BACKGROUND
inferring the processes generating large-scale patterns of biodiversity, especially those shaping adaptive radiations, are fascinating areas of past and current biological research .
current debates on diversification focus on speciation modes in the temporal dimension , the spatial dimension , and increasingly on the general mechanisms driving divergence .
life history traits have equally long been discussed as drivers of speciation.
changes in these traits might act as key innovations, promoting ecological opportunity for the emergence of adaptive radiations in the absence of habitat changes .
body size of animal species is such a life history trait that has numerous ecological consequences  <cit> .
for instance, in many taxa body size seems to determine the size of distribution ranges, with smaller taxa having more restricted ranges  <cit> .
this might, at least in some taxa, be simply related to lower dispersal capabilities of smaller species; for instance, a phylogenetically independent positive correlation between home range and body size has been found in ferungulate mammals  <cit> , a result later refined using optimization methods  <cit> .
adaptive radiation likely evolves in stages  <cit> , with body size being one of the first proposed axes of morphological change leading to new ecological opportunity .
body size frequency distributions of animals are generally right-skewed, i.e., most species are generally small  <cit> , as for example in 99% of the world's major lizard groups  <cit>  and often the right-skewed shape is kept after log-transformation.
in lizards, often there is a strong negative correlation between body size within families and species richness: groups containing mainly small species are more species rich  <cit> .
similar unimodal body size distributions have been found in other poikilotherm vertebrates , with right-skewedness of the distributions increasing towards the equator  <cit> .
other researchers found that large radiations of species tend to be small bodied  <cit> , which is in agreement with the hypothesis that the number of ecological niches is potentially greater for small-bodied taxa , which suggests a correlation between small body size and species richness.
despite the intuitive nature of the hypothesis that small organisms should have elevated net rates of diversification, an influence of body size on clade richness has been refuted on several taxonomic levels  <cit> .
in a recent study on toads of the cosmopolitan family bufonidae, van bocxlaer and colleagues  <cit>  found that speciose clades were composed mainly of large species with large range sizes.
they used a combination of various traits to define an optimal expansion phenotype  which they invoked to explain the diversification and success of these amphibians.
in fact, much of the evolutionary success of bufonids is related to the fact that their radiation was intimately connected to colonization of vast new areas, i.e., continents on which these toads were previously absent  <cit> .
this pattern is opposite than would be expected from a classical explanation for adaptive radiation, where small ranges and specialization by occupation of specific ecological niches are thought to promote diversification within a given area  <cit> .
to test correlation among traits  or influences of such traits on diversification rates, phylogenetic comparative methods  are applied, ensuring phylogenetic independence of data  <cit> .
usually, pcm rely on ancestral character state reconstruction along a phylogenetic tree .
a generally more robust alternative is the direct comparison of sister species, with each pair of sister species being a phylogenetically independent data point , but in most data sets there are not sufficient sister species pairs for adequate statistical analysis  <cit> .
frogs of the family mantellidae are a highly diverse clade restricted to madagascar and the comoroan island of mayotte, with 100% species-level endemism and over  <dig> species and candidate species  <cit> .
a large number of mantellid species are small sized, i.e., below  <dig> mm snout-vent length , and many are microendemic, i.e., restricted to a very small geographic area.
mantellids have evolved a variety of ecological adaptations, including arboreal, semiaquatic and fully terrestrial species.
given that they diversified within a single geographic setting, these frogs can serve as an excellent model group to test mechanisms of diversification and correlates of diversity, and their large species diversity allows to base pcm calculations on pairs of sister species additional to tree-based methods.
because most amphibian lineages at the genus or family level are restricted to major biogeographic areas  <cit> , and show a strong pattern of regional diversification similar to the mantellids , we assume that observations on these frogs in madagascar are more representative for overall amphibian diversification patterns than are bufonid toads with their high dispersal capacity.
in this paper, we reconstruct a near-complete species-level phylogeny of the mantellidae from nuclear and mitochondrial markers .
we integrate this phylogeny with  <dig> geo-referenced gis records for  <dig> species-locality records  <cit>  and with bioclimatic information.
based on this phylogeny we identify well-supported pairs of sister species.
these are then used in sister species comparisons, along with whole-tree based pcm, to identify geographical patterns of speciation, to test the influence of body size on range size, and the combined effect of these two traits on diversification and molecular rates of evolution.
we hypothesize that, equivalent to the oep  characterized by large body and range sizes and observed in successful colonizing lineages in the bufonidae, the majority of malagasy mantellid frogs might exhibit a microendemic phenotype  with small range sizes as a result of their small body size.
we also develop the hypothesis that this mep may have influenced rapid diversification in the group, making them one of the most speciose amphibian radiations to date  <cit> .
