Genetic correlations between cartilage regeneration and degeneration reveal an inverse relationship.

OBJECTIVE: The etiology of osteoarthritis (OA) is unknown, however, there appears to be a significant contribution from genetics. We have identified recombinant inbred strains of mice derived from LG/J (large) and SM/J (small) strains that vary significantly in their ability to repair articular cartilage and susceptibility to post-traumatic OA due to their genetic composition. Here, we report cartilage repair phenotypes in the same strains of mice in which OA susceptibility was analyzed previously, and determine the genetic correlations between phenotypes. DESIGN: We used 12 recombinant inbred strains, including the parental strains, to test three phenotypes: ear-wound healing (n = 263), knee articular cartilage repair (n = 131), and post-traumatic OA (n = 53) induced by the surgical destabilization of the medial meniscus (DMM). Genetic correlations between various traits were calculated as Pearson's correlation coefficients of strain means. RESULTS: We found a significant positive correlation between ear-wound healing and articular cartilage regeneration (r = 0.71; P = 0.005). We observed a strong inverse correlation between articular cartilage regeneration and susceptibility to OA based on maximum (r = -0.54; P = 0.036) and summed Osteoarthritis Research Society International (OARSI) scores (r = -0.56; P = 0.028). Synovitis was not significantly correlated with articular cartilage regeneration but was significantly positively correlated with maximum (r = 0.63; P = 0.014) and summed (r = 0.70; P = 0.005) OARSI scores. Ectopic calcification was significantly positively correlated with articular cartilage regeneration (r = 0.59; P = 0.021). CONCLUSIONS: Using recombinant inbred strains, our study allows, for the first time, the measurement of genetic correlations of regeneration phenotypes with degeneration phenotypes, characteristic of OA (cartilage degeneration, synovitis). We demonstrate that OA is positively correlated with synovitis and inversely correlated with the ability to repair cartilage. These results suggest an addition to the risk paradigm for OA from a focus on degeneration to regeneration.

RNA-seq analysis of chondrocyte transcriptome reveals genetic heterogeneity in LG/J and SM/J murine strains.

OBJECTIVE: To investigate the transcriptomic differences in chondrocytes obtained from LG/J (large, healer) and SM/J (small, non-healer) murine strains in an attempt to discern the molecular pathways implicated in cartilage regeneration and susceptibility to osteoarthritis (OA). DESIGN: We performed RNA-sequencing on chondrocytes derived from LG/J (n = 16) and SM/J (n = 16) mice. We validated the expression of candidate genes and compared single nucleotide polymorphisms (SNPs) between the two mouse strains. We also examined gene expression of positional candidates for ear pinna regeneration and long bone length quantitative trait loci (QTLs) that display differences in cartilaginous expression. RESULTS: We observed a distinct genetic heterogeneity between cells derived from LG/J and SM/J mouse strains. We found that gene ontologies representing cell development, cartilage condensation, and regulation of cell differentiation were enriched in LG/J chondrocytes. In contrast, gene ontologies enriched in the SM/J chondrocytes were mainly related to inflammation and degeneration. Moreover, SNP analysis revealed that multiple validated genes vary in sequence between LG/J and SM/J in coding and highly conserved noncoding regions. Finally, we showed that most QTLs have 20-30% of their positional candidates displaying differential expression between the two mouse strains. CONCLUSIONS: While the enrichment of pathways related to cell differentiation, cartilage development and cartilage condensation infers superior healing potential of LG/J strain, the enrichment of pathways related to cytokine production, immune cell activation and inflammation entails greater susceptibility of SM/J strain to OA. These data provide novel insights into chondrocyte transcriptome and aid in identification of the quantitative trait genes and molecular differences underlying the phenotypic differences associated with individual QTLs.

Rate of evolutionary change in cranial morphology of the marsupial genus Monodelphis is constrained by the availability of additive genetic variation.

We tested the hypothesis that the rate of marsupial cranial evolution is dependent on the distribution of genetic variation in multivariate space. To do so, we carried out a genetic analysis of cranial morphological variation in laboratory strains of Monodelphis domestica and used estimates of genetic covariation to analyse the morphological diversification of the Monodelphis brevicaudata species group. We found that within-species genetic variation is concentrated in only a few axes of the morphospace and that this strong genetic covariation influenced the rate of morphological diversification of the brevicaudata group, with between-species divergence occurring fastest when occurring along the genetic line of least resistance. Accounting for the geometric distribution of genetic variation also increased our ability to detect the selective regimen underlying species diversification, with several instances of selection only being detected when genetic covariances were taken into account. Therefore, this work directly links patterns of genetic covariation among traits to macroevolutionary patterns of morphological divergence. Our findings also suggest that the limited distribution of Monodelphis species in morphospace is the result of a complex interplay between the limited dimensionality of available genetic variation and strong stabilizing selection along two major axes of genetic variation.

