Constraints on mammalian forelimb development: Insights from developmental

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disparity

Darcy Ross1,2, Jonathan D. Marcot1, Keith J. Betteridge3, Nanette Nascone-Yoder4, C. Scott Bailey5, and Karen E. Sears1,6*

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School of Integrative Biology, 505 South Goodwin Avenue, University of Illinois, Urbana IL 61801

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Current affiliation: Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637 3

Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada

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Department of Molecular Biomedical Sciences, Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh NC 27607 5

Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh NC 27607 6

Institute for Genomic Biology, 1206 West Gregory Drive, University of Illinois, Urbana IL 61801

*Corresponding Author: Dr. Karen E. Sears, Assistant Professor, Department of Animal Biology, School of Integrative Biology, 465 Morrill Hall, 505 South Goodwin Avenue, Urbana IL Email: [email protected]; Phone: 217-244-7855

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/evo.12204.

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Running title: Developmental disparity of mammalian forelimbs Text word count: 3,932; Tables: 2; Figures: 2

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Data archival location: DRYAD (http://datadryad.org), doi:10.5061/dryad.9m59b

Abstract Tetrapod limb development has been studied extensively for decades, yet the strength

and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint.

Key Words: constraints, diversity, limb, morphometrics, mammal, ontogeny

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Mammalian limbs have diversified in structural complexity to a degree otherwise unseen in vertebrates, having evolved specialized adaptations that enable aerial, fossorial,

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aquatic, and cursorial lifestyles (Polly 2007). Limb skeletal elements are readily observable during embryonic development and well represented in the fossil record, making the limbs of mammals a powerful model for understanding the evolution of morphology in macroevolutionary, microevolutionary, and functional contexts. Although much progress has been made toward understanding limb evolution and development, there remain fundamental unanswered questions about the processes guiding limb morphogenesis. In this study we investigate when differences in limb development arise between mammalian species, and use the resulting data to evaluate the existence of constraints on this process. Inductive interactions among embryo components are hypothesized to peak around

the time of digit condensation (e.g., paddle stage; Figure 1) (Galis and Metz 2001; Kalinka et. al. 2010). Developmental changes at this stage are therefore predicted to incur serious negative pleiotropic effects, and be evolutionarily constrained (Galis et al 2001). If this constraint exists, then limb development in organisms with diverse adult limb forms should not diverge until after digit condensation. The process of mammalian digit reduction (i.e., loss in digit size or number) has been

cited in support of the constraint hypothesis. Galis and Metz (2001) suggest that digit reduction usually occurs by construction of mesenchymal condensations for the ancestral number of digits (i.e., five) during early limb development, followed by destruction of tissues in digits that are reduced or lost. However, mammalian digit reduction has also been used as evidence against the constraint hypothesis. For example, Hamrick (2002) proposed that digit reduction can occur via changes in early limb development (e.g., digit condensations never form). In the first experimental test of the constraint hypothesis in mammals, Sears et al. (2011) observed changes in pig limb development that are consistent with subtle changes in early limb development underlying pig digit reduction. Because of these conflicts and minimal relevant experimental data, the existence of an evolutionary constraint on mammalian limb development at the time of digit condensation remains unresolved.

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This study takes a geometric morphometric approach to test the hypothesis that mammalian limb development is constrained at the time of digit condensation (e.g., paddle stage; Figure 1). In this approach, levels and patterns of morphological diversity (disparity)

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are quantified and compared over the course of early limb morphogenesis (i.e., from the appearance of the forelimb ridge to the regression of the interdigital tissue). If mammalian limb development is constrained at the paddle stage of development, then disparity among mammalian limbs should be lower at this stage than at the subsequent stages of their development. Mammalian limb disparity could also be low before the paddle stage, as a constraint on limb disparity at the paddle stage could limit the evolution of increased disparity at earlier stages. If early limb development is not constrained, then mammalian limb disparity is likely to exhibit a different pattern such as high disparity from the earliest developmental stages. These quantifications are performed in four mammals: mouse (Mus musculus), gray

short-tailed opossum (Monodelphis domestica), domestic pig (Sus scrofa), and horse (Equus

