Table Of ContentZOOLOGICAL SCIENCE 11: 337-342 (1994) 1994ZoologicalSocietyofJapan
Polymorphism of Lampbrush Chromosomes in Japanese
Populations ofRana nigromaculata
Hiromi Ohtani
LaboratoryforAmphibian Biology, Faculty ofScience,
Hiroshima University, Higashi-hiroshima 724, Japan
—
ABSTRACT The 13pairsofthelampbrushchromosomesofR. nigromaculataarecharacterizedbyonetofivelandmarks
situatedatspecificpositionsontheaxisofeachchromosome. InR. nigromaculatacollectedfrom28sitesinJapan,eight
chromosome pairs did not show any variations, whereas the remaining five pairsconsisted oftwo forms ofchromosome
which differed in the number or type of the landmarks. The variations in chromosomal polymorphism indicated that
Japanese R. nigromaculata hasgeneticallydifferentiated intofourgroupswhichwereformed by abreakinthe migration
due perhaps to geographic obstacles and the expansion of new genetic materials provided from continental R.
nigromaculata in the Wiirm glacial stage. By comparing the distribution of the variations and the lampbrush
chromosomes ofcontinental R. nigromaculata, the history ofthe migration ofR. nigromaculata into Japan is discussed.
INTRODUCTION MATERIALS AND METHODS
Rananigromaculata isdistributedoverthe north-eastern Lampbrush chromosomes were removed from the ova of 199
area of China, the whole of the Korean Peninsula, and female Rana nigromaculata Hallowell collected from 28 sites in
Kyushu, Shikoku and Honshu (except the Kanto and Sendai Japan. Figure 1 shows the collection sites and the number of
plains) in Japan [5]. This species is supposed to have females. In addition, lampbrushchromosomeswere removedfrom
evolved from an ancestral species population common to three females from Beijing, China, and six females from Suwon,
Ranaplancyi in the continent, to adapt to the arid and cold Korea, which had been bred in the Laboratory for Amphibian
climates [6]. After its own evolution was sufficiently com- Biology, Hiroshima University.
Lampbrush chromosomes were prepared by means of a slight
pleted, R. nigromaculata came to Japan through the Korean
modification of Gall's method [2, 13]. Because the size of the
Peninsula when Japan was still a part of the continent [6]. landmarksvarieswiththestagesofoogenesis,tenormorelampbrush
Recently, Nishioka et al. [12] found that R. nigromaculata chromosome preparationsperfemalewere examinedunderaphase-
collected from 45 sites in Japan have differentiated into four contrast microscope to avoid misjudgements on the presence or
groups on the basis of Nei's genetic distances obtained from absence oflandmarks.
electrophoretic analyses.
Thegeneticdifferentiationwasreflectedinthecharacter- RESULTS
istics of its lampbrush chromosomes which are composed of
13 pairs of bivalent chromosomes at the diplotene stage of Previously, Nishioka et al. [10] described a map of the
oogenetic meiosis. The lampbrush chromosomes ofR. nig- lampbrush chromosomes of R. nigromaculata, which repre-
romaculata are characterized by one to five landmarks, sented the positions and typesoflandmarks oneach chromo-
consisting of simple-type giant loops, compound-type giant somal axis. The map was constructed from the lampbrush
loops, spheres,andanoval-likestructure, atspecificpositions chromosomes of three female offspring of a pair collected
of each chromosome axis [10], though the landmarks grow from Hiroshima. According to that map, 13 lampbrush
conspicuous by accumulating their own gene product around chromosomespossessonetofivelandmarkseach,oratotalof
their axes [1]. However, some lampbrush chromosomes 35 landmarks, andtheyarealldistinguishablebypositionand
showed variations in the landmarks among populations ofR. type.
nigromaculata. These variations are presumed to relate to In this study, eight ofthe 13 lampbrush chromosomes of
theprocesses ofthe habitatexpansion ofR. nigromaculata in all the populations of Japanese R. nigromaculata examined
Japaninthe same wayasthe mitoticchromosomalmutations showed the same characteristics (2-5, 8-10, and 12). By
ofRattus species [15-17]. contrast, the remaining five, 1, 6, 7, 11, and 13, showed two
Herein, the geographical distribution and the history of forms which differed in the numberortype ofthe landmarks
the polymorphism of the lampbrush chromosomes of they carried. In chromosome 1, one form possessed two
Japanese R. nigromaculata in comparison with those of simple-type giant loops, one on the short arm and the other
continental species are described. on the long arm; the other form possessed one compound-
typegiant loopon the longarm in addition to these (Fig. 2a).
