The evolution of host associations in the parasitic wasp genus Ichneumon (Hymenoptera: Ichneumonidae): convergent adaptations to host pupation sites
© Tschopp et al.; licensee BioMed Central Ltd. 2013
Received: 7 August 2012
Accepted: 20 March 2013
Published: 27 March 2013
The diversification of organisms with a parasitic lifestyle is often tightly linked to the evolution of their host associations. If a tight host association exists, closely related species tend to attack closely related hosts; host associations are less stable if associations are determined by more plastic traits like parasitoid searching and oviposition behaviour. The pupal-parasitoids of the genus Ichneumon attack a variety of macrolepidopteran hosts. They are either monophagous or polyphagous, and therefore offer a promissing system to investigate the evolution of host associations. Ichneumon was previously divided into two groups based on general body shape; however, a stout shape has been suggested as an adaptation to buried host pupation sites, and might thus not represent a reliable phylogenetic character.
We here reconstruct the first molecular phylogeny of the genus Ichneumon using two mitochondrial (CO1 and NADH1) and one nuclear marker (28S). The resulting phylogeny only supports monophyly of Ichneumon when Ichneumon lugens Gravenhorst, 1829 (formerly in Chasmias, stat. rev.) and Ichneumon deliratorius Linnaeus, 1758 (formerly Coelichneumon) are included. Neither parasitoid species that attack hosts belonging to one family nor those attacking butterflies (Rhopalocera) form monophyletic clades. Ancestral state reconstructions suggest multiple transitions between searching for hosts above versus below ground and between a stout versus elongated body shape. A model assuming correlated evolution between the two characters was preferred over independent evolution of host-searching niche and body shape.
Host relations, both in terms of phylogeny and ecology, evolved at a high pace in the genus Ichneumon. Numerous switches between hosts of different lepidopteran families have occurred, a pattern that seems to be the rule among idiobiont parasitoids. A stout body and antennal shape in the parasitoid female is confirmed as an ecological adaptation to host pupation sites below ground and has evolved convergently several times. Morphological characters that might be involved in adaptation to hosts should be avoided as diagnostic characters for phylogeny and classification, as they can be expected to show high levels of homoplasy.
KeywordsIdiobionts Parasitoid wasp Phylogeny Homoplasy Host relations
The evolution of host ranges in parasitic life forms deserves special attention, not only because it allows the investigation of numerous questions central to evolutionary biology, but also because of the tremendous ecological and economic importance of ecosystem functions delivered by these species. The time-scales over which processes like host-switching and co-speciation take place are of immediate interest as they not only help us understand current host ranges, but also predict future developments and adaptability of parasitic species. Insect parasitoids represent a special case of parasitic organisms because they ultimately kill their hosts during development. They are often classified ecologically into idiobionts and koinobionts. Idiobionts prevent further development of the host after initially immobilizing it, while koinobionts allow the host to continue its development after parasitization, often over several host life stages [1, 2]. While many koinobionts show high degrees of specialization and host fidelity, idiobionts are usually generalists and can even vary in their host ranges even at the population level. In such generalists, individuals often show a high level of behavioural and developmental plasticity as a response to an inconstant environment, and this plasticity can be crucial for their persistence . On a macro-evolutionary level, such plasticity can result in a high rate of host switching. If host switches are common in the evolutionary history of a group, then the phylogenies of hosts and parasitoids show low concordance . The opposite pattern, i.e., high concordance between host and parasitoid phylogenies, can result from very tight associations and a correspondingly low frequency of host switches, and in the extreme even co-speciation between host and parasites or parasitoids [5–7]. An intermediate level of phylogenetic concordance can be expected if host ranges evolve according to the “host-ecology hypothesis” [3, 8–10]. This hypothesis assumes that parasitoid species are able to broaden their host ranges by recruiting new hosts that exist within the parasitoids searching niche, and that this process can eventually lead to the appearance of a new, specialist species. Specialization thus takes place on the level of the host’s niche instead of its taxonomic or phylogenetic identity.
