BACKGROUND
the debate about the relative importance of ecological speciation in species diversification spans several decades  <cit> .
however, concrete cases based on empirical evidence remain relatively scarce  <cit> .
in essence, ecological speciation is related to the "ecological species concept", which was defined as follows  <cit> : "a species is a lineage , which occupies an adaptive zone minimally different from that of any other lineage in its range and which evolves separately from all lineages outside its range".
the driving force behind ecological speciation is thus divergent natural selection between environments or, in other words, reproductive isolation of populations by means of adaptation to different environments or niches  <cit> .
ecological selection is a consequence of individual-based interactions with the environment.
from this interaction follows that divergent selection between ecological niches is a major driving force differentiating lineages until reproductive isolation occurs  <cit> .
ecologically divergent pairs of populations will show higher levels of reproductive incompatibility and lower levels of gene flow than ecologically more similar population pairs  <cit> .
a resulting corollary is that ecological speciation is more likely to arise in regions with patchworks of contrasting habitats and/or distinct environmental gradients.
the number of taxa within the insect order coleoptera exceeds that of any known plant or animal group  <cit> .
more than half of the beetles are phytophagous, including the species rich superfamilies curculionoidea and chrysomeloidea, of which a majority feeds on angiosperms  <cit> .
the increase in phytophagous beetle diversity was facilitated by the rise of flowering plants  <cit> .
the family chrysomelidae currently consists of more than thirty-five thousand recognized species including economically important pest species such as the colorado potato beetle , the northern corn rootworm , the cereal leaf beetle , and the striped turnip flea beetle .
the biological and economic importance of the superfamily chrysomeloidea make it vital to understand the factors that drive diversification in this group.
here, we present a case of ecological niche differentiation in the alpine leaf beetle oreina speciosissima that may represent the early stages of ecological speciation.
the genus oreina currently includes twenty-eight species, of which only seven early-diverging taxa do not exclusively occur in high forbs   <cit> .
according to current knowledge  <cit> , the most parsimonious explanation is that high forbs vegetation is the ancestral niche for the remaining twenty-one oreina lineages, among which only our focal taxon oreina speciosissima shows a partial reversal, since it is found both in high forbs and stone run vegetation.
oreina speciosissima is distributed across nearly the entire range of the genus oreina  through a wide altitudinal gradient .
at lower elevations it generally colonizes the very abundant high forbs vegetation whereas at higher elevations it is found in stone run habitats across a small portion of its distribution range  <cit> .
kippenberg  <cit>  and personal observations suggest that oreina speciosissima feeds exclusively on asteraceae  and colonizes four distinct habitats represented by well-established plant associations: two occurring in high forbs - petasition officinalis and adenostylion - and two in stone run -androsacion alpinae on siliceous bedrock and petasition paradoxi on calcareous bedrock -  <cit> .
these plant communities are often patchily distributed due to the myriad of spatially proximate microclimates that occur in the alps, especially sites with calcareous bedrocks which regularly present a mosaic of microhabitats.
for instance, sinkholes or dolines, formed through water erosion in so-called karstic areas represent ecological islands inhabited by high forbs vegetation surrounded by areas covered by stone run vegetation .
beetles inhabiting the highly divergent habitats have been categorized in two different subspecies, namely oreina speciosissima sensu stricto  and oreina speciosissima troglodytes , on the basis of differences in elytral coloration and the shape of male genitalia   <cit> .
oreina speciosissima sensu stricto beetles are bright metallic green or blue whereas the coloration in oreina speciosissima troglodytes is generally darker and mat  <cit> .
it is not known whether the morphological differences between ecotypes are in any way adaptive and/or have a genetic basis, although color patterns in another species of oreina are known to influence natural selection through predation pressure  <cit> .
like most other members of the genus, oreina speciosissima sensu stricto can be found feeding or mating throughout the day on, or in the vicinity of its host plants.
in contrast, oreina speciosissima troglodytes is usually found adjacent to its host plants, concealed in crevices and under loose rocks.
previous studies by the authors  <cit>  greatly challenged the existence of clear species boundaries within the genus: it is therefore realistic from a biological point of view to refer to these taxonomic entities as ecotypes rather than subspecies.
leaf beetles from the genus oreina are generally thought to make only limited use of their dispersal capabilities  <cit>  even though kalberer et al.
