To be published in the Royal Entomological Society’s 20th International Symposium

volume ‘Insect movement: mechanisms and consequences’. Version 8th June, 2000.

Use of genetic diversity in movement studies of

flying insects
HUGH D. LOXDALE1 & GUGS LUSHAI2*1

1, Entomology and Nematology Dept., IACR-Rothamsted, Harpenden, Herts., AL5 2JQ. U.K.; 2,
Biodiversity and Ecology Division, School of Biological Sciences, Basset Crescent East,
Southampton University, Southampton, SO16 7PX, U.K

1. General introduction

The power of flight probably evolved in semi-aquatic insects in the Middle and Upper
Devonian when watery habitats regularly dried out (Wigglesworth, 1976; Kurkalova-
Peck, 1978). This necessitated dispersal in search of new habitats, resources and mates
and may have selected for morphs using articulated gill-plates as wings (Wigglesworth,
1976; Kurkalova-Peck, 1978; Marden & Kramer, 1995; Thomas & Norberg, 1996; see
also Wootton this volume). Flight dispersal to other localities also served to mix
populations, thereby increasing the likelihood of favourable genotypes arising from
these distinct, quite literal gene pools in a constantly changing environment.

Whatever the mode of displacement, movement from an established resource to

another potential site is often a gamble that is necessitated by abiotic and biotic factors
that select against continual habitation of the original location. The ‘gamble’ that
dispersing individuals face is that they may fail to reach new habitats or resources if
these are distant, separated by geographical barriers, because of navigational errors,
due to insufficient fuel sources to maintain movement between habitats or, if they are
predated en route. Even if they do reach a suitable niche, sexually reproducing
organisms still need to find a mate to begin to establish a new population, unless they
have mated prior to dispersal. Over geological time, there has been a selection for
optimal habitat-resource and mate location (visual, olfactory and navigational),
although interestingly, there are only a few insects which show true migration, i.e.,
innate, directed, long distance two-way movements. Perhaps this is because there are
undesirable genetic consequences in terms of loss of ‘adaptive flexibility’ to the
environment. In contrast, the majority of insect species undergo random changes of
spatial scale, classed here as dispersal, and often these movement events occur over
several generations (Loxdale & Lushai, 1999).

With winged insects, because of the inherent difficulty in re-establishment after

population movements have occurred, the chances of successfully recapturing marked

*
Present address: Crop Protection Program, Eastern Cereal & Oilseed Research Centre, K.W. Neatby
Building, C.E.F., Ottawa, Ontario, CANADA, K1A 0C6.

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individuals is rare. This is especially so if: 1, the population size is small; 2, the spatial
scale over which migrants move is geographically extensive or 3, the small size of the
insects themselves means that their movement involves inert or random transport on
the wind above their flight speed in still air. As a consequence, individuals are usually
collected from the field with little prior knowledge of where they have come from,
thereby undermining what can be learnt from these arbitrary collections.

In the present article, we discuss how patterns of genetic variation derived

from the use of molecular markers can be used to infer the extent and nature of
population movements in flying insects. We highlight how in some cases this
information provides insights into biological processes as well as how these patterns
are influenced over time in an evolutionary sense. We confine ourselves to descriptions
of aerial movement which involves the quest on the part of the insect or insect
population concerned to find new habitat and/ or resources, rather than local
movements such as foraging activities (e.g. as with bees) which do not have
reproduction as a possible outcome.

2. Markers used to study genetic variation and to infer population movement

Amongst the methods used to explore variation across an insect population are those
that can be considered non-molecular ‘tags’, i.e. that describe phenotypic
polymorphisms. These include behavioural differences e.g. insect song (Henry, 1994);
wing pattern (Dowdeswell & Ford, 1953; Roskam & Brakefield, 1999); wing size
(Zera & Denno, 1997); eye colour (Dobzhansky & Wright, 1943); and body pigments
(Odonald & Majerus, 1992). Use has also been made of element markers (see Sherlock
et al., 1985), radio-labelling (Taimr & Kříž, 1978) and other innovative markers such
as pollen from unique sites (Westbrook et al., 1998), fluorescent dust dyes
(Dobzhansky & Powell, 1974; Byrne et al., 1996), as well as radar (Smith & Riley,
1996) (see Reynolds et al., 1997 for a detailed review of these aspects). Pheromone
lures (Suckling et al., 1999) are, physiologically-speaking, molecular substances, but
they differ from the next group of markers in that population data may be collected
with little further processing. Molecular ‘tags’ require the collection of samples and
their subsequent molecular processing before a population can be typed. These include
proteins, especially allozymes, and DNA markers, both of which are separated
electrophoretically; chromosomal markers (White, 1978; Bullini & Nascetti, 1989),
e.g. chromosome number, nuclear organising regions (NORs) and C-banding (Berry et
al., 1992 and references therein), and fluorescent in situ hybridisation (FISH) markers
(Blackman et al., 1995; Bizzaro et al., 1996). Allozyme and DNA based markers are
the most commonly used as molecular tags in entomological studies of population
structure and movement and hence we concentrate on these, whilst paying tribute to
the important findings made using the aforementioned other markers. Some of the
most informative studies have utilised a two-pronged approach, for example using
mark-release-recapture (MRR) protocols in conjunction with molecular analysis of the
re-trapped samples to study movement (e.g. Lewis et al., 1997).