Accepted January 19, 1994 In chromosome 6, one form hadonecompound-type andone
Received October26, 1993 simple-typegiantlooponthe longarm;theotherhadanother
i
338 H. Ohtani
FukuiOO)
Kanazawa(IO)
ToyamaO)
Joetsu(15)
Oita(3)
MunakataO) SutamaO)
Kumamoto(IO) Mishima(6)
KagoshimaOO) Okaya(5)
da(10)
Sasebo(10)
Nagoya(4)
Maibara(4)
Fig. 1. Map showing the collection sites of Rana nigromaculata and the number offemales studied. Bold lines mark the
mountain ranges and a dotted line marks the location ofthe presumptive coastline about 2x104years ago [3].
OO
lOO
S\JJ ^Q^fl
•.jjQ*•**!(^ *
~JL v
3 q T?
WJtoffij,
J-
-
<::
* *J v r ' HHOI chhi
d A B
Fig. 2. Variations in lampbrush chromosomes 1(a), 6(b), 7(c), 11(d), and 13(e) of R. nigromaculata. In diagrammatic
representation,simple-andcompound-typegiantloopsarerepresentedbysolidanddottedlines,respectively,andspheres
areshownwith blackcircles. Asegmentbetweentwoshort parallel linesshowsarangeconsistingoflargernormal loops
than any other parts and including a centromere. A, B, and C indicate A-, B-, and C-form, respectively. In
photographs,simple-typegiant loopsappearasastiffloopwhichbecomesthickbycoveringitsaxiswithalargequantityof
matrixes and it has a smooth outline. Compound-type giant loopsconsist oftwo or more simple-type giant loopswhich
aremoreslenderthanthe independentsimple-typegiantloopsandhaveanotchedoutline. Scalebarrepresents 100^m.
Polymorphism of Lampbrush Chromosomes 339
simple-type giant loop on the long arm in addition to these In chromosome 6, the two forms were somewhat similar
(Fig. 2b). In chromosome 7, one form had two simple-type in distributional pattern to those of chromosome 1. The
andonecompound-typegiantlooponthelongarm; theother A-form was found in the Chubu group at a fairly high
had another compound-type giant loop on the short arm in frequency, not being so predominant as the A-form of
addition to these (Fig. 2c). In chromosome 11, one form chromosome 1. The B-form was predominant in the East-
had one compound-type giant loop on the long arm and two ern Honshu and Western Honshu-Shikoku groups, like
spheres on both the arms; the other had three simple-type chromosome 1. The KyushugroupalsoinvolvedtheB-form
giant loops in place of the compound-type giant loop of the at high frequency. It was remarkable, however, that five
former (Fig. 2d). In chromosome 13, one form had one females from Kagoshima were homozygous for the A-form
compound-type giant loop on the short arm and one simple- and four were heterozygous for the A- and B-forms.
type giant loop on the long arm; the other had another Inchromosome7, allthefemalesoftheEasternHonshu,
simple-type giant loop on the short arm in addition to these Chubu, and Western Honshu-Shikoku groups had the only
(Fig. 2e). In each of these five chromosomes, the former A-form. On the other hand, the Kyushu group had the
was named the A-form, and the latter was the B-form. B-form at anearlyequalfrequencytothatoftheA-form. It
Based upon the frequencies of the A- and B-forms in was remarkable, however, that all females from Kagoshima
each ofthe 28 collection sites, the R. nigromaculata popula- had the homologous A-form.
tions were divided into four groups, named the Eastern In chromosome 11, the B-form was distributed in a
Honshu, Chubu, Western Honshu-Shikoku, and Kyushu similar manner to the A-form of chromosome 7. The B-
groups, respectively (Fig. 3; Table 1). In chromosome 1, form was found in all the females of the Eastern Honshu,
the A-form was predominant in the Chubu group, while the Chubu, and Western Honshu-Shikoku groups in the homolo-
B-formwaspredominantintheEasternHonshuandWestern gous condition. The Kyushu group involved both the A-
Honshu-Shikokugroups. IntheKyushugroup,thefrequen- and B-forms at nearly equal frequencies.
cy ofthe B-form was a little higher than that ofthe A-form. In chromosome 13, all the females of the Eastern Hon-
Itwasremarkable, however, thatnineofthe 10femalesfrom shu and Chubu groups were homologous for the A-form.
Kagoshima had the homologous A-form. On the other hand, most of the females of the Western
Western Honshu-Shikoku Eastern Honshu
group group
Kyushu ' Chromosome no.
group
/
Form
Chubu
group
Fig.3. FrequenciesoftheA-andB-formoffivelampbrushchromosomes, 1,6,7, 11,and 13,ineachcollectingsite. Areas
partitioned with broken lines represent groups ofR. nigromaculata females with about the same characteristics in their
lampbrush chromosomes. A, Akita. B, Sakata. C, Shibata. D, Joetsu. E, Toyama. F, Kanazawa. G, Fukui.