<cit>  reported an average flight dispersal of approximately one hundred meters for oreina cacaliae beetles during autumn migration.
rowell-rahier  <cit>  showed that low vagility in concert with a patchy host plant distribution resulted in a low level of genetic structuring in oreina speciosissima.
oreina speciosissima sensu stricto beetles inhabit high forbs patches that are generally larger in size, lie closer together and harbor more beetles per unit of surface area than stone run patches inhabited by oreina speciosissima troglodytes .
the present work investigates  <dig> populations representative of the two ecotypes, using sequencing of nuclear  and mitochondrial   dna regions as well as aflp genome fingerprinting in a way to address the following questions:
<dig>  are ecotypes monophyletic?
<dig>  is adaptation to different habitats and host plants associated with genetic divergence?
RESULTS
phylogenetic reconstruction of the dna sequence data sets
the nuclear its <dig> region showed no variation for oreina speciosissima and was thus excluded from further analyses.
in contrast, the three mtdna regions were polymorphic with a total alignment length of  <dig> bp;  <dig> bp for 16s,  <dig> for coi and  <dig> bp for coii.
excluding the outgroup,  <dig> characters were potentially parsimony informative  among  <dig> variable characters.
the three mtdna regions contributed as follows: 16s , coi  and coii .
the best substitution models were hasegawa-kishino-yano   <cit>  for 16s and hasegawa-kishino-yano plus gamma   <cit>  for coi and coii.
the alignments of the mtdna markers were combined in a total evidence approach, after pairwise incongruence length difference ild test  <cit>  revealed no incongruence among the three mtdna markers .
the resulting dataset was investigated using maximum parsimony  and bayesian phylogenetic inference methods  <cit> .
both approaches produced highly congruent topologies with the same major nodes.
the mp topology with bremer supports  <cit>  and corresponding bayesian posterior probabilities from the bayesian analysis  are shown in figure  <dig>  the ingroup is well supported with a bremer support of  <dig> and a bpp of  <dig> .
the ingroup splits into two groups, a well-supported clade  containing all individuals from the gra population and a polytomy  containing all other individuals.
apart from a clade containing all individuals of gsb and one with two individuals from cdm and one from tsc, there is no resolution within the polytomy.
sample nuf_ <dig> failed to amplify and is therefore not shown in figure  <dig>  only samples that rendered both satisfactory dna sequences and aflp fingerprints were used for phylogenetic analysis.
aflp
the aflp analysis produced a total of  <dig> bands  with an average of  <dig> bands per individual and an average reproducibility rate of  <dig> %.
among  <dig> variable characters,  <dig> were potentially parsimony informative.
just as the mtdna data, the aflp dataset was investigated using mp and bayesian phylogenetic inference methods.
again, both approaches were highly congruent as the mp and bayesian trees shared the same major nodes.
consequently, only the bayesian phylogeny  is displayed in figure  <dig>  due to the lack of an outgroup, we present an unrooted topology , which led to a separation of specimens into two well supported clans sensu wilkinson et al.
<cit>  , each with a bpp of  <dig>  .
clan i includes three sub-clans supported with bpp values of  <dig>  ,  <dig>   and  <dig>   respectively.
clan i contains nine specimens with a strict oreina speciosissima sensu stricto morphology  and six specimens with an intermediate morphology .
within clan ii, two sub-clans were well supported with a bpp of  <dig>   and  <dig>   respectively.
clan ii contains eight individuals with strict oreina speciosissima troglodytes morphology  and five individuals with an intermediate morphology .
notably, specimens with intermediate morphologies were sorted close to the midpoint root of the tree topology.
the aflp dataset was further investigated using a bayesian  and a distance-based  clustering algorithm.
the approaches produced fully congruent relationships and only results of the former are provided here.
the structure analysis showed highly likely clusters when considering k values ranging between two and five .
the obtained results were largely congruent across k values  and highly compatible with the phylogenetic relationships.
the only incongruence that could be observed when considering all k values, or when comparing structure results with the tree topology, involved specimens with an intermediate morphology.
