BACKGROUND
ants of the northern-hemispheric, temperate genus lasius  are scientifically significant, in terms of relative abundance and ecological impact  <cit> .
because of the diversity of their signal and defense chemistry, lasius ants are organisms widely used in chemical ecology  <cit>  and the wide range of colony organisations makes the genus an ideal system for exploring social evolution  <cit> .
two further complex traits found in lasius are yet to be adequately understood: social parasitism and fungiculture.
social parasitism implies that one eusocial species depends on the labour force of another  <cit> .
the social parasitism exhibited in lasius is temporary in that it is confined to the early stages of the parasite's colony: the parasitic queen founds her colony through entering a host colony where she kills the resident queen and takes over the worker force  <cit> .
the study of social parasitism has become virtually a little discipline of entomology in itself  <cit> , but the conditions for social parasitism to arise remain poorly understood  <cit> .
social parasitism has evolved many times independently in ants  <cit> , but the evolutionary trajectories at finer systematic scale, e.g., whether it evolved once or multiply within genera, have only recently received detailed attention  <cit> .
fungiculture by ants, termites and beetles, on the other hand, provides a powerful study system for studying the origin and maintenance of mutualism  <cit> .
in ants, fungiculture has evolved independently at least twice: in attines , which culture the fungi for food, and in lasius ants, which use fungi to build composite nest walls  <cit> .
the patterns of diversification in the intensely studied attine fungiculture are only gradually starting to be understood, as brought out by recent papers on leaf-cutter fungiculture which reverse earlier impressions of certainty for some important issues  <cit> .
for inferences on the evolution of the outstanding ecological and social traits including social parasitism and fungiculture in lasius, a well-founded phylogeny of the genus is needed.
there have been three previous studies to resolve the phylogeny of lasius  <cit> , but these have disagreed with each other in significant respects .
in the present study we attempt to establish a robust phylogenetic framework for the relationships of the lasius subgenera.
we apply bayesian analysis, a powerful tool in phylogenetic reconstruction of combined data  <cit>  and not previously applied to lasius.
in addition, we also apply maximum parsimony analysis ; mp represents a completely different computational technique for phylogenetic reconstruction  <cit>  and agreement of the reconstructions by the two independent methods would increase confidence in the tree.
our approach comprised five steps.
we combined evidence from different data sets, which for many organisms, including ants, often yields a stronger phylogenetic signal than using the data sets singly  <cit> ; specifically, we combined mitochondrial dna sequence and morphological data.
we explored potential causes of distortion of the molecular phylogenetic signal, namely substitution saturation  <cit> , positive selection  <cit> , and compositional heterogeneity  <cit> .
we also assessed which morphological characters may be functionally coupled with social parasitism  <cit> , because similarities in those characters could reflect convergent adaptations to social parasitic life rather than reflect common ancestry  <cit> .
we then excluded any suspected cause of distortion in the phylogenetic reconstruction.
to address potential issues of character exclusion  <cit> , we explored the effect of excluding those morphological characters suspected to be functionally coupled with social parasitism by repeating the reconstruction when including them.
we explored whether the topologies as inferred from the different data sets are in statistically significant conflict with the phylogenetic framework inferred from the concatenated data which would tend to reduce confidence in the latter  <cit> .
our approach was to handle any conflict arising by collapsing the affected node.
we explored whether any of the previous lasius phylogenies fit our data set as well as our resulting topology.
finally, we used posterior mapping  <cit>  to define hypotheses on the evolution of social parasitism and fungiculture in lasius.
in all, the resulting topology provides a solid basis for studying the evolution of the various ecologically and sociobiologically relevant traits of lasius across subgenera.
RESULTS
we found no evidence for saturation of substitutions, neither for the 16s ribosomal rna  nor for any of the codon positions, single or in combination, of the cytochrome c oxidase subunit i  data.
tests for positive selection within the cox <dig> data indicated that none of the sites was subject to positive selection.
but we did detect compositional heterogeneity of sequences for the third codon position of cox <dig>  recoding the nucleotides of these sites to purines and pyrimidines successfully eliminated this effect and we used the recoded sequences for all phylogenetic reconstructions.
scrutinising the morphological data set for characters potentially coupled functionally with social parasitism  <cit>  yielded  <dig> characters which we hence excluded from our morphological data set.
the final data set consisted of  <dig> samples of  <dig> species including three outgroup species  for which a total of  <dig>  base pairs , and  <dig> morphological characters were used for phylogenetic reconstruction .
voucher specimens have been deposited at the national science museum, tokyo, with the numbers indicated and a reference to this publication .
abbreviations of lasius subgenera are acanthomyops , austrolasius , cautolasius , chthonolasius , dendrolasius , lasius sensu stricto .
subgenus was not applicable  for the outgroup taxa, myrmecocystus mimicus and m. mendux, and formica japonica.
ry purine + pyrimidine coding, otherwise the original nucleotide sequence was used.
"-" under model selection indicates that aic and hlrt model selection were not applicable for the partition.
topology, branch lengths, and bayesian posterior probabilities of the markov chain monte carlo  analyses of the concatenated data  are given in fig.
