Non-random segregation of an autosomal gene in males of the flea beetle, Phyllotreta nemorum:...

Non-random segregation of an autosomal gene in malesof the flea beetle, Phyllotreta nemorum: implicationsfor colonization of a novel host plantJens Kvist Nielsen*Department of Agriculture and Ecology, Section of Botany, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej

21, DK-1958 Frederiksberg C, Denmark

Accepted: 9 March 2012

Key words: Barbarea vulgaris, Brassicaceae, Cruciferae, Coleoptera, Chrysomelidae, Alticinae, insect–

plant relationships, host plant selection, genetics, inheritance, neo-Y, sex linkage

Abstract The flea beetle, Phyllotreta nemorum (L.) (Coleoptera: Chrysomelidae: Alticinae), is currently

expanding its host plant range in Europe. The ability to utilize a novel host plant, Barbarea vulgaris

R. Br. (Brassicaceae), is controlled by major dominant genes named R-genes. The present study used

extensive crossing experiments to illustrate a peculiar mode of inheritance of the R-gene in a popula-

tion from Delemont (Switzerland). When resistant males from Delemont are mated with recessive

females from a laboratory line, the female F1 offspring contains the R-allele and is able to utilize

B. vulgaris, whereas the male offspring contains the r-allele and is unable to utilize the plant. This out-

come suggests X-linkage of the R-gene, but further crossing experiments demonstrated that this was

not the case. When the R-gene is present in offspring from males from a laboratory line that origi-

nates from Taastrup (Denmark), it is transmitted to female and male offspring in equal proportions

as a normal autosomal gene. The results demonstrate a polymorphism in segregation patterns of an

autosomal R-gene in P. nemorum males. Males from Delemont contain a factor which causes non-

random segregation of the R-gene (NRS-factor). This factor is inherited patrilineally (from fathers to

sons). Males with the NRS-factor transmit the R-gene to their female offspring, whereas males with-

out the NRS-factor transmit the R-gene to female and male offspring in equal proportions. Various

models for the non-random segregation of autosomes in P. nemorum males are discussed – e.g.,

fusions between autosomes and sex chromosomes, and genomic imprinting. The implications of var-

ious modes of inheritance of R-genes for the ability of P. nemorum populations to colonize novel

patches of B. vulgaris are discussed.

Introduction

Plant-feeding insects exhibit an extraordinary diversity in

feeding habits (Schoonhoven et al., 2005). Most species

feed only on a limited number of the plant species that are

available in their habitat. A major obstacle to the utiliza-

tion of many plant species is the content of secondary plant

substances that may be toxic or have deleterious effects on

the behaviour of non-adapted insects (Hartmann, 1996).

On the other hand, many insects have evolved traits that

circumvent the deleterious effects of specific secondary

compounds, and adaptations to secondary compounds

are often involved in the evolution of novel host plant use

in phytophagous insects (Schoonhoven et al., 2005;

Thompson, 2005). Evolutionary processes involved in the

acquisition of novel host plant use can be studied in cases

where plant-feeding insects use different plant species in

different habitats or in different parts of their geographical

range and there is a growing body of evidence that these

processes can lead to host race formation and eventually to

ecological speciation (Matsubayashi et al., 2010).

Novel host plant use may require genetic adaptations in

traits influencing insect behaviour and performance as

natural selection is supposed to favour those individuals

that choose host plants that are suitable for survival and

growth of their offspring (Thompson, 1988a, 2005). Intra-

specific genetic differences in traits influencing host plant

selection have been documented in several cases (Nielsen,

1997b; Sezer & Butlin, 1998; de Jong et al., 2000; Jones,*Correspondence: E-mail: [email protected]

� 2012 The Author Entomologia Experimentalis et Applicata 143: 301–312, 2012

Entomologia Experimentalis et Applicata � 2012 The Netherlands Entomological Society 301

DOI: 10.1111/j.1570-7458.2012.01262.x

2005; Nygren et al., 2006). Crosses between closely related

species have provided further insight into the factors that

control differential host use. These studies have docu-

mented that genes influencing host plant selection may be

located on autosomes as well as on the sex chromosomes

(Thompson, 1988b; Thompson et al., 1990; Scriber, 1994;

Xue et al., 2009).

Host shifts or host range expansions are likely to be

facilitated if there are genetic correlations between genes

influencing behaviour and performance on the new host

plant (Hawthorne & Via, 2001; Jones, 2005; Matsubay-

ashi et al., 2010) or if the same genes influence behav-

iour and performance on the new host plant (Nielsen,

1996, 1997b; Nielsen et al., 2010). It is therefore impor-

tant to study the modes of inheritance of genes influenc-

ing host plant selection and to determine how genes

with different effects and modes of inheritance influence

the colonization of novel host plants. The present study

documents the aberrant inheritance of a gene that con-

fers the ability of the flea beetle, Phyllotreta nemorum

(L.) (Coleoptera: Chrysomelidae: Alticinae), to utilize a

novel host plant, Barbarea vulgaris ssp. arcuata (Opiz.)

Simkovics (Brassicaceae).

Phyllotreta nemorum is an intermediate specialist (oligo-

phage) which lives on a small number of plant species

belonging to Brassicaceae (formerly Cruciferae). One very

common host plant in Denmark and Switzerland is Sinapis

arvensis L., whereas B. vulgaris is used less commonly.

Barbarea vulgaris is a variable plant, and two subspecies,

vulgaris and arcuata, have been recognized. Furthermore,

the subspecies arcuata can be divided into two types which

differ in suitability as host for flea beetle larvae: the P-type

is susceptible to all known flea beetle genotypes, and the

G-type is resistant to the most abundant flea beetle geno-

types (Nielsen, 1997a; Agerbirk et al., 2003). Resistance in

the G-type is caused by the presence of triterpenoid sapo-

nins which inhibit feeding in adult beetles and prevent

larval development (Kuzina et al., 2009, 2011; Nielsen

et al., 2010). However, the saponins are not effective

against all flea beetle genotypes and the plant is used as a

host plant by a few Danish flea beetle populations (Nielsen

& de Jong, 2005). Detailed investigations on the genetics of

the ability to utilize the G-type have been performed in

two Danish flea beetle populations (Nielsen, 1997b; de

Jong et al., 2000). In both cases major, dominant genes

(named R-genes) were involved, but the mode of inheri-

tance was different. In one population (Kværkeby) only

autosomal R-genes were found (de Jong et al., 2000),

whereas autosomal and sex-linked genes (X and Y) were

found in a population from Ejby (Nielsen, 1997b). Beetles

that contained two copies (RR, XRXR, and XRYR) or one

copy of the R-allele (Rr, XRXr, XRYr, and XrYR) could

survive on the G-type, whereas rr, XrXr, and XrYr could

not. The Y-linked R-gene from Ejby and the autosomal

genes from Ejby and Kværkeby had similar effects on

behavioural responses of flea beetles to saponins (Nielsen

et al., 2010). The R-genes confer the ability to utilize B.

vulgaris and a few other Barbarea spp., and they are con-

sidered to be counteradaptations to defences in these

potential host plants (Nielsen, 1999; Nielsen et al., 2010).

