Mitochondrial DNA variation and GIS analysis confirm a secondary ...

Molecular Ecology (2001) 10, 603–611 

Mitochondrial DNA variation and GIS analysis 

Blackwell Science, Ltd 

confirm a secondary origin of geographical variation 

in the bushcricket Ephippiger ephippiger (Orthoptera: 

Tettigonioidea), and resurrect two subspecies 

MICHAEL G. RITCHIE,* DAVID M. KIDD† and JENNIFER M. GLEASON* 

*Environmental & Evolutionary Biology, Bute Medical Building, University of St Andrews, St Andrews, Fife KY16 9TS, UK, 

†Department of Geography, University of Portsmouth, Buckingham Building, Lion Terrace, Portsmouth PO1 3HE, UK 

Abstract 

Geographic variation within species can originate through selection and drift in situ (primary 

variation) or from vicariant episodes (secondary variation). Most patterns of subspecific 

variation within European flora and fauna are thought to have secondary origins, reflecting 

isolation in refugia during Quaternary ice ages. The bushcricket Ephippiger ephippiger has 

an unusual pattern of geographical variability in morphology, behaviour and allozymes in 

southern France, which has been interpreted as reflecting recent primary origins rather than 

historical isolation. Re-analysis of this variation using Geographical Information Systems 

(GIS) suggests a possible zone of hybridization within a complex pattern of geographical 

variation. Here we produce a genetic distance matrix from restriction fragment length polymorphism 

(RFLP) bandsharing of an approximately 4.5 kb fragment of mitochondrial DNA 

(mtDNA), and compare this with predictions resulting from the GIS analysis. The mtDNA 

variation supports a postglacial origin of geographical variation. Partial Mantel test comparisons 

of genetic distances with matrices of geographical distance, relevant environmental characteristics 

and possible refugia show refugia to be the best predictors of genetic distance. 

There is no evidence to support isolation by distance. However, environmental contrasts do 

explain significant variation in genetic distance after allowing for the effect of refugial origin. 

Also, a neighbour-joining tree has a major division separating eastern and western forms. We 

conclude that the major source of variation within the species is historical isolation in glacial 

refugia, but that dispersal, hybridization and selection associated with environmental features 

has influenced patterns of mtDNA introgression. At least two valid subspecies can be defined. 

Keywords: genetic distance, GIS, geographical variation, hybrid zone, Mantel analysis, mtDNA 

Received 4 June 2000; revision received 23 August 2000; accepted 23 August 2000 

Introduction 

The study of geographical variation and systematics has 

played a major role in the development of theories of 

speciation. Geographic variation can arise in a number of 

ways but, following from Mayr (1942), two major causes 

have been distinguished. Primary variation arises in situ 

because of contemporary or recent selection and drift. In 

contrast, secondary variation arises from historical vicariant 

events. Hewitt and colleagues have argued that most major 

Correspondence: Michael G. Ritchie. Fax: 01334 463 6000; E-mail: 

mgr@st-and.ac.uk 

hybrid zones in Europe reflect divergence during Quaternary 

ice ages (e.g. Barton & Hewitt 1985), with periodic expansion 

and contraction cycles accentuating and accumulating 

genetic differences (Hewitt 1988, 1989, 1996, 1999; see also 

Remington 1968; Taberlet et al. 1998). 

Despite the common assumption that most hybrid zones 

result from secondary contact, steep coincident clines in 

organisms can have a primary origin, having arisen in 

response to exogenous selection across ecotones (Endler 

1977; Harrison 1990). It is not possible to clearly distinguish 

primary or secondary origins of hybrid zones from an 

examination of their geographical context, because many 

areas of secondary contact are likely to occur across ecotones 

© 2001 Blackwell Science Ltd

604 M. G. RITCHIE, D. M. KIDD and J . M . GLEASON 

(Hewitt 1989). Also, theory suggests that the characteristics 

of primary and secondary clines will be identical (Endler 

1977; Barton & Hewitt 1985; but see Durrett et al. 2000). Distinguishing 

primary and secondary origins of clines and 

hybrid zones remains a major, and relatively poorly met, aim 

of studies of geographical variation (Thorpe 1984; Harrison 

1990, 1993; Willett et al. 1997; Schilthuizen 2000). 

