BACKGROUND
limb reduction has occurred repeatedly during tetrapod evolution, affecting several morphologically and ecologically disparate clades   <cit> .
among these clades, squamata  is regarded generally as an exemplary model for studying modes and causes of this potentially dramatic evolutionary phenomenon .
at least  <dig> squamate lineages have been identified as having independently lost one or more bones of the fore- or hindlimb  <cit> .
many of these lineages are closely related and, accordingly, offer excellent material for comparative study.
more significantly, a number of squamate clades include extant species displaying a range of intermediate states between pentadactyl and limbless conditions, affording the possibility of reconstructing patterns and rates of limb reduction and loss.
aside from their potential for elucidating the mechanisms and causes of limb reduction itself, such inferences of patterns and rates may contribute to an improved understanding of the substantial transformations of body form accompanying the emergence of higher taxa and, concomitantly, the relationship of macro- and microevolutionary phenomena .
previous studies have examined several aspects of limb reduction in squamates.
wiens and slingluff  <cit>  presented an explicitly phylogenetic analysis of limb reduction and associated body elongation in the morphologically diverse clade anguidae.
ancestral character state reconstructions provided evidence for three independent transitions to an elongate, functionally limbless body form, each of which was inferred to have occurred gradually over a period of more than  <dig> million years.
shapiro  <cit>  and shapiro et al.
<cit>  demonstrated that digit loss in the scincid clade hemiergis is not explained by simple truncation of a putative ancestral  developmental program.
instead, structurally reduced limbs exhibit abbreviated embryonic expression of the gene sonic hedgehog and a presumably related decrease in the proliferation of limb mesenchyme.
wiens et al.
<cit>  considered the roles of biogeography and competition in determining the frequency of transitions to an elongate, limb-reduced body form across squamates, proposing that discontinuity of habitats, dispersal limitation, and interspecific competition may be as significant as functional and developmental constraints in explaining the number of times such transitions have occurred.
kohlsdorf and wagner  <cit>  used phylogenetic methods to examine the evolution of limb morphology in the gymnophthalmid clade bachia.
their analyses indicated that digits have been regained in some species of this clade and, accordingly, that the ability to develop digits may be preserved for an extended period after their loss.
this conclusion challenges the traditional perspective that limb reduction is irreversible, and emphasises the need for further study of additional squamate clades to evaluate the generality of the above results.
most recently, brandley et al.
<cit>  employed a time-calibrated phylogeny for  <dig> species to examine patterns in the evolution of an elongate, limbless body form across squamates.
their analyses revealed a consistent association of decreases in limb length, digit loss, and body elongation across ecologically and phylogenetically diverse squamate clades, indicating an influence of shared functional and developmental constraints on the evolution of body form.
transitions to a highly elongate, limbless body plan were estimated to have occurred over periods as brief as  <dig> million years, although intermediate morphologies  were inferred to persist for considerably greater lengths of time in some instances.
this result intimates that such intermediate morphologies may be evolutionarily stable and, accordingly, are not necessarily transitory stages in an incomplete process of body elongation and limb loss.
although confirming the pervasiveness of structural reduction in the evolution of squamate limb morphology , brandley et al.
's  <cit>  analyses identified at least six instances in which digits have been reacquired, supporting kohlsdorf and wagner's  conclusion that limb reduction may be reversible.
while brandley et al.
's  <cit>  study is notable for its broad phylogenetic scope, the relatively sparse taxon sampling for several clades dictated  by such an inclusive analysis potentially limits the resolution that can be attained in inferring the mode and tempo of evolutionary transitions.
as brandley et al.
<cit>  note, denser taxon sampling may reveal that substantial limb reduction is possible over intervals considerably shorter than those indicated by their analyses.
similarly, inferred frequencies of digit loss and gain may be significantly altered by the inclusion of additional species; that brandley et al.
's  <cit>  analyses, which include only three of the  <dig> species considered by kohlsdorf and wagner  <cit> , provide no evidence for reversals of limb reduction in bachia serves to illustrate this point.
accordingly, detailed studies of comparatively recent clades are needed to establish general patterns and rates of squamate limb reduction and loss.
a primary concern is whether the pattern indicated by analyses such as those of wiens and slingluff  <cit>  and brandley et al.
