Eur. J. Entomol. 105: 185–196, 2008
http://www.eje.cz/scripts/viewabstract.php?abstract=1321
ISSN 1210-5759 (print), 1802-8829 (online)

Geographical patterns of chromosomal differentiation in the brachypterous
grasshopper Podisma sapporensis (Orthoptera: Acrididae)
ELĪBIETA WARCHAàOWSKA-ĝLIWA 1, HARUKI TATSUTA2, SHIN-ICHI AKIMOTO3, ANNAMARYAēSKA-NADACHOWSKA 1, MAREK KOWALCZYK1 and ALEXANDER BUGROV 4
1

Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences,
Sáawkowska 17, 31-016 Kraków, Poland; e-mail: warchalowska@isez.pan.krakow.pl
2
Laboratory of Ecological Risk Assessment, Research Centre for Environmental Risk, National Institute for Environmental Studies,
16-2, Onogawa, Tsukuba, Ibaraki 305-8506, Japan
3
Systematic Entomology, Department of Ecology and Systematics, Graduate School of Agriculture, Hokkaido University, Sapporo,
060-8589, Japan
4
Novosibirsk State University, 630090 Novosibirsk and Institute of Systematics and Ecology of Animals, Siberian Branch of
Russian Academy of Sciences, 630091 Novosibirsk, Russia
Key words. Grasshoppers, Acrididae, Podisma, chromosomes, evolution, polymorphism, chromosome races
Abstract. The distribution patterns of the X0/XX and neo-XY/neo-XX chromosome races, subraces, and “hybrids” between
subraces of the grasshopper P. sapporensis were analyzed. The origin of the observed variation is Robertsonian translocations
between a sex chromosome and an autosome, and chromosome rearrangements. The fixation levels of inversions varied dependingon geographic regions. No hybrid population is known implying that a strong reproductive isolation system exists in hybrids between
the different chromosomal races. The probable reasons for the purity of X0 and neo-XY chromosome races and high chromosome
polymorphism in contact zones between chromosomal subraces are discussed. The presence of isolating barriers between chromosome races indicates a review of the taxonomic structure of P. sapporensis is required. It is proposed to divide P. sapporensis into
two sibling species, which differ in the chromosome mechanisms of the sex determination system.The analysis of the distribution ofchromosomal races and subraces of P. sapporensis allows a reconstruction of the history of this species in the Okhotsk sea region.
INTRODUCTION

Chromosome rearrangements and their role in speciation are well known phenomena in various organisms, but
only a few grasshopper species show complex chromosomal polymorphism across their geographical distributions (White, 1973, 1974; Shaw & Wilkinson, 1980;
Shaw et al., 1976; Moran & Shaw, 1977; Coates & Shaw,
1982; Hewitt, 1979; John, 1983; Gosalvez et al., 1997).
Two species of brachypterous grasshoppers belonging
to the genus Podisma Berthold, namely P. pedestris andP. sapporensis, show chromosome polymorphism. P.
pedestris is widely distributed in the Palearctic region
from Western Europe to Eastern Siberia (Mistschenko,
1952). This species consists of two distinct chromosomal
races. In a large part of its distribution, in Europe andAsia, P. pedestris has the X0/XX sex chromosome determination system with a diploid chromosome number of
23 in the male and 24 in the female, similar to many othergrasshoppers (White, 1973; Hewitt, 1979; John, 1983)
and therefore considered to be an ancestral type.
However, several local populations of this species in theSouthern French Alps have the neo-XY/neo-XX systemderived from a Robertsonian translocation of the X chromosome to an autosome with a diploid number of 22 ineach sex (John & Hewitt, 1970; Hewitt & John, 1972;
Barton & Hewitt, 1985).

Three species belonging to the genus Podisma are distributed on the islands of Okhotsk and in the Japan Sea
region. P. sapporensis Shir. occurs on Hokkaido, Sakhalin and Kunashiri Islands (Shiraki, 1910; Bey-Bienko,
1949; Storozhenko, 1993), P. tyatiensis Bugrov et Sergeev is endemic to Tyatya volcano (northern part ofKunashiri Island) (Bugrov & Sergeev, 1997), whereas P.
kanoi Storozhenko occurs in the central part of Honshu(Storozhenko, 1993). P. sapporensis is conspicuously different, especially in its morphology (Tatsuta et al., 2000)
and cytological features (Bugrov, 1995; Bugrov et al.,2000, 2001, 2003; Warchaáowska-ĝliwa et al., 2001).
This species consists of many geographic races (Akimoto
et al., 1993; Tatsuta & Akimoto, 1994, 1998; Tatsuta etal., 2000) and their taxonomic status is still a matter ofcontroversy (Bey-Bienko, 1949; Storozhenko, 1993;
Bugrov & Sergeev, 1997; Tchernykh & Bugrov, 1997;
Tatsuta et al., 2000). This polytypic species consists offour subspecies, and two of them, P. sapporensis sapporensis (Shir.) and P. s. ashibetsuensis Storozhenko,
occur on Hokkaido (Storozhenko, 1993). The distributionof P. sapporensis krylonensis Storozhenko is restricted to
the Krylion peninsula of Sakhalin, and P. sapporensis
kurilenensis (Bey-Bienko) inhabits the central andsouthern parts of Kunashiri (Storozhenko, 1993).
In previous work, two chromosome races of P. sapporensis were detected (Bugrov et al., 2000, 2001). The

185

Fig. 1. Distribution of the localities where Podisma sapporensis was sampled. Numbers are the same as in Table 1. Races/subraces
are indicated by different colours (yellow – X0/XX-Standard; violet – X0/XX-Sakhalin; blue – X0/XX-Yotei; grey – X0/XXStandard × Yotei; dark-green – X0/XX-Naganuma; light-green – X0/XX-Naganuma × Yotei; brown – X0/XX-Daisengen; red –XY/XX-Standard; white – XY/XX-Tanno/Oketo).

