Gabriel Nève
email: gabriel.neve@univ-provence.fr

J Settele

T G Shreeve

R L H Dennis

Eds H Van Dyck

Population Genetics of Butterflies

Continental scale -Most species show differences of phenotypes between different regions. What are the causes of such differences? At the local scale, population genetics theory recognises four parameters to explain the local makeup of any given population: mutation, genetic drift, migration and selection. Mutation is generally regarded as of minor importance in natural populations over short time scales. Genetic drift is the result of random changes of allelic frequencies due to the small size of an isolated population. Ultimately, drift may lead to loss or fixation of alleles. Migration involves movement of individuals originating in one population to another where reproduction occurs. This results in the immigration or emigrations of individuals to or from the population of interest. If different genotypes in a given population reproduce at different rates, then selection will be operating. This can be a major factor in shaping the population structure of organisms. The effect of selection or drift may be counterbalanced by migration processes but if the population is small and immigration is not occurring, genetic drift is likely to be an important factor. Thus the ability of butterflies to disperse is a key factor in shaping population structure. The individuals of some species rarely move more than a few hundred meters from their natal location, (e.g., Cupido minimus;

Introduction

In a world where natural habitats are fast disappearing and where the landscape is intensively managed, even in nature reserves, it is important to understand the consequences of management and large-scale processes on populations. In this respect, studies of butterflies have been seminal in understanding how landscape structure affects populations [START_REF] Saccheri | Genetics of cherspot populations[END_REF]Chapters D1 & D2). From a genetic perspective, the population structure of a given species in a particular landscape depends on a series of parameters : population size in each patch of habitat, movements between these patches, and the level of immigration and emigration to and from the system, together with their respective points of origin or destination. The two key factors determining the population structure of butterfly populations are the spatial distribution of their habitats and the ability of each species to disperse through the different components of the habitat matrix in the landscape. Different species may respond differently from the same change in habitat structure, depending on their movement ability and their habitat choice mainly, but not exclusively, determined by their choice of host plants.

When examining populations, it is important to define the scale at which processes occur. For example, post-glacial dispersal does not occur at the same spatial and temporal scales as emigration between habitat patches within a metapopulation.

Depending on the scale, different population structures emerge. Population geneticists interested in spatial structure may ask different questions, which may be classified according to the scale at which they are tackled. These are: (1) Local scale -What are the causes of within population variation? Not two butterflies are exactly alike and how is this variation maintained? (2) Landscape scale -Are populations of a given species distinct entities within a landscape, or do they form a single genetic unit ? If populations are different within the landscape, what could be the causes of such differences? (3) Regional scale -To what extent do populations from different landscapes (i. e. river systems or mountain ranges) interact within a given region? (4) numerous subspecies (e.g. [START_REF] Capdeville | Les races géographiques de Parnassius apollo[END_REF]. Caution must be applied when looking at subspecies recognised only on the basis of morphological characters : in the Australian butterfly Ogyris amaryllis , the different subspecies were found to be less relevant than host plant choice in the partitionning of the among populations genetic variability [START_REF] Schmidt | Genetic affinities among subspecies of a widespread Australian lycaenid butterfly, Ogyris amaryllis (Hewitson)[END_REF]. Individual aberrations, more or less frequent, were often formally named (e.g. [START_REF] Courvoisier | Ueber Zeichnungs-Aberrationen bei Lycaeniden[END_REF]review in Russwurm 1978), and their genetic basis have sometimes been described [START_REF] Robinson | Genetics of European butterflies[END_REF][START_REF] Harmer | Variation in British butterflies[END_REF].

How are these forms first described by ardent collectors related to the population structure of these butterflies ? The firsts to give a biological interpretation of individual variation were [START_REF] Ford | Fluctuation in numbers and its influence on variation in Melitaea aurinia Rott. (Lepidoptera)[END_REF]. They showed that, in Euphydryas aurinia, the individual variation was dependent on local population trends: during phases of population increase, phenotypic variation increased and included a series of aberrant individuals, whereas in periods of population stasis, the individuals were much closer to a uniform phenotype. This process was interpreted as an increase of genetic variability due to a decrease of selection during population increase phases.

Thus, selection seems to play a key role in the variability of individuals within populations. The other key role is played by genetic drift, especially in small populations. Drift has two major impacts on populations : it is one of the main factors differentiating populations among which there is no gene flow, and through an increase in homozygosity of individuals it may have a deleterious effect on individual fitness. This was shown to be the case in at least two European butterflies of which populations had undergone bottlenecks : Melitaea cinxia and Coenonympha hero (see below).

Genetic differentiation among populations

The second level of variation is what happens among populations. For mountain species, numerous subspecies have often been described, and their distribution corresponds roughly to the distribution of mountain ranges (e.g. Parnassius apollo; [START_REF] Glassl | Parnassius apollo. Seine Unterarten[END_REF]). In Europe, many species have a wide range and show wing pattern variation between northern and southern populations, such as the Mediterranean and northern subspecies of Pararge aegeria [START_REF] Sbordoni | The World of Butterflies[END_REF], Brakefield & Shreeve 1992), or variations within France of Melanargia galathea [START_REF] Descimon | Mélanisme et facteurs climatiques[END_REF][START_REF] Mérit | Diversité et variations chez Melanargia galathea (Linné) en France[END_REF]. These species are classically described as sedentary, moving at the most a few kilometres out of their habitats. For migratory species, such as Aglais urticae or Vanessa cardui, hardly any within Europe variation can be phenotypically recognised, apart from some island forms (e.g. A. urticae ishnusa of Corsica and Sardinia). This contrast of migratory habits corresponds to the ecological classification of butterflies into erratic or migrant species, and sedentary species. The latter have local populations, which may persist year after year. By contrast, migratory species usually occur in a wide range of habitat, but their presence at any given locality is more difficult to predict. [START_REF] Thomas | The conservation of butterflies in temperate countries: past efforts and lessons for the future[END_REF] stated that about 85% of British Butterfly species have "closed" populations, i.e. have viable colonies in distinct habitat patches, whereas the remaining 15 % have open or migratory populations.

Long-term studies on the distribution of these butterflies have shown that species which have "closed" populations may disperse out of their habitat patches, as in the case of Hesperia comma, which recolonized many habitat patches from remnant populations [START_REF] Davies | The re-expansion and improving status of the silver-spotted skipper butterfly (Hesperia comma) in Britain: a metapopulation success story[END_REF]. This questions the relationship between ecological data, either from population survival data or from MRR and the genetic make-up of population in a spatial context. By essence, dispersal events are rare and difficult to Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008[START_REF] Hedrick | Genetics of Populations[END_REF] page 4

record; this is a major drawback to comparative data among species [START_REF] Bennetts | Methods for estimating dispersal probabilities and related parameters using marked animals[END_REF]). The tools of population genetics may be used in this context to assess the levels of gene flow among populations.

From a genetics point of view, a population is a group of individuals which share a common gene pool [START_REF] Dobzhansky | Mendelian populations and their evolution[END_REF]) and populations will be different if they do not share a common gene pool. Such differences may be quantified using genetical and statistical techniques. From a statistical point of view, two populations are different from each other if their allele frequencies, as estimated from the samples, are statistically different.

Without the exchange of individuals populations may become differentiated. In cases of complete isolation, each population has its own history of genetic drift and/or selection and over time the populations become more and more differentiated. (Box 1). Movements among populations do not need to be abundant to counteract the effect of genetic drift; an exchange of only one individual per generation is sufficient to avoid population differentiation [START_REF] Hartl | Principles of Population Genetics[END_REF]. Obviously, such a low movement rate is difficult to detect in the field by MRR studies. Furthermore, it is not possible to infer the probabilities of long distance dispersal from the analysis of within-patch short-distance dispersal, as such movement follow different ecological clues. Usually an individual engaged in dispersal behaviour outside its preferred habitat flies higher and quicker [START_REF] Baguette | Sex-biased density-dependent dispersal in a metapopulation of the butterfly Proclossiana eunomia[END_REF]. Such movements are hard to detect in the field by direct observation. Two kinds of data may be useful in this respect. Firstly, ecological data on colonisation gives evidence that a movement from an occupied to an unoccupied patch has occurred. Colonisation of empty patches is a key component of the metapopulation dynamics of many butterfly species (see Chapter C1). Secondly, genetic data may tell how different populations are from each other. Generally, the more the population are differentiated, the less individuals they have exchange, directly or indirectly.

Genetic indices of population differentiation may be used to infer the level of migration of individuals between populations (Box 1). As such, the relationships between the genetic differentiation of populations and their geographical distances may be compared between areas within a species, or between species. Using this approach, [START_REF] Britten | A test for isolation-by-distance in central rocky mountain and great basin populations of edith's checkerspot butterfly (Euphydryas editha)[END_REF] showed that the isolation by distance in Euphydryas editha populations was much stronger in the Rocky Mountains than in the Great Basin, resulting from stronger barriers to dispersal in mountain areas compared to the plains.