hereafter, we will consider and discuss results based on k =  <dig>  given that they are the most informative.
when viewed from a host plant perspective it becomes apparent that all leaf beetle specimens in clan ii occur in the same stone run habitat with individuals from sub-clans iia and iib developing respectively in alkaline petasition paradoxi and in acidic androsacion alpinae habitats .
in contrast all individuals from clan i occurred in high forbs, in adenostylion or petasites officinalis habitat, either on alkaline, neutral or slightly acidic soils  <cit>  .
discussion
are ecotypes monophyletic?
the phylogenetic tree based on mtdna markers provides high support for the monophyly of oreina speciosissima sensu lato .
however, very little polymorphism and genetic structure are revealed within the ingroup, although the mtdna markers proved variable enough to reconstruct intra-specific phylogenetic relationships in other arthropod systems .
indeed, the resulting topology supports neither geographical nor ecotypic grouping of the beetles, possibly suggesting a recent divergence of oreina speciosissima lineages, with the exception of specimens from the gra population, which cluster as the sister lineage to all other specimens.
beetles from this latter population thus form an orphan clade  <cit> , which may correspond to an isolated refugial lineage.
aflp data on the other hand shows a clear-cut differentiation of specimens .
however, this pattern does not appear to have a geographical basis  and instead correlates with the beetle ecotypic definition, or in other words, to the plant habitat .
in contrast, aflp genetic structuring only partly correlates with morphotypes .
ecotypes per se are thus not monophyletic, although there is a strong tendency for specimens and populations to cluster within the boundaries set by plant associations and their intrinsic ecologies.
is adaptation to different habitats and host plants associated with genetic divergence?
our results showed that genetic differentiation among oreina speciosissima lineages was clearly associated with plant communities .
accordingly, clustering in oreina speciosissima is well explained by differences in bedrock type and host plants  .
while specimens feeding in the petasition paradoxi association  cluster in sub-clan iia, specimens developing in the androsacion alpinae association  are restricted to sub-clan iib.
the effect of soil acidity is less striking in clan i, probably because the adenostylion and petasition officinalis associations, which are characteristic of all specimens within this clan, are defined by intermediate soil phs.
these two plant communities include species showing an intermediate tolerance to acidic-alkaline variation, such as achillea macrophylla, adenostyles alliarae and petasites albus  <cit> .
whereas the latter two represent the main host plant species of oreina speciosissima in high forbs habitat , other species  could play or have been playing the role of subalpine bridge species between the montane high forbs and alpine stone runs .
we are confident that these results are robust to potential shortcomings inherent to our limited sampling size .
first, specimens were collected throughout the common geographical range of both ecotypes, a strategy that maximized both the phylogeographic and ecological representativity of our sampling.
second, robust and consistent results were obtained using both phylogenetic and clustering algorithms.
towards a scenario of ecological speciation in alpine oreina speciosissima
although our data does not allow for divergence time estimates between oreina speciosissima ecotypes, it seems likely that they diverged relatively recently.
indeed, the current distribution of oreina populations suggest that the ecotype divergence might have arisen after one of the last glacial maxima, given that populations were probably not able to survive cold periods at high altitudes due to the presence of ice caps .
this hypothesis is consistent with the low level of genetic variation observed in nuclear sequences and the low resolution in the mtdna topology, as well as with a preliminary dating of the oreina genus, in which the origin of oreina speciosissima is estimated at circa  <dig>  million years ago  <cit> .
our results suggest that from an ancestral niche associated with high forbs  beetle populations were able to colonize new habitats along an altitudinal gradient  and invaded the acidic siliceous stone run habitat , which is typical for alpine regions in central europe.
we propose that this habitat change could have been associated with host shifting events.
accordingly, the plant communities on which oreina ecotypes feed appear to be connected by phylogenetically related host species.
in a framework of plant-insect coevolution  <cit> , adaptation to a given plant species might allow beetles to spread to other similarly-defended congeneric species  <cit> .
accordingly, doronicum species occur in the petasition paradoxi and the androsacion alpinae, petasites species link the petasition officinalis to the petasition paradoxi and finally, adenostyles species are shared among the adenostylion, the petasition paradoxi and the androsacion alpinae.
assuming host-plant conservatism, the connections described above might represent "shifting" routes that could explain how oreina speciosissima lineages transited among habitats via host switching.
furthermore, these connections could account for the presence of putatively admixed specimens showing intermediate morphologies , thereby outlining a possible ongoing migration of beetles from one habitat to the other.