Of the two main categories of molecular marker, allozymes (Richardson et al.,

1986) are the products of genes (alleles) coding for enzymes and for many insects,
provide very useful Mendelian markers. However, the number of allelic variants
recorded per locus is often not large (< 10). In some insect groups, i.e. Hymenoptera

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and Aphididae, allozyme loci are found to be generally invariant (Loxdale, 1994).
When allozyme variability is found to be low over a range of loci in the insect species
under study, DNA markers are usually sought (Carvalho, 1998; Loxdale & Lushai,
1998). DNA analysis is based on six basic practices of molecular biology: 1,
acquisition of DNA (DNA release or extraction); 2, cutting (restricting) the DNA; 3,
joining (ligating) the DNA; 4, probing the DNA with a labelled sequence to investigate
sequence homology between samples; 5, amplifying the DNA in a polymerase chain
reaction (PCR) and 6, sequencing the DNA.

Involved with one or a number of these basic processes are the following
molecular marker types: restriction fragment length polymorphisms (RFLPs);
minisatellites (DNA ‘fingerprinting’ using single and multilocus probes); PCR
involving mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), and scrutinised
using primer pairs which amplify hypervariable regions, e.g. rDNA, introns and
microsatellites (single sequence repeats, SSRs); random amplified polymorphic DNA
markers (RAPDs); amplified fragment length polymorphisms (AFLPs), and
transposons. Each marker has its advantages and disadvantages for population studies,
as discussed in detail elsewhere (Hoy, 1994; Carvalho, 1998; Karp et al., 1998;
Loxdale et al., 1996; Loxdale & Lushai, 1998). In particular, whether the marker
produced is dominant or co-dominant, the latter thereby allowing heterozygotes to be
screened for analysing population structure and inbreeding, etc. (see Loxdale &
Lushai, 1998).

Allozymes mutate at rates of around 10-6 to 10-9 per gene per generation
whereas with microsatellites, rates are two to three orders of magnitude greater (Jarne
& Lagoda, 1996; Luikart & England, 1999). As a consequence of this relatively high
mutation rate, microsatellites are less useful for assessing historical patterns of
population structure and dynamics (Jarne & Lagoda, 1996). Within this group of
rapidly evolving marker, the different repeat classes (di and tri nucleotides) have
differential rates of evolution thereby complicating the picture derived when using a
range of these markers in conjunction (Rubinsztein et al., 1999). Similarly, mtDNA has
several regions which evolve at different rates (Zhang & Hewitt, 1997) and this has to
be taken into account when making conclusions derived from such markers. Zhang &
Hewitt (1996) have also highlighted another problem, that of multiple gene-copies for
a given mtDNA region, with some of these integrating within the nuclear genome.
Even so, mitochondrial markers are considered extremely versatile because of their
predominantly maternal inheritance, low recombination rate (Wallis, 1999) and fast rate of
evolution (Avise, 1994; Zhang & Hewitt, 1997). They have been employed in studies
where genetic bottlenecks may have had a role in shaping population structure (Loxdale et
al., 1996; Carvalho, 1998). However, whilst they are often found to show low variation in
migration studies, this could be as a direct result of the populations tending to move
together, i.e. the 'moving-deme' hypothesis (Loxdale & Lushai, 1999) rather than the
influence of a bottleneck. Concerted evolution occurs where multiple genes exist as if
they represent a single locus. Hence, homogeneity may occur among repeats within
individuals and populations, but heterogeneity among the tandem arrays from
divergent populations and species (Rand, 1994). Examples are found in nuclear rDNA
cistron genes (Black, 1993) and variable number of tandem repeats (VNTR) in the
control region of mitochondrial genes (Rand, 1994). However, in some cases there

3
have intraspecific, even intraclonal variation at these sites, which can give as much
variation within an individual as in a population (Shufran et al., 1992).

Bossart & Pashley (1998) discuss the shortcomings of various molecular

markers and their interpretations in population genetic studies including estimates of
gene flow (views that have been challenged by Bohonak et al., 1998). Suffice to say
that there are examples where different markers, i.e. DNA fingerprinting, RAPDs and
microsatellites describe the same population genetic phenomena, but reveal
increasingly greater ecological and evolutionary information. Different markers may
provide a contrasting picture of the ecological and evolutionary forces shaping the
genetic patterns observed, as demonstrated in the Peach-potato aphid, Myzus persicae
(Sulzer) by Brookes & Loxdale (1987) and Fenton et al., (1998) using allozymes and
rDNA-IGS (intergenic spacer) markers, respectively. Interestingly, minisatellites,
RAPDs and microsatellites appear to show host-plant-based adaptation in aphids,
whereas, the IGS markers do not. This maybe because these former markers are
integrated at random along the genome, closely linked by ‘hitchhiking’ (Maynard
Smith & Haigh, 1974) to genes under selection (Lushai et al., 2000). In contrast, the
rDNA IGS region are confined to the ends of the arms of the X-chromosomes
(Blackman & Spence, 1996) where it is possible that they are not under the same kind
of molecular selection.

When markers are employed to elucidate patterns of movement, assessment

always involves a survey of sample populations and specific gene and genotype
frequencies. In non-molecular genetic methodologies, it is often assumed that a single
gene or very closely associated groups of genes (under similar selection pressure) are
associated with a particular trait. Although allozyme and DNA based allelic markers
are thought to be neutral (not selected for) (Jarne & Lagoda, 1996), they are also often
assumed to be genetically independent, i.e one-gene-one trait. This can be statistically
proved by testing for linkage disequilibrium, i.e. whether alleles at different loci
screened are independently assorted or linked (Parkin, 1979; Richardson et al., 1986).
Only allozymes, microsatellites and RFLPs of nDNA provide heterozygote banding
patterns from which, in the case of the former two markers, deviations of Hardy-
Weinberg (H-W) expectations (Richardson et al., 1986), and heterozygosity per locus
and over all loci (average heterozygosity) can be deduced (Luikart & England, 1999).
Daly (1989) and Slatkin (1985a, 1987, 1993, 1995) discuss how estimates of gene flow are
derived from allele frequency data, e.g. contingency χ2 tests, F-statistics (FST) and RST in
microsatellites, Nm (effective population size, N x migration rate, m), Slatkin’s (1985b) rare
allele methods, genetic distances, etc. All these analyses can be related to the extent of
movement (see also Mallet, this volume). Measures of direct sequence similarity-
relatedness or nucleotide divergence within and between groups can also give useful
inference to movement. Genotypes may be assumed to be moving to or from an area if
a known genotype of a sample population group is traced from a source by subsequent
re-trapping away from the origin. These approaches depend on the ability to
differentiate genotypes in a statistical manner. If genotypes are demographically
distinct, for example, an invasion of an insecticide-resistant genotype into an
insecticide-susceptible population, then a more definitive statement about movement
can be made, assuming that such a genotype has not secondarily evolved by mutation
in the residing population. Some caution is needed in analysing across developmental