<cit> , specifically, moderately frequent limb loss occurring over extended intervals  with occasional re-elaboration of limbs, accurately represents the evolution of body form in squamates.
if analyses of more recent clades  indicate consistently higher frequencies and rates of limb loss , there may be cause to query the perspective offered by these broader-scale studies.
a number of diverse genera composed of species exhibiting varying degrees of limb reduction constitute ideal candidates for such analyses.
the australian scincid clade lerista is pre-eminent among these, comprising more than  <dig> species displaying at least  <dig> distinct limb bone configurations, from that considered plesiomorphic for squamates  to entirely limbless  <cit> .
although lerista has been considered the best available model for investigating squamate limb reduction  <cit> , lack of a well-resolved phylogeny has impeded study of the pattern and mode of limb reduction and loss within the clade.
greer  <cit> , who examined intra- and interspecific variation in phalangeal configurations for more than half of the species of lerista then described, relied on an arrangement of observed configurations entailing the minimum amount of change between each in reconstructing sequences of phalanx loss, while noting that 'it would be most desirable to be able to order variation on the basis of a hypothesis of intrageneric relationships for lerista based on characters independent of those under study, but unfortunately, this is not yet possible'  <cit> .
more than two decades later, the phylogeny of lerista remains largely obscure.
a modest number of species groups have been diagnosed explicitly on the basis of proposed apomorphic character states, implying monophyly  <cit> , however, relationships within and among these species groups are unknown.
in this paper, we present the first comprehensive phylogeny for lerista, inferred from nucleotide sequences for a nuclear intron and six mitochondrial genes.
this phylogeny is employed in reconstructing ancestral digit configurations, which, in conjunction with estimates of absolute ages for nodes, provide insight into the pattern and rate of limb reduction within the clade.
RESULTS
phylogenetic relationships were inferred from atp synthetase-β subunit intron, 12s rrna, 16s rrna, and nd <dig> and adjacent trna-his, trna-ser, and trna-leu nucleotide sequences  for  <dig> species of lerista  and three outgroup taxa .
a majority-rule consensus of trees sampled in a bayesian analysis of the combined sequence data  includes many significantly-supported clades .
several of these clades correspond with putative monophyletic groups recognised previously on the basis of morphology, including the bipes species group  <cit> , the nichollsi species group , and the orientalis species group .
however, the majority of recovered relationships have never been hypothesised by earlier authors .
the four species with well-developed, pentadactyl limbs  compose two distant clades, neither of which is positioned basally within lerista.
those species in which limbs are greatly reduced  or absent  similarly are polyphyletic.
to investigate the pattern and rate of limb reduction within lerista, we reconstructed numbers of digits for internal nodes assuming a markov model of character evolution with separate parameters describing instantaneous rates of digit gain and loss.
the number of digits for the manus and pes were considered independently.
for the manus, the ratio of maximum likelihood rates of digit gain and loss  indicates that increases in the number of digits are far less probable than reductions.
maximum likelihood reconstructions of ancestral states imply  <dig> instances of digit loss with no reversals to an increased number of digits .
all rates of digit gain not significantly less likely than the maximum likelihood estimate yield maximum likelihood ancestral states identical to or only insignificantly more likely than those reconstructed assuming the maximum likelihood rate .
for the pes, the estimated rate of digit gain exceeds that of digit loss , with maximum likelihood reconstructions of ancestral states indicating  <dig> increases in the number of digits and  <dig> reductions .
although suggesting several reversals of limb reduction in lerista, we regard this result as equivocal for two reasons.
firstly, a range of rates of digit gain for the pes are nearly as likely as the maximum likelihood estimate , and some of these rates yield ancestral state reconstructions implying no increases in the number of digits.
specifically, rates of  <dig> – <dig>  are not significantly less likely than the maximum likelihood rate and yield maximum likelihood ancestral states indicating  <dig> cases of digit loss but no instances in which digits are gained .
secondly, when considered in conjunction with states inferred for the manus, reconstructions of numbers of pedal digits  imply implausible digit configurations for many internal nodes.
in particular, configurations of either five or four digits for the manus and two digits for the pes are implied for the majority of basal nodes, however, neither is represented among extant species of lerista .
ancestral states reconstructed assuming rates of digit gain of  <dig> – <dig> , by contrast, imply implausible digit configurations for very few internal nodes.
moreover, in all instances, the ancestral states implying implausible digit configurations are not significantly more likely than alternative states implying configurations observed among extant species of lerista .
thus, we are disinclined to conclude that limb reduction has been reversed in lerista, preferring the ancestral states reconstructed assuming rates of pedal digit gain of  <dig> – <dig>  .