RESULTS
molecular phylogeny of the mantellidae
as the backbone of our species-level phylogeny, we used a reduced-taxa phylogeny recovered for  <dig> mantellid species of all genera, subgenera and species groups, based on  <dig> base pairs of six mitochondrial and nuclear genes.
the intrageneric relationships in this mantellid phylogeny were well resolved and highly supported in the bayesian analysis, with a similar topology recovered by maximum parsimony  and maximum likelihood  analyses .
all mantellid genera were monophyletic with mp/ml bootstrap support values > <dig>  and bayesian posterior probabilities > <dig>  with the exception of mantidactylus and blommersia, whose ml bootstrap values were 93%.
the bayesian analysis of  <dig> species and confirmed candidate species in the all-taxa data set, including  <dig> bp from three mitochondrial genes and using the preferred partitioning strategy , yielded a well-supported phylogenetic tree, with high posterior probability values on the levels of species groups .
most of the subgenera in the mantellidae  <cit> , especially of mantidactylus and gephyromantis, were found to be monophyletic.
basal relationships among genera in this analysis were constrained on the basis of the previous reduced-taxa topology.
the 50% majority-rule consensus tree obtained from the all-taxa bayesian analysis was converted into an ultrametric timetree using the software pathd <dig> , based on a combination of primary and secondary calibrations .
alternative runs including either only primary or only secondary calibrations recovered similar node ages .
we thus used the combined analysis with all calibrations to obtain evolutionary ages for mantellid sister species, and used these age data in further analyses.
among the  <dig> mantellid species in the phylogeny, we identified  <dig> sister species pairs  with high bayesian posterior probabilities  and within subclades in which we consider taxonomy and distribution ranges to be sufficiently understood.
the majority of these  showed no range overlap .
<dig> species pairs  showed partial or full overlap of their distribution ranges .
in  <dig> of these sympatric pairs we ascertained syntopic occurrence  in the field, and in two further pairs we ascertained occurrence in the same area, yet at different altitudes.
correlation between evolutionary age and range  of sister species pairs was not significant .
range overlap among sister species pairs was log-normally distributed, with a mean percentage of range overlap of  <dig> %.
this value is significantly higher than the mean overlap among all possible combinations of non-sister species in mantellids , as indicated by a sign test .
mean range overlap of the sympatric sister species is generally high , with a significantly higher median than all possible combinations of mantellid species when sister species were excluded.
the initial diversification of mantellids was estimated at  <dig> mya from our time-calibrated phylogeny.
the  <dig> pairs of mantellid sister species were remarkably old, with evolutionary ages ranging from  <dig>  mya to  <dig>  mya with an arithmetic mean of  <dig>  mya, and with node ages following a normal distribution .
spatial characteristics of diversification
most mantellids had small body sizes and small to very small range sizes , and this was true also for the subset of species included in the tip contrast comparisons .
we found a positive correlation between evolutionary age and centroid distance of ranges of pairs of sister species , which indicates lineage diversification in close spatial proximity.
patterns related to range size were separately analyzed for two range size estimates  that differed in the extent of buffer zones assigned to single-locality species .
for the  <dig> pairs of sister species included in the analysis we found a trend of increasing absolute range size with evolutionary age .
all species with most recent splits from their sister species  had small range sizes and range size differences , with larger range sizes and range size differences between sister species being present only in older species .
under peripatric speciation, a triangular or no correlation among evolutionary age and range size  is expected; we therefore performed quantile regression.
quantile regression showed that the 90% quantile slope for absolute range size was positive , a pattern that is not expected under peripatric speciation where we expected the slope to be negative or zero .
regression of the 90% quantile found the slopes for absolute differences in rsa  and rsb  to be positive.
congruent with these results, the absolute differences in rsa and rsb also increased with centroid distance .
body size as a predictor for range size in the mantellidae
range and body sizes for all mantellid species were highly positively correlated .
to ensure phylogenetic independence of these results, standardized independent contrasts for body size and range size estimates were subsequently computed for pairs of sister species  <cit> .
factorial regression through the origin found contrasts in svl per mantellid sister species pair to significantly predict tip contrasts of rsa and rsb .
sister species with small contrasts of svl had small range size contrasts .
we used the software coevol to assess the coupled evolution of svl and rsa/rsb in a probabilistic framework  <cit> .
partial regression revealed a significantly positive effect of svl on both estimates of range size, although the correlation was not particularly strong .
multiple regressions performed with each range size estimate and substitution rate did not alter the positive direction of the correlation, however, the posterior probabilities remained only significant for the smaller range size estimate , but not for rsa .
univariate regression through the origin results to determine the effect of standardized tip contrasts in svl on range size estimators using large  and small  buffer zones for one- or two locality species for mantellid sister species pairs.
significant p-values in bold.
matrix of covariances, correlation coefficients, and posterior probabilities for maximal male svl, range size estimates and synonymous substitution rate ds obtained with coevol.
negative covariances indicate negative correlations, posterior probabilities close to zero indicate significant negative correlation, close to  <dig> indicate significant positive correlations.
<dig> generations were sampled and burnin was set =  <dig> after visual inspection of the trace file as showing stability in estimated parameters.
the role of body size and range size influencing mantellid diversification
to understand whether body size and range size affect the rate and mode of diversification in the mantellidae, we first tested whether geographical proximity of ranges and similarities of bioclimatic envelopes of sister species are related to their range and body sizes.
a factorial regression analysis revealed standardized contrasts in range size  and body size to be functions of spatial characteristics: microendemic and miniaturized sister species have more proximate ranges and climatically more similar niches than widely distributed or larger species .
univariate results for the single predictors and their interaction terms can be found in additional file  <dig>  table s <dig>
results of factorial regressions through the origin to determine the effect of evolutionary age, of geographic distance and bioclimatic distance, and their interaction terms on body size and range size contrasts using large  and small  buffer zones for one- or two locality species in mantellid sister species pairs.
we used the software macrocaic as a whole-tree based pcm to test whether mantellid lineages with smaller svl are more speciation-prone than lineages showing larger svl  <cit> ).
both lineage richness and svl contrasts were normally distributed , so we could perform standard regression through the origin  <cit> .
the null hypothesis of small svl not influencing clade diversification rate could not be rejected with this test .
however, the negative slope indicates that clades exhibiting smaller svl have a trend of being more species-rich .