The bushcricket (katydid) Ephippiger ephippiger (Feibig 

1784) is a flightless tettigoniid with unusual patterns of 

geographical variation and, as a result, has been subject to 

a number of taxonomic surveys and revisions. The species 

is patchily distributed and highly variable in morphology 

and behaviour. This is most pronounced in southern 

France, from the Pyrenees to the Alps, where a number of 

species and subspecies have been described. Harz (1969), a 

standard taxonomic reference for European tettigoniids, 

recognized three species within the group under consideration 

here. E. ephippiger is a medium sized, predominantly 

green bushcricket with a monosyllabic calling song found 

in central Europe, with the subspecies E. e. vitium (Serville 

1831) in western Europe and E. e. ephippiger (Fiebig 1784) in 

the east (an additional five subspecies have been described, 

Hartley & Warne 1984). E. cunii (Bolivar 1877) is small, almost 

black, with a polysyllabic calling song, found in Catalonia. 

E. cruciger (Fieber 1853, syn. E. bitterensis) is large, grey or 

green, usually with a melanized sulcus on the pronotum 

and a mono- or polysyllabic calling song, found around 

the Languedoc (see Duijm & Oudman 1983; Kidd & 

Ritichie 2000, for distribution maps). Recently, studies have 

reduced these forms in taxonomic status. Duijm & Oudman 

(1983), Duijm et al. (1983) and Hartley & Warne (1984), after 

analyses of morphology and copulation compatabilities, 

concluded that only the cunii form was valid (as a subspecies), 

and combined E. e. cruciger, E. e. vitium and E. e. ephippiger 

into a single highly variable ‘superspecies’ which they 

termed E. ephippiger diurnus Kruseman (referred to here as 

E. ephippiger for simplicity). Duijm and colleagues (Oudman 

et al. 1989, 1990; Duijm 1990) further questioned whether 

cunii was valid even as a subspecies. They found low genetic 

divergence between forms (Nei’s D, from four allozyme 

loci, was around 0.03 between forms). They also examined 

the geographical pattern of clines for the morphological 

traits used to define the various forms. Visual inspection 

implied the clines were usually noncoincident geographically 

and extremely wide. Some were 50–100 km wide, which 

compares with an average cline width for similar traits of 

5–10 km in the Pyrenean hybrid zone of Chorthippus parallelus 

(Hewitt 1989; Butlin 1998), an organism which probably 

has a higher dispersal than E. ephippiger. Oudman et al. 

(1990) concluded that the geographical variation within 

E. ephippiger was unlikely to reflect secondary contact: ‘We 

see the E. ephippiger complex as a subspecies … with sufficient 

gene flow to restrict differentiation. In the south of 

France the species has met conditions that have led to 

greater differentiation. This has on the one hand the character 

of shallow clines, perhaps along environmental gradients 

and probably adaptive, and on the other hand a chance 

character as the result of genetic drift and random dispersal 

effects.’ Oudman et al. (1990) also concluded that no valid 

subspecies could be distinguished, the local forms being 

‘without taxonomic status’. This interpretation has been 

adopted by other authorities (Ragge & Reynolds 1998; p. 71). 

We have re-examined E. ephippiger from southern France 

from two perspectives. Geographical Information Systems 

(GIS) facilitate the detailed examination of spatially patterned 

data. We employed GIS to examine concordance between 

interpolated trait clines and ecotones, to ask if the broad, 

nonconcordant clines are coincident with ecotones, as 

would be expected if they result from primary selection. 