an examination of ancestral state reconstructions across  <dig> trees  sampled after attaining stationarity in the bayesian analysis indicates that the above conclusions are largely insensitive to uncertainty in the inferred phylogeny.
for all nodes, maximum likelihood numbers of manual digits reconstructed for more than 90% of trees are consistent with  those inferred assuming the bayesian majority-rule consensus , and reversals of limb reduction are inferred for only an insignificant proportion  of trees.
similar results are obtained when rates of digit gain considerably higher than those estimated by maximum likelihood are assumed.
for example, a rate of  <dig> , although significantly less likely than the  maximum likelihood rates for 90% of trees, yields maximum likelihood ancestral states for  <dig> of  <dig> internal nodes consistent with those reconstructed assuming the bayesian majority-rule consensus in more than 92% of trees, with more than 62% of trees exhibiting no reversals of limb reduction .
thus, the inferred ancestral states for the manus in figure  <dig> are not substantially affected by uncertainty in tree topology, branch lengths, and rates of character state transition.
ancestral state reconstructions for the pes are similarly stable when uncertainty in the phylogeny is considered.
assuming estimated rates of digit gain, maximum likelihood numbers of pedal digits for all nodes are consistent with those for the bayesian majority-rule consensus  in more than 90% of trees.
as for the bayesian majority-rule consensus , ancestral state reconstructions for nearly all trees  imply one or more instances of reversed limb reduction.
nonetheless, a range of rates of pedal digit gain only insignificantly lower than those estimated for a considerable proportion of trees yield ancestral state reconstructions very similar to those obtained for the bayesian majority-rule consensus assuming rates of digit gain of  <dig> – <dig>  .
a rate of  <dig> , for example, is not significantly less likely than the estimated rates for 64% of trees and, for all except a single node, yields maximum likelihood ancestral states for more than 97% of trees that are consistent with the preferred ancestral states in figure  <dig>  in almost all cases, numbers of pedal digits reconstructed for the single exceptional node  entail no increases in the number of digits, and reversed limb reduction is implied in less than 5% of trees.
the phylogeny in figure  <dig> thus implies extraordinary evolutionary mutability of limb morphology in lerista.
although this conclusion does not depend on a preference for a specific set of ancestral states, the alternative reconstructions for the pes presented in figure  <dig> and additional file  <dig> evidently entail disparate patterns of limb evolution .
as discussed above, there is no cogent statistical basis for favouring either of these reconstructions; ancestral states inferred assuming the maximum likelihood rate of digit gain  are only insignificantly more likely than those reconstructed assuming rates of digit gain of  <dig> – <dig>  .
however, when the very robust ancestral states for the manus are considered, a compelling argument can be made for accepting reconstructions for the pes implying no reversals of limb reduction.
whereas ancestral states entailing repeated re-elaboration of the pes imply digit configurations for many internal nodes differing fundamentally from those displayed by extant species of lerista, ancestral digit configurations implied by reconstructions entailing no reversals of pedal digit loss are nearly invariably represented among observed phenotypes.
although we prefer ancestral state reconstructions for the pes implying repeated, unreversed digit loss, we acknowledge that our results could be considered to indicate the reacquisition of pedal digits, and so provide further evidence for kohlsdorf and wagner's  <cit>  proposal that limb reduction is potentially reversible.
perhaps significantly, the inferred age of lerista  is comparable to estimates of the period over which unexpressed developmental genes may preserve their functionality , rendering the re-elaboration of structurally reduced limbs at least plausible.
nonetheless, it should be noted that such re-elaboration would entail two instances in which a highly reduced pes  has regained an entire or nearly entire complement of phalanges .
this contrasts with the inferred cases of digit reacquisition in bachia, where species having putatively re-evolved digits exhibit unusual phalangeal complements , possibly reflecting the activation of a novel  developmental pathway .
allowing that pedal digit loss has not been reversed in lerista, our analyses indicate a remarkably high frequency of limb reduction.
ancestral states reconstructed assuming the bayesian majority-rule consensus, rates of digit gain of  <dig> – <dig>  for the pes, and maximum likelihood estimates for all other rates imply  <dig> independent reductions in the number of digits from a pentadactyl condition .
a further seven reductions are inferred to have proceeded independently from a tetradactyl condition derived from one of these reductions .
four independent losses of all manual and pedal digits are inferred, three from pentadactyl or tetradactyl conditions.
assuming an age of  <dig>  million years for lerista, estimated using molecular dating methods , the loss of all digits from a pentadactyl condition in ameles, apoda, and stylis has occurred within a period of  <dig>  million years.
the most recent tetradactyl ancestor of ameles and stylis is no more than  <dig>  million years old, while that of apoda is no more than  <dig>  million years old.
the highest rate of complete loss of digits from a pentadactyl or tetradactyl condition is inferred for humphriesi and praepedita; the most recent shared ancestor of these species  is separated from an inferred pentadactyl ancestor by no more than  <dig>  million years .
this is considerably more rapid than the rate implied by brandley et al.