However, it is still uncertain whether they originated as

independent mutations on different chromosomes or

whether they have a common origin and their current

position is due to translocation events.

The geographic variation in modes of adaptation of flea

beetles to B. vulgaris in Denmark prompted a search for

modes of inheritance of adaptation to this plant in other

geographic regions. During this search one population in

Delemont (Switzerland) seemed at first to differ from the

Danish populations, as X-linkage of a dominant R-allele

seemed to predominate. The present investigation reports

on detailed genetic investigations on this Swiss population

and demonstrates that the R-gene from Delemont is not

X-linked. The apparent X-linkage was caused by aberrant

segregation of the R-gene in offspring from males from

Delemont.

The sex determination system in P. nemorum is based

on a normal XY system. Males are heterogametic and

contain 15 pairs of autosomes and the Xyp arrangement,

where a large X and a small Y chromosome form a

parachute-like structure during meiotic metaphase I (Pet-

itpierre, 1988; Segarra & Petitpierre, 1990). The Xyp

arrangement is common in Chrysomelidae and assumed

to be the ancestral state in the family as a whole as well

as in P. nemorum (Petitpierre, 1988). Karyological mech-

anisms which can explain non-random segregation of

autosomes or autosomal genes during spermiogenesis

have not yet been described in P. nemorum, but fusions

between sex chromosomes and autosomes have been

found in other members of Chrysomelidae (Petitpierre,

1988; Petitpierre et al., 1988; Virkki, 1988).

The present report describes the presence of non-ran-

domly segregating flea beetle males (NRS-males) from

Delemont and explains how an R-gene can be inherited as

an X-linked gene in offspring from NRS-males, whereas

the same gene is inherited as an autosomal gene in off-

spring from males from a laboratory line that originates

from Taastrup (Denmark).

Materials and methods

Insects

Sinapis arvensis leaves containing leaf-mining P. nemorum

third instars were collected in Delemont in 1995 and 1998.

302 Nielsen

Leaves with larvae were placed in 500-ml plastic containers

containing a moist peat-vermiculite mixture. Full grown

larvae pupated in the peat-vermiculite mixture and adult

beetles emerged ca. 2 weeks later.

The ST-line (i.e., the susceptible line from Taastrup)

was reared in the laboratory. This line does not contain R-

alleles, and the larvae do not survive on the G-type of B.

vulgaris ssp. arcuata. The ST-line was developed from a

population living on radish in Taastrup as described previ-

ously (Nielsen, 1999). Imagines of the ST-line were kept in

groups of 15–25 beetles at 24 ± 2 �C and a L18:D6

photoperiod in 500-ml plastic containers with a moist gyp-

sum-charcoal layer (Nielsen, 1997a). They were fed radish

cotyledons or leaves. Larvae were reared on radish. Pupa-

tion and adult emergence occurred as described above for

field-collected larvae.

Crosses

Mating pairs (families) were kept separately at 24 ± 2 �C

and a L18:D6 photoperiod in plastic 158-ml containers

with a moist gypsum-charcoal layer. They were fed radish

cotyledons or leaves. Eggs were laid in crevices in the gyp-

sum-charcoal. Larvae emerged from the eggs 5–6 days

later. Inexperienced neonate larvae were used in bioassays

(see below) before they were 24 h old.

Larval offspring from the crosses were reared on the G-

type of B. vulgaris ssp. arcuata in order to determine the

sex ratio of the survivors. Detached leaves were exposed to

neonate larvae that initiated leaf mines in them. Leaves

with larvae were transferred to larger plastic containers

(500 ml) with a moist peat–vermiculite mixture. New

leaves were added every 3–4 days. The larvae continued

feeding in the leaves; sometimes they left unsuitable leaves

and initiated a new leaf mine in a fresh leaf. Full-grown lar-

vae left the mines and pupated in the peat–vermiculite

mixture. The sex ratio was determined from the adult bee-

tles that emerged from these containers ca. 2 weeks later.

F1 and backcrosses

Males from the 1995 sample from Delemont were mated

with virgin females from the ST-line (F1). Four males from

Delemont proved to have R-genes, because their offspring

survived on the G-type of B. vulgaris ssp. arcuata (Figure 1,

Table 1). The transmission of R-genes solely to the female

offspring could be explained by X-linkage of the R-gene or

by non-random segregation of autosomes carrying the

R-gene. In an attempt to distinguish between these mecha-

nisms, a series of backcrosses was initiated. Each of the

four field-collected males became the founder of a line that

was produced by repeated backcrosses to the ST-line.

F1XrXr and ArAr XRYr or ARNr

ST ♀ ×

×

×

♂

♀ ♂X-linked: XRXr (50%) XrYr (50%)

Non-random, autosomal: ARAr (50%) ArNr (50%)

F1 ♀ ♂XrYr and ArArXRXr or ARAr

B1

♀ ♀ ♂ ♂X-linked: XrXr (25%) XRXr (25%) XrYr (25%) XRYr (25%)

Autosomal: ArAr (25%) ARAr (25%) ArAr (25%) ARAr (25%)

ST ♀ B1 ♂XrXr and ArAr XRYr or ARAr

B2

♀ ♀ ♂ ♂X-linked: XRXr (50%) XrYr (50%)

Autosomal: ArAr (25%) ARAr (25%) ArAr (25%) ARAr (25%)

DE

STFigure 1 Genetic investigations of four

resistant Phyllotreta nemorum males col-

lected in Delemont in 1995. In initial

crosses between these four males (DE) and

susceptible females (ST), resistance genes

(R-genes) were solely transferred to the

female offspring. This segregation pattern

can be explained by two mechanisms:

X-linkage and non-random segregation of

autosomes. Putative genotypes of parents

and offspring based on these two mecha-

nisms are illustrated [A: autosome or allo-

X-chromosome (see text); N: neo-Y, i.e.,

autosome fused with the Y-chromosome;

X: X-chromosome; Y: Y-chromosome],

and further backcross experiments (B1 and

B2) that can distinguish between them are

outlined.

Non-random segregation of autosomes in flea beetle males 303

Virgin F1-females from the lines were mated with ST-

males in order to produce the first backcross generation

(B1). Resistant B1-males (XRYr or ARAr) were subsequently

mated with ST-females in order to produce B2, and B2-

males were mated with ST-females to produce B3, etc.

(Figure 1, Table 2).

Search for NRS-males in the 1998 sample

The first step was an F1-cross similar to the one described

above for the 1995 sample. Results from F1 demonstrated

that there were four males without any R-alleles (Table 1).