Isolation by distance

In a particular species, isolation by distance (IBD) may be observed or not at the same scale, depending on geographic area. In Parnassius apollo, [START_REF] Descimon | Diversité génétique, variation géographique et flux géniques chez quelques Lépidoptères Rhopalocères français[END_REF] showed that IBD was highly significant in the high Alps, but that populations from the southern Alps do not present such a pattern. This is because at some point in a recent past, the populations from the southern Alps were linked with each other in a single neighbourhood, and that recent barriers between these populations have not yet lead to isolation by distance. This is due to the large size of populations, and occasional migrations between them. By contrast, populations from the high Alps are more differentiated, due to the individual history of each of these populations, and post glacial colonization occurring in a stepping-stone fashion, leading to a greater IBD (Box 2).

The spatial structure of butterfly populations depends on where individuals of each sex have come from when they mate, and where females lay eggs. As the adult stage is usually the only one when long distance dispersal is possible, population structure is strongly related to dispersal in the adult stage. In a habitat patch network where individuals all have the same movement potential, if there is a negative relationship between the distances from emergence to reproduction sites and their frequencies, a pattern of isolation by distance emerges [START_REF] Wright | Isolation by distance[END_REF][START_REF] Epperson | Geographical Genetics[END_REF]. When a large number of populations have been sampled, it is possible to infer the spatial structure of the population from the genetic make-up of the individual populations and their geographical location (Box 2). Spatial statistics give information on how populations are alike to each other depending on their location. Because most adult butterflies move more or less freely within their natal habitat patch, population structure is generally studied at the between-population level. The isolation by distance model is only applicable if movements between neighbouring patches are more frequent than between patches further apart, as would be expected in sedentary species. Indeed, many studies were started suspecting that movement out of the natal patch would be rare, as very few individuals were ever sighted outside the preferred habitat (e.g. the mitochondrial DNA of this species, showed that from Europe to Japan, there is a high genetic diversity with wide distribution of both common and rare haplotypes (Vandewoestijne et al. 2004). This corroborates high gene flow, and hence the strong dispersal power of this species. In Maniola jurtina, local densities are generally high, and the global fixation indexes (F ST ) are in the range of 0.015 to 0.065, without any isolation by distance effect either a the local (Birmingham, Islaes of Scilly) or at the continental scales (Europe) (Table 1) ; this implies frequent individual migration between populations of this species. [START_REF] Porter | Limitations to the inference of gene flow at regional geographic scalesan example from the Pieris napi group (Lepidoptera: Pieridae) in Europe[END_REF] used the genetic approach to assess movements in the erratic species Pieris napi. In their study of 38 populations distributed throughout Europe, the isolation by distance model followed the relationship F ST =0.03-0.45/(4x+1), which for F ST =0 gives a estimated value of x=3.5 km, which is thus the estimated radius of the neighbourhood area for this species, and the estimated F ST for the whole continent was 0.0887 (SE=0.0076), giving estimated numbers of migrants between populations at 2.6 (CI : 1.6 -5.5). Such values suggest that there is a significant gene flow across the whole continent, which is not surprising, given the erratic behaviour of individuals and the wide distribution of their reproductive habitats. However, the populations of the Nordic montane subspecies Pieris napi adalwinda and the lowland subspecies (P. napi napi) are ecologically and genetically separated [START_REF] Espeland | Ecomorphological and genetic divergence between lowland and montane forms of the Pieris napi species complex (Pieridae, Lepidoptera)[END_REF]).

The situation for many species of European butterflies is very different from this.

Most occur in patchily distributed habitats, from which dispersal is a rare event. If a species is distributed in a series of discrete patches with metapopulation dynamics (see chapter C1), the total effective size of its population will be much smaller than if each population were long lived. Even a low extinction probability will have a dramatic effect on the total effective population size [START_REF] Whitlock | Fixation probability and time in subdivided populations[END_REF].

Selection

Population differentiation may be caused by substantially different selection pressures occurring in different habitat patches. The identification of the cause of the selection pressure is always difficult [START_REF] Manly | The statistics of natural selection on animal populations[END_REF][START_REF] Endler | Natural Selection in the Wild[END_REF]. In butterflies the identified causes of selection concern primarily temperature, host plant availability and there is evidence that habitat structure may also be a selective factor. E.B. Ford studied

Maniola jurtina in different habitat structures in the Isles of Scilly. Populations from small islands (<16 ha) were either unimodal with 0 or 2 wing spots, or bimodal at 0 and 2 spots, whereas the populations from the bigger islands were more evenly distributed. Evidence of constancy of spot pattern distribution in individual populations, even after a bottleneck, strongly suggested that spot pattern was under selection pressure rather than the result of random genetic drift in small island populations. Spot pattern frequencies changed after habitat changes, such as the removing of cattle grazing, rather than with population bottlenecks (studies summarized in [START_REF] Ford | Ecological genetics[END_REF][START_REF] Brakefield | The ecological genetics of quantitative characters of Maniola jurtina and other butterflies[END_REF]& 1990). More recently, other evidence was found for selection in this species. Different PGM alleles were favoured in different areas of its English distribution [START_REF] Goulson | Allozyme variation in the butterfly Maniola jurtina (Lepidoptera: Satyrinae) (L.): evidence for selection[END_REF]. Indirectly, this explains why the relationship between genetic similarity and geographic distance between populations is steeper in Britain than for the whole of Europe [START_REF] Thomson | Enzyme variation at morphological boundaries in Maniolia and related genera (Lepidoptera: Nymphalidae: Satyrinae)[END_REF]. As several of the loci studied by the latter author are probably under selection, similarity between regions under equivalent ecological conditions is expected to occur, thus counterbalancing the general isolation by distance effect, which has not been found

for this species in more recent studies at the regional or continent scales [START_REF] Goulson | Allozyme variation in the butterfly Maniola jurtina (Lepidoptera: Satyrinae) (L.): evidence for selection[END_REF]Schmitt et al. 2005b;[START_REF] Grill | Genetic differentiation and natural hybridization between the Sardinian endemic Maniola nurag and the European Maniola jurtina[END_REF]).

In the American species Euphydryas editha, the natural host plant at Schneider's Meadow (Nevada, USA) used to be the native plant Collinsia parviflora. Over a decade, the European plant Plantago lanceolata spread through the habitat. [START_REF] Singer | Rapid human-induced evolution of insect host associations[END_REF] showed conclusively that the host plant choice switched from the native species to the introduced one. Associated with this host plant switch will be changes of selection regimes related to host plant quality and phenology.

For several American Colias species, temperature is a major factor affecting the polymorphism of the enzyme phosphoglucoisomerase (PGI). [START_REF] Watt | Adaptation at specific loci. III. Field behaviour and survivorship differences among Colias PGI genotypes are predictable from in vitro biochemistry[END_REF] showed that the alleles present in different individuals were related to the temperature at which they fly, according to the optimal temperature of the PGI enzyme, as different PGI polymorphisms depending on the habitat they occupy (above tree line tundra vs. below tree line steppe), irrespective of the distance between these populations [START_REF] Watt | Adaptation at specific loci. VII. Natural selection, dispersal and the diversity of molecular-functional variation patterns among butterfly species complexes (Colias : Lepidoptera, Pieridae)[END_REF]. The populations of these different habitats may be either isolated or exchange many individuals each year, including between the two habitat types. Nevertheless there is consistent 10 to 20 % difference in PGI frequencies between the two habitat types; such a pattern may only be explained through continuous strong selection at the PGI locus. This key enzyme of glucose metabolism affects flight capacity. In Melitaea cinxia, the dispersal ability of the individual bearing the different PGI alleles were significantly different, and that this in turn affected population growth and dispersal pattern [START_REF] Haag | A candidate locus for variation in dispersal rate in a butterfly metapopulation[END_REF][START_REF] Hanski | Molecular-level variation affects population growth in a butterfly metapopulation[END_REF]). Using single nucleotide polymorphisms, [START_REF] Saastamoinen | Genotypic and environmental effect on flight activity and oviposition in the Granville fritillary butterfly[END_REF] showed that the two most common alleles for PGI in the Åland islands (Finland) populations of M. cinxia were linked with different temperature preferenda;

the individuals with the PGI-f genotypes flew at lower temperature and laid 32 % larger clutch size than PGI-non-f females because they tend to initiate oviposition during the warmest time of the day when clutched tend to be larger.. As this leads to a strong selection against PGI-non-f alleles, the question then remains as to what favours the PGI-non-f alleles in the population system.

Behaviour may also be subject to selection: individuals from isolated patches of habitat from which emigration would be extremely unlikely to be successful may be selected against. In the UK, severe isolation of the last remnant populations of both Maculinea arion and Papilio machaon resulted in decrease of thorax size in recent museum specimens compared to older ones [START_REF] Dempster | Fragmentation, isolation and mobility of insect populations[END_REF]. A more thorough study on the effect of isolation on flight ability was conducted in UK populations of the silver-spotter skipper (Hesperia comma), which was once widespread in southern and eastern England. It declined to its smallest range in the 1970s and 1980s, because a decrease of grazing rabbit populations, due to myxomatosis, led to a loss of habitat areas. With the recovery of rabbit populations from the beginning of the 1980, the species has recolonised some areas [START_REF] Thomas | Partial recovery of a skipper butterfly (Hesperia comma) from population refuges -lessons for conservation in a fragmented landscape[END_REF]. [START_REF] Hill | Flight morphology in fragmented populations of a rare British butterfly, Hesperia comma[END_REF] related morphology to colonization and demonstrated that thorax size was bigger in the area where recolonisation had been the quickest (East Sussex), than where it was slower (Surrey). They suggested that selection had operated more strongly against large thorax size and mobility in Surrey where the species had persisted in small (<1ha) species with strong flight abilities, such as Pieris napi, such a phenomenon does not occur. The case of Colias meadii where there is a significant difference of PGI allele frequencies according to altitude [START_REF] Watt | Adaptation at specific loci. VII. Natural selection, dispersal and the diversity of molecular-functional variation patterns among butterfly species complexes (Colias : Lepidoptera, Pieridae)[END_REF]) may be due to individual actively seeking a habitat according to their individual temperature requirements. The question remains open as to how often this may occur in other species. The number of generations per year for Aglais urticae may also be under a similar selection pressure, although at a larger scale, as A. urticae is trivoltine in central France, mostly bivoltine in England and univoltine in northern Scotland, with local variation according to altitude (Brakefield & Shreeve, 1992b).