CRHR1 genotypes, neural circuits and the diathesis for anxiety and depression.

The corticotrophin-releasing hormone (CRH) system integrates the stress response and is associated with stress-related psychopathology. Previous reports have identified interactions between childhood trauma and sequence variation in the CRH receptor 1 gene (CRHR1) that increase risk for affective disorders. However, the underlying mechanisms that connect variation in CRHR1 to psychopathology are unknown. To explore potential mechanisms, we used a validated rhesus macaque model to investigate association between genetic variation in CRHR1, anxious temperament (AT) and brain metabolic activity. In young rhesus monkeys, AT is analogous to the childhood risk phenotype that predicts the development of human anxiety and depressive disorders. Regional brain metabolism was assessed with (18)F-labeled fluoro-2-deoxyglucose (FDG) positron emission tomography in 236 young, normally reared macaques that were also characterized for AT. We show that single nucleotide polymorphisms (SNPs) affecting exon 6 of CRHR1 influence both AT and metabolic activity in the anterior hippocampus and amygdala, components of the neural circuit underlying AT. We also find evidence for association between SNPs in CRHR1 and metabolism in the intraparietal sulcus and precuneus. These translational data suggest that genetic variation in CRHR1 affects the risk for affective disorders by influencing the function of the neural circuit underlying AT and that differences in gene expression or the protein sequence involving exon 6 may be important. These results suggest that variation in CRHR1 may influence brain function before any childhood adversity and may be a diathesis for the interaction between CRHR1 genotypes and childhood trauma reported to affect human psychopathology.

Fine-mapping quantitative trait loci affecting murine external ear tissue regeneration in the LG/J by SM/J advanced intercross line.

External ear hole closure in LG/J mice represents a model of regenerative response. It is accompanied by the formation of a blastema-like structure and the re-growth of multiple tissues, including cartilage. The ability to regenerate tissue is heritable. An F34 advanced intercross line of mice (Wustl:LG,SM-G34) was generated to identify genomic loci involved in ear hole closure over a 30-day healing period. We mapped 19 quantitative trait loci (QTL) for ear hole closure. Individual gene effects are relatively small (0.08 mm), and most loci have co-dominant effects with phenotypically intermediate heterozygotes. QTL support regions were limited to a median size of 2 Mb containing a median of 19 genes. Positional candidate genes were evaluated using differential transcript expression between LG/J and SM/J healing tissue, function analysis and bioinformatic analysis of single-nucleotide polymorphisms in and around positional candidate genes of interest. Analysis of the set of 34 positional candidate genes and those displaying expression differences revealed over-representation of genes involved in cell cycle regulation/DNA damage, cell migration and adhesion, developmentally related genes and metabolism. This indicates that the healing phenotype in LG/J mice involves multiple physiological mechanisms.

Relationship of age and body mass index to the expression of obesity and osteoarthritis-related genes in human meniscus.

