Reintroduction（2011）1:77 – 80

Yoshihiro Yamamoto: New mitochondrial haplotypes of Oriental white stork

SHORT COMMUNICATION

New haplotypes in the mitochondrial control region of Oriental
White Storks, Ciconia boyciana* Yoshihiro Yamamoto1
Abstract Previous studies have shown 17 haplotypes ingenome of the Oriental White Stork has been determined

the mitochondrial D-loop region of Oriental White Stork,(Accession number AB₀₂₆₁₉₃), and haplotypes in theCiconia boyciana, in Japan (Yamamoto et al. 2000; Murata

control region without repeated sequences (₁,₂₀₃ bp) haveet al. 2004). We found 7 additional haplotypes while ana-been analyzed (Yamamoto et al. ₂₀₀₀). Nine haplotypes

lyzing 11 samples; 2 mounted skin specimens, 5 blood

were found in captive Oriental Storks (Yamamoto et al.

samples and 4 fallen feathers. Altogether, 24 haplotypes

₂₀₀₀), while an additional ₈ have been reported from cap-have been identiﬁed in the Japanese population from live

tive Oriental White Storks and stuffed samples (Murata etcaptive individuals and wild white storks, and stuffedal. ₂₀₀₄). Therefore, ₁₇ Oriental White Stork haplotypesspecimens. The haplotype diversity (h) and the nucleotide

were known previously. In this study, I analyzed ₂diversity (π) of Japanese captive and re-introduced Orien-

mounted skin specimens (SCB₆₀₁ & SCB₆₀₂), ₅ bloodtal White Storks in 2010 were 0 . 8319±0 . 0113 andsamples (Stud number ₁₉, ₂₁, ₂₀₈, ₈₁₈ and ₈₁₉), and ₄0.00357±0.00196, respectively. The population size offeather roots, and I was able to identify ₇ novel haplo-captive white storks in Japan has increased in recenttypes. The ₄ feather samples (FCB₄₁₂, FCB₄₁₄, FCB₄₁₅

years, however, the haplotype distribution among breed-and FCB₆₀₃) were derived from wild Oriental Whiteing centers should be improved. I suggest that new found-Storks during the wintering season in Japan.

ers should be introduced from Russian or Chinese wild

Whole cellular DNA was extracted from blood by thepopulation.

standard method with protease K and phenol (SambrookKey words Mitochondria, Haplotype, D-loop, Oriental

et al. ₁₉₈₉). DNA extraction from feather roots was per-White Stork, Ciconia boyciana

formed with the DNeasy Blood & Tissue Kit (QIAGEN).
The PCR conditions and sequencing methods were theThe re-introduction of Oriental White Storks in thesame as described previously (Yamamoto et al. ₂₀₀₀;

Toyooka-city began on April ₂₄ in ₂₀₀₅. The last wild

Murata et al. ₂₀₀₄). Three PCR primer sets (STM₃ &Oriental White Stork had been captured ₃₅ years ago for

STM₄; STM₅ & STM₆; STM₇ & STM₈) were used tobreeding. The re-introduction was possible because of thecover ₁,₂₄₈ bp which corresponds to the control region

recovery of some suitable habitats in Toyooka, and(D-loop) excluding the repeated sequences. Combined

because of the increased number of captive storks follow-

nucleotide sequences of ₁,₂₁₀ bp without overlaps anding successful breeding in Hyogo Park of the Oriental

primer sequences were treated as haplotype sequences

White Stork. In breeding programs, ₂ points should be(Yamamoto et al. ₂₀₀₀), and were analyzed further. Allconsidered to prevent a decline in the genetic diversity ofdetermined sequences were compared and analyzed using

captive stork populations. The first is that founders

the GENETYX software (GENETYX Co., Tokyo, Japan).

belonging to the same mitochondrial haplotype should not

Seven new haplotypes (₁₈–₂₄) were found after a com-form breeding pairs because they might be relatives. The

parison of ₁₁ nucleotide sequences, and they have been

second is that the pedigree status of the offspring should

listed with their nucleotide differences in Table ₁. The

be monitored to avoid inbreeding.

previously reported haplotypes (₁–₁₇) are also shown in

The entire nucleotide sequences of the mitochondrial
₁Department of Genetics, Hyogo College of Medicine, ₁-₁
Mukogawa-cho, Nishinomiya, Hyogo, ₆₆₃-₈₅₀₁ Japan* E-mail:

