BACKGROUND
what evolutionary mechanisms are responsible for differences in the relative size of body parts among organisms?
this basic question has long been confronted by biologists, for example, by j. s. huxley, in his problems of relative growth  <cit> .
when considering the tetrapod limb, one might ask why the forelimbs are relatively larger or smaller than the hindlimbs in some species, and how these differences have arisen during evolution.
tetrapods exhibit great diversity in limb structures among species and in differences between fore- and hindlimbs within species, which typically are correlated with locomotor modes and life history  <cit> .
among mammals, the relatively large wings of an adult bat exhibit a striking contrast in size and proportions to its legs, whereas the disparity in most other living eutherians  is more modest.
kangaroos represent the opposite situation, having relatively massive hindlimbs.
these differences are not restricted to mammals but characterize tetrapods as a whole, as evidenced when considering a bird or a frog or a turtle, or fossils such as tyranosaurus rex, which has huge hindlimbs and tiny forelimbs.
a largely unanswered question is how these differences are reflected in the ontogenetic development of the limbs.
limbs are one of the best studied systems in both evolution and development  <cit>  and display a sequence of well-defined temporal events, such as formation of the apical ectodermal ridge  and the chondrification of skeletal elements.
we examine here the extent to which features of early limb development, especially chondrogenesis, might be associated with obvious differences in forelimb and hindlimb size or function in the adult.
we concentrate on heterochrony, the evolutionary change in developmental timing, a process which is thought to be important and common in evolution  <cit> .
in particular, we examine the relative timing of developmental events during ontogeny across the phylogeny of the species examined .
RESULTS
the average event-pair score  was plotted for each species .
the two anurans  show average eps scores significantly less than one, indicating that hindlimb development generally precedes that of the forelimb .
the two birds  also tend to show an advance in hindlimb development – small differences between fore- and hindlimb timing can be detected visually  – but are not significantly different from forelimb-hindlimb synchrony together with the remaining diapsids lacerta and emys.
among mammals, the marsupials  and eulipotyphlan insectivores  all show significant advances in forelimb development.
the generally smaller forelimb advances among the remaining eutherian mammals were not significantly different from synchronous development.
the event-pair score  may range from  <dig>  to  <dig> .
two-tailed student's t-tests were used to determine if the eps was significantly different from  <dig>  n is the number of stages of limb development identified and avg.
stage is the mean number of events that occurred at each stage.
an analysis of variance in combination with fisher's plsd test detected four major clades among the tetrapod species we examined :
<dig>  anuran amphibians: strong, significant hindlimb acceleration with an average eps of  <dig>  ±  <dig>  ;
<dig>  diapsids : slight, but insignificant hindlimb acceleration with an average eps of  <dig>  ±  <dig> ;
<dig>  eutherian mammals: slight, but insignificant forelimb acceleration with an average eps of  <dig>  ±  <dig> ;
<dig>  marsupial mammals: strong, significant forelimb acceleration with an average eps of  <dig>  ±  <dig> .
discussion
the relative timing of fore- and hindlimb development is labile in evolution, but with the observed pattern of change in timing showing a strong phylogenetic component.
different clades  each have evolved characteristic timing relationships  that show no apparent correlation to the diverse lifestyles and adult morphologies of the species within each clade.
the relative conservation of timing relationships within clades in the face of disparate adaptive needs is particularly striking among eutherians, which included a flying species , a glider , and a terrestrial species .
although the eulipotyphlan insectivores erinaceus and talpa do show a significant forelimb advance within and with respect to other eutherian mammals, this pattern is also consistent for the clade despite the different locomotory modes of the exemplars .
in short, the strong selection for modification of the adult limb morphology in eutherians did not produce correlated changes in the relative timing of forelimb-hindlimb development.
the only exceptions to the general pattern of clade-dependent conservation across tetrapods that we are aware of occur in lissamphibians.