effects of rsa and rsb on clade diversification were, however not detectable at all with this test .
a strong influence of small body and range sizes not on the number of lineages but on the mitochondrial substitution rate itself was detected by phylogenetic regression in coevol  <cit>  .
the approximated synonymous substitution rate of the all-taxa dataset was, given the ultrametric phylogeny, significantly negatively correlated with svl; small body size turned out to be associated with high rates of molecular substitution.
this result remained unchanged if multiple regressions including rsa and rsb were performed .
in contrast, the two range size estimates were positively correlated with the substitution rate , which means that in extant species that are both small and have small range sizes , substitution rate would experience a relative slow-down compared to lineages that have small body sizes but large range sizes.
these results were supported in a multiple regression by high posterior probabilities for rsb, but not for the larger minimal range size estimate rsa .
discussion
characteristics of the mantellid radiation
most mantellid sister species lineages diversified between  <dig> and  <dig> mya.
these generally old ages  indicate that pleistocene speciation mechanisms as they have been proposed for lemurs  <cit>  cannot be invoked to explain the bulk of mantellid speciation, and instead unveil an older speciation timing similar to that detected in a diverse range of neotropical taxa  <cit>  and in other malagasy animals  <cit> .
about 64% of mantellid sister species did not show range overlap, a percentage that is consistent with an average value of 72% allopatric species estimated for other animal clades  <cit> .
mantellid frogs show a right-skewed body size species richness pattern, contrary to what has been observed in malagasy mammals  <cit> .
speciation in close spatial proximity and rejection of peripatric speciation
although methods that attempt to quantify the frequency of sympatric versus allopatric speciation exist, e.g., age-range overlap correlation  <cit> , these have been widely criticized when applied to fast-dispersing groups of taxa due to the high probability of post-speciation range shifts and because their results usually do not differ from null models  <cit> .
most amphibians, and many mantellids in particular, are probably no fast dispersers, so conclusions drawn from age-range overlap correlation in this group might thus be less compromised than in other groups.
we found sympatric and allopatric sister species to be present in all major mantellid lineages and to span similar evolutionary age ranges.
youngest pairs of sister species were both distributed in sympatry and in allopatry, which resulted in the absence of a correlation between evolutionary age and range overlap.
such absence of age-range overlap correlation can either be caused by post-speciation range shifts, or by a mix of different geographical modes of speciation  <cit> .
in fact, our results bear visual similarities to the modeled data under multiple speciation modes  <cit> .
alternatively, pleistocene climate oscillations that postdate the time of maximal lineage diversification in mantellid frogs can be thought of having triggered post-speciation range shifts  <cit> .
besides the many microendemic mantellids, also widespread species do exist in this family  <cit> , so we cannot conclusively distinguish between the hypotheses of  a mix of speciation modes, or  absence of a clear pattern due to range shifts.
however, a clear conclusion derived from our data is that lineage diversification in mantellid frogs typically happens in close spatial proximity, since youngest mantellid sister species pairs were also the most geographically proximate and spatial distance gradually increased with evolutionary age.
under sympatric speciation, the geographical proximity is obvious, and under allopatric speciation, the initial geographic separation of populations of the ancestral species is likely to be small in non-vagile animals - except for cases of overseas dispersal  <cit>  or speciation of relict populations in isolated habitats.
in fact, in several species pairs with almost fully overlapping ranges, the currently available evidence would favor sympatric or parapatric species formation, and we flag them for future detailed study.
some of these are old species, such as gephyromantis azzurrae and g. corvus , both of which are endemic to a very small range in the isalo sandstone massif in south-western madagascar, or spinomantis bertini and s. sp.
<dig>  which occupy different altitudinal bands on the andohahela massif in the south-east.
boophis majori and b. sp.
<dig>  occur in syntopy in ranomafana national park in the southern central east, and differ strongly by advertisement calls and tadpole morphology  <cit> .
and one of the youngest mantellid sister species pairs, gephyromantis eiselti and g. thelenae , two species with endotrophic tadpole development, even form mixed choruses at some sites near andasibe in the northern central east, with pronounced differences in advertisement calls  <cit> .
despite these examples, the high proportion of allopatric pairs of sister species in the mantellidae as apparent from our analysis suggests that allopatric modes of species formation probably have played a predominant role in the diversification of these frogs.
range sizes of species included in the sister species comparisons increased with evolutionary age, as did range size differences between sister species.
this is not trivial as it firmly rejects peripatric speciation  <cit>  as predominant speciation mode for mantellid frogs .
speciation in mantellids mostly resulted in microendemic sister species , a pattern not expected under predominance of species formation based on peripheral, allopatric isolation of small subpopulations as in peripatric speciation.
a probable explanation for some mantellid frogs being widespread is that their ecological tolerance is wider, which would make the interruption of gene flow to peripheral populations by ecogeographic barriers less likely.
in contrast, narrow-range species are likely habitat specialists, which increases the probability of allopatric populations to become isolated, e.g.
by habitat changes through climatic shifts.
small adaptive changes in such a population can already confer a significant shift in habitat preference, thus leading to genetic isolation in parapatry or sympatry.
a role for a microendemic phenotype in lineage diversification?
as a first hypothesis we tested whether our data support the existence of a microendemic phenotype , i.e., whether frogs of small body size also have particularly small range sizes.
if this pattern is phylogenetically independent, we can conclude evolutionary convergence of body size and small range sizes forming a distinct mep.
using robust tip-based pcm based on a high number of mantellid sister species pairs we found svl to be a significant predictor of range size.
