New data on the cytology of parthenogenetic weevils (Coleoptera, Curculionidae).

Abstract

Parthenogenesis and, in particular, polyploidy are rare in animals. A number of cases, known among weevils, represent apomictic parthenogenesis--a reproductive mode in which eggs undergo one maturation division, the chromosomes divide equationally, and no reduction takes place. Among parthenogenetic weevils there are two diploids, 48 triploids, 18 tetraploids, six pentaploids, three hexaploids and one decaploid. Eight examined parthenogenetic species are triploids with 33 chromosomes of different morphology, confirming that triploidy is the most common level of ploidy in weevils. The karyotypes are heterogeneous with the presence of meta-, submeta-, subtelo- and acrocentric chromosomes. The C-banding method showed that only two species possess a large amount of heterochromatin visible as a band around the centromere during mitotic metaphase. This agrees with observations that weevils are characterized by a small amount of heterochromatin, undetectable in metaphase plates after C-banding. In three species an atypical course of apomictic oogenesis occurs with stages similar to meiosis, in which chromosomes form bivalents and multivalent clusters. This association of chromosomes probably represents the remnants of meiosis, although these events have nothing to do with recombination. The results support the hypothesis that the evolution of apomictic parthenogenesis in weevils has proceeded through a stage of automixis.

Full text

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New data on the cytology of parthenogenetic weevils (Coleoptera,
Curculionidae)
Dorota Lachowska Æ Æ Maria Ro_ zek Æ Æ Milada Holecova ´
Received: 19 January 2007/Accepted: 12 November 2007/Published online: 27 November 2007
? Springer Science+Business Media B.V. 2007
Abstract
are rare in animals. A number of cases, known among
weevils, represent apomictic parthenogenesis—a repro-
ductive mode in which eggs undergo one maturation
division, the chromosomes divide equationally, and no
reduction takes place. Among parthenogenetic weevils
there are two diploids, 48 triploids, 18 tetraploids, six
pentaploids, three hexaploids and one decaploid. Eight
examined parthenogenetic species are triploids with 33
chromosomes of different morphology, confirming that
triploidy is the most common level of ploidy in weevils. The
karyotypes are heterogeneous with the presence of meta-,
submeta-, subtelo- and acrocentric chromosomes. The
C-banding method showed that only two species possess a
large amount of heterochromatin visible as a band around
the centromere during mitotic metaphase. This agrees with
observations that weevils are characterized by a small
amount of heterochromatin, undetectable in metaphase
plates after C-banding. In three species an atypical course of
apomictic oogenesis occurs with stages similar to meiosis,
in which chromosomes form bivalents and multivalent
clusters. This association of chromosomes probably repre-
sents the remnants of meiosis, although these events have
nothing to do with recombination. The results support the
hypothesis that the evolution of apomictic parthenogenesis
in weevils has proceeded through a stage of automixis.
Parthenogenesis and, in particular, polyploidy
Keywords
Chromosome number ? C-bands ? Karyotype ?
Parthenogenesis
Coleoptera ? Curculionidae ?
Introduction
Sexual reproduction is the predominant genetic system in
eukaryotes, whereas parthenogenesis is an alternative
genetic system where all individuals are female and inher-
itance is typically clonal (Maynard Smith 1978). Organisms
that reproduce by parthenogenesis offer many potential
insights into cytology, genetics, ecology, and evolution, and
therefore have received a great deal of attention from
biologists. Among insects, the weevils, mainly from sub-
family Entiminae, are well known for having a large
number of parthenogenetic lineages (Suomalainen et al.