Techniques in population genetics

Wing pattern

The first techniques used in population genetics concerned phenotypic variations, using these as surrogates for genetic information. The number and size of spots of several species of Satyrinae often display variable numbers of spots on their wings.

The number and size of spots in Coenonympha tullia vary with sex (females have more spots than males) and with locality [START_REF] Turner | A quantitative study of a Welsh colony of the Large Heath Butterfly, Coenonympha tullia Müller (Lepidoptera)[END_REF][START_REF] Dennis | Ocellation in Coenonympha tullia (Mőller) (Lepidoptera: Satyridae) I. Structures in correlation matrices[END_REF]. Spot pattern also varies among localities in Maniola jurtina and it was used to study population differentiation [START_REF] Dowdeswell | The influence of isolation on variability in the butterfly Maniolia jurtina L[END_REF][START_REF] Dowdeswell | The Life of the Meadow Brown[END_REF]. It was assumed that the wing spotting was heritable, on the basis that the pattern was consistent among years. However, heritability of this character was only formally demonstrated later, and was found to be sex linked: it was first tentatively estimated at 0.14 in males and 0.63 in females [START_REF] Mcwhirter | Heritability of spot number in Scillonian strains of the Meadow Brown Butterfly (Maniola jurtina)[END_REF], and later at 0.66 in males and 0.89 in females [START_REF] Brakefield | The genetics of spot pattern characters in the meadow brown butterfly Maniola jurtina (Lepidoptera, Satyrinae)[END_REF].

Protein electrophoresis

Upon the general availability of protein electrophoresis from the 1960s [START_REF] Johnson | Analysis of enzyme variation in natural populations of the butterfly Colias eurytheme[END_REF], this technique has been widely used for population genetic studies of butterflies, from the pioneering studies of Handford (1973a & b) to date. Nowadays this technique still remains the most widely used in population genetics studies of butterflies. The main reason for this choice is a combination of relative ease of scoring, and a fairly low price (Wynne et al. 1992). The scoring of the resulting zymograms is usually straightforward [START_REF] Richardson | Allozyme Electrophoresis[END_REF], and the Mendelian basis of the observed polymorphism may be checked using the known quaternary structure of the given protein, by experimental crosses or by Hardy-Weinberg equilibrium expectations. This technique proved powerful as many species exhibit a high degree of polymorphism. Studies usually focus on 3 to 25 polymorphic loci.

Most of the studies on protein electrophoresis assume that allele variation is neutral, or at least that no selection could be detected [START_REF] Besold | Allozyme polymorphisms in the small heath, Coenonympha pamphilus: recent ecological selection or old biogeographical signal?[END_REF]. As the proteins of interest have all definite functions, this is unlikely to be true [START_REF] Van Oosterhout | On the neutrality of molecular genetic markers: pedigree analysis of genetic variation in fragmented populations[END_REF]) but population differentiation based on protein electrophoresis has been, and still is, widely studied.

Some species (e.g. Lycaena helle) or life-stages (e.g. caterpillars) have sometimes proved difficult to be studied by protein electrophoresis, due to toxic compounds (e.g. oxalic acid, phenols) which interfere with enzyme activity. In this case the homogenization procedure should extract or neutralize these compounds.

Polyvinylpolypyrrolidone (PVPP) and a few grains of instant coffee have been mentioned as compounds which may improve enzyme stability by removing phenolic compounds during the grinding and homogenization procedures [START_REF] Hebert | Methodologies for Allozyme Analysis Using Cellulose Acetate Electrophoresis[END_REF]. For adult butterflies, I have used the following homogenizing solution : 50 mM Tris-HCl, 0.5 % (v/v) triton X-100 (optional), 15 % (w/v) sucrose, adjusted to pH 7.1

with HCl (Wynne & Brookes 1992). For 4th and 5th instar Melitaea cinxia larvae, the following homogenizing solution has been used : 100 ml distilled water, 10 mg NADP, 100 µl β-mercaptoethanol (Saccheri pers. com. ; solution from [START_REF] Richardson | Allozyme Electrophoresis[END_REF].

Molecular techniques

Protein electrophoresis has the major drawback that a common protein migration rate may result from two different alleles, hiding heterogeneity [START_REF] Johnson | Assessing electrophoretic similarity: the problem of hidden heterogeneity[END_REF]. Recent genetic studies on butterflies often rely on DNA-based molecular techniques. These are rapidly improving tools, and the choice of a method depends primarily on the questions asked, the scale of the study and on the chosen organism. Several good reviews of methods are currently available (e.g. [START_REF] Parker | What molecules can tell us about populations: Choosing and using a molecular marker[END_REF][START_REF] Avise | Molecular Markers, Natural History and Evolution[END_REF][START_REF] Behura | Molecular marker systems in insects: current trends and future avenues[END_REF]).

Mitochondrial DNA sequencing

Variation of mt DNA is studied by sequencing one or several genes of the short strand of mt DNA. The most studied parts are the control region (CR), cytochrome oxydase I or II (COI and COII). The region of interest is amplified by PCR and then sequenced for each individual [START_REF] Avise | Molecular Markers, Natural History and Evolution[END_REF]). Sequences of mitochondrial DNA are usually scored for phylogeography studies aiming at an understanding the pattern of colonization at the continental scale (e.g. Vandewoestijne et al. 2004). Variation in mt DNA sequence has also been used to assess levels of genetic variation in cases where the conservation of frozen specimens would have been difficult. Diversity of populations of Mycalesis orseis within forest fragments in Malayan Borneo, assessed by the number of mt haplotypes, was negatively affected by isolation of their habitat patch, but not by population size of patch size [START_REF] Benedick | Impacts of habitat fragmentation on genetic diversity in a tropical forest butterfly on Borneo[END_REF]). The low level of mutation within the mitochondrial DNA allow the study of long term processes: in the North American Parnassius smintheus, the local variation of mt DNA haplotypes could be linked with the range expansion and retraction during glacial-interglacial cycles. During warm periods, populations persisted at mountain tops, whereas they expanded during cold spells. As a result, populations from an area within a mountain range have a series of possible refugias during warm periods, and end up being more diverse than those from an area with fewer refugias (DeChaine & Martin 2004).

Randomly Amplified Polymorphic DNA (RAPD)

This technique uses short PCR primers (ca. 10 bp) to amplify DNA fragments. This primer length is short enough to find several annealing sites in the genome by chance alone, but long enough as not to amplify too many fragments. Usually several possible primers are tested, and the ones yielding recognisable and repeatable banding patterns are then selected for the study. The major drawback of this method is that it is not possible to identify from which genome region each band is amplified. Furthermore, the banding pattern is very sensitive to laboratory conditions. Due to these drawbacks, [START_REF] Vandewoestijne | The genetic structure of endangered populations in the Cranberry Fritillary, Boloria aquilonaris (Lepidoptera, Nymphalidae): RAPDs vs allozymes[END_REF] showed that RAPD on 18 polymorphic loci in Boloria aquilonaris yielded significant IBD, while isozymes (4 polymorphic loci) on the same populations did not. This draws attention to the fact that the lack of genetic differentiation found with one marker does not necessarily mean that the populations are not differentiated.

From a statistical point of view, it is simply that the hypothesis that the populations are not differentiated may not be rejected [START_REF] Bossart | Genetic estimates of population structure and gene flow: limitations, lessons and new directions[END_REF].

Amplified fragment length polymorphism (AFLP)

To avoid the drawbacks of RAPD, primer pairs are used to amplify known regions of the nuclear genome and then digested by restriction enzymes. These generate a series of bands, according to the length of the amplified regions. Three primer pairs used on 190 specimens from across the North American range of Lycaedes melissa yielded a total of 143 bands ranging in size from 71 to 481 bp [START_REF] Gompert | Identifying units for conservation using molecular systematics: the cautionary tale of the Karner blue butterfly[END_REF].