's  <cit>  estimate of the minimum interval over which limb loss has occurred in more than  <dig> squamate lineages .
as brandley et al.
<cit>  note, however, their estimates represent maximum values, and so may significantly overestimate the amount of time necessary for this radical evolutionary transition.
accordingly, it is at least conceivable that the higher rates of limb loss inferred here for lerista are representative of those for squamates generally.
this perspective suggests a pattern of body form evolution differing markedly from that envisaged by wiens and slingluff  <cit>  for anguids , and, if substantiated, would emphasise the significance of brandley et al.
's  <cit>  finding that intermediate phenotypes may persist for tens of millions of years.
almost invariably, species with greatly reduced limbs are separated from inferred pentadactyl or tetradactyl ancestors by very few nodes, so that, assuming limb reduction has not been saltational , reconstruction of complete sequences of digit loss for individual lineages is not possible.
nonetheless, if digit loss is presumed to proceed similarly across all  lineages, general conclusions concerning the mode of limb reduction may be derived from a consideration of inferred losses for lineages exhibiting varying degrees of reduction.
thus, a plot of the numbers of manual and pedal digits lost in all reductions from a pentadactyl or tetradactyl condition versus the total number of  digits lost  may be considered to indicate that in the initial stages of limb reduction rates of digit loss are similar for the manus and pes, however, as limb reduction progresses, manual digits are lost more readily than pedal digits.
this is consistent with a more significant role of the hindlimb in limb-mediated locomotion and selection for some ability to employ limb-mediated locomotion  in limb-reduced species.
particular intermediate digit configurations, most conspicuously that of no digits for the manus and two digits for the pes, generally originate relatively rapidly and persist for extended periods.
this pattern intimates that these configurations do not represent transitory stages in a continuing process of limb reduction and provides further evidence for adaptive retention of digits, consistent with brandley et al.
's  <cit>  proposal that intermediate phenotypes may be targets of selection.
CONCLUSIONS
many authors have noted the recurring evolution of an elongate, limb-reduced body form in squamates, however, even within this clade, the frequency and rate of limb reduction inferred here for lerista are exceptional.
ancestral state reconstructions imply  <dig> instances of limb reduction , almost as many as reported by greer  <cit>  for all remaining scincids, and nearly half the number of reductions inferred for all other squamates.
an interval of no more than  <dig>  million years for the complete loss of limbs in one lineage of lerista is less than one fourth of brandley et al.
's  <cit>  minimum estimate of the time required for this substantial phenotypic transition.
these results emphasise the potential for extensive alteration of body form in squamates over  brief periods.
an absence of cogent evidence for reversals of digit loss in lerista contrasts with kohlsdorf and wagner's  <cit>  proposal that digits have been regained in some species of bachia, possibly reflecting an influence of differing environmental and genetic contexts on the evolution of limb morphology in these clades.
future study of the genetic, developmental, and ecological bases of limb reduction and loss in lerista promises the elucidation of not only this phenomenon in squamates, but also the dramatic evolutionary transformations of body form that have produced the extraordinary diversity of multicellular organisms.