Additionally, GIS has been used to try to identify clusters in 

the geographical variability, which might reflect secondary 

origins, separate from or additional to the primary variation. 

If geographical clusters truly reflect secondary subdivisions 

within the species, we would expect these to be supported 

by similar patterns in genetic differentiation. A study of 

random amplified polymorphic DNA (RAPD) markers 

broadly separated the cunii form from cruciger and vitium 

(Ritchie et al. 1997), but this survey was geographically 

limited. Here we report restriction fragment length polymorphism 

(RFLP) analysis of mitochondrial DNA (mtDNA) 

using more extensive samples. Partial Mantel test analyses 

(Thorpe 1996) of matrices of genetic distance compared 

with geographical distance, ‘environmental distance’ and 

potential ice age refugial origins imply that most genetic 

variation reflects refugia, though significant partitioning is 

also associated with environmental variation. 

Background: Geographical Information Systems analysis 

of geographical variation in E. ephippiger 

Full details of our GIS analysis of geographical variation in 

E. ephippiger are available elsewhere (Kidd & Ritchie 2000, 

2001). Briefly, data was compiled on geographical variation 

in morphological, allozyme and behavioural traits, 

as well as abiotic environmental parameters. Individual 

and multivariate trait surfaces were interpolated, compared 

with environmental data, and examined for covariation 

and concordance. Environmental features explained little 

other than body size of the organism. In contrast to the 

interpretation of these data by Oudman et al. (1990), some 

concordance between patches was observed in the interpolated 

surfaces, and fairly sharp, coincident transitions in 

some traits were detected. Figure 1 indicates the centre of a 

relatively steep multivariate cline, which broadly follows 

the path of the Aude river. Multivariate discriminant analyses 

(with resampled null models) confirmed that the regions 

either side of the cline represent significant clusters within 

a complex pattern of geographical variability. Kidd & 

© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 603–611

GEOGRAPHIC VARIATION IN A BUSHCRICKET 605 

Fig. 1 Location of collecting sites for the mtDNA analysis in southern France. Also indicated is the position of the cryptic hybrid zone 

inferred in Kidd & Ritchie (2000) from more extensive samples, plus geographical features referred to in the text. Assignment of specimens 

to the forms is indicated (V = vitium, Cu = cunii, Cr = cruciger). Inset shows details of the eastern Pyrenees, and the locations of the 

Cerdagne, Capcir and Conflent regions. 

Ritchie (2000) concluded that the cline represented a 

‘cryptic hybrid zone’ where some traits, notably genitalia, 

probably reflected secondary contact between forms originating 

from western (Iberian) and an unidentified eastern refugia. 

This was broadly but imperfectly coincident with the vitium 

and cunii forms. The cruciger form was more difficult to fit 

into this reconstruction. Some traits, notably the frequency of 

some allozymes and the third principal component, broadly 

matched the distribution of crickets identified as cruciger, 

but this was around both sides of the eastern end of the 

potential hybrid zone, near Narbonne (around the mouth 

of the Aude). Kidd & Ritchie (2000) suggested three possibilities; 

cruciger was not valid, cruciger was a hybrid form, 

or cruciger was from a distinct western refugium. 