In parasitoid wasps, our knowledge of host range evolution is very limited due to a lack both of reliable host records in many groups and of sound species-level phylogenies [1, 11]. Very few studies have examined the evolution of host ranges and thus the prevalence of different macro-evolutionary processes from a phylogenetic perspective [8, 12–15]. The specious parasitic wasp genus Ichneumon Linnaeus, 1758 (Hymenoptera: Ichneumonidae, Ichneumoninae) consists mainly of endoparasitoids that attack the pupal stage of their macro-lepidopteran hosts [16, 17]. After parasitization, the hosts do not continue to grow and the parasitoid larvae thus have to develop on the host resources present at the time of oviposition; most Ichneumon species thus follow the idiobiont strategy of development . Several exceptions however exist in the genus, e.g., Ichneumon eumerus Wesmael, 1857 and Ichneumon caloscelis Wesmael, 1845 that attack the larva of their hosts, while emerging from the pupa [18, 19]. These species clearly are koinobionts and might show a closer association with their hosts. Within Ichneumon, some species are highly polyphagous as is typical for idiobionts, while other species are known only from a single host species ; this genus therefore offers an interesting system to study the evolution of host association patterns and host specificity.
Here, we build the first molecular phylogeny of the genus Ichneumon including 38 species using two mitochondrial markers, cytochrome oxydase 1 (CO1) and NADH dehydrogenase 1 (NADH1), and the nuclear 28S rRNA (D2-D3 region). The molecular phylogeny was reconstructed using maximum likelihood (ML) and Bayesian approaches. To investigate whether parasitoids that attack host species of the same family cluster together, we plotted host family associations onto the parasitoids phylogeny. Additionally, we tested for monophyly of the butterfly parasitoids under a likelihood-based and a Bayesian approach. To test the host-ecology hypothesis for Ichneumon, the evolution of the parasitoids’ searching niche was reconstructed. Finally, we tested for correlated evolution between antennal shape and the host pupation site.
Species, specimen numbers and origins, and Genbank accession numbers
Ichneumon albiger Wesmael, 1845
Ichneumon alius Tischbein, 1879
SWITZERLAND/Graubünden/Sur, Alp Flix/16.06.2003
Ichneumon alpinator Aubert, 1964
SWITZERLAND/Graubünden/Sur, Alp Flix/28.07.2003
Ichneumon amphiboles Kriechbaumer, 1888
SWEDEN/Stochholms län/Haninge, Tyresta/21.07.2003
Ichneumon bucculentus Wesmael, 1845
SWEDEN/Stockholms län/Södertälje, Tullgarn/19.08.2004
Ichneumon caloscelis Wesmael, 1845
SWEDEN/Kalmar län/Högsby, Hornsö kronopark/10.08.2003
Ichneumon computatorius Müller, 1776
SWEDEN/Kalmar län/Nybro, Bäckebo/19.06.2005
Ichneumon confusor Gravenhorst, 1820
SWEDEN/Kalmar län/Nybro, Alsterbo/10.06.2006
Ichneumon delator Wesmael, 1845
SWEDEN/Västerbottens län/Vindeln, Kulbäckslidens försökspark/03.09.2004
Ichneumon dilleri Heinrich, 1980
SWITZERLAND/Graubünden/Sur, Alp Flix/15.07.2006
Ichneumon emancipatus Wesmael, 1845
SWEDEN/Uppsala län/Håbo, Biskops-Arnö/18.07.2005
Ichneumon extensorius Linnaeus, 1758
Ichneumon formosus microcephalus Stephens, 1835
SWEDEN/Hallands län/Laholm, Mästocka/04.10.2003
Ichneumon fulvicornis Gravenhorst, 1829
SWEDEN/Västerbottens län/Vindeln, Kulbäckslidens försökspark/22.09.2003
Ichneumon gracilentus Wesmael, 1845
SWEDEN/Kronobergs län/Älmhult, Stenbrohult/20.07.2005
Ichneumon gracilicornis Gravenhorst, 1829
SWITZERLAND/Graubünden/Sur, Alp Flix/27.07.2006
Ichneumon cf. gracilicornis Gravenhorst, 1829
Ichneumon grandicornis Thomson, 1886
SWEDEN/Hallands län/Halmstad, Gardshult/13.07.2005
Ichneumon ignobilis Wesmael, 1855
SWEDEN/Västerbottens län/Vindeln, Kulbäckslidens försökspark/22.09.2003
Ichneumon inquinatus Wesmael, 1845
Ichneumon karpaticus Heinrich, 1951
SWEDEN/Norbottens län/Jokkmokk, Muddus nationalpark/18.06.2004
Ichneumon ligatorius Thunberg, 1822
SWEDEN/Västerbottens län/Vindeln, Kulbäcken meadow/20.08.2004
Ichneumon melanosomus Wesmael, 1855
SWEDEN/Gävleborgs län/Hudiksvall, Stensjön/11.08.2004
Ichneumon minutorius Desvignes, 1856
SWEDEN/Stochholms län/Haninge, Tyresta/20.07.2004
Ichneumon oblongus oblongus Schrank, 1802