4
stages and morph types as these may show different genetic patterns within an
individual lineage (cf. Loxdale, 1994; Loxdale et al., 1996; Lushai et al., 1997).

The above detail provides a framework of suitable markers along with analytical
approaches to address topics of spatial scale and gene flow. In the next section we show
how these have been applied to insect examples which reveal patterns of spatial distribution
across various geographic scales.

3. Genetic variation: patterns of range expansion and factors affecting spatial

distribution.

Theoretical aspects of spatial and temporal genetic patterns of animal

populations in relation to gene flow-movement have been recently detailed by Baur &
Schmid (1996) and in insects, by Roderick (1996) (see also Waser & Strobeck, 1998
and Dieckmann et al., 1999). We briefly describe contemporary methods of spatial
sampling and concentrate on environmental and behavioural factors that influence
patterns of aerial displacements in insects.

Collections of insect samples in the field has been performed by hand, by

vacuum sampling, or by using nets or sticky, pheromone and fixed suction traps, some
suction trap networks e.g. Rothamsted Insect Survey being national and operated daily.
Many of these methods have been employed for molecular ecological studies in aphids by
Shufran et al. (1991), De Barro et al. (1995a,b) and Llewellyn et al., (1997, 1999) and
have involved collecting insects at local and geographic scales over a growing season to
assess spatial and temporal trends. Recently, an ingenious MRR method has been employed
to examine the flight behaviour of individual white fly, Bemisia tabaci (Gennadius), at local
scales in the field (Isaacs & Byrne, 1998). MRR studies carried out in conjunction with
molecular markers by Lewis et al. (1997) have also provided effective ways of mapping
genetic variation over different spatial and temporal scales. For more accurate spatial
experiments, a concentric collection approach from a fixed point spreading out at geometric
distances is optimal (e.g. Byrne et al., 1996; Byers, 1999). Even with all this innovative
methodology, sampling small flying insects is always a problem. Whilst small insects like
aphids are carried by fast directional jet streams above 900m in some parts of the
world (e.g. USA and India), more usually they are borne on lower altitude winds.
Since these air masses may quickly change direction within a few hours, it is difficult to
make typical sampling regimes comprehensive (Riley et al., 1995; see also Gatehouse,
1997), and indeed thereby be sure of orientation and scale of displacement.

In general, the directed, innate behavioural type of migratory behaviour is the

exception rather than the rule (Loxdale & Lushai, 1999). Most insects undergo
geographical displacements by random changes of spatial scales, sometimes over
several generations and this pattern is thus the basic one for most species.
Unfortunately, there is little direct evidence from molecular marker data demonstrating
the genetic consequences of such invasions.

One study indicating displacement, and not migration, concerns the movement
of the cynipid Knopper gall wasp, Andricus quercuscalicis (Burgsdorf), which host
alternates between Turkey Oak, Quercus cerris and the pedunculate oak, Q. robur, in
spring and summer, respectively (Stone & Sunnucks, 1993). The wasp has tracked its

5
obligate host, Turkey Oak, in a typical radiating pattern of colonisation since this was
planted throughout Western Europe outside its native range over the last three to four
hundred years. Since the planted Turkey oak trees are rarer and more widely scattered
outside the native range than within in it, this was seen as likely to influence the
population structure of the insect. Allozyme analysis of 39 populations failed to detect
any new alleles in the invaded range, although allelic diversity and mean heterozygosity
decreased significantly with distance from the original refugia (Stone & Sunnucks,
1993). Spatial analysis revealed that genetic differences were greater in the invaded
compared with the native area. This suggested that genetic changes across Europe
were probably the result of strong directional movement followed by limited gene flow
between populations, rather than due to selection. There was also evidence that genetic
patterns of populations were founded sequentially from the east spreading westward by
a process of random invasion, from which new populations invaded further, with a
concomitant dilution of genetic diversity. Another recent study tracking invasion is
described by Taberner et al. (1997). Here RAPD markers were used to deduce the
spread of a pest sugar beet weevil, Aubeonymus mariaefranciscae (Roudier), in
southern Spain. A combination of phylogenetic and genetic distance versus
geographical distance approaches enabled the spread of the beetle to be ascertained in
relation to its ecology and evolution. This genetic information affirmed that the beetle
populations had arisen from a unique colonisation event, probably dating from 1979.

When geographic barriers are present such as mountains, expanses of water,

deserts or other inhospitable regions, the movement of individuals between populations
can be prevented and hence gene flow influenced to the extent that populations may
not show homogenous gene frequencies over time. In the case of the Knopper gall
wasp, the English Channel and Irish sea have proven to be partial barriers to the rate of
insect colonisation, ~ 80 years in the former case (Stone & Sunnucks, 1993). With the
grain aphid, Sitobion avenae (F.) populations from Britain and Spain separated by 800
km of sea and the Pyrenees mountains showed a degree of genetic differentiation in
terms of allozyme derived genetic distances (Loxdale et al., 1985).