Microsatellites

Given the highly functional nature of enzymes studied by allozyme electrophoresis, and the proven selection which may occur on these loci, a neutral marker is desirable for population studies. Microsatellites are tandem repeats of 1 to 6 bp motifs such as ACACACAC. This marker seemed to be the "Holy Grail" for population geneticists, as microsatellites are non-coding lengths of repetitive DNA. It was thought that these would be neutral and, by providing high levels of polymorphism, could end up as excellent tools for population genetics. Although it has been shown that they may be linked by hitch-hiking to a gene under selection pressure or that they may even be under selection themselves [START_REF] Estoup | Microsatellite evolution : inferences from population data[END_REF], they are still regarded as the first choice of neutral markers (Golstein & Schlötterer 1999). Compared with allozymes, which require fresh or frozen material, microsatellite analysis can be conducted with dry material. This facilitates the study of museum material (Meglécz et al. 1998b[START_REF] Harper | Analysis of museum specimens suggests extreme genetic drift in the adonis blue butterfly (Polyommatus bellargus)[END_REF] or the use of non-lethal sampling [START_REF] Lushai | Application of molecular techniques to non-lethal tissue samples of endangered butterfly populations (Parnassius apollo L.) in Norway for conservation management[END_REF][START_REF] Keyghobadi | Genetic differentiation and gene flow among populations of the alpine butterfly, Parnassius smintheus, vary with landscape connectivity[END_REF]. From a practical point of view, each microsatellite locus is specifically amplified by PCR using locus specific primer pairs which recognise the flanking region of each side of the studied loci. The identification of these microsatellite loci with the design of primers is the most time consuming task for the set-up for microsatellite based studies. This has to be done for each new species studied, but workable pairs of loci in one species are typically tested in other congeneric species, with various success. From a series of 17 loci identified for Papilio zelicaon, between 5 and 14 loci could be amplified in other Papilio species but their polymorphism in these other species still remains to be tested [START_REF] Zakharov | Characterization of 17 polymorphic microsatellite loci in the Anise swallowtail, Papilio zelicaon (Lepidoptera: Papilionidae), and their amplification in related species[END_REF]). In the 1990s, many butterfly biologists tried to develop microsatellite methods for butterfly population biology studies. By the end of the decade, it became apparent that the recurrent problems faced by the butterfly geneticists might be linked to the structure of the Lepidoptera genome rather than to the expertise of the involved laboratories (Meglécz & Solignac 1998 Euphydryas aurinia and Parnassius apollo showed that many microsatellites loci could be grouped by similar flanking regions. Thus the numbers of microsatellites with unique flanking regions were drastically reduced [START_REF] Meglécz | High similarity between flanking regions of different microsatellites detected within each of two species of Lepidoptera: Parnassius apollo and Euphydryas aurinia[END_REF].

Subsequently this was found to be the case for many other Lepidoptera species, and also other insects [START_REF] Meglécz | Microsatellite flanking region similarities among different loci within insect species[END_REF].

Single Nucleotide Polymorphism

Due to problems in the use of microsatellite loci in Lepidoptera, other markers useful for population genetics markers were desirable. [START_REF] Orsini | SNP discovery by mismatch-targeting of Mu transposition[END_REF]2008) identified a series of single nucleotide polymorphisms (SNP) in Melitaea cinxia. Among these two could be identified with known variants of the Phosphoglucoisomerase (PGI) locus, a key enzyme in the glycolysis cycle, which was already known to affect dispersal rate in this species [START_REF] Haag | A candidate locus for variation in dispersal rate in a butterfly metapopulation[END_REF]. As the use of SNP could be done without killing the individuals (a 2 mm diameter part of the hind wing was enough), the PGI genotypes could be studied on individuals which were later followed for their behaviour, with collected data on their flight body temperature, oviposition time and clutch size.

The new partial sequencing of the coding region of the M. cinxia genome gave sequence information on over half of the genes of this species (Vera et al. 2008;[START_REF] Ellegren | Sequencing goes 454 and takes large-scale genomics into the wild[END_REF]). As the method used a pool of ca. 80 individuals (caterpillars, pupae and adults from 8 families), this approach provided unprecedented access to M. cinxia genome polymorphism, leading to the identification of numerous SNP and to future detailed quantitative trait loci studies. No doubt the future of butterfly population genetics will increasingly use SNPs in their approach.

Differentiation among butterfly populations

In Europe, a total of 87 studies of spatial aspects of population genetics have been located (Table 1). Of these, the great majority involved allozyme electrophoreses (80 cases), 3 studies involved RAPD and 5 involved microsatellites. Generally the authors give a value of population differentiation, either F ST , or one of its derived estimates (θ, G ST ). Each study species has its own ecological needs, and history of postglacial colonisation, from one or several refugia. These species may have widespread populations with frequent movements, as is the case of migratory species such as Aglais urticae [START_REF] Vandewoestijne | Spatial and temporal population structure of the butterfly Aglais urticae (Lepidoptera Nymphalidae)[END_REF], whereas others are very sedentary, such as Plebejus argus [START_REF] Brookes | Genetic analysis of founder bottlenecks in the rare British butterfly Plebejus argus[END_REF].

Most habitat choice of many species, butterflies of the families Papilionidae and Nymphalinae tend to have higher F ST than those of the families Lycaenidae, Pieridae and Satyrinae (Figure 8). This is also due to a bias in the studied species, as e.g. many

Lycaenidae species have narrow ecological requirements, and probably a low colonization power. Within four of these families (Lycaenidae, Papilionidae, Pieridae, Nymphalinae), there is no trend between the size of a study area and the observed F ST (P>0.3). For Satyrinae, however, the size of the study area, in logarithmic scale, is correlated with the observed F ST (t=3.07, 22 df, P=0.006, Figure 3). Such a trend is

Dispersal ability and population differentiation

Species with low dispersal abilities show larger F ST values. Parnassius apollo is known to be very vagile and it is therefore not surprising that population from the same mountain massif are hardly different from one another, with a non-significant isolation-by-distance effect in the southern Alps. However, when all French populations are included, the slope of F ST against distance is -0.54 indicating that areas between mountain ranges act as effective barriers to dispersal [START_REF] Descimon | Diversité génétique, variation géographique et flux géniques chez quelques Lépidoptères Rhopalocères français[END_REF], even if vagrants sometimes occur there [START_REF] Lafranchis | Les papillons de jour de France, Belgique et Luxembourg et leurs chenilles[END_REF]. In the case of large scale disturbances (such as fires or drought) the local genetic diversity of a population will depend on the scale at which migration and colonization events take place. In the tropical species Drupadia theda, populations in areas near to undisturbed habitats tend to be more diverse then more isolated populations [START_REF] Fauvelot | Short-term impact of disturbance on genetic diversity and structure of Indonesian populations of the butterfly Drupadia theda in East Kalimantan[END_REF].

When a range of species within a single habitat network are studied, their dispersal abilities may effectively be compared. With a MRR survey, [START_REF] Baguette | Population spatial structure and migration of three butterfly species within the same habitat network: consequences for conservation[END_REF] showed that Cupido minimus had much less dispersal ability than the sympatric Melanargia galathaea and Aporia craetegi. Both MRR and genetic approaches showed that Euphydryas aurinia is less prone to inter patch movement between patches than Melitaea phoebe (Wang et al. 2003[START_REF] Wang | Contrasting movement patterns in two species of chequerspot butterflies, Euphydryas aurinia and Melitaea phoebe, in the same patch network[END_REF]. Genetic studies of three Thymelicus species in Luxembourg and Germany showed that the three species have [START_REF] Porter | Limitations to the inference of gene flow at regional geographic scalesan example from the Pieris napi group (Lepidoptera: Pieridae) in Europe[END_REF]. Similarly, populations of Proclossiana eunomia from the Pyrenees display more differentiation than those from the Ardennes, as the slope of the IBD is -0.91 in the Pyrenees and -0.53 in Ardenne (Fig. 4).

Population differentiation generally occurs when population are isolated from each other in space. In some cases, however, there seem to be isolation through ecological preferences. In Carterocephalus palaemon, two morphotypes (Carterocephalus palaemon palaemon and C.p. tolli), probably originating from distinct glacial refugia, are found in BiałowisŜa primeval forest (NE Poland). These are maintained because of their ecological differentiation both in habitat and phenology, resulting in assortative mating [START_REF] Ratkiewicz | Allopatric origins of sympatric forms: the skippers Carterocephalus palaemon palaemon, C. p. tolli and C. silvicolus[END_REF]. The two subspecies of Proclossiana eunomia at BiałowisŜa [START_REF] Krzywicki | Fauna Papilionoidea i Hesperioidea (Lepidoptera) Puszczy Bialowieskiej[END_REF]) may show the same phenomenon.

Population isolation

For conservation biology, the consequences of population isolation set challenges. An isolated population may evolve locally according to local conditions, but it may also undergo genetic drift, leading to loss of genetic variability, or to stochastic demographic extinction. If populations are long lived, the total effective size of the population is higher if it is the sum of a series of isolated demes [START_REF] Whitlock | The effective size of a subdivided population[END_REF]. However, in a metapopulation system (see Chapter F3), as local populations result from an equilibrium between extinction and colonisation, there is high gene flow between the populations, and little room for local adaptation to take place; selection will then operate more at the metapopulation scale. Genetic drift and selection are difficult to distinguish in population differentiation. In Erebia triaria, the isolated population of Xistral (NW Spain), named Erebia triaria pargapondalense, is as different from Cordillera Cantabrica populations (ca. 120 km apart) as from Pyrenean populations (720 km away), according to a study using four microsatellite loci and mitochondrial DNA. In this case, the genetic approach confirmed ecological and morphological data [START_REF] Vila | The importance of time scale and multiple refugia: Incipient speciation and admixture of lineages in the butterfly Erebia triaria (Nymphalidae)[END_REF][START_REF] Vila | A new conservation unit in the butterfly Erebia triaria (Nymphalidae) as revealed by nuclear and mitochondrial markers[END_REF].