 limb reduction has occurred repeatedly during tetrapod evolution, affecting several morphologically and ecologically disparate clades. among these clades, squamata is regarded generally as an exemplary model for studying modes and causes of this potentially dramatic evolutionary phenomenon. at least squamate lineages have been identified as having independently lost one or more bones of the fore- or hindlimb. many of these lineages are closely related and, accordingly, offer excellent material for comparative study. more significantly, a number of squamate clades include extant species displaying a range of intermediate states between pentadactyl and limbless conditions, affording the possibility of reconstructing patterns and rates of limb reduction and loss. aside from their potential for elucidating the mechanisms and causes of limb reduction itself, such inferences of patterns and rates may contribute to an improved understanding of the substantial transformations of body form accompanying the emergence of higher taxa and, concomitantly, the relationship of macro- and microevolutionary phenomena. previous studies have examined several aspects of limb reduction in squamates. wiens and slingluff presented an explicitly phylogenetic analysis of limb reduction and associated body elongation in the morphologically diverse clade anguidae. ancestral character state reconstructions provided evidence for three independent transitions to an elongate, functionally limbless body form, each of which was inferred to have occurred gradually over a period of more than million years. shapiro and shapiro et al. demonstrated that digit loss in the scincid clade hemiergis is not explained by simple truncation of a putative ancestral developmental program. instead, structurally reduced limbs exhibit abbreviated embryonic expression of the gene sonic hedgehog and a presumably related decrease in the proliferation of limb mesenchyme. wiens et al. considered the roles of biogeography and competition in determining the frequency of transitions to an elongate, limb-reduced body form across squamates, proposing that discontinuity of habitats, dispersal limitation, and interspecific competition may be as significant as functional and developmental constraints in explaining the number of times such transitions have occurred. kohlsdorf and wagner used phylogenetic methods to examine the evolution of limb morphology in the gymnophthalmid clade bachia. their analyses indicated that digits have been regained in some species of this clade and, accordingly, that the ability to develop digits may be preserved for an extended period after their loss. this conclusion challenges the traditional perspective that limb reduction is irreversible, and emphasises the need for further study of additional squamate clades to evaluate the generality of the above results. most recently, brandley et al. employed a time-calibrated phylogeny for species to examine patterns in the evolution of an elongate, limbless body form across squamates. their analyses revealed a consistent association of decreases in limb length, digit loss, and body elongation across ecologically and phylogenetically diverse squamate clades, indicating an influence of shared functional and developmental constraints on the evolution of body form. transitions to a highly elongate, limbless body plan were estimated to have occurred over periods as brief as million years, although intermediate morphologies were inferred to persist for considerably greater lengths of time in some instances. this result intimates that such intermediate morphologies may be evolutionarily stable and, accordingly, are not necessarily transitory stages in an incomplete process of body elongation and limb loss. although confirming the pervasiveness of structural reduction in the evolution of squamate limb morphology, brandley et al.'s analyses identified at least six instances in which digits have been reacquired, supporting kohlsdorf and wagner's conclusion that limb reduction may be reversible. while brandley et al.'s study is notable for its broad phylogenetic scope, the relatively sparse taxon sampling for several clades dictated by such an inclusive analysis potentially limits the resolution that can be attained in inferring the mode and tempo of evolutionary transitions. as brandley et al. note, denser taxon sampling may reveal that substantial limb reduction is possible over intervals considerably shorter than those indicated by their analyses. similarly, inferred frequencies of digit loss and gain may be significantly altered by the inclusion of additional species; that brandley et al.'s analyses, which include only three of the species considered by kohlsdorf and wagner, provide no evidence for reversals of limb reduction in bachia serves to illustrate this point. accordingly, detailed studies of comparatively recent clades are needed to establish general patterns and rates of squamate limb reduction and loss. a primary concern is whether the pattern indicated by analyses such as those of wiens and slingluff and brandley et al., specifically, moderately frequent limb loss occurring over extended intervals with occasional re-elaboration of limbs, accurately represents the evolution of body form in squamates. if analyses of more recent clades indicate consistently higher frequencies and rates of limb loss, there may be cause to query the perspective offered by these broader-scale studies. a number of diverse genera composed of species exhibiting varying degrees of limb reduction constitute ideal candidates for such analyses. the australian scincid clade lerista is pre-eminent among these, comprising more than species displaying at least distinct limb bone configurations, from that considered plesiomorphic for squamates to entirely limbless. although lerista has been considered the best available model for investigating squamate limb reduction, lack of a well-resolved phylogeny has impeded study of the pattern and mode of limb reduction and loss within the clade. greer, who examined intra- and interspecific variation in phalangeal configurations for more than half of the species of lerista then described, relied on an arrangement of observed configurations entailing the minimum amount of change between each in reconstructing sequences of phalanx loss, while noting that'it would be most desirable to be able to order variation on the basis of a hypothesis of intrageneric relationships for lerista based on characters independent of those under study, but unfortunately, this is not yet possible'. more than two decades later, the phylogeny of lerista remains largely obscure. a modest number of species groups have been diagnosed explicitly on the basis of proposed apomorphic character states, implying monophyly, however, relationships within and among these species groups are unknown. in this paper, we present the first comprehensive phylogeny for lerista, inferred from nucleotide sequences for a nuclear intron and six mitochondrial genes. this phylogeny is employed in reconstructing ancestral digit configurations, which, in conjunction with estimates of absolute ages for nodes, provide insight into the pattern and rate of limb reduction within the clade.  