ferus caballus). These species are targeted for study as their limbs have several salient distinguishing features, including variation in the degree of digit reduction. Mouse and opossum are pentadactylous, while pig digits I, II, and V are reduced to varying degrees (Sears et al. 2011), and the horse has lost all but digit III, and small vestiges of digits II and IV (Matthew 1926; Prothero and Schoch, 2002). Additionally, the forelimbs of opossums develop at an accelerated rate compared to the rest of the body, relative to the other study species (Bininda-Emonds et al. 2007; Sears 2009). These focal species encompass a substantial diversity of developmental trajectories and adult limb morphologies found in living mammals.

Methods Data Collection and Staging Embryonic mice (N = 13) and opossums (N = 16) were obtained from timed matings in the breeding colonies housed in the Sears Lab at the University of Illinois. Pig embryos (N = 15) were obtained through timed ovulations following inseminations at the University of

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Illinois pig farm. Embryonic horses (N = 13) were obtained through timed ovulations following inseminations at the University of Guelph and North Carolina State University. These collections were supplemented through examination of published figures of embryonic

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limbs (e.g., Agarwal 2003, Allan and Lohnes 2000, Fowles et al. 2003, Hockman 2008, Hughes et al. 2000, Keyte and Smith 2010, Wanek et al. 1989, Zülch 2001). Dorsal views of embryonic forelimbs were captured using a Leica M205 C

microscope and a Leica DFC425 digital microscope camera (Leica Microsystems Inc., Buffalo Grove, IL, USA). Specimens were sorted into five comparable stages of forelimb development, based on forelimb morphology (Figure 1): emergence of the limb ridge (stage 1), limb bud (stage 2), paddle stage (stage 3), formation of the digit ridges (stage 4), and the regression of interdigital webbing (“tissue regression” stage, stage 5) using various staging guides (Acker et al. 2001, Butler and Juurlink 1987, Franciolli et al. 2011, Patten 1948, Theiler 1972). The first three stages, limb ridge through paddle formation, are readily recognizable across all species, and the fourth and fifth stages (i.e., formation of the digit ridge and tissue regression) in all species but horse. The adult horse bears only one digit, homologous to digit III in pentadactylous mammals (Prothero and Schoch 2002). Following the paddle stage (stage 3), the next discernable morphological “step” in horse forelimb development is the formation of the footpad that is akin to the digit ridge (stage 4), followed by a distinct tapering of the distal limb that is comparable to the tissue regression stage (stage 5) (Acker et al. 2001).

Landmark Acquisition During forelimb development, there are few discrete, homologous points that are

recognizable in every stage for every species (i.e., homologous landmarks). In the limb ridge and limb bud stages (1 and 2, respectively), the forelimb extends primarily in a single lateral plane, so relatively little morphological information is lost by treating it in two dimensions. In subsequent stages, the arms often curve out of the plane and are significantly warped when projected onto the plane. For this reason, only the laterally flattened hand plate (from wrist to distal tips of digits) could be analyzed for stages 3, 4 and 5.

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Five semi-homologous landmarks (LMs) were selected to capture the overall shape of the developing forelimb at each stage. As the form of the limb changes over its development, available landmarks subtly differ between earlier and later limb stages. However, the effect of

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the difference in landmarks on results is minimized by the standardization of stage-level disparity (described below), and exclusion of principal component 1 (which captures the variation among stages due to landmarks, described below) from result interpretation. For the limb ridge and the limb bud stages (1 and 2, respectively), landmarks are defined as follows (Figure 1): 1) anterior junction of limb and body wall, 2) anterior endpoint of line halfway between the tip and base of the bud along its proximal-distal axis. 3) most distal point of limb, 4) posterior endpoint of line halfway between the tip and base of the bud along its proximaldistal axis, 5) posterior junction of limb and body wall. Landmark definitions for the paddle, digit ridge, and tissue regression stages (3, 4, and 5, respectively) are as follows (Figure 1): 1) anterior base of the wrist, 2) anterior endpoint of widest anterior-posterior length across hand plate, 3) most distal point of paddle/tip of digit III, 4) Posterior endpoint of widest anteriorposterior length across hand plate, 5) posterior base of the wrist. Landmarks were digitized using tpsDig (version 2.16) from images imported using