These four males were mated with virgin females from the

four lines described above (Table 2) in an attempt to recon-

struct the ARNr genotype that was assumed to occur in

resistant males from Delemont (Figures 1 and 2; C-

crosses). Each male was initially mated with a female from

one line. When this female had started oviposition, the male

was mated with a new female from another line until it died.

Male 1 was mated with females from all four lines, whereas

male 4 was mated with females from lines 3 and 4 (Table 3).

Male offspring from the C-crosses that were able to sur-

vive on the G-type of B. vulgaris ssp. arcuata (ARNr) had

received the R-allele from their mothers and non-random

segregation would be demonstrated if it could be shown

that they passed the R-allele on to their daughters, but not

to their sons. These males were therefore mated with virgin

ST-females in the diagnostic D crosses (Figure 2, Table 3).

One male from the 1998 sample produced only female

offspring in F1 that was able to utilize the G-type of B. vul-

garis ssp. arcuata (Table 1). This male was therefore

another good candidate to be an NRS-male (ARNr). F1 off-

spring from this male (crossed with an ST-female) was

reared on the P-type of B. vulgaris ssp. arcuata on which

both males (ArNr) and females (ARAr) survived. F1 males

were subsequently crossed with females from the four

lines (Table 2) in the E-crosses (parallel to the C-crosses;

Figure 2). Male offspring from the E-crosses reared on the

G-type of B. vulgaris ssp. arcuata (ARNr) were mated with

ST-females in the diagnostic F-crosses (parallel to the

D-crosses; Figure 2).

Bioassays

A detached leaf of the G-type of B. vulgaris ssp. arcuata was

placed in a 25-ml plastic container together with a piece of

moist filter paper. Five flea beetle larvae were transferred

to each individual leaf by means of a fine brush. The leaves

were left for 3–4 days at 24 ± 2 �C and a L18:D6 photope-

riod. The surviving larvae in leaf mines after 3–4 days were

counted by means of a stereomicroscope. Surviving resis-

tant larvae were at that time close to their first moult, and

there was a pronounced difference between them and sus-

ceptible larvae that had died apparently without any size

increase. Survival rates were determined based on 50–100

larvae per family.

Survival until the 3rd or 4th day is a good indication for

the presence of R-alleles (Nielsen, 1997b; de Jong et al.,

2000). Genotypes of beetles could be determined by the

results of two generations of crosses to the ST-line (F1 and

B1) as outlined previously (Nielsen, 1997b; de Jong et al.,

2000). If the survival on the G-type of B. vulgaris ssp. arcu-

ata in F1 is close to 100%, the beetle is considered to be

homozygous (RR or the equivalent if R-genes are not

found on regular autosomes), whereas the beetle is consid-

ered to be heterozygous (Rr or the equivalent) if survival

rates are close to 50% on the G-type, but close to 100% on

alternative host plants such as radish and the P-type of

Table 1 Classification of Phyllotreta nemorum males collected in Delemont in 1995 and 1998 into genotypes based on the survival of larval

offspring (F1) on the G-type of Barbarea vulgaris ssp. arcuata and the sex ratio of the survivors. The field-collected males were mated with

females from a laboratory line (ST) that did not survive on the plant

No.

males

No. larval

offspring

tested

% survival of

larvae in bioassay

No. adult

offspring sexed

Offspring

sex ratio (% $)

Possible genotypes based on

two assumptions1

X-linkage

Non-random segregation

of autosomes in males2

1995 4 515 40.8 92 100 XRYr ARNr

6 514 0 0 – XrYr ArNr

1998 1 100 48.0 9 100 XRYr ARNr

2 163 46.6 20 0 – –

1 75 96.0 9 44.4 – –

4 365 2.5 0 – XrYr ArNr

1Both assumptions can explain the data in Table 1, but X-linkage can be ruled out based on further crossing experiments (Table 2).2N is a neo-Y chromosome with a fusion between the original Y chromosome and an autosome; A is the allo-X-chromosome, e.g., the

homologue to the autosome that is fused with the Y-chromosome (see text).

304 Nielsen

B. vulgaris ssp. arcuata. The R-allele is considered to be

absent if there is no survival on the G-type of B. vulgaris

ssp. arcuata. Skewed sex ratios in offspring from heterozy-

gous males indicate sex linkage of the R-alleles: Y-linkage

when only male offspring survive on the G-type and X-

linkage when only females survive (Nielsen, 1997b; de Jong

et al., 2000). The present results demonstrate that female-

biased sex ratios do not necessarily prove X-linkage of the

R-allele.

Results

The flea beetle population from Delemont turned out to

be polymorphic with respect to the ability to use the

G-type of B. vulgaris ssp. arcuata as a host plant. From the

sample collected in 1995, four males proved to contain

genes which conferred resistance to defences in B. vulgaris

ssp. arcuata, whereas six males did not contain such genes

(Table 1). Survival rates on the G-type among the F1 off-

spring from resistant males were close to 50% as expected

when males from Delemont are heterozygous for the

R-allele (Nielsen, 1997b; de Jong et al., 2000). Only females

from F1 survived on the G-type (Table 1). This outcome

would occur if the males from Delemont had an X-linked

major R-gene similar to those previously reported from a

Danish population in Ejby (Nielsen, 1997b), i.e., the geno-

type of males from Delemont would be XRYr and when

mated with susceptible ST females (XrXr), the female off-

spring would be XRXr and survive on the G-type of B. vul-

garis ssp. arcuata whereas the male offspring would be

XrYr and be unable to survive on this plant (Figure 1).

Both genotypes survived on radish and on the P-type of B.

vulgaris ssp. arcuata, and the sex ratio among survivors

was ca. 50% (data not shown).

The possible X-linkage of a major R-gene was investi-

gated in further backcross experiments using four isogenic

lines that originated from the four resistant males

(Figure 1, Table 2). Survival in the G-type among progeny

from backcross B1 (F1-females mated with ST-males) and

B2–B5 (e.g., B1 males mated with ST-females to produce

B2, etc.) was close to 50% (Table 2), as expected if one

major R-gene was present in the resistant parent. Sex ratios

in B1–B5 were close to 50%. This observation shows that

the R-gene from Delemont is not X-linked as suggested in

F1 (Table 1). If the R-gene had been X-linked, there would

have been 100% females in offspring from B2 that survived

on the G-type (Figure 1). However, the sex ratio among

offspring from B2 was not significantly different from 50%

of each sex in any of the lines (v2 test: P>0.05), and the

possibility of X-linkage is therefore clearly ruled out.

