This comprehensive analysis of sexual and asexual parasitoids of the LFG using population genetic and phylogenetic approaches provided the following main insights: (1) These aphid parasitoids represent a closely related group in which thelytoky predominates, and in which the occurrence of sexual reproduction shows a strong pattern of host association, as well as geographic variation. (2) Reproductive modes tend to be aggregated on the mitochondrial tree, as previously reported , albeit with important exceptions. (3) Nuclear genetic differentiation between reproductive modes was generally strong, but lowest for wasps collected from A. hederae, the only host on which arrhenotokous and thelytokous parasitoids commonly co-occur. (4) Nuclear genotypic diversity is very high in asexuals, indicating frequent transitions to asexuality and/or the frequent occurrence of 'cryptic sex'. (5) Nuclear differentiation among parasitoids collected from different aphids indicates host specialization. (6) Characters used traditionally in the taxonomy of LFG parasitoids poorly reflect their genetic relationships. We discuss these findings in more detail below.
Genetic relationships between reproductive modes
The incomplete genetic separation at nuclear and mitochondrial loci clearly indicates that the gene pools of sexual and asexual parasitoids of the LFG are not fully independent (Figures 1 & 4A). But how does gene flow take place between reproductive modes?
One feasible route is the experimentally demonstrated formation of new thelytokous lines via males introgressing the recessive thelytoky-inducing genetic factor into sexual populations . This field survey indeed provides evidence for contagious parthenogenesis: Few thelytokous mitochondrial haplotypes fell into a mainly sexual clade (Figure 1), and corresponding individuals were identified as the few asexual microsatellite MLGs interspersed in the large sexual clade of the NJ tree (Figure 4A). This supports the view that recessive thelytoky requires inbreeding after introgression into sexual lineages to be expressed . However, if this route was very common, it should rapidly erode any association of mitochondrial variation and reproductive mode. Hence, contagious parthenogenesis is unlikely to be the only or most important route of gene flow between reproductive modes.
An alternative route is introgression from sexuals into asexual lineages, as proposed previously . If thelytokous females mate with males from sexual populations, they may include paternal alleles rather than complete meiotic parthenogenesis . For the parasitoid Venturia canescens, which also exhibits central fusion automixis , this route of introgression has been documented and shown to strongly affect the overall genetic relationships among sexual and asexual populations [80, 81]. We suspect such genetic exchange to also occur in LFG parasitoids (Table 6 & Figure 3), as males from sexual lines readily mated with thelytokous females in the laboratory (CS, pers. obs.). However, the recessiveness of thelytoky  poses a major challenge to predict the effective direction of gene flow between reproductive modes and evolutionary outcomes in general. Both pathways of introgression would increase the frequency of the recessive thelytoky-inducing factor in sexual populations and may thus operate synergistically in elevating asexual genotypic diversity, but they cannot explain the strong genetic differentiation between reproductive modes (Figures 1 & 4A).
A third route reconciling the high genotypic diversity in asexuals with the still substantial genetic differentiation from sexuals is cryptic sex within thelytokous populations of the LFG. It also relies on the occasional production of males by thelytokous lines (i.e., male carriers of a thelytoky-inducing allele ). In most instances, the only females these males will encounter are thelytokous females. Occasional sperm incorporation via fertilization rather than automictic diploidy restoration in thelytokous females [15, 81] would then result in genetic exchange between individuals of thelytokous origin. This third mechanism of covert sex can readily maintain a high genotypic diversity in asexual populations without gene flow from sexuals, thereby allowing the buildup of genetic differentiation between the two groups, consistent with our observations and previously reported patterns .
Probably all three mechanisms of genetic exchange play a role in shaping the overall genetic architecture of the sexual-asexual LFG complex, but their relative importance is likely to differ regionally. In mixed populations and on shared hosts there appears to be ample opportunity for gene flow between reproductive modes. In purely thelytokous populations the third mechanism may be the only one available. All pathways rely on males produced by thelytokous lines being functional  and on thelytokous females being able to use sperm occasionally, for which central fusion automixis seems to be especially eligible [15, 81, 82]. This indicates that this sexual-asexual complex is an evolutionarily young system, which is also supported by the shallow mitochondrial genealogy of LFG parasitoids.