<dig>  monophyly of all subgenera was strongly supported , as were all nodes defining subgenus relationships , with exception of one , connecting cautolasius to .
strictly applying a cutoff for node support of >  <dig> , we retrieved two major lineages,  + chthonolasius) + lasius pallitarsis) and , but the position of cautolasius relative to these two lineages remains unresolved.
the three individual data set phylogenies  differed considerably in phylogenetic resolution at subgenus level and above and none of them achieved resolution of all nodes scored by the concatenated data reconstruction.
repeating reconstruction of cox <dig> without the third codon position did not yield any well supported nodes that contradicted well supported nodes in the reconstruction using all three codon positions, which confirms the reliability of the result from the applied test for substitution saturation.
comparison of the individual data set topologies with the concatenated data topology at subgenus level and above revealed not a single significant  disagreement but rather agreement on well supported nodes.
the following nodes of the concatenated topology were supported by the individual data set topologies: cox <dig> - cautolasius monophyly, chthonolasius monophyly, and monophyly of the two myrmecocystus outgroup species; 16s rrna - cautolasius monophyly, , and ); morphology - dendrolasius monophyly and .
including those morphological characters suspected to be functionally coupled with social parasitism, which we therefore had excluded before, resulted in an identical topology, with very similar node support values , but with a decrease of the value for the node connecting cautolasius to  from  <dig>  to  <dig> .
mp reconstructions based on the concatenated data set significantly  confirmed all significant nodes of the bayesian reconstructions, including retrieving as significant the node connecting cautolasius to .
this pattern persisted in the mp analysis when using the concatenated data including those morphological characters suspected to be functionally coupled with social parasitism, with one exception: the node connecting dendrolasius to lasius sensu stricto was then no longer supported .
considering the lack of significant disagreement between the signals in the individual data set phylogenies and the confirmation of the bayesian topology by maximum parsimony reconstructions, we regard the bayesian concatenated data topology as a phylogenetic framework sufficiently robust for use in subsequent evolutionary hypotheses .
subsequently, we also made a bayesian reconstruction of the concatenated molecular data sets , without any morphological data, to allow for dna based estimates of branch length.
this tree  was in large agreement with the other bayesian trees, in topology and branch lengths, but in addition to the node connecting cautolasius to , three previously well supported nodes now lacked significant support ; lasius sensu stricto monophyly).
in additional rounds of reconstruction, we enforced the subgenus relationships recovered in three previous phylogenetic analyses  <cit>  as constraints  on our concatenated data.
all reconstructions with constraints had lower likelihood values than the reconstruction without constraint  and, on the basis of our concatenated data, bayes factor analysis revealed very strong evidence against all previously suggested subgenus relationships .
ngens  and burnin are given in units of a million; ar and hr refer to the arithmetic and harmonic means, averaged over the simultaneous runs; asdsf = average standard deviation of split frequencies; 99% refers to the number of trees sampled from the 99% credible set.
summary of bayes factor comparisons and interpretation after  <cit> .
the bayesian posterior probabilities for the occurrence of social parasitism and fungiculture at the nodes of the phylogenetic framework above subgenus level  are shown in table  <dig>  for eight of the ten possible inferences a state was significantly inferred , for the remaining two the probability values were  <dig>  and  <dig> .
"no" indicates absence, "yes" indicates presence; the posterior probabilities were estimated using simmap and the last  <dig> post-burnin trees of the  <dig>  used to derive the consensus tree of fig.
<dig>  significantly positive values  are given in bold.
node numbers refer to those shown in fig.
<dig>
discussion and
CONCLUSIONS
the new phylogenetic framework
the new phylogenetic framework we present clarifies the relationships of all but one subgenus, namely the position of cautolasius relative to the other subgenera: mp reconstructions significantly support the topology, cautolasius + , and bayesian reconstructions do not contradict it.
given the lack of significant support in the bayesian tree, we nevertheless subsequently refrain from considering the node as resolved .
this lack of significant resolution does not affect considerations on the evolution of social parasitism and fungiculture, though, because the ancestral state of lasius was absence of both traits , and because also cautolasius displays neither trait.
a significantly supported sister-status to  thus would not alter any conclusions .
the previous phylogenies were recovered by various methods and we discuss below how the methods applied may have influenced the respective results.
taken together, the reasons for the increased robustness that we postulate for the new phylogenetic framework, which robustness is highlighted also by the confirmation of our bayesian topology by our mp reconstructions, may be that  we excluded potential causes of phylogenetic distortion and  used additional analysis methods, that  the phylogenetic signal of combined data is potentially stronger than that of individual data sets  <cit> , especially when applying bayesian inference  <cit> , that  all nodes but one at subgenus level or above were significantly supported in our concatenated analysis , and that  we did not recover any disagreement between the individual data set phylogenies and the concatenated data topologies.
nevertheless, because the molecular data of this paper all derive from mitochondrial dna, we cannot absolutely exclude that the tree is influenced by introgression at very shallow levels.