Materials and methods 

mtDNA analysis 

Samples were collected throughout southern France over 

several years by M.G. Ritchie (Fig. 1). During collection, 

crickets were provisionally assigned to the forms cunii, 

cruciger or vitium according to morphology and behaviour 

(a single assignment per locale). Fifty-three individuals 

were analysed, including individuals of Ephippiger perforatus, 

E. terrestris and Uromenus rugosicollis as outgroup specimens. 

DNA was extracted from frozen or ethanol preserved 

hind femora using the Puregene DNA Isolation Kit (Gentra 

Systems). Based on the Locusta migratoria mtDNA sequence, 

we designed primers to amplify the region including cytochrome 

oxidase (CO) subunits II and III, ATPase6, NADH 

subunits 3 and 5, as well as nine tRNAs, for a total length of 

approximately 4.5 kb. Polymerase chain reaction (PCR) conditions 

were denaturing 92 °C for 2 min followed by 10 cycles 

of 92 °C for 10 s, 55 °C for 15 s, 69 °C for 2 min; and then 30 

cycles of 92 °C for 10 s, 60 °C for 15 s; 68 °C for 2 min extended 

by 20 s per cycle. Reactions were done in 100 µL volumes 

with 3.5 mm MgCl 2 

, 0.1 mm dNTP, 1× Optibuffer (Bioline) 

and 2 units Bio-X-Act DNA polymerase (Bioline). The forward 

primer was located within cytochrome oxidase I (COI) and 

the reverse primer within ND5. Their sequences for the 

first, C2-J-3120: was GGCAACYTGATCYAMTTTRAATTT- 

ACAAAAATAGTGC and for the second, N5-N-7700, was 

ACAGCTTTGTCAAATCGYRTTGGGGATGT. 



Following PCR, the products were digested singly by eight 

different restriction enzymes: DraI, EcoRI, HaeIII, HpaII, 

Hsp92II, RsaI, StyI and TaqI (Promega). This resulted in a total 

of 119 fragments that were scored for presence and absence. 

A similarity (band sharing) index S (Nei & Li 1979) was 

calculated from the restriction profiles of each individual as 

S = 2Na,b/(Na + Nb), where Na, Nb = number of bands in 

individual a, b and Na,b = number of shared bands. A genetic 

distance matrix was constructed from 1-S. A phenogram 

was produced from this matrix using the neighbour-joining 

algorithm (default conditions, randomised input order) in 

phylip 3.57c (Felsenstein 1995). Bootstrap values are derived 

from 100 resamples (with replacement) of the RFLP data. 

Matrix comparisons 

Matrix comparisons provide a powerful method of assessing 

the association between biological or environmental features 

and variation within a species (Douglas & Endler 1982; 

Smouse et al. 1986; Thorpe 1996). In order to assess the best 

predictors of genetic distance, a series of indicator matrices 

were constructed and compared with the genetic distance 

matrix. Outgroups and multiple samples from individual 

populations were removed, reducing the number of samples 

to 30. Within populations, individuals were first removed 

if they had missing data (across all samples, the average 

proportion of missing data was 1.4%), then randomly if more 

than one individual remained. Because replicate individuals 

usually group together it was not thought necessary to 

bootstrap this process. 

Predictor matrices were constructed from geographical 

distance, environmental dissimilarity, and two different 

vicariant scenarios. Inter-site distances were calculated 

from a user-defined network by network tracing in ArcInfo 

(Environmental Systems Research Institute 1997). This 

method produces a distance matrix not ‘as the crow flies’ 

but ‘as the cricket crawls’, taking into account the real barriers 

of the Pyrenees and Mediterranean (Kidd & Ritchie 2001). 

A measure of ‘environmental distance’ between sites was 

derived from Kidd & Ritchie’s (2000) multiple linear regression 

model of body size. This explained 80% of the variation 

in body size using a combination of latitude, altitude, 

irradiation, precipitation and distance from the coast (no 

other traits were significantly correlated with environmental 

parameters). The fitted values of this model were used 

as a proxy measure of relevant environmental variance. 

Euclidean similarity measures for each population were 

calculated and (1-environmental similarity) used to produce 

an environmental dissimilarity matrix. 

The first matrix constructed to reflect potential refugial 

history, the ‘east vs. west refugia’ (E–WR) matrix, simply 

contrasted populations which probably originated in eastern 

vs. western refugia. Thus, all vitium populations were 

assumed to originate in an eastern refugium, and cunii and 

cruciger from a western refugium (if pairs of populations 

arose from the same refugium they were ascribed a contrast 

of 0, 1 if from different refugia). The second matrix, the ‘three 

refugia’ (3-R) matrix, was constructed to include the possibility 

of a separate western refugium for the cruciger crickets. 