the correlation between range size and svl was positive, supporting that the combination of small svl and small range size defines a mep.
whole-tree based methods support this pattern: coevol found a phylogenetically independent, significant positive correlation between svl and rsb, reflecting the fact that most mantellids are both small and have small range sizes, and hinting at replicated evolution of this phenotype within the mantellid radiation.
as a second hypothesis, we posit that lineages exhibiting mep will be less dispersal-prone and therefore will be less able to maintain gene flow among populations , leading to increased rates of speciation.
if the mep drives diversification in the mantellidae, clades exhibiting mep are thus expected to be more species rich and to inhabit ranges in close spatial proximity.
using pcm based on mantellid sister species pairs we found small body and range size contrasts to be prevalent in proximate and young sister species, thus supporting the hypothesis.
the application of tree-based pcm revealed more ambiguous results to this question.
macrocaic found the expected negative regression slope between diversification rate and svl, but the regression through the origin could not reject the null hypothesis of no effect of svl on diversification rate .
the effect size of the correlation, however, almost reached significance , but no effects of range size on clade diversification could be detected.
while these analyses using clade diversity remained ambiguous, we found effects of both svl and range size  on the synonymous mitochondrial substitution rate.
while small svl predicted a high rate of substitution, range size in contrast was positively associated with the substitution rate: larger-range lineages show higher rates of substitution.
mitochondrial substitution rates have been found to be positively correlated with speciation rates and contemporary species richness before  <cit> .
if we assume a similar relationship in the mantellidae, the positive correlation between range sizes and substitution rate would contradict the hypothesis that mep  promotes diversification.
small-bodied species that also have small range sizes  would experience a net reduction in substitution rate, and presumably in diversification rate, as compared to lineages with small svl but less microendemic distributions.
a slow-down of speciation rate is, however, implied in the definition of adaptive radiations - after environmental niches are occupied in the later stages of adaptive radiation, lineages cannot continue to diversify as rapidly  <cit> .
a similar slow down of diversification rate with decreasing range size has been predicted  <cit>  under the scenario that small range sizes are indicative of strong ecological competition from close relatives in adaptive radiations.
however, a stronger rate slow-down in a bird dataset has been found be related to larger ranges  <cit> , contrary to the expectation.
in general, the association of rates of clade diversification and molecular substitution clearly require further study.
another factor possibly related to the pattern observed is population density: in small frogs, a viable population could be established in smaller ranges than in large frogs  <cit> , potentially driving the correlated evolution of microendemism.
however, the lack of data on population densities for mantellid frogs  does not permit testing this hypothesis at present.
from our results, we conclude that our data support an association of small range and body size and thus the existence of a mep.
we furthermore find indications that not the mep, but instead small body sizes might be crucial in promoting lineage diversification.
extant mantellids show a high frequency of mep lineages, but instead of having a combined high diversification rate, these lineages might experience a net slow down of diversification and substitution rates, as expected in the late stages of adaptive radiation.
we can back up the results derived from tree-based pcm  with tip-based pcm using a high number of sister species pairs.
although we implicitly assume that the sister lineages are characterized by the extant state since they diverged at the nodes in tip-based pcm, these can complement tree-based pcm in near-complete phylogenies.
CONCLUSIONS
the mep hypothesis complements the optimal expansion phenotype  hypothesis  <cit> , residing at the other end of a phenotypic continuum.
the phenotypes at both extremes of this continuum promote fast successful speciation, but are based on different mechanisms.
the oep promotes speciation by providing the ability to colonize vast ranges that create ecological opportunity  <cit>  while the mep promotes rapid speciation on a smaller spatial scale because of its association with low dispersal capacity, and restriction of gene flow by making geographic barriers relatively larger  <cit> .
the oep contradicts the expectations from a classical adaptive radiation  <cit> , where small ranges and specialization by occupation of specific ecological niches are thought to promote diversification within a given area .
our results provide evidence for the existence of an mep in the mantellidae, but contrary to the expectation of the faster diversification in microendemic phenotype species, the majority of extant mantellids  that show small body sizes combined with small range sizes can be thought of experiencing a relative slowed-down rates of speciation compared to small but not microendemic species.
a joint analysis of various species-rich regional anuran radiations might provide enough species with all combinations of range and body sizes for a more conclusive test of the influence of body and range size on the diversification rate in amphibians.