nuclear pseudogenes are also a concern, but we consider it unlikely that we amplified these because neither reading frame shifts nor sequence ambiguities were apparent.
the possibilities of incomplete lineage sorting and of selection driven by symbionts which are in disequilibrium with mtdna  <cit>  cannot be ruled out, but such effects are unlikely to confound clade history at a deeper level, such as that of subgenera.
it remains true that future studies using multiple nuclear genes are desirable to confirm our findings.
the new phylogenetic framework presented here confirms the monophyly of the six subgenera.
the new framework also affirms the taxonomic placement of acanthomyops as a subgenus of lasius  <cit> .
moreover, there is additional evidence for treating lasius pallitarsis as a separate, monotypic subgenus, as suggested earlier  <cit> .
detailed morphological characterisation of the new subgenus and taxonomic implications will be followed up elsewhere in the frame of a formal taxonomic revision.
the evolution of the lasius subgenera occurred in two major lineages, the first lineage comprising acanthomyops, austrolasius, chthonolasius, and lasius pallitarsis, and the second lineage comprising dendrolasius, and lasius sensu stricto, with cautolasius probably belonging to the second lineage .
within the first lineage acanthomyops and austrolasius form a crown-group which is sister to chthonolasius.
comparison with previous phylogenies
the new framework disagrees with previous topologies  <cit> .
given that we actively sought to exclude potential factors causing phylogenetic distortion from our data, and that the previous phylogenies disagreed with each other , we suggest that where our topology disagrees with previous ones the new one is preferable .
examination of the reasons for disagreements between the new topology and previous schemes is desirable and we now address this.
the phylogeny by wilson published in  <dig>  <cit>   is the only one of the previous phylogenies that hypothesised the existence of two major lineages as recovered in the new framework.
in terms of subgenera, the two w <dig> lineages agree with those of the new framework with the exception of lasius pallitarsis .
however, there are disagreements within the two lineages.
within the first lineage, the situation is ambiguous because austrolasius, found in the new framework to be sister to acanthomyops, was treated as part of chthonolasius.
within the second lineage, the relations differ in that lasius sensu stricto is considered sister to cautolasius in w <dig>  whereas it is sister to dendrolasius in the new framework.
reasons for the disagreements could include that w <dig> is based on morphological information only, and that it lacked a formal reconstruction algorithm.
in the phylogeny by hasegawa of  <dig>  <cit>  , there was only one significantly supported  subgenus relationship, i.e., that cautolasius, chthonolasius, and lasius sensu stricto form a crown-group sister to dendrolasius, and this is not supported by the new framework.
reasons for the disagreement could include that h <dig> had very limited taxon sampling, and that no check for compositional heterogeneity was undertaken: the reconstruction methods applied  could also contribute as the genetic distance based neighbour joining reconstruction is known to have the limitation that rate variation among sites cannot be accurately accounted for  <cit> , and as mp ignores the possible existence of a range of alternative topologies that are not significantly less likely than the most parsimonious one, even though they would require more evolutionary changes  <cit> .
the subgenus relationships recovered by janda and coworkers in  <dig>  <cit>  , placing lasius sensu stricto in a sister clade to the rest, and within this clade lasius pallitarsis sister to the rest, are not supported by the new framework.
however, our data confirm two important aspects of j <dig>  the allocation of acanthomyops to the genus lasius, and the discovery that lasius pallitarsis does not belong to lasius sensu stricto or any other of the established subgenera.
reasons for the disagreements with the new framework could include that the dna data of j <dig> add up to a total of only  <dig> bp and that no check for compositional heterogeneity was undertaken.
moreover, while the combination of molecular with other data by janda et al.
marks an advance in the history of lasius phylogeny, the morphological data included  <dig> characters  that, as indicated by other studies  <cit> , may possibly be coupled functionally with social parasitism.
scrutiny in morphological character selection was recently confirmed as crucial in phylogenetic reconstruction  <cit> .
also, the occurrence of social parasitism itself was included as character.
these characters might contribute to grouping all parasitic subgenera together in an unresolved crown group as indeed is the case in j <dig> , whereas they were distributed over the two major lineages in the new framework.
the phylogenetic reconstruction methods applied  could possibly have contributed to suboptimal reconstruction as compared to bayesian inference, especially for combined data sets, demonstrated after the publication of janda et al.