SWEDEN/Kronobergs län/Älmhult, Stenbrohult/06.05.2004
Ichneumon oblongus picticollis Holmgren, 1864
SWEDEN/Västerbottens län/Vindeln, Svartbergets försökspark/22.09.2003
Ichneumon parengensis Kiss, 1929
SWITZERLAND/Graubünden/Sur, Alp Flix/21.06.2003
Ichneumon primatorius Forster, 1771
SWITZERLAND/Graubünden/Sur, Alp Flix/15.07.2006
Ichneumon pseudocaloscelis Heinrich, 1949
SWITZERLAND/Graubünden/Sur, Alp Flix/09.06.2003
Ichneumon simulans Tischbein, 1873
SWEDEN/Kalmar län/Nybro, Bäckebo/18.05.2006
Ichneumon simulans 2 Tischbein, 1873
Ichneumon spurius Wesmael, 1848
SWEDEN/Västa Götalands län/Stenungsund/25.05.2004
Ichneumon stigmatorius Zetterstedt, 1838
SWEDEN/Västerbottens län/Vindeln, Kulbäckslidens försökspark/22.09.2003
Ichneumon stramentarius Gravenhorst, 1820
Ichneumon stramentor Rasnitsyn, 1981
SWEDEN/Kronobergs län/Älmhult, Stenbrohult/01.11.2003
Ichneumon submarginatus Gravenhorst, 1829
SWEDEN/Uppsala län/Älvkarleby, BatforSweden/01.07.2004
Ichneumon suspiciosus Wesmael, 1845
SWEDEN/Skåne län/Klippans, Skäralid/06.08.2004
Ichneumon terminatorius Gravenhorst, 1820
SWEDEN/Kronobergs län/Älmhult, Stenbrohult/01.08.2003
Ichneumon tuberculipes Wesmael, 1848
SWEDEN/Stockholms län/Haninge, Tyresta/20.07.2004
Ichneumon sp. 1
France/Hautes-AlpeSweden/Col du Lautaret/summer 2008
Coelichneumon cyaniventris (Wesmael, 1859)
Ichneumon deliratorius Linnaeus, 1758 (former Coelichneumon)
SWEDEN/Stockholms län/Södertälje, Tullgarn/17.07.2005
Ichneumon lugens Gravenhorst, 1829 (former Chasmias)
Diplazon flixi Klopfstein, 2013
SWITZERLAND/Graubünden/Sur, Alp Flix/17.07.2006
Maximum likelihood and Bayesian analyses all only support the monophyly of the genus Ichneumon when it is expanded to include Chasmias lugens and Coelichneumon deliratorius. The support for the monophyly of such an Ichneumon s. l. was high in both analyses (bootstrap support: 0.85, posterior probability: 0.89) (Figure 2), while monophyly of the genus excluding C. lugens and C. deliratorius proved to be very unlikely (SH test, p<0.001).
Evolution of host ranges
Parasitoid species that attack hosts that belong to a single lepidopteran family do not cluster together, as shown in Figure 2, but instead appear in distant parts of the tree. Sister species often attack hosts from different families, and parasitoids of none of the host families were recovered as monophyletic. Also the parasitoids of butterfly hosts were recovered as paraphyletic in all our analyses, and the hypothesis of monophyly of these species was highly rejected both by a Bayesian approach (Bayes factor: 195.28) and by the Shimodaira-Hasegawa test  (p< 0.001).
Morphological adaptations to host pupation site
Ichneumon species, their antennal shape and host pupation site
Ichneumon cf. gracilicornis
Ichneumon formosus microcephalus*
Ichneumon oblongus picticollis
Ichneumon simulans 2
Ichneumon sp. 1
Phylogeny of Ichneumonand implications for taxonomy
We here present the first molecular phylogeny of the genus Ichneumon. It will serve as a robust starting point for future investigations of this specious genus, both in terms of phylogenetic and evolutionary research. Our molecular dataset provided good resolution of most of the nodes in the tree, but proved not to be variable enough to resolve some of the more recent relationship. Even the mitochondrial locus used for DNA barcoding , cytochrome oxidase 1, did not allow distinguishing among all the included Ichneumon species, with identical barcodes observed at least in two species pairs, and pairwise distances below 1% in many more. A similar observation has been made in the ichneumonid subfamily Diplazontinae [29, 30], but CO1 has proven very useful in other groups of parasitic wasps [13, 31]. The failure of DNA barcoding in Ichneumon might be due to imperfect taxonomy, insufficient variability of the markers to detect relatively recent speciation events, or in some of the cases due to incomplete lineage sorting or introgression [32, 33]. More data, including several fast-evolving nuclear markers like introns will probably be necessary, as non-monophyly of biological species in mitochondrial DNA has been convincingly demonstrated already in several cases, and might concern up to a third of all species in nature [34, 35].