phylogenetic relationships were inferred from atp synthetase-β subunit intron, 12s rrna, 16s rrna, and nd and adjacent trna-his, trna-ser, and trna-leu nucleotide sequences for species of lerista and three outgroup taxa. a majority-rule consensus of trees sampled in a bayesian analysis of the combined sequence data includes many significantly-supported clades. several of these clades correspond with putative monophyletic groups recognised previously on the basis of morphology, including the bipes species group, the nichollsi species group, and the orientalis species group. however, the majority of recovered relationships have never been hypothesised by earlier authors. the four species with well-developed, pentadactyl limbs compose two distant clades, neither of which is positioned basally within lerista. those species in which limbs are greatly reduced or absent similarly are polyphyletic. to investigate the pattern and rate of limb reduction within lerista, we reconstructed numbers of digits for internal nodes assuming a markov model of character evolution with separate parameters describing instantaneous rates of digit gain and loss. the number of digits for the manus and pes were considered independently. for the manus, the ratio of maximum likelihood rates of digit gain and loss indicates that increases in the number of digits are far less probable than reductions. maximum likelihood reconstructions of ancestral states imply instances of digit loss with no reversals to an increased number of digits. all rates of digit gain not significantly less likely than the maximum likelihood estimate yield maximum likelihood ancestral states identical to or only insignificantly more likely than those reconstructed assuming the maximum likelihood rate. for the pes, the estimated rate of digit gain exceeds that of digit loss, with maximum likelihood reconstructions of ancestral states indicating increases in the number of digits and reductions. although suggesting several reversals of limb reduction in lerista, we regard this result as equivocal for two reasons. firstly, a range of rates of digit gain for the pes are nearly as likely as the maximum likelihood estimate, and some of these rates yield ancestral state reconstructions implying no increases in the number of digits. specifically, rates of – are not significantly less likely than the maximum likelihood rate and yield maximum likelihood ancestral states indicating cases of digit loss but no instances in which digits are gained. secondly, when considered in conjunction with states inferred for the manus, reconstructions of numbers of pedal digits imply implausible digit configurations for many internal nodes. in particular, configurations of either five or four digits for the manus and two digits for the pes are implied for the majority of basal nodes, however, neither is represented among extant species of lerista. ancestral states reconstructed assuming rates of digit gain of –, by contrast, imply implausible digit configurations for very few internal nodes. moreover, in all instances, the ancestral states implying implausible digit configurations are not significantly more likely than alternative states implying configurations observed among extant species of lerista. thus, we are disinclined to conclude that limb reduction has been reversed in lerista, preferring the ancestral states reconstructed assuming rates of pedal digit gain of –. an examination of ancestral state reconstructions across trees sampled after attaining stationarity in the bayesian analysis indicates that the above conclusions are largely insensitive to uncertainty in the inferred phylogeny. for all nodes, maximum likelihood numbers of manual digits reconstructed for more than 90% of trees are consistent with those inferred assuming the bayesian majority-rule consensus, and reversals of limb reduction are inferred for only an insignificant proportion of trees. similar results are obtained when rates of digit gain considerably higher than those estimated by maximum likelihood are assumed. for example, a rate of, although significantly less likely than the maximum likelihood rates for 90% of trees, yields maximum likelihood ancestral states for of internal nodes consistent with those reconstructed assuming the bayesian majority-rule consensus in more than 92% of trees, with more than 62% of trees exhibiting no reversals of limb reduction. thus, the inferred ancestral states for the manus in figure are not substantially affected by uncertainty in tree topology, branch lengths, and rates of character state transition. ancestral state reconstructions for the pes are similarly stable when uncertainty in the phylogeny is considered. assuming estimated rates of digit gain, maximum likelihood numbers of pedal digits for all nodes are consistent with those for the bayesian majority-rule consensus in more than 90% of trees. as for the bayesian majority-rule consensus, ancestral state reconstructions for nearly all trees imply one or more instances of reversed limb reduction. nonetheless, a range of rates of pedal digit gain only insignificantly lower than those estimated for a considerable proportion of trees yield ancestral state reconstructions very similar to those obtained for the bayesian majority-rule consensus assuming rates of digit gain of –. a rate of, for example, is not significantly less likely than the estimated rates for 64% of trees and, for all except a single node, yields maximum likelihood ancestral states for more than 97% of trees that are consistent with the preferred ancestral states in figure in almost all cases, numbers of pedal digits reconstructed for the single exceptional node entail no increases in the number of digits, and reversed limb reduction is implied in less than 5% of trees. the phylogeny in figure thus implies extraordinary evolutionary mutability of limb morphology in lerista. although this conclusion does not depend on a preference for a specific set of ancestral states, the alternative reconstructions for the pes presented in figure and additional file evidently entail disparate patterns of limb evolution. as discussed above, there is no cogent statistical basis for favouring either