1987). Parthenogenetic weevils are apomictic (i.e. they lack
meiosis and recombination). In these broad-nosed curcu-
lionids, apomictic parthenogenesis occurs in connection
with polyploidy, confirmed cytologically in over 75 species
(Smith and Virkki 1978; Suomalainen et al. 1987). The
grade of polyploidy may be different in various species, or
even in various forms of one species, also the degree of
polyploidy increase progressively in the forms distributed
more distantly from the ancestral bisexual form (Korotyaev
1994). In weevils, parthenogenesis and polyploidy are
derived phenomena. All parthenogenetic weevils are
flightless because of the reduction or absence of the meta-
thoracic wings, thus flightlessness seems to be a pre-
condition for parthenogenesis (Takenouchi 1970; Saura
et al. 1993). Parthenogenetic races have a much broader
distribution and occasionally coexist with sexual ones
exhibiting geographical parthenogenesis (Suomalainen and
D. Lachowska (&) ? M. Ro_ zek
Institute of Systematics and Evolution of Animals Polish
Academy of Science, Sławkowska 17, Krako ´w 31-016, Poland
e-mail: lachowska@isez.pan.krakow.pl
M. Holecova ´
Department of Zoology, Comenius University, Mlynska ´ dolina
B-1, Bratislava 842-15, Slovakia
123
Genetica (2008) 134:235–242
DOI 10.1007/s10709-007-9230-x

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Saura 1973; Takenouchi 1976). The cytology of partheno-
genetic weevils was the subject of intense research by
Suomalainen (1940, 1947, 1969), Seiler (1947), Mikulska
(1949, 1950, 1953, 1960), Petryszak (1972), Takenouchi
(1969, 1976, 1978, 1983, 1986), Saura et al. (1993), Nor-
mark (1996a). The main goal of this paper was to undertake
an in depth cytological approach to the eight parthenoge-
netic curculionid weevils.
Materials and methods
The list of species from which chromosome preparations
were obtained, as well as localities, taxonomical position
and number of specimens are listed in Table 1. The spe-
cies are classified according to Alonzo-Zarazaga and Lyal
(1999), Wanat and Mokrzycki (2005). Female gonads
(number of specimens is given in Table 1) were dissected
under a stereomicroscope in several drops of hypotonic
0.9% sodium citrate solution containing 0.005% colchi-
cine. The ovaries were transferred into a small volume of
the same solution and incubated for 30–45 min at room
temperature, next they were fixed according to the method
described by Ro_ zek (1994) with minor modification
(Ro_ zek and Lachowska 2001). C-banding was performed
using the procedure described by Sumner (1972) with
some modifications (Lachowska et al. 2005). Evaluation
of chromosome morphology was based on ten mitotic
metaphases, and the chromosome classification system
follows Levan et al. (1964). Chromosome lengths were
calculated as a percentage of total chromosome length of
the haploid set (%TCL) (Table 2). The chromosomes
were analyzed and photographed with a Nikon Eclipse
400 light microscope and CCD DS-U1 camera (Nikon)
using the software Lucia Image version 5.0 (Laboratory
Imaging, Prague, Czech Republic).
Results
Cytological analyses were carried out on eight partheno-
genetic species from Poland and Slovakia. Five of these
have never been examined karyologically. One of the
species, Otiorhynchus raucus, is known to be partheno-
genetic, while in a population from Slovakia (Pezinska
Baba) males were also found. Preliminary cytological
observations suggested some disturbance in the sper-
matogenesis of these males. In Otiorhynchus raucus,
Polydrusus inustus and Strophosoma melanogrammum an
atypical course of apomictic oogenesis was observed with
stages similar to meiosis. In some oogonial cells, con-
centrations of 2, 3 or more chromosomes undergoing
spiralization were visible. Next, the chromosomes formed
ring and cross associations as in automictic diakinesis
(Figs. 1a–d). All eight species are triploids with 33
chromosomes (3n = 33), although some variation in
chromosome number in mitotic metaphases was visible.
Very often the plates contained 27–31 chromosomes. The
resultsofindividual chromosome
description of their morphology and percentage partici-
pation in karyotype length are given in Table 2. The C-
banding method revealed that only two species, Sciaphilus
asperatus and Strophosoma melanogrammum, possess a
large amount of heterochromatin visible as a band around
the centromere during mitotic metaphase (Figs 6, 10). The
rest of the species are characterized by a small amount of
heterochromatin, undetectable in metaphase plates after
C-banding, and only short heterochromatic bands were
visible in prophase (Fig. 2).