To reflect the fact that such a refugium was more proximal 

to the main western refugium, contrasts were 0 if populations 

probably arose from the same refugium (i.e. if the samples 

were considered of the same form), 1 if from the main Iberian 

refugium vs. the eastern refugium, 0.2 if from the two separate 

western refugia, and 0.8 if from the eastern vs. ‘cruciger’ 

refugium. Note that in constructing these matrices, the 

original assignment of specimens to type during collection 

was used, not the position of the sample in the mtDNA tree. 

We, therefore, have five matrices: genetic distance is the 

response matrix and there are four predictor matrices. The 

magnitude of the partial regression coefficients indicates 

the relative importance of the different predictors. If differences 

in mtDNA reflects simple isolation by distance, geographical 

distance would be the best predictor. If primary 

selection is most important, then environmental distance 

would be the best predictor. If vicariance is the most important, 

one of the refugial contrasts would be the best predictor 

(and the 3-R matrix best if the cruciger form was from a 

distinct refugium). All matrices were transformed to a 

mean of zero and unit variance before completing partial 

Mantel analyses (Thorpe 1996; Smouse et al. 1986). Individual 

Mantel comparisons between genetic distance and 

the predictor matrices were each strongly significant. Probability 

values are derived from 10 000 randomizations of the 

response matrix. Matrix calculations were carried out using 

genstat (Genstat Committee 1993) and customised software. 

Results 

Matrix comparisons 

The regression coefficients and associated probability 

values for the predictor matrices are influenced by the 

number of matrices included in the analysis, and the two 

predictor matrices derived from refugial scenarios are 

themselves highly correlated. We, therefore, present three 

analyses, one with all four predictor matrices, and two 

with only one of the refugium contrasts (Table 1). As three 

tests were carried out, the critical value is 0.017 (2-tailed) or 

0.034 (1-tailed, perhaps justified for tests comparing such 

distances). There is no support for any contribution of 

isolation by distance. The environmental contrast approaches 

significance in each test, but the greatest matrix regression 

coefficients are always associated with the refugial contrasts. 

Both refugial models are similar, but that involving 3-R has 

a greater coefficient. When both are entered, E–WR is not 

significant. The value of the matrix coefficient is much 

greater for 3-R than the environmental contrast. 



Table 1 Partial regression coefficients from three Mantel analyses (E–WR is the matrix constrasting eastern and western refugia, 3-R is that 

with an additional refugium for cruciger) 

Analysis Matrix Geographic Distance Environment E–WR 3-R 

Full model Partial regression coefficient 0.027 0.109 −0.155 0.588 

(probability) (ns) (0.032) (ns) (0.018) 

E–WR excluded Partial regression coefficient 0.030 0.107 — 0.434 

(probability) (ns) (0.027) (0.001) 

3-R excluded Partial regression coefficient 0.066 0.102 0.395 — 

(probability) (ns) (0.035) (0.001) 

(ns), nonsignificant. 

Fig. 2 Neighbour-Joining tree produced 

from mtDNA RFLP profiles of all individual 

bushcrickets used in the study. The assignment 

to form initially made in the field and 

the population code number from Figs 1 

and 3 are indicated. The consensus tree 

from the bootstrapped data had a virtually 

identical topology. Values are shown for 

clades discussed in the text. 

mtDNA phylogeography 

A neighbour-joining tree based on the mtDNA RFLPs is 

not well resolved (Fig. 2). This is probably not surprising 

given the high number of samples from regions of 

hybridization. Despite this, some clades are well supported. 

The deepest division within Ephippiger ephippiger almost 

perfectly separates individuals identified as vitium from 

the remainder of the samples (Fig. 2). This division fairly 

closely corresponds with the location of the putative Kidd 



Fig. 3 The branching pattern of a neighbourjoining 

tree superimposed on the geography 

of the region. This tree contained only one 

sample per collecting site. Branch lengths are 

determined by geography, but the branching 

pattern reflects the genetic distances. 