 inferring the processes generating large-scale patterns of biodiversity, especially those shaping adaptive radiations, are fascinating areas of past and current biological research. current debates on diversification focus on speciation modes in the temporal dimension, the spatial dimension, and increasingly on the general mechanisms driving divergence. life history traits have equally long been discussed as drivers of speciation. changes in these traits might act as key innovations, promoting ecological opportunity for the emergence of adaptive radiations in the absence of habitat changes. body size of animal species is such a life history trait that has numerous ecological consequences. for instance, in many taxa body size seems to determine the size of distribution ranges, with smaller taxa having more restricted ranges. this might, at least in some taxa, be simply related to lower dispersal capabilities of smaller species; for instance, a phylogenetically independent positive correlation between home range and body size has been found in ferungulate mammals, a result later refined using optimization methods. adaptive radiation likely evolves in stages, with body size being one of the first proposed axes of morphological change leading to new ecological opportunity. body size frequency distributions of animals are generally right-skewed, i.e., most species are generally small, as for example in 99% of the world's major lizard groups and often the right-skewed shape is kept after log-transformation. in lizards, often there is a strong negative correlation between body size within families and species richness: groups containing mainly small species are more species rich. similar unimodal body size distributions have been found in other poikilotherm vertebrates, with right-skewedness of the distributions increasing towards the equator. other researchers found that large radiations of species tend to be small bodied, which is in agreement with the hypothesis that the number of ecological niches is potentially greater for small-bodied taxa, which suggests a correlation between small body size and species richness. despite the intuitive nature of the hypothesis that small organisms should have elevated net rates of diversification, an influence of body size on clade richness has been refuted on several taxonomic levels. in a recent study on toads of the cosmopolitan family bufonidae, van bocxlaer and colleagues found that speciose clades were composed mainly of large species with large range sizes. they used a combination of various traits to define an optimal expansion phenotype which they invoked to explain the diversification and success of these amphibians. in fact, much of the evolutionary success of bufonids is related to the fact that their radiation was intimately connected to colonization of vast new areas, i.e., continents on which these toads were previously absent. this pattern is opposite than would be expected from a classical explanation for adaptive radiation, where small ranges and specialization by occupation of specific ecological niches are thought to promote diversification within a given area. to test correlation among traits or influences of such traits on diversification rates, phylogenetic comparative methods are applied, ensuring phylogenetic independence of data. usually, pcm rely on ancestral character state reconstruction along a phylogenetic tree. a generally more robust alternative is the direct comparison of sister species, with each pair of sister species being a phylogenetically independent data point, but in most data sets there are not sufficient sister species pairs for adequate statistical analysis. frogs of the family mantellidae are a highly diverse clade restricted to madagascar and the comoroan island of mayotte, with 100% species-level endemism and over species and candidate species. a large number of mantellid species are small sized, i.e., below mm snout-vent length, and many are microendemic, i.e., restricted to a very small geographic area. mantellids have evolved a variety of ecological adaptations, including arboreal, semiaquatic and fully terrestrial species. given that they diversified within a single geographic setting, these frogs can serve as an excellent model group to test mechanisms of diversification and correlates of diversity, and their large species diversity allows to base pcm calculations on pairs of sister species additional to tree-based methods. because most amphibian lineages at the genus or family level are restricted to major biogeographic areas, and show a strong pattern of regional diversification similar to the mantellids, we assume that observations on these frogs in madagascar are more representative for overall amphibian diversification patterns than are bufonid toads with their high dispersal capacity. in this paper, we reconstruct a near-complete species-level phylogeny of the mantellidae from nuclear and mitochondrial markers. we integrate this phylogeny with geo-referenced gis records for species-locality records and with bioclimatic information. based on this phylogeny we identify well-supported pairs of sister species. these are then used in sister species comparisons, along with whole-tree based pcm, to identify geographical patterns of speciation, to test the influence of body size on range size, and the combined effect of these two traits on diversification and molecular rates of evolution. we hypothesize that, equivalent to the oep characterized by large body and range sizes and observed in successful colonizing lineages in the bufonidae, the majority of malagasy mantellid frogs might exhibit a microendemic phenotype with small range sizes as a result of their small body size. we also develop the hypothesis that this mep may have influenced rapid diversification in the group, making them one of the most speciose amphibian radiations to date.  molecular phylogeny of the mantellidae as the backbone of our species-level phylogeny, we used a reduced-taxa phylogeny recovered for mantellid species of all genera, subgenera and species groups, based on base pairs of six mitochondrial and nuclear genes. the intrageneric relationships in this mantellid phylogeny were well resolved and highly supported in the bayesian analysis, with a similar topology recovered by maximum parsimony and maximum likelihood analyses. all mantellid genera were monophyletic with mp/ml bootstrap support values > and bayesian posterior probabilities > with the exception of mantidactylus and blommersia, whose ml bootstrap values were 93%. the bayesian analysis of species and confirmed candidate species in the all-taxa data set, including bp from three mitochondrial genes and using the preferred partitioning strategy, yielded a well-supported phylogenetic tree, with high posterior probability values on the levels of species groups. most of the subgenera in the mantellidae, especially of mantidactylus and gephyromantis, were found to be monophyletic. basal relationships among genera in this analysis were constrained on the basis of the previous reduced-taxa topology. the 50% majority-rule consensus tree obtained from the all-taxa bayesian analysis was converted into an ultrametric timetree using the software pathd, based on a combination of primary and secondary calibrations. alternative runs including either only primary or only secondary calibrations recovered similar node ages. we thus used the combined analysis with all calibrations to obtain evolutionary ages for mantellid sister species, and used these age data in further analyses. among the mantellid species in the phylogeny, we identified sister species pairs with high bayesian posterior probabilities and within subclades in which we consider taxonomy and distribution ranges to be sufficiently understood. the majority of these showed no range overlap. species pairs showed partial or full overlap of their distribution ranges. in of these sympatric pairs we ascertained syntopic occurrence in the field, and in two further pairs we ascertained occurrence in the same area, yet at different altitudes. correlation between evolutionary age and range of sister species pairs was not significant. range overlap among sister species pairs was log-normally distributed, with a mean percentage of range overlap of %. this value is significantly higher than the mean overlap among all possible combinations of non-sister species in mantellids, as indicated by a sign test. mean range overlap of the sympatric sister species is generally high, with a significantly higher median than all possible combinations of mantellid species when sister species were excluded. the initial diversification of mantellids was estimated at mya from our time-calibrated phylogeny. the pairs of mantellid