Duplouy Anne

The Glanville fritillary (<i>Melitaea cinxia</i>) butterfly is a model system for metapopulation dynamics research in fragmented landscapes. Here, we provide a chromosome level assembly of the butterfly's genome produced from Pacific Biosciences sequencing of a pool of males, combined with a linkage map from population crosses. The final assembly size of 484 Mb is an increase of 94 Mb on the previously published genome. Estimation of the completeness of the genome with Benchmarking Universal Single-Copy Orthologs (BUSCO) indicates that the genome contains 93 - 95% of the BUSCO genes in complete and single copies. We predicted 14,810 genes using the MAKER pipeline and manually curated 1,232 of these gene models. The genome and its annotated gene models are a valuable resource for future comparative genomics, molecular biology, transcriptome and genetics studies on this species.

ABSTRACTThe success of maternally transmitted endosymbiotic bacteria, such as Wolbachia, is directly linked to their host reproduction but in direct conflict with other parasites that kill the host before it reaches reproductive maturity. Therefore, symbionts that have evolved strategies to increase their host’s ability to evade lethal parasites may have high penetrance, while detrimental symbionts would be selected against, leading to lower penetrance or extinction from the host population. In a natural population of the parasitoid wasp Hyposoter horticola in the Åland Islands (Finland), the Wolbachia strain wHho persists at an intermediate prevalence (∼50%). Additionally, there is a negative correlation between the prevalence of Wolbachia and a hyperparasitoid wasp, Mesochorus cf. stigmaticus, in the landscape. Using a manipulative field experiment, we addressed the persistence of Wolbachia at this intermediate level, and tested whether the observed negative correlation could be d...

Habitat loss and fragmentation threaten the long-term viability of innumerable species of plants and animals. At the same time, habitat fragmentation may impose strong natural selection and lead to evolution of life histories with possible consequences for demographic dynamics. The Baltic populations of the Glanville fritillary butterfly (Melitaea cinxia) inhabit regions with highly fragmented habitat (networks of small dry meadows) as well as regions with extensive continuous habitat (calcareous alvar grasslands). Here, we report the results of common garden studies on butterflies originating from two highly fragmented landscapes (FL) in Finland and Sweden and from two continuous landscapes (CL) in Sweden and Estonia, conducted in a large outdoor cage (32 by 26 m) and in the laboratory. We investigated a comprehensive set of 51 life-history traits, including measures of larval growth and development, flight performance, and adult reproductive behavior. Seventeen of the 51 traits sh...

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia) from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have high heritability and w...

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393 Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140 My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics high...

We investigated inbreeding depression and genetic load in a small ( N e ∼ 100) population of the Glanville fritillary butterfly ( Melitaea cinxia ), which has been completely isolated on a small island [Pikku Tytärsaari (PT)] in the Baltic Sea for at least 75 y. As a reference, we studied conspecific populations from the well-studied metapopulation in the Åland Islands (ÅL), 400 km away. A large population in Saaremaa, Estonia, was used as a reference for estimating genetic diversity and N e . We investigated 58 traits related to behavior, development, morphology, reproductive performance, and metabolism. The PT population exhibited high genetic load ( L = 1 − W PT /W ÅL ) in a range of fitness-related traits including adult weight ( L = 0.12), flight metabolic rate ( L = 0.53), egg viability ( L = 0.37), and lifetime production of eggs in an outdoor population cage ( L = 0.70). These results imply extensive fixation of deleterious recessive mutations, supported by greatly reduced d...

Reproductive parasites such as Wolbachia can spread through uninfected host populations by increasing the relative fitness of the infected maternal lineage. However, empirical estimates of how fast this process occurs are limited. Here we use nucleotide sequences of male‐killing Wolbachia bacteria and co‐inherited mitochondria to address this issue in the island butterfly Hypolimnas bolina. We show that infected specimens scattered throughout the species range harbour the same Wolbachia and mitochondrial DNA as inferred from 6337 bp of the bacterial genome and 2985 bp of the mitochondrial genome, suggesting this strain of Wolbachia has spread across the South Pacific Islands at most 3000 years ago, and probably much more recently.