H, Okaya. I, Sutama. J, Mishima. K, Iida. L, Nagoya. M, Maibara. N, Shingu. O, Tottori. P, Gotsu. Q,
Yamaguchi. R,Hiroshima. S,Konko. T,Himeji. U,Takamatsu. V,Nangoku. W,Matsuyama. X,Munakata.
Y, Sasebo. Z, Kumamoto. a, Oita. b, Kagoshima.
340 H. Ohtani
Table 1. Frequencies ofbivalent chromosomal types in four groups ofR. nigromaculata
Figures in parentheses show expected values based on Hardy-Weinberg hypothesis.
Type HEaosntsehrun Chubu WestSehrinkoHkounshu Kyushu Total
Chromosome 1
AA
31 19 51
( 0.2) (31.0) ( 0.4) ( 9.7)
AB
6 8 22
( 5.7) ( 1.0) ( 9.1) (25.5)
BB 53 47 26 126
(53.2) (0.0) (46.4) (16.7)
Total 59 32 56 52 199
Chromosome 6
AA
12 5 17
(10.1) ( 0.0) ( 1.7)
AB 12 2 9 23
(15.8) ( 2.0) (15.5)
BB 59 8 54 38 159
( 6.1) (54.0) (34.7)
Total 59 32 56 52 199
Chromosome 7
AA
59 32 56 18 165
(14.0)
AB
18 18
(26.0)
BB 16 16
(12.0)
Total 59 32 56 52 199
Chromosome 11
AA
17 17
(11.5)
AB
15 15
(25.9)
BB 59 32 56 20 167
(14.5)
Total 59 32 56 52 199
Chromosome 13
AA
59 32 4 4 99
( 3.0) ( 1.9)
AB 18 12 30
(20.0) (16.2)
BB 34 36 70
(33.0) (33.9)
Total 59 32 56 52 199
Honshu-Shikoku and Kyushu groups were homozygous for found in the A- and B-forms, and the latter in the B-form.
the B-form. In chromosome 13 ofthree females from Beijing, there was a
The lampbrush chromosomesofcontinental R. nigroma- variation which was not found in Japanese R. nigromaculata.
culata were identical in characteristics with those ofJapanese This variation had two compound-type giant loops on the
R. nigromaculata, except forchromosome 1. The character- short and long arms and was named the C-form (Fig. 2e).
istics ofchromosomes 6, 7, 11, and 13, in which Japanese R. Two females were homologous for the C-form and one was
nigromaculata had variations, were ofthe A-form. Howev- heterologous for the A- and C-forms.
er, of the six females from Suwon, two had the homozygous
and heterozygous B-form in chromosome 6 and one was
DISCUSSION
homozygous for the B-form in chromosome 11. Chromo-
some 1 of all the females from Beijing and Suwon had one Nishiokaetal. [12] found that R. nigromaculatacollected
simple-type giant loop on the short arm and one compound- from all over Japan can be divided into four groups by a
type giant loop on the long arm (Fig. 2a). This was named cluster analysisofthe geneticdistances between local popula-
the C-form. Of the two landmarks, the former was also tions. The geographic areas of the four groups are as
Polymorphism of Lampbrush Chromosomes 341
follows: 1) the Tohoku area comprising the prefectures of situated in the Pacific side ofeastern Honshu, are divided in
Aomori, Akita, and Niigata; 2) the Chubu area comprising the dendrograms from those in the other areas. Conse-
the prefectures ofShizuoka, Aichi, Nagano, and Yamanashi; quently, the absence of R. nigromaculata in the Kanto and
3) the Hokuriku, Kinki, Sanyo, Shikoku, and Kagoshima Sendai plains implies that its ancestral population reached
areas comprising the prefectures of Toyama, Ishikawa, Japan through the Korean Peninsula between about 0.5 and
Fukui, Shiga, Mie, Wakayama, Osaka, Hyogo, Okayama, 0.12 million years ago. The area of the north end of the
Hiroshima, Kagawa, Kochi, Ehime, and Kagoshima; and 4) eastern Honshu region could not provide another entrance
the San-in and Kyushu areas comprising the prefectures of because the mountain ranges virtually reached the shore.