Table ₁. Nucleotide polymorphisms are present at ₃₄ sites,and ₂₄ haplotypes have been determined. Haplotypes ₁₈,₁₉, and ₂₀ were found in captive storks. Haplotype ₂₁

contains ₃ unique polymorphic sites and a deletion of ₃77

78

－－－

A

G－－－A－

AC
A
A
CAA

A

A
AA
A
A
A
A

－－－A
A－－－－－－－－－C－

G－C－－T－－－－－－－－－－T－－－－－－－－－A－－－－－－－GGG－－－－－－－－－－G－－－－－G－－－－－－－－－－－－－－－－－－－－TT－－－－－－－－－－－－－－－－－－CT－－－－－C－－－－－－－－－－－T－－TT－－CTA－－－－－－－－－－

GG
GG－－－－－－－－－G－－－－－－－－－－AAAA－A－－－－－－－T－－－C－－－－－－－－－－－－－－－－－－－G－－－－－－－－－－AAAA－－－－－－－－－G－－－－A－－－－－－－－－－－－－－－－－－T－C－C－－CCCC－－－－C－－CC－C－－G－－－A－－－－－－－－－－－－－－－－－－－G－－－－－A－－－－－－－－－－－－－－－

A－T－－－G－－－－－－－－－－－－－－－－－－－A－－－－－G－－－－－－－－－－－－－－－G－T－

CCG－－－－GG－－－－－－－－－－C
G－A－－GG－G－－－－－－－－G－G－－－－G－

A－－－－－－－GG

G－－－－－－－－－－G－－－－－－－－－－－－－－－－－－－－－－－－

G
A

G－－－－－－－－－－－

A
A
A－－－－－－－－－－

C
C
C
C
C
C
C
C

C

G
CC
C
C
C
C
C
C
C
C
C
C
C

A－－－－－G－－－－－－－－－－－－－－－G－G－－－－－－A－－－－－－－－－－－－－－－－G－－AA－－－－－－－－－－A－A－－－－－－T－C－－－－－－CC－－－－－－－－－－C－－A－

C－－－－－－－－－－－－－－－－－－－－－T－C－－－－－－－－－－－－－－－－－－－－－C－T－－－－－－－T－－－－－－－－－－－－－T－－－－－－－CC－C－－－－－－－－－－－－T－－－－－－－－－－－C－C－－－－－－－－－

₁₉
₂₁
₂₀₈
₈₁₈, ₈₁₉
FCB₄₁₄
FCB₆₀₃
SCB₆₀₂

(A)
(A)
(A)
(A)
(A)
(A)
(A)
(A)
(A)
(B)
(B)
(B)
(B)
(B),
SCB₆₀₁(B),
FCB₄₁₂,
FCB₄₁₅(B)(B)

sampleb)

₂₂

₂

₂₄

₃₁
₄₃
₁₆
₁₈
₃₀
₆₉Nc)

b)

Polymorphic sites found within a ₁,₂₁₀ bp region of the mitochondrial genome of Oriental white storks are displayed (Accession number AB₀₂₆₁₉₃).
Samples analyzed in this paper are indicated by their individual numbers. Previously reported haplotypes are indicated by (A) or (B). (A) Yamamoto et al. ₂₀₀₀. (B) Murata et al. ₂₀₀₄.
c)
The numbers of live captive and wild Oriental white storks belonging to each haplotype in Japan. The haplotype diversity (h) and nucleotide diversity (π) were estimated using Arlequin (Schneider etal. ₂₀₀₀).

a)－－－－－－－－－

₁₆
₁₇
₁₈
₁₉
₂₀
₂₁
₂₂
₂₃
₂₄

C

AAA
A－A－－－－－－－－－

A－A－－－ACA－－－－－－－－－T－－－－－－－

G
A

₁₅₁₂₁₃₁₄₁₂₃₄₅₆₇₈₉₁₀₁₁TYPE ₁₄₆ ₁₈₁ ₁₈₉ ₂₀₃ ₂₂₂ ₂₃₇ ₂₈₉ ₂₉₂ ₃₀₇ ₃₁₂ ₃₁₈ ₃₁₉ ₃₃₄ ₃₃₆ ₃₄₂ ₃₄₈ ₃₅₁ ₃₅₂ ₃₆₃ ₃₆₄ ₃₇₁ ₃₉₃ ₃₉₆ ₄₁₇ ₄₄₆ ₄₆₃ ₅₀₅ ₅₄₆ ₇₅₆ ₉₃₅ ₉₉₅ ₁₀₀₀ ₁₁₄₂ ₁₁₄₅

Table ₁. Nucleotide differences and haplotypes of the Oriental white storks in the control region (D-loop) of the mitochondrial genome.a)

Reintroduction（2011）1:77 – 80

Yoshihiro Yamamoto: New mitochondrial haplotypes of Oriental white stork

bp between ₃₅₄ and ₃₅₆. It is interesting that haplotype ₂₁

has been recorded that ₁₈₃ storks were in captivity in

was only found in specimens ₈₁₈ and ₈₁₉, which wereJapanese zoos in ₂₀₁₀ (Regional studbook for the Oriental

captured from the same nest in Khabarovsk and wereWhite Stork ₂₀₁₀). Mitochondrial haplotypes of almost alltransferred to Japan in ₂₀₀₃. These individuals had been

captive individuals had been identified and recorded,transferred in order to increase the nucleotide diversity

including those of the re-introduced (wild) white storks.