tpsUtil (version 1.47). Landmarks were digitized in replicate three times for each specimen and subsequently averaged. Error among replicates, presumably due to digitizing error, was minimal (data not shown). All resulting data has been archived in DRYAD (http://datadryad.org, doi:10.5061/dryad.9m59b). Analysis of landmark configurations was performed using the R statistical environment (http://www.r-project.org) using the shapes package. Before analysis, all landmark configurations of left forelimb were reflected about the horizontal axis so that the left and right forelimb could be compared. Landmark coordinates from corresponding forelimbs of a single specimen were then subjected to a Generalized Procrustes Analysis (GPA) for alignment and averaged. GPA translates the coordinates to a common centroid (the center of the shape), scales the coordinates to a centroid size of 1, and then rotates the set of coordinates to minimize the sum of squared distances between configurations (Bookstein 1991). This procedure minimizes differences due to size and orientation, leaving differences primarily due to shape. A second GPA was

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performed on all specimens (after averaging left and right limbs) of each stage. The magnitude of left-right differences in shape was minimal when compared to amongindividual shape variation (data not shown). Mean species shapes at each stage were

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calculated using registered configurations of all constituent specimens at the corresponding stage. These mean species shapes were used to quantify the level and patterns of morphological variation among species.

Morphological Diversity Mean pairwise dissimilarity (MPWD) was used to quantify morphological diversity.

Among disparity metrics, MPWD has been shown to be relatively insensitive to variation in sample size (Foote 1993, 1994; Ciampaglio et al. 2001). MPWD was calculated at each stage as the averaged squared Procrustes distance (the sum of squared distances) between the mean shapes of pairs of species (i.e., MPWD among species means) and between the mean shapes of individual specimens (i.e., MPWD among specimens). Procrustes distance is a measure of the net shape dissimilarity between two LM configurations that have been superimposed by GPA as described above. As mentioned above, the landmarks used in this study differ between early and later

developmental stages. To minimize the effect of these differences on results, stage-level disparity was standardized. To perform this standardization, the Procrustes distances between all pairs of specimens within each species were calculated for each developmental stage. These distances were then averaged to yield the MPWD within species for a given stage. The ratio of MPWD among species (described above) to average MPWD within species (i.e., MPWD ratio) was calculated at each stage to standardize the among species disparity by the within species disparity (Gower 1971, Kaufman and Rousseeuw 1990, Struyf et al. 1997). However, it should be noted that because different landmarks were used to quantify early (1 and 2) and later (3 to 5) limb stages, disparity of early and later stages of limb development are not directly comparable even after this standardization. The raw MPWD among species (i.e., not divided by the average MPWD within species) was also compared to investigate the relative contribution of changes in MPWD among and within species to the MPWD ratio.

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To investigate the relative contribution of each species to the overall disparity, the partial disparity (PD) of each species was calculated for each stage (Foote 1993). To obtain confidence intervals for all disparity metrics, for each stage, specimens

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within species were resampled with replacement 10,000 times, and all disparity metrics were recalculated. Observed differences in disparity were conservatively judged to be significant if their respective 95% confidence intervals do not overlap (Sokal and Rohlf, 1995). All disparity analyses were performed in R using custom written code available on request.

Shape variation To investigate patterns of shape variation, Procrustes registered configurations were

analyzed in Principal Components Analyses (PCA). This technique identifies those aspects of morphology that contribute most to overall morphological variation (Foote, 1993). Separate PCAs were run for the early (1 and 2) and later (3, 4, and 5) stages of limb development because of the non-homologous nature of their landmarks. All specimens were plotted on the resulting principal component axes, and TPS (thin-plate spline) grids were used to visualize the morphologies that extremities along each axis represented (Bookstein, 1991). PCA was performed in R and TPS grids were generated using PAST software (http://folk.uio.no/ohammer/past).