Tottori, Shimane, Yamaguchi, Fukuoka, Nagasaki, Kuma- The formation of the Eastern Honshu, Chubu, and
moto, Oita, and Miyazaki. In this study, it was found that WesternHonshu-Shikokugroupsisattributabletotherelease
the spreadofvariationsin five lampbrushchromosomes, 1, 6, ofmigration due tomarine regression duringthe glacial stage
7, 11, and 13, is also coincident with this grouping, though and the geographical isolation due to marine transgression
there were slight discrepancies in dividing lines. In the four during the interglacial stages. The formation ofthe Kyushu
groupsbasedonthe chromosomalvariations, thegeographic- group is attributed to the introgression of new genetic mate-
al area of the Eastern Honshu group comprises the Tohoku rials provided from continental R. nigromaculata which in-
and Hokuriku areas, that of the Chubu group comprises the vadedagainduringtheWurmglacialstagewhentheJapanese
Chubu area, that of the Western Honshu-Shikoku group Islandsweretemporarilyreconnectedwiththe KoreanPenin-
comprises the Kinki, Sanyo, San-in (except Yamaguchi Pre- sula (between about20,000and 18,000yearsago) [14]. This
fecture) and Shikoku areas, and that of the Kyushu group introgression is typified by the presence of the A-form of
comprises the Kyushu area and Yamaguchi and Kagoshima chromosome 11 which is found in continental R. nigromacu-
Prefectures. lata.
Ineachgroup, thefrequenciesoftheformsoflampbrush Ineachofthe lampbrushchromosomes6,7,11 and 13, it
chromosomes agreed well with expected values based on the isquite certain that the A-formisolderthanthe B-form, that
Hardy-Weinberg hypothesis (P>0.05), except for chromo- is, the B-form was derived from the A-form by mutations.
somes 1,6, and 11 ofthe Kyushugroup. Thisseemstoshow The reasons are, firstly, that continental R. nigromaculata is
thatthreegroupsotherthantheKyushugrouparegeographi- of the A-form in these lampbrush chromosomes and the
cally isolated from one another by major mountain ranges. Chubu group, ofwhich the R. nigromaculata migrates to the
The Kyushu group, however, is not partitioned from the furthest area in Japan, also has the A-form in the lampbrush
adjoininggroupbyacleartopographicalobstacle,sinceapart chromosomes 6, 7, and 13. Secondly, the lampbrush
of this group encroaches on the western Honshu area. chromosomes 6, 11 and 13 of R. brevipoda, which diverged
Moreover, this group seems to include a heterogeneous from an ancestral species common to R. nigromaculata [6],
subgroup,becauseinchromosomes 1 and6manyR. nigroma- have the same characteristics as the A-form [10, 13]. In the
culata from Kagoshima had the A-forms which were also twoformsoflampbrush chromosome 1, an age comparison is
found in the Chubu group. The Kyushu group may be difficult because continental R. nigromaculata and R. brevi-
dividedintotwosubgroupsbyincreasingthesitesofexamina- poda have forms that differ from these. However, both
tion further. forms of chromosome 1 somewhat resembled those of
It seems that the estimation of the period when the chromosome 6 in their distribution pattern. Therefore, the
ancestralpopulationofR. nigromaculatainvadedJapangives A-form is also probably older than the B-form in chromo-
an important clue to the process ofthe formation ofthe four some 1.
groups. Japan was a part ofthe continent up to the end of Inview ofthe probable migration course ofR. nigroma-
the Riss glacial stage [4]. It is reasonably certain that the culata and the distributional extent of the B-forms, the
complete separation of Japan occurred in the Riss-Wurm B-form ofeach lampbrush chromosome is presumed to have
interglacial stage (about 0.12 million years ago) [8]. After occurred in the order of chromosomes 11, 1 and 6, 13, and
enteringJapan, R. nigromaculata is supposed to have moved lastly 7. The B-form of chromosome 7 seems to have
along the coastal plains without passing over the highly occurred toward the Wurm glacial stage, because it is very
upheaved mountain ranges; the coastal plains which had similar in distribution pattern to the A-form of chromosome
widened owing to marine regression in the glacial periods 11. The B-form of chromosome 13 probably occurred be-
were more favorable. The population which had reached fore the Wurm glacial stage and spread in this stage, judging
theChubuarea, however, couldnotgofurtherthanthisarea. from the absence in the Eastern Honshu and Chubu groups.
The reason is that a collision between the central Honshu This is because the western Honshu, Shikoku, and Kyushu
region and Izu island (presently a peninsula) broke down the areas united during the Wurm glacial stage, since the Inland
coastalplain (about0.5 millionyearsago) [7]. The difficulty Seadriedupowingtomarineregression [4]. TheB-formsof
ofmigrationinthisregionisevidencedbytheclusteranalyses chromosomes 1 and 6 probably spread during the glacial
ofgenetic distances in the populations ofRana rugosa, Rana stages before the Wurm glacial stage. The B-form of
japonica, and Rana brevipoda [9, 11, 12]. The populations chromosome 11 spreadatthebeginningofthemigrationof/?.
of these species in the Kanto and Sendai plains, which are nigromaculata.
342 H. Ohtani
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