western group of populations on Hokkaido and the population on Sakhalin (Krylion peninsula) have the X0/XX
sex chromosome determination. The eastern group of
populations on Hokkaido and the population on Kunashiri
Is. (Golovnin volkano) belong to the neo-XY/XX race,
which differs from the X0 race by a Robertsonian translocation between the originally acrocentric X chromosome
and M5 autosome in a homozygous state (sex determination is neo-XY male/neo-XX female, 2n = 22). Moreover,in several populations, the pericentric inversion is fixed in
one or more pairs of chromosomes, a key character distinguishing discrete karyomorphs (chromosome subraces).
Interestingly, various levels of polymorphism in both
chromosome races result from pericentric inversions,C-banding variation and the occurrence of additional (B)chromosomes (Bugrov et al., 2001, 2003; Warchaáowskaĝliwa et al., 2001). The level of diversity in the P. sap186

porensis karyotypes is substantially higher than in otherspecies of Podismini grasshoppers distributed in the Palearctic region, suggesting a unique model of cytogenetic
evolution in Orthoptera. In this paper, the pattern of chromosome divergence across the range of P. sapporensis isreviewed. We aimed to summarize the distribution patterns of each chromosomal race and subrace, and determine the border-zones of distribution of the chromosome
races and subraces. The results were used to consider thenumber of potential areas of contact between different
chromosomal races and the phylogeographical scenario
for explaining chromosomal diversification in P. sapporensis.
MATERIAL AND METHODS
During July/August in the year 1999–2005, a total of 1500
males and 250 females of P. sapporensis sapporensis were col-

TABLE 1. Summary of the locations, year of collection, samples size, and chromosome race/subrace type of Podisma sapporensis. Locality number(No.) refers to Table 1 and Fig. 1; e – embryos.
No.
Locality nameLatitudeLongitude 1999
2000 2001 2002 2003 2004 2005 Total
Chromosome type1
Nakatombetsu
44.967142.267
7
8
X0/XX-Standard
2
Bifuka44.45
142.517
6
6
X0/XX-Standard
3
Shimokawa-D
44.417142.6
17e
8
X0/XX-Standard
4
Shimokawa-B
44.383142.617
10
10
X0/XX-Standard
5
Shimokawa-C
44.3667
142.555
5
X0/XX-Standard
6
Shimokawa-A
44.35
142.6
11
11
X0/XX-Standard
7
Shimokawa (2005)
44.283142.667
68
68
X0/XX-Standard
8
Moshiri
44.35
142.25
11
11
X0/XX-Standard
9
Hinata Spa
44.267142.333
8
8
X0/XX-Standard
10
Nishi Okoppe-1
44.333142.833
5,3e
8
X0/XX-Standard
11
Nishi Okoppe-3
44.333142.817
3
3
X0/XX-Standard
12
Togeshita
43.85
141.817
4
4
X0/XX-Standard
13
Bibai
43.283141.833
14
14
X0/XX-Standard
14
Mt Teine
43.1
141.217
30
10e
44
84
X0/XX-Standard
15
Ishiyama
42.967141.3
28
28
X0/XX-Standard
16 Krylion Penninsula
46.983142.91737,54e81
X0/XX-Sakhalin
17
Iwaonobori
42.867140.6519
19
X0/XX-Yotei
18
Mt Yotei42.817140.8
1020,16e46
X0/XX-Yotei
19
Hyakumatsuzawa
42.967141.2
20
20
X0/XX-Yotei
20
Kannonzawa
42.967141.267
13
13
X0/XX-Standard × Yotei
21
Toyotaki
42.95
141.217
20
20
X0/XX-Standard × Yotei
22
Kuriyama
43.033
141,783
18,5e
23
X0/XX-Naganuma
23
Naganuma43
141.683
20
6
15
10
51
X0/XX-Naganuma
24
Tsukigata43.333141.667
11
11
X0/XX-Naganuma × Yotei
25 Yuubari (Takinoue)
42.9
141.967
24
24
X0/XX-Naganuma × Yotei
26
Kamikawa-1
43.833142.767
7
7
X0/XX-Naganuma × Yotei
27
Sounkyo43.767142.9
8
8
X0/XX-Naganuma × Yotei
28
Kamishihoro
43.333143.15
8
8
X0/XX-Naganuma × Yotei
29
Iwamizawa
43.183141.767
17
17
X0/XX-Naganuma × Yotei
30
Ochiai43.117142.667
14
14
X0/XX-Naganuma × Yotei
31
Sahoro43.117142.8
3
3
X0/XX-Naganuma × Yotei
32
Shirakaba
43.133143.133
3
3
X0/XX-Naganuma × Yotei
33
Memuro42.917143.017
2
2
X0/XX-Naganuma × Yotei
34
Obihiro42.867143.2
5
5
X0/XX-Naganuma × Yotei
35
Urakawa
42.183142.767
24
24
X0/XX-Naganuma × Yotei
36
Inasato42.75
142.117
16
16
X0/XX-Naganuma × Yotei
37
Biratori
42.567142.083
2
2
X0/XX-Naganuma × Yotei
38
Kitayuzawa
42.617
141
11
11
X0/XX-Naganuma × Yotei
39
Mt Daisengen
41.583140.15
5
17
22
X0/XX-Daisengen
40
Nishi Okoppe-2,444.283142.96722
22
XY/XX-Standard
41
Takinoue
44.217142.983
9
9
XY/XX-Standard
42
Sakkuru-Toge
44.233142.983
2
2
XY/XX-Standard
43
Tsubetsu
43.667143.917
4
4
XY/XX-Standard
44
Ashoro-A
43.433144.017
2
2
XY/XX-Standard
45
Ashoro-B
43.433143.983
11
11
XY/XX-Standard
46
Akan-A
43.433144.033
105,7e
22
XY/XX-Standard
47
Akan-B
43.45
144.05
11
11
XY/XX-Standard
48
Senpuku-A
43.45
144.017
18
18
XY/XX-Standard
49
Senpuku-B
43.483144
17
17
XY/XX-Standard
50
Senpuku-C
43.467144.033
14
14
XY/XX-Standard
51
Senpuku-D
43.517143.967
7
7
XY/XX-Standard
52
Teshikaga-A
43.467
144.3
12
22
34
XY/XX-Standard
53
Teshikaga-B
43.483144.333
11
11
XY/XX-Standard
54
Teshikaga-C
43.483144.367
3
3
XY/XX-Standard
55
Teshikaga-2005
43.467144.3
9
9
XY/XX-Standard
56
Mt. Shari
43.767144.7
24
24
XY/XX-Standard
57
Mt Etombi
43.75
144.667
4
4
XY/XX-Standard
58
Kiyosato
43.683144.6
10
10
XY/XX-Standard
59
Golovnin
43.733145.5175
7 12
XY/XX-Standard
60
Kamirubetsu
44.217143.317
14,5e
19
XY/XX-Tanno/Oketo
61
Kami-koonomai
44.1
143.359
9
XY/XX-Tanno-Oketo
62
Hakuryu
44.083143.4
24
24
XY/XX-Tanno/Oketo
63
Kimpachi-toge
44.05
143.3
3
3
XY/XX-Tanno-Oketo
64
Rukushi-Toge
43.667143.733
4
4
XY/XX-Tanno/Oketo
65
Kitami43.95
143.7515
15
XY/XX-Tanno/Oketo
66
Rubeshibe
43.9
143.683
5
5
XY/XX-Tanno/Oketo
67
Tanno
43.833143.9578,4e
19
XY/XX-Tanno/Oketo
68
Maruseppu
43.733143.2542
42
XY/XX-Tanno/Oketo
69
Oketo
43.683143.567
73e
919
XY/XX-Tanno/Oketo
70
Kamikawa-2
43.583143.117
2
2
XY/XX-Tanno/Oketo
TOTAL
1133