Clouded Apollo butterflies, Parnassius mnemosyne (L.), in the Alps appear to

consist of ‘closed’ populations as shown following behavioural observations
(Napolitano et al., 1988). Survey of four such populations using 24 allozyme loci
showed these populations to have differing levels of heterozygosity and significant
allele frequency differences at some polymorphic loci, suggesting that migration was
restricted among these colonies separated by mountainous terrain (Napolitano et al.,
1988). In a later study, 24 populations of the butterfly collected over a wider area in
southern France revealed that distinct populations from the Pyrenees and the Massif
Central were similar to those collected in the Prealps. Genetic and clustering analysis
indicated that these far spread populations may have been colonised from north-eastern
Europe via the central Prealps during warm interglacial periods and thereafter, become
isolated during subsequent climate cooling (Napolitano & Descimon, 1994). Thus, the
genetic structuring of these outlying populations represents the probable historical
pattern of gene flow. However, the fact that the average FST over all 24 loci was high
(0.135), revealing significant population heterogeneity and hence substructuring
comparable with a low dispersal rate, emphasised the role of geographical and
ecological barriers in shaping population structure at small geographic scales (~30 km)
(Napolitano & Descimon, 1994). Meglecz et al., (1998, 1999) have also studied this

6
butterfly in north-east Hungary using both MRR and molecular markers (allozymes
and microsatellites) and utilising field and historical collections. Their work showed
that even large colonies appear to have small effective reproductive population sizes
with a lack of heterozygotes compared with H-W expectations, indicative of uneven
sex ratio, recent bottlenecks and/or founder effects. Therefore, the genetic approach
differentiates between subpopulations from some regions having metapopulation
structure (Hanski & Gilpin, 1997); some with homogeneous genetic structure within
regions; and some geographically widespread populations that are genetically
differentiated, indicative of restricted movement and genetic drift.

Population genetic structure and gene flow is also influenced by elevation.

Leibherr (1988) studied beetles at different altitudes in North America, analysing allele
frequency data using FST and private allele approaches. He showed that in five carabid
species of varying vagility, FST values were positively correlated with elevation from 0
to 1500 m above sea level, indicating more population substructuring with increased
elevation. At first instance this was thought to be a result of low vagility at greater
elevation, but closer analysis revealed that one of the key factors influencing this trend
was greater habitat fragmentation with increasing altitude. It was concluded that lower
levels of gene flow associated with greater population subdivision occurred in upland
regions due to habitat fragmentation i.e. topographical diversity and habitat persistence
leading to a reduced population extinction rate (Liebherr, 1988). Again, this study
highlights the fact that although population genetic markers reveal a lot of ecological
detail, correct interpretation of this depends on full assessment of flight behaviour
correlated with the various ecosystem parameters.

For some species, the apparent high estimates of gene flow are artifactual
rather than real and probably reflect historical patterns of movement. This leads to a
very important point of gene flow versus vagility. For many insects, even truly migratory
species, the further populations are away from each other, even without the problems of
physical barriers to gene flow, the more likely they are to diverge genetically, i.e. show more
heterogeneous genetic population patterns.

The relationship of isolation by distance (IBD) and dispersal ability of insects has
recently been studied in detail by Peterson & Denno (1998a). They analysed genetic data
versus IBD for 43 phytophagous species and races of insects and tested two opposing
hypotheses. In the first, IBD slopes in log plots of gene flow versus distance, were
unchanged by distance yet the intercepts increase with mobility. In the second, IBD slopes
varied with dispersal ability. They found that mobility trends tended to fit the second
hypothesis. In sedentary and highly mobile species, IBD appeared weak over distances from
10 - 1,000 km and both species categories had shallow slopes and markedly different
intercepts. In moderately mobile species, the relationship was more pronounced, i.e. there
was greater gene flow with distance. From the analysis of the genetic data they concluded
that divergence amongst most populations of sedentary species over the distances tested
was due to limited gene flow. The genetic homogeneity found in intermediate dispersers
over small spatial scales was due to appreciable gene flow, whilst the heterogeneity at larger
spatial scales was thought to be a result of its restriction. The homogeneous genetic
structure of highly mobile species at all spatial scales was considered to be due to high gene
flow. Hence, genetic structure was to some degree directly correlated with the ability of the
insect to disperse providing valuable insights and demonstrating the usefulness of the genetic

7
marker approach in arriving at these conclusions. However, there are numerous factors that
influence population genetic structure, some of which have already been outlined (cf. also
Peterson & Denno, 1998b). In some instances, genetic structuring is strongly influenced by
biotic factors. In the next section we relate evolutionary and biological processes of various
host-herbivore interactions, host ranges and fidelity to genetic variation.

4. Genetic patterns and biotic factors

There is a growing body of evidence for host association of adaptive-genetic patterns

in insects at a variety of spatial scales (e.g. Lepidopteran leaf miners; Mopper, 1998),
both small (e.g. scale insects; Alstad and Corbin, 1990) and large (e.g. wood boring
beetles; Stock et al., 1979; but see Peterson & Denno, 1998b). Whether this apparent
association is due to differential survival on hosts rather than to differential host
preference of genotypes per se (Langor & Spence, 1991) needs to be verified. Whilst it
is difficult to relate these patterns to the beginning of evolutionary divergence below
the species level, i.e. biotypes, race formation, ecotypes, etc. (Cobb & Witham, 1998),
host-dependent traits suggest co-evolutionary trade-offs (Thompson, 1994) and may
be the fundamental mechanisms of such divergence (Nuismer et al., 1999).

