pan. Importantly, in contrast to the analysis of morphological integration using morphometric approaches, network analyses do not support the prediction that forelimb and hindlimb are more dissimilar in species with functionally divergent limbs such as bipedal humans.

Primate modularity and evolution: first anatomical network analysis of primate head and neck musculoskeletal system.

Network theory is increasingly being used to study morphological modularity and integration. Anatomical network analysis (AnNA) is a framework for quantitatively characterizing the topological organization of anatomical structures and providing an operational way to compare structural integration and modularity. Here we apply AnNA for the first time to study the macroevolution of the musculoskeletal system of the head and neck in primates and their closest living relatives, paying special attention to the evolution of structures associated with facial and vocal communication. We show that well-defined left and right facial modules are plesiomorphic for primates, while anthropoids consistently have asymmetrical facial modules that include structures of both sides, a change likely related to the ability to display more complex, asymmetrical facial expressions. However, no clear trends in network organization were found regarding the evolution of structures related to speech. Remarkably, the increase in the number of head and neck muscles - and thus of musculoskeletal structures - in human evolution led to a decrease in network density and complexity in humans.

Anatomical network analysis of the musculoskeletal system reveals integration loss and parcellation boost during the fins-to-limbs transition.

Tetrapods evolved from within the lobe-finned fishes around 370 Ma. The evolution of limbs from lobe-fins entailed a major reorganization of the skeletal and muscular anatomy of appendages in early tetrapods. Concurrently, a degree of similarity between pectoral and pelvic appendages also evolved. Here, we compared the anatomy of appendages in extant lobe-finned fishes (Latimeria and Neoceratodus) and anatomically plesiomorphic amphibians (Ambystoma, Salamandra) and amniotes (Sphenodon) to trace and reconstruct the musculoskeletal changes that took place during the fins-to-limbs transition. We quantified the anatomy of appendages using network analysis. First, we built network models-in which nodes represent bones and muscles, and links represent their anatomical connections-and then we measured network parameters related to their anatomical integration, heterogeneity, and modularity. Our results reveal an evolutionary transition toward less integrated, more modular appendages. We interpret this transition as a diversification of muscle functions in tetrapods compared to lobe-finned fishes. Limbs and lobe-fins show also a greater similarity between their pectoral and pelvic appendages than ray-fins do. These findings on extant species provide a basis for future quantitative and comprehensive reconstructions of the anatomy of limbs in early tetrapod fossils, and a way to better understand the fins-to-limbs transition.

Musculoskeletal networks reveal topological disparity in mammalian neck evolution.

BACKGROUND: The increase in locomotor and metabolic performance during mammalian evolution was accompanied by the limitation of the number of cervical vertebrae to only seven. In turn, nuchal muscles underwent a reorganization while forelimb muscles expanded into the neck region. As variation in the cervical spine is low, the variation in the arrangement of the neck muscles and their attachment sites (i.e., the variability of the neck's musculoskeletal organization) is thus proposed to be an important source of neck disparity across mammals. Anatomical network analysis provides a novel framework to study the organization of the anatomical arrangement, or connectivity pattern, of the bones and muscles that constitute the mammalian neck in an evolutionary context. RESULTS: Neck organization in mammals is characterized by a combination of conserved and highly variable network properties. We uncovered a conserved regionalization of the musculoskeletal organization of the neck into upper, mid and lower cervical modules. In contrast, there is a varying degree of complexity or specialization and of the integration of the pectoral elements. The musculoskeletal organization of the monotreme neck is distinctively different from that of therian mammals. CONCLUSIONS: Our findings reveal that the limited number of vertebrae in the mammalian neck does not result in a low musculoskeletal disparity when examined in an evolutionary context. However, this disparity evolved late in mammalian history in parallel with the radiation of certain lineages (e.g., cetartiodactyls, xenarthrans). Disparity is further facilitated by the enhanced incorporation of forelimb muscles into the neck and their variability in attachment sites.

Comparison of musculoskeletal networks of the primate forelimb.

Anatomical network analysis is a framework for quantitatively characterizing the topological organization of anatomical structures, thus providing a way to compare structural integration and modularity among species. Here we apply this approach to study the macroevolution of the forelimb in primates, a structure whose proportions and functions vary widely within this group. We analyzed musculoskeletal network models in 22 genera, including members of all major extant primate groups and three outgroup taxa, after an extensive literature survey and dissections. The modules of the proximal limb are largely similar among taxa, but those of the distal limb show substantial variation. Some network parameters are similar within phylogenetic groups (e.g., non-primates, strepsirrhines, New World monkeys, and hominoids). Reorganization of the modules in the hominoid hand compared to other primates may relate to functional changes such as coordination of individual digit movements, increased pronation/supination, and knuckle-walking. Surprisingly, humans are one of the few taxa we studied in which the thumb musculoskeletal structures do not form an independent anatomical module. This difference may be caused by the loss in humans of some intrinsic muscles associated with the digits or the acquisition of additional muscles that integrate the thumb more closely with surrounding structures.

Bone Fusion in Normal and Pathological Development is Constrained by the Network Architecture of the Human Skull.

