BACKGROUND
the profound effect of pleistocene glaciation upon species distributions in the northern hemisphere has been well documented  <cit> .
as continental ice sheets advanced into temperate areas, rendering once suitable areas uninhabitable, many northern taxa experienced extreme range reduction and fragmentation into refugia, while other populations and taxa were decimated entirely.
subsequently, as the ice sheets receded, recolonization of these historically glaciated areas by northernmost populations in refugia occurred rapidly.
the genetic population structure in these organisms often reflects geological patterns  <cit> .
rapid, post-glacial expansions distribute internal  haplotypes throughout large, previously glaciated, geographic areas.
furthermore, exponential growth of founding populations and subsequent resource competition may prevent other refugial populations from expanding simultaneously  <cit> .
taxa currently occupying previously glaciated and historically unsuitable areas are often characterized by low levels of genetic diversity  <cit> .
approximately  <dig>  to  <dig>  years before present, global temperatures were significantly lower than today and the volume of land-based ice was at its maximum  <cit> .
recently, niche-based species' distribution modeling has been applied to construct habitat suitability models for taxa during the last glacial maximum  <cit> .
increasing availability of high-resolution temporal and spatial paleoclimatic data have greatly aided and improved fine-scale modeling of species' historical geographic movements.
the number of studies showing the presence of genetic signatures indicative of glacial refugia among terrestrial and aquatic species has increased dramatically since the 1990's  <cit> .
these models provide a relatively novel approach to exploring historical distributions of species, as they often coincide with previously hypothesized refugial regions  <cit> .
the millipede genus narceus rafinesque  <dig>  is widely distributed throughout eastern north america with populations currently located in both historically glaciated and un-glaciated regions  <cit> .
narceus ranges from southern quebec south through the florida keys and west to central texas.
narceus specimens  are relatively large, cylindrical millipedes commonly found in appalachian mixed mesophytic deciduous forests.
they are primarily nocturnal, burrowing in rotten logs or leaf litter during the day and roaming about the forest floor or climbing trunks of large trees throughout the night.
during the winter, they burrow deep into soil and re-emerge in the spring to feed on dead tree material and mate.
narceus are some of the most commonly encountered millipedes in the appalachian mountains .
they are often seen in large numbers just after rainfall or sometimes during the night.
whereas most millipedes are intolerant of dry, warm temperatures, narceus species seem to have a wide range of moisture and heat tolerance and are often encountered crossing asphalt roads during the summer.
consequently their dispersal and colonization capabilities are likely greater than other co-distributed millipede groups .
with the exception of one morphologically distinct species in florida, populations of narceus appear phenotypically homogeneous throughout their range.
however, their rather simplified morphology and current nomenclature belie a number of taxonomic complexities that have historically plagued this geographically widespread genus.
at present, the genus comprises three species , which contain no fewer than  <dig> synonyms.
although resolving the taxonomy of this group is not our primary focus, the complex history of the genus  has likely contributed to confusing species boundaries within this ubiquitous and interesting group of millipedes.
given the history  of the genus, as suggested by shelley  <cit> , molecular data are likely needed to begin to properly disentangle the species limits within narceus.
the union of phylogeographic and population genetic principles provides the toolkit necessary to investigate the historical distribution of these lineages and, ultimately, helps disclose the mechanisms that maintain current levels of diversity and population structuring.
here we present the first phylogeographic study of the genus narceus.
we show that the genetic structure and current distribution of the genus narceus  reflects a complex history of refugia in the appalachian mountains and coastal plain of north america.
we examine our data for patterns that should be consistent with a pleistocene refugia hypothesis.
first, there should be a profile of phylogeographic and genetic diversity that distinguishes coastal plain versus all other populations.
secondly, populations currently occupying historically unsuitable habitat should show genetic signatures of rapid range expansion.
and last, high probability of habitat suitability from paleo-models of historical distributions should be coincident with those populations having relatively higher levels of genetic diversity.