187

Fig. 2. Distribution of the Podisma sapporensis karyotypes in seven chromosome races/subraces belonging to X0/XX (ring) andneo-XY/XX (square) races.
lected from 68 localities on Hokkaido. In August 2002 and2004, 37 males and 25 females of P. sapporensis krylonensis
from Sakhalin Is. (Krylion peninsula), and 12 males of P. sapporensis kurilensis from Kunashiri Is. (Golovnin volcano) werecollected. The collection sites are named after the closest
town/village or geographical region (Fig. 1, Table 1). Twentynine of these localities are already reported in two previous
reports (Bugrov et al., 2000, 2001), and the cytogenetic datafrom these studies are also included in the present study. A totalof 1133 males and embryos were studied cytologically andincluded in the present study (Table 1). The method of preparing chromosome slides of testes and embryos, and the
C-banding methods were as previously reported (Bugrov et al.,2000, 2001).
RESULTSThe results of the cytogenetic analyses of populations
from Hokkaido, Sakhalin and Kunshir are given in Table
1. These results are concordant with previous data on
chromosome divergence in P. sapporensis and indicate
two main chromosomal races. The X0/XX race occurs in
the western region of Hokkaido and southern Sakhalin,
whereas the neo-XY/XX occurs in the eastern part of
Hokkaido and Kunashiri (Figs 1 and 2). These races havea complex polymorphism in terms of pericentric inversions, C-heterochromatin content, additional (B) chromosomes and C-positive second arms, as well as interchanges between B chromosomes and autosomes.
188

The X0 chromosome race
In order to describe the distribution area of the X0 race,
individuals from 39 localities of P. sapporensis were analysed (Table 1, Fig. 1). On the basis of chromosome polymorphism, this race was subdivided into seven categories:
five subraces and two “hybrid” types.
(1) Samples from 15 localities (Table 1, nos 1–15) had
the standard chromosome complement of the genus
Podisma, i.e., 22 acrocentric autosomes and the acrocentric X chromosome in males (2n% = 23, X0) and two
acrocentric X chromosomes in females (2n& = 24, XX).
This type of chromosome set corresponds to the chromosome complement of P. sapporensis from the vicinities of
Shimokawa and Sapporo (Mt Teine) (see Fig. 2 inBugrov et al., 2001, Fig. 3). This chromosome morphotype is denoted as “X0/XX-Standard” subrace of P. sapporensis (Table 1). This race is distributed from northernto central Hokkaido (Figs 1 and 2).
(2) P. sapporensis from the Sakhalin (Krylion peninsula) population is described as P. sapporensis krylonensis (Storozhenko, 1993) (Table 1, Fig. 1, no. 16).
The karyotype of this population has 23 chromosomes in
the male and 24 in the female and a X0%/XX& sex determination system. All autosome pairs are acrocentric,
whereas the X-chromosome is subacrocentric. The
euchromatic nature of the short arm of the X chromosome

Fig. 3. The frequency distribution of heterozygotes and homozygotes for the pericentric inversion at twenty localities of the
X0/XX race of Podisma sapporensis. Locality number and names of localities correspond to individuals of races/subraces in Table 1
and Fig. 1. Diagrams present results where there are more then 5 individuals.