The rare but geographically widespread detection of triploid females was interesting. Such females may originate either from thelytokous females occasionally fertilizing their diploid eggs with haploid sperm, or else from sexual females fertilizing their haploid eggs with diploid sperm produced by diploid males . The fact that in our survey, triploid females were detected exclusively in all-females samples, suggests that they were produced by the former route. Nevertheless, diploid males were also detected in three cases. Diploid males are known to occur under inbreeding in sexual Hymenopterans with complementary sex determination (CSD, reviewed in ). Inbreeding increases the probability of homozygosity at the CSD locus (in species with single locus CSD) or at all CSD loci, respectively (in species with multilocus CSD), which results in male development of diploid offspring. Diploid males produced by thelytokous lineages highlight an interesting interaction that can occur between automixis and sex determination in thelytokous Hymenopterans (discussed in ). Just like inbreeding in sexuals, automixis in asexuals increases offspring homozygosity, which in turn might result in thelytokous females producing some proportion of diploid males if the CSD loci are situated in recombining regions of the genome. We have preliminary evidence that the sex determination system in LFG parasitoids corresponds to multilocus CSD, and that laboratory-generated thelytokous lines may indeed produce some diploid male offspring that are functional and able to sire daughters (CS & CV, unpublished data). In principle, such males could be efficient vehicles to spread the recessive, thelytoky-inducing allele into sexual populations, but our field data show that male production by thelytokous LFG parasitoids is generally rare, and diploid male production even rarer than haploid male production. Note that the mechanism leading to the production of haploid males (occasional failure of central fusion) is different from the mechanism we propose for diploid male production (homozygosity at CSD loci). Thus, it appears that thelytokous lineages producing a noteworthy proportion of diploid male offspring are disfavoured by selection, as expected . Clearly, the interplay between the genetic determination of thelytoky and the sex determination system as well as the role of triploids in this interesting group of parasitoids deserves further research.
Apart from the observed differentiation between reproductive modes, nuclear markers also indicated limitations to gene flow between parasitoids collected from different aphids, i.e., host-associated differentiation (HAD). This was most obvious for the exclusively sexual wasps collected from B. cardui, which were clearly differentiated from all asexual groups as well as other sexuals. Their separate status was recognized in a previous genetic investigation  and is further supported by their possession of characteristic cuticular hydrocarbon profiles . Yet, no clear divergence is evident from mitochondrial data. Haplotypes found in wasps from B. cardui were either shared with or closely related to wasps collected from Aphis hosts (Figures 1 & 2). This suggest a recent acquisition of B. cardui as a host. Further, the close similarity of B. cardui-attacking wasps from geographically distant locations indicates that this host switch did not occur independently in different regions (Figures 3 & 4A).
Other well-differentiated groups were the thelytokous wasps collected from A. farinosa and A. ruborum, and the sexual wasps from A. hederae (Table 6). This strongly indicates the evolution of host specialization in the LFG. Host fidelity due to imprinting during development is known from Aphidius parasitoids [86–88]. They prefer the same aphid-host plant assemblages on which they developed for oviposition, presumably based on olfactory cues . Lysiphlebus wasps also tend to exhibit better performance after conditioning  and they mate and oviposit very soon after emergence on or close by their natal patch . Genetic exchange between parasitoids associated with different hosts will be further restricted if higher fitness on a particular aphid host entails reduced performance on others. There is evidence for such trade-offs from host switch and selection experiments in other aphid parasitoids [88, 92]. Indeed, on certain plants, mixed host colonies of B. cardui and A. f. cirsiiacanthoidis are common, suggesting that HAD in LFG parasitoids is held up despite ample opportunities for interbreeding [93, 94]. Additional indirect evidence for trade-offs in host performance was gained by establishing laboratory cultures of field-collected wasps on A. f. fabae. Establishing wasps collected from A. f. fabae or A. f. cirsiiacanthoides is generally easy, establishing wasps from A. hederae, A. urticata and A. ruborum is more difficult but possible, and establishing wasps from A. farinosa is near-impossible (, CS & CV, unpubl.).
Regarding the close relationships among sexual Lysiphlebus affiliated with different Aphis hosts in southern France (Figure 3), little gene flow may be sufficient to erode patterns of HAD at neutral markers in sexual parasitoids [31, 92]. Yet, many of the common thelytokous MLGs had very restricted host ranges (Table 4). This indicates strong specialization which may primarily emerge in thelytokous LFG parasitoids, because a genotype that is particularly well adapted to a certain host will not be broken up by recombination. Possibly, the strong host specialization of certain genotypes is related to Lysiphlebus' strategy of chemical camouflage to avoid detection by tending ants , which might only work on a single aphid host. Nevertheless, Table 4 also shows that strongly restricted host ranges are by no means an unavoidable evolutionary outcome. Some of the most common thelytokous MLGs were collected from various hosts. The remarkable host range variation of different thelytokous lineages in the LFG clearly deserves further investigation.