RESULTS
summary of data and phylogenetic analyses
mitochondrial sequence data  were obtained for individuals  from  <dig> localities;  <dig> positions were parsimony informative .
the average ingroup sequence divergence was  <dig> % with a range of <  <dig> % –  <dig> %.
most localities are represented by >  <dig> individuals.
however at  <dig> localities we were only able to obtain a single individual.
one hundred and fifty-three unique haplotypes were recovered following alignment.
twelve of these haplotypes were found at multiple localities, one of which occurred at  <dig> widely distributed northern localities.
of the  <dig> localities surveyed where more than a single individual was sampled ,  <dig> contained a single haplotype.
for the bayesian analysis, gtr + i + Γ model of molecular evolution was inferred for the 16s region and hky + i for both the trna-val and 12s partitions.
of  <dig> million generations,  <dig>  were discarded as burnin, resulting in a tree with a mean ln-likelihood value of - <dig> .
the bayesian phylogeny  shows four distinct groups: 1) a texas and louisiana clade, 2) a southern appalachian clade, 3) a clade corresponding to the morphologically distinct narceus gordanus , and 4) a large, widely distributed sister clade to n. gordanus  <cit> .
we used statistical parsimony at a 95% confidence level, implemented by tcs  <cit> , to subdivide this larger clade.
with a 95% reconnection limits most clades were connected, however, by relaxing the confidence level to 90%  the networks were more inclusive and reflected the major clades recovered in the bayesian tree with a higher degree of precision.
six networks  and two unconnected haplotypes were recovered, all of which agreed with the topology of the bayesian phylogeny: a northwestern clade, a northern clade, two small mississippi/louisiana networks, a widespread southeastern clade, a florida panhandle/southern appalachian clade, and two haplotypes that fall out on a single branch sister to the northern clade .
most nodes are highly supported with posterior probabilities >  <dig>  and, with the exception of two interior nodes and several very shallow nodes, all others are well supported .
the haplotype networks of both northern clades reveal star-like patterns around each of the widespread haplotypes, a pattern indicative of the rapid expansion of ancestral haplotypes over a large geographic area  <cit> .
phylogeography
tests for isolation by distance were significant in all clades except the northwestern clade  and a subclade of the northern clade .
although it is one of the more widespread clades, isolation by distance was not predicted in the northwestern clade as it exhibits low levels of sequence divergence relative to the other clades in the phylogeny.
mismatch distribution plots  were multimodal in most cases, although the above-mentioned subclade of the northern clade showed a smooth, unimodal curve.
fu's fs was significant in the northwestern clade , southeastern clade , and the northern clade  and non-significant in all other clades .
the coastal plain character optimization  shows a complex phylogeographic pattern with nine independent colonizations into the appalachian highlands and six re-colonizations back to the coastal plain.
when we optimize the character "coastal plain" onto the haplotype phylogeny and then compare it to the optimization of suitable habitat during the pleistocene  we find a very high level of similarity ; we only recovered a single haplotype with a high probability of paleo-model habitat suitability not located in the coastal plain.
a large majority of the coastal plain haplotype lineages occupy areas that would have been suitable refugia.
furthermore, each clade with a coastal plain interior node has a non-coastal plain sister clade.
the mississippi/louisiana clade is sister to the exclusively non-coastal, shallowly divergent northwestern clade; likewise, the florida panhandle/southern appalachian clade is sister to the southeastern clade, which comprises a non-coastal sister to a subclade showing a radiation from and back to the coastal plain.
genetic diversity for the coastal clade consisting of only two haplotypes  could not be calculated due to limited sampling.
however, we do see a pattern similar to other sister clades within the tree.
discussion
using an integrative approach that employs genetic and geospatial data, this study provides a straightforward example of pleistocene refugia and post-glacial colonization.
the star-like patterns and widely distributed haplotypes in our phylogeographic analyses taken together with significantly negative fu's fs values, support two major rapid expansions from coastal refugia into northern, historically glaciated areas.
the southern populations of narceus are characterized by relatively high levels of genetic diversity, isolation by distance, multimodal mismatch distributions, and an absence of widely distributed ancestral haplotypes.
similar genetic patterns across geographic regions attributed to pleistocene related phenomena have been detected in numerous animals including salamanders  <cit> , frogs  <cit> , voles  <cit> , mice  <cit> , bears  <cit> , and grasshoppers  <cit> .
our niche-based paleodistribution model also provides compelling evidence for southern refugia, specifically relegated to the coastal plain.
pleistocene refugia and post-glacial expansion
glacial pulses during the pleistocene profoundly affected animal distributions in north america, and species in the genus narceus are no exception.
as continental glaciers advanced, the destruction of habitat necessitated the displacement or extinction of northern taxa.
populations of displaced northern taxa suffered severe bottlenecks and hence decreased genetic diversity, while southern taxa that persisted during glacial advances were able to maintain high levels of diversity  <cit> .
an example of this phenomenon was provided by church et al.