Population size

Small and isolated populations undergo genetic drift,due to the low numbers of reproducing individuals, and the homozygosity of such populations tends to increase. Isolated populations also suffer from several ecological effects, which affects their reproduction and hence their long term survival: demographic stochasticity, a low buffering effect due to the small habitat patch size and micro-habitat diversity, behaviour alterations due to low population density or close proximity to habitat boundaries, and often an overall lower habitat quality. These phenomena are often exarcerbated by the position on the edge of the relevant species' range (see Chapter E2). Such situation lead to increased homozygosity in individuals, as observed on westernmost populations of Coenonympha hero [START_REF] Cassel | Allozyme variability in central, peripheral and isolated populations of the scarce heath (Coenonympha hero : Lepidoptera, Nymphalidae): Implications for conservation[END_REF] or

Polyommatus bellargus [START_REF] Harper | Molecular evidence for a recent founder event in the UK populations of the Adonis blue butterfly (Polyommatus bellargus)[END_REF]. By contrast phenotypic variation may increase, due to lower canalization in populations with low genetic variation [START_REF] Debat | Mapping phenotypes: canalization, plasticity and developmental stability[END_REF], as seen in peripheral populations of Polyommatus icarus [START_REF] Artemyeva | Clinal variation in populations of the common blue butterfly Polyommatus icarus Rott. (Lepidoptera, Lycaenidae)[END_REF]).

In the Åland islands (Finland), the main factors affecting Melitaea cinxia population survival are population size, density of neighbouring populations, patch size and cattle grazing [START_REF] Hanski | Multiple equilibria in metapopulation dynamics[END_REF]. A genetic study conducted on individuals caught in 1996

showed that heterozygosity had a significant extra effect on the extinction risk of the 42 genetically studied M. cinxia populations: the seven populations which went extinct between 1995 and 1996 had both ecological factors affecting their survival and a lower than average heterozygosity [START_REF] Saccheri | Inbreeding and extinction in a butterfly metapopulation[END_REF]. A low heterozygosity was thus shown to be a significant extra factor affecting population survival (Fig. 5).

Further evidence that population heterozygosity affects survival was given by an experiment in which the founder individuals of each populations were either full sibs, generating individuals with an inbreeding coefficient of 0.25, or outbred individuals from parents from two different populations, thus having a zero inbreeding coefficient. Three larval groups from either outbred or inbred individuals were introduced into one of 12 unoccupied habitat patches. Only two of the inbred populations attained adulthood and reproduction, and went extinct by the next year, while four of the six outbred populations survived until the next year [START_REF] Nieminen | Experimental confirmation that inbreeding depression increases extinction risk in butterfly populations[END_REF]). As inbreeding is deleterious, mate choice could avoid inbreeding, but experimental result showed that individual butterflies are unable to recognize sibs from non-sibs for mating [START_REF] Haikola | Has inbreeding depression led to avoidance of sib mating in the Glanville fritillary butterfly (Melitaea cinxia)?[END_REF]). As a result, inbreeding is more likely in small than in large populations, and this affects egg hatching rate, larval surviving rate (Haag & de Araú 1994[START_REF] Haikola | Inbreeding depression and the maintenance of genetic load in Melitea cinxia metapopulations[END_REF], and adult survival rate [START_REF] Saccheri | Inbreeding and extinction in a butterfly metapopulation[END_REF]; furthermore, a second generation of inbreeding, by pairing full sibs, further decreased clutch size [START_REF] Haikola | Effects of inbreeding in the Glanville fritillary butterfly (Melitaea cinxia)[END_REF]. A comparison of inbreeding effect conducted on French and Finnish individuals showed that a reduction of hatching rate from full sibs occurred in all cases, but was more pronounced in French individuals than in Finnish individuals. The genetic load thus seems to be less severe in Finnish populations than in French ones. This is probably due to the repeated bottlenecks through which Finnish populations have been through, which have purged them from a number of deleterious recessives [START_REF] Haikola | Inbreeding depression and the maintenance of genetic load in Melitea cinxia metapopulations[END_REF]. Data on low reproductive output in isolated populations of Parnassius apollo also indicates that inbreeding was the main factor affecting small populations of this species as well [START_REF] Witkowski | Extinction and reintroduction of Parnassius apollo in the Pieniny National Park (Polish Carpathians)[END_REF] ; in some cases the reproductive power of an isolated population was enhanced by the introduction of individuals from nearby populations with which exchanges are now unlikely to occur [START_REF] Nakonieczny | Apollo Butterfly (Parnassius apollo L.) in Europe -its history, decline and perspective of conservation[END_REF]).

Coenonympha hero is a species which has already vanished from most of its former European range (van Swaay & Warren 1999). In Sweden, some local populations are Festuca ovina. The hatching rate of eggs from small populations was lower, and their death rate and proportion of unfertilized eggs larger, compared to eggs from larger populations (Fig. 6). This phenomenon is most likely to be related to the increased homozygosity of the small populations, due to local inbreeding. Furthermore females from small populations had a higher probability of not being mated, and thus to be effectively infertile. It is unlikely that small populations of this species will remain viable, both for the genetic reasons of increased homozygosity, and for ecological reasons such as a reduced microhabitat variability which will not effectively buffer against environmental variation. Furthermore, Swedish populations of C. hero already have a heterozygosity which is much lower (Hobs=0.017) that in the more central populations of Estonia of the Urals (Hobs=0.052; [START_REF] Cassel | Allozyme variability in central, peripheral and isolated populations of the scarce heath (Coenonympha hero : Lepidoptera, Nymphalidae): Implications for conservation[END_REF].

The studies of inbreeding effects on Melitaea cinxia and Coenonympha hero indicate that inbreeding depression can have a significant effect on small and isolated populations. As reintroduction schemes are being considered for a number of species in parts of their range from which they have disappeared, there is therefore a requirement for genetic diversity to be considered to avoid inbreeding effects.

Host plant range

Populations which use a number of host plants tend to be more varied than ones using a single host plants. In this respect the case of Euphydryas aurinia in France is spectacular. In the Atlantic and continental part of the country it feeds on one or two Dipsacacae species: mainly Succisa pratensis and sometimes on some Knautia species. Sampled populations, up to 700 km apart, show a low F ST value of 0.0648, whereas populations from South France (up to ca. 650 km apart) show a F ST value of 0.112. [START_REF] Descimon | Diversité génétique, variation géographique et flux géniques chez quelques Lépidoptères Rhopalocères français[END_REF] concluded that the high F ST obtained from southern populations is due to the large number of food plants used in this part of the range:

Dipsacacae (Cephalaria, Knautia, Scabiosa, Succisa), Caprifoliacae (Lonicera), Valerianacae (Centrentus) and Gentianacae (Gentiana). Each local population seems to use only one food plant in a given locality [START_REF] Mazel | Structure et évolution du peuplement d'Euphydryas aurinia Rotemburg (Lepidoptera) dans le sud-ouest européen[END_REF]). In the South of France, a neighbour-joining dendrogram (Fig. 7) shows that populations mainly cluster according to their geographical origin, i.e. according to distance. However some populations show strong difference from this tendency. The population from Sommail is more similar to ones in southwest France, than to others in Languedoc, and this population is the only sampled Languedoc population which feeds on Succisa pratensis, like the ones in the southwest, and unlike the others in Languedoc, which feed on Cephalaria. Similarly, two populations from the Pyrenees, found feeding on Succisa, do not group with the other ones from the same area feeding on Lonicera, but with the ones from Languedoc, also feeding on Succisa. In a study of 11 populations of E. aurinia from south France and north Spain, scored by AFLP markers, larvae of Euphydryas aurinia found on Succisa or on Lonicera at the same site were shown to be as different as two allopatric populations feeding on different hosts are [START_REF] Singer | Spatial pattern in checkerspot butterfly-host plant association at local, metapopulation and regional scales[END_REF]. Differences between individuals at the same site, whether on the same or on different hosts, are generally smaller within than between populations. A high level of differentiation between larvae found on different host plants will be more likely to occur in allopatry than in sympatry, but the latter may be relevant in some cases (Wee 2004, Singer unpubl.) This tends to confirm Descimon's interpretation that E. aurinia may be undergoing speciation in South France. However, such a differentiation pattern in not the rule. Euphydryas editha caterpillars at Sonora junction (California, USA), were not genetically different whether they came from eggs laid on Penstemon or Castilleja, thereby discarding any speciation event between the individuals on the two hosts. Furthermore, the selection pressure put forward by the different hosts may affect the variability of the population. [START_REF] Singer | Spatial pattern in checkerspot butterfly-host plant association at local, metapopulation and regional scales[END_REF] showed that larvae of E. editha developing on Castilleja (from eggs naturally laid on this host) had a higher heterozygosity (0.137, SE=0.007) than larvae from Pedicularis (0.119, SE=0.007) (p<0.001, Mann-Whitney U test) at the same site (T-junction, Ca, USA). As female E.

editha from this site all accept both hosts to lay their eggs, a process of sympatric speciation may be ruled out. The only cause seems to be that the two hosts induce different mortality rates between egg hatch and time of sampling, thereby indicating different selection pressure on the populations.

