Species coexistence and oviposition strategies
Five or six Chiastocheta species were present in most populations sampled. This result confirms previous studies concerning coexistence of Chiastocheta spp. in T. europaeus populations ( Pellmyr, 1989; Johannesen & Loeschcke, 1996a). In Scandinavia, only four species were described: Pellmyr (1989) found all coexisting in different meadows along a river in Finland and Johannesen & Loeschcke (1996a) found the four species coexisting at 12 sites out of 18 in Denmark. In the Alps, six species were found to coexist in most populations (this study). Moreover, we found that 2/3 of the flowers harboured eggs from more than one species. This indicates that larvae of different species do interact within flower heads most of the time. In another study we showed that competition occurs between larvae for seed consumption (Jaeger et al., unpublished data). So far, no general mechanism has been demonstrated to promote coexistence of the Chiastocheta species ( Johannesen & Loeschcke, 1996a) and the hypothesis of niche partitioning by specific stereotyped larval mining patterns ( Pellmyr, 1989) remains to be tested.
The six Chiastocheta spp. can be characterized by their ovipositing behaviour (distribution of eggs among flowers and age of flowers visited) as being more or less beneficial to the host plant. C. rotundiventris rarely deposits more than one egg per flower and visits only young flowers (mostly first-day flowers) that are more likely to be unpollinated. It is worth noticing that pollination levels obtained at the end of the first day of flowering represent a significant part of total seed production (30% at Cherlieu and Lac, 50% at Som), reflecting the important role played by C. rotundiventris in globeflower pollination. At the opposite, C. dentifera tends to highly aggregate its eggs and was shown to oviposit mostly on fruits, thus after pollination took place. This parasite species was found in almost all populations and represents the highest number of eggs laid, presumably leading to the highest predation costs in terms of seeds consumed among all Chiastocheta species. The four other species have intermediate strategies, aggregating eggs in 2–7-day-old flowers, thus potentially contributing to pollination as they visit flowers and not fruits. Under the pollination conditions observed in this study (i.e. full pollination not achieved before days 4–6 depending on population), pollen deposition during early oviposition of C. setifera and C. macropyga females is more likely to lead to seed production than during late oviposition of C. inermella and C. trollii females.
Evolution of oviposition strategies in Chiastocheta spp.
Three clades are strongly supported by high bootstrap values whatever the phylogenetic method used to build the tree: the first clade groups the two vicariant species C. rotundiventris and C. abruptiventris, the second clade includes all the C. dentifera haplotypes and the third clade groups the four species with intermediate oviposition strategies.
The mitochondrial phylogeny obtained strongly supports the basal position of C. rotundiventris and its northern vicariant C. abruptiventris. These two species could not be distinguished in this study: they shared the same haplotype, but also the same egg morphology and oviposition strategy. We could not find any morphological differences on the male genitalia of individuals collected in Sweden and Russia and those collected in the Alps: they all had the morphological traits specific to C. rotundiventris as described and drawn by Hennig (1976). Pellmyr (1989) called the species he found in Finland C. sp. aff. rotundiventris and only in his 1992 paper did he accept C. abruptiventris as a valid species, without giving any specific diagnostic character. The validity of C. abruptiventris as a distinct species can be questioned as no morphological description of this species has been published so far. The haplotype common to C. rotundiventris and C. abruptiventris was well differentiated from all the other haplotypes with divergences ranging from 3.65 to 4.15% (Table 2). In a study based on allozyme electrophoresis, Johannesen & Loeschcke (1996b) established interspecific Nei’s genetic distance between the four species they found in Denmark: C. abruptiventris was the most distinct species and C. dentifera, C. inermella and C. trollii were grouped in one cluster. The present mtDNA rooted phylogenetic tree with C. rotundiventris and C. abruptiventris as basal species confirms this result.
In the present study, despite the high number of sites sequenced (1320 bp), the molecular divergence observed is low (0–4.15%), particularly among species with intermediate strategies (0–1.23%). As a comparison, pairwise sequence divergences ranged from 0.1 to 17% between 16 species of the genus Greya (Lepidoptera, COI-COII, Brown et al., 1994 ), from 0.1 to 13.1% between seven species of the genus Phratora (Coleoptera, COI, Köpf et al., 1998 ), and from 2 to 4.8% between six species of the Drosophila affinis subgroup, the most recent radiation in the D. obscura group (Diptera, COII, Beckenbach et al., 1993 ). In this latter study, authors gave a rough estimation of 1.6–3.5 Ma for this radiation. As Chiastocheta spp. show divergences of the same order of magnitude, and under the assumption that the evolutionary rate of mtDNA is not totally different between these two Diptera genera, the diversification of Chiastocheta spp. may have begun in the late Pliocene/early Pleistocene (about 2 Ma). According to Doroszewska (1974), T. europaeus came to Europe from Asia through the Balkans during the Miocene (23.5–5.3 Ma). Other taxa are thought to have migrated from Asia to Europe at this period (e.g. Dactylis spp., Stebbins & Zohary, 1959). The low molecular divergence found among Chiastocheta spp. associated with T. europaeus suggests that diversification of these species is much more recent than Trollius migration to Europe and that it did take place within the host plant. An alternative hypothesis is that speciation within the Chiastocheta genus occurred before or throughout radiation of a plant host clade including four Trollius Asiatic species and T. europaeus ( Pellmyr, 1992). Further molecular phylogenetic studies including Asiatic Chiastocheta species sequences would be necessary to test whether the European and Asiatic species form two separate clades, or are clustered together.