<cit>  in eastern tiger salamanders.
they showed a distinct split between eastern and western populations.
within the eastern lineage, two possible refugia were identified: the blue ridge mountains and the coastal plain of the carolinas.
they hypothesized that the populations located in mountain refugia have remained isolated after deglaciation whereas the coastal plain refugia were the source for northern post-glacial expansion along the east coast.
we found a similar pattern in narceus.
the basal-most clade in the phylogeny  occurs south of any previously glaciated areas in the southern appalachians, with populations from the blue ridge mountains of virginia to the southern extent of appalachia in eastern alabama.
this clade also exhibits isolation by distance and relatively high levels of genetic diversity with no unique haplotypes occupying more than a single locality.
the texas/louisiana clade, the next basal-most clade, also demonstrates the genetic signature of a refugial past.
within the texas/louisiana clade, mismatch distributions  indicate highly fragmented populations  <cit> , with each locality sampled having at least one unique haplotype – another common characteristic of refugia populations  <cit> .
with our sampling scheme, we found a single distributional extension into northern indiana , at first believed to be an anthropogenic introduction.
however, we did detect isolation by distance within this clade.
without further sampling, the phylogenetic placement of this population remains enigmatic.
a similar pattern of radiation was detected between the mississippi/louisiana clade and its sister, the northwestern clade .
the mississippi/louisiana clade exhibits isolation by distance, relatively high levels of genetic diversity, and a lack of shared haplotypes among localities – all characteristics consistent with a deep refugial history  <cit> .
its sister, the northwestern clade, is widely distributed west of the appalachians .
this clade exhibits relatively low levels of genetic divergence and is distributed across a large geographic distance.
it also contains a single haplotype  found in eight localities in four states .
the haplotype network indicates six other haplotypes connected to iloj514ii by =  <dig> mutational steps, resulting in a star-like pattern .
a pattern also observed for rapidly expanded populations of spotted salamanders  <cit> , wood frogs  <cit> , and other taxa  <cit> .
the star-like pattern and the mismatch plot of pairwise differences in the northwestern clade  are both consistent with relatively rapid expansion, as might be expected for a quick founding colonization into a newly deglaciated suitable area.
indeed, the results of the paleodistribution models  show that the majority of habitat currently occupied by the northwestern clade was likely unsuitable  <dig>  years ago.
this pattern reflects a northern radiation from the gulf coast northward and west of the appalachians, resembling that of the texas/louisiana clade.
similar to patterns found in eastern tiger salamanders  <cit> , we detected a radiation from the mid-atlantic coastal plain north through upstate new york; however, this radiation appears to have extended west as well, like that seen in the spotted salamander   <cit> .
the northern clade has three widely distributed haplotypes all of which show star-like tcs networks .
fu's fs, an indicator of rapid range expansion  <cit> , was significant and negative for the northern clade.
despite multiple lines of evidence indicating rapid range expansion, isolation by distance was detected for this clade.
although long-distance dispersal can also account for patterns similar to those seen for this clade, millipedes are non-vagile and thus unlikely to disperse, via a single event, across a long distance.
therefore, the phylogeographic pattern is more likely the result of rapid range expansion.
the southeastern and florida panhandle/southern appalachian clades represent separate refugial populations that have remained isolated.
the southeastern clade indicates a rather complex phylogeographic pattern in which mismatch plots and isolation by distance analyses were consistent with those of refugial populations.
however, fu's fs was significant and negative in the southeastern clade, suggesting a rapid expansion.
this clade is widespread throughout the southeast, and character map reconstructions suggest multiple radiations to and from the coastal plain within this clade.
these pulses may have been fairly recent, a scenario consistent with significant negative values for fs obtained here.
the florida panhandle/southern appalachian area represents a coastal refugium with an east-to-west phylogeographic break at the apalachicola/chattahoochee/flint  river basin, a well documented geographic barrier to gene flow for multiple taxa  <cit> .
there is a single radiation of these millipedes forming a monophyletic group extending southeast of the acf barrier into the southern appalachians.
the fall line  does not seem to prevent radiations of these millipedes; the area immediately east and running north-to-south of the acf basin may serve as a riparian corridor from the coastal plain to the southern appalachians  <cit> .
paleodistribution modeling
high-resolution models of past distributions for species provide a promising new tool in the field of phylogeography  <cit> .
a recent study by waltari et al.
