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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: jeni@life.ku.dk
� 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.
Non-random segregation of autosomes in flea beetle males 305
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.
Non-random segregation of autosomes in flea beetle males 307
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
Non-random segregation of autosomes in flea beetle males 309
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.
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