C E

D F

B ♀ ♂ARAr ArNr

♀ ♀ ♂ ♂ArAr ARAr ArNr ARNr

B ♀ 1 ♂ARAr ArNr

♀ ♀ ♂ ♂ArAr ARAr ArNr ARNr

ST ♀ ♂ArAr ARNr

ST ♀ ♂ArAr ARNr

♀ ♂ARAr ArNr

♀ ♂ARAr ArNr

×

× ×

×DE

C E

F

Figure 2 Genetic investigations used in a search for non-randomly segregating males (NRS-males) among four susceptible (C, D) and one

resistant (E, F) male Phyllotreta nemorum collected in Delemont in 1998. Susceptible males from Delemont (DE) were mated with females

that carried the R-gene from Delemont (B-females) in order to reconstruct the ARNr genotype (C-crosses). One resistant male from Dele-

mont (ARNr) was initially mated with an ST female (data not shown), and F1 males (ArNr) were mated with ST females in order to recon-

struct the ARNr genotype (E-crosses). Diagnostic crosses (D and F) could distinguish between random (equal sex ratios) and non-random

(female-biased sex ratios) segregation of the R-allele in sons (D) and grandsons (F) of males collected in Delemont.


An alternative hypothesis that can explain the skewed

sex-ratios in F1 and equal sex ratios in B2 is therefore

needed. These results could occur if segregation in a pair of

autosomes during meiosis in males from Delemont is

non-random, i.e., that one homologue (obtained from the

mother) invariably segregate with the X-chromosome,

whereas the other homologue (obtained from the father)

segregates with the Y-chromosome. One possibility is that

in these NRS-males there is a fusion between an autosome

and the Y-chromosome (neo-Y), whereas there is no

fusion between the homologous autosome (allo-X) and

the X-chromosome. If the R-gene is located on the allo-

X-chromosome it will be transmitted together with the

X-chromosome to daughters of NRS-males, whereas the

neo-Y-chromosome with the r-allele will be transmitted to

the sons.

According to this hypothesis, the genotype of resistant

NRS-males can be described as ARNr, as they contain the

R-allele on the allo-X chromosome and the r-allele on the

neo-Y chromosome (Figure 1). When these males are

mated with females from the laboratory line (ArAr, because

they carry the r-allele on an autosome that is equivalent to

the allo-X), there would be two genotypes in the offspring

in equal proportions: ARAr (females that survive on the

G-type) and ArNr (males that do not survive on the

G-type) (Figure 1). This is in agreement with results from

F1 (Table 1).

In order to investigate this hypothesis, a new sample of

larvae was collected on S. arvensis in Delemont in 1998.

This second sample proved to differ somewhat from the

first sample, as only one male seemed to contain the allo-

X-linked gene (50% survival and 100% females among off-

spring reared on the G-type) (Table 1). Two males seemed

to contain a Y-linked gene (50% survival and 100% males

among offspring reared on the G-type), and one male was

apparently homozygous. Four susceptible males from the

1998 sample did not contain R-alleles (Table 1). The males

that were apparently homozygous or contained Y-linked

genes were not investigated further, whereas the search

for NRS-males was restricted to the susceptible males

(assumed to be ArNr) and to the single male with an appar-

ently allo-X-linked gene (assumed to be ARNr) (Table 1).

The search for NRS-males in the 1998 sample from Del-

emont started with the four susceptible males without any

R-alleles. This was done in a series of crosses (C and D)

among which the D-cross was the diagnostic one which

would enable a distinction between NRS-males and males

with a Mendelian segregation of the autosomes (Figure 2).

The C-crosses were made in order to reconstruct the

putative ARNr genotype that was assumed to be present in

resistant males from Delemont and that would explain

non-random segregation of the R-allele. In the C-crosses,

susceptible males (ArNr) were mated with heterozygous

females from the four isogenic lines (ARAr) developed pre-

viously by repeated backcrossings to the ST line (Table 2).

The C-crosses were assumed to produce four genotypes in

equal proportions: ARAr (females that survive on the G-

type), ArAr (females that do not survive on the G-type),

Table 2 Backcross experiments used for classification of Phyllotreta nemorum males collected in Delemont in 1995. Backcross 1 (B1) was

made between F1-females and ST-males. The diagnostic B2 cross was made between B1-males and ST-females. Sex ratios among survivors

in B2 should be 100% females if the R-gene is X-linked and 50% if the gene is autosomal

Line no.1 No. families

No. offspring

tested in bioassays % survival

No. adult

offspring

Offspring sex

ratio (% $)

Backcross 1 (B1) 1 5 493 41.4 280 51.1

2 2 218 31.7 57 36.8

3 6 661 45.5 232 51.7

4 6 665 39.9 262 53.1

Backcross 2 (B2) 1 5 557 39.9 138 53.6

2 6 509 36.0 98 58.2

3 8 655 41.7 208 56.7

4 5 452 50.2 180 55.0

Backcross 3 (B3) 1 2 53 47.2 75 46.7

2 3 145 44.8 121 52.9

3 2 182 44.0 86 44.2

4 3 247 42.9 139 56.8

Backcross 4–5 (B4–B5) 1 3 291 35.4 137 44.5

2 5 483 43.5 278 45.7

3 – – – – –

4 4 280 42.5 150 55.3

1Each line was founded by one resistant male from the 1995 sample from Delemont.

306 Nielsen

ARNr (males that survive on the G-type), and ArNr (males

that do not survive on the G-type) (Figure 2). The

observed survival rates among offspring from the C crosses

on the G-type were in agreement with these expectations,

i.e., 50% survival and 50% females (Table 3).

For the diagnostic D-crosses, resistant males from the

C-crosses (ARNr; selected by rearing the offspring on the

G-type) were mated with susceptible ST-females (ArAr)

(Figure 2). In the D-cross, survival rates among offspring

reared on the G-type were once more close to 50%, and

almost all the survivors were females (Table 3). These

results support the hypothesis that the four susceptible

males from the 1998 sample from Delemont are NRS-

males. All the males used in the D-crosses had received the

R-allele from their mothers, and in agreement with the

assumptions on non-random segregation, this R-allele was

transmitted to daughters. R-alleles from all four lines were

transmitted to daughters, i.e., there is no evidence for the

presence of R-alleles at different loci on different chromo-

somes in the flea beetle population from Delemont.

Further support for non-random segregation was

obtained based on results from one resistant male from the

1998 sample that produced solely female offspring that

survived on the G-type (Table 1). When offspring from

this male (F1) was reared on the P-type of B. vulgaris ssp.

arcuata, males (ArNr) as well as females (ARAr) survived

(data not shown). These F1 males (ArNr) were subse-

quently crossed with females from the four isogenic lines

(ARAr) in order to produce the putative ARNr genotype

(E-cross) (Figure 2, Table 4). Male offspring from the

E-crosses that survived on the G-type (ARNr) were selected

and used in diagnostic crosses with ST-females (F-cross,

Table 4). The F-crosses confirmed earlier findings from

the D-crosses (Table 3) that the male parent contained an

R-gene which they had inherited from their mother and

passed on to their daughters, i.e., the sex ratios among sur-

vivors were almost 100% females (Table 4). Comparisons

of results from the D- and F-crosses (Tables 3 and 4) with

those of B2 (Table 2) unequivocally demonstrate two seg-

regation patterns of an R-gene in P. nemorum males, i.e.,

non-random in males originating from Delemont and ran-

dom (Mendelian) in males from Taastrup. The results do

not prove that the non-random segregation is caused by

chromosome fusions, as the observed segregation patterns

could be explained by other mechanisms (see below).