CONCLUSIONS
our study reveals a genetic structure in oreina speciosissima as a function of the plant community in which beetles develop.
we discussed several possible ecological features that could cause the divergence between ecotypes, among which the habitat and host-plant switches seem key factors.
these results could be consistent with an ecological speciation scenario.
still, non-adaptive processes such as genetic drift, founder events and population bottlenecks might also have produced the observed pattern.
hence, further investigation is needed, for instance, fine scale studies relying on genomic approaches and targeting populations from a patchy distribution of the two ecotypes following an approach such as described by  <cit>  could provide a powerful framework for detecting adaptive signatures associated to ecological speciation.
additionally reciprocal transplantation experiments in concert with crossings using local and non-local beetles could possibly reveal performance differences between locally adapted and non-adapted beetles and strengthen our argument for the existence of host races and ongoing or incomplete speciation .

 the debate about the relative importance of ecological speciation in species diversification spans several decades. however, concrete cases based on empirical evidence remain relatively scarce. in essence, ecological speciation is related to the "ecological species concept", which was defined as follows: "a species is a lineage, which occupies an adaptive zone minimally different from that of any other lineage in its range and which evolves separately from all lineages outside its range". the driving force behind ecological speciation is thus divergent natural selection between environments or, in other words, reproductive isolation of populations by means of adaptation to different environments or niches. ecological selection is a consequence of individual-based interactions with the environment. from this interaction follows that divergent selection between ecological niches is a major driving force differentiating lineages until reproductive isolation occurs. ecologically divergent pairs of populations will show higher levels of reproductive incompatibility and lower levels of gene flow than ecologically more similar population pairs. a resulting corollary is that ecological speciation is more likely to arise in regions with patchworks of contrasting habitats and/or distinct environmental gradients. the number of taxa within the insect order coleoptera exceeds that of any known plant or animal group. more than half of the beetles are phytophagous, including the species rich superfamilies curculionoidea and chrysomeloidea, of which a majority feeds on angiosperms. the increase in phytophagous beetle diversity was facilitated by the rise of flowering plants. the family chrysomelidae currently consists of more than thirty-five thousand recognized species including economically important pest species such as the colorado potato beetle, the northern corn rootworm, the cereal leaf beetle, and the striped turnip flea beetle. the biological and economic importance of the superfamily chrysomeloidea make it vital to understand the factors that drive diversification in this group. here, we present a case of ecological niche differentiation in the alpine leaf beetle oreina speciosissima that may represent the early stages of ecological speciation. the genus oreina currently includes twenty-eight species, of which only seven early-diverging taxa do not exclusively occur in high forbs. according to current knowledge, the most parsimonious explanation is that high forbs vegetation is the ancestral niche for the remaining twenty-one oreina lineages, among which only our focal taxon oreina speciosissima shows a partial reversal, since it is found both in high forbs and stone run vegetation. oreina speciosissima is distributed across nearly the entire range of the genus oreina through a wide altitudinal gradient. at lower elevations it generally colonizes the very abundant high forbs vegetation whereas at higher elevations it is found in stone run habitats across a small portion of its distribution range. kippenberg and personal observations suggest that oreina speciosissima feeds exclusively on asteraceae and colonizes four distinct habitats represented by well-established plant associations: two occurring in high forbs - petasition officinalis and adenostylion - and two in stone run -androsacion alpinae on siliceous bedrock and petasition paradoxi on calcareous bedrock -. these plant communities are often patchily distributed due to the myriad of spatially proximate microclimates that occur in the alps, especially sites with calcareous bedrocks which regularly present a mosaic of microhabitats. for instance, sinkholes or dolines, formed through water erosion in so-called karstic areas represent ecological islands inhabited by high forbs vegetation surrounded by areas covered by stone run vegetation. beetles inhabiting the highly divergent habitats have been categorized in two different subspecies, namely oreina speciosissima sensu stricto and oreina speciosissima troglodytes, on the basis of differences in elytral coloration and the shape of male genitalia. oreina speciosissima sensu stricto beetles are bright metallic green or blue whereas the coloration in oreina speciosissima troglodytes is generally darker and mat. it is not known whether the morphological differences between ecotypes are in any way adaptive and/or have a genetic basis, although color patterns in another species of oreina are known to influence natural selection through predation pressure. like most other members of the genus, oreina speciosissima sensu stricto can be found feeding or mating throughout the day on, or