<cit>  addressed concordance between pleistocene refugia predicted using traditional biogeographic methods and niche-based paleodistribution modeling.
fourteen of the twenty north american terrestrial taxa tested showed significant spatial correlations between traditional and niche-based modeling methods for identifying pleistocene refugia  <cit> .
agreement between these methods suggests that paleodistribution models may well provide useful guidelines for biogeographic studies.
our last glacial maximum model for narceus  indicates a major reduction in habitat suitability that stands in stark contrast to the present-day model.
hypotheses of refugia along the gulf and atlantic coastal plains are consistent with indicated areas of highest predicted suitability in the niche-based distribution models.
the character map of haplotypes by region, illustrates a disproportionate number of haplotypes from these areas on the phylogeny .
a correlation between genetic diversity and habitat suitability from our pleistocene model suggests that the model's highest predicted probabilities, in fact, do show higher levels of genetic diversity and are consistent with those patterns expected from refugial areas.
these refugia hypotheses are consistent with all mismatch plots and isolation by distance tests for clades occurring in coastal areas.
somewhat inconsistent with the refugia hypotheses was the low habitat suitability modeled in the southern appalachian and texas coastal plain regions of our paleodistribution model.
although these areas show very low probability  of having suitable habitat during the last glacial maximum , it is apparent that millipede populations were able to survive there.
during the last glacial maximum, suitable habitat in these areas may have been reduced into numerous microrefugia within the overall southern appalachian refugium that could lead to the diversity currently found in these clades.
under this hypothesis, overall habitat suitability would appear to be very low; however, populations may have persisted within suitable microrefugia.
taxonomic considerations
as discussed in the background section, narceus presently comprises three nominal species, two of which appear to be morphologically homogenous.
narceus gordanus is the only species that appears to be morphologically distinct and has a rather limited distribution, endemic to peninsular florida and extending north to south carolina  <cit> .
individuals assigned to n. gordanus are represented in the phylogeny  by a clade nested squarely within populations that are morphologically indistinguishable and clades that likely represent the remaining two species.
this phylogenetic placement renders n. americanus and n. annularis  paraphyletic.
moreover, as has been demonstrated for other millipede groups , cryptic species may be a problem for species delineation within this genus.
further work will be needed to resolve these issues, both in terms of careful morphological analyses and intensive collecting west of the mississippi.
an integrative approach that takes into account biogeographic patterns, analyses of morphology, phylogenetic history, and climatic envelope will likely provide the framework necessary to investigate species boundaries in this widespread, morphologically homogeneous genus .
CONCLUSIONS
the signature of climatic change on the distribution of the millipede genus narceus is evident in the mtdna data presented here.
niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying areas of probable pleistocene refugia.
this study provides the first molecular phylogeographic and ecological modeling data for the millipede genus narceus.
the methods used here provide an integrative biogeographic approach to understand processes generating patterns of biodiversity, and an approach to explore the evolutionary diversification of species, populations, and organismal distributions.