Discussion

The present study demonstrates a polymorphism in segre-

gation patterns of a gene in males of the flea beetle, P.

nemorum. The gene originates from a population from

Delemont (Switzerland) and the R-allele confers the ability

of P. nemorum larvae (ARAr and ARNr) to develop on the

G-type of B. vulgaris ssp. arcuata, whereas conspecifics

without the allele (ArAr and ArNr) cannot live on this

plant. The R-allele has different modes of inheritance

depending on the type of male that it occurs in. In males

originating from Delemont (NRS-males), the R-allele is

passed on solely to the female offspring, whereas in males

originating from Taastrup (Denmark), it is passed on to

the male and female offspring in equal proportions. The

segregation pattern observed in crosses using males from

Table 3 Crossing experiments used for demonstration of non-random segregation of an autosomal R-gene in offspring from four suscepti-

ble Phyllotreta nemorum males collected in Delemont in 1998. The C-cross was made in order to produce the ARNr genotype. Skewed sex

ratios in the D-cross indicate non-random segregation of the R-allele in sons of field-collected males

Male no. Line no.

C-cross1 D-cross (C # · ST $)2

% survival3 Sex ratio4 (% $) % survival3 Sex ratio4 (% $)

1 1 46.0 (100) 40.0 (35) 52.3 (44) 100 (19)

1 2 42.3 (132) 50.0 (22) 35.1 (154) 100 (44)

1 3 45.6 (90) 30.0 (10) 48.7 (199) 100 (27)

1 4 41.8 (79) 45.0 (40) 48.9 (135) 100 (39)

2 2 45.6 (90) 43.2 (37) 48.3 (240) 100 (81)

2 4 40.0 (120) 40.6 (32) 43.3 (201) 100 (6)

3 2 43.8 (105) 53.1 (32) 45.7 (210) 98.8 (84)

4 3 39.4 (127) 34.3 (35) 42.0 (100) 100 (11)

4 4 52.5 (80) 48.8 (41) 39.9 (293) 100 (5)

1The C-cross was produced between four susceptible males from Delemont (ArNr) and females (ARAr) from lines containing the R-gene

from Delemont (Figure 2, Table 2).2ARNr males were selected from the C-cross by rearing the offspring on the G-type of Barbarea vulgaris ssp. arcuata.3Numbers of larvae tested shown in parentheses.4Numbers of adult beetles sexed shown in parentheses.


Taastrup is evidence for simple Mendelian inheritance of

an autosomal gene. These results rule out that the R-gene

could be X-linked, although X-linkage could also explain

the skewed sex ratios observed in offspring from NRS-

males. The skewed sex ratios observed in offspring from

NRS-males must then be explained as non-random segre-

gation of an autosome that carries the R-gene in NRS-

males. It could be shown that NRS-males contain a factor

(NRS-factor) that is inherited patrilineally (from fathers to

sons). Resistant males which contain the NRS-factor

(ARNr) transmit the R-gene to their female offspring (Fig-

ure 2D and F, Tables 3 and 4), whereas resistant males

without the NRS-factor transmit the R-gene to female and

male offspring in equal proportions (Figure 1, Table 2,

B2–B5). Evidence for the presence of the NRS-factor was

found in nine males (100%) from Delemont that were

investigated in sufficient detail (Tables 1, 3 and 4). The

NRS-factor can rely on several mechanisms, e.g., (1) fusion

of the Y-chromosome to one (or more) autosomes, (2)

associations between groups of chromosomes during sper-

miogenesis without physical connections between them,

and (3) genomic imprinting.

Fusions between sex chromosomes and autosomes to

form so-called neo-X and neo-Y chromosomes have been

reported from several insect taxa, such as Coleoptera

(Smith & Virkki, 1978; Petitpierre et al., 1988; Virkki,

1988; Macaisne et al., 2006), Diptera (Berlocher, 1984;

McPheron & Berlocher, 1985; Bachtrog, 2006; Flores

et al., 2008; McAllister et al., 2008), Lepidoptera (Nilsson

et al., 1988; Raijmann et al., 1997; Yoshido et al., 2011),

Orthoptera (John & Hewitt, 1970; Barton, 1980; Tatsuta

et al., 2006; Castillo et al., 2010), and Heteroptera (Bressa

et al., 2009), and seems to be a common event in the evo-

lution of sex chromosomes (Charlesworth & Charles-

worth, 2005; Kaiser & Bachtrog, 2010). Fusions can

involve the Y-chromosome and an autosome (Bachtrog,

2006), the X-chromosome and an autosome (John & He-

witt, 1970; Tatsuta et al., 2006; McAllister et al., 2008), or

both (Macaisne et al., 2006; Castillo et al., 2010). In

Lepidoptera where females are heterogametic, fusions

have been reported between the W-chromosome and an

autosome (Nilsson et al., 1988) as well as between both

sex chromosomes and an autosome (Yoshido et al.,

2011). The segregation pattern observed in NRS-males of

P. nemorum can be explained by a fusion between the Y-

chromosome and an autosome (neo-Y) whereas there is

no fusion between the X-chromosome and the homo-

logue to the autosome (allo-X) that is fused with the Y-

chromosome. Fusions between sex chromosomes and

autosomes have not yet been demonstrated in P. nemo-

rum, but they are known from several related species of

Alticinae (Petitpierre et al., 1988; Virkki, 1988; Segarra &

Petitpierre, 1990). The karyotype of P. nemorum has been

reported to be 15 + Xyp (Segarra & Petitpierre, 1990).

The Xyp with a small Y-chromosome and a larger X-chro-

mosome forming a parachute-like structure during mei-

otic metaphase I is common in Chrysomelidae and is

supposed to be the ancestral sex chromosome arrange-

ment in P. nemorum. This ancestral state may have been

maintained in males from Taastrup, but not in males

from Delemont.