testifies to its inversion origin. The karyotype of P. s. krylonensis is more similar to the standard chromosome
complement of the populations at the eleven localities
described above, but differs slightly in the fixed pericentric inversion in the X-chromosome (see Fig. 1 in Bugrov,
1995; Fig. 3). This chromosome subrace, “X0/XXSakhalin”, is separated by the Laperuz strait from otherpopulations of P. sapporensis on Hokkaido and the
Okhotsk Sea from the Kunashiri population (Fig. 2).
(3) The “X0/XX-Yotei” subrace differs from othersubraces of P. sapporensis in having a fixed pericentric
inversion resulted in a short euchromatic arm on M4, and
the X-chromosome as well as heterochromatic short arms
on M5 and M8–S10 pairs. Additionally, a polymorphism inthe short euchromatic short arm on L1, L2, M3, and M6
was revealed (Fig. 3). In contrast, the vast majority ofsamples possessed short second heterochromatic arms on

the M7 and S11 pairs (see Fig. 3 in Bugrov et al., 2001).
This subrace occurs at three localities: Mt Yotei, Iwaonobori and Hyakumatsuzawa (Fig. 1, Table 1, nos 17–19).
These localities are adjacent to the distribution of the
X0/XX-standard subrace and no conspicuous geographic
barriers occur between the two subraces, except for Mt
Yotei (Fig. 2).
(4) The samples from Naganuma and Kuryama (Figs 1
and 2, Table 1, nos 22 and 23) were homozygous for the
pericentric inversion on M6. The inverted segment of M6
forms the euchromatic arm, resulting in the derived morphology of M6 from acrocentric to submetacentric. This
pair of autosomes can be used as specific markers. A highfrequency of heterozygotes and homozygotes for the
inversion on M5, and low frequency of heterozygotes forthe inversion on M4, as well as the presence of additional
short C-heterochromatic arms on two pairs of autosomes
189

Fig. 4. The frequency distribution of heterozygotes and homozygotes for the pericentric inversion at nine localities of the XY/XX
race of Podisma sapporensis. Locality number and names of localities correspond to individuals of races/subraces in Table 1 andFig. 1. Diagrams present results where there are more then 5 individuals.

and the X chromosome were revealed (see Fig. 6 inBugrov et al., 2001, Figs 2 and 3). The Ishikari river running through the Naganuma area seems to constitute abarrier between the “X0-Standard” and “X0-Naganuma”
subrace.
(5) The population on Mt Daisengen – the “X0/XXDaisengen” subrace (Figs 1 and 2, Table 1, no. 39), differs greatly from other populations belonging to the X0
chromosome race, not only in the morphology of its chromosomes but also in the localization and amount ofC-heterochromatin. All chromosome pairs in representatives of this population had two arms (Fig. 3). Short,second euchromatic arms were observed only on M6 autosomes and X-chromosomes. On the remaining chromosomes the short arms were mostly heterochromatic (seeFig. 4 in Bugrov et al., 2001). The population on Mt
Daisengen is completely isolated from other populations
and is relatively small. Hence, the inbreeding rate is considered to be extremely high and this novel karyotype
may be rapidly fixed due to random genetic drift.
Sixteen out of the 39 localities distributed in thewestern part and in the centre of the X0/XX race area
were highly polymorphic for short chromosome arms. A
substantial amount of chromosome polymorphism associated with pericentric inversions and the additional heterochromatic arm may imply that hybridisation between
neighbouring subraces has occurred. According to chromosome markers, the composition of “hybrids” were as
follows: 1) between X0/XX-standard and X0/XX-Yotei –“X0/XX-standard × Yotei hybrid” and 2) X0/XXNaganuma and X0/XX-Yotei – “Naganuma × Yotei
hybrid”. They are described below.
190(6) Thirty-three specimens from two localities (Kannonzawa and Toyotaki) distributed along the border ofthe X0-Standard and X0 Yotei subraces can be classified
as “X0/XX-Standard × Yotei hybrids” (Fig. 1, Table 1,
nos 20 and 21). Most of the autosomes (with the exception of L2 pair) and the X chromosome are polymorphic,
thus the frequency of subacrocentric chromosomes variedamong samples as well as chromosome pairs. The vast
majority of specimens are heterozygous for chromosome
markers characteristic of the “X0 Yotei” subrace (shorteuchromatic arm on M6, short second heterochromatic
arms on M7 and S11 pairs, and morphology of the X chromosome). On Toyotaki (Fig. 1, no. 21, Fig. 5A, B) an
acrocentric X chromosome was found, which is morphologically similar to X0-Standard, whereas on Kannonzawa (Fig. 1, Table 1, no. 20; Fig. 5C, D) two of 11
individuals possessed subacrocentric X chromosomes
similar to individuals of X0 Yotei. The diagram shows
the frequency of polymorphism in this subrace (Fig. 3).
These localities are on the border of X0/XX-standard and
X0/XX-Yotei and there is no geographic barrier in thisarea.
(7) An analysis of 90 individuals from 13 localities(Fig. 1, Table 1, nos 24–38) revealed high polymorphism
for pericentric inversions in each diagnostic chromosome,including the specific markers on chromosome M6 (submetacentric chromosome) and the X chromosome (acrocentric or subacrocentric with heterochromatic arm) in the“X0 Naganuma” subrace as well as an inversion polymorphism on L1–L3, M6 (subacrocentric) and the X chromosome marker (subacrocentric with short euchromatic
second arms) in the X0 Yotei subrace. These individuals

Fig. 5. Meiotic chromosomes of Podisma sapporensis males stained for C-banding. (A–D). X0/XX “Standard × Yotei hybrids”
from Toyotaki (A, B) and Kannonzawa (C, D) localities. (A, B) Diakinesis, heterozygous and homozygous for chromosome markers
characteristic of the X0-Yotei subrace; (A) short heterochromatic arms in heterozygous M5, M7 and S11 pairs (arrows), homozygous
for S9, S10, short euchromatic arm on M6 (white arrow) and (B) heterozygous for heterochromatic arms on the M5, M6, M7 and S9pairs, homozygous for S11, as well as acrocentric for M4. (arrows). The acrocentric X chromosome is morphologically similar to X0Standard. (C) Diakinesis with the X chromosome subacrocentric with euchromatic short arm and (D) anaphase I with acrocentric X
chromosome, additionally, M5–S9 pairs are heterozygous for short arms (arrows). (E–H). X0/XX “Naganuma × Yotei hybrid” fromthe Kitayuzawa (E, F) and Urokawa (G, H) localities. Diakinesis with (E, H) heterozygous or (F, G) homozygous for submetacentric
M6 chromosome (chromosome markers for X0-Naganuma) (arrows) and the X chromosome with euchromatin short arm (the marker
for X0-Yotei). Additionally, (G, H) homozygous for pericentric inversion on L2, M6 (G) and L2, M4 (H), as well as heterozygous for
pericentric inversion on M4, M5, M7, M8 (G) and L1 M3, M4–M8 (H) (arrows). Scale bar = 10 µm.