The genus Ichneumon as it is currently defined was not retrieved as monophyletic (Figure 2), unless Chasmias lugens and Coelichneumon deliratorius were included. The relations of these species to Ichneumon have been discussed controversially in the past, and the morphological definition of the genus is based mainly on characters that might well be plesiomorphic [16, 17, 36]. Chasmias lugens does not fit well into the genus Chasmias morphologically, and based on our result should definitely be treated as part of the genus Ichneumon, where we transfer it hereby (stat. rev.). Coelichneumon deliratorius, based on morphology and on the results of the current study, has recently been re-included in the genus Ichneumon.
The molecular phylogeny recovered here clearly refutes the ad hoc hypothesis of the evolution of this genus as it was put forward by Hilpert . The synapomorphies that Hilpert suggested to support his cladogram are mostly mere trends  and included several character states that are putative adaptations to parasitizing particular hosts. As one example, Hilpert used a stout versus elongated shape of the female body and antennae to support an early split within Ichneumon. We could demonstrate here that a stout body shape is probably an adaptation to searching for hosts below ground. Character states associated with host relations can be misleading for classification and phylogenetic reconstruction, as has been shown for various groups of parasitoid wasps [21–25, 38–40]. In brief, such characters are only reliable if the switch to a particular host group happened only once during the evolutionary history of a group of parasitoids, but are prone to be homoplasious if it has been colonized several times in parallel.
Numerous switches between host families and between host searching niches
Host ranges in Ichneumon have undergone numerous switches during the evolution of this genus, and there was no sign of a conservative evolution of host associations among the species examined here (Figure 2). On the other hand, the Ichneumon species known to be polyphagous are usually restricted to hosts from a single family, demonstrating specialization at a low taxonomic level. Our taxon sampling was too sparse to predict how often host families are retained across speciation events, even though some of the included species might be closely related. Reliable host records are only available for a small fraction of the known Ichneumon species, and well-identified material suited for DNA extraction is difficult to get. The 38 species sampled here only represent a small fraction of the total species diversity of the genus, and if minor radiations have taken place within a host group, they might have been overlooked with our limited taxon sampling. In any case, our study provides a conservative estimate of the minimum number of host switches that took place during the evolution of this genus.
Although similar studies are scarce, a prominent role for host switching in shaping the host ranges of parasitoid wasps has been demonstrated in several cases. Sime & Wahl (ref. 2002), based on a morphological phylogeny, observed separate origins of butterfly parasitism in the Callajoppa genus group (Ichneumonidae, Ichneumoninae), and stated that host ranges in these parasitoids were dominated by comparatively recent host switches. A similar scenario was put forward by Shaw , again based on a morphological phylogeny. Zaldivar-Riveron et al.  used molecular markers in combination with a calibrated relaxed clock analysis to show that host associations changed quickly during the evolution of rogadinae braconids, and that the radiation of the wasps took place dozens of millions of years after the radiation of their hosts.
In terms of the niche where Ichneumon females search for their hosts, we observe a similar pattern. Polyphagous species only attack hosts that can be found either above or below ground, but no conservatism was apparent on a higher phylogenetic level (Figure 3), as it would be predicted under the host-ecology hypothesis [3, 8, 41]. Again, our taxon sampling does not exclude the possibility of smaller radiations within one searching niche, as it has been demonstrated for the braconid wasp genus Aleiodes. In this genus, closely related species tend to parasitize hosts with similar physical and ecological properties but which do not need to be closely related.
A high level of behavioural plasticity in host searching and host selection could be an explanatory factor for the macro-evolutionary patterns that we observed here, especially as behavioural traits have been shown to be less stable than physiological or morphological traits on evolutionary time-scales [42, 43]. Shaw  suggested that a new host association resulting from behavioural plasticity of a female parasitoid wasp might even be passed on to its progeny through post-eclosion or pre-adult experience [44, 45]. These mechanisms could enable the parasitoids to respond quickly to changes in host availability. They might be especially important in idiobiont parasitoids that only spend a short period of time in close association with the living host and thus do not need to adapt as much to the host’s physiological environment as koinobionts. Anecdotal evidence for the importance of host searching behaviour in comparison to host physiology stems from a laboratory experiment with Ichneumon hinzi, a supposedly monophagous parasitoid of Xestia speciosa (Hübner, 1813). In the laboratory, the parasitoid females also accepted the pupae of other, not closely related noctuids, and their progeny could successfully complete development in these non-host species . These hosts are probably excluded from the natural host range of I. hinzi through a narrow search strategy of the female that is focussed on its primary host.