The segregation patterns observed in offspring from

NRS-males could also be explained by an association

between sex chromosomes and autosomes that does not

involve physical connections between them. This type of

association has been described in a mole cricket (Ortho-

ptera) (Kubai & Wise, 1981), but not yet in Chrysomeli-

dae. It is therefore considered to be a less likely mechanism

for the non-random segregation that was observed in

P. nemorum.

A third mechanism that can explain non-random segre-

gation is genomic imprinting. Genomic imprinting is a

mechanism that places different parent-of-origin specific

marks (imprints) on parts of the genome, and these marks

determine the subsequent fate of the imprinted parts

(Goday & Esteban, 2001; Khosla et al., 2006). Genomic

Table 4 Crossing experiments used for

demonstration of non-random segregation

of an autosomal R-gene in offspring from a

resistant Phyllotreta nemorum male col-

lected in Delemont in 1998. The E-cross

was made in order to produce the ARNr

genotype. Skewed sex ratios in the F-cross

indicate non-random segregation of the R-

allele in grandsons of the field-collected

male

Line no.

E-cross1 F-cross (E # · ST $)2

% survival3 Sex ratio4 (% $) % survival3 Sex ratio4 (% $)

1 35.8 (187) 66.6 (12) – 100 (22)

2 43.5 (115) 53.3 (15) 43.4 (175) 100 (86)

3 48.0 (200) 66.7 (45) 44.4 (117) 99.1 (105)

4 46.5 (245) 37.2 (68) 39.4 (246) 100 (80)

1The E-cross was produced between F1 males (ArNr; Figure 2) and females (ARAr) from

four lines containing the R-allele from Delemont as described in Table 2.2ARNr males were selected from the E-cross by rearing the offspring on the G-type of

Barbarea vulgaris ssp. arcuata.3Numbers of larvae tested shown in parentheses.4Numbers of adult beetles sexed shown in parentheses.

308 Nielsen

imprinting can act on individual genes and on whole chro-

mosomes and chromosome sets. In sciarid flies (Diptera)

and in mealybugs (Heteroptera: Coccoidea) genomic

imprinting is associated with elimination of whole chro-

mosomes of paternal origin during spermiogenesis (Goday

& Esteban, 2001; Khosla et al., 2006). Genomic imprinting

followed by chromosome elimination cannot explain non-

random segregation in P. nemorum, as offspring from

NRS-males do receive genes of paternal as well as maternal

origin, but only the maternally derived allele (R) confers

the ability to live on the G-type, whereas the paternally

derived allele (r) does not. However, it cannot be ruled out

that other types of genomic imprinting could act on genes

or chromosomes and lead to parent-of-origin specific gene

expression that could explain non-random segregation in

P. nemorum males.

Whereas there is little information on the role of geno-

mic imprinting in Coleoptera, it is well known that chro-

mosome fusions between autosomes as well as between

autosomes and sex chromosomes are common (Smith &

Virkki, 1978; Petitpierre, 1988; Petitpierre et al., 1988;

Virkki, 1988). A clear distinction between these mecha-

nisms will require careful cytological investigations and ⁄ or

methods to study the expression of relevant genes in P.

nemorum. These types of investigations have not yet been

carried out.

The mechanism which maintains non-random segrega-

tion in NRS-males is very stable when judged from the

low proportion of males (<1%) that occurred in the diag-

nostic crosses, D and F (Tables 3 and 4). If the hypothesis

assuming a fusion between the Y-chromosome and an

autosome is correct, the presence of males would provide

evidence of crossing over between the allo-X and the

neo-Y chromosome in NRS-males. If such crossing over

occurred, the R-allele would become Y-linked. Unfortu-

nately, the few males that appeared in the diagnostic

crosses were not investigated further, and it is not known

whether their presence in low numbers indicates cross-

ing-over events, variability in levels of defences in plants,

errors due to contamination, or other factors. There is

weak evidence for the presence of Y-linked genes in males

from the 1998 sample from Delemont. However, the

numbers of offspring were low and the presence of

Y-linked genes in Delemont needs confirmation. In sum-

mary, it can be concluded that exchange of the R-allele

between paternal (e.g., neo-Y) and maternal (e.g., allo-X)

parts of the genome is very low in NRS-males. Low

recombination rates are often found among sex chromo-

somes in the heterogametic sex (Charlesworth & Charles-

worth, 2005), and the present results suggest that

recombinations are also rare on the chromosome part

where the R-gene is located.

Non-random segregation of R-genes on autosomes has

not been documented from any Danish flea beetle popula-

tion. Sex-linked genes (X and Y) were reported from a

population that lived on the G-type of B. vulgaris ssp. arcu-

ata in Ejby (Nielsen, 1997b) and X-linked genes have been

reported in low frequencies in populations living on other

host plants (de Jong & Nielsen, 1999). However, the previ-

ous reports did not continue the backcrossings long

enough to make the distinction between true X-linkage

and the non-random segregation of autosomes that is

documented here. It is therefore uncertain whether true

X-linkage and ⁄ or non-random segregation of autosomes

exists in Danish flea beetle populations. More recent

attempts to find the X-linked gene in Ejby have failed (JK

Nielsen & PW de Jong, unpubl.). This fact could be

explained in two ways: (1) the gene has disappeared from

the locality, e.g., during a bottleneck in 1996–1997 where

populations of B. vulgaris and flea beetles in Ejby were

extremely low (JK Nielsen, unpubl.) or (2) X-linked genes

have never been present, but previous results can be

explained by non-random segregation of an autosomal

gene in offspring from some males. These questions will be

dealt with in a separate paper.

The presence of NRS-males in a Swiss flea beetle popula-

tion also merits attempts to understand how this arrange-

ment is maintained and influenced by natural selection. It

has been suggested that fusions between sex-chromosomes

and autosomes could be favoured by selection if there is

heterosis in small inbreeding populations (Charlesworth &

Wall, 1999). Heterosis effects have been found in P. nemo-

rum, as both homozygous genotypes (RR and rr) had lower

fitness than heterozygotes under certain circumstances (de

Jong & Nielsen, 2000, 2002). However, there is no informa-

tion yet on how these processes influence the abundance of

R-genes in natural flea beetle populations.

The study by Charlesworth & Wall (1999) did not con-

sider consequences of the presence of genes controlling

ecologically important traits on neo-X or neo-Y chromo-

somes. The present study seems to be the first report where

a gene controlling an ecologically important trait might be

located on an autosome that is involved in fusions with a

sex chromosome. The R-gene confers resistance to

defences in the G-type of B. vulgaris ssp. arcuata and a few

related taxa, i.e., B. vulgaris ssp. vulgaris, B. intermedia, and

B. verna (Agerbirk et al., 2003), but not to defences in

other crucifers (Nielsen, 1999). R-genes are found in high

frequencies in a few populations living on the G-type of

B. vulgaris ssp. arcuata (Nielsen & de Jong, 2005). How-

ever, most Barbarea patches are not utilized by flea beetles

although R-alleles are found at low frequencies in neigh-

bouring populations living on other host plants. Coloniza-

tion of novel Barbarea patches by immigrants from


neighbouring populations living on alternative host

plants is therefore likely to have immediate evolutionary

consequences, as frequencies of R-genes will increase

(Nielsen & de Jong, 2005). In this process, the mode of

inheritance of the R-gene might influence the success rates

of the colonization. The presence of NRS-males would lead

to female-biased sex ratios among genotypes that are able

to colonize Barbarea spp., whereas equal sex ratios would

occur if the R-allele is autosomal. If population growth is

more depending on female numbers than on male num-

bers or total population size (Rankin & Kokko, 2007), the

increased relative abundance of females might increase

population growth in a population that is in the process of

colonization of a novel Barbarea patch. The population

from Delemont was collected on S. arvensis, and B. vulgaris

was growing in the neighbourhood. However, there are no

data that can illustrate whether the presence of Barbarea

has influenced the structure of populations living on

S. arvensis or whether the presence of NRS-males has influ-

enced the colonization of nearby Barbarea patches. The

limited data available suggest that most beetles in Dele-

mont are NRS-males, and that the R-allele is found in ca.