OBJECTIVE: Aging and obesity contribute to the initiation and progression of osteoarthritis with little information on their relation to gene expression in joint tissues, particularly the meniscus. Here, we test the hypothesis that patient age and body mass index (BMI) correlate with the expression of osteoarthritis- and obesity-related gene signatures in the meniscus. DESIGN: Meniscus was obtained from patients (N=68) undergoing arthroscopic partial meniscectomy. The mRNA expression of 24 osteoarthritis-related and 4 obesity-related genes in meniscus was assessed by quantitative real-time PCR. The relationship between gene expression and patient age and BMI was analyzed using Spearman's rank-order correlation. Hierarchical cluster dendrogram and heat map were generated to study inter-gene associations. RESULTS: Age was negatively correlated (P<0.05) with the expression of MMP-1 (r=-0.447), NFκB2 (r=-0.361), NFκBIA (r=-0.312), IκBA (r=-0.308), IL-8 (r=-0.305), ADAMTS-4 (r=-0.294), APLN (apelin) (r=-0.250) and IL-6 (r=-0.244). Similarly, BMI was negatively correlated with the expression of APLN (r=-0.328), ACAN (r=-0.268) and MMP-1 (r=-0.261). After adjusting for the correlation between age and BMI (r=0.310; P=0.008), the only independent effect of BMI on gene expression was for APLN (r=-0.272). However, age had an independent effect on the expression on ADAMTS-4 (r=-0.253), MMP-1 (r=-0.399), IL-8 (r=-0.327), COL1A1 (r=-0.287), NFκBIA (r=-0.278), NFκB2 (r=-0.312) and IκBA (r=-0.299). The gene correlation analysis identified four clusters of potentially relevant genes: transcription factors, matrix-degrading enzymes, cytokines and chemokines, and obesity genes. CONCLUSION: Age and BMI were negatively correlated with several osteoarthritis- and obesity-related genes. Although the bulk of these changes appeared to be driven by age, expression of APLN was related to BMI. Inter-gene correlation analysis implicated a common role for strongly correlated genes. Although age-related variations in gene expression appear to be more relevant than obesity-related differences for the role of the meniscus in osteoarthritis development, further investigation into the role of APLN in meniscus and joint health is warranted.

Cartilage and bone changes during development of post-traumatic osteoarthritis in selected LGXSM recombinant inbred mice.

INTRODUCTION: Little evidence is available on the natural course of osteoarthritis (OA) development and the genes that protect and predispose individuals to it. This study was designed to compare strain-dependent development of OA and its association with tissue regeneration in mice. Two recombinant inbred lines LGXSM-6 and LGXSM-33 generated from LG/J and SM/J intercross were used. Previous studies indicated that LGXSM-6 can regenerate both articular cartilage and ear hole punch while LGXSM-33 cannot. METHODS: Transection of the medial meniscotibial ligament was performed on 10-week-old male mice to induce OA. Cartilage damage was analyzed by histology and bone morphology was evaluated using micro-computed tomography (CT). Ear punches were performed and evaluated by measurement of residual hole diameter. RESULTS: Cartilage analysis showed that LGXSM-33 developed a significantly higher grade of OA than LGXSM-6. Bone analysis showed that LGXSM-33 had substantial subchondral bone and trabecular bone thickening 8 weeks post-surgery, while LGXSM-6 showed bone loss over time. We also confirmed that LGXSM-6 can heal ear tissues significantly better than LGXSM-33. CONCLUSIONS: OA was found to be negatively correlated with the degree of tissue regeneration. LGXSM-33, a poor healer of ear tissues (and articular cartilage), developed more OA compared to LGXSM-6, which had better regenerative ability for ear tissues and articular cartilage. The phenotypic differences observed here are due to genetic differences further suggesting that similar sets of physiological processes and gene variants may mediate variation in OA development and tissue regeneration.

Genotype-dependent responses to levels of sibling competition over maternal resources in mice.

Research on phenotypic plasticity has often focused on how a given genotype responds to the changing physical environments such as temperature or diet. However, for many species the social environment has an equally important role because of competition for resources. During early development, the level of competition for limited (maternally provided) resources will often depend critically on the number of siblings. Therefore, competition among siblings should drive the evolution of genes that allow flexible responses to realized levels of competition and maternal resource availability. However, it is unknown whether genetically based differences between individuals exist in their response to the social environment that affect their future development. Using a quantitative trait locus approach in an experimental population of mice we demonstrate that effects of sibling number on body weight depend on individual genotype at seven loci, over and above the general negative litter size effect. Overall, these litter size-by-genotype interactions considerably modified the degree to which increasing litter size caused reduced weight. For example at one locus this effect leads to a 7% difference in body weight at week 7 between individuals experiencing the extremes of the normal range of litter sizes in our population (five to nine litter mates). The observed interaction between genotype and the competitive environment can produce differences in body weight that are similar in magnitude to the main effect of litter size on weight. Our results show that different genotypes respond to the social environment differentially and that interaction effects of genotype with litter size can be as important as genotype-independent effects of litter size.

Healing quantitative trait loci in a combined cross analysis using related mouse strain crosses.