<cit> .
possibly none of these various causes had a strong effect per se, as suggested by rather minor differences between the bayesian trees based on the concatenated data excluding versus including those morphological characters suspected to be coupled with social parasitism as well as between the bayesian and the mp trees when excluding those morphological characters .
the various causes may, however, have added up to a significant effect, as is suggested by the lack of significant support for the sister-group relationship in the mp tree when including those morphological characters , which node scored a value of  <dig>  in the bayesian tree independently of excluding or including the characters.
hypotheses on the evolution of social parasitism and fungiculture
the new phylogenetic framework  and the reconstructed ancestral states  suggest that both social parasitism and fungiculture evolved two times independently within lasius, once in each of the major lineages .
for social parasitism such parallel evolution within a monophyletic group is not surprising in general.
it has long been established that the combination of certain traits resulted in a predisposition for social parasitism in two  of the  <dig>  <cit>  ant subfamilies  <cit> .
evidence that social parasitism also evolved multiple times within tribes  <cit>  or within genera  <cit>  confirms the principle for lower taxonomic levels.
having a reliable phylogenetic framework for lasius facilitates examination of temporary social parasitism, considered less derived than other types of social parasitism  <cit> .
one factor that may have favoured the rise of parasitism in lasius may derive from their colony organisation: in several species of the non-parasitic subgenera, lasius sensu stricto and cautolasius, the lack of aggression between different single-queened colonies has been reported  <cit> .
whereas this behaviour concerns intraspecific interactions, the predisposition to reduce aggression may have been important for social parasitism to arise.
the paramount significance of reduced aggression is illustrated by the chemical disguise of founding queens of chthonolasius to appease host workers  <cit>  and the exceptional winter activity of l.  mixtus to ease entry to the then less aggressive host colonies  <cit> .
characterisation of such potentially preadaptive traits and search for them in extant non-parasitic lasius species might help in finding potential early stages of incipient social parasitism.
such discoveries would then contribute to resolving mechanisms in the evolution of social parasitism in general  <cit> .
fungiculture on the other hand only is known from one other group of ants, the attini .
it is established that fungiculture arose only once in attines  <cit>  and the lasius situation might thus indicate a stronger predisposition to evolve fungiculture for ants generally.
for the evolution of both fungiculture and social parasites two pairs of alternative hypotheses remain , both concerning the lineage of )).
this is because for both traits the probability value for the ancestral state for one node was below  <dig>  .
for each trait there is one scenario considering an earlier origin with subsequent loss in the respective lineage, and the other considering an origin at a later stage in the respective lineage.
concerning both traits the hypothesis of a reversal is less likely from the point of view of biological realism because it is less parsimonious, and also because the probability values for the respective nodes  indicate, though not significantly, that social parasitism and fungiculture had not yet evolved at those stages .
still, we cannot formally refute the reversal scenarios here, given the lack of statistical support, and briefly discuss them in the below.
for social parasitism, a reversal has been considered generally unlikely  <cit> .
on the other hand, reversal from temporary social parasitism may be more likely than reversal from any other, more derived type of social parasitism.
for example, temporarily socially parasitic ant species of formica sensu stricto that are capable of alternative nest foundation through colony budding or adoption into another colony show a certain flexibility in the nest foundation mode in this type of social parasitism  <cit> .
on the other hand, the fact that acanthomyops, austrolasius, and chthonolasius form an entirely socially parasitic clade shows that temporary social parasitism may in fact be a stable trait and makes reversal appear less likely.
once arisen, fungiculture is not known to have been lost in insects and loss has been regarded as improbable  <cit> , which provides an additional argument against the scenario involving a loss in lasius.
it may, however, be more probable in the lasius case: fungiculture for nest building is probably less likely to entail a high dependence of the ants as compared to fungiculture for nutrition, which involves physiological adaptations  <cit> .
it is not possible to definitely decide in favour of any of the alternative hypotheses at present, neither concerning social parasitism nor fungiculture, but it is worth considering the question further because a plausible loss of either would be of considerable evolutionary significance.
for both traits the fossil record may offer answers in combination with a sound molecular clock for lasius, for social parasitism by revealing the queen morphology of the ancestor of the entire major lineage, for fungiculture by a preserved fungal nest structure.
laboratory experiments may also be helpful for the analysis of both traits, for social parasitism by checking whether any of the temporary social parasites of the lineage are capable of independent colony foundation, and for fungiculture whether chthonolasius colonies can be maintained when deprived of their fungus.
this study provides a basis for studying the evolution of the various ecologically and sociobiologically relevant traits across the ca.
<dig>  <cit>  species of lasius, by establishing a phylogenetic framework and resolving the position of six of the seven lineages at subgenus level.
we have used the framework to define hypotheses of the evolution of two outstanding traits, social parasitism and fungiculture, the evolution of which continues to pose riddles to evolutionary biology.
our results suggest that both traits arose twice in lasius which opens up new opportunities for comparative analyses in a close phylogenetic relationship.
we present competitive hypotheses that either do or do not involve reversal from the traits.