We here present evidence that the evolution of host ranges in the parasitoid wasp genus Ichneumon included multiple transitions between host families and between microhabitats where the hosts can be found. Similar studies are scarce due to a lack of well-supported phylogenies for most groups and, more importantly, a lack of reliable host records for most parasitoid species. New molecular techniques, e.g., the DNA barcoding of host and parasitoid remains, or even of the gut contents of adult parasitoid wasps , might in the future complement time-intensive field observations and rearing as a means to document host-parasitoid associations and will thus allow for a more detailed picture of the evolution of host ranges in Ichneumon as well as in other parasitoid wasps. A better understanding of the dynamics and speed of the evolution of host associations will be crucial in order to predict adaptability of parasitoids to changes in the environment. Furthermore, it has important implications for risk assessments in bio-control, and for the comprehension of the tremendous diversity of parasitoid wasps.
We included 40 individuals of 38 Ichneumon species and subspecies in our study (Table 1). For two species, we sequenced two individuals for different reasons. A male of I. gracilicornis, a species that can only be determined with certainty in the female sex, was added to check the identification. Second, two I. simulans females showed large size differences and were collected in different countries. The genus Ichneumon is defined by a number of plesiomorphic characters, but also by several probably derived characters . Chasmias lugens was in the past variously combined with the genera Ichneumon or Chasmias. Because morphologically, it takes a rather isolated position within Chasmias, we also included it in our analysis. Moreover, Coelichneumon deliratorius shares several morphological and colour traits with Ichneumon species, but does not hibernate as an adult , which represents the only marked difference from Ichneumon. This species was also included in our analysis to investigate its phylogenetic position. As outgroups, we included representatives from the genera Barichneumon and Coelichneumon from the same subfamily, and the more distantly related Diplazon as a functional outgroup. We could obtain sequences of 42, 43 and 20 individuals from the markers NADH1, CO1 and 28S, respectively, and Genbank accession numbers are given in Table 1.
The specimens used were either preserved in 80% ethanol or air dried. Genomic DNA was extracted either from whole insects or, if the specimens were larger than 1.5 cm, from the metasoma, using the Promega Wizard kit for blood and tissue extraction. DNA samples are kept at the Natural history Museum in Bern (NMBE), vouchers at NMBE and at the Naturhistoriska Riksmuseet in Stockholm (NRM) (Table 1). Approximately 600 base pairs (bp) from the 5′ end of the mitochondrial CO1 gene were amplified using the primers designed by Folmer et al. . From NADH1, the second mitochondrial gene, we amplified 390 bp using the primers described by Smith et al. . To obtain about 650 bp of the nuclear 28S rRNA, the D2 and partial D3 region were amplified utilising primers designed by Belshaw and Quicke  and Mardulyn and Whitfield .
Polymerase chain reactions (PCR) were done in 20 μl final volumes using Promega GoTaq Flexi DNA Polymerase kits. Final volumes contained 30 pmol MgCl2, 16 pmol of each primer, 4 pmol of each dNTP, 0.3 U Taq polymerase and 2 μl genomic DNA. PCR conditions were: 94°C for 5 min, 37 cycles of 30s at 94°C, 30s at the respective annealing temperature (51°C for CO1, 48°C for NADH1 and 52°C for 28S), and 45 s at 72°C. PCR products were purified by the purification service of Macrogen Korea. The PCR products were sequenced on an ABI 377 automated sequencer using Big Dye Terminator technology (Applied Biosystems). Half of the taxa showed superimposed parts of the 28S sequences, probably due to the existence of different alleles due to incomplete concerted evolution of the ribosomal DNA; they were excluded from the analyses. The remaining 28S sequences are distributed over the whole tree and provided good resolution of the backbone, which is why we decided to include them despite a high level of missing data.