Barriers to dispersal

In Germany, populations of Chazara briseis have declined. Present populations on igneous and calcarious hills are separated from each other by farmland and urban development. Most individuals of this species stay in their natal patch, with a mean distance between capture events of 80 m (n=191), while about 2% (n=5) of individuals moved more than 1000 m. The question then is: are these occasional long distance movements efficient in maintaining gene flow within the landscape? An analysis of 165 individuals from 9 populations up to ca. 10 km apart gave a mean F ST of 0.022 for the 15 polymorphic loci. [START_REF] Johannesen | Analysis of gene flow and habitat patch network for Chazara briseis (Lepidoptera: Satyridae) in an agricultural landscape[END_REF] suggested that these populations show limited substructure and that the agricultural landscape does not constitute a barrier for this species. Dispersive individuals moving out of their preferred habitat tended to move to a neighbouring habitat patch, leading to an isolation by distance effect, which was indeed detected.

Post-glacial dispersal pattern

Since the last glacial maximum (18,000 yr BP), the climate of Europe has changed dramatically, and this has lead to the colonisation of northern areas from southern refugia [START_REF] Huntley | Migration: Species' response to climatic variations caused by changes in the earth's orbit[END_REF]. This change in distribution of organisms can have lasting consequences for the genetic make-up of populations. For species with a low dispersal ability, post-glacial colonisation event took place slowly, and usually by stepping stone patterns (see Chapter E2). The main consequence for the genetic diversity is that the centres of origin of this post-glacial northern migration are in southern Europe or Asia. Consequently there is generally a decrease of genetic diversity from the centre of origin to the edge of a species range [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF] European stock during historic time, and that this colonization was subjected to a bottleneck [START_REF] Harper | Molecular evidence for a recent founder event in the UK populations of the Adonis blue butterfly (Polyommatus bellargus)[END_REF]).

Open questions and conclusion

Generally, populations of butterflies show differentiation according to the geographical distance separating them, but the pattern may differ widely between species within a given landscape. As expected, widely distributed and highly mobile species display less differentiation than more sedentary species. The high fragmentation of habitats decreases gene flow between populations. As most species of European butterflies occur in discrete populations, this effect is of major consequence, and may lead to the quick decline of populations once a critical threshold of habitat connectivity has been reached [START_REF] With | Critical thresholds in species' responses to landscape structure[END_REF]. However, the type of habitat between patches also has crucial importance in the dispersal ability of individuals. For example, forest habitats are effective barriers to dispersal for open habitat species such as Erebia medusa [START_REF] Schmitt | Forests as dispersal barriers for Erebia medusa (Nymphalidae, Lepidoptera)[END_REF] and Parnassius smintheus [START_REF] Keyghobadi | Genetic differentiation and gene flow among populations of the alpine butterfly, Parnassius smintheus, vary with landscape connectivity[END_REF], while the reverse is true for several forest species, such as the moth Operophtera brumata (van Dongen 1994) or Pararge aegeria, as it seems that individuals from forest areas need to perceive the presence of a forest to move towards it [START_REF] Merckx | The evolution of movements and behaviour at boundaries in different landscapes: a common arena experiment with butterflies[END_REF]. How other habitat structures affect butterfly dispersal remains an open, but critical, question. The response of individuals will also depend on regional habitat structure, as dispersal behaviour is expected to be selected against in very fragmented habitats, while such behaviour will be more common in more continuous habitats [START_REF] Baguette | Effect of habitat fragmentation on dispersal in the butterfly Proclossiana eunomia[END_REF]. As dispersal ability is heritable -it was recently shown to be heritable from mother to female offspring in Melitaea cinxia [START_REF] Saastamoinen | Heritability of dispersal rate and other life history traits in the Glanville fritillary butterfly[END_REF]) -, the selection pressure of landscape structure on individual dispersal abilities may occur, and result in differential dispersal abilities according to regional landscape structure [START_REF] Schtickzelle | Dispersal depression with habitat fragmentation in the bog fritillary butterfly[END_REF][START_REF] Van Dyck | Dispersal behaviour in fragmented landscapes: Routine or special movements?[END_REF], as was first suggested by difference in thorax width among Hesperia comma populations [START_REF] Hill | Flight morphology in fragmented populations of a rare British butterfly, Hesperia comma[END_REF].

In detecting significant barriers to dispersal, Bayesian methods may prove to be very useful. The emerging field of landscape genetics uses techniques of Bayesian clustering to identify clusters of individuals, and hence the barriers between clusters (Software GENELAND, [START_REF] Guillot | GENELAND: a computer package for landscape genetics[END_REF], or EASYPOP, Balloux 2001). Bayesian methods may also help to delineate populations in very mobile species such as Aglais urticae or various Colias species. Graphical methods using multivariate analyses offer some grouping, but do not allow confidence to be made about the barriers. In contrast, Bayesian methods may prove useful in this respect, as these may combine the information from the genotypes of the sampled individuals and tests which of several barriers may be the most likely [START_REF] Corander | BAPS 2: enhanced possibilities for the analysis of genetic population structure[END_REF]). Unfortunately, such important studies involving population genetics and landscape ecology need high research investment, and may be carried out only in few, carefully chosen study systems.

The inbreeding consequences shown in Finnish Melitaea cinxia populations most probably occur generally. As more and more populations become isolated, they end up occurring in non-equilibrium metapopulations, where extinctions are more frequent than colonisation, eventually leading to the total extinction, due to both local inbreeding and demographic disequilibrium. In extreme cases where active management aims at rescuing a declining population, on top of habitat restoration, "genetic restoration" should also take place by pairing individuals which come from populations which used to be part of the same metapopulation, even if populations in between have now become extinct.

The distribution of many European butterflies is likely to change dramatically over the coming decades as a result of global warming. Several species have already started a northward shift [START_REF] Parmesan | Poleward shifts in geographical ranges of butterfly species associated with regional warming[END_REF]. The future of this pattern is likely to have far reaching consequences, as populations of the mountains of southern Europe will move upwards, which will result in their increased isolation, and in many cases eventual extinction [START_REF] Wilson | Changes to the elevation limits and extent of species ranges associated with climate change[END_REF]. Furthermore, species should not be viewed generally as adapted to one particular environment; they generally include populations genetically adapted to a range of environmental conditions (see Chapters B5 and D1).

Populations from the middle of the range, will also be affected, as these will have to adapt to changing conditions, in a typical "Red Queen" fashion: they will have to adapt to new environmental conditions to stay in the same place [START_REF] Lythgoe | Catching the Red Queen? The advice of the rose[END_REF], through selection of genotypes adapted to warmer temperatures, as has been shown for numerous other species [START_REF] Parmesan | Ecological and evolutionary responses to recent climate change[END_REF]. In erratic species, alleles adapted to warm conditions presently present in the south of the range will migrate north, through a change in the selective gradient due to climatic conditions getting warmer.

For more sedentary species, this process may be more difficult, as most species occur in patchy habitats, which are now isolated from each other [START_REF] Bridle | Limits to evolution at range margins: when and why does adaptation fail?[END_REF].

In such species, the genetic variation on the edges of the distribution is generally lower than at its center, as shown in Erynnis propertius and Papilio zelicaon [START_REF] Zakharov | Genetic differentiation across a latitudinal gradient in two cooccurring butterfly species: revealing population differences in a context of climate change[END_REF]. This in turn may affect the future shift in distribution, as the species with less gene flow (E. propertius) will be more affected than the more vagile species (P. zelicaon).

For species responding to photoperiod to time specific life-stages, this adaptation is under a strong selection pressure. With climate change the environmental conditions associated with specific photoperiods will alter, imposing new selection pressures and requirements for changes of responses to specific photoperiods. Populations of butterflies will then be affected in numerous ways, involving a large set of ecological and physiological characters, ultimately depending on their genetic make-up governing their response to photoperiod, dispersal behaviour and temperature range for adult and larval activities. How these strong selection pressures will globally affect European butterflies is largely unknown, as such diverse and large-scale environmental change has not been observed before.

Box 1

Genetics drift and population differentiation

The population genetics of population differentiation are based on the simple principle that two opposite forces act on this phenomenon: genetic drift tends to differentiate populations, whereas gene flow tends to homogenize populations. If two small populations are isolated from each other, they tend to diverge in allele frequencies more quickly than do large populations (Fig. 1). This principle is used backward to evaluate how population are different from each other, from their allele frequency differences using different methods of calculating a genetic distance between populations (Hartl & Clarck 1989[START_REF] Hedrick | Genetics of Populations[END_REF]. Within populations, individuals may mate at random, in which case the number of heterozygotes in the population will be dependant solely on allele frequency. For an allele frequency of p, the frequency of heterozygotes is 2p(1-p). If populations do not show heterozygote deficiency (in which cases the individual samples may actually result from a local deviance of random mating), the degree of difference among populations may be measured by means of Wright's F statistics, now easily computed using programs such as Hierfstat [START_REF] Goudet | Hierfstat, a package for R to compute and test hierarchical F-statistics[END_REF] or Genepop [START_REF] Rousset | GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux[END_REF]. When a large number of populations are studied, F statistics may be ranked to study hierarchical clustering of populations, to study differentiation within a cluster of habitat patches, or among them, which may be river systems, mountain tops or otherwise discrete habitats.