 ants of the northern-hemispheric, temperate genus lasius are scientifically significant, in terms of relative abundance and ecological impact. because of the diversity of their signal and defense chemistry, lasius ants are organisms widely used in chemical ecology and the wide range of colony organisations makes the genus an ideal system for exploring social evolution. two further complex traits found in lasius are yet to be adequately understood: social parasitism and fungiculture. social parasitism implies that one eusocial species depends on the labour force of another. the social parasitism exhibited in lasius is temporary in that it is confined to the early stages of the parasite's colony: the parasitic queen founds her colony through entering a host colony where she kills the resident queen and takes over the worker force. the study of social parasitism has become virtually a little discipline of entomology in itself, but the conditions for social parasitism to arise remain poorly understood. social parasitism has evolved many times independently in ants, but the evolutionary trajectories at finer systematic scale, e.g., whether it evolved once or multiply within genera, have only recently received detailed attention. fungiculture by ants, termites and beetles, on the other hand, provides a powerful study system for studying the origin and maintenance of mutualism. in ants, fungiculture has evolved independently at least twice: in attines, which culture the fungi for food, and in lasius ants, which use fungi to build composite nest walls. the patterns of diversification in the intensely studied attine fungiculture are only gradually starting to be understood, as brought out by recent papers on leaf-cutter fungiculture which reverse earlier impressions of certainty for some important issues. for inferences on the evolution of the outstanding ecological and social traits including social parasitism and fungiculture in lasius, a well-founded phylogeny of the genus is needed. there have been three previous studies to resolve the phylogeny of lasius, but these have disagreed with each other in significant respects. in the present study we attempt to establish a robust phylogenetic framework for the relationships of the lasius subgenera. we apply bayesian analysis, a powerful tool in phylogenetic reconstruction of combined data and not previously applied to lasius. in addition, we also apply maximum parsimony analysis; mp represents a completely different computational technique for phylogenetic reconstruction and agreement of the reconstructions by the two independent methods would increase confidence in the tree. our approach comprised five steps. we combined evidence from different data sets, which for many organisms, including ants, often yields a stronger phylogenetic signal than using the data sets singly; specifically, we combined mitochondrial dna sequence and morphological data. we explored potential causes of distortion of the molecular phylogenetic signal, namely substitution saturation, positive selection, and compositional heterogeneity. we also assessed which morphological characters may be functionally coupled with social parasitism, because similarities in those characters could reflect convergent adaptations to social parasitic life rather than reflect common ancestry. we then excluded any suspected cause of distortion in the phylogenetic reconstruction. to address potential issues of character exclusion, we explored the effect of excluding those morphological characters suspected to be functionally coupled with social parasitism by repeating the reconstruction when including them. we explored whether the topologies as inferred from the different data sets are in statistically significant conflict with the phylogenetic framework inferred from the concatenated data which would tend to reduce confidence in the latter. our approach was to handle any conflict arising by collapsing the affected node. we explored whether any of the previous lasius phylogenies fit our data set as well as our resulting topology. finally, we used posterior mapping to define hypotheses on the evolution of social parasitism and fungiculture in lasius. in all, the resulting topology provides a solid basis for studying the evolution of the various ecologically and sociobiologically relevant traits of lasius across subgenera.  we found no evidence for saturation of substitutions, neither for the 16s ribosomal rna nor for any of the codon positions, single or in combination, of the cytochrome c oxidase subunit i data. tests for positive selection within the cox data indicated that none of the sites was subject to positive selection. but we did detect compositional heterogeneity of sequences for the third codon position of cox recoding the nucleotides of these sites to purines and pyrimidines successfully eliminated this effect and we used the recoded sequences for all phylogenetic reconstructions. scrutinising the morphological data set for characters potentially coupled functionally with social parasitism yielded characters which we hence excluded from our morphological data set. the final data set consisted of samples of species including three outgroup species for which a total of base pairs, and morphological characters were used for phylogenetic reconstruction. voucher specimens have been deposited at the national science museum, tokyo, with the numbers indicated and a reference to this publication. abbreviations of lasius subgenera are acanthomyops, austrolasius, cautolasius, chthonolasius, dendrolasius, lasius sensu stricto. subgenus was not applicable for the outgroup taxa, myrmecocystus mimicus and m. mendux, and formica japonica. ry purine + pyrimidine coding, otherwise the original nucleotide sequence was used. "-" under model selection indicates that aic and hlrt model selection were not applicable for the partition. topology, branch lengths, and bayesian posterior probabilities of the markov chain monte carlo analyses of the concatenated data are given in fig. monophyly of all subgenera was strongly supported, as were all nodes defining subgenus relationships, with exception of one, connecting cautolasius to. strictly applying a cutoff for node support of >, we retrieved two major lineages, + chthonolasius) + lasius pallitarsis) and, but the position of cautolasius relative to these two lineages remains unresolved. the three individual data set phylogenies differed considerably in phylogenetic resolution at subgenus level and above and none of them achieved resolution of all nodes scored by the concatenated data reconstruction. repeating reconstruction of cox without the third codon position did not yield any well supported nodes that contradicted well supported nodes in the reconstruction using all three codon positions, which confirms the reliability of the result from the applied test for substitution saturation. comparison of the