Population genetic homogeneity in highly dispersive species may relate to the

fact that the transient presence of the host plants selects for enhanced dispersive
behaviour compared with related insects that colonise perennial hosts. Hence, with
high dispersers, host adaptation seems less likely to evolve. Even so, in at least one
case, the tiger swallowtail butterfly, Papilio glaucus L., there is evidence that even in
such a vagile species, differential selection for host plant has contributed to significant
genetic differentiation of populations. Bossart & Scriber (1995) have shown that in
North American P. glaucus populations sampled in southern Ohio, north central
Georgia and southern Florida from three hosts (Liriodendra tulipifera, Magnolia
virginiana and Prunus serotina), differentiation existed among populations for both
oviposition preference and larval performance. However, using a range of allozyme
markers, no evidence was found for population differentiation matching these traits.
This argues for significant gene flow between populations on the various hosts
(Bossart & Scriber, 1995), but alternatively, other markers such as microsatellites
could perhaps resolve the host-based populations.

In aphids with high vagility, plant-host related differentiation is less likely to

evolve. It also appears that in species which do not host alternate, i.e. utilise a single
plant group, even spatial scale of the niche seems to have little effect on genetic
structuring. An example is the sycamore aphid, Drepanosiphum platanoidis (Schrank),
which lives all year around on sycamore, Acer pseudoplatanus in both asexual and
sexual phases. In this species, allozyme studies reveal a range of genotypes (eight at
the phosphoglucomutase, PGM locus) and genetic homogeneity at both local and
national scales in southern England (Wynne et al., 1994). The fact that as much
genotypic variation is found at the leaf scale as the national scale suggests that ‘trivial’
flight’, i.e. local flight within or close to the canopy of the host tree itself, (Dixon,
1969), prevents clonal formation on individual sycamore leaves, as well as
homogenising aphid genetic patterns between clumps of trees and trees in the same
area. Whilst the aphid appears to be highly dispersive, since it is commonly found in
the summer in Rothamsted 12.2. m. high suction trap samples (Woiwod et al., 1988),

8
the homogeneous national genetic pattern observed may reflect a historical
colonisation rather than inter-population migration over seasons (Wynne et al.,1994).
The green bug, Schizaphis graminum (Rondani), a serious predominanntly asexual
aphid pest of cereals in north America studied using rDNA IGS markers, also showed
population genetic homogeneity at various spatial scales (Shufran et al., 1991).
Numerous genotypes were detected, yet the populations appeared as genetically
diverse at the lowest spatial (sorghum leaf) scale as at the County scale (Kansas),
possibly the result of long distance dispersal (see also Fenton et al., 1994).

If insects have low vagility, they are perhaps more likely to be subject to local
differentiating selective forces (Cobb & Whitham, 1998) and produce host adapted
demes with little gene flow between them (Mopper & Strauss, 1998). There is some
genetic evidence that this is occurring in the holocyclic (with sexual phase) cereal
aphid, Sitobion fragariae (Walker). Local British populations of this species (< 30 km
apart) showed genetic heterogeneity and stable genotypes frequencies at an allozyme
locus (glutamate-oxaloacetate transaminase, GOT) over a five year sampling period
during the summer asexual phase of reproduction (Loxdale & Brookes, 1990).

Sometimes genetic patterns are disrupted such that a discontinuity or transition

occurs in gene frequencies between populations. Usually, such patterns have a spatial
dimension although occasionally, they have a temporal one. At the extreme end of the
spectrum, discontinuous allele frequency patterns may relate to the presence of two
adjacent species populations. An example of this situation is found in two noctuid
moth species, the native hop vine borer, Hydraecia immanis (Guenée), and the
introduced potato stem borer, H. micacea Esper. These populations are separated by
the North American Great Lakes host range differences in Wisconsin and Michigan
(Scriber et al., 1992). The former species is more specialised in its diet and generally
occurs south of the plant community transition zone, whereas the latter species is
polyphagous and occurs in corn north of this zone. Of 19 enzyme loci screened, 6
showed fixed or nearly fixed allelic differences. Hybridisation of the two Hydraecia
species “may contribute to wider larval host plant use abilities, altered voltinism
patterns, reduced developmental temperature thresholds of the larva and possibly
changes in oviposition preferences" (Scriber et al, 1992 and references therein). Here
migration and subsequent colonisation of either species into adjacent ecosystems is
restricted, unless moths can adapt to the new ecological challenges presented. In other
cases, such separation has been shown to be due to the presence of morphologically-
cryptic species e.g. crickets of the genus Allonemobius Hebard (Howard, 1983) or
indeed to the phenomenon of sympatric speciation. Somewhere in this spectrum of
diversification is also the phenomenon of hybridisation. The influence of hybrid zones
on genetic patterning and gene flow is detailed by Mallet (this volume). Boecklen &
Howard (1997) discuss the choice of molecular marker, number required and their
power of resolution when studying such zones.

Much more rarely, as supported by scientific evidence, population divergence

occurs sympatrically instead of resulting from para- and allopatric separation (White,
1978). In the tortricid larch budworm moth, Zeiraphera diniana Guenée, eleven larch
and pine feeding populations were studied in western Europe using 24 allozyme loci.
(Emelianov et al., 1995). From these data, genetic analysis showed that the larch and

9
pine forms of the moth were either host races or sympatric species that hybridized
rarely.