The sequences of the two protein-coding genes (CO1 and NADH1) were aligned after translation into amino acids using CLUSTAL  as implemented in Mega 4.0  with default settings. For both genes, no indels were detected. The D2-D3 region of the large subunit of 28S rRNA was aligned according to published secondary structure maps of ichneumonids , identifying the stem regions for partitioning and the pairing nucleotide position for the application of the doublet model in MrBayes and RAxML (see below). Of the identified non-pairing regions, only those that were length-conserved across the alignment were included in the analyses, while length-variable stretches were excluded. We thus obtained a 616 bp fragment of CO1, a 389 bp fragment of NADH1and 571 unambiguously alignable basepairs of 28S. Variability patterns of the different molecular partitions were obtained from PAUP* , where we also conducted a test for compositional heterogeneity. As none of the partitions showed significant heterogeneity, we proceeded to analyse the data under homogeneous models of nucleotide substitution (see next paragraph).
Properties of molecular partitions
CO1 first and second codon positions
CO1 third codon positions
NADH1 first and second codon positions
NADH1 third codon postitions
all markers combined
Comparison of partitioning strategies
partitioned according to mitochondrial (CO1 and NADH1) and nulclear (28S) gene identity
28S unpartitioned, mitochondrial markers partitioned into first and second versus third codon position
partitioned according to gene identity (CO1, NADH1 and 28S)
28S unpartitioned, mitochondrial markers separately partitioned into first and second versus third codon position
mitochondrial genes partitioned as under P5 and 28S partitioned into stem and loop
as P6, but with doublet model for the pairing stem partition of 28S
The final likelihood analysis of the joint dataset was conducted using RaxML  under a GTR+Г+I model with 1,000 nonparametric bootstrap iterations, adopting the partitioning strategy preferred by Bayes factor comparisons and using a 16-state secondary structure model for the stem regions of 28S. Final Bayesian analyses were run for 2*107 generations, and convergence was assessed as above. The matrix and resulting trees are deposited on TreeBASE http://purl.org/phylo/treebase/phylows/study/TB2:S13911.
Evolution of host ranges
We obtained information on host families for the included Ichneumon species from the literature [17, 64] and mapped them onto the consensus tree resulting from the Bayesian analysis (Figure 2) to look for host switching events. The five known butterfly parasitoids included in this study were recovered as paraphyletic in all our analyses. To test if this non-monophyly is statistically supported, we used a Bayes-factor and a likelihood-based approach. For the first, we conducted another Bayesian MCMC analysis, but imposing monophyly of the butterfly parasitoids as a phylogeny constraint, and compared the resulting marginal likelihood as estimated by the harmonic means. In addition, we applied the Shimodaira-Hasegawa test  as implemented in PAUP*  to the two maximum-likelihood phylogenetic hypotheses obtained with and without imposing the monophyly constraint.
Morphological adaptation to hosts that are attacked below ground
To investigate the evolution of searching niches, we scored all species for the pupation sites of their hosts [65–67]. We distinguished between hosts pupating below ground, i.e. among plant roots or in the soil, and species whose pupae can be found above the ground, e.g., in the vegetation or fully exposed. For the larval-pupal parasitoid I. caloscelis that attacks the caterpillars of its host well before feeding has finished , the search habitat is certainly above ground where the hosts can be found feeding and resting, although one of its five known hosts, Hipparchia semele, pupates below ground. I. fulvicornis has been reared from Phenagria caterpillars found in ant nests. It is not entirely clear whether already the young caterpillars are attacked prior to the adoption by ants, i.e. above ground, but seems more likely that the female searches for last-instar caterpillars in the ant nests like I. eumerus. We thus scored these two species according to the place where the last-instar larvae are found. We used parsimony and maximum likelihood to reconstruct ancestral states in the Ape package of the R statistical environment [68, 69]. To test for correlated evolution of parasitoid body shape and hosts pupation sites, we used BayesDiscrete from the BayesTraits package , comparing a model of independent with one assuming dependent evolution. Likelihoods obtained under the two models with 50 ML attempts per tree were compared by a likelihood ratio test. Posterior probabilities of the dependent and independent models and harmonic means of the likelihoods for Bayes-factor comparison were obtained by Markov-chain Monte Carlo approaches. For this calculation, we applied an exponential reversible-jump hyperprior within the interval between zero and 30 and set the ratedev parameter that controls the proposal rate of new values, to 8. This resulted in an acceptance rate between 20% and 40%, which falls inside the recommended range .
We would like to thank the Swedish Malaise Trap Project for providing specimens for this study. We acknowledge Christopher Sherry and Stefan Bachofner for technical support. A previous version of the manuscript was considerably improved by constructive comments by Mark Shaw and two anonymous reviewers.
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