 the profound effect of pleistocene glaciation upon species distributions in the northern hemisphere has been well documented. as continental ice sheets advanced into temperate areas, rendering once suitable areas uninhabitable, many northern taxa experienced extreme range reduction and fragmentation into refugia, while other populations and taxa were decimated entirely. subsequently, as the ice sheets receded, recolonization of these historically glaciated areas by northernmost populations in refugia occurred rapidly. the genetic population structure in these organisms often reflects geological patterns. rapid, post-glacial expansions distribute internal haplotypes throughout large, previously glaciated, geographic areas. furthermore, exponential growth of founding populations and subsequent resource competition may prevent other refugial populations from expanding simultaneously. taxa currently occupying previously glaciated and historically unsuitable areas are often characterized by low levels of genetic diversity. approximately to years before present, global temperatures were significantly lower than today and the volume of land-based ice was at its maximum. recently, niche-based species' distribution modeling has been applied to construct habitat suitability models for taxa during the last glacial maximum. increasing availability of high-resolution temporal and spatial paleoclimatic data have greatly aided and improved fine-scale modeling of species' historical geographic movements. the number of studies showing the presence of genetic signatures indicative of glacial refugia among terrestrial and aquatic species has increased dramatically since the 1990's. these models provide a relatively novel approach to exploring historical distributions of species, as they often coincide with previously hypothesized refugial regions. the millipede genus narceus rafinesque is widely distributed throughout eastern north america with populations currently located in both historically glaciated and un-glaciated regions. narceus ranges from southern quebec south through the florida keys and west to central texas. narceus specimens are relatively large, cylindrical millipedes commonly found in appalachian mixed mesophytic deciduous forests. they are primarily nocturnal, burrowing in rotten logs or leaf litter during the day and roaming about the forest floor or climbing trunks of large trees throughout the night. during the winter, they burrow deep into soil and re-emerge in the spring to feed on dead tree material and mate. narceus are some of the most commonly encountered millipedes in the appalachian mountains. they are often seen in large numbers just after rainfall or sometimes during the night. whereas most millipedes are intolerant of dry, warm temperatures, narceus species seem to have a wide range of moisture and heat tolerance and are often encountered crossing asphalt roads during the summer. consequently their dispersal and colonization capabilities are likely greater than other co-distributed millipede groups. with the exception of one morphologically distinct species in florida, populations of narceus appear phenotypically homogeneous throughout their range. however, their rather simplified morphology and current nomenclature belie a number of taxonomic complexities that have historically plagued this geographically widespread genus. at present, the genus comprises three species, which contain no fewer than synonyms. although resolving the taxonomy of this group is not our primary focus, the complex history of the genus has likely contributed to confusing species boundaries within this ubiquitous and interesting group of millipedes. given the history of the genus, as suggested by shelley, molecular data are likely needed to begin to properly disentangle the species limits within narceus. the union of phylogeographic and population genetic principles provides the toolkit necessary to investigate the historical distribution of these lineages and, ultimately, helps disclose the mechanisms that maintain current levels of diversity and population structuring. here we present the first phylogeographic study of the genus narceus. we show that the genetic structure and current distribution of the genus narceus reflects a complex history of refugia in the appalachian mountains and coastal plain of north america. we examine our data for patterns that should be consistent with a pleistocene refugia hypothesis. first, there should be a profile of phylogeographic and genetic diversity that distinguishes coastal plain versus all other populations. secondly, populations currently occupying historically unsuitable habitat should show genetic signatures of rapid range expansion. and last, high probability of habitat suitability from paleo-models of historical distributions should be coincident with those populations having relatively higher levels of genetic diversity.  summary of data and phylogenetic analyses mitochondrial sequence data were obtained for individuals from localities; positions were parsimony informative. the average ingroup sequence divergence was % with a range of < % – %. most localities are represented by > individuals. however at localities we were only able to obtain a single individual. one hundred and fifty-three unique haplotypes were recovered following alignment. twelve of these haplotypes were found at multiple localities, one of which occurred at widely distributed northern localities. of the localities surveyed where more than a single individual was sampled, contained a single haplotype. for the bayesian analysis, gtr + i + Γ model of molecular evolution was inferred for the 16s region and hky + i for both the trna-val and 12s partitions. of million generations, were discarded as burnin, resulting in a tree with a mean ln-likelihood value of -. the bayesian phylogeny shows four distinct groups: 1) a texas and louisiana clade, 2) a southern