On small isolated islands, natural selection is expected to reduce the dispersal capacity of organisms, as short distances do not require a high rate of dispersal, which might lead to accidental emigration from the population. In addition, individuals foregoing the high cost of maintaining flight capacity may instead allocate resources to other functions. However, in butterflies and many other insects, flight is necessary not only for dispersal but also for most other activities. A weakly flying individual would probably do worse and have an elevated rather than reduced probability of accidental emigration. Here, we report results consistent with the hypothesis that a butterfly population on an isolated island, instead of having lost its flight capacity, has evolved better grip to resist the force of wind and to avoid being blown off the island. Our study suggests that local adaptation has occurred in this population in spite of its very small size (Ne∼ 100), complete isolation, low...

The success of maternally transmitted endosymbiotic bacteria, such as Wolbachia, is directly linked to their host reproduction but in direct conflict with other parasites that kill the host before it reaches reproductive maturity. Therefore, symbionts that have evolved strategies to increase their host's ability to evade lethal parasites may have high penetrance, while detrimental symbionts would be selected against, leading to lower penetrance or extinction from the host population. In a natural population of the parasitoid wasp Hyposoter horticola in the Åland Islands (Finland), the Wolbachia strain wHho persists at an intermediate prevalence (∼50%). Additionally, there is a negative correlation between the prevalence of Wolbachia and a hyperparasitoid wasp, Mesochorus cf. stigmaticus, in the landscape. Using a manipulative field experiment, we addressed the persistence of Wolbachia at this intermediate level, and tested whether the observed negative correlation could be due to Wolbachia inducing either susceptibility or resistance to parasitism. We show that infection with Wolbachia does not influence the ability of the wasp to parasitize its butterfly host, Melitaea cinxia, but that hyperparasitism of the wasp increases in the presence of wHho. Consequently, the symbiont is detrimental, and in order to persist in the host population, must also have a positive effect on fitness that outweighs the costly burden of susceptibility to widespread parasitism.

The maternally transmitted bacterium Wolbachia pipientis is well known for spreading and persisting in insect populations through manipulation of the fitness of its host. Here, we iden- tify three new Wolbachia pipientis strains, wHho, wHho2 and wHho3, infecting Hyposoter horticola, a specialist wasp parasitoid of the Glanville fritillary butterfly. The wHho strain (ST435) infects about 50% of the individuals in the Åland islands in Finland, with a different infection rate in the two mitochondrial (COI) haplotypes of the wasp. The vertical transmis- sion rate of Wolbachia is imperfect, and lower in the haplotype with lower infection rate, sug- gesting a fitness trade-off. We found no association of the wHho infection with fecundity, longevity or dispersal ability of the parasitoid host. However, preliminary results convey spa- tial associations between Wolbachia infection, host mitochondrial haplotype and parasitism of H. horticola by its hyperparasitoid, Mesochorus cf. stigmaticus. We discuss the possibility that Wolbachia infection protects H. horticola against hyperparasitism

1. The Glanville fritillary butterfly (Melitaea cinxia L.) has a small
population (Ne ∼ 100) on the small island of Pikku Tytärsaari (PT) in the Gulf of
Finland. The population has remained completely isolated for ∼100 generations, which
has resulted in greatly reduced genetic variation and high genetic load (low fitness). In
particular, females lay small egg clutches with a low egg-hatching rate in comparison
with a large reference population in the Åland Islands (ÅL).
2. In the present study, to what extent egg clutch size and egg-hatching rate are
influenced by male population and spermatophore size was analysed.
3. Spermatophore size increases with male body size, is smaller after the first mating,
and is smaller in the small PT population. In the ÅL population but not in the PT
population, the egg-hatching rate increases with spermatophore size. The egg-hatching
rate of PT females is higher when mated with ÅL males than when mated with PT males
(heterosis), but there is no such effect on clutch size. The clutch size of ÅL females is,
however, reduced when mated with PT males.
4. These results indicate that both male and female traits contribute to reduced
reproductive fitness in the small isolated population.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved,
yet the number of haploid chromosomes varies widely from 5 to 223. Here we report
the genome (393Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely
recognized model species in metapopulation biology and eco-evolutionary research, which has
the putative ancestral karyotype of n¼31. Using a phylogenetic analyses of Nymphalidae and of
other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude
that the ancestral lepidopteran karyotype has been n¼31 for at least 140My. We show that
fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements
have occurred across the fusion sites. The same, shortest ancestral chromosomes
have independently participated in fusion events in species with smaller karyotypes. The short
chromosomes have higher rearrangement rate than long ones. These characteristics highlight
distinctive features of the evolutionary dynamics of butterflies and moths.