Results Data are consistent with an overall pattern of MPWD through limb development

(Figure 1) in which disparity slightly decreases or stays the same from stage 1 to stage 2, and increases sharply from stages 3 to 4 to 5. Of the later stages of limb development, stage 3 displays the lowest MPWD, and 5 the highest. The 2.75% and 97.5% CI of stage 1 (limb ridge) overlap with those of stage 2 (limb

bud) for the among species, among specimens, and MPWD ratio analyses (Table 1). This indicates that the disparities of limb stages 1 and 2 do not significantly differ. For all analyses, the disparity in stage 3 significantly differs (i.e., the 2.75% and 97.5% CI do not overlap) from those of stages 4 and 5, with the disparities of stages 4 and 5 being higher than that of

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stage 3. Analysis of the raw MPWD among species (i.e., not divided by within species MPWD) yields results that are comparable to those of the MPWD ratio, with a decrease in MPWD from limb stage 1 to 2, and an increase in MPWD from limb stages 3 to 4 to 5. This

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suggests that MPWD ratio patterns are the result of changes in MPWD among rather than within species. Variation in shape through development was evaluated using principal component

analyses (PCA). The first three principal components from these analyses describe most of the morphological variation during limb development. For the early limb stages (1 and 2), PC1 explains 93.2% of the variation, PC2 3.23%, and PC3 1.80% (Figures 2A and 2B). PC1 separates the wider and flatter limb ridge specimens (more positive) from the narrower and longer limb bud specimens (more negative). Note that, because size is removed in the Procrustes analysis, PC1 here does not correspond to size variation among stages, as it often does in PCA of linear measurements. Rather, this result indicates most of the variation among early limb shapes occurs between ontogenetic stages, rather than among species within them. In contrast, distributions of the limb ridge and limb bud specimens overlap for PC2 and PC3. Relative position of the middle, distal-most landmark (LM3) along the anterior-posterior limb axis is the primary determinant of specimen distribution along PC2, and relative width of the limb at its midpoint (i.e., distance between LMs 2 and 4) is the primary determinant for PC3. Specimens with more anterior LM3s tend to have more negative PC2s, while specimens with wider limbs at LMs 2 and 4 tend to have more negative PC3s. For the later limb stages (3, 4, and 5), PC1 explains 47.4% of the variation, PC2

24.2%, and PC3 13.3% (Figures 2C and 2D). As in the early limb stages, PC1 separates the narrower and longer specimens (more negative) from the wider and flatter specimens (more positive) at later limb stages. However, unlike the early limb stages, distributions of the later limb stages overlap on PC1. As a result, PC1 does not separate limb stages, but rather separates the older horse and, to a lesser degree, opossum specimens (from stages 4 and 5) from the others. Relative position of LMs 2 and 4 along the limb’s proximal to distal axis (with more proximal being more positive) is the primary determinant of specimen distribution along PC2, and relative width of the limb at its midpoint (i.e., distance between LMs 2 and 4)

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and base (i.e., distance between LMs 1 and 5) is the primary determinant of PC3. Specimens with uniform widths at their midpoints and bases tend to have more negative PC3s, while

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specimens with wider midpoints than bases tend to have more positive PC3s. Analysis of partial disparity (PD) was used to investigate the contribution of each

species to patterns of total disparity (Table 2). As in the MPWD analyses above, the PD of two species significantly differ (P < 0.05) if their 2.75% and 97.5% CI do not overlap. The mouse PD is significantly lower than those of horse or opossum in stages 1 and 3, and significantly lower than those of all other species in stage 2. In stages 1 and 2, the pig PD is significantly lower than that of horse. The opossum PD is significantly lower than that of all other species in stage 4. Finally, the horse PD is significantly higher than that of all other species in stage 5.