appalachian clade, 3) a clade corresponding to the morphologically distinct narceus gordanus, and 4) a large, widely distributed sister clade to n. gordanus. we used statistical parsimony at a 95% confidence level, implemented by tcs, to subdivide this larger clade. with a 95% reconnection limits most clades were connected, however, by relaxing the confidence level to 90% the networks were more inclusive and reflected the major clades recovered in the bayesian tree with a higher degree of precision. six networks and two unconnected haplotypes were recovered, all of which agreed with the topology of the bayesian phylogeny: a northwestern clade, a northern clade, two small mississippi/louisiana networks, a widespread southeastern clade, a florida panhandle/southern appalachian clade, and two haplotypes that fall out on a single branch sister to the northern clade. most nodes are highly supported with posterior probabilities > and, with the exception of two interior nodes and several very shallow nodes, all others are well supported. the haplotype networks of both northern clades reveal star-like patterns around each of the widespread haplotypes, a pattern indicative of the rapid expansion of ancestral haplotypes over a large geographic area. phylogeography tests for isolation by distance were significant in all clades except the northwestern clade and a subclade of the northern clade. although it is one of the more widespread clades, isolation by distance was not predicted in the northwestern clade as it exhibits low levels of sequence divergence relative to the other clades in the phylogeny. mismatch distribution plots were multimodal in most cases, although the above-mentioned subclade of the northern clade showed a smooth, unimodal curve. fu's fs was significant in the northwestern clade, southeastern clade, and the northern clade and non-significant in all other clades. the coastal plain character optimization shows a complex phylogeographic pattern with nine independent colonizations into the appalachian highlands and six re-colonizations back to the coastal plain. when we optimize the character "coastal plain" onto the haplotype phylogeny and then compare it to the optimization of suitable habitat during the pleistocene we find a very high level of similarity; we only recovered a single haplotype with a high probability of paleo-model habitat suitability not located in the coastal plain. a large majority of the coastal plain haplotype lineages occupy areas that would have been suitable refugia. furthermore, each clade with a coastal plain interior node has a non-coastal plain sister clade. the mississippi/louisiana clade is sister to the exclusively non-coastal, shallowly divergent northwestern clade; likewise, the florida panhandle/southern appalachian clade is sister to the southeastern clade, which comprises a non-coastal sister to a subclade showing a radiation from and back to the coastal plain. genetic diversity for the coastal clade consisting of only two haplotypes could not be calculated due to limited sampling. however, we do see a pattern similar to other sister clades within the tree. discussion using an integrative approach that employs genetic and geospatial data, this study provides a straightforward example of pleistocene refugia and post-glacial colonization. the star-like patterns and widely distributed haplotypes in our phylogeographic analyses taken together with significantly negative fu's fs values, support two major rapid expansions from coastal refugia into northern, historically glaciated areas. the southern populations of narceus are characterized by relatively high levels of genetic diversity, isolation by distance, multimodal mismatch distributions, and an absence of widely distributed ancestral haplotypes. similar genetic patterns across geographic regions attributed to pleistocene related phenomena have been detected in numerous animals including salamanders, frogs, voles, mice, bears, and grasshoppers. our niche-based paleodistribution model also provides compelling evidence for southern refugia, specifically relegated to the coastal plain. pleistocene refugia and post-glacial expansion glacial pulses during the pleistocene profoundly affected animal distributions in north america, and species in the genus narceus are no exception. as continental glaciers advanced, the destruction of habitat necessitated the displacement or extinction of northern taxa. populations of displaced northern taxa suffered severe bottlenecks and hence decreased genetic diversity, while southern taxa that persisted during glacial advances were able to maintain high levels of diversity. an example of this phenomenon was provided by church et al. in eastern tiger salamanders. they showed a distinct split between eastern and western populations. within the eastern lineage, two possible refugia were identified: the blue ridge mountains and the coastal plain of the carolinas. they hypothesized that the populations located in mountain refugia have remained isolated after deglaciation whereas the coastal plain refugia were the source for northern post-glacial expansion along the east coast. we found a similar pattern in narceus. the basal-most clade in the phylogeny occurs south of any previously glaciated areas in the southern appalachians, with populations from the blue ridge mountains of virginia to the southern extent of appalachia in eastern alabama. this clade also exhibits isolation by distance and relatively high levels of genetic diversity with no unique haplotypes occupying more than a single locality. the texas/louisiana clade, the next basal-most clade, also demonstrates the genetic signature of a refugial past. within the texas/louisiana clade, mismatch distributions indicate highly fragmented populations, with each locality sampled having at least one unique haplotype – another common characteristic of refugia populations. with our