sister species were remarkably old, with evolutionary ages ranging from mya to mya with an arithmetic mean of mya, and with node ages following a normal distribution. spatial characteristics of diversification most mantellids had small body sizes and small to very small range sizes, and this was true also for the subset of species included in the tip contrast comparisons. we found a positive correlation between evolutionary age and centroid distance of ranges of pairs of sister species, which indicates lineage diversification in close spatial proximity. patterns related to range size were separately analyzed for two range size estimates that differed in the extent of buffer zones assigned to single-locality species. for the pairs of sister species included in the analysis we found a trend of increasing absolute range size with evolutionary age. all species with most recent splits from their sister species had small range sizes and range size differences, with larger range sizes and range size differences between sister species being present only in older species. under peripatric speciation, a triangular or no correlation among evolutionary age and range size is expected; we therefore performed quantile regression. quantile regression showed that the 90% quantile slope for absolute range size was positive, a pattern that is not expected under peripatric speciation where we expected the slope to be negative or zero. regression of the 90% quantile found the slopes for absolute differences in rsa and rsb to be positive. congruent with these results, the absolute differences in rsa and rsb also increased with centroid distance. body size as a predictor for range size in the mantellidae range and body sizes for all mantellid species were highly positively correlated. to ensure phylogenetic independence of these results, standardized independent contrasts for body size and range size estimates were subsequently computed for pairs of sister species. factorial regression through the origin found contrasts in svl per mantellid sister species pair to significantly predict tip contrasts of rsa and rsb. sister species with small contrasts of svl had small range size contrasts. we used the software coevol to assess the coupled evolution of svl and rsa/rsb in a probabilistic framework. partial regression revealed a significantly positive effect of svl on both estimates of range size, although the correlation was not particularly strong. multiple regressions performed with each range size estimate and substitution rate did not alter the positive direction of the correlation, however, the posterior probabilities remained only significant for the smaller range size estimate, but not for rsa. univariate regression through the origin results to determine the effect of standardized tip contrasts in svl on range size estimators using large and small buffer zones for one- or two locality species for mantellid sister species pairs. significant p-values in bold. matrix of covariances, correlation coefficients, and posterior probabilities for maximal male svl, range size estimates and synonymous substitution rate ds obtained with coevol. negative covariances indicate negative correlations, posterior probabilities close to zero indicate significant negative correlation, close to indicate significant positive correlations. generations were sampled and burnin was set = after visual inspection of the trace file as showing stability in estimated parameters. the role of body size and range size influencing mantellid diversification to understand whether body size and range size affect the rate and mode of diversification in the mantellidae, we first tested whether geographical proximity of ranges and similarities of bioclimatic envelopes of sister species are related to their range and body sizes. a factorial regression analysis revealed standardized contrasts in range size and body size to be functions of spatial characteristics: microendemic and miniaturized sister species have more proximate ranges and climatically more similar niches than widely distributed or larger species. univariate results for the single predictors and their interaction terms can be found in additional file table s results of factorial regressions through the origin to determine the effect of evolutionary age, of geographic distance and bioclimatic distance, and their interaction terms on body size and range size contrasts using large and small buffer zones for one- or two locality species in mantellid sister species pairs. we used the software macrocaic as a whole-tree based pcm to test whether mantellid lineages with smaller svl are more speciation-prone than lineages showing larger svl ). both lineage richness and svl contrasts were normally distributed, so we could perform standard regression through the origin. the null hypothesis of small svl not influencing clade diversification rate could not be rejected with this test. however, the negative slope indicates that clades exhibiting smaller svl have a trend of being more species-rich. effects of rsa and rsb on clade diversification were, however not detectable at all with this test. a strong influence of small body and range sizes not on the number of lineages but on the mitochondrial substitution rate itself was detected by phylogenetic regression in coevol. the approximated synonymous substitution rate of the all-taxa dataset was, given the ultrametric phylogeny, significantly negatively correlated with svl; small body size turned out to be associated with high rates of molecular substitution. this result remained unchanged if multiple regressions including rsa and rsb were performed. in contrast, the two range size estimates were positively correlated with the substitution rate, which means that in extant species that are both small and have small range sizes, substitution rate would experience a relative slow-down compared to lineages that have small body sizes but large range sizes. these results were supported in a multiple regression by high posterior probabilities for rsb, but not for the larger minimal range size estimate rsa. discussion characteristics of the mantellid radiation most mantellid sister species lineages diversified between and mya. these generally old ages indicate that pleistocene speciation mechanisms as they have been proposed for lemurs cannot be invoked to explain the bulk of mantellid speciation, and instead unveil an older speciation timing similar to that detected in a diverse range of neotropical taxa and in other malagasy animals. about 64% of mantellid sister species did not show range overlap, a percentage that is consistent with an average value of 72% allopatric species estimated for other animal clades. mantellid frogs show a right-skewed body size species richness pattern, contrary to what has been observed in malagasy mammals. speciation in close spatial proximity and rejection of peripatric speciation although methods that attempt to quantify the frequency of sympatric versus allopatric speciation exist, e.g., age-range overlap correlation, these have been widely criticized when applied to fast-dispersing groups of taxa due to the high probability of post-speciation range shifts and because their results usually do not differ from null models. most amphibians, and many mantellids in particular, are probably no fast dispersers, so conclusions drawn from age-range overlap correlation in this group might thus be less compromised than in other groups. we found sympatric and allopatric sister species to be present in all major mantellid lineages and to span similar evolutionary age ranges. youngest pairs of sister species were both distributed in sympatry and in allopatry, which resulted in the absence of a correlation between evolutionary age and range overlap. such absence of age-range overlap correlation can either be caused by post-speciation range shifts, or by a mix of different geographical modes of speciation. in fact, our results bear visual similarities to the modeled data under multiple speciation modes. alternatively, pleistocene climate oscillations that postdate the time of maximal lineage diversification in mantellid frogs can be thought of having triggered post-speciation range shifts. besides the many microendemic mantellids, also widespread species do exist in this family, so we cannot conclusively distinguish between the hypotheses of a mix of speciation modes, or absence of a clear pattern due to range shifts. however, a clear conclusion derived from our data is that lineage diversification in mantellid frogs typically happens in close spatial proximity, since youngest mantellid sister species pairs were also the most geographically proximate and spatial distance gradually increased with evolutionary age. under