of these reconstructions; ancestral states inferred assuming the maximum likelihood rate of digit gain are only insignificantly more likely than those reconstructed assuming rates of digit gain of –. however, when the very robust ancestral states for the manus are considered, a compelling argument can be made for accepting reconstructions for the pes implying no reversals of limb reduction. whereas ancestral states entailing repeated re-elaboration of the pes imply digit configurations for many internal nodes differing fundamentally from those displayed by extant species of lerista, ancestral digit configurations implied by reconstructions entailing no reversals of pedal digit loss are nearly invariably represented among observed phenotypes. although we prefer ancestral state reconstructions for the pes implying repeated, unreversed digit loss, we acknowledge that our results could be considered to indicate the reacquisition of pedal digits, and so provide further evidence for kohlsdorf and wagner's proposal that limb reduction is potentially reversible. perhaps significantly, the inferred age of lerista is comparable to estimates of the period over which unexpressed developmental genes may preserve their functionality, rendering the re-elaboration of structurally reduced limbs at least plausible. nonetheless, it should be noted that such re-elaboration would entail two instances in which a highly reduced pes has regained an entire or nearly entire complement of phalanges. this contrasts with the inferred cases of digit reacquisition in bachia, where species having putatively re-evolved digits exhibit unusual phalangeal complements, possibly reflecting the activation of a novel developmental pathway. allowing that pedal digit loss has not been reversed in lerista, our analyses indicate a remarkably high frequency of limb reduction. ancestral states reconstructed assuming the bayesian majority-rule consensus, rates of digit gain of – for the pes, and maximum likelihood estimates for all other rates imply independent reductions in the number of digits from a pentadactyl condition. a further seven reductions are inferred to have proceeded independently from a tetradactyl condition derived from one of these reductions. four independent losses of all manual and pedal digits are inferred, three from pentadactyl or tetradactyl conditions. assuming an age of million years for lerista, estimated using molecular dating methods, the loss of all digits from a pentadactyl condition in ameles, apoda, and stylis has occurred within a period of million years. the most recent tetradactyl ancestor of ameles and stylis is no more than million years old, while that of apoda is no more than million years old. the highest rate of complete loss of digits from a pentadactyl or tetradactyl condition is inferred for humphriesi and praepedita; the most recent shared ancestor of these species is separated from an inferred pentadactyl ancestor by no more than million years. this is considerably more rapid than the rate implied by brandley et al.'s estimate of the minimum interval over which limb loss has occurred in more than squamate lineages. as brandley et al. note, however, their estimates represent maximum values, and so may significantly overestimate the amount of time necessary for this radical evolutionary transition. accordingly, it is at least conceivable that the higher rates of limb loss inferred here for lerista are representative of those for squamates generally. this perspective suggests a pattern of body form evolution differing markedly from that envisaged by wiens and slingluff for anguids, and, if substantiated, would emphasise the significance of brandley et al.'s finding that intermediate phenotypes may persist for tens of millions of years. almost invariably, species with greatly reduced limbs are separated from inferred pentadactyl or tetradactyl ancestors by very few nodes, so that, assuming limb reduction has not been saltational, reconstruction of complete sequences of digit loss for individual lineages is not possible. nonetheless, if digit loss is presumed to proceed similarly across all lineages, general conclusions concerning the mode of limb reduction may be derived from a consideration of inferred losses for lineages exhibiting varying degrees of reduction. thus, a plot of the numbers of manual and pedal digits lost in all reductions from a pentadactyl or tetradactyl condition versus the total number of digits lost may be considered to indicate that in the initial stages of limb reduction rates of digit loss are similar for the manus and pes, however, as limb reduction progresses, manual digits are lost more readily than pedal digits. this is consistent with a more significant role of the hindlimb in limb-mediated locomotion and selection for some ability to employ limb-mediated locomotion in limb-reduced species. particular intermediate digit configurations, most conspicuously that of no digits for the manus and two digits for the pes, generally originate relatively rapidly and persist for extended periods. this pattern intimates that these configurations do not represent transitory stages in a continuing process of limb reduction and provides further evidence for adaptive retention of digits, consistent with brandley et al.'s proposal that intermediate phenotypes may be targets of selection.  many authors have noted the recurring evolution of an elongate, limb-reduced body form in squamates, however, even within this clade, the frequency and rate of limb reduction inferred here for lerista are exceptional. ancestral state reconstructions imply instances of limb reduction, almost as many as reported by greer for all remaining scincids, and nearly half the number of reductions inferred for all other squamates. an interval of no more than million years for the complete loss of limbs in one lineage of lerista is less than one fourth of brandley et al.'s minimum estimate of the time required for this substantial phenotypic transition. these results emphasise the potential for extensive alteration of body form in squamates over brief periods. an absence of cogent evidence for reversals of digit loss in lerista contrasts with kohlsdorf and wagner's proposal that digits have been regained in some species of bachia, possibly reflecting an influence of differing environmental and genetic contexts on the evolution of limb morphology in these clades. future study of the genetic, developmental, and ecological bases of limb reduction and loss in lerista promises the elucidation of not only this phenomenon in squamates, but also the dramatic evolutionary transformations of body form that have produced the extraordinary diversity of multicellular organisms. 