in anurans, the limbs develop approximately in synchrony in rana temporaria  <cit>  in contrast to the hindlimb acceleration found in the two species we examined.
among other lissamphibians, it is known that in some urodeles such as the smooth newt triturus vulgaris, the forelimb is so accelerated relative to the hindlimb that it is chondrified before the bud of the latter has even appeared  <cit> .
forelimb advanced over the hindlimb also characterizes the iberian ribbed newt pleurodeles watl  <cit> .
in other urodeles, such as the siberian newt salamandrella keyserlingii  <cit>  and the clawed salamander onychodactylus japonica  <cit> , the forelimb is only marginally advanced if at all or, as in the mexican plethodontid chiropterotriton magnipes, the two limbs reportedly develop more or less simultaneously  <cit> .
increased species sampling may reveal yet additional variation within lissamphibia.
altogether, there was little evidence that adaptations of the adult tetrapod limb were associated with fore-/hindlimb sequence heterochrony during chondrogenesis.
instead, it appears that post-chondrogenic differences in growth rate  play a more deciding role in shaping the final adult morphology of the tetrapod limb.
a cogent example are marsupials, which often display a much larger hindlimb in adults despite the extreme acceleration of forelimb development in embryos.
similarly, the bat actually displayed the least degree of forelimb acceleration among the eutherians studied, despite the forelimbs being greatly enlarged and modified as wings in the adult.
this morphology appears to derive from allometric heterochrony instead: the initially conserved pattern of chondrogenesis is followed by accelerated chondrocyte proliferation and differentiation, with increased bmp <dig> expression and bmp signalling  <cit> .
a similar phenomenon of allometric and not sequence heterochrony was hypothesized by jeffery et al.
<cit>  to account for eye development in the spectral tarsier , a nocturnal primate with the largest relative eye-size among mammals.
an important question, therefore, is what is driving the changes in the early pattern of chondrogenesis among the major tetrapod clades, especially in light of the apparent conservation of this trait within most of them.
similar developmental pathways are responsible for the patterning and growth of fore- and hindlimbs, and covariation between both has been recorded statistically at different levels of the phylogenetic hierarchy in adults  <cit> .
yet, our study shows that heterochronic changes and dissociation have still occurred across major tetrapod clades and within lissamphibians to occasionally override the common mechanisms underlying limb development.
one possible explanation for the inferred transformations is that the different patterns are directly adaptive for the embryo.
this has been hypothesized for marsupials and monotremes, where the greatly accelerated forelimb development has been linked to the necessity for the embryo to climb to the mother's marsupium to attach to a teat for further development  <cit> .
similarly, marsupials show another one of the few, clear instances of sequence heterochrony in mammals, with many cranial structures associated with feeding in the pouch also showing advanced development  <cit> .
similar adaptive explanations are lacking for the remaining species, however.
this is particularly true for the two anurans examined here , which show a conserved pattern despite their very different developmental modes .
altogether, adaptive explanations seem difficult to postulate in general and for the amniotes in particular, the embryos of which develop in the protected environment of the cleidoic egg and so might be evolutionarily 'privileged'  and shielded from diversifying selection.
an alternative, but not mutually exclusive, explanation is the presence of developmental 'constraints'  <cit>  or that the overall pattern is the 'by-product' of other developmental processes.
although our findings are consistent with the existence of such constraints, they in no way can be taken as evidence of their existence.
we would add that the general pattern of fore- and hindlimb developmental timing we present based on  <dig> developmental landmarks belies its actual complexity.
in dasyurus, for example, although the forelimb buds appear before those of the hindlimb, a clear hindlimb bud is present throughout most of forelimb development.
however, chondrogenesis does not start in the hindlimb buds until forelimb development is nearly complete.
the initial timing difference between the appearance of fore- and hindlimb buds correlates with the strong craniocaudal developmental gradient observed at early stages of development  <cit> .
however, the failure of the hindlimb bud to progress after its initial formation , is likely to be caused by more local factors, effectively maintaining a viable limb-bud whilst temporarily suspending outgrowth.