In particular, patterns of allozyme genotype frequencies allow the use of Sewall

Wright's F ST index, which gives a concise way of expressing the degree of population differentiation between populations [START_REF] Wallis | Population genetics and conservation in New-Zealand -a hierarchical synthesis and recommendations for the 1990s[END_REF]. In a group of populations, the population differentiation is measured by the fixation index (symbolized F ST ), which is estimated as

T s T ST H H H F / ) ( - =
, with s H the mean expected heterozygosity of an individual in an equivalent population mating randomly, and T H the expected heterozygosity of an individual in a total population mating randomly. [START_REF] Wright | Variability within and among Natural Populations[END_REF] suggested some guidelines to interpret the resulting values. A value of F ST smaller than 0.05 may be considered as indicating little genetic differentiation, the range 0.05 to 0.15 indicates moderate genetic differentiation, 0.15 to 0.25 great genetic differentiation and over 0.25 very great genetic differentiation. The higher the value of F ST is, the lower the number of migrant among populations per generation would be.

In an island model, where individuals moving out of a population may move with an equal probability to any other population, not just to the ones nearby, the migration rate among populations may be expressed as [START_REF] Slatkin | Isolation by distance in equilibrium and non-equilibrium populations[END_REF] with m the migration rate and N the effective population size.

        - ≈ 1 1 4 1 ST F Nm
This equation allows an estimate of the number of migrating individuals from the F ST differentiation statistics. The given value always has to be taken with caution. The island model assumptions are rarely valid with field data; the model assumes that (1) there is no selection, (2) there is no mutation, (3) all populations host the same number of individuals and contribute equally to the migration pool, (4) migration is random (i.e. irrespective of the distance between the populations), (5) the system is at equilibrium [START_REF] Whitlock | Indirect measures of gene flow and migration: F ST ≠1/(4Nm+1)[END_REF]. In practice most of these assumptions are violated with field studies, but comparative analyses using F ST nevertheless yield valuable information on among-population migration [START_REF] Bohonak | Dispersal, gene flow, and population structure[END_REF]. Furthermore, if the number of populations from which F ST is estimated is small, the variance of F ST is large [START_REF] Douwes | Selective versus stochastic processes in the genetic differentiation of populations of the butterfly Erebia embla (Thunberg) (Lepidoptera, Satyridae)[END_REF], rendering the estimate of the number of migrants impossible to infer. However, as F ST increases or decreases monotonously with the number of migrants, F ST values or the estimate number of variants may still be used to compare different population system, either within a species or among species [START_REF] Wang | Estimating effective population size and migration rates from genetic samples over space and time[END_REF].Other indexes of population differentiation have been implemented since the description of the F ST index. G ST is a generalisation of F ST for the study of different loci simultaneously [START_REF] Hartl | Principles of Population Genetics[END_REF]. These indices both study the population differentiation, irrespective of the relative spatial positions of the relevant populations.

[Figure 1]

Box 2

Spatial aspect of population genetics

The two main parameters of spatial population genetics are the genetic neighbourhood and the isolation by distance. The isolation by distance approach compares populations two at a time, and assess whether there is a correlation between geographic distance and genetic differentiation of populations. The slope of the curve is then an indication of the level of gene flow between the populations, and this parameter varies both among species and among region within species, according to the distribution of habitat patches [START_REF] Nève | Gene flow rise with habitat fragmentation in the bog fritillary butterfly (Lepidoptera, Nymphalidae)[END_REF].

If migration occurs more frequently between neighbouring populations than between distant ones, one would expect the allele frequencies to be more similar between neighbouring populations then between more distant ones. The genetic neighbourhood is the area within which the individuals mate at random [START_REF] Wright | Variability within and among Natural Populations[END_REF]. Isolation by distance occurs if the genetic relatedness between populations decreases as the geographic distance between them increases. This may be estimated using Moran's I [START_REF] Legendre | Numerical Ecology[END_REF], which computes the correlation between the allelic frequencies in pairs of populations according to the geographical distance between them. In cases of isolation by distance, the correlation decreases continuously with distance, going from a strong positive correlation for populations close to each other to a nil or negative correlation for populations the furthest away (Fig. 2a). The pattern showing a decrease of autocorrelation with an asymptotic approach to zero suggests that populations from a given area are similar to each other, but with no correlation at a wider scale (Fig. 2c).

The spatial pattern of differentiation may also reveal the different scales at which the different kinds of movements occur: in sedentary species most individuals reproduce within a small genetical neighbourhood, whereas the dispersal of a few individuals out of this area, and the distance to which they will eventually move determines the level of isolation by distance between populations. The local effective population size, and hence the neighbourhood size, may be estimated by using the y-intercept of the relationship between Nm and geographic distance, using logarithmic scales on both axes [START_REF] Slatkin | Isolation by distance in equilibrium and non-equilibrium populations[END_REF]. For sedentary species, this would be equivalent to their natal habitat patch. The slope of the isolation-by-distance, on the other hand, reveals the extent to which individuals move away to other patches. In migratory or erratic species, both values are of high interest. As it would be expected that populations which are closer to each other would be more similar than more distant ones, there is a need to address the relationship between the degree of population differentiation and the distance between populations. The first approach on this topic is to assess the isolation by distance, by a study of the relationship between the geographical distance between populations and their index of differentiation. This is usually done by using the estimated number of migrants per generation (Nm) between any two populations, using the relationship G ST =1/4(Nm+1) and the geographic distance between these two populations, usually both log-transformed [START_REF] Slatkin | Isolation by distance in equilibrium and non-equilibrium populations[END_REF]. The estimated number of migrants should not be taken literally; [START_REF] Whitlock | Indirect measures of gene flow and migration: F ST ≠1/(4Nm+1)[END_REF] stressed that the relationship between Nm and G ST or F ST is based on a number of assumptions, most of which are unrealistic in natural populations. The most important concern here is that this relationship is based on an island model, where the number of migrants between populations is not correlated with the distance between populations. This is obviously not true in most butterfly populations. In consequence, the estimated number of migrants between two populations should be taken as an index of their differentiation, but not at face value [START_REF] Neigel | Is F ST obsolete?[END_REF][START_REF] Whitlock | Indirect measures of gene flow and migration: F ST ≠1/(4Nm+1)[END_REF].

This approach is usually applied on each studied allele separately (e.g. [START_REF] Porter | Limitations to the inference of gene flow at regional geographic scalesan example from the Pieris napi group (Lepidoptera: Pieridae) in Europe[END_REF], Nève et al. 2000[START_REF] Descimon | Diversité génétique, variation géographique et flux géniques chez quelques Lépidoptères Rhopalocères français[END_REF], but may also be applied using the information from the different loci simultaneously [START_REF] Smouse | Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure[END_REF], as performed by [START_REF] Harper | Microsatellite markers to assess the influence of population size, isolation and demographic change on the genetic structure of the UK butterfly Polyommatus bellargus[END_REF] on English populations of Polyommatus bellargus.

  isolated refuges, compared to East Sussex where the population had persisted in a large (18 ha) refuge. The stronger flight ability of East Sussex populations resulted in a higher colonisation rate and gene flow, whereas the lower flight ability of Surrey populations resulted in a higher isolation by distance effect. The identification of selection pressure implicitly asks the question of what populations are. The East Sussex and Surrey populations of Hesperia comma had suffered different selection pressures according to the characteristics of the two regions. However, such clear-cut situations are infrequent, as most butterflies exhibit gene flow between habitat patches. The question of the identification of whatconstitutes a population is central to many problems in ecology, and -as seen abovethe genetic makeup in a population is under selection from its environmental conditions. The scale at which selection will affect butterfly populations will depend on gene flow among these populations. H. comma displays discrete populations which suffered a bottleneck in the 1970s and 1980s[START_REF] Hill | Flight morphology in fragmented populations of a rare British butterfly, Hesperia comma[END_REF], and a differential effect of selection could be detected between Surrey and East Sussex. In widely distributed Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version25 March 2008[START_REF] Hedrick | Genetics of Populations[END_REF] page 8

  probably the consequence of similar open structures of most studied Satyrinae populations, and therefore may be linked with a global isolation by distance process affecting Satyrinae species in a similar way, despite the absence of IBD in Maniola jurtina. The choice of study species may also have biased these results. It is noteworthy that only two genetic studies, involving four species, could be found on Hesperidae, despite numerous studies on their ecology and distribution. Within Papilionidae, only isolated populations or mountain species have been studied. The genetic structures of e.g. the widespread Iphiclides podalirius and Papilio machaon have not been worked out. The only study of P. machaon has been carried out in Britain where the species is localized and threatened. In Pieridae the bias is the other way, only the widespread species Pieris napi has been thoroughly investigated. The diverse Lycaenidae family has been studied both in localised threatened species, such as the Maculinea species and in widespread species such as Polyommatus icarus and Aricia agestis. With the exception of the widespread and migratory Aglais urticae, studies on the Nymphalinae have focussed on species with localised populations, often with vulnerable and decreasing distributions. The various studies on Satyrinae, like those of Lycaenidae, have involved both common species such as Maniola jurtina and Coenonympha pamphilus, and species with very restricted ranges such as Erebia triaria and Coenonympha hero.