future study of the genetic, developmental, and ecological bases of limb reduction and loss in lerista promises the elucidation of not only this phenomenon in squamates, but also the dramatic evolutionary transformations of body form that have produced the extraordinary diversity of multicellular organisms. the inferred phylogeny reveals extraordinary evolutionary mutability of limb morphology in lerista. ancestral state reconstructions indicate at least ten independent reductions in the number of digits from a pentadactyl condition, with a further seven reductions proceeding independently from a tetradactyl condition derived from one of these reductions. four independent losses of all digits are inferred, three from pentadactyl or tetradactyl conditions. these conclusions are not substantially affected by uncertainty in assumed rates of character state transition or the phylogeny. the australian scincid clade lerista provides perhaps the best available model for studying limb reduction in squamates, comprising more than species displaying a remarkable variety of digit configurations, from pentadactyl to entirely limbless conditions. at the highest rate, complete loss of digits from a pentadactyl condition is estimated to have occurred within million years. we investigated the pattern and rate of limb reduction and loss in lerista, employing a comprehensive phylogeny inferred from nucleotide sequences for a nuclear intron and six mitochondrial genes. the exceptionally high frequency and rate of limb reduction inferred for lerista emphasise the potential for rapid and substantial alteration of body form in squamates. an absence of compelling evidence for reversals of digit loss contrasts with a recent proposal that digits have been regained in some species of the gymnophthalmid clade bachia, possibly reflecting an influence of differing environmental and genetic contexts on the evolution of limb morphology in these clades. an estimated age of million years for lerista entails that limb reduction has occurred not only repeatedly, but also very rapidly.
 the australian scincid clade lerista provides perhaps the best available model for studying limb reduction in squamates, comprising more than species displaying a remarkable variety of digit configurations, from pentadactyl to entirely limbless conditions. we investigated the pattern and rate of limb reduction and loss in lerista, employing a comprehensive phylogeny inferred from nucleotide sequences for a nuclear intron and six mitochondrial genes.  the inferred phylogeny reveals extraordinary evolutionary mutability of limb morphology in lerista. ancestral state reconstructions indicate at least ten independent reductions in the number of digits from a pentadactyl condition, with a further seven reductions proceeding independently from a tetradactyl condition derived from one of these reductions. four independent losses of all digits are inferred, three from pentadactyl or tetradactyl conditions. these conclusions are not substantially affected by uncertainty in assumed rates of character state transition or the phylogeny. an estimated age of million years for lerista entails that limb reduction has occurred not only repeatedly, but also very rapidly. at the highest rate, complete loss of digits from a pentadactyl condition is estimated to have occurred within million years.  the exceptionally high frequency and rate of limb reduction inferred for lerista emphasise the potential for rapid and substantial alteration of body form in squamates. an absence of compelling evidence for reversals of digit loss contrasts with a recent proposal that digits have been regained in some species of the gymnophthalmid clade bachia, possibly reflecting an influence of differing environmental and genetic contexts on the evolution of limb morphology in these clades. future study of the genetic, developmental, and ecological bases of limb reduction and loss in lerista promises the elucidation of not only this phenomenon in squamates, but also the dramatic evolutionary transformations of body form that have produced the extraordinary diversity of multicellular organisms. 