individual data set topologies with the concatenated data topology at subgenus level and above revealed not a single significant disagreement but rather agreement on well supported nodes. the following nodes of the concatenated topology were supported by the individual data set topologies: cox - cautolasius monophyly, chthonolasius monophyly, and monophyly of the two myrmecocystus outgroup species; 16s rrna - cautolasius monophyly,, and ); morphology - dendrolasius monophyly and. including those morphological characters suspected to be functionally coupled with social parasitism, which we therefore had excluded before, resulted in an identical topology, with very similar node support values, but with a decrease of the value for the node connecting cautolasius to from to. mp reconstructions based on the concatenated data set significantly confirmed all significant nodes of the bayesian reconstructions, including retrieving as significant the node connecting cautolasius to. this pattern persisted in the mp analysis when using the concatenated data including those morphological characters suspected to be functionally coupled with social parasitism, with one exception: the node connecting dendrolasius to lasius sensu stricto was then no longer supported. considering the lack of significant disagreement between the signals in the individual data set phylogenies and the confirmation of the bayesian topology by maximum parsimony reconstructions, we regard the bayesian concatenated data topology as a phylogenetic framework sufficiently robust for use in subsequent evolutionary hypotheses. subsequently, we also made a bayesian reconstruction of the concatenated molecular data sets, without any morphological data, to allow for dna based estimates of branch length. this tree was in large agreement with the other bayesian trees, in topology and branch lengths, but in addition to the node connecting cautolasius to, three previously well supported nodes now lacked significant support; lasius sensu stricto monophyly). in additional rounds of reconstruction, we enforced the subgenus relationships recovered in three previous phylogenetic analyses as constraints on our concatenated data. all reconstructions with constraints had lower likelihood values than the reconstruction without constraint and, on the basis of our concatenated data, bayes factor analysis revealed very strong evidence against all previously suggested subgenus relationships. ngens and burnin are given in units of a million; ar and hr refer to the arithmetic and harmonic means, averaged over the simultaneous runs; asdsf = average standard deviation of split frequencies; 99% refers to the number of trees sampled from the 99% credible set. summary of bayes factor comparisons and interpretation after. the bayesian posterior probabilities for the occurrence of social parasitism and fungiculture at the nodes of the phylogenetic framework above subgenus level are shown in table for eight of the ten possible inferences a state was significantly inferred, for the remaining two the probability values were and. "no" indicates absence, "yes" indicates presence; the posterior probabilities were estimated using simmap and the last post-burnin trees of the used to derive the consensus tree of fig. significantly positive values are given in bold. node numbers refer to those shown in fig. discussion and  the new phylogenetic framework the new phylogenetic framework we present clarifies the relationships of all but one subgenus, namely the position of cautolasius relative to the other subgenera: mp reconstructions significantly support the topology, cautolasius +, and bayesian reconstructions do not contradict it. given the lack of significant support in the bayesian tree, we nevertheless subsequently refrain from considering the node as resolved. this lack of significant resolution does not affect considerations on the evolution of social parasitism and fungiculture, though, because the ancestral state of lasius was absence of both traits, and because also cautolasius displays neither trait. a significantly supported sister-status to thus would not alter any conclusions. the previous phylogenies were recovered by various methods and we discuss below how the methods applied may have influenced the respective results. taken together, the reasons for the increased robustness that we postulate for the new phylogenetic framework, which robustness is highlighted also by the confirmation of our bayesian topology by our mp reconstructions, may be that we excluded potential causes of phylogenetic distortion and used additional analysis methods, that the phylogenetic signal of combined data is potentially stronger than that of individual data sets, especially when applying bayesian inference, that all nodes but one at subgenus level or above were significantly supported in our concatenated analysis, and that we did not recover any disagreement between the individual data set phylogenies and the concatenated data topologies. nevertheless, because the molecular data of this paper all derive from mitochondrial dna, we cannot absolutely exclude that the tree is influenced by introgression at very shallow levels. nuclear pseudogenes are also a concern, but we consider it unlikely that we amplified these because neither reading frame shifts nor sequence ambiguities were apparent. the possibilities of incomplete lineage sorting and of selection driven by symbionts which are in disequilibrium with mtdna cannot be ruled out, but such effects are unlikely to confound clade history at a deeper level, such as that of subgenera. it remains true that future studies using multiple nuclear genes are desirable to confirm our findings. the new phylogenetic framework presented here confirms the monophyly of the six subgenera. the new framework also affirms the taxonomic placement of acanthomyops as a subgenus of lasius. moreover, there is additional evidence for treating lasius pallitarsis as a separate, monotypic subgenus, as suggested earlier. detailed morphological characterisation of the new subgenus and taxonomic implications will be followed up elsewhere in the frame of a formal taxonomic revision. the evolution of the lasius subgenera occurred in two major lineages, the first lineage comprising acanthomyops, austrolasius, chthonolasius, and lasius pallitarsis, and the second lineage comprising dendrolasius, and lasius sensu stricto, with cautolasius probably belonging to the second lineage. within the first lineage acanthomyops and austrolasius form a crown-group which is sister to chthonolasius. comparison with previous phylogenies the new framework disagrees with previous topologies. given that we actively sought to exclude potential factors causing phylogenetic distortion from our data, and that the previous phylogenies disagreed with each other, we suggest that where our topology disagrees with previous ones the new