CONCLUSIONS
we find that the relative timing of forelimb versus hindlimb development varies widely between major vertebrate clades.
however, such forelimb-hindlimb sequence heterochronies are largely absent within major clades, the lissamphibia forming a notable exception.
the sequence differences that we did note between fore- and hindlimbs were not easy to explain in terms of morphological differences in the adult fore- and hindlimbs.
rather, the latter were more likely explained by allometric growth differences.
the apparently conservative nature of forelimb versus hindlimb timing may mean that of skeletal chondrogenesis sequences could provide useful phylogenetic characters at higher taxonomic levels in tetrapods.
our data, and those summarized in rabl  <cit>  and richardson  <cit>  allow an initial attempt to reconstruct the evolution of forelimb-hindlimb heterochrony across tetrapods .
the primitive tetrapod condition appears to be for the forelimb to be advanced over the hindlimb .
this condition characterizes the primitive condition for the homologous structures  in chondrichthyes and osteichthyes  <cit> .
thereafter, the evolution of this trait is equivocal , with two equally parsimonious solutions.
one solution is for the forelimb advance to be retained ancestrally throughout tetrapods, with the individual groups  independently deviating from this pattern.
the other solution is for a hindlimb advance to be a shared derived feature  of amniotes, with marsupials and eulipotyphlan insectivores independently regaining the primitive tetrapod hindlimb advance.
additional data will help clarify this picture, both by testing the reality of the apparent clade-specificity of forelimb-hindlimb heterochrony and by hopefully resolving outstanding regions of uncertainty.
key among the latter is within urodeles, where there is evidence that the pattern we present might not hold in unsampled species  <cit> .

 what evolutionary mechanisms are responsible for differences in the relative size of body parts among organisms? this basic question has long been confronted by biologists, for example, by j. s. huxley, in his problems of relative growth. when considering the tetrapod limb, one might ask why the forelimbs are relatively larger or smaller than the hindlimbs in some species, and how these differences have arisen during evolution. tetrapods exhibit great diversity in limb structures among species and in differences between fore- and hindlimbs within species, which typically are correlated with locomotor modes and life history. among mammals, the relatively large wings of an adult bat exhibit a striking contrast in size and proportions to its legs, whereas the disparity in most other living eutherians is more modest. kangaroos represent the opposite situation, having relatively massive hindlimbs. these differences are not restricted to mammals but characterize tetrapods as a whole, as evidenced when considering a bird or a frog or a turtle, or fossils such as tyranosaurus rex, which has huge hindlimbs and tiny forelimbs. a largely unanswered question is how these differences are reflected in the ontogenetic development of the limbs. limbs are one of the best studied systems in both evolution and development and display a sequence of well-defined temporal events, such as formation of the apical ectodermal ridge and the chondrification of skeletal elements. we examine here the extent to which features of early limb development, especially chondrogenesis, might be associated with obvious differences in forelimb and hindlimb size or function in the adult. we concentrate on heterochrony, the evolutionary change in developmental timing, a process which is thought to be important and common in evolution. in particular, we examine the relative timing of developmental events during ontogeny across the phylogeny of the species examined.  the average event-pair score was plotted for each species. the two anurans show average eps scores significantly less than one, indicating that hindlimb development generally precedes that of the forelimb. the two birds also tend to show an advance in hindlimb development – small differences between fore- and hindlimb timing can be detected visually – but are not significantly different from forelimb-hindlimb synchrony together with the remaining diapsids lacerta and emys. among mammals, the marsupials and eulipotyphlan insectivores all show significant advances in forelimb development. the generally smaller forelimb advances among the remaining eutherian mammals were not significantly different from synchronous development. the event-pair score may range from to. two-tailed student's t-tests were used to determine if the eps was significantly different from n is the number of stages of limb development identified and avg. stage is the mean number of events that occurred at each stage. an analysis of variance in combination with fisher's plsd test detected four major clades among the tetrapod species we examined: anuran amphibians: strong, significant hindlimb acceleration with an average eps of ±; diapsids: slight, but insignificant hindlimb