Chromosomesof
Otiorhynchus
metaphase plates, consist of 33 chromosomes (n = 11) of
different morphology. In the asymmetrical karyotype, the
first metacentric pair is the longest and constituted 23.13%
of the whole chromosome set, the remaining chromosomes
are shorter with no differences in length. Seven pairs are
measurements,a
raucus—oogonial
Table 1 Chromosomally examined species of weevils
Species Geographic source and date of collectionNumber of specimens
Tribe: Otiorhynchini Otiorhynchus raucus (Fabricius, 1776)SW Slovakia, Male ´ Karpaty Mts., Pezinska ´ Baba 10
Tribe: Polydrusini Polydrusus mollis (Stro ¨em, 1768)SW Slovakia, Zvolenska ´ kotlina, Jakub - Roha ´c ˇovo8
Tribe: Polydrusini Polydrusus inustus Germar, 1824S Poland, Wielickie Foothils, Wieliczka12
Tribe: Sciaphilini Sciaphilus asperatus (Bonsdorff, 1785)SW Slovakia, Male ´ Karpaty Mts., Pezinska ´ Baba7
Tribe: Sciaphilini Eusomus ovulum Germar, 1824SE Poland, Przemyskie Foothils, Przemys ´l16
Tribe: Sciaphilini Brachysomus echinatus (Bonsdorff, 1785)SW Slovakia, Male ´ Karpaty Mts., Naha ´c ˇ Katarı ´nka5
Tribe: Sciaphilini Parafoucartia squamulata (Herbst, 1795) SE Poland, Przemyskie Foothils, Przemys ´l13
Tribe: Brachyderini Strophosoma melanogrammum (Forster, 1771) SW Slovakia, Male ´ Karpaty Mts., Pezinska ´ Baba9
236Genetica (2008) 134:235–242
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metacentric, one pair is submetacentric, one subtelocentric,
and one acrocentric (Fig. 3, Table 2).
Polydrusus inustus—in most plates the chromosome
numberis33,andthekaryotypeisasymmetricalwiththefirst
metacentricpairthelongest(21.81%).The2ndpairissmaller
and metacentric (14.03%), the remaining pairs are clearly
shorter (9.67–3.14%). Among them the 5th and 11th pairs
have a subtelocentric morphology (Fig. 4, Table 2).
Polydrusus mollis—during oogonial division 33 chro-
mosomes of similar size are observed. The well marked
centromere position distinguishes the following morpho-
logical types: four pairs of metacentric chromosomes
(13.44–11.93%), three smaller metacentric pairs (8.31–
9.44%), and the shortest four pairs of meta-, and sumeta-
centric chromosomes (4.73–6.42%) (Fig. 5, Table 2).
Sciaphilus asperatus—the accurate number of chromo-
somes is 33, although in most plates the chromosome
number varied around 30. The karyotype is symmetric with
no differences in length, the longest first pair is metacentric
(16.79%), whereas the remaining pairs are slightly shorter
representing metacentric (five pairs), submetacentric (three
pairs), and subtelocentric (two pairs) morphology. The
heterochromatin is visible as dark bands in all chromo-
somes around centromeres, with the exception of pair
seven which possesses a larger amount (Fig. 6, Table 2).
Eusomus ovulum—the chromosome number is 33. The
karyotype is clearly asymmetric with different morphology
of chromosomes. The metacentric chromosomes of the first
pair are twice as long as the second and third pairs, and
they represent 22.63%. The 2nd–11th pairs are similar in
size but the localization of their centromeres varies. The
chromosomes of the 3rd, 5th, 8th, 9th, and 10th pairs are
metacentric, the 7th pair is submetacentric, the 2nd and
11th are subtelocentric, however, the 4th and 6th pairs
consist of acrocentric chromosomes (Fig. 7, Table 2).
Brachysomus echinatus—the number of chromosomes is
33, the karyotype is symmetric with the prevalence of
metacentric chromosomes (1st, 2nd, 4–9th pairs), only
Table 2 Relative length (%TCL) and centromeric index (AR) of particular chromosome pairs
Pair No.
Otiorhynchus raucus Polydrusus mollis Polydrusus inustusSciaphilus asperatus
Mitotic metaphase Mitotic metaphase Mitotic metaphase Mitotic metaphase
%TCLAR %TCL AR%TCLAR %TCLAR
123.13 1.19 13.441.24 21.811.2816.79 1.24
210.78– 12.44 1.2014.031.1713.321.04
39.91 1.5512.451.529.67 1.3110.511.74
48.89 1.2411.93 1.06 8.861.59 9.78 1.81
5 8.871.12 9.441.04 8.643.75 9.43 1.07
7.82 1.51 9.141.167.981.22 9.151.55
77.182.08 8.31 1.37 7.06 1.608.17 3.56
8 6.673.416.42 1.96 7.28 1.407.45 1.25
9 6.241.33 5.92 2.33 6.58 1.266.201.84
105.52 1.615.761.574.95 1.414.66 1.45
114.98 1.84 4.73 1.933.14 3.544.534.16
Pair No.