sympatric speciation, the geographical proximity is obvious, and under allopatric speciation, the initial geographic separation of populations of the ancestral species is likely to be small in non-vagile animals - except for cases of overseas dispersal or speciation of relict populations in isolated habitats. in fact, in several species pairs with almost fully overlapping ranges, the currently available evidence would favor sympatric or parapatric species formation, and we flag them for future detailed study. some of these are old species, such as gephyromantis azzurrae and g. corvus, both of which are endemic to a very small range in the isalo sandstone massif in south-western madagascar, or spinomantis bertini and s. sp. which occupy different altitudinal bands on the andohahela massif in the south-east. boophis majori and b. sp. occur in syntopy in ranomafana national park in the southern central east, and differ strongly by advertisement calls and tadpole morphology. and one of the youngest mantellid sister species pairs, gephyromantis eiselti and g. thelenae, two species with endotrophic tadpole development, even form mixed choruses at some sites near andasibe in the northern central east, with pronounced differences in advertisement calls. despite these examples, the high proportion of allopatric pairs of sister species in the mantellidae as apparent from our analysis suggests that allopatric modes of species formation probably have played a predominant role in the diversification of these frogs. range sizes of species included in the sister species comparisons increased with evolutionary age, as did range size differences between sister species. this is not trivial as it firmly rejects peripatric speciation as predominant speciation mode for mantellid frogs. speciation in mantellids mostly resulted in microendemic sister species, a pattern not expected under predominance of species formation based on peripheral, allopatric isolation of small subpopulations as in peripatric speciation. a probable explanation for some mantellid frogs being widespread is that their ecological tolerance is wider, which would make the interruption of gene flow to peripheral populations by ecogeographic barriers less likely. in contrast, narrow-range species are likely habitat specialists, which increases the probability of allopatric populations to become isolated, e.g. by habitat changes through climatic shifts. small adaptive changes in such a population can already confer a significant shift in habitat preference, thus leading to genetic isolation in parapatry or sympatry. a role for a microendemic phenotype in lineage diversification? as a first hypothesis we tested whether our data support the existence of a microendemic phenotype, i.e., whether frogs of small body size also have particularly small range sizes. if this pattern is phylogenetically independent, we can conclude evolutionary convergence of body size and small range sizes forming a distinct mep. using robust tip-based pcm based on a high number of mantellid sister species pairs we found svl to be a significant predictor of range size. the correlation between range size and svl was positive, supporting that the combination of small svl and small range size defines a mep. whole-tree based methods support this pattern: coevol found a phylogenetically independent, significant positive correlation between svl and rsb, reflecting the fact that most mantellids are both small and have small range sizes, and hinting at replicated evolution of this phenotype within the mantellid radiation. as a second hypothesis, we posit that lineages exhibiting mep will be less dispersal-prone and therefore will be less able to maintain gene flow among populations, leading to increased rates of speciation. if the mep drives diversification in the mantellidae, clades exhibiting mep are thus expected to be more species rich and to inhabit ranges in close spatial proximity. using pcm based on mantellid sister species pairs we found small body and range size contrasts to be prevalent in proximate and young sister species, thus supporting the hypothesis. the application of tree-based pcm revealed more ambiguous results to this question. macrocaic found the expected negative regression slope between diversification rate and svl, but the regression through the origin could not reject the null hypothesis of no effect of svl on diversification rate. the effect size of the correlation, however, almost reached significance, but no effects of range size on clade diversification could be detected. while these analyses using clade diversity remained ambiguous, we found effects of both svl and range size on the synonymous mitochondrial substitution rate. while small svl predicted a high rate of substitution, range size in contrast was positively associated with the substitution rate: larger-range lineages show higher rates of substitution. mitochondrial substitution rates have been found to be positively correlated with speciation rates and contemporary species richness before. if we assume a similar relationship in the mantellidae, the positive correlation between range sizes and substitution rate would contradict the hypothesis that mep promotes diversification. small-bodied species that also have small range sizes would experience a net reduction in substitution rate, and presumably in diversification rate, as compared to lineages with small svl but less microendemic distributions. a slow-down of speciation rate is, however, implied in the definition of adaptive radiations - after environmental niches are occupied in the later stages of adaptive radiation, lineages cannot continue to diversify as rapidly. a similar slow down of diversification rate with decreasing range size has been predicted under the scenario that small range sizes are indicative of strong ecological competition from close relatives in adaptive radiations. however, a stronger rate slow-down in a bird dataset has been found be related to larger ranges, contrary to the expectation. in general, the association of rates of clade diversification and molecular substitution clearly require further study. another factor possibly related to the pattern observed is population density: in small frogs, a viable population could be established in smaller ranges than in large frogs, potentially driving the correlated evolution of microendemism. however, the lack of data on population densities for mantellid frogs does not permit testing this hypothesis at present. from our results, we conclude that our data support an association of small range and body size and thus the existence of a mep. we furthermore find indications that not the mep, but instead small body sizes might be crucial in promoting lineage diversification. extant mantellids show a high frequency of mep lineages, but instead of having a combined high diversification rate, these lineages might experience a net slow down of diversification and substitution rates, as expected in the late stages of adaptive radiation. we can back up the results derived from tree-based pcm with tip-based pcm using a high number of sister species pairs. although we implicitly assume that the sister lineages are characterized by the extant state since they diverged at the nodes in tip-based pcm, these can complement tree-based pcm in near-complete phylogenies.  the mep hypothesis complements the optimal expansion phenotype hypothesis, residing at the other end of a phenotypic continuum. the phenotypes at both extremes of this continuum promote fast successful speciation, but are based on different mechanisms. the oep promotes speciation by providing the ability to colonize vast ranges that create ecological opportunity while the mep promotes rapid speciation on a smaller spatial scale because of its association with low dispersal capacity, and restriction of gene flow by making geographic barriers relatively larger. the oep contradicts the expectations from a classical adaptive radiation, where small ranges and specialization by occupation of specific ecological niches are thought to promote diversification within a given area. our results provide evidence for the existence of an mep in the mantellidae, but contrary to the expectation of the faster diversification in microendemic phenotype species, the majority of extant mantellids that show small body sizes combined with small range sizes can be thought of experiencing a relative slowed-down rates of speciation compared to small but not microendemic species. a joint analysis of various species-rich regional anuran radiations might provide enough species with all combinations of range and body sizes for a more conclusive test of the influence of body and range size on the diversification rate in amphibians. 