acceleration with an average eps of ±; eutherian mammals: slight, but insignificant forelimb acceleration with an average eps of ±; marsupial mammals: strong, significant forelimb acceleration with an average eps of ±. discussion the relative timing of fore- and hindlimb development is labile in evolution, but with the observed pattern of change in timing showing a strong phylogenetic component. different clades each have evolved characteristic timing relationships that show no apparent correlation to the diverse lifestyles and adult morphologies of the species within each clade. the relative conservation of timing relationships within clades in the face of disparate adaptive needs is particularly striking among eutherians, which included a flying species, a glider, and a terrestrial species. although the eulipotyphlan insectivores erinaceus and talpa do show a significant forelimb advance within and with respect to other eutherian mammals, this pattern is also consistent for the clade despite the different locomotory modes of the exemplars. in short, the strong selection for modification of the adult limb morphology in eutherians did not produce correlated changes in the relative timing of forelimb-hindlimb development. the only exceptions to the general pattern of clade-dependent conservation across tetrapods that we are aware of occur in lissamphibians. in anurans, the limbs develop approximately in synchrony in rana temporaria in contrast to the hindlimb acceleration found in the two species we examined. among other lissamphibians, it is known that in some urodeles such as the smooth newt triturus vulgaris, the forelimb is so accelerated relative to the hindlimb that it is chondrified before the bud of the latter has even appeared. forelimb advanced over the hindlimb also characterizes the iberian ribbed newt pleurodeles watl. in other urodeles, such as the siberian newt salamandrella keyserlingii and the clawed salamander onychodactylus japonica, the forelimb is only marginally advanced if at all or, as in the mexican plethodontid chiropterotriton magnipes, the two limbs reportedly develop more or less simultaneously. increased species sampling may reveal yet additional variation within lissamphibia. altogether, there was little evidence that adaptations of the adult tetrapod limb were associated with fore-/hindlimb sequence heterochrony during chondrogenesis. instead, it appears that post-chondrogenic differences in growth rate play a more deciding role in shaping the final adult morphology of the tetrapod limb. a cogent example are marsupials, which often display a much larger hindlimb in adults despite the extreme acceleration of forelimb development in embryos. similarly, the bat actually displayed the least degree of forelimb acceleration among the eutherians studied, despite the forelimbs being greatly enlarged and modified as wings in the adult. this morphology appears to derive from allometric heterochrony instead: the initially conserved pattern of chondrogenesis is followed by accelerated chondrocyte proliferation and differentiation, with increased bmp expression and bmp signalling. a similar phenomenon of allometric and not sequence heterochrony was hypothesized by jeffery et al. to account for eye development in the spectral tarsier, a nocturnal primate with the largest relative eye-size among mammals. an important question, therefore, is what is driving the changes in the early pattern of chondrogenesis among the major tetrapod clades, especially in light of the apparent conservation of this trait within most of them. similar developmental pathways are responsible for the patterning and growth of fore- and hindlimbs, and covariation between both has been recorded statistically at different levels of the phylogenetic hierarchy in adults. yet, our study shows that heterochronic changes and dissociation have still occurred across major tetrapod clades and within lissamphibians to occasionally override the common mechanisms underlying limb development. one possible explanation for the inferred transformations is that the different patterns are directly adaptive for the embryo. this has been hypothesized for marsupials and monotremes, where the greatly accelerated forelimb development has been linked to the necessity for the embryo to climb to the mother's marsupium to attach to a teat for further development. similarly, marsupials show another one of the few, clear instances of sequence heterochrony in mammals, with many cranial structures associated with feeding in the pouch also showing advanced development. similar adaptive explanations are lacking for the remaining species, however. this is particularly true for the two anurans examined here, which show a conserved pattern despite their very different developmental modes. altogether, adaptive explanations seem difficult to postulate in general and for the amniotes in particular, the embryos of which develop in the protected environment of the cleidoic egg and so might be evolutionarily'privileged' and shielded from diversifying selection. an alternative, but not mutually exclusive, explanation