Eusomus ovulumBrachysomus echinatus Parafoucartia squamulataStrophosoma melanogrammum
Mitotic metaphaseMitotic metaphase Mitotic metaphaseMitotic metaphase
%TCLAR %TCLAR%TCL AR%TCLAR
122.63 1.01 14.171.48 12.862.4714.58 1.09
2 10.873.7812.60 1.2211.74 1.6214.181.57
3 9.721.0812.29– 11.571.2311.75 1.41
4 8.38– 10.001.17 11.351.03 11.67 1.54
5 8.541.45 9.581.20 10.851.0210.12 1.16
6 7.74– 8.231.12 10.651.11 9.641.20
77.261.937.081.058.831.158.311.49
86.52 1.05 6.981.107.601.068.24 1.83
96.36 1.296.87 1.076.551.265.24 1.43
106.281.126.25–4.47 4.524.702.37
115.683.535.94–3.52–2.12–
Genetica (2008) 134:235–242 237
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three pairs are acrocentric (3rd, 10–11th pairs) (Fig. 8,
Table 2).
Parafoucartia squamulata—the number of chromo-
somes in the oogonia is 33 forming a symmetric karyotype
with different localization of centromeres. In the first pair it
is situated submedially, in the 2nd–9th pairs it is medial,
whereas the 10th pair is subtelocentric, and the 11th is
acrocentric (Fig. 9, Table 2).
Strophosoma melanogrammum—the asymmetric karyo-
type consists of 33 chromosomes comprised of metacentric
pairs 1–7 and 9, submetacentric 8th and 10th pairs while
pair 11 is acrocentric (Fig. 10, Table 2). C-banding shows
heterochromatin localized around centromeres in all chro-
mosomes with the exception of the 3rd pair which is
entirely euchromatic. The size of the C-bands is almost the
same, only the short arm of chromosomes of the 8th pair is
heterochromatic (Fig. 10).
Discussion
Excluding the European Polydrusus mollis and Japanese
Scepticus insularis with 22 chromosomes, among parthe-
nogenetic weevils there are 48 triploids having 33 or 30
chromosomes, 18 tetraploids with 44 chromosomes, six
pentaploidswith55chromosomes,threehexaploidscarrying
66 chromosomes and one decaploid (Saura et al. 1993).
Eight examined species are parthenogenetic triploids,
showing that triploidy is the most common level of ploidy.
Soumalainen (1940) and Mikulska (1949) described
Fig. 1 Oogonial nuclei, the arrows show chromosomes undergoing to
spiralization and association forming rings, crosses and clusters: (a)
Otiorhynchus raucus, (b) Polydrusus inustus, (c-d) Strophosoma
melanogrammum
Fig. 2 Mitotic prometaphase in Polydrusus inustus. Small arrows
show short C-bands, the big arrow shows heteropycnotic parts of
three X chromosomes. The bar equals 10 lm
Fig. 3 Mitotic metaphase (3n) and haploid set of chromosomes in
Otiorhynchus raucus
238 Genetica (2008) 134:235–242
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parthenogenetic Polydrusus mollis
Finland and Poland, respectively. The same species from
Slovakia represents triploid races which probably evolved
from diploid ancestors (Lokki 1976a, present study). It is
assumed that triploid weevils may originate when the pro-
cessofoogenesisisdisturbedandadiploidgameteisformed.
The union of this diploid ovum with a haploid sperm pro-
duces a triploid embryo (Stenberget al. 2000). Transition of
ploidy is caused by chance fertilization of unreduced eggs.
An alternative hypothesis states that new degrees of poly-
poidy can arise through disjunction events that involve one
or several haploid chromosomes. Such events have been
observed in the mitosis that accompanies oogenesis in par-
thenogenetic weevils (Saura et al. 1993). However, some
molecularandmorphologicalresultsinAramigustessellates
support evidence that parthenogenetic lineages of weevils
are of hybrid origin (Normark and Lanteri 1998). The
polyploid forms are characterized by the basic number of 11
chromosomes. The exception is Listroderes costirostris and
Eusomus ovulum from Poland with 30 chromosomes, sug-
gesting a basic number of 10 (Mikulska 1953; Takenouchi
1969).Thereisdiscrepancybetweenthepreviousresultsand
this study because the number of chromosomes in Eusomus
ovulum does not match our data. The karyotype of Eusomus
as diploid races in
Fig. 4 Mitotic metaphase (3n) and haploid set of chromosomes in
Polydrusus inustus. Bar equals 10 lm
Fig. 5 Mitotic metaphase (3n) and haploid set of chromosomes in
Polydrusus mollis
Fig. 6 Mitotic metaphase (3n) and haploid set of chromosomes in
Sciaphilus asperatus
Genetica (2008) 134:235–242239
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ovulum from Slovakia consists of 33 chromosomes follow-
ing the rule of multiplication of 11 chromosomes. The
difference in chromosome number probably results from
difficulties in discerning each single chromosome in the
follicular cells. Another explanation is that the chromosome
numberinpolyploidparthenogeneticweevils,whetherinthe
oogonial and maturation metaphases of a specimen or a
species, is not always an exact multiple of the basic number
Fig. 7 Mitotic metaphase (3n) and haploid set of chromosomes in
Eusomus ovulum
Fig. 8 Mitotic metaphase (3n) and haploid set of chromosomes in
Brachysomus echinatus. Bar equals 10 lm
Fig. 9 Mitotic metaphase (3n) and haploid set of chromosomes in
Parafoucartia squamulata
Fig. 10 Mitotic metaphase (3n) and haploid set of chromosomes in
Strophosoma melanogrammum. The arrows indicate chromosomes
without C-bands. The bar equals 10 lm
240 Genetica (2008) 134:235–242
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11. This phenomenon is quite common in the polyploid
parthenogenetic weevils studied so far (Takenouchi 1978).