Discussion The goal of this study was to use a comparative, morphometric approach to test the

hypothesis that mammalian limb development is constrained at the time of digit condensation (i.e., paddle stage). Consistent with this hypothesis, this study found that disparity is significantly lower at the paddle stage of limb development (stage 3) than at later stages (4 and 5). Results also suggest that the earliest stages of limb outgrowth (i.e., limb ridge) exhibit considerable disparity, consistent with Sears et al. (2011) and Hamrick (2002), as do the later stages of limb development (i.e., digit ridges, tissue regression). PCA results suggest that primary axis of variation differs in early (1 and 2) and later

(3, 4, and 5) stages of limb development. Morphological differences among stages (e.g., stages 1 and 2) define the primary axis of variation during early limb development, while differences among species (e.g., horse and others) define the primary axis of variation later in limb development. However, closer inspection of the early PCA results reveals that the general pattern observed during later development, with horse and opossum specimens representing extremes of PC1, is present if limb stages 1 and 2 are considered separately. Furthermore, although horse and opossum generally represent the extremes for PC1, opossum specimens overlap with those of other species at the early and, to a much lesser degree, later

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stages of limb development, while horse specimens generally do not. Taken together, these results are consistent with: (1) the existence of species-specific differences in morphology by the ridge stage of limb development, and (2) the extreme digit reduction observed in horses

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playing a considerable role in the generation of patterns of limb disparity during development. This latter hypothesis is further supported by the significantly higher partial disparities of horse than other study species at multiple developmental stages (e.g., stages 1 and 5). The overall pattern of mammalian limb disparity, and, in particular, the low limb

disparity in Stage 3 relative to later stages, is reminiscent of the proposed embryo-wide stage of maximum morphological constraint, suggested to be the result of critical inductive interactions acting across the embryo (Raff 1994, Galis and Metz 2001). The distinctive pattern of opossum limb development may offer a means to investigate this possibility further. Relative to placental mammals and other tetrapods, marsupial mammals such as opossums accelerate forelimb development relative to the rest of the body (Sharman 1970, Sears 2009). This shift in the timing of limb development might be expected to decouple (or possibly result from the decoupling of) the process of limb development from the proposed embryowide critical inductive interactions. As a result, the morphology of developing opossum limbs might be expected to be freer to vary, and thus be an outlier in the disparity analyses. Although the morphologies of some opossum specimens fall at the edge of the range of occupied morphospace, the morphologies of others overlap with those of other species (e.g., mouse). Furthermore, partial disparity results suggest that the morphology of opossum forelimbs is not disproportionately contributing to the disparity among species at any limb stage. In fact, the partial disparity of opossum limbs is significantly lower than that of all other species at stage 4 (digit ridges), when overall disparity significantly increases. As a result, there is no indication that the precocial appearance of the opossum limb has dramatically altered its morphological development relative to other mammalian species, which suggests that embryo-wide critical inductive interactions may not be driving the reduced disparity documented at the paddle stage (stage 3), and/or that opossum forelimb development has somehow been decoupled from that constraint. The limb’s physical position away from the rest of the body isolates it to some degree from developmental and genetic

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interactions with other organs and developing systems, and perhaps decouples its development from inductive genetic interactions acting elsewhere in the developing embryo (Raff 1996, Shubin 2002, Hamrick 2002, Stopper and Wagner, 2005). Although results of

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this study are not consistent with the existence of a developmental constraint due to embryowide inductive interactions, they are consistent with forelimb development being constrained during the paddle stage (stage 3). It is possible that this constraint is the result of limbspecific inductive interactions, rather than interactions of the limb with other structures in the embryo (i.e., trunk). There remains the question of how divergence in early stages of limb development

occurs without disturbing subsequent processes. Stopper and Wagner (2005) suggest that there are two types of mechanisms governing limb development: core mechanisms and ancillary mechanisms. Core mechanisms may guide the formation of the three limb regions of autopod, zeugopod, and stylopod (hand, forearm, and upper arm in human forelimbs) and direct the growth of muscles, blood vessels, and nerves, without which the limb would not be functional. Ancillary mechanisms may control processes such as rates of growth or cell death to prune limbs into diverse morphologies. In theory, ancillary processes could be modified at early as well as late stages of development with relatively fewer consequences, but core processes would be expected to exhibit a period of inductive interactions resistant to change due to the fitness cost of a non-functioning limb (Stopper and Wagner 2005). It is therefore possible that evolutionary modifications in ancillary processes are driving the significant disparity of earliest limb development (stages 1 and 2), and that the reduced disparity at the paddle stage (stage 3) results from the conservation of core processes among mammals. Further comparative studies are needed to identify the types of cellular and molecular processes shaping limb morphogenesis at each developmental stage, and thereby generate a mechanistic understanding of the pattern of disparity observed among these species (Sears 2011).