191

TABLE 2. Frequency of the six B chromosomes and translocation between M3 and B in Podisma sapporensis at different localities; unclear type ofB (Bn). Locality number (No.) refers to Table 1 and Fig. 1. BB – standard homozygote; BS – translocation heterozygote; SS – translocation homozygote; e – embryos.
B Chromosomes
M3 translocation
No.
Locality name
Chromosome type
Total
BS
SS
0B
B1
B2
B3
B4
B5
B6
Bn
BB
3
Shimokawa-A
X0/XX-Standard
11
9
2
10
1
4
Shimokawa-B
X0/XX-Standard
10
8
2
9
1
6
Shimokawa-D
X0/XX-Standard
8
44e
7
Shimokawa (2005)
X0/XX-Standard
68
56
2
3
7
9
Hinata Spa
X0/XX-Standard
85
3
10
Nishi Okoppe-1
X0/XX-Standard
8
62
12
Togeshita
X0/XX-Standard
4
3
1iso/S9
14
Mt Teine
X0/XX-Standard
84
81
3
81
3
15
Ishiyama
X0/XX-Standard
28
20
8
16 Krylonian Penninsula
X0/XX-Sakhalin
81
65
3,13e
18
Mt Yotei
X0/XX-Yotei
46
40
4,2e
22
Kuriyama
X0/XX-Naganuma
23
19
1
3
18
5
23
Naganuma
X0/XX-Naganuma
51
37
10
2
1
1
45
6
25 Yuubari (Takinoue) X0/XX-Naganuma × Yotei
24
20
4
22
2
26
Kamikawa-1
X0/XX-Naganuma × Yotei
7
6
1
6
1
27
Sounkyo
X0/XX-Naganuma × Yotei
85329
Iwamizawa
X0/XX-Naganuma × Yotei
17
16
1?
30
Ochiai
X0/XX-Naganuma × Yotei
14
10
4
32
Shirakaba
X0/XX-Naganuma × Yotei
32
1
36
Inasato
X0/XX-Naganuma × Yotei
16
12
4
14
2
37
Biratori
X0/XX-Naganuma × Yotei
202
44
Akan-AXY/XX-Standard
22
21
1
15
7
45
Ashoro-B
XY/XX-Standard
11
8
3
48
Senpuku-A
XY/XX-Standard
18
17
1
13
5
49
Senpuku-B
XY/XX-Standard
17
16
6
50
Senpuku-C
XY/XX-Standard
14
11
3
51
Senpuku-D
XY/XX-Standard
7
6
1
52
Teshikaga-A
XY/XX-Standard
34
15
1,11e
2e
1
1
2,1e
28 1,5e53
Teshikaga-B
XY/XX-Standard
11
10
1
8
3
58
Kiyosato
XY/XX-Standard
10
9
1
7
3
60
Kamirubetsu
XY/XX-Tanno/Oketo
19
18
1
61
Kami-koonomai
XY/XX/Tanno-Oketo
9b
7
1
1
62
Hakuryu
XY/XX-Tanno/Oketo
24
21
1
2
63
Kimpachi-toge
XY/XX-Tanno/Oketo
3b
2
1
65
Kitami
XY/XX-Tanno/Oketo
15
12
1
1
1
67
Tanno
XY/XX-Tanno/Oketo
19
15
8e
68
Maruseppu
XY/XX-Tanno/Oketo
42
38
4
40
2
69
Oketo
XY/XX-Tanno/Oketo
19
18
1e

are classified as “Naganuma × Yotei hybrids” (Figs 1 and
3). The frequencies of chromosomes with two arms varyconsiderably between different localities within this geographical zone. There are some interesting features in thegeographical distribution of chromosomal types within
this admixed area. In general, these populations arehomozygous or heterozygous for the submetacentric M6chromosome (Fig. 5E–G). Additionally, the acrocentric/
subacrocentric (with heterochromatic arm) form of the X
chromosome generally has an eastern-northerly distribution; it is acrocentric on Iwamizawa (no. 29), Tsukigata(no. 24) and Kamikawa-1 (no. 26) or heterozygous(acrocentric/subacrocentrc) on Sounkyu (no. 27) andYuubari (no. 25). The subacrocentric with an euchromatic
short arm form of the X chromosome is found mainly inthe south-western part of the distribution of the“Naganuma – Yotei hybrids” (Fig. 5F–H) and includes
eight localities. Some of them lie across the Hidaka
Range (Fig. 1, Table 1, nos 28, 30, 31, 32, 33, 34, and
19235). There appears to be a cline of integradation of frequencies within the subrace in the area between localities
where the alternative X chromosome morphs belonging to
the “Yotei” subrace are known to be fixed. Grasshoppers
in hybrid areas are distributed on the western and eastern
slopes of the Hidaka Range.
The neo-XY race
Samples from 31 localities from the eastern part of
Hokkaido and Kunashiri have the neo-XY in the male (2n= 22) and neo-XX in the female (2n = 22) type of sex
determination. The range of this race could be divided
into two chromosome subraces: (1) “XY/XX-Standard”(including the Golovnin population) and (2) “XY/XXTanno/Oketo” subraces (Fig. 1, Table 1, nos 40–70).
The Kunashiri (Golovnin volcano) population was firstdescribed as P. kurilensis (Bey-Bienko, 1949). Subsequently, Storozhenko (1993) changed the taxonomic
status to P. sapporensis kurilensis. Specimens with the
karyotype of the Kunashiri population also occur at