Phylogeography and geographic parthenogenesis
We observed a geographic signal in the distribution of sexual and asexual populations of Lysiphlebus associated with Aphis hosts, apparently reflecting geographic parthenogenesis [95, 96]: on most hosts in northern and eastern Europe thelytokous populations dominate, while sexuals are prevalent in southern France, where they use a large host range. The fact that central European sexuals associated with A. hederae represent a subset of the haplotypic diversity of southern populations (Figure 1) suggests that glacial refuges may have been located in Mediterranean or Iberian areas. Range expansion from these regions is also indicated in gallwasps, for example . Assuming that both reproductive modes were already co-residing in former refuges, higher colonization abilities of asexuals  with subsequent monopolization of the habitats  might have influenced this pattern. Yet, present ecological forces could be relevant as well. Shorter growth seasons in more temperate regions, coupled with 'boom-and-bust' dynamics of aphid hosts may favour asexuals in balancing frequent local extinction events with stochastic recolonization . Indeed, populations associated with B. cardui indicate that in the absence of asexual competitors, sexual parasitoids prevail.
However, the phylogenetic aggregation of reproductive modes and low levels of asexual haplotype diversity in southern France (Figure 1) suggest that many thelytokous lineages residing in northern and eastern Europe originally stem from other geographic sources, not considered here. A similar pattern is indicated in V. canescens, where sexual populations are only known from southern France . Indeed, LFG parasitoids have been also reported from the Balkans, Anatolia and the Near East [8, 23, 93, 101–104], including morphologically variable sexual populations from various hosts. Some of these areas were shown to represent major hot spots of genetic diversity, e.g. in gallwasps . In that group, there is evidence that south-western Europe was colonized from Iberian refuges after the last ice age while other European populations could be traced back to south-eastern refuges [97, 105]. Assuming that Lysiphlebus exhibited similar range expansions, we strongly recommend including samples from south-eastern areas in future assays to allow more detailed inferences on phylogeographic patterns and the evolutionary history of reproductive modes.
The morphological variation in LFG parasitoids was certainly informative ecologically. The three morphotypes (Table 1) tended to be associated with certain host aphids, although these associations were rarely exclusive. Genetic analyses showed, however, that morphological variation carried little phylogenetic information, as previously suggested by Belshaw et al. . Thelytokous parasitoids of different morphotypes were widely mixed in the mitochondrial genealogy with some haplotypes found across all three morphotypes (Figure 1). Moreover, morphotypes were strongly admixed in the NJ-tree based on microsatellite genotypes (Figure 4B). On the other hand, some host associated groups of the same morphotype displayed strong nuclear divergence (Table S3 [Additional file 5]).
It appears that to a limited extent, the morphological characters used in LFG taxonomy exhibited variable expression. We had rare cases in which different individuals from the same thelytokous MLG were classified as different morphotypes (Table 4), suggesting some degree of plasticity in these traits. Generally, however, the characters underlying morphotype definitions breed true and are stably expressed over many generations in laboratory cultures (CS & CV, pers. obs.). Hence, it is likely that morphological differences among asexual lineages represent 'frozen' variation that was captured when they split from sexual, morphologically variable, source populations. Indeed, crossing experiments using sexual Lfa and Lco morphotypes indicate that the relevant traits are under nuclear genetic control [see ]. Thus, the observed variation within thelytokous populations, which may well be ecologically relevant, can also be expected to be fed by genetic exchange resulting from 'cryptic sex' as described above (Figure 4B).
Implications for taxonomy
According to current taxonomy, the three distinguishable morphotypes (Table 1) are treated as distinct species within the LFG [22, 24, 106–109]. However, taxonomists are well aware of their problematic status [8, 22]. In accordance with Belshaw et al. , we conclude that these species boundaries cannot be upheld. This is for two main reasons: First, the morphological characters used for species definitions are not phylogenetically conservative. Second, the mitochondrial sequence divergence of no more than 1.54% at COI across the whole LFG is well within what is considered a normal level of within-species variability in molecular taxonomy [51, 110]. Only the group of sexual wasps collected from B. cardui might well deserve a separate taxonomic status, as already proposed by Starý . However, this would solely be based on their nuclear differentiation and their specific host use, but could not be justified with mtDNA divergence (Figures 1 & 2). We are aware that simply challenging current taxonomic agreements does not improve this issue. With their remarkable reproductive mode variation and patterns of host specialization, these parasitoids clearly deserve further study. Treating them as a single unit under the umbrella 'Lysiphlebus fabarum group' might be the least contentious approach for the time being.
The relationships of the outgroup taxa in our mitochondrial phylogeny were largely consistent with existing phylogenies of the Aphidiinae [111–113]. The only surprise was the placement of Adialytus salicaphis between Palearctic and Nearctic representatives of the genus Lysiphlebus according to both mitochondrial genes. This is in contrast to a previous phylogeny based on the mitochondrial 16S rRNA gene . However, A. salicaphis used to be placed in the genus Lysiphlebus [e.g. ], and Sanchis et al.  also found a member of Adialytus falling inside the genus Lysiphlebus using nuclear 18S rRNA while others branched outside. It is thus recommended that the phylogenetic status of Adialytus be revisited.