The ‘classic’ example of allelic discontinuities in geographically coexisting

populations is demonstrated in tephritid fruitflies of the genus Rhagoletis Loew.
Sympatric populations on apple/hawthorn and blueberry appeared to be reproductively
distinct, probably the result of host plant recognition acting as pre-mating barriers to
gene flow, as shown by allozyme separation at 11 out of 29 loci indicating host specific
alleles. The molecular separation supports the status of these two host-adapted forms
as R. pomonella (Walsh) and R. mendax (Curran) respectively, despite their close
morphological similarity, overlapping geographic distributions and inter-fertility in
laboratory crosses (Feder et al., 1989). In addition, populations of R. pomonella
showed geographic differentiation. Populations from the eastern USA, the native range
of the fly, were highly polymorphic and showed homogeneity within and among
populations. In contrast, populations from the western USA where it has colonised
recently (c. 1980), generally lacked genetic variation (McPheron, 1990), although at
the remaining polymorphic loci, showed spatial heterogeneity suggestive of founder
events. Other data by Feder et al. (1990) (6 enzyme loci) also showed that flies (R.
pomonella) not only differed at loci between hosts (apple or hawthorn) but that allele
frequencies were influenced by latitude. This produced a north-south cline in the
eastern USA and Canada superimposed on inter-host patterns. It appears that adult
flies do not migrate between host species, but rather, tend to infest the same host that
they colonised as larvae. Hence genetic markers were able to differentiate individuals
with specific host preference and conditioning that caused host fidelity, whilst selection
may be responsible for geographic patterns observed. Therefore, these fly populations
are strong contenders for the sympatric speciation model because they are most likely
to successfully colonise new hosts in the area of their birth, and this must restrict the
dispersal ambit of these insects (Feder et al., 1998).

Patterns of genetic diversity and gene flow of insects are influenced by niche
patchiness and behaviour-related patch fidelity even when obvious plant host influences
appear to be absent. Some butterflies with low vagility such as the Glanville fritillary,
Melitaea cinxia L. show metapopulation structure. Genetic studies on island
populations in south-western Finland using both allozyme and microsatellite markers
(Saccheri et al., 1998) have revealed that the risk of extinction of subpopulations
increased significantly with decreasing heterozygosity, a sign of inbreeding. These
deleterious effects were manifest in larval survival, adult longevity and egg hatch rate,
which in turn appeared to be the fitness components responsible for the relationship
between inbreeding and extinction (Saccheri et al., 1998) (see also Sutcliffe et al.,
1997). In a lyceanid blue butterfly, Euphilotes enoptes (Boisduval) subpopulations
from Washington State and Oregon, USA were studied using allozyme markers. The
apparent low vagility of the butterfly was thought to be the cause of genetic
heterogeneity found between subpopulations (Peterson, 1996). This was described by
clustering analysis of genetic distances as well as a regression analysis of gene flow
versus geographical distance. The two parameters were as expected, negatively
correlated. Even so, estimates of gene flow among population pairs separated by more
than 100 km were greater than 10 individuals per generation, a value higher than
predicted from the butterfly's apparent aerial dispersion (rarely more than 1 km). Yet
neighbourhood size was estimated at around 40 individuals, consistent with poor

10
vagility (Peterson, 1996). Thus it appears that in sedentary insects, the magnitude of
gene flow described by the 'stepping stone' hypothesis (Peterson, 1996) has a
significant influence on genetic homogenisation over large spatial scales. This is
precisely the scenario in the Knopper gall wasp. These studies again emphasise the
potential utility of molecular markers for deriving behavioural and ecological trends.
Spatial heterogeneity within a population can also have a temporal component to it. As
described below, these include lifecycle synchrony, with annual (short-term) temporal
aspects, and very much longer historical patterns of genetic variation.

5. Temporal changes in genetic variation (not independent of spatial variation)

In the satyrid butterfly, the inornate ringlet, Coenonympha tullia Mőller ssp.
inornata Edwards, populations in the northern USA and Canada showed a step-
latitudinal cline in some allozyme frequencies monitored as well as wing characters
(Wiernasz, 1989). In the old, more northern part of the range, the insects are
predominantly univoltine, but in the south-western and eastern United States they are
bivoltine. The eastern region is an area of recent range expansion. The level of genetic
variability is strongly correlated with life history so that bivoltine populations tended to
have higher levels of polymorphism and heterozygosity with a higher average number
of alleles than univoltine populations, and were differentiated using genetic distance
and wing pattern data. Although interpopulation gene flow (movement) is high,
selection is the probable cause maintaining the cline. In bivoltine populations,
asynchrony between larval diapause and parents leads to alternating selection, thereby
increasing the level of genetic variation present (cf. Wiernasz, 1989).

Aphids also show latitudinal distribution of certain lifecycle strategies (obligate

asexual and sexual) correlated with climate. In French populations of the bird cherry-
oat aphid, Rhopalosiphum padi (L.) studied using allozyme markers, sexual
populations were found to be polymorphic early in the season, whereas asexual
populations were relatively less varied, and possibly monoclonal in origin (Simon et
al., 1996a). On seasonal analysis within a year using mtDNA markers, sexual insects
with haplotypes II and III were differentiated from asexuals, most of which were of
haplotype I (Simon et al., 1996b; Martinez-Torres et al., 1996, 1997). This last type
showed a north-south latitudinal cline in frequency in France, decreasing from the
southwest of the country (c. >90%) to the north (c. 15 – 45%) in various samples.
This trend coincides with a climatic gradient that may reflect selection in the north for
aphids with the sexual phase. These forms are favoured because the egg stage is more
resistant compared with living aphids to the harsher winter conditions prevailing there
(cf. Martinez-Torrez et al., 1997). Recent microsatellites analysis at five polymorphic
loci in S. avenae by Simon et al. (1999) has revealed widespread genotypes
throughout France and demonstrated similar lifecycle structuring. Presumably, the
genotypic structuring of aphids of different lifecycle strategy impacts on the dispersal
ability of this species. If too great a displacement occurs, climatic parameters will
select against forms with maladapted behavioural responses including lifecycle
synchrony in tune with particular photoperiodic responses (Smith & MacKay, 1990;
Loxdale et al, 1993; Lushai et al., 1996). Both the aforementioned butterfly example
and aphid work exemplify the degree of resolution obtainable using molecular markers,
which enables intraspecific variation to be teased apart in relation to biological and

11
ecological parameters. It also emphasises the complexity that has to be unravelled if
the biology of a given species is to be better understood.