Acknowledgements

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We thank the members of the Sears Lab for discussion of ideas, J. O’Boyle (www.jamesoart.com) for executing the limb drawings of Figure 1, and J. Mitchell (Univ. of Chicago) for assistance with R. We also thank the University of Illinois Pig Farm for

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assistance with timed inseminations; and Drs. R. O. Waelchli and Allison Schroeder for help in collecting horse specimens at the University of Guelph. This research was supported by NSF grants 0104927 and 125873 to K. Sears, NIH grant HD050042-01 to K. Sears, and by a North Carolina State University College of Veterinary Medicine Grant to C. Bailey and J. Gadsby.

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Figure 1. Analysis of the level of disparity at each stage via mean pairwise dissimilarity (MPWD) calculations. MPWD among species means was divided at each stage by the average within-species MPWD to yield a more conservative estimate of disparity. Disparity decreases from the limb ridge stage to limb bud stage, and rapidly diverges from the paddle stage through digit ridge and tissue regression stages. The results for early (Stages 1 and 2) and later (Stages 3, 4, and 5) cannot be directly compared because of landmark differences. The landmarks used in this study are displayed on the limb stage drawings.

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Accepted Article

Figure 2. Geometric morphometric analysis of mammalian forelimb morphology through five stages of development. Early (A and B) and later (C and D) stages of limb development were analyzed in separate PCAs. For early limb development (stages 1 and 2), axes in (A) display principal components I (93.2% of the variation) and II (3.23% of the variation), and in (B) principal components II (2.3% of the variation) and III (1.80% of the variation). For later limb development (stages 3, 4, and 5), axes in (C) display principal components I (47.4% of the variation) and II (24.2% of the variation), and in (D) principal components II (24.2% of the variation) and III (13.3% of the variation). Shape deformation drawings indicate the mean shape and the shapes represented by a given point in morphospace.

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19

Accepted Article

Table 1. Disparity of developing mammalian limbs. Stage = limb stage, CI = confidence interval (calculated by resampling).

CI Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

MPWD among species means 2.75% 50% 97.50% 0.0189 0.0236 0.0299 0.0109 0.0162 0.0289 0.0060 0.0075 0.0151 0.0468 0.0557 0.0716 0.0575 0.0694 0.0897

MPWD Ratio (within to among species) 2.75% 50% 97.50% 2.9912 5.2400 8.7052 0.5260 5.0354 12.2876 0.4611 1.7853 2.9655 3.2172 25.9015 50.3607 12.4335 16.3346 27.5750

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MPWD within species 2.75% 50% 97.50% 0.0075 0.0091 0.0110 0.0036 0.0120 0.0196 0.0039 0.0057 0.0072 0.0174 0.0214 0.0237 0.0218 0.0248 0.0276

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Accepted Article

Table 2. Partial disparities of developing mammalian limbs. Stage = limb stage, CI = confidence interval (calculated by resampling).

CI Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

2.75% 0.0034 0.0024 0.0010 0.0055 0.0135

Horse 50% 0.0048 0.0058 0.0017 0.0089 0.0144

97.50% 0.0066 0.0099 0.0023 0.0097 0.0155

2.75% 0.0014 0.0001 0.0011 0.0013 0.0032

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Opossum 50% 0.0027 0.0052 0.0025 0.0016 0.0045

97.50% 0.0036 0.0091 0.0034 0.0019 0.0057

2.75% 0.0000 0.0000 0.0005 0.0026 0.0024

Mouse 50% 0.0003 0.0000 0.0007 0.0040 0.0030

97.50% 0.0008 0.0000 0.0009 0.0060 0.0037

2.75% 0.0006 0.0006 0.0005 0.0053 0.0015

Pig 50% 0.0010 0.0008 0.0009 0.0069 0.0029

97.50% 0.0018 0.0016 0.0014 0.0090 0.0042

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Constraints on Mammalian forelimb development: insights from developmental disparity.

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unr...
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