is the presence of developmental'constraints' or that the overall pattern is the'by-product' of other developmental processes. although our findings are consistent with the existence of such constraints, they in no way can be taken as evidence of their existence. we would add that the general pattern of fore- and hindlimb developmental timing we present based on developmental landmarks belies its actual complexity. in dasyurus, for example, although the forelimb buds appear before those of the hindlimb, a clear hindlimb bud is present throughout most of forelimb development. however, chondrogenesis does not start in the hindlimb buds until forelimb development is nearly complete. the initial timing difference between the appearance of fore- and hindlimb buds correlates with the strong craniocaudal developmental gradient observed at early stages of development. however, the failure of the hindlimb bud to progress after its initial formation, is likely to be caused by more local factors, effectively maintaining a viable limb-bud whilst temporarily suspending outgrowth.  we find that the relative timing of forelimb versus hindlimb development varies widely between major vertebrate clades. however, such forelimb-hindlimb sequence heterochronies are largely absent within major clades, the lissamphibia forming a notable exception. the sequence differences that we did note between fore- and hindlimbs were not easy to explain in terms of morphological differences in the adult fore- and hindlimbs. rather, the latter were more likely explained by allometric growth differences. the apparently conservative nature of forelimb versus hindlimb timing may mean that of skeletal chondrogenesis sequences could provide useful phylogenetic characters at higher taxonomic levels in tetrapods. our data, and those summarized in rabl and richardson allow an initial attempt to reconstruct the evolution of forelimb-hindlimb heterochrony across tetrapods. the primitive tetrapod condition appears to be for the forelimb to be advanced over the hindlimb. this condition characterizes the primitive condition for the homologous structures in chondrichthyes and osteichthyes. thereafter, the evolution of this trait is equivocal, with two equally parsimonious solutions. one solution is for the forelimb advance to be retained ancestrally throughout tetrapods, with the individual groups independently deviating from this pattern. the other solution is for a hindlimb advance to be a shared derived feature of amniotes, with marsupials and eulipotyphlan insectivores independently regaining the primitive tetrapod hindlimb advance. additional data will help clarify this picture, both by testing the reality of the apparent clade-specificity of forelimb-hindlimb heterochrony and by hopefully resolving outstanding regions of uncertainty. key among the latter is within urodeles, where there is evidence that the pattern we present might not hold in unsampled species. 
the apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. we aim to examine the potential relation of changes in developmental timing to the origin of limb morphological diversity in an explicit comparative and quantitative framework. this pattern is either retained or re-evolved in eulipotyphlan insectivores and taken to its extreme in marsupials. major heterochronic changes in early limb development and chondrogenesis were absent within major clades except lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. tetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology. in the primitive condition, as seen in chondrichthyes and osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. an overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development.
 tetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. we aim to examine the potential relation of changes in developmental timing to the origin of limb morphological diversity in an explicit comparative and quantitative framework. in particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in embryos of tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. whole-mounts and histological sections were used to code the appearance of developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb.  an overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. in the primitive condition, as seen in chondrichthyes and osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. this pattern is either retained or re-evolved in eulipotyphlan insectivores and taken to its extreme in marsupials. although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. all other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs.  major heterochronic changes in early limb development and chondrogenesis were absent within major clades except lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. the apparently conservative nature of this trait means that changes in chondrogenetic patterns may serve as useful phylogenetic characters at higher taxonomic levels in tetrapods. our results highlight the more important role generally played by allometric heterochrony in this instance to shape adult morphology. 