Aphids also exemplify another aspect of short-term seasonal variation. In

southern British populations of S. avenae, analysis of patterns of genetic variation
indicated that population movements were very localised (De Barro et al., 1995a;
Sunnucks et al, 1997). In these studies, the range sampled covered mainly asexual
clones showing a strong genetic differentiation between various species of Gramineae
early in the field season (De Barro et al., 1995b,c; Sunnucks et al, 1997). The host-
based genotype heterogeneity dissipated as the growing season progressed, probably
associated with increasing local movements, both within and between fields (De Barro
et al., 1995b). Similarly, genetic variation was also seen to fall within a field as the
result of adaptive performance, measured by increases in fitness, which selected for
particular genotypes which came to dominate greater areas as they spread from
founder sites (De Barro et al., 1995a). In the damson-hop aphid, Phorodon humuli
(Schrank) sampled within the commercial-hop growing regions of England, highly
elevated esterase variants (associated with resistance to insecticides) were common in
spring populations on the overwintering host, Prunus spp. compared with summer
populations on wild, uncultivated hops, Humulus lupulus (Loxdale et al., 1998). These
data suggested that autumn fliers leave cultivated hop gardens and move only a short
distance to the primary host whereas summer fliers moving in the opposite direction
come from relatively further afield. This result and the local heterogeneity at esterase
loci and at another enzyme locus (6-phosphogluconate dehydrogenase, 6-PGD),
support both suction trap data and wind tunnel experiments that this aphid is a short
range migrant (probably usually < 20 km; Loxdale et al., 1998). Comparisons of the
lifecycle history and host selection data confirm the importance of long-term studies in
order to move away from ‘time-slice’ analysis of ecosystems which may fail to resolve
informative population detail.

An example of such detail is given by the diurnal behavioural responses of

migratory butterflies. Changes in enzyme phenotype or activity at particular loci as a
function of flight periodicity have been recorded in the Monarch butterfly, Danaus
plexippus L. A higher proportion of heterozygotes at the PGI (phosphoglucose
isomerase) and PGM loci were found in butterflies captured earlier than later in the day
(Carter et al., 1989). Further, using PGI alone, Hughes & Zalucki (1993) showed that
animals with a particular allele (M) were more likely to fly at low temperatures, and so
were active earlier in the day. Differences were also observed between males and
females in the effect of PGI alleles on flight activity. Work on clouded yellow
butterflies of the genus Colias F. demonstrated that young adults in the early part of
their non-overlapping generations showed genotype frequencies in Hardy-Weinberg
equilibrium, although a heterozygote excess developed as the insects aged (Watt,
1977; Watt et al., 1983). Of four frequent alleles carried by butterflies in culture, major
differences were observed in heat stability and various kinetic parameters among the
ten possible genotypes, with heterosis for kinetic parameters in some cases (Watt,
1977). Later, it was shown that certain heterozygotes began flights earlier in the day
compared with others. Among the most common genotypes, these could be arranged
in order of heterotic advantage, time of flight initiation, duration and overall flight
density throughout the day. Under heat stress conditions, butterflies with the most
thermally stable enzyme phenotypes survived the best. Such experiments suggest that

12
selection operates on these glycolytic enzymes, which clearly influences the time and
temperature at which butterflies first become active, and in migratory species, when
they are liable to disperse. They also appear to affect populations in relation to
genotype and sex and hence, these enzymes play a pivotal functional role in
determining population structuring and dynamics. So clearly without molecular
markers, and even more so, the right molecular marker (in this case a relevant
functional one), such biological intricacy for flight behaviour would allude us.

In the context of historical trends, allele frequency patterns are sometimes seen
to be rather homogeneous, even at large spatial scales. Such patterns may or may not
relate to concomitant large-scale aerial movements of the insects concerned. For
example, the fruit fly, Drosophila willistoni Sturtevant displays similar allele
frequencies at a range of allozyme loci over much of Mexico, Florida, Central
America, the West Indies and tropical South America (Ayala et al., 1972). Likewise,
two noctuid moth species examined also display homogenous among population allele
frequencies, i.e. the Velvetbean Caterpillar, Anticarsia gemmatalis Hőbner in
Louisiana and middle America (Pashley & Johnson, 1986), and the African armyworm,
Spodoptera exempta (Walker) in east Africa (Den Boer, 1978), as does the Cabbage
butterfly, Pieris rapae (L.) along the eastern seaboard of the USA (Vawter &
Brussard, 1983). In the case of D. willistoni, this homogeneity of pattern is thought to
be the result of locally differentiating forces, i.e. stabilising selection rather than the
result of dispersal counteracting the effects of drift and mutation (e.g. Nevo et al.,
1998). Although, Drosophila are known to be capable of dispersing up to 15 km.
across inhospitable desert in California (Coyne et al., 1982), it is unlikely that the
homogeneity observed is due to long range movements. The effect here is probably a
historical pattern of expansion and colonisation, reinforced to some degree by selection
and occasional migrants between populations (Slatkin, 1987). In the Velvet bean
Caterpillar, etc., the homogeneity observed is considered to be predominantly the
result of large-scale inter-population movement over long distances, and high levels of
gene flow.