Inbred mouse strains MRL and LG share the ability to fully heal ear hole punches with the full range of appropriate tissues without scarring. They also share a common ancestry, MRL being formed from a multi-strain cross with two final backcrosses to LG before being inbred by brother-sister mating. Many gene-mapping studies for healing ability have been performed using these two strains, resulting in the location of about 20 quantitative trait loci (QTLs). Here, we combine two of these crosses (N = 638), MRL/lpr × C57BL/6NTac and LG/J × SM/J, in a single combined cross analysis to increase the mapping power, decrease QTL support intervals, separate multiple QTLs and establish allelic states at individual QTL. The combined cross analysis located 11 QTLs, 6 affecting only one cross (5 LG × SM and 1 MRL × B6) and 5 affecting both crosses, approximately the number of common QTLs expected given strain SNP similarity. Amongst the five QTLs mapped in both crosses, three had significantly different genetic effects, additive in one cross and over or underdominant in the other. It is possible that allelic states at these three loci are different in SM and B6 because they lead to differences in dominance interactions with the LG and MRL alleles. QTL support intervals are 40% smaller in the combined cross analysis than in either of the single crosses. Combined cross analysis was successful in enhancing the interpretation of earlier QTL results for these strains.

The effect of a population bottleneck on the evolution of genetic variance/covariance structure.

It is well known that standard population genetic theory predicts decreased additive genetic variance (V(a) ) following a population bottleneck and that theoretical models including interallelic and intergenic interactions indicate such loss may be avoided. However, few empirical data from multicellular model systems are available, especially regarding variance/covariance (V/CV) relationships. Here, we compare the V/CV structure of seventeen traits related to body size and composition between control (60 mating pairs/generation) and bottlenecked (2 mating pairs/generation; average F = 0.39) strains of mice. Although results for individual traits vary considerably, multivariate analysis indicates that V(a) in the bottlenecked populations is greater than expected. Traits with patterns and amounts of epistasis predictive of enhanced V(a) also show the largest deviations from additive expectations. Finally, the correlation structure of weekly weights is not significantly different between control and experimental lines but correlations between necropsy traits do differ, especially those involving the heart, kidney and tail length.

Reply to Lawler: feeding competition, cooperation, and the causes of primate sociality.

This is a reply to Richard Lawler's commentary on our previous work [Lawler, 2011; this issue] in which he develops a set of operational models to test socioecological theories of the evolutionary importance of feeding competition. We strongly agree that we need to critically re-evaluate the basic assumptions of all models of primate sociality, and to verify the explanatory power of alternative models. We also feel Lawler's commentary provides an important opportunity to broaden the debate concerning the fundamental roles of cooperation, competition, and aggression in understanding primate social systems. Lawler provides a number of suggestions as to how models developed in primate socioecology might be tested. We agree with these suggestions, make further suggestions, and call for specific operational definitions so that researchers might begin to develop and test various methodologies. However, we also call for testing alternative theories. Current socioecological theory is based on the assumption that competition and positive selection is always in operation and has driven the evolution of living organisms. We believe that this "explanation of choice" often is treated as an assumed truth to which data are forced to fit, rather than being seen as a theory to be tested. Furthermore, we agree with Weiss and Buchanan [2009. The Mermaid's Tale: Four Billion Years of Cooperation in the Making of Living Things] that on ecological and developmental scales, where organisms actually live out their lives, cooperation may play a more fundamental role than competition.

Epistasis and the evolutionary dynamics of measured genotypic values during simulated serial bottlenecks.

The evolutionary effects of epistasis have been primarily explored analytically and most empirical studies have utilized yeast, viral and bacterial populations. Empirical analyses in multi-cellular organisms are rare because of experimental constraints. Here, we report the results of a genome-wide scan for two-way epistasis in 16 traits related to body size and composition in F(2) mice from the LG/J by SM/J intercross. We analyze two-locus genotypic values at quantitative trait loci (QTL), which provides an especially detailed view of epistatic architectures, to evaluate their predicted evolutionary consequences via Monte Carlo simulations. Epistatic profiles vary, but all traits show complicated genetic architectures which are largely hidden in single locus QTL scans. On average, detected epistatic effects are comparable in size to marginal effects. Simulations demonstrate an expected preservation, and often inflation, of heritable variance across several generations of small effective population size for many identified epistatic pairs over a range of starting allele frequencies.

Sex-specific quantitative trait loci linked to autoresuscitation failure in SWR/J mice.