in the vicinity of its host plants. in contrast, oreina speciosissima troglodytes is usually found adjacent to its host plants, concealed in crevices and under loose rocks. previous studies by the authors greatly challenged the existence of clear species boundaries within the genus: it is therefore realistic from a biological point of view to refer to these taxonomic entities as ecotypes rather than subspecies. leaf beetles from the genus oreina are generally thought to make only limited use of their dispersal capabilities even though kalberer et al. reported an average flight dispersal of approximately one hundred meters for oreina cacaliae beetles during autumn migration. rowell-rahier showed that low vagility in concert with a patchy host plant distribution resulted in a low level of genetic structuring in oreina speciosissima. oreina speciosissima sensu stricto beetles inhabit high forbs patches that are generally larger in size, lie closer together and harbor more beetles per unit of surface area than stone run patches inhabited by oreina speciosissima troglodytes. the present work investigates populations representative of the two ecotypes, using sequencing of nuclear and mitochondrial dna regions as well as aflp genome fingerprinting in a way to address the following questions: are ecotypes monophyletic? is adaptation to different habitats and host plants associated with genetic divergence?  phylogenetic reconstruction of the dna sequence data sets the nuclear its region showed no variation for oreina speciosissima and was thus excluded from further analyses. in contrast, the three mtdna regions were polymorphic with a total alignment length of bp; bp for 16s, for coi and bp for coii. excluding the outgroup, characters were potentially parsimony informative among variable characters. the three mtdna regions contributed as follows: 16s, coi and coii. the best substitution models were hasegawa-kishino-yano for 16s and hasegawa-kishino-yano plus gamma for coi and coii. the alignments of the mtdna markers were combined in a total evidence approach, after pairwise incongruence length difference ild test revealed no incongruence among the three mtdna markers. the resulting dataset was investigated using maximum parsimony and bayesian phylogenetic inference methods. both approaches produced highly congruent topologies with the same major nodes. the mp topology with bremer supports and corresponding bayesian posterior probabilities from the bayesian analysis are shown in figure the ingroup is well supported with a bremer support of and a bpp of. the ingroup splits into two groups, a well-supported clade containing all individuals from the gra population and a polytomy containing all other individuals. apart from a clade containing all individuals of gsb and one with two individuals from cdm and one from tsc, there is no resolution within the polytomy. sample nuf_ failed to amplify and is therefore not shown in figure only samples that rendered both satisfactory dna sequences and aflp fingerprints were used for phylogenetic analysis. aflp the aflp analysis produced a total of bands with an average of bands per individual and an average reproducibility rate of %. among variable characters, were potentially parsimony informative. just as the mtdna data, the aflp dataset was investigated using mp and bayesian phylogenetic inference methods. again, both approaches were highly congruent as the mp and bayesian trees shared the same major nodes. consequently, only the bayesian phylogeny is displayed in figure due to the lack of an outgroup, we present an unrooted topology, which led to a separation of specimens into two well supported clans sensu wilkinson et al., each with a bpp of. clan i includes three sub-clans supported with bpp values of, and respectively. clan i contains nine specimens with a strict oreina speciosissima sensu stricto morphology and six specimens with an intermediate morphology. within clan ii, two sub-clans were well supported with a bpp of and respectively. clan ii contains eight individuals with strict oreina speciosissima troglodytes morphology and five individuals with an intermediate morphology. notably, specimens with intermediate morphologies were sorted close to the midpoint root of the tree topology. the aflp dataset was further investigated using a bayesian and a distance-based clustering algorithm. the approaches produced fully congruent relationships and only results of the former are provided here. the structure analysis showed highly likely clusters when considering k values ranging between two and five. the obtained results were largely congruent across k values and highly compatible with the phylogenetic relationships. the only incongruence that could be observed when considering all k values, or when comparing structure results with the tree topology, involved specimens with an intermediate morphology. hereafter, we will consider and discuss results based on k = given that they are the most informative. when viewed from a host plant perspective it becomes apparent that all leaf beetle specimens in clan ii occur in the same stone run habitat with individuals from sub-clans iia and iib developing respectively in alkaline petasition paradoxi and in acidic androsacion alpinae habitats. in contrast all individuals from clan i occurred in high forbs, in adenostylion or petasites officinalis habitat, either on alkaline, neutral or slightly acidic soils. discussion are ecotypes monophyletic? the phylogenetic tree based on mtdna markers provides high support for the monophyly of oreina speciosissima sensu lato. however, very little polymorphism and genetic structure are revealed within the ingroup, although the mtdna markers proved variable enough to reconstruct intra-specific phylogenetic