50% of the males. Unfortunately, there are no data on fre-

quencies of R-alleles in females from Delemont, and it is

not possible to confirm the assumption that the NRS-fac-

tor increases the relative abundance of females with

R-alleles in a natural population.

The R-gene from Delemont has similar effects on larval

survival in bioassays as the Y-linked gene and several auto-

somal genes found in Denmark (Nielsen, 1997b; de Jong

et al., 2000). Furthermore, these genes have similar effects

on behavioural responses of adult beetles to saponins

(Nielsen et al., 2010). These observations suggest that all

known R-genes in P. nemorum are adaptations to defensive

saponins in Barbarea, but further studies are needed to

unravel whether they are identical by descent, belong to

the same gene family, or have more independent origins.

In offspring from NRS-males, the autosomal R-gene

segregates as an X-linked gene. Both autosomal and sex-

linked genes have previously been found to control traits

that influence host plant use in insects (Thompson, 1988b;

Thompson et al., 1990; Scriber, 1994; Sperling, 1994;

Jones, 2005; Nygren et al., 2006), and it is not well under-

stood how location of genes on different chromosomes

influences the evolutionary processes that lead to differen-

tiation of feeding habits and novel host plant use in phy-

tophagous insects (Thompson, 2005; Matsubayashi et al.,

2010). The interaction between Barbarea and P. nemorum

offers excellent opportunities to investigate these topics

and perform experimental studies on natural selection on

genes with similar effects, but different modes of inheri-

tance, i.e., (1) Y-linked, (2) autosomal, and (3) autosomal

in combination with a factor in non-randomly segregating

males that lead to apparent X-linkage.

Acknowledgements

The work was supported by the Danish Agency for Science

and Technology.

References

Agerbirk N, Ørgaard M & Nielsen JK (2003) Glucosinolates, flea

beetle resistance and leaf pubescence as taxonomic characters

in the genus Barbarea (Brassicaceae). Phytochemistry 63: 69–

80.

Bachtrog D (2006) Expression profile of a degenerating neo-Y

chromosome in Drosophila. Current Biology 16: 1694–1699.

Barton NH (1980) The fitness of hybrids between two chromo-

somal races of the grasshopper, Podisma pedestris. Heredity 45:

47–59.

Berlocher SH (1984) Segregation and linkage of allozymes of the

walnut husk fly. Journal of Heredity 75: 392–396.

Bressa MJ, Papeschi AG, Vitkova M, Kubickova S, Fukova I et al.

(2009) Sex chromosome evolution in cotton stainers of the

genus Dysdercus (Heteroptera: Pyrrhocoridae). Cytogenetic

and Genome Research 125: 292–305.

Castillo ERD, Bidau CJ & Marti DA (2010) Neo-sex chromosome

diversity in Neotropical melanopline grasshoppers (Melano-

plinae, Acrididae). Genetica 138: 775–786.

Charlesworth D & Charlesworth B (2005) Sex chromosomes:

evolution of the weird and wonderful. Current Biology 15:

R129–R131.

Charlesworth B & Wall JD (1999) Inbreeding, heterozygote

advantage and the evolution of neo-X and neo-Y sex chromo-

somes. Proceedings of the Royal Society of London B 266:

51–56.

Flores SV, Evans AL & McAllister BF (2008) Independent ori-

gins of new sex-linked chromosomes in the melanica and

robusta species groups of Drosophila. BMC Evolutionary

Biology 8: 33.

Goday C & Esteban MR (2001) Chromosome elimination in sci-

arid flies. BioEssays 23: 242–250.

Hartmann T (1996) Diversity and variability of plant secondary

metabolism: a mechanistic view. Entomologia Experimentalis

et Applicata 80: 177–188.

Hawthorne DJ & Via S (2001) Genetic linkage of ecological spe-

cialization and reproductive isolation in pea aphids. Nature

412: 904–907.

John B & Hewitt GM (1970) Inter-population sex chromosome

polymorphism in the grasshopper Podisma pedestris. Chromo-

soma 31: 291–308.

Jones CD (2005) The genetics of adaptation in Drosophila sechel-

lia. Genetica 123: 137–145.

de Jong PW & Nielsen JK (1999) Polymorphism in a flea beetle

for the ability to use an atypical host plant. Proceedings of the

Royal Society of London B 266: 103–111.

310 Nielsen

de Jong PW & Nielsen JK (2000) Reduction in fitness of flea

beetles which are homozygous for an autosomal gene confer-

ring resistance to defences in Barbarea vulgaris. Heredity 84:

20–28.

de Jong PW & Nielsen JK (2002) Host plant use of Phyllotreta

nemorum: do coadapted gene complexes play a role? Entomo-

logia Experimentalis et Applicata 104: 207–215.

de Jong PW, Frandsen HO, Rasmussen L & Nielsen JK (2000)

Genetics of resistance against defences of the plant Barbarea

vulgaris in a Danish flea beetle population. Proceedings of the

Royal Society of London B 267: 1663–1670.

Kaiser VB & Bachtrog D (2010) Evolution of sex chromosomes in

insects. Annual Review of Genetics 44: 91–112.

Khosla S, Mendiratta G & Brahmachari V (2006) Genomic

imprinting in the mealybugs. Cytogenetic and Genome

Research 113: 41–52.

Kubai DF & Wise D (1981) Nonrandom chromosome segrega-

tion in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural

study. Journal of Cell Biology 88: 281–293.

Kuzina V, Ekstrom CT, Andersen SB, Nielsen JK, Olsen CE &

Bak S (2009) Identification of defense compounds in Bar-

barea vulgaris against the herbivore Phyllotreta nemorum by

an ecometabolomic approach. Plant Physiology 151: 1977–

1990.

Kuzina V, Nielsen JK, Augustin JM, Torp AM, Bak S & Andersen

SB (2011) Barbarea vulgaris linkage map and quantitative trait

loci for saponins, glucosinolates, hairiness and resistance to the

herbivore Phyllotreta nemorum. Phytochemistry 72: 188–198.