& Ritchie contact zone (compare Fig. 3). The only samples 

in the ‘incorrect’ clades are one from Lodeve, which is from 

the area of transition between the vitium and other forms, 

and one from the Col du Pourtalet. The ‘pure’ vitium clade 

is reasonably well supported. 

There are other striking features of this tree. The deepest 

node within the nonvitium samples separate off a branch 

containing cunii samples from the Segre river valley (the 

Cerdagne region of the Pyrenees) from the remainder of 

the cunii and cruciger samples. A second node separates 

off cunii samples from the Têt river valley (the Conflent 

region). The remainder of the tree includes samples from a 

third Pyrenean valley, that of the river Aude (the Capcir 

region) and samples from the Languedoc. This branch, 

therefore, contains samples identified as cunii and nearly 

all the cruciger samples. The Aude valley is the northernmost 

of the eastern Pyrenean valleys sampled here (north of the 

main watershed), and leads down into the Languedoc. 

Discussion 

The large morphological and behavioural variability 

within Ephippiger ephippiger has lead to a confusing series of 

taxonomic classifications and revisions (Harz 1969; Hartley 

& Warne 1984). Previous studies concluded that none of the 

described species or subspecies were taxonomically valid, 

and that the variation reflected primary differentiation 

(Oudman et al. 1989, 1990; Duijm 1990; but see Grandcolas 

1987). Our current analyses lead us to conclude that the 

major source of variation is historical isolation during 

recent glaciations, followed by secondary contact between 

eastern and western forms, as has commonly shaped 

subspecific variation in European flora and fauna (Taberlet 

et al. 1998; Hewitt 1999; Vogel et al. 1999). However, in partial 

Mantel analyses, genetic variability is correlated with environmental 

contrasts even after allowing for the influence of 

refugia, and significant environmental influences are also 

detectable on body size (Kidd & Ritchie 2000). Both 

secondary contact and environmental selection have therefore 

influenced the pattern of genetic variability, though 

secondary contact has had the greater effect. 

Evidence supporting a vicariant origin of the differentiation 

comes from the consistently high Mantel regression 

coefficients associated with the refugial contrast matrices 

(Table 1), which is also reflected in the deepest node within 

the mtDNA relationships separating the samples into eastern 

and western forms (Figs 2 and 3). The multivariate 

transition identified by Kidd & Ritchie (2000, 2001) lies 

within the same geographical region as the division in 

mtDNA, but the two are not exactly coincident (Figs 1 and 

3). The division in mtDNA occurs to the north of the multivariate 

transition, somewhere south of Lodeve and west of 

Montpellier. This lack of coincidence could be due to primary 

selection patterning differential introgression of traits (see 

below), although cytoplasmic DNA introgression through 

hybrid zones is not uncommon (Shaw et al. 1990; Whittemore 

& Schaal 1991). 

Vitium, therefore, represents a form that probably originates 

from a refugium to the east of the study area. Many eastern 

European subspecies are thought to have originated in 

the Balkans or Caucasus (Hewitt 1996, 1999; Taberlet et al. 

1998), or perhaps in the Carpathians or Ural mountains 

(Lagercrantz & Ryman 1990). Vitium presumably occupies 

most of central Europe now, and extends as far west as the central 

Pyrenees. One sample from here (from site 2) falls within 

the western clade of the mtDNA tree (Fig. 3). E. ephippiger 

from this area has been claimed to represent yet another 

subspecies, E. moralesagacinoi (Harz 1969), but it seems 

more likely that there is another rapid transition between 

the eastern and western forms here (Oudman et al. 1990). 