relationships in other arthropod systems. indeed, the resulting topology supports neither geographical nor ecotypic grouping of the beetles, possibly suggesting a recent divergence of oreina speciosissima lineages, with the exception of specimens from the gra population, which cluster as the sister lineage to all other specimens. beetles from this latter population thus form an orphan clade, which may correspond to an isolated refugial lineage. aflp data on the other hand shows a clear-cut differentiation of specimens. however, this pattern does not appear to have a geographical basis and instead correlates with the beetle ecotypic definition, or in other words, to the plant habitat. in contrast, aflp genetic structuring only partly correlates with morphotypes. ecotypes per se are thus not monophyletic, although there is a strong tendency for specimens and populations to cluster within the boundaries set by plant associations and their intrinsic ecologies. is adaptation to different habitats and host plants associated with genetic divergence? our results showed that genetic differentiation among oreina speciosissima lineages was clearly associated with plant communities. accordingly, clustering in oreina speciosissima is well explained by differences in bedrock type and host plants. while specimens feeding in the petasition paradoxi association cluster in sub-clan iia, specimens developing in the androsacion alpinae association are restricted to sub-clan iib. the effect of soil acidity is less striking in clan i, probably because the adenostylion and petasition officinalis associations, which are characteristic of all specimens within this clan, are defined by intermediate soil phs. these two plant communities include species showing an intermediate tolerance to acidic-alkaline variation, such as achillea macrophylla, adenostyles alliarae and petasites albus. whereas the latter two represent the main host plant species of oreina speciosissima in high forbs habitat, other species could play or have been playing the role of subalpine bridge species between the montane high forbs and alpine stone runs. we are confident that these results are robust to potential shortcomings inherent to our limited sampling size. first, specimens were collected throughout the common geographical range of both ecotypes, a strategy that maximized both the phylogeographic and ecological representativity of our sampling. second, robust and consistent results were obtained using both phylogenetic and clustering algorithms. towards a scenario of ecological speciation in alpine oreina speciosissima although our data does not allow for divergence time estimates between oreina speciosissima ecotypes, it seems likely that they diverged relatively recently. indeed, the current distribution of oreina populations suggest that the ecotype divergence might have arisen after one of the last glacial maxima, given that populations were probably not able to survive cold periods at high altitudes due to the presence of ice caps. this hypothesis is consistent with the low level of genetic variation observed in nuclear sequences and the low resolution in the mtdna topology, as well as with a preliminary dating of the oreina genus, in which the origin of oreina speciosissima is estimated at circa million years ago. our results suggest that from an ancestral niche associated with high forbs beetle populations were able to colonize new habitats along an altitudinal gradient and invaded the acidic siliceous stone run habitat, which is typical for alpine regions in central europe. we propose that this habitat change could have been associated with host shifting events. accordingly, the plant communities on which oreina ecotypes feed appear to be connected by phylogenetically related host species. in a framework of plant-insect coevolution, adaptation to a given plant species might allow beetles to spread to other similarly-defended congeneric species. accordingly, doronicum species occur in the petasition paradoxi and the androsacion alpinae, petasites species link the petasition officinalis to the petasition paradoxi and finally, adenostyles species are shared among the adenostylion, the petasition paradoxi and the androsacion alpinae. assuming host-plant conservatism, the connections described above might represent "shifting" routes that could explain how oreina speciosissima lineages transited among habitats via host switching. furthermore, these connections could account for the presence of putatively admixed specimens showing intermediate morphologies, thereby outlining a possible ongoing migration of beetles from one habitat to the other.  our study reveals a genetic structure in oreina speciosissima as a function of the plant community in which beetles develop. we discussed several possible ecological features that could cause the divergence between ecotypes, among which the habitat and host-plant switches seem key factors. these results could be consistent with an ecological speciation scenario. still, non-adaptive processes such as genetic drift, founder events and population bottlenecks might also have produced the observed pattern. hence, further investigation is needed, for instance, fine scale studies relying on genomic approaches and targeting populations from a patchy distribution of the two ecotypes following an approach such as described by could provide a powerful framework for detecting adaptive signatures associated to ecological speciation. additionally reciprocal transplantation experiments in concert with crossings using local and non-local beetles could possibly reveal performance differences between locally adapted and non-adapted beetles and strengthen our argument for the existence of host races and ongoing or incomplete speciation. 