In the geometrid November moth, Epirrita dilutata Denis & Schiffermőller, a

weakly flying woodland species, different inferences can be drawn from genetic pattern
depending on spatial scale and the sample size examined (Wynne, 1997). At the
national scale in Britain, a range of enzyme markers tested showed subpopulations to
have homogeneous allele frequency distributions. However, when subpopulations from
woods on the Rothamsted estate (330 ha) were tested using large sample sizes (n
>200), significant differentiation was observed, even at the commonest alleles. This
suggested a restriction of inter-population gene flow between populations less than 2
km apart, supported by MRR techniques. It was concluded that only in years of large
population size was there enough gene flow to lead to homogenisation of populations
locally. At the national scale, the similarity of population gene frequencies probably
reflects a historical pattern of colonisation. These data exemplify the necessity of
examining spatial patterns using representative sample sizes for the scale involved, so
that the molecular markers utilised are able to provide a credible measure. Historical
events have also been further elucidated using non-recombinant molecular markers.
The invasion of the medfly, Ceratitis capitata (Weidemann) into California in the late
1980s was thought to have originated in Hawaii; however, this premise was not
supported by mtDNA evidence (Sheppard et al., 1992). Similarly, in the brown rice

13
plant hopper, Nilaparvata lugens (Stål), it was shown, again using mtDNA markers,
that populations which annually invade Korea most probably arise from populations in
China which share haplotypes in common, rather than those from the Indochina
Peninsula, which do not (Mun et al., 1999).

The population genetics of the Monarch, D. plexippus, and how this is

influenced by historical pattern of movement is also of considerable interest. The
butterfly annually undergoes innate, long distance, two-way directed movement, i.e.
true migration from overwintering sites in southern California (Western population)
and Mexico (eastern population) northward to southern Canada and back (Urquhart
and Urquhart, 1977). Here the insect is tracking its milkweed host, Asclepias spp.
Interestingly, allozyme and mtDNA analysis of populations have shown a generally low
level of genetic diversity (Eanes & Koehn, 1978; Brower & Boyce, 1991; Brower,
1996). This invariance probably reflects the fact that the two populations, separated
geographically by the Rocky Mountains, move en masse as large, discrete panmictic
populations (Eanes & Koehn, 1978), and with gene flow apparently restricted between
them (Brower & Boyce, 1991). A low rate of mtDNA mutation, natural selection, a
high effective fecundity per female and possible population bottle effects may all
contribute to the genetic pattern observed (Brower & Boyce, 1991). Another possible
explanation for the low genetic variation is that the two populations are effectively
'moving demes' which breed together in close groups. As a consequence such lack of
variance, tantamount to specialisation, is indicative of a potential adaptive dead-end
and the species is thus ‘trapped’ in an evolutionary sense because of its very specific
behaviour and life history (Loxdale & Lushai, 1999).

6. Concluding remarks

In this synthesis, we have endeavoured to present some of the clearest

examples of the use of molecular markers in the interpretation of movement of flying
insects. From these, we have attempted to emphasise the complexity of biological
systems which molecular markers can assist in elucidating. It is only now in hindsight,
looking back at studies of insect migration and dispersal over the last century that new
challenges and objectives can be advanced in a field of study which is, after all, still
relatively young.

Study of the aerial movements of insects in relation to their behaviour, ecology

and population biology perhaps rank amongst the most difficult in zoology. This is
especially so for small insects that cannot be readily tagged (except using radio
labelling) making it difficult to estimate flight duration/distance, direction and speed.
With larger insects where direct physical tagging is possible, dilution effects and
displacement by air currents still hinder recapture so that such studies can also be
inconclusive over large spatial scales. Consequently molecular markers are our best
solution to attempt to resolve population structure and dynamics. Nevertheless, the
analysis of molecular genetic patterns still has to be performed in the light of
knowledge of the natural history of the insect concerned, otherwise as outlined in this
synthesis, there is tremendous room for misinterpretation.

Prior to the 1970s, even though a wealth of information was collected and
analysed for various moving insects (e.g. Johnson, 1969), molecular tools were not

14
available. Today however, a plethora of such markers exist. Despite this utility and
availability, whilst there is copious and random gathering of information, yesterday’s
questions remain largely unanswered, even for the most well studied migratory species,
including various international pests. Major questions that are largely unresolved are:
1, what is the orientation and defining range of a given moving species population?; 2,
are there for given species, 'true' migratory genotypes and populations, separate from
genotypes of established perennial populations of the same species?; 3, what are the
rates of movement of species individuals and populations?; 4, to what extent do flying
insects travel individually or collectively, initiating gene flow between isolated
populations?; and 5, does mating take place in some groups producing moving demes?
It is not even well known in most instances whether homogeneous allelic patterns
reflect the influence of recent gene flow or are the result of stabilising selection on
historical demographic patterning. Whilst it may be argued that these are the
fundamental and applied questions that we strive and seek to answer to illuminate
adaptation and evolution, it may also be timely to reflect upon the words of Sir Isaac
Newton (1642-1727), “....I seem to have been only like a boy playing on the sea-shore
and diverting myself in now and then finding a smoother pebble or a prettier shell than
ordinary, whilst the great ocean of truth lay all undiscovered before me” (Partington,
1992). With the advent of the Millennium, our endeavours should perhaps be focussed
on the above questions more clearly in a case specific way to move the science forward
e.g. Lenormand et al., (1999). Our knowledge of the movement of flying insects is far
from complete in spite of the massive efforts of those of the preceding fifty years in
particular. Further advances will undoubtedly result from the application of the right
molecular marker and careful consideration of the questions asked.

7. Acknowledgements

This paper was supported by the Ministry of Agriculture, Fisheries and Food, MAFF (H.D.L) and The
Leverhulme Trust (G.L.; F/180/AP). IACR-Rothamsted receives grant-aided support from the
BBSRC. We are most grateful to Ian Woiwod, Ian Denholm and Jim Mallet for their helpful
comments on the manuscript.

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