The western form, comprising nearly all the samples 

identified as cunii or cruciger, has geographically structured 

mtDNA variation. Samples from around the Pyrenean 

watershed form distinct groups, with one from each of the 



three main valleys (Cerdagne, Capcir and Conflent). This 

provides a striking contrast with previous conclusions of 

extensive gene flow based on allozyme, morphological and 

RAPD markers (Oudman et al. 1990; Ritchie et al. 1997). The 

two deepest nodes within the western form separate off 

individuals from Cerdagne and Conflent. The remainder of 

the samples comprise a group including the northernmost 

cunii and almost all the cruciger samples, and geographically 

corresponds to Capcir and the Languedoc. Clearly, there is 

evidence of a shared mtDNA history to these samples, 

despite their extensive range (over 150 km). The distinctness 

of the cunii from different valleys, and the presence 

of cruciger within this clade, disrupts cunii as a 

genetic form. The 3-refugium matrix in the Mantel test was 

designed to test the distinctness of cruciger, and it always 

provided the greatest partial regression coefficient. So 

there is some evidence to support the existence of a distinct 

cruciger genotype, but the historical processes underlying 

the relationship between cunii and cruciger are obscure. 

We consider there to be two possible explanations whereby 

cruciger may represent a distinct form. The first is that 

cruciger was confined to a third refugium east of the Pyrenees, 

possibly around the Mediterranean coast. Cruciger would 

then have been subject to hybridization, first with the 

northernmost samples of cunii expanding from a Spanish 

refugium, then latterly with vitium expanding from an eastern 

refugium. Cruciger could be facing extinction via introgression 

due to this pincer movement. Most reconstructions of 

Quaternary glacial refugia in southern Europe only include 

major ones in southern Spain, Italy and the Balkans (Taberlet 

et al. 1998; Hewitt 1999). Huntley & Birks 1983 (see also Vogel 

et al. 1999) suggest that there was another refugium around 

the Alpes Maritimes, and there is a suggestion of Pleistocene 

coastal Mediterranean refugia including one around the 

mouth of the Rhône (Comes & Abbott 1998), but no other 

phylogenetic studies support the presence of distinct genotypes 

from around this area (Comes & Kadereit 1998). 

The second scenario is that cunii and cruciger are from 

partially distinct southern refugia, both in Iberia, but have 

a long history of hybridization and introgression during 

repeated expansion/contraction cycles, with the Languedoc 

being most recently invaded by a form already having 

undergone hybridization with northern cunii. Repeated 

hybridization could explain the imprecision of many of 

the taxonomic traits. This scenario is perhaps more likely 

given a very high level of Iberian endemism within the 

ephippigerinae in general (Harz 1969; Gangwere & Morales 

Agacino 1970). Also, several other studies have found 

evidence for multiple refugia or variation within Iberia 

(Cooper et al. 1995; Comes & Abbott 2000). 

Although the main genetic divisions reflect secondary 

contact, we also detect evidence supporting primary selection 

influencing Ephippiger. This is most apparent in body size 

and body ratios (Kidd & Ritchie 2000). However, the 

partial Mantel tests show that, after allowing for refugia, 

environmental contrasts are still correlated with variation 

in mtDNA. Environmental conditions are unlikely to select 

directly on mtDNA, it is more likely that indirect selection 

influences the introgression of genotypes including mtDNA. 

Cunii is probably more cold adapted or robust to inclement 

environmental conditions than vitium. Hybrid zones with 

a broad front are likely to act as semi-permeable barriers, 

with recombination separating out genotypes under direct 

selection from linked genetic variation (Barton & Gale 1993), 

but patchy, mosaic or intermittent episodes of hybridization 

may produce more opportunity for persistence of associations 

across different types of genetic markers (Harrison 

& Rand 1989; Rieseberg & Wendel 1993; Arnold 1997). 

Our studies provide a good example of the value of an 

integrative approach, involving techniques from geographical 

analysis as well as spatial statistical analysis and 

molecular phylogeography, in unravelling the pattern and 

historical processes contributing to geographical variation. 