We characterize allelic and gene expression variation between populations of the Glanville fritillary butterfly (Melitaea cinxia)
from two fragmented and two continuous landscapes in northern Europe. The populations exhibit significant differences in
their life history traits, e.g. butterflies from fragmented landscapes have higher flight metabolic rate and dispersal rate in the
field, and higher larval growth rate, than butterflies from continuous landscapes. In fragmented landscapes, local
populations are small and have a high risk of local extinction, and hence the long-term persistence at the landscape level is
based on frequent re-colonization of vacant habitat patches, which is predicted to select for increased dispersal rate. Using
RNA-seq data and a common garden experiment, we found that a large number of genes (1,841) were differentially
expressed between the landscape types. Hexamerin genes, the expression of which has previously been shown to have
high heritability and which correlate strongly with larval development time in the Glanville fritillary, had higher expression in
fragmented than continuous landscapes. Genes that were more highly expressed in butterflies from newly-established than
old local populations within a fragmented landscape were also more highly expressed, at the landscape level, in fragmented
than continuous landscapes. This result suggests that recurrent extinctions and re-colonizations in fragmented landscapes
select a for specific expression profile. Genes that were significantly up-regulated following an experimental flight treatment
had higher basal expression in fragmented landscapes, indicating that these butterflies are genetically primed for frequent
flight. Active flight causes oxidative stress, but butterflies from fragmented landscapes were more tolerant of hypoxia. We
conclude that differences in gene expression between the landscape types reflect genomic adaptations to landscape
fragmentation.

Habitat loss and fragmentation threaten the long-term viability of innumerable
species of plants and animals. At the same time, habitat fragmentation may
impose strong natural selection and lead to evolution of life histories with possible
consequences for demographic dynamics. The Baltic populations of the Glanville
fritillary butterfly (Melitaea cinxia) inhabit regions with highly fragmented habitat
(networks of small dry meadows) as well as regions with extensive continuous
habitat (calcareous alvar grasslands). Here, we report the results of common garden
studies on butterflies originating from two highly fragmented landscapes (FL)
in Finland and Sweden and from two continuous landscapes (CL) in Sweden and
Estonia, conducted in a large outdoor cage (32 by 26 m) and in the laboratory.
We investigated a comprehensive set of 51 life-history traits, including measures
of larval growth and development, flight performance, and adult reproductive
behavior. Seventeen of the 51 traits showed a significant difference between fragmented
versus CL. Most notably, the growth rate of postdiapause larvae and several
measures of flight capacity, including flight metabolic rate, were higher in
butterflies from fragmented than CL. Females from CL had shorter intervals
between consecutive egg clutches and somewhat higher life-time egg production,
but shorter longevity, than females from FL. These results are likely to reflect the
constant opportunities for oviposition in females living in continuous habitats,
while the more dispersive females from FL allocate more resources to dispersal
capacity at the cost of egg maturation rate. This study supports theoretical predictions
about small population sizes and high rate of population turnover in fragmented
habitats selecting for increased rate of dispersal, but the results also
indicate that many other life-history traits apart from dispersal are affected by the
degree of habitat fragmentation.