Autoresuscitation (AR) is a highly conserved response among mammals, which allows survival from transient extreme hypoxia. During hypoxia, bradycardia, and hypoxic gasping develop after a brief period of hyperactivity. Normally, AR occurs if oxygen is restored during the gasping period where an initial heart rate increase is rapidly followed resumption or eupneic breathing. Humans and other mammals can survive multiple immediately repeated AR. A defective AR capacity has been implicated in Sudden Infant Death Syndrome. We had reported earlier that inbred strains of mice such as BALB/cJ could survive a characteristic number of immediately repeated AR trials, but that SWR/J mice failed to AR from a single hypoxic episode. We now report that strains closely related to SWR/J, FVB/N and SJL/J exhibit partial resuscitation defects relative to BALB/cJ or other mouse strains, establishing a genetic basis for variation in AR failure. The AR trial phenotype of BALB/cJ x SWR/J intercross F(1) and F(2) mice was consistent with BALB/cJ dominance and a discrete number of loci. Genome-wide mapping conducted with 60 intercross F(2) animals linked two loci to the number of AR trials survived, including one sex-specific locus with male expression, consistent with the observed 50% male bias for Sudden Infant Death Syndrome in humans. A locus carried on SWR/J chromosome 10 seems to be particularly important in AR failure and was confirmed in a partial consomic line. These results establish a genetic basis for AR failure phenotype in mice, with relevance to Sudden Infant Death Syndrome.

A search for quantitative trait loci exhibiting imprinting effects on mouse mandible size and shape.

Genomic imprinting refers to the pattern of monoallelic parent-of-origin-dependent gene expression where one of the two alleles at a locus is expressed and the other silenced. Although some genes in mice are known to be imprinted, the true scope of imprinting and its impact on the genetic architecture of a wide range of morphometric traits is mostly unknown. We therefore searched for quantitative trait loci (QTL) exhibiting imprinting effects on mandible size and shape traits in a large F(3) population of mice originating from an intercross of the LG/J (Large) and SM/J (Small) inbred strains. We discovered a total of 51 QTL affecting mandible size and shape, 6 of which exhibited differences between reciprocal heterozygotes, the usual signature of imprinting effects. However, our analysis showed that only one of these QTL (affecting mandible size) exhibited a pattern consistent with true imprinting effects, whereas reciprocal heterozygote differences in the other five all were due to maternal genetic effects. We concluded that genomic imprinting has a negligible effect on these specific morphometric traits, and that maternal genetic effects may account for many of the previously reported instances of apparent genomic imprinting.

Epistasis and its contribution to genetic variance components.

We present a new parameterization of physiological epistasis that allows the measurement of epistasis separate from its effects on the interaction (epistatic) genetic variance component. Epistasis is the deviation of two-locus genotypic values from the sum of the contributing single-locus genotypic values. This parameterization leads to statistical tests for epistasis given estimates of two-locus genotypic values such as can be obtained from quantitative trait locus studies. The contributions of epistasis to the additive, dominance and interaction genetic variances are specified. Epistasis can make substantial contributions to each of these variance components. This parameterization of epistasis allows general consideration of the role of epistasis in evolution by defining its contribution to the additive genetic variance.

Is the genotype-phenotype map modular? A statistical approach using mouse quantitative trait loci data.

Various theories about the evolution of complex characters make predictions about the statistical distribution of genetic effects on phenotypic characters, also called the genotype-phenotype map. With the advent of QTL technology, data about these distributions are becoming available. In this article, we propose simple tests for the prediction that functionally integrated characters have a modular genotype-phenotype map. The test is applied to QTL data on the mouse mandible. The results provide statistical support for the notion that the ascending ramus region of the mandible is modularized. A data set comprising the effects of QTL on a more extensive portion of the phenotype is required to determine if the alveolar region of the mandible is also modularized.

Genetic architecture of mandible shape in mice: effects of quantitative trait loci analyzed by geometric morphometrics.

This study introduces a new multivariate approach for analyzing the effects of quantitative trait loci (QTL) on shape and demonstrates this method for the mouse mandible. We quantified size and shape with the methods of geometric morphometrics, based on Procrustes superimposition of five morphological landmarks recorded on each mandible. Interval mapping for F(2) mice originating from an intercross of the LG/J and SM/J inbred strains revealed 12 QTL for size, 25 QTL for shape, and 5 QTL for left-right asymmetry. Multivariate ordination of QTL effects by principal component analysis identified two recurrent features of shape variation, which involved the positions of the coronoid and angular processes relative to each other and to the rest of the mandible. These patterns are reminiscent of the knockout phenotypes of a number of genes involved in mandible development, although only a few of these are possible candidates for QTL in our study. The variation of shape effects among the QTL showed no evidence of clustering into distinct groups, as would be expected from theories of morphological integration. Further, for most QTL, additive and dominance effects on shape were markedly different, implying overdominance for specific features of shape. We conclude that geometric morphometrics offers a promising new approach to address problems at the interface of evolutionary and developmental genetics.