sampling scheme, we found a single distributional extension into northern indiana, at first believed to be an anthropogenic introduction. however, we did detect isolation by distance within this clade. without further sampling, the phylogenetic placement of this population remains enigmatic. a similar pattern of radiation was detected between the mississippi/louisiana clade and its sister, the northwestern clade. the mississippi/louisiana clade exhibits isolation by distance, relatively high levels of genetic diversity, and a lack of shared haplotypes among localities – all characteristics consistent with a deep refugial history. its sister, the northwestern clade, is widely distributed west of the appalachians. this clade exhibits relatively low levels of genetic divergence and is distributed across a large geographic distance. it also contains a single haplotype found in eight localities in four states. the haplotype network indicates six other haplotypes connected to iloj514ii by = mutational steps, resulting in a star-like pattern. a pattern also observed for rapidly expanded populations of spotted salamanders, wood frogs, and other taxa. the star-like pattern and the mismatch plot of pairwise differences in the northwestern clade are both consistent with relatively rapid expansion, as might be expected for a quick founding colonization into a newly deglaciated suitable area. indeed, the results of the paleodistribution models show that the majority of habitat currently occupied by the northwestern clade was likely unsuitable years ago. this pattern reflects a northern radiation from the gulf coast northward and west of the appalachians, resembling that of the texas/louisiana clade. similar to patterns found in eastern tiger salamanders, we detected a radiation from the mid-atlantic coastal plain north through upstate new york; however, this radiation appears to have extended west as well, like that seen in the spotted salamander. the northern clade has three widely distributed haplotypes all of which show star-like tcs networks. fu's fs, an indicator of rapid range expansion, was significant and negative for the northern clade. despite multiple lines of evidence indicating rapid range expansion, isolation by distance was detected for this clade. although long-distance dispersal can also account for patterns similar to those seen for this clade, millipedes are non-vagile and thus unlikely to disperse, via a single event, across a long distance. therefore, the phylogeographic pattern is more likely the result of rapid range expansion. the southeastern and florida panhandle/southern appalachian clades represent separate refugial populations that have remained isolated. the southeastern clade indicates a rather complex phylogeographic pattern in which mismatch plots and isolation by distance analyses were consistent with those of refugial populations. however, fu's fs was significant and negative in the southeastern clade, suggesting a rapid expansion. this clade is widespread throughout the southeast, and character map reconstructions suggest multiple radiations to and from the coastal plain within this clade. these pulses may have been fairly recent, a scenario consistent with significant negative values for fs obtained here. the florida panhandle/southern appalachian area represents a coastal refugium with an east-to-west phylogeographic break at the apalachicola/chattahoochee/flint river basin, a well documented geographic barrier to gene flow for multiple taxa. there is a single radiation of these millipedes forming a monophyletic group extending southeast of the acf barrier into the southern appalachians. the fall line does not seem to prevent radiations of these millipedes; the area immediately east and running north-to-south of the acf basin may serve as a riparian corridor from the coastal plain to the southern appalachians. paleodistribution modeling high-resolution models of past distributions for species provide a promising new tool in the field of phylogeography. a recent study by waltari et al. addressed concordance between pleistocene refugia predicted using traditional biogeographic methods and niche-based paleodistribution modeling. fourteen of the twenty north american terrestrial taxa tested showed significant spatial correlations between traditional and niche-based modeling methods for identifying pleistocene refugia. agreement between these methods suggests that paleodistribution models may well provide useful guidelines for biogeographic studies. our last glacial maximum model for narceus indicates a major reduction in habitat suitability that stands in stark contrast to the present-day model. hypotheses of refugia along the gulf and atlantic coastal plains are consistent with indicated areas of highest predicted suitability in the niche-based distribution models. the character map of haplotypes by region, illustrates a disproportionate number of haplotypes from these areas on the phylogeny. a correlation between genetic diversity and habitat suitability from our pleistocene model suggests that the model's highest predicted probabilities, in fact, do show higher levels of genetic diversity and are consistent with those patterns expected from refugial areas. these refugia hypotheses are consistent with all mismatch plots and isolation by distance tests for clades occurring in coastal areas. somewhat inconsistent with the refugia hypotheses was the low habitat suitability modeled in the southern appalachian and texas coastal plain regions of our paleodistribution model. although these areas show very low probability of having suitable habitat during the last glacial maximum, it is apparent that millipede populations were able to survive there. during the last glacial maximum, suitable habitat in these areas may have been reduced into numerous microrefugia within the overall southern appalachian refugium that could lead to the diversity