Our results also confirm these observations, i.e. the triploid
races have 27–31 chromosomes, but the most frequent
number was 33. The variability of this number could be
affected by fragmentation or fusion of single chromosomes
(Mikulska 1960). The basic chromosomal number in bisex-
ual speciesofweevilsis
multiplication of this haploid set should have occurred in
parthenogenetic, polyploid races. Karyotypes with a preva-
lence of meta- and submetacentric chromosomes with a
small proportion of heterochromatin are the rule in the
karyotypic architecture of curculionids (Lachowska et al.
1998; Ro_ zek et al. 2004). Meanwhile eight examined par-
thenogeneticspeciesarecharacterizedbydiversekaryotypes
in which acrocentric chromosomes also occurred, but the
heterochromatin was visualized around centromeres only in
two species. Another interesting fact is the occurrence of
several parthenogenetic species with two or three races of
different degrees of polyploidy, in addition to the diploid
bisexual races. This phenomenon is called geographic par-
thenogenesis. One example is the European Otiorhynchus
scaber, which has tetraploid, triploid, and diploid bisexual
races (Suomalainen et al. 1987; Stenberg et al. 2000). The
presence of diploid males of Otiorhynchus raucus in a popu-
lation from Slovakia could be explained by geographic
parthenogenesis, but in this case both lineages occur toge-
ther.Thecoexistenceofsexualandparthenogeneticlineages
was proven by molecular analysis in Aramigus pallidus, in
which parthenogenetic and bisexual forms are morphologi-
cally indistinguishable(Normark
japonicusisanotherspecieswherebothdiploidbisexualand
parthenogeneticracesco-existed(Takenouchi1976).Allthe
parthenogeneticweevilssofarstudiedcytologicallyareofan
apomictic, thelytokus type in which eggs undergo one mat-
uration division, and the chromosomes divide equationally,
so that no reduction take place. Eggs that develop only
through mitoses, but show vestiges of meiosis, are called
rudimentaryreductiondivisions,firstobservedintheeggofa
triploid parthenogenetic weevil, Otiorhynchus sulcatus, by
Seiler (1947). In contrast automixis retains meiosis with
restoration of diploidy by duplication or fusion of the
gametes. According to Simon et al. (2003), this kind of
parthenogenesisoccursinsomeweevils,buttheseauthorsdo
not give an example. Our observations confirm apomixes of
parthenogenetic weevils and the phenomenon of a rudi-
mentary reduction division.
chromosomes arrange in bivalents and multivalents resem-
bling bivalents observed in normal meiotic prophase. These
chromosome associations probably represent remnants of
meiosis, supporting the hypothesis that the evolution of
apomicticparthenogenesisinweevilshasproceededthrough
n = 11suggestingthat
1996b).
Blosyrus
During oogenesisthe
a stage of automixis (Suomalainen 1969). Some meiotic
events among ameiotic parthenogenetic insects have been
described for Diptera (e.g. Chironomidae, Agromyzidae),
but in all cases the remnants of meiosis have nothing to do
with recombination (Block 1969).
Acknowledgements
Ministry of Science and Information Society Technologies, grant
number 3PO4C 085 to Dorota Lachowska and by VEGA (Scientific
Grant Agency of the Ministry of Education and the Slovak Academy
of Sciences), grant number 1/3277/06 for Milada Holecova ´.
This research was supported by the Polish
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