within the captive Japanese population of Oriental White

The individual numbers of ₁₀ haplotypes in captive andStorks. It is conceivable that these individuals also con-wild Oriental White Storks in Japan are listed in Table ₁.

tain nucleotide differences in nuclear DNA relative toThe genetic diversity of the Japanese population in ₂₀₁₀

other captive birds, making them suitable for the purpose

was estimated to be ₀.₈₃₁₉±₀.₀₁₁₃ for haplotype diver-of introduction.

sity (h) and ₀.₀₀₃₅₇±₀.₀₀₁₉₆ for nucleotide diversity (π)

Haplotype analysis of ₄ wild Oriental White Stork

using Arlequin ₂.₀₀₀ (Schneider et al. ₂₀₀₀). The lowfeathers revealed ₂ new haplotypes; ₂₂ and ₂₃. Haplotype

nucleotide diversity is considered to be attributable to the

₂₂ was found in feather FCB₄₁₄, which was obtained insmall number of haplotypes, which is present within thethe Chiba Prefecture in ₂₀₀₅, while haplotype ₂₃ wascaptive population. Although the genetic diversity of thefound in FCB₆₀₃, which was collected at Okinoshima inJapanese population in ₂₀₁₀ was relatively lower than that

the Shimane Prefecture in ₂₀₀₆. Two additional feathers,of the Chinese population (Zan et al. ₂₀₀₈), it was higher

FCB₄₁₂ (collected at the Kameoka, Kyoto Prefecture inthan that reported for threatened or near-threatened avian₂₀₀₄) and FCB₄₁₅ (collected in the Chiba Prefecture inpopulations such as the Red-crowned crane population of₂₀₀₅), were found to belong to haplotype ₁₅. This haplo-

East Asia population (Hasegawa et al. ₁₉₉₉; h is ₀.₇₈) ortype was previously reported in ₃ mounted specimens,

the Resplendent Quetzal (Solorzano et al. ₂₀₀₄; h is

SS₁₃, SS₁₄ and SS₁₅, stored in the Izushi Village near the₀.₈₁₈₀).

Toyooka city (Murata et al. ₂₀₀₄). It is remarkable thatA minimum-spanning network among ₂₄ haplotypes ofOriental White Storks with the same haplotype ₁₅ as indi-

the Oriental White Stork was estimated using the TCS₁.₂₁

viduals that visited in the past are still coming to Japansoftware (Clement et al. ₂₀₀₀), as shown in Fig. ₁. Thesefor wintering. Haplotype ₂₄ was found in a mounted spec-

haplotypes were obtained from a mixed population con-imen with no available information stored at Museum oftaining captive storks, mounted specimens and fallen

Nature and Human Activities, Hyogo.

feathers of wild birds. Therefore, the circle size is ﬁxed

The genetic structure of Chinese populations of Orien-

because it is not meaningful to compare the number oftal White Storks in new breeding areas was recently

individuals or samples belong to each haplotype. Fifteen

described by Zan et al. (₂₀₀₈). New breeding colonies

haplotypes were found within the captive Japanese popu-

were found in wintering areas and at some stopover sites.

lation, while ₄ and ₉ haplotypes were reported from wild

Zan et al. (₂₀₀₈) reported that ₃₇ haplotypes were foundand mounted specimens, respectively. In addition, ₅ hap-in ₆₆ individuals from the southern and northern breedingareas, and from zoo colonies. It has also been noted that
the Chinese population has greater genetic diversity thanthe Japanese population, with a haplotype diversity (h) of
₀.₉₅₃±₀.₀₁₃ and a nucleotide diversity (π) of
₀.₀₁₃±₀.₀₀₇. However, since previously reported haplo-types and haplotype frequencies within the Japanese population (Yamamoto et al. ₂₀₀₀; Murata et al. ₂₀₀₄) were
determined from captive and mounted specimens, it may
not be appropriate to compare these values directly.
Regional and International Studbooks for the Oriental
White Stork have been published every year by the Japanese
Association of Zoological Gardens and Aquariums, and itFig. ₁. Haplotype network of the Japanese population of Oriental
white storks.
79