Background: The endosymbiont Wolbachia pipientis causes diverse and sometimes dramatic phenotypes in its
invertebrate hosts. Four Wolbachia strains sequenced to date indicate that the constitution of the genome is
dynamic, but these strains are quite divergent and do not allow resolution of genome diversification over shorter
time periods. We have sequenced the genome of the strain wBol1-b, found in the butterfly Hypolimnas bolina,
which kills the male offspring of infected hosts during embyronic development and is closely related to the
non-male-killing strain wPip from Culex pipiens.
Results: The genomes of wBol1-b and wPip are similar in genomic organisation, sequence and gene content, but
show substantial differences at some rapidly evolving regions of the genome, primarily associated with prophage
and repetitive elements. We identified 44 genes in wBol1-b that do not have homologs in any previously
sequenced strains, indicating that Wolbachia’s non-core genome diversifies rapidly. These wBol1-b specific genes
include a number that have been recently horizontally transferred from phylogenetically distant bacterial taxa. We
further report a second possible case of horizontal gene transfer from a eukaryote into Wolbachia.
Conclusions: Our analyses support the developing view that many endosymbiotic genomes are highly dynamic,
and are exposed and receptive to exogenous genetic material from a wide range of sources. These data also
suggest either that this bacterial species is particularly permissive for eukaryote-to-prokaryote gene transfers, or that
these transfers may be more common than previously believed. The wBol1-b-specific genes we have identified
provide candidates for further investigations of the genomic bases of phenotypic differences between
closely-related Wolbachia strains.

On small isolated islands, natural selection is expected to reduce the dispersal
capacity of organisms, as short distances do not require a high rate of dispersal,
whichmight leadto accidental emigrationfromthe population. In addition, individuals
foregoing the high cost of maintaining flight capacity may instead
allocate resources to other functions. However, in butterflies and many other
insects, flight is necessary not only for dispersal but also formost other activities.
A weakly flying individual would probably do worse and have an elevated
rather than reduced probability of accidental emigration.Here,we report results
consistent with the hypothesis that a butterfly population on an isolated island,
instead of having lost its flight capacity, has evolved better grip to resist the force
of wind and to avoid being blown off the island. Our study suggests that local
adaptation has occurred in this population in spite of its very small size
(Ne ~100), complete isolation, low genetic variation and high genetic load.

We investigated inbreeding depression and genetic load in a small
(Ne ∼ 100) population of the Glanville fritillary butterfly (Melitaea
cinxia), which has been completely isolated on a small island
[Pikku Tytärsaari (PT)] in the Baltic Sea for at least 75 y. As a reference,
we studied conspecific populations from the well-studied
metapopulation in the Åland Islands (ÅL), 400 km away. A large
population in Saaremaa, Estonia, was used as a reference for estimating
genetic diversity and Ne.We investigated 58 traits related
to behavior, development, morphology, reproductive performance,
and metabolism. The PT population exhibited high genetic
load (L = 1 − WPT/WÅL) in a range of fitness-related traits including
adult weight (L = 0.12), flight metabolic rate (L = 0.53), egg viability
(L = 0.37), and lifetime production of eggs in an outdoor population
cage (L = 0.70). These results imply extensive fixation of
deleterious recessive mutations, supported by greatly reduced diversity
in microsatellite markers and immediate recovery (heterosis)
of egg viability and flight metabolic rate in crosses with other
populations. There was no significant inbreeding depression in
most traits due to one generation of full-sib mating. Resting metabolic
rate was significantly elevated in PT males, which may be
related to their short lifespan (L = 0.25). The demographic history
and the effective size of the PT population place it in the part of
the parameter space in which models predict mutation accumulation.
This population exemplifies the increasingly common situation
in fragmented landscapes, in which small and completely
isolated populations are vulnerable to extinction due to high genetic
load.

Symbionts are widespread and might have a substantial effect on the outcome of interactions between species, such as in
host-parasitoid systems. Here, we studied the effects of symbionts on the outcome of host-parasitoid interactions in a fourpartner
system, consisting of the parasitoid wasp Leptopilina boulardi, its two hosts Drosophila melanogaster and D.
simulans, the wasp virus LbFV, and the endosymbiotic bacterium Wolbachia. The virus is known to manipulate the
superparasitism behavior of the parasitoid whereas some Wolbachia strains can reproductively manipulate and/or confer
pathogen protection to Drosophila hosts. We used two nuclear backgrounds for both Drosophila species, infected with or
cured of their respective Wolbachia strains, and offered them to L. boulardi of one nuclear background, either infected or
uninfected by the virus. The main defence mechanism against parasitoids, i.e. encapsulation, and other important traits of
the interaction were measured. The results showed that virus-infected parasitoids are less frequently encapsulated than
uninfected ones. Further experiments showed that this viral effect involved both a direct protective effect against
encapsulation and an indirect effect of superparasitism. Additionally, the Wolbachia strain wAu affected the encapsulation
ability of its Drosophila host but the direction of this effect was strongly dependent on the presence/absence of LbFV. Our
results confirmed the importance of heritable symbionts in the outcome of antagonistic interactions.