Conventional population genetic methods for estimating 

substructure within species allow little more than rejection 

of the hypotheses that populations are panmictic or show 

isolation by distance (Neigel 1997; Bossart & Prowell 1998; 

Whitlock & McCauley 1998). Techniques are required 

which allow detection of more realistic historical scenarios. 

GIS allows independent identification of clusters in population 

structure, and phylogeographic and Mantel tests, or 

hierarchical analysis of F ST 

or its derivatives can assess the 

validity of these externally derived predictors of clustering 

(Kidd & Ritchie 2001). 

These analyses have resurrected two forms of the 

bushcricket E. ephippiger from a taxonomic hinterland, and 

we conclude that vitium is a valid subspecies. Whether 

cruciger and cunii warrant separate subspecies status is 

more problematical and will rely on further analyses. The 

Mantel tests support their distinction, and several of the 

trait surfaces of Kidd & Ritchie (2000) show clusters around 

Narbonne. In contrast, the mtDNA relationships imply 

that cruciger mtDNA is a derived form of the cunii genotype, 

though this might be confused by hybridization 

between the northern cunii and cruciger forms. It is difficult 

to predict the effect of hybridization on phylogenetic trees 

(Arnold 1997). 

Finally, what lessons might E. ephippiger have for systematics? 

We think it appropriate to designate at least two 

forms of E. ephippiger as subspecies because they hybridize in 

nature, yet are distinguishable on multiple traits throughout 

the majority of their ranges. Some species definitions, particularly 

the Phylogenetic (Cracraft 1989) and, depending on 

details of hybrid populations, the Cluster species definitions 

(Mallet 1995) might justify the elevation of these forms 

to species status. We do not accept this due to the obvious 

hybridization and lack of obvious incompatibilities between 

the forms. However, does the systematic term ‘subspecies’ 



have genuine biological validity? Mayr (1942, p. 106) thought 

not: ‘The taxonomist is an orderly person whose task it is 

to assign every specimen to a definite category (or museum 

drawer!). This necessary process of pigeonholing has led to 

the erroneous belief … that subspecies are clear-cut units 

which can easily be separated from one another.… But subspecies 

intergrade almost unnoticeably in nearly all the 

cases in which there is distributional continuity’. This reflects 

Mayr’s (possibly essentialist, Mallet et al. 1998) view that 

species were the only ‘true’ biological categories within 

organisms. The increasing body of evidence that hybrid 

zones result from historical vicariance episodes between 

forms that have accumulated multiple concordant genetic 

differences over a time scale possibly stretching as far back 

as the Pliocene, must support a valid general concept of the 

subspecies (Avise & Ball 1990). 

Acknowledgements 

Matthijs Duijm and Leendert Oudman provided very generous 

help during the initial stages of this project. Bill Black, Hans Peter 

Comes, Colin Hartley, and Roger Thorpe provided advice, and 

several people helped with fieldwork. Klaus-Gerhardt Heller 

kindly supplied the specimen of E. perforatus, and Roger Butlin 

and Jeff Graves gave advice on the manuscript. The work was 

funded by the NERC, UK, via a research fellowship and grant to 

MGR. 

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Mike Ritchie completed his PhD with Godfrey Hewitt, studying 

the Chorthippus parallelus hybrid zone in the Pyrenees. There he 

encountered Ephippiger ephippiger, and carried out a postdoctoral 

fellowship in Hewitt’s laboratory studying the behavioural variation 

of this species. Dave Kidd has collaborated with MGR in applying 

GIS methods to the study of geographical variation in E. ephippiger. 

Jenny Gleason studies molecular evolution and quantitative genetics, 

and collaborated with MGR to develop the mtDNA RFLP analysis 

of E. ephippiger.

Mitochondrial DNA variation and GIS analysis confirm a secondary ...

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