Quantitative trait loci for murine growth.

Body size is an archetypal quantitative trait with variation due to the segregation of many gene loci, each of relatively minor effect, and the environment. We examine the effects of quantitative trait loci (QTLs) on age-specific body weights and growth in the F2 intercross of the LG/J and SM/J strains of inbred mice. Weekly weights (1-10 wk) and 75 microsatellite genotypes were obtained for 535 mice. Interval mapping was used to locate and measure the genotypic effects of QTLs on body weight and growth. QTL effects were detected on 16 of the 19 autosomes with several chromosomes carrying more than one QTL. The number of QTLs for age-specific weights varied from seven at 1 week to 17 at 10 wk. The QTLs were each of relatively minor, subequal effect. QTLs affecting early and late growth were generally distinct, mapping to different chromosomal locations indicating separate genetic and physiological systems for early and later murine growth.

Differential response to dietary fat in large (LG/J) and small (SM/J) inbred mouse strains.

The "large" (LG/J) and "small" (SM/J) inbred mouse strains differ for a wide variety of traits related to body size and obesity. Ninety-three LG/J and SM/J mice were divided into two treatment categories and fed a moderately high-fat diet (21% kcal fat) or a low-fat diet (12% kcal fat) from weaning to necropsy. Strain differences in obesity-related traits and differential response to dietary fat increases were analyzed using ANOVA. LG/J animals grow faster from 3 to 10 wk, have longer tails, and have heavier body weight, liver weight, and fat pad weight than SM/J animals. SM/J animals grow faster after 10 wk of age and have higher fasting glucose levels than LG/J animals. SM/J mice were more responsive to increased dietary fat than LG/J mice for growth after 10 wk, necropsy weight, liver weight, fat pad weights, and fasting glucose levels (in males). The growth from 3 to 10 wk had a much greater response in the LG/J strain, whereas tail length had no response. This pattern of dietary response is similar to that expected under the "thrifty" phenotype hypothesis. Genes affecting strain differences and the differential response of the strains to dietary fat can be successfully mapped in the intercross of the LG/J and SM/J strains. This intercross provides an excellent multigenic model for the genetic basis of complex traits and diseases related to body size and obesity.

A comparison of phenotypic variation and covariation patterns and the role of phylogeny, ecology, and ontogeny during cranial evolution of new world monkeys.

Similarity of genetic and phenotypic variation patterns among populations is important for making quantitative inferences about past evolutionary forces acting to differentiate populations and for evaluating the evolution of relationships among traits in response to new functional and developmental relationships. Here, phenotypic co variance and correlation structure is compared among Platyrrhine Neotropical primates. Comparisons range from among species within a genus to the superfamily level. Matrix correlation followed by Mantel's test and vector correlation among responses to random natural selection vectors (random skewers) were used to compare correlation and variance/covariance matrices of 39 skull traits. Sampling errors involved in matrix estimates were taken into account in comparisons using matrix repeatability to set upper limits for each pairwise comparison. Results indicate that covariance structure is not strictly constant but that the amount of variance pattern divergence observed among taxa is generally low and not associated with taxonomic distance. Specific instances of divergence are identified. There is no correlation between the amount of divergence in covariance patterns among the 16 genera and their phylogenetic distance derived from a conjoint analysis of four already published nuclear gene datasets. In contrast, there is a significant correlation between phylogenetic distance and morphological distance (Mahalanobis distance among genus centroids). This result indicates that while the phenotypic means were evolving during the last 30 millions years of New World monkey evolution, phenotypic covariance structures of Neotropical primate skulls have remained relatively consistent. Neotropical primates can be divided into four major groups based on their feeding habits (fruit-leaves, seed-fruits, insect-fruits, and gum-insect-fruits). Differences in phenotypic covariance structure are correlated with differences in feeding habits, indicating that to some extent changes in interrelationships among skull traits are associated with changes in feeding habits. Finally, common patterns and levels of morphological integration are found among Platyrrhine primates, suggesting that functional/developmental integration could be one major factor keeping covariance structure relatively stable during evolutionary diversification of South American monkeys.