Maternally inherited insect symbionts often manipulate host reproduction for their own benefit. Symbionts are transmitted to the next host generation through the female hosts, and as such males represent dead ends for transmission. Natural selection therefore favors symbiont-induced phenotypes that provide a reproductive advantage to infected females, regardless of possible negative selective effects on males. Male-killing (MK) is one such phenotype, in which symbionts kill the male progeny of infected females. Compared with other symbiont-associated reproductive phenotypes, MK is relatively unexplored mechanistically as well as ecologically. A male-killing Wolbachia bacterium strain named wBol1 has been described in the tropical butterfly Hypolimnas bolina. By reviewing the different features of this association it is possible to summarize what is already known about the biology and evolution of MK symbionts, as well as highlight the current gaps in our understanding of this striking reproductive phenotype.

Reproductive parasites such as Wolbachia can spread through uninfected host populations by increasing the relative fitness of the infected maternal lineage. However, empirical estimates of how fast this process occurs are limited. Here we use nucleotide sequences of male-killing Wolbachia bacteria and co-inherited mitochondria to address this issue in the island butterfly Hypolimnas bolina. We show that infected specimens scattered throughout the species range harbour the same Wolbachia and mitochondrial DNA as inferred from 6337 bp of the bacterial genome and 2985 bp of the mitochondrial genome, suggesting this strain of Wolbachia has spread across the South Pacific Islands at most 3000 years ago, and probably much more recently.

Background The interaction between the Blue Moon butterfly, Hypolimnas bolina, and Wolbachia has attracted interest because of the high prevalence of male-killing achieved within the species, the ecological consequences of this high prevalence, the intensity of selection on the host to suppress the infection, and the presence of multiple Wolbachia infections inducing different phenotypes. We examined diversity in the co-inherited marker, mtDNA, and the partitioning of this between individuals of different infection status, as a means to investigate the population biology and evolutionary history of the Wolbachia infections. Results Part of the mitochondrial COI gene was sequenced from 298 individuals of known infection status revealing ten different haplotypes. Despite very strong biological evidence that the sample represents a single species, the ten haplotypes did not fall within a monophyletic clade within the Hypolimnas genus, with one haplotype differing by 5% from the other nine. There were strong associations between infection status and mtDNA haplotype. The presence of w Bol1 infection in association with strongly divergent haplotypes prompted closer examination of w Bol1 genetic variation. This revealed the existence of two cryptic subtypes, w Bol1a and w Bol1b. The w Bol1a infection, by far the most common, was in strict association with the single divergent mtDNA haplotype. The w Bol1b infection was found with two haplotypes that were also observed in uninfected specimens. Finally, the w Bol2 infection was associated with a large diversity of mtDNA haplotypes, most often shared with uninfected sympatric butterflies. Conclusion This data overall supports the hypothesis that high prevalence of male-killing Wolbachia (w Bol1) in H. bolina is associated with very high transmission efficiency rather than regular horizontal transmission. It also suggests this infection has undergone a recent selective sweep and was introduced in this species through introgression. In contrast, the sharing of haplotypes between w Bol2-infected and uninfected individuals indicates that this strain is not perfectly transmitted and/or shows a significant level of horizontal transmission.

Wolbachia and other intracellular bacteria that manipulate reproduction are widespread and can have major consequences on the ecology and evolution of their hosts. Several studies have attempted to assess the host range of these bacteria based on polymerase chain reaction assays on material preserved and collected using a variety of methods. While collecting in the field, mass storage in ethanol before sorting specimens in the laboratory is by far the easiest technique, and an integral component of Malaise trapping. This implicitly relies on the assumption that mass ethanol storage does not produce cross-contamination of Wolbachia DNA among specimens. Here we test this assumption. The absence of cross contamination between known positive and negative samples stored within a vial indicate there is no reason to believe collective storage of specimens creates artefactual increases in the incidence of Wolbachia or other intracellular bacteria.