here we specifically focus on two ecotypes of the alpine leaf beetle oreina speciosissima, which have been shown to exhibit morphological differences in male genitalia roughly corresponding to the subspecies oreina speciosissima sensu stricto and oreina speciosissima troglodytes. through use of gene sequencing and genome fingerprinting we analyzed the genetic structure and habitat use of oreina speciosissima populations from the swiss alps to examine whether the two ecotypes have a genetic basis. while little phylogenetic resolution was observed based on the sequencing of four dna regions, the topology and clustering resulting from aflp genotyping grouped specimens according to their habitat, mostly defined by plant associations. the prominent role of leaf beetles in the insect world, their omnipresence across all terrestrial biomes and their economic importance as common agricultural pest organisms make this family particularly interesting for studying the mechanisms that drive diversification. both host plants and leaf beetles have a patchy geographical distribution. these results were discussed in an ecological speciation framework. a few specimens with intermediate morphologies clustered with one of the two ecotypes or formed separate clusters consistent with habitat differences. in general the two ecotypes segregate along an elevation gradient and by host plants: oreina speciosissima sensu stricto colonizes high forb vegetation at low altitude and oreina speciosissima troglodytes is found in stone run vegetation at higher elevations.
 within the coleoptera, the largest order in the animal kingdom, the exclusively herbivorous chrysomelidae are recognized as one of the most species rich beetle families. the evolutionary processes that have fueled radiation into the more than thirty-five thousand currently recognized leaf beetle species remain partly unresolved. the prominent role of leaf beetles in the insect world, their omnipresence across all terrestrial biomes and their economic importance as common agricultural pest organisms make this family particularly interesting for studying the mechanisms that drive diversification. here we specifically focus on two ecotypes of the alpine leaf beetle oreina speciosissima, which have been shown to exhibit morphological differences in male genitalia roughly corresponding to the subspecies oreina speciosissima sensu stricto and oreina speciosissima troglodytes. in general the two ecotypes segregate along an elevation gradient and by host plants: oreina speciosissima sensu stricto colonizes high forb vegetation at low altitude and oreina speciosissima troglodytes is found in stone run vegetation at higher elevations. both host plants and leaf beetles have a patchy geographical distribution. through use of gene sequencing and genome fingerprinting we analyzed the genetic structure and habitat use of oreina speciosissima populations from the swiss alps to examine whether the two ecotypes have a genetic basis. by investigating a wide range of altitudes and focusing on the structuring effect of habitat types, we aim to provide answers regarding the factors that drive adaptive radiation in this phytophagous leaf beetle.  while little phylogenetic resolution was observed based on the sequencing of four dna regions, the topology and clustering resulting from aflp genotyping grouped specimens according to their habitat, mostly defined by plant associations. a few specimens with intermediate morphologies clustered with one of the two ecotypes or formed separate clusters consistent with habitat differences. these results were discussed in an ecological speciation framework.  the question of whether this case of ecological differentiation occurred in sympatry or allopatry remains open. still, the observed pattern points towards ongoing divergence between the two ecotypes which is likely driven by a recent shift in host plant use. 