one is preferable. examination of the reasons for disagreements between the new topology and previous schemes is desirable and we now address this. the phylogeny by wilson published in is the only one of the previous phylogenies that hypothesised the existence of two major lineages as recovered in the new framework. in terms of subgenera, the two w lineages agree with those of the new framework with the exception of lasius pallitarsis. however, there are disagreements within the two lineages. within the first lineage, the situation is ambiguous because austrolasius, found in the new framework to be sister to acanthomyops, was treated as part of chthonolasius. within the second lineage, the relations differ in that lasius sensu stricto is considered sister to cautolasius in w whereas it is sister to dendrolasius in the new framework. reasons for the disagreements could include that w is based on morphological information only, and that it lacked a formal reconstruction algorithm. in the phylogeny by hasegawa of, there was only one significantly supported subgenus relationship, i.e., that cautolasius, chthonolasius, and lasius sensu stricto form a crown-group sister to dendrolasius, and this is not supported by the new framework. reasons for the disagreement could include that h had very limited taxon sampling, and that no check for compositional heterogeneity was undertaken: the reconstruction methods applied could also contribute as the genetic distance based neighbour joining reconstruction is known to have the limitation that rate variation among sites cannot be accurately accounted for, and as mp ignores the possible existence of a range of alternative topologies that are not significantly less likely than the most parsimonious one, even though they would require more evolutionary changes. the subgenus relationships recovered by janda and coworkers in, placing lasius sensu stricto in a sister clade to the rest, and within this clade lasius pallitarsis sister to the rest, are not supported by the new framework. however, our data confirm two important aspects of j the allocation of acanthomyops to the genus lasius, and the discovery that lasius pallitarsis does not belong to lasius sensu stricto or any other of the established subgenera. reasons for the disagreements with the new framework could include that the dna data of j add up to a total of only bp and that no check for compositional heterogeneity was undertaken. moreover, while the combination of molecular with other data by janda et al. marks an advance in the history of lasius phylogeny, the morphological data included characters that, as indicated by other studies, may possibly be coupled functionally with social parasitism. scrutiny in morphological character selection was recently confirmed as crucial in phylogenetic reconstruction. also, the occurrence of social parasitism itself was included as character. these characters might contribute to grouping all parasitic subgenera together in an unresolved crown group as indeed is the case in j, whereas they were distributed over the two major lineages in the new framework. the phylogenetic reconstruction methods applied could possibly have contributed to suboptimal reconstruction as compared to bayesian inference, especially for combined data sets, demonstrated after the publication of janda et al.. possibly none of these various causes had a strong effect per se, as suggested by rather minor differences between the bayesian trees based on the concatenated data excluding versus including those morphological characters suspected to be coupled with social parasitism as well as between the bayesian and the mp trees when excluding those morphological characters. the various causes may, however, have added up to a significant effect, as is suggested by the lack of significant support for the sister-group relationship in the mp tree when including those morphological characters, which node scored a value of in the bayesian tree independently of excluding or including the characters. hypotheses on the evolution of social parasitism and fungiculture the new phylogenetic framework and the reconstructed ancestral states suggest that both social parasitism and fungiculture evolved two times independently within lasius, once in each of the major lineages. for social parasitism such parallel evolution within a monophyletic group is not surprising in general. it has long been established that the combination of certain traits resulted in a predisposition for social parasitism in two of the ant subfamilies. evidence that social parasitism also evolved multiple times within tribes or within genera confirms the principle for lower taxonomic levels. having a reliable phylogenetic framework for lasius facilitates examination of temporary social parasitism, considered less derived than other types of social parasitism. one factor that may have favoured the rise of parasitism in lasius may derive from their colony organisation: in several species of the non-parasitic subgenera, lasius sensu stricto and cautolasius, the lack of aggression between different single-queened colonies has been reported. whereas this behaviour concerns intraspecific interactions, the predisposition to reduce aggression may have been important for social parasitism to arise. the paramount significance of reduced aggression is illustrated by the chemical disguise of founding queens of chthonolasius to appease host workers and the exceptional winter activity of l. mixtus to ease entry to the then less aggressive host colonies. characterisation of such potentially preadaptive traits and search for them in extant non-parasitic lasius species might help in finding potential early stages of incipient social parasitism. such discoveries would then contribute to resolving mechanisms in the evolution of social parasitism in general. fungiculture on the other hand only is known from one other group of ants, the attini. it is established that fungiculture arose only once in attines and the lasius situation might thus indicate a stronger predisposition to evolve fungiculture for ants generally. for the evolution of both fungiculture and social parasites two pairs of alternative hypotheses remain, both concerning the lineage of )). this is because for both traits the probability value for the ancestral state for one node was below. for each trait there is one scenario considering an earlier origin with subsequent loss in the respective lineage, and the other considering an origin at a later stage in the respective lineage. concerning both traits the hypothesis of a reversal is less likely from the point of view of biological realism because it is less parsimonious, and also because the probability values for the respective nodes indicate, though not significantly, that social parasitism and fungiculture had not yet evolved at those stages. still, we cannot formally refute the reversal