Given the phylogeny obtained, the most parsimonious hypothesis is that the ancestor of European Chiastocheta spp. did lay eggs on flowers (Fig. 4). This is consistent with the fact that most if not all other Chiastocheta species described so far lay eggs during flowering ( Pellmyr, 1992). From this flower-ovipositing ancestor, there was (1) separation of an oviposition strategy on young flowers, (2) separation of an oviposition strategy on fruits and (3) diversification of the four intermediate strategies.
At the beginning of its development, a larva of C. rotundiventris uses the basal part of the flower where there is no room for several larvae. This may explain why females of this species lay no more than one egg/flower in young flowers, which are more likely to be unoccupied. We do not know whether the use of receptacle is an ancestral or a derived trait in Chiastocheta. However, adoption or abandonment of the use of receptacle is likely to have profound effects on the evolution of the oviposition strategy. Plant specialization on a seed parasitic-pollinator requires external conditions like rarity of copollinators ( Thompson & Pellmyr, 1992) and also particular traits of the insect like local host specificity, pollination during oviposition and nonexcessive seed destruction ( Pellmyr et al., 1996b ). T. europaeus was associated early with a particularly mutualistic species, suggesting that favourable conditions for increasing plant specialization were present early in the association history. Specialization ultimately led to an obligate association: T. europaeus has a totally closed corolla which is unique in its genus, excluding all visitors but Chiastocheta.
The parasitic species ovipositing on fruits arose from a mutualistic species ovipositing on flowers. Similarly, cheaters ovipositing on fruits rather than on flowers were found in several yucca species ( Addicott, 1996; Pellmyr et al., 1996a ) and molecular data show that they derive from pollinators ( Pellmyr et al., 1996a ). It has been argued that a cheater strategy evolving within an active pollinator species should invade because it is less costly ( Pellmyr & Huth, 1994; Pellmyr et al., 1996a ), leading to reciprocal extinction, so that sympatric speciation within the same host-plant could not occur. However, pollination by Chiastocheta is presumably passive (no special pollen transport structures have been found so far) so that the cost of pollination could be very low. In such a system, the age of flower when oviposition takes place will determine a pollinating or a nonpollinating behaviour with no obvious advantage to one or the other strategy. Since species coexist in the same fruits and competition among larvae has been shown (Jaeger et al., unpublished data), delay in oviposition may be costly because larvae will have late access to seeds and thus will suffer higher interspecific competition costs. However, there may be an advantage to lay eggs only on well-developed fruits and thus decrease the risk of laying eggs on unpollinated or poorly pollinated flowers. Such balance between the advantage of choosing already well-pollinated fruits to lay several eggs and the cost of increased larval competition may be a major stabilizing selective factor explaining coexistence of flower- and fruit-oviposition strategies.
If specialization on different flower parts or stages (receptacle use by larva, fruit- vs. flower-oviposition) can underlie sympatric speciation in Chiastocheta, it is unlikely to be involved in speciation of the four intermediate species for which oviposition dates largely overlap. The several Pleistocene ice ages have produced a series of changes in climate which have repeatedly altered the habitat and range of living organisms ( Hewitt, 1993, 1996). The four intermediate strategies may have evolved under such a situation, in geographically isolated T. europaeus populations, their actual presence in the same populations in the Alps being the result of recent secondary northern recolonization following the last ice age. This hypothesis is supported by two facts. First, although all the individuals from C. inermella sequenced were collected in our alpine populations, this species appears clearly paraphyletic, which may reflect a previous fragmentation of its geographical distribution. Second, the four intermediate species are not found in all parts of T. europaeus distribution: only two were found in Scandinavia ( Pellmyr, 1989; Johannesen & Loeschcke, 1996a), and three in Great Britain ( Collin, 1954). Unfortunately, no reports of Chiastocheta community in other parts of the globeflower range are available. Sampling in the Pyrenees, Italian Appenines and Balkan mountains would be particularly interesting as these three mountain ranges are at the southern limit of T. europaeus distribution and are well known post ice age refuges for several alpine organisms ( Hewitt, 1993, 1996; Konnert & Bergmann, 1995; Taberlet et al., 1998 ). The four intermediate species appear to be very similar in terms of molecular divergence, and only slightly differ in the age of the flowers visited. However, they are well differentiated by the morphology of the male genitalia, a character known to evolve rapidly in Dipteras ( True et al., 1997 ), presumably under sexual selection. Strong male genitalia differences observed between the otherwise morphologically very similar four intermediate species could be the result of reinforcement of reproductive isolation through sexual selection after secondary contact of these very closely related species.
In conclusion, the highly diverse Chiastocheta community found on T. europaeus is likely to be the result of a recent radiation during the Pleistocene climatic events. The specialization of the plant on seed-parasites as pollinators (and thus establishment of an obligate mutualism) was favoured by the mutualistic behaviour of the most ancestral species. Nonpollinating behaviour evolved from the pollinator. Unlike in other plant–insect systems where diversification is usually thought to be driven by cospeciation or host shifts, it is more likely that Chiastocheta speciation took place within the host plant, reproductive isolation between species being reinforced by niche partitioning for oviposition and/or sexual selection.