Macaisne N, Dutrillaux AM & Dutrillaux B (2006) Meiotic behav-

iour of a new complex X-Y-autosome translocation and ampli-

fied heterochromatin in Jumnos ruckeri (Saunders) (Coleoptera,

Scarabaeidae, Cetoniinae). Chromosome Research 14: 909–918.

Matsubayashi KW, Ohshima I & Nosil P (2010) Ecological speci-

ation in phytophagous insects. Entomologia Experimentalis et

Applicata 134: 1–27.

McAllister BF, Sheeley SL, Mena PA, Evans AL & Schlotterer C

(2008) Clinal distribution of a chromosomal rearrangement: a

precursor to chromosomal speciation? Evolution 62: 1852–

1865.

McPheron BA & Berlocher SH (1985) Segregation and linkage of

allozymes of Rhagoletis tabellaria. Journal of Heredity 76: 218–

219.

Nielsen JK (1996) Intraspecific variation in adult flea beetle

behaviour and larval performance on an atypical host plant.

Entomologia Experimentalis et Applicata 80: 160–162.

Nielsen JK (1997a) Variation in defences of the plant Barbarea

vulgaris and in counteradaptations by the flea beetle Phyllotreta

nemorum. Entomologia Experimentalis et Applicata 82: 25–35.

Nielsen JK (1997b) Genetics of the ability of Phyllotreta nemorum

larvae to survive in an atypical host plant, Barbarea vulgaris

ssp. arcuata. Entomologia Experimentalis et Applicata 82:

37–44.

Nielsen JK (1999) Specificity of a Y-linked gene in the flea beetle

Phyllotreta nemorum for defences in Barbarea vulgaris. Entom-

ologia Experimentalis et Applicata 91: 359–368.

Nielsen JK & de Jong PW (2005) Temporal and host-related vari-

ation in frequencies of genes that enable Phyllotreta nemorum

to utilize a novel host plant, Barbarea vulgaris. Entomologia

Experimentalis et Applicata 115: 265–270.

Nielsen JK, Nagao T, Okabe H & Shinoda T (2010) Resistance in

the plant, Barbarea vulgaris and counter-adaptations in flea

beetles mediated by saponins. Journal of Chemical Ecology 36:

277–285.

Nilsson N-O, Lofstedt C & Davring L (1988) Unusual sex chromo-

some inheritance in six species of small ermine moths (Ypono-

meuta, Yponomeutidae, Lepidoptera). Hereditas 108: 259–265.

Nygren GH, Nylin S & Stefanescu C (2006) Genetics of host use

and life history in the comma butterfly across Europe: varying

modes of inheritance as a potential reproductive barrier. Jour-

nal of Evolutionary Biology 19: 1882–1893.

Petitpierre E (1988) Cytogenetics, cytotaxonomy and genetics of

Chrysomelidae. Biology of Chrysomelidae (ed. by P Jolivet,

E Petitpierre & TH Hsiao), pp. 131–159. Kluwer Academic

Publishers, Dordrecht, The Netherlands.

Petitpierre E, Segarra C, Yadav JS & Virkki N (1988) Chromo-

some numbers and meioformulae of Chrysomelidae. Biology

of Chrysomelidae (ed. by P Jolivet, E Petitpierre & TH Hsiao),

pp. 161–186. Kluwer Academic Publishers, Dordrecht, The

Netherlands.

Raijmann LEL, van Ginkel WE, Heckel DG & Menken SBJ (1997)

Inheritance and linkage of isozymes in Yponomeuta padellus

(Lepidoptera, Yponomeutidae). Heredity 78: 645–654.

Rankin DJ & Kokko H (2007) Do males matter? The role of males

in population dynamics. Oikos 116: 335–348.

Schoonhoven LM, van Loon JJA & Dicke M (2005) Insect-Plant

Biology. Oxford University Press, Oxford, UK.

Scriber JM (1994) Climatic legacies and sex chromosomes: latitu-

dinal patterns of voltinism, diapause, size, and host-plant selec-

tion in two species of swallowtail butterflies at their hybrid

zone. Insect Life-cycle Polymorphism (ed. by HV Danks),


Netherlands.

Segarra C & Petitpierre E (1990) Chromosomal survey in three

genera of Alticinae (Coleoptera, Chrysomelidae). Cytobios 64:

169–174.

Sezer M & Butlin RK (1998) The genetic basis of host plant adap-

tation in the brown planthopper (Nilaparvata lugens). Hered-

ity 80: 499–508.

Smith SG & Virkki N (1978) Coleoptera. Animal Cytogenetics

Vol. 3, Insecta 5 (ed. by B John). Gebruder Borntraeger, Berlin,

Germany.

Sperling FAH (1994) Sex-linked genes and species differences in

Lepidoptera. Canadian Entomologist 126: 807–818.

Tatsuta H, Hoshizaki S, Bugrov AG, Warchalowska-Sliwa E,

Tatsuki S & Akimoto S-I (2006) Origin of chromosomal

rearrangement: Phylogenetic relationship between X0 ⁄ XX

and XY ⁄ XX chromosomal races in the brachypterous

grasshopper Podisma sapporensis (Orthoptera: Acrididae).

Annals of the Entomological Society of America 99: 457–

462.


Thompson JN (1988a) Evolutionary ecology of the relationship

between oviposition preference and performance of offspring

in phytophagous insects. Entomologia Experimentalis et

Applicata 47: 3–14.

Thompson JN (1988b) Evolutionary genetics of oviposition pref-

erence in swallowtail butterflies. Evolution 42: 1223–1234.

Thompson JN (2005) The Geographic Mosaic of Coevolution.

University of Chicago Press, Chicago, IL, USA.

Thompson JN, Wehling W & Podolsky R (1990) Evolutionary

genetics of host use in swallowtail butterflies. Nature 344: 148–

150.

Virkki N (1988) Cytotaxonomy of Alticinae. Biology of Chryso-

melidae (ed. by P Jolivet, E Petitpierre & TH Hsiao),


Netherlands.

Xue H-J, Li W-Z & Yang X-K (2009) Genetic analysis of feeding

preference in two related species of Altica (Coleoptera: Chryso-

melidae: Alticinae). Ecological Entomology 34: 74–80.

Yoshido A, Sahara K, Marec F & Matsuda Y (2011) Step-by-step

evolution of neo-sex chromosomes in geographical popu-

lations of wild silkworms, Samia cynthia spp. Heredity 106:

614–624.

312 Nielsen

Non-random segregation of an autosomal gene in males of the flea beetle, Phyllotreta nemorum:...

Documents

Transcript of Non-random segregation of an autosomal gene in males of the flea beetle, Phyllotreta nemorum:...