Figures

  Figures

  Example of simulation of allele frequencies of 20 populations of 20 individuals each, with an initial allele frequency of 0.5. a. With no migrant between populations, most populations loose one of the two alleles within 100 generations. In this run only 4 of the 20 populations retain both alleles, with a highly variable proportion. b. With one migrant per population per generation, all populations retain their variability in the long run, as each event of allele fixation (arrows) is subsequently rescued through migration of an individual from another population. Example of simulation of allele frequencies of 20 populations of 100 individuals each, with an initial allele frequency of 0.5. c. With no migrant between populations, some populations loose one of the two alleles within 100 generations. In this run 16 of the 20 populations retain both alleles, with a highly variable proportion. d. With one migrant per population per generation, all populations retain their variability in the long run, with allele frequencies closer to each other than if no migration occurred.

Figure 2 .Figure 3 .

 23 Figure 2. Spatial population structures. Black squares depict suitable habitat patches, and circles the neighbourhood sizes. (a) Each of the 25 populations exchanges individuals with its direct neighbours at each generation. (b) Each of the populationsexchanges individuals with its direct and indirect neighbours, resulting in a much higher gene flow throughout the system. (c) The neighbourhood size is the same as in (a), but with a matrix of half of the habitat patches, each population ends up being isolated, and is subjected to genetic drift independently of its neighbours.

Figure 4 .Figure 5 .

 45 Figure 4. The estimated pairwise number of migrants Nm, as a function of the geographical distances in Proclossiana eunomia populations in Ardennes and Pyrenees. The slopes indicate the levels of isolation by distance, while the intercept indicate the neighbourhood size (from Nève et al. 2008).

Figure 6 .

 6 Figure 6. Percentages of total number of eggs per female Coenonympha hero females(± 1 SE) that hatched, died as zygotes, or remained unfertilized according on whether they came fro large or small Swedish populations (from[START_REF] Cassel | Effects of population size and food stress on fitnessrelated characters in the scarce heath, a rare butterfly in western Europe[END_REF]

  checked in vitro. At a broad scale, populations of the Alpine Colias meadii have

	Population genetics of European butterflies
	G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008	page 7

  of the values of the global fixation index (F ST ) among populations of European butterflies show that generally populations show little genetic differenciation (sensu Wright 1978): the median F ST value is 0.053. The lowest F ST value (0.004) is found in Polyommatus icarus, a common and widely distributed butterfly which thus shows numerous movements among its populations. The highest value (0.291) is found among isolated populations of the mountain species E. epiphron, showing the ancient separation of its populations and the lack of movements between mountain massifs.

	The 86 studies of spatial population genetics on European butterflies show a general
	relationship between F

ST and the size (log scale) of the study area for all species combined, which is close to significance (Figure 3, t=1.94, 84 df, P=0.055). However, global F ST tends to vary among the five butterfly (sub)families (excluding the four species of Hesperidae ; F=2.88, 4 and 79 df, P=0.03). Probably due to the narrow Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 12

  different genetic structures as a result of their different dispersal ranges and habitat requirements. Thymelicus lineola displays a high dispersal ability and has broad habitat requirements, resulting in a panmictic genetic structure at the regional scale; Thimelicus sylvestris displays a lower dispersal ability in the same habitat matrix, which results in isolation-by-distance effect. The third species, Thymelicus acteon, has narrow habitat requirements in combination with a low dispersal ability, resulting in populations being more isolated from each other, as reflected by this species having the highest F ST value of the three species in the same habitat patch network, without any isolation by distance effect[START_REF] Louy | Strongly diverging population genetic patterns of three skipper species: the role of habitat fragmentation and dispersal ability[END_REF]. T. acteon has declined in many European countries, and is of conservation concern, while the other two are stable (van Swaay and Warren 1999). Population in southern mountain areas tend to be more variable than low elevation ones. Pieris bryonae populations within the Swiss Alps are more isolated from each other than Pieris napi populations between south France, Germany and Hungary

Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 13 very

  with an estimated size of 7 to 15 individuals during the flight period, while others are larger, with estimated population sizes from 51 to 128 individuals. Furthermore, large populations are connected to each other by suitable habitat corridor structures, such as grassy roadbanks.[START_REF] Cassel | Effects of population size and food stress on fitnessrelated characters in the scarce heath, a rare butterfly in western Europe[END_REF] collected eggs from the different populations, and placed them in semi-natural conditions on tussocks of

Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 15 extremely small,

Table 1 .

 1 Population genetics studies of butterflies conducted in Europe.

					Number of	Approximate
	Species and study area	Methods	N populations	F ST	polymorphic loci	longest distance	IBD	Reference
	Hesperidae
	Carterocephalus palaemon
	BiałowisŜa forest, Poland Allozymes	2	0.191	16	(sympatry)	NA	Ratkiewicz & Jaroszewicz 2006
	Thymelicus acteon
	GD Luxemburg +
	neighbouring areas Allozymes	12	0.053	18	110 km	NA	Louy et al. 2007
	Thymelicus sylvestris
	GD Luxemburg +
	neighbouring areas Allozymes	12	0.023	18	110 km	*	Louy et al. 2007
	Thymelicus lineola
	GD Luxemburg +
	neighbouring areas Allozymes	12	0.008 NS	18	110 km	NA	Louy et al. 2007
	Papilionidae
	Parnassius mnemosyne
	Hungary Allozymes	8	0.075	3	80 km	*	Meglécz et al. 1998a
	Hungary Microsatellites	8	0.051	3	80 km	NA	Meglécz et al. 1998b
	Alps+ Pyrenees Allozymes	24	0.135	9	500 km	NA	Napolitano & Descimon 1994
	S Alps (France) Allozymes	4	0.035 -0.175	9	90 km	NA	Napolitano et al. 1988
	Parnassius apollo
	French Alps + Jura Allozymes	17	0.0548	14	350 km	**	Descimon et al. 2001
	French southern Alps Allozymes	10	0.041	14	120 km	NS	Descimon et al. 2001
	Jura Allozymes	2	0.124	12	100 km	NA	Descimon et al. 2001
	Pyrenees Allozymes	7	0.087	14	220 km	NA	Descimon et al. 2001
	Massif central Allozymes	3	0.266	10	200 km	NA	Descimon et al. 2001
	Spain Allozymes	2	0.152	12	NA	NA	Descimon et al. 2001
	Parnassius phoebus
	French Alps Allozymes	12	0.255	11	200 km	NA	Descimon 1995
	Papilio hospiton
	Corsica Allozymes	6	0.015 NS	8	100 km	NS	Aubert et al. 1997

Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 36 Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 37 Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 38 Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 39 Population genetics of European butterflies G. Nève (gabriel.neve@univ-provence.fr) final version 25 March 2008, revised 19 Dec. 2008 page 40 NA : not available, NS : not significant, * : P<0.05, ** : P<0.01, *** : P<0.001, § F ST and isolation by distance computed by present author

Acknowledgements

This chapter benefited from the constructive comments of Emese Meglécz, Sören Nylin, Thomas Schmitt and the editors.


David Roche

Nathalie Perrot

Thomas Chabin

Angélique Villière

Ronan Symoneaux

Thierry Thomas-Danguin

Alice Roche
email: alice.roche@inra.fr

HOW TO PREDICT THE ODOR PROFILE OF WINE FROM ITS CHEMICAL COMPOSITION ? IN SILICO MODELLING USING EXPERTS' KNOWLEDGE, FUZZY LOGIC AND OPTIMIZATION

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Odor description of the odorants

The odor component of food represents the identity, exemplarity, acceptability and the recognition by consumers of the food product 1 Escape The analysis of food odor provides a list of molecules 2 , but no information about the perceptual influence of mixed compounds Escape Escape The perception of a mixture of odorants is not the simple sum of the odor of each odorants but the results of highly complex perceptual interactions 3,4 Predictive approaches are now developing to predict the odor characteristics of molecules: detection thresholds 5,6 , odor quality 7,8 , odor intensity 9,10,11 , odor pleasantness 12,13 However there are few papers focusing on the odor characteristics of mixtures of molecules 14 The average of the prediction accuracy for the 15 sensory descriptors is higher than 96 %. But the wine ranking has to be improved.

CONCLUSION

  , et al., 2006, Journal of agricultural and food chemistry. 2: Brattoli et al., 2013, Sensors. 3: Atanasova et al., 2005, Food quality and preference. 4: Le Berre et al., 2008, Chemical senses. 5: Abraham et al., 2012, Chemical senses. 6: Hau and Connell, 1998, Indoor air. 7: Keller et al., 2017, Science. 8: Kumar et al., 2015, Plos one. 9: Yan et al., 2015, Sensors. 10: Mainland et al., 2014, Trends in neurosciences. 11: Thomas-Danguin and Chastrette, 2002, Compte rendus biologies. 12: Lapid et al., 2008, Chemical senses. 13: Kahn et al., 2007, Journal of neurosciences. 14: Snitz et al., 2013, Plos computational biology. 15: Arctander, 1969, "Perume and Flavor Chemicals" vols I and II. 16: http://www.leffingwell.com. 17: http://www.thegoodscentscompany.com. 18: Hansen and Ostermeier, 2001, Evolutionary computation.

  Analysis: PCA(Sensory evaluation)=PCA(Sensory predicted score) ?

  Promising results => validation of our modelling strategy espace Strengths:Considering the mixture as a whole rather than as a sum of constituents Integration of flavorists' expertise along the flavor analytical path espace espace Perspectives: databases aggregation, optimization parameters PCA(Sensory evaluation) PCA(Sensory predicted score)Food product ONTOLOGY