TABLE 3. Polymorphism of additional chromosome segments in Podisma sapporensis. Locality number (No.) refers to Table 1 and Fig. 1. BB –homozygous for the chromosome without extra heterochromatin; BS – heterozygous; SS – homozygous for the chromosome with extra heterochromatin; interstitial C-bands located near the centromere (c) or near the distal (d) part of the chromosome.
M6/M5
M6 neo/ M6neo/
M7neo/
M7/8/
L1 interst
S10/9
S11 dist
interst
inter
dist
dist
distalNo.
Locality name
Chromosome type
Tota
BB BS SS BB BS SS BB BS SS BB BS SS BB BS SS BB BS SS BB BS SS BB BS SS
4
Shimokawa-B
X0/XX-Standard
10 9 1c 0 9 0 1
6Shimokawa-A
X0/XX-Standard
11 8 1c 1c,1d 10 0 1
7 Shimokawa (2005)
X0/XX-Standard
68
43 3 22 32 5 31
10 Nishi Okoppe-1
X0/XX-Standard
8
7 1 0 7 1 0
13Bibai
X0/XX-Standard
14
13 1 015
Ishiyama
X0/XX-Standard
28
26 2 022
Kuriyama
X0/XX-Naganuma
23
22 0 1
23
Naganuma
X0/XX-Naganuma
51
49 2 024
Tsukigata
X0/XX-Naganuma × Yotei 11
5 4 2
6 5 0
26
Kamikawa-1
X0/XX-Naganuma × Yotei 7 6 0 1d
28
Kamishihoro
X0/XX-Naganuma × Yotei 8
7 1 030
Ochiai
X0/XX-Naganuma × Yotei 14
10 3 1
13 0 135
Urakawa
X0/XX-Naganuma ×Yotei 24
14 9 140 Nishi Okoppe-2,4
XY/XX-Standard
22
20 2 041
Takinoue
XY/XX-Standard
9 4 5 044
Ashoro-A
XY/XX-Standard
2 1 0 1 2 0 0
0 1 1
45
Ashoro-B
XY/XX-Standard
11 9 0 2d 9 0 2
10 1 046
Akan-AXY/XX-Standard
22 19 0 3d 17 0 5 20 2 0 19 3 0 20 2 0
21 1 048
Senpuku-A
XY/XX-Standard
18 13 2d 3d49
Senpuku-B
XY/XX-Standard
17 11 2d 4d 16 1 0
14 3 050
Senpuku-C
XY/XX-Standard
14
13 1
13 1 051
Senpuku-D
XY/XX-Standard
7 6 0 1d 6 1 0
52
Teshikaga-A
XY/XX-Standard
34
33 0 1 30 3 1 32 0 2 33 1 0
31 0 353
Teshikaga-B
XY/XX-Standard
11 7 0 4d
55
Teshikaga
XY/XX-Standard
9 4 5 0twenty localities of the “XY/XX-Standard” chromosome
subrace possessing 10 pairs of acrocentric chromosomes
and two sex chromosomes (metacentric neo-X and acrocentric neo-Y in the male and double metacentric neo-Xin the female (see Fig. 2 in Bugrov, 1995). This subrace isdistributed throughout the eastern part of Hokkaido(Table 1, nos 40–59). This subrace on Hokkaido isdivided into two separate populations. One is situatednear the northeastern border of the distribution of the
X0/XX-Standard subrace (Fig. 1, nos 40–42). The second
group of populations occur in the southeast part of Hokkaido (Fig. 1, nos 43–58 and Fig. 2).
At eleven localities studied around Oketo and Tanno(Fig. 1, nos 60–70, Fig. 2) P. sapporensis differ slightly
from those at localities with standard neo-XY karyotype
in possessing a high frequency of heterozygotes andhomozygotes for the inversions in some large andmedium sized autosomes (Fig. 4). However, a fixed pericentric inversion with short euchromatic arms on M3 andneo-M5 was observed only at the Tanno locality (see Figs
8 and 9 in Bugrov et al., 2001). This chromosome morphotype is denoted as the “XY/XX-Tanno/Oketo”
subrace. This subrace is located between two areas of distribution of the XY/XX-Standard subrace. There is noconspicuous geographic barrier between XY/XX-standard
and XY/XX-Tanno/Oketo subrace. The diagrams show
the frequency of polymorphism in this subrace (Fig. 4).
Possible contact zone of X0 and neo-XY races
In 2005, additional material from the northeastern partof Hokkaido was collected in order to find the contact

zone between the X0/XX and neo-XY/XX races. A totalof 350 adult male and female grasshoppers were collectedfrom 12 new localities. The natural hybrid zones between
these races have not yet been discovered. However, specimens from Nishi-Okoppe-1,3 and Nishi-Okoppe-2,4included X0-Standard or neo-XY-Standard races (Table
1, nos 10, 11, and 40, Fig. 1). These populations are separated by about 14 km and thus the width of this transect is
not very long. Moreover, 11 individuals from two localities (26 and 70, respectively) in Kamikawa-1 andKamikawa-2 consisted of X0/XX-Naganuma/Yotei and
XY/XX-standard (Fig. 1, Table 1) races. These are separated by about 24 km.
B chromosomes, translocations between Bs andautosomes, and C-heterochromatin polymorphism
B chromosomes were found in 120 specimens
belonging to both chromosome races from 38 out of 70
localities (Table 2). These Bs were then subdivided into
seven categories according to the structure, size andC-banding content (see Fig. 1 in Warchaáowska-ĝliwa etal., 2001). The localities and the number of specimens foreach category are given in Table 2. The highest frequency
was observed for B1 and B5iso variants, and reached 3%
and 2.03% in X0 samples and 0.61% and 1.06% in XY
individuals, respectively. Only single individuals of othertypes (B2, B4 and B6iso) were found. The B4 variant was
found at the Naganuma and Yuubari localities (X0 race),
whereas the B6iso variant was detected only at Teshikaga(XY/XX race).