currently found in these clades. under this hypothesis, overall habitat suitability would appear to be very low; however, populations may have persisted within suitable microrefugia. taxonomic considerations as discussed in the background section, narceus presently comprises three nominal species, two of which appear to be morphologically homogenous. narceus gordanus is the only species that appears to be morphologically distinct and has a rather limited distribution, endemic to peninsular florida and extending north to south carolina. individuals assigned to n. gordanus are represented in the phylogeny by a clade nested squarely within populations that are morphologically indistinguishable and clades that likely represent the remaining two species. this phylogenetic placement renders n. americanus and n. annularis paraphyletic. moreover, as has been demonstrated for other millipede groups, cryptic species may be a problem for species delineation within this genus. further work will be needed to resolve these issues, both in terms of careful morphological analyses and intensive collecting west of the mississippi. an integrative approach that takes into account biogeographic patterns, analyses of morphology, phylogenetic history, and climatic envelope will likely provide the framework necessary to investigate species boundaries in this widespread, morphologically homogeneous genus.  the signature of climatic change on the distribution of the millipede genus narceus is evident in the mtdna data presented here. niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying areas of probable pleistocene refugia. this study provides the first molecular phylogeographic and ecological modeling data for the millipede genus narceus. the methods used here provide an integrative biogeographic approach to understand processes generating patterns of biodiversity, and an approach to explore the evolutionary diversification of species, populations, and organismal distributions. 
using phylogeographic, population, and paleoclimatic data, we show that the distribution and mitochondrial data for the millipede genus narceus are consistent with classical examples of pleistocene refugia and subsequent post-glacial population expansion seen in other organismal groups. recently, paleoclimatic data have been combined with niche-based distribution modeling to locate possible refugia during the last glacial maximum. such interdisciplinary biogeographic studies provide a comprehensive approach to understanding these processes that generate and maintain biodiversity as well as the framework necessary to explore questions regarding evolutionary diversification of taxa. niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying probable refugia. many studies indicate that northern animal populations exhibit low levels of genetic diversity over geographically widespread areas whereas southern populations exhibit relatively high levels. we found a strong correlation between these zones of ecological suitability inferred from our paleo-model with levels of genetic diversity derived from phylogenetic and population estimates of genetic structuring. distributional models of narceus during the last glacial maximum show a dramatic reduction from the current distribution, with suitable ecological zones concentrated along the gulf and atlantic coastal plain. evidence for refugial populations were found in the southern appalachian mountains and in the coastal plain. the signature of climatic change, during and after the pleistocene, on the distribution of the millipede genus narceus is evident in the genetic data presented.
 species that are widespread throughout historically glaciated and currently non-glaciated areas provide excellent opportunities to investigate the role of pleistocene climatic change on the distribution of north american biodiversity. many studies indicate that northern animal populations exhibit low levels of genetic diversity over geographically widespread areas whereas southern populations exhibit relatively high levels. recently, paleoclimatic data have been combined with niche-based distribution modeling to locate possible refugia during the last glacial maximum. using phylogeographic, population, and paleoclimatic data, we show that the distribution and mitochondrial data for the millipede genus narceus are consistent with classical examples of pleistocene refugia and subsequent post-glacial population expansion seen in other organismal groups.  the phylogeographic structure of narceus reveals a complex evolutionary history with signatures of multiple refugia in southeastern north america followed by two major northern expansions. evidence for refugial populations were found in the southern appalachian mountains and in the coastal plain. the northern expansions appear to have radiated from two separate refugia, one from the gulf coastal plain area and the other from the mid-atlantic coastal region. distributional models of narceus during the last glacial maximum show a dramatic reduction from the current distribution, with suitable ecological zones concentrated along the gulf and atlantic coastal plain. we found a strong correlation between these zones of ecological suitability inferred from our paleo-model with levels of genetic diversity derived from phylogenetic and population estimates of genetic structuring.  the signature of climatic change, during and after the pleistocene, on the distribution of the millipede genus narceus is evident in the genetic data presented. niche-based historical distribution modeling strengthens the conclusions drawn from the genetic data and proves useful in identifying probable refugia. such interdisciplinary biogeographic studies provide a comprehensive approach to understanding these processes that generate and maintain biodiversity as well as the framework necessary to explore questions regarding evolutionary diversification of taxa. 