Craniosynostosis, the premature fusion of cranial bones, affects the correct development of the skull producing morphological malformations in newborns. To assess the susceptibility of each craniofacial articulation to close prematurely, we used a network model of the skull to quantify the link reliability (an index based on stochastic block models and Bayesian inference) of each articulation. We show that, of the 93 human skull articulations at birth, the few articulations that are associated with non-syndromic craniosynostosis conditions have statistically significant lower reliability scores than the others. In a similar way, articulations that close during the normal postnatal development of the skull have also lower reliability scores than those articulations that persist through adult life. These results indicate a relationship between the architecture of the skull and the specific articulations that close during normal development as well as in pathological conditions. Our findings suggest that the topological arrangement of skull bones might act as a structural constraint, predisposing some articulations to closure, both in normal and pathological development, also affecting the long-term evolution of the skull.

Challenges in identifying and interpreting organizational modules in morphology.

Form is a rich concept that agglutinates information about the proportions and topological arrangement of body parts. Modularity is readily measurable in both features, the variation of proportions (variational modules) and the organization of topology (organizational modules). The study of variational modularity and of organizational modularity faces similar challenges regarding the identification of meaningful modules and the validation of generative processes; however, most studies in morphology focus solely on variational modularity, while organizational modularity is much less understood. A possible cause for this bias is the successful development in the last twenty years of morphometrics, and specially geometric morphometrics, to study patters of variation. This contrasts with the lack of a similar mathematical framework to deal with patterns of organization. Recently, a new mathematical framework has been proposed to study the organization of gross anatomy using tools from Network Theory, so-called Anatomical Network Analysis (AnNA). In this essay, I explore the potential use of this new framework-and the challenges it faces in identifying and validating biologically meaningful modules in morphological systems-by providing working examples of a complete analysis of modularity of the human skull and upper limb. Finally, I suggest further directions of research that may bridge the gap between variational and organizational modularity studies, and discuss how alternative modeling strategies of morphological systems using networks can benefit from each other.

In search of morphological modules: a systematic review.

Morphological modularity arises in complex living beings due to a semi-independent inheritance, development, and function of body parts. Modularity helps us to understand the evolvability and plasticity of organismal form, and how morphological variation is structured during evolution and development. For this reason, delimiting morphological modules and establishing the factors involved in their origins is a lively field of inquiry in biology today. Although it is thought that modularity is pervasive in all living beings, actually we do not know how often modularity is present in different morphological systems. We also do not know whether some methodological approaches tend to reveal modular patterns more easily than others, or whether some factors are more related to the formation of modules or the integration of the whole phenotype. This systematic review seeks to answer these type of questions through an examination of research investigating morphological modularity from 1958 to present. More than 200 original research articles were gathered in order to reach a quantitative appraisal on what is studied, how it is studied, and how the results are explained. The results reveal an heterogeneous picture, where some taxa, systems, and approaches are over-studied, while others receive minor attention. Thus, this review points out various trends and gaps in the study of morphological modularity, offering a broad picture of current knowledge and where we can direct future research efforts.

Musculoskeletal anatomy of the pelvic fin of Polypterus: implications for phylogenetic distribution and homology of pre- and postaxial pelvic appendicular muscles.

As a member of the most basal clade of extant ray-finned fishes (actinopterygians) and of one of the most basal clades of osteichthyans (bony fishes + tetrapods), Polypterus can provide insights into the ancestral anatomy of both ray-finned and lobe-finned fishes, including those that gave rise to tetrapods. The pectoral fin of Polypterus has been well described but, surprisingly, neither the bones nor the muscles of the pelvic fin are well known. We stained and dissected the pelvic fin of Polypterus senegalus and Polypterus delhezi to offer a detailed description of its musculoskeletal anatomy. In addition to the previously described adductor and abductor muscles, we found preaxial and postaxial muscles similar to those in the pectoral fin of members of this genus. The presence of pre- and postaxial muscles in both the pectoral and pelvic fins of Polypterus, combined with recent descriptions of similar muscles in the lobe-finned fishes Latimeria and Neoceratodus, suggests that they were present in the most recent common ancestor of bony fishes. These results have crucial implications for the evolution of appendicular muscles in both fish and tetrapods.

Characteristic tetrapod musculoskeletal limb phenotype emerged more than 400 MYA in basal lobe-finned fishes.

Previous accounts of the origin of tetrapod limbs have postulated a relatively sudden change, after the split between extant lobe-finned fish and tetrapods, from a very simple fin phenotype with only two muscles to the highly complex tetrapod condition. The evolutionary changes that led to the muscular anatomy of tetrapod limbs have therefore remained relatively unexplored. We performed dissections, histological sections, and MRI scans of the closest living relatives of tetrapods: coelacanths and lungfish. Combined with previous comparative, developmental and paleontological information, our findings suggest that the characteristic tetrapod musculoskeletal limb phenotype was already present in the Silurian last common ancestor of extant sarcopterygians, with the exception of the autopod (hand/foot) structures, which have no clear correspondence with fish structures. Remarkably, the two major steps in this long process - leading to the ancestral fin anatomy of extant sarcopterygians and limb anatomy of extant tetrapods, respectively - occurred at the same nodes as the two major similarity bottlenecks that led to the striking derived myological similarity between the pectoral and pelvic appendages within each taxon. Our identification of probable homologies between appendicular muscles of sarcopterygian fish and tetrapods will allow more detailed reconstructions of muscle anatomy in early tetrapods and their relatives.