193

Additionally, a potential interchange between the B
chromosome and one autosome from M3 (Fig. 9 inWarchaáowska-ĝliwa et al., 2001) was observed at 18 of
the 70 localities examined. Fifty-nine males and embryosfrom the two main races were heterozygous, whereas two
specimens were homozygous for this translocation (Table
2).
Moreover, polymorphism of additional segments associated with the occurrence of interstitial or distal extra
heterochromatin was identified in individuals from different localities, polymorphic for the presence of six additional C-bands (see Figs 14–17 in Warchaáowska-ĝliwa etal., 2001; Table 3 in the present paper). In most cases,
three different types of bivalents exist: BB – homozygous
for the chromosome without supernumerary heterochromatin, BS – heterozygous and SS – homozygous for thechromosome with extra heterochromatin. For chromosome L1, 0.44% of males from the X0 race and 2.82%
from the neo-XY race were either heterozygous or homozygous for this supernumerary segment. Variation in the
C-banding pattern was due to the presence or absence ofthin C-bands located near centromeres (c) or near the
distal parts of chromosomes (d). The interstitial segment
on M6 (X0), M5 (XY), S10 (X0) and S9 (XY) and additionally a distal segment on the last pair showed the samestructural basis in the two chromosome races. This differ
from the polymorphism in the interstitial and distal
C-heterochromatin on M6 and distal C-heterochromatinon M7, which occurred only in the neo-XY race. These
polymorphic C-bands were observed with low frequency
at different localities (Table 3).
DISCUSSION

Among orthopteroid insects, P. sapporensis is characterized by extremely high chromosome variation. Thechanges in chromosome set (e.g. translocations, inversions, additional chromosome elements) might be easily
fixed and accumulated as a result of random genetic drift.
The recognition of the distribution and nature of geographical barriers results in a better understanding of theinfluence of dispersal on population genetic structure anddefines factors responsible for the distribution of favourable mutations (Mantel et al., 2003).
The Naganuma subrace (with a fixed inversion on theM6 autosome) has no clear borders of distribution. The
Ishikari river, running through the Naganuma area, seems
to be a potential barrier separating X0-Standard and X0
Naganuma subraces, which may have facilitated karyotype differentation. Moreover, two types of “hybrids”
contain mixed karyotypes a possible result of hybridization between neighbouring X0-Standard and X0-Yotei
subraces. Beginning in the Naganuma and Yotei areas,
populations are highly fragmented throughout the south
to the centre of Hokkaido. On the basis of the polymorphism in two armed chromosomes, the frequency of heterozygotes, and the geographical distribution of these
localities, two separate areas of hybridization are proposed: the “X0-Standard × Yotei” (Fig. 1, nos 20, 21) andthe “X0-Naganuma × Yotei” (Fig. 1, nos 25–38). The
194

occurrence of chromosome markers characterizing individual subraces and the frequency of heterozygotes may
indicate that neighbouring chromosome subraces havehybridized. However, it cannot be excluded, that theextensive karyotypic variation within the X0 race is a
result of the polymorphism observed within the race,
which is more or less continuously variable geographically.
Bugrov et al. (2001) found that P. sapporensis is represented by two main allopatric chromosome races, namely
X0/XX and neo-XY/XX, however, the geographic borders between these races have not yet been identified. In aprevious paper, we put forward the hypothesis that the X0
and neo-XY chromosome races may be geographicallyisolated by a mountainous expanse consisting of theDaisetsu Mts and Hidaka Range, occupying the central
part of Hokkaido, and by the sea straits between Hokkaido, Sakhalin and Kunashiri islands. The present
investigations have shown that the X0 race is distributed
across the Hidaka Range from their western to eastern
slopes (Fig. 1). Thus, it appears that the mountainous
system does not represent a geographic barrier that promotes allopatric chromosomal speciation. On thecontrary, no conspicuous geographic barrier exists in thearea between different chromosomal races. This factstrongly suggests that spatial isolation is not necessary forchromosomal speciation.
In spite of the stability of the X-A Robertsonian translocation leading to strong territorial isolation of the X0
and neo-XY chromosomal races of P. sapporensis, polymorphism in pericentric inversions and additional
chromosome elements are common in this species. In
some populations the pericentric inversion chromosomechanges are fixed in one (Sakhalin population) or somepairs of chromosomes (Mt Daisengen population, Mt
Yotei and Naganuma populations), which enables theidentification of separate chromosome subraces. Consequently, we conclude that P. sapporensis can be divided
into six chromosome subraces, each of which has of atleast a single fixed chromosome change in theirkaryotype.
The majority of the fixed changes in the X0 race werediscovered at two isolated localities: (1) the top of Mt
Daisengen (1072 m a.s.l., X0 Daisengen subrace) and (2)around Mt Yotei (about 1800 m a.s.l., X0 Yotei subrace)(Fig 1). Obviously these populations have been presumably isolated from other populations for a long time.
Each chromosome in the karyotype of this subrace differsfrom analogous chromosomes in the X0 Standard subracein having pericentric inversions (M5, X) or a C-positive
short arm. To our knowledge, P. sapporensis occurs only
on the summit of Mt Daisengen on the Oshima Peninsula.
Genetic structure is determined not only by current evolutionary processes but also modelled by the history of
populations (Avise, 2000). An analysis of the distributionof chromosomal races and subraces of P. sapporensis
may be used to reconstruct the history of this species in
the Okhotsk Sea region.