scenarios here, given the lack of statistical support, and briefly discuss them in the below. for social parasitism, a reversal has been considered generally unlikely. on the other hand, reversal from temporary social parasitism may be more likely than reversal from any other, more derived type of social parasitism. for example, temporarily socially parasitic ant species of formica sensu stricto that are capable of alternative nest foundation through colony budding or adoption into another colony show a certain flexibility in the nest foundation mode in this type of social parasitism. on the other hand, the fact that acanthomyops, austrolasius, and chthonolasius form an entirely socially parasitic clade shows that temporary social parasitism may in fact be a stable trait and makes reversal appear less likely. once arisen, fungiculture is not known to have been lost in insects and loss has been regarded as improbable, which provides an additional argument against the scenario involving a loss in lasius. it may, however, be more probable in the lasius case: fungiculture for nest building is probably less likely to entail a high dependence of the ants as compared to fungiculture for nutrition, which involves physiological adaptations. it is not possible to definitely decide in favour of any of the alternative hypotheses at present, neither concerning social parasitism nor fungiculture, but it is worth considering the question further because a plausible loss of either would be of considerable evolutionary significance. for both traits the fossil record may offer answers in combination with a sound molecular clock for lasius, for social parasitism by revealing the queen morphology of the ancestor of the entire major lineage, for fungiculture by a preserved fungal nest structure. laboratory experiments may also be helpful for the analysis of both traits, for social parasitism by checking whether any of the temporary social parasites of the lineage are capable of independent colony foundation, and for fungiculture whether chthonolasius colonies can be maintained when deprived of their fungus. this study provides a basis for studying the evolution of the various ecologically and sociobiologically relevant traits across the ca. species of lasius, by establishing a phylogenetic framework and resolving the position of six of the seven lineages at subgenus level. we have used the framework to define hypotheses of the evolution of two outstanding traits, social parasitism and fungiculture, the evolution of which continues to pose riddles to evolutionary biology. our results suggest that both traits arose twice in lasius which opens up new opportunities for comparative analyses in a close phylogenetic relationship. we present competitive hypotheses that either do or do not involve reversal from the traits. 
both social parasitism and fungiculture evolved twice in lasius, once in each major lineage, which opens up new opportunities for comparative analyses. possible causes for previous problems with reconstructing the lasius phylogeny include use of other reconstruction techniques, possibly more prone to instabilities in some instances, and the inclusion of phylogenetically distorting characters. one includes acanthomyops, austrolasius, chthonolasius, and lasius pallitarsis, which we confirm to represent a seventh subgenus, the other clade contains dendrolasius, and lasius sensu stricto. the subgenus cautolasius, displaying neither social parasitism nor fungiculture, probably belongs to the second clade, but its phylogenetic position is not resolved at the cutoff values of node support we apply. here we employed two mitochondrial genes, comprising bp, together with morphological characters, to recover the phylogeny of lasius by bayesian and maximum parsimony inference after exploration of potential causes of phylogenetic distortion. by establishing an updated phylogenetic framework, our study provides the basis for a later formal taxonomic revision of subgenera and for studying the evolution of various ecologically and sociobiologically relevant traits of lasius, although there is need for future studies to include nuclear genes and additional samples from the nearctic. the repeated evolution of social parasitism has been established for other groups of ants, though not for temporary social parasitism as found in lasius. ants of the genus lasius are ecologically important and an important system for evolutionary research. we use the resulting framework to infer evolutionary pathways for social parasitism and fungiculture. we recovered two well supported major lineages.
 ants of the genus lasius are ecologically important and an important system for evolutionary research. progress in evolutionary research has been hindered by the lack of a well-founded phylogeny of the subgenera, with three previous attempts disagreeing. here we employed two mitochondrial genes, comprising bp, together with morphological characters, to recover the phylogeny of lasius by bayesian and maximum parsimony inference after exploration of potential causes of phylogenetic distortion. we use the resulting framework to infer evolutionary pathways for social parasitism and fungiculture.  we recovered two well supported major lineages. one includes acanthomyops, austrolasius, chthonolasius, and lasius pallitarsis, which we confirm to represent a seventh subgenus, the other clade contains dendrolasius, and lasius sensu stricto. the subgenus cautolasius, displaying neither social parasitism nor fungiculture, probably belongs to the second clade, but its phylogenetic position is not resolved at the cutoff values of node support we apply. possible causes for previous problems with reconstructing the lasius phylogeny include use of other reconstruction techniques, possibly more prone to instabilities in some instances, and the inclusion of phylogenetically distorting characters.  by establishing an updated phylogenetic framework, our study provides the basis for a later formal taxonomic revision of subgenera and for studying the evolution of various ecologically and sociobiologically relevant traits of lasius, although there is need for future studies to include nuclear genes and additional samples from the nearctic. both social parasitism and fungiculture evolved twice in lasius, once in each major lineage, which opens up new opportunities for comparative analyses. the repeated evolution of social parasitism has been established for other groups of ants, though not for temporary social parasitism as found in lasius. for fungiculture, the independent emergence twice in a monophyletic group marks a novel scenario in ants. we present alternative hypotheses for the evolution of both traits, with one of each involving loss of the trait. though less likely for both traits than later evolution without reversal, we consider reversal as sufficiently plausible to merit independent testing. 