based on these results we define a testable hypothesis under which small body sizes result in limited dispersal capabilities and low physiological tolerances, causing smaller and more strongly fragmented ranges. range sizes of species included in the sister species comparisons increased with evolutionary age, as did range size differences between sister species, which rejects peripatric speciation. a joint analysis of various species-rich regional anuran radiations might provide enough species with all combinations of range and body sizes for a more conclusive test of this hypothesis. based on this phylogeny we identified well-supported pairs of sister species that we used for phylogenetic comparative analyses, along with whole tree-based phylogenetic comparative methods. sister species within the mantellidae diverged at - million years ago and more recently diverged sister species had geographical range centroids more proximate to each other, independently of their current sympatric or allopatric occurrence. the largest number of sister species pairs had non-overlapping ranges, but several examples of young microendemic sister species occurring in full sympatry suggest the possibility of non-allopatric speciation. for the majority of mantellid sister species and the whole mantellid radiation, range and body sizes were associated with each other and small body sizes were linked to higher mitochondrial nucleotide substitution rates and higher clade diversity. contrary to the expectation of the faster speciation of such microendemic phenotype species, we only found small body sizes of mantellid frogs to be linked to higher diversification and substitution rates, but not small range sizes. in contrast, small range sizes were unexpectedly associated with a slow-down of mitochondrial substitution rates. we integrate a newly reconstructed near-complete species-level timetree of the mantellidae with georeferenced distribution records and maximum male body size data to infer the influence of these life-history traits on each other and on mantellid diversification. we reconstructed a molecular phylogeny based on nuclear and mitochondrial dna for species and candidate species of the mantellid frog radiation. in madagascar, many groups of organisms are characterized by tiny distribution ranges and small body sizes, and this high degree of microendemism and miniaturization parallels a high species diversity in some of these groups. the rate and mode of lineage diversification might be shaped by clade-specific traits.
 the rate and mode of lineage diversification might be shaped by clade-specific traits. in madagascar, many groups of organisms are characterized by tiny distribution ranges and small body sizes, and this high degree of microendemism and miniaturization parallels a high species diversity in some of these groups. we here investigate the geographic patterns characterizing the radiation of the frog family mantellidae that is virtually endemic to madagascar. we integrate a newly reconstructed near-complete species-level timetree of the mantellidae with georeferenced distribution records and maximum male body size data to infer the influence of these life-history traits on each other and on mantellid diversification.  we reconstructed a molecular phylogeny based on nuclear and mitochondrial dna for species and candidate species of the mantellid frog radiation. based on this phylogeny we identified well-supported pairs of sister species that we used for phylogenetic comparative analyses, along with whole tree-based phylogenetic comparative methods. sister species within the mantellidae diverged at - million years ago and more recently diverged sister species had geographical range centroids more proximate to each other, independently of their current sympatric or allopatric occurrence. the largest number of sister species pairs had non-overlapping ranges, but several examples of young microendemic sister species occurring in full sympatry suggest the possibility of non-allopatric speciation. range sizes of species included in the sister species comparisons increased with evolutionary age, as did range size differences between sister species, which rejects peripatric speciation. for the majority of mantellid sister species and the whole mantellid radiation, range and body sizes were associated with each other and small body sizes were linked to higher mitochondrial nucleotide substitution rates and higher clade diversity. in contrast, small range sizes were unexpectedly associated with a slow-down of mitochondrial substitution rates.  based on these results we define a testable hypothesis under which small body sizes result in limited dispersal capabilities and low physiological tolerances, causing smaller and more strongly fragmented ranges. this can be thought to facilitate reproductive isolation and thus favor speciation. contrary to the expectation of the faster speciation of such microendemic phenotype species, we only found small body sizes of mantellid frogs to be linked to higher diversification and substitution rates, but not small range sizes. a joint analysis of various species-rich regional anuran radiations might provide enough species with all combinations of range and body sizes for a more conclusive test of this hypothesis. 