Reintroduction（2011）1:77 – 80

lotypes, ₇, ₈, ₉, ₁₅ and ₁₉ have been recently lost by deathwithin the captive Japanese population. Therefore, individuals belong to these haplotypes are absent in Table ₁.
The rest ₁₀ haplotypes contained ₁₈₃ individuals in captivity in ₂₀₁₀ (Regional studbook for the Oriental White
Stork ₂₀₁₀). However, the distribution of haplotypes ofthe captive population is biased toward the Japanese zooand Hyogo Park of the Oriental White Stork. For instance,each one individual belong to haplotype ₃ and ₄ is rearedin Hyogo Park of the Oriental White Stork, while ₁₅ for
haplotype ₃ and ₁₇ for haplotype ₄ in other Japanese zoo.
Exchanges of individuals between breeding centers andintroduction of new founders from the Russian and/orChinese wild populations are required to increase thegenetic diversity of the Japanese captive population ofOriental White Storks. The re-introduction program ofWhite Stork, Ciconia boyciana (₂₀₀₀) Tama ZoologicalPark, Hino.
Sambrook J, Fritsch EF, Maniatis T (₁₉₈₉) Molecular Cloning—a Laboratory Manual, ₂nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. ₄₅₈ – ₄₅₉.
Schneider S, Roessli D, Excofﬁer L (₂₀₀₀) Arlequin: A software
for population genetics data analysis. Ver. ₂.₀₀₀. Genetics
and Biometry Lab, Dept. of Anthropology, University ofGeneva.
Solórzano S, Baker AJ, Oyama K (₂₀₀₄) Conservation priorities
for resplendent quetzals based on analysis of mitochondrial
DNA control region sequences. Condor, ₁₀₆: ₄₄₉ – ₄₅₆.
Yamamoto Y, Murata K, Matsuda H, Hosoda T, Tamura K,Furuyama J (₂₀₀₀) Determination of the complete nucleotide
sequence and haplotypes in the D-loop region of the mitochondrial genome in the Oriental White Stork, Ciconia boyciana. Genes and Genetic Systems, ₇₅: ₂₅ – ₃₂.
Zan S, Zhou L, Jiang H, Zhang B, Wu Z, Hou Y (₂₀₀₈) Genetic
structure of the Oriental White Stork (Ciconia boyciana):
implications for a breeding colony in a non-breeding area.
Integrative Zoology, ₃: ₂₃₅ – ₂₄₄.

Oriental White Storks is now successfully in progress.

(Accepted: ₂₉ September ₂₀₁₁)

Additional studies will be required to assess the genetic
diversity of captive and wild populations by using nuclear
DNA in addition to mitochondrial DNA.

ニホンコウノトリにおける新しいミトコンドリアのハプ
ロタイプ

Acknowledgement
I am grateful to M. Sato and Y. Mitsuhashi from Hyogo
Park of the Oriental White Stork for assistance with sampling of the Oriental White Stork populations. I would
also like to thank S. Satake from NPO Sicchi Net for his
support during the sampling.

* 山本義弘1₁

兵庫医科大学遺伝学講座
₆₆₃-₈₅₀₁

兵庫県西宮市武庫川町₁-₁* E-mail:
ニホンコウノトリ（Ciconia boyciana）の遺伝的多様性については，ミトコンドリアの D-loop 領域のハプロタイプの解析により₁₇の遺伝系統が見いだされていた．

References
Clement M, Posada D, Crandall KA (₂₀₀₀) TCS: a computer
program to estimate gene genealogies. Molecular Ecology,
₉: ₁₆₅₇ – ₁₆₆₀.
Hasegawa O, Takada S, Yoshida MC, Abe S (₁₉₉₉) Variations ofmitochondrial control region sequences in three crane species, the Red-crowned Crane Grus japonensis, the Common
Crane G. grus and the Hooded Crane G. monacha. ZooScience, ₁₆: ₆₈₅ – ₆₉₂.
Murata K, Satou M, Matsushima K, Satake S, Yamamoto Y(₂₀₀₄) Retrospective estimation of genetic diversity of anextinct Oriental White Stork (Ciconia boyciana) populationin Japan using mounted specimens and implications for reintroduction programs. Conservation Genetics, ₅: ₅₅₃ – ₅₆₀.
Tama Zoological Park (₂₀₀₀) Regional Studbook for the Oriental80

今回の分析では，₁₁の解析用試料，すなわち ₂ 体の剥製， ₅ 体の血液試料，及び ₄ 本の羽根を調べることにより，新たに ₇ つのハプロタイプを見いだした．従って日本のコウノトリのハプロタイプは₂₄となった．しかしながら，遺伝的多様性の解析ではハプロタイプ多様度（₀.₈₃₁₉±₀.₀₁₁₃）は他種集団と比較しても低くはなかったが，ヌクレオチド多様度（₀.₀₀₃₅₇±₀.₀₀₁₉₆）が低い値を示した．総飼育個体数は増加しているので，飼育施設間でのハプロタイプの偏りを解消することに加えて，ロシアや中国からの新しい血統の導入が望まれる．
キーワード

ミトコンドリア，ハプロタイプ， D -ループ，コウノトリ，Ciconia boyciana