In the Miocene, territories that subsequently transformed into the present-day Kuril Islands, Sakhalin andHokkaido constituted the eastern end of the Asian continent (Lebedev, 1968). This area was influenced by amoist and warm climate and dominated by deciduous forests (Kryshtofovich, 1955). By the beginning of the Quaternary (ca 2.0 mln years BP), the outlines of theshoreline in the Okhotsk Sea region became closer to thepresent-day situation. From that time, further basin evolution was controlled mostly by global climatic changes. At
the Last Glacial Maximum (15–18 thousand years ago),the sea level decreased by 130 m and the majority of thepresent-day shelf was drained, while Sakhalin, Hokkaido,
Kunashir and presumably Iturup were integrated into an
extensive mountain ridge. This period is characterized bya cooling of the atmosphere that caused a growth of themountain glaciers on Sikhote-Alin’, Hokkaido andHonshu (Bezverkhyi et al., 2002).
Due to these processes, the present-day biodiversity of
the Podismimi grasshoppers on Hokkaido is much impoverished compared with the continental biodiversity on theother shore of the Japan and Okhotsk seas. Yet the native
species may have originated from here, for example ourmodel species – P. sapporensis. The Last Glacial
Maximum had a major affect on the biogeocenoses of the
Okhotsk region. In this period forest-tundra dominated in
the north of the region and forest-steppe landscapes in thesouth (Kryshtofovich, 1955). Representatives of relatively thermophilic fauna could have survived only in refuges. During the period of mountain glaciation on Hokkaido, the metapopulation of P. sapporensis was probably
divided into two main refuges on two sides of the central
mountain system separated by the ridges Daisetsu andHidaka. Owing to the small population sizes in these refuges, neutral or selectively significant evolutionary transformations of the genome may have rapidly spread in the
populations of this wingless species with a small radius of
reproductive activity. P. sapporensis evolved into a
unique acridid species, in which chromosome rearrangements frequently appeared and were fixed. It is likely thatby virtue of stochastic mechanisms, translocations
between the sex chromosome and the fifth pair of autosomes became fixed in the eastern part of the range of thisspecies and resulted in the occurrence of the neo-XY
chromosome sex determination. It should be noted that apolymorphism involving additional (B) chromosomes
probably occurred in the initial metapopulation of P. sapporensis before it separated into X0 and XY races.
Unique B morphotypes inherent only to a specific race
were not detected in either the standard X0 or XY race.
FISH analysis also has confirmed the unique origin ofclusters of repetitive DNA sequences (18S rDNA) in allmorphotypes of B chromosomes and in additional chromosome elements (Bugrov et al., 2003, 2004).
The evolutionary differentiation of the XY chromosome race in Eastern Hokkaido suggests one more step inthe long-term isolation of several populations (XYStandard and XY-Tanno subraces). In the western part of
the range, the initial Podismini type of chromosome set

was maintained, yet differentiation of the X0 chromosome race into subraces was more intensive in this area.
One of the probable reasons for this differentiation is thelong-term isolation of the present-day Oshima peninsula(southern part of Western Hokkaido) as an island(Yasuda, 1984). In the southern and northern parts of thispeninsula, the chromosome subraces (X0-Daisengen and
X0-Yotei, respectively) differ from the X0-Standard
subrace in more than 5 fixed arrangements. During thepostglacial transgression of the southern islands of theKuril ridge, Sakhalin island and Hokkaido were isolated(14,000–11,000 years BP). On the basis of geological andgeophysical data, the isolation of Hokkaido island from
Sakhalin can be dated to around 12,000–11,000 years BP(Bezverkhyi et al., 2002). It is likely that the formation ofanother chromosome subrace (X0-Sakhalin, inversion in
the X-chromosome) may be associated with this period.
According to all known palaeogeographic reconstructions, Kunashiri island was the last to become isolated from Hokkaido. Importantly, the Kunashiri
population in a cytological sense is identical to the XYstandard chromosome subrace on Hokkaido.
In summary, the present study describes the distribution
pattern of the chromosome races and subraces of P. sapporensis. The observed variation is most likely due to a
Robertsonian translocation between a sex chromosome
and an autosome and also the result of chromosome rearrangements. The fixation level of inversions varied
depending on geographic region. Hybrids between the
X0/XX and neo-XY/XX race have not been discovered,presumably implying the evolution of a postzygotic
reproductive isolation system. The presence of isolating
barriers between chromosome races provides an interpretation of the taxonomic structure of P. sapporensis thatdiffers in terms of the sex determination system.
ACKNOWLEDGEMENTS. The authors thank T. Hironaga forcollecting samples of Podisma sapporensis from Nakatombetsutown and Y. Sugano for collecting samples of this species from
Tanno-town in 2000. The study was supported partially by theState Committee for Scientific Research (KBN), Project No.
2P04C 088 26 to E. Warchaáowska-ĝliwa and A.G. Bugrov; bya JSPS Fellowship for Research in Japan, Project No.
RC30026002 to A.G. Bugrov; by JSPS Fellowship for JapaneseYoung Scientists Project No. 08608 to H. Tatsuta; by a grant of
the Russian Fund of the Basic Researches (RFBR) No. 02-0449398 for A.G. Bugrov; by the research program conductedunder the 21st Century COE Program on “Neo-Science of
Natural History” at the Graduate School of Science, Hokkaido
University to E. Warchaáowska-ĝliwa, S. Akimoto, and H. Tatsuta. A.G. Bugrov would like to express thanks to M. Ohara of
the Hokkaido University Museum for organization of the cytogenetic laboratory during his stay as visiting professor in 2005.
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Received August 22, 2007; revised and accepted October 23, 2007

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