Biological Journal of the Linnean Society, 2010, 99, 570–581. With 4 figures
Multiple origins of Locusta migratoria (Orthoptera:
Acrididae) in the Japanese Archipelago and the
presence of two major clades in the world:
evidence from a molecular approach
MAKOTO TOKUDA1*, SEIJI TANAKA1 and DAO-HONG ZHU2
1
Locust Research Laboratory, National Institute of Agrobiological Sciences at Ohwashi, 305-8634
Tsukuba, Ibaraki, Japan
2
Laboratory of Entomology, College of Resources and Environment – Central South Forestry
University, Changsha, 412005 Hunan, China
Received 4 August 2009; accepted for publication 2 October 2009
bij_1386
570..581
Phylogenetic relationships among migratory locust (Locusta migratoria) populations in different climatic regions
were analysed by sequencing four mitochondrial DNA regions, with special reference to the origin of Japanese
populations. The populations are clearly separated into two clades: one consists of individuals from temperate and
cold-temperate areas of Japan and the Chinese continent, and the other comprises those from subtropical islands
of Japan, Hainan Island in China, Timol Leste, Australia, Ethiopia, France, and some individuals from Tsushima
Island and Honshu of Japan. The divergence time between the two clades is estimated to be 0.86–1.89 Mya. The
phylogenetic analysis revealed that Japanese L. migratoria populations were composed of individuals of six
different origins: (1) Hokkaido populations possibly from the Russian continent; (2) Honshu–Kyushu populations
from the Chinese continent; (3) Southwest Island populations from Hainan Island or adjacent areas; (4) Ogasawara
populations that might have originated from Micronesia; (5) part of the Tsushima population that originated from
somewhere in the Asian tropics; and (6) a possible relict population of ancient southern haplotypes that exists in
western areas of northern Honshu. The Tsugaru Straits and Tokara Straits have acted as effective geographical
barriers, as in other organisms, isolating locust populations for a few thousand years. © 2010 The Linnean Society
of London, Biological Journal of the Linnean Society, 2010, 99, 570–581.
ADDITIONAL KEYWORDS: biogeography – migratory locust – mitochondrial DNA – molecular phylogeny.
INTRODUCTION
Certain locusts such as the migratory locust Locusta
migratoria L. (Orthoptera: Acrididae: Oedipodinae)
and the desert locust Schistocerca gregaria Forskål
(Acrididae: Cyrtacanthacridinae) are potentially the
most destructive agricultural pest insects worldwide.
A notable trait of both species is density-dependent
phase polyphenism, which involves graded changes in
*Corresponding author. Current address: Center for
Research and Advancement in Higher Education, Kyushu
University, Fukuoka 819-0395, Japan.
E-mail: [email protected]
570
morphological, physiological, and behavioural traits
(Uvarov, 1966, 1977; Pener, 1991). At low population
density, individuals are solitary and sedentary (solitarious phase), whereas, at high population density,
they become gregarious, travelling long distances in
massive swarms (gregarious phase). This trait is considered to be important in distribution patterns and
population dynamics of these insects (Lovejoy et al.,
2006; Chapuis et al., 2008).
Locusta migratoria is distributed widely from the
tropics to cold-temperate zones (Uvarov, 1966, 1977;
Farrow & Colless, 1980) in the Old World and populations alternate between reproductive isolation in
the solitarious phase and massive gene flow in the
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
PHYLOGENY OF THE MIGRATORY LOCUST
gregarious phase. It is therefore interesting to elucidate the evolutionary history of L. migratoria, including the processes of geographic expansion and genetic
homogenization that should follow the gregarious
phase, and local isolation and adaptation in the solitarious phase.
Locusta migratoria occurs commonly throughout
the Japanese archipelago where occasional outbreaks
have been recorded at various localities (Miyashita,
1963). Recent outbreaks, however, have occurred at a
small scale and have been limited to small islands
and artificially created grasslands, such as those
found at airports and on reclaimed land (Ito & Yamagishi, 1976; Yamagishi & Tanaka, 2009). Locusta
migratoria shows latitudinal variation in voltinism
and various physiological traits in Japan (Hakomori
& Tanaka, 1992; Tanaka, 1994a, b). The life cycle is
univoltine in the north, bivoltine in central Japan,
and multivoltine in the south, as observed on the
European and Chinese continents (Verdier, 1972;
Tanaka & Zhu, 2008). Although the local adaptations
of this locust have been intensively studied, little
information is available regarding their genetic structure and variation, as well as the origin of the
Japanese populations.
To reveal biogeographical aspects of the migratory
locust in the Japanese archipelago, we analysed mitochondrial DNA sequences and examined the phylogenetic relationships among L. migratoria populations.
Most locust samples were collected at various localities within Japan, but some were obtained from other
countries on different continents to elucidate the
origin of the Japanese locusts.
MATERIAL AND METHODS
Locusta migratoria specimens were collected at
various locations primarily between 2005 and 2008,
for molecular phylogenetic analysis (Table 1). They
were kept at -80 °C, or in pure ethanol or acetone
until DNA extraction (Fukatsu, 1999). Two
laboratory-reared strains, originally collected on
Okinawa Island, Japan in March 1990 (Tanaka, 1993)
and an African strain collected in Ethiopia (J. Huybrechts, personal communication), were also included
in the analysis. A single individual was chosen from
each strain for DNA extraction. Voucher specimens
will be deposited at Kyushu University, Fukuoka,
Japan (Table 1).
An adult of S. gregaria from a laboratory-reared
strain was used in the analysis as an outgroup taxon.
This strain was originally collected in Ethiopia
(Tanaka & Yagi, 1997; Maeno & Tanaka, 2004). Two
species belonging to the subfamily Oedipodinae,
Aiolopus thalassinus tamulus (Fabricius) collected on
Miyako Island, Okinawa, Japan in January 2008, and
571
Oedaleus infernalis Saussure collected in Tsukuba,
Ibaraki, Japan in October 2006, were also included in
the analysis (Table 1).
Total DNA was extracted from femoral muscle
tissue of a middle or hind leg of adults, fifth or third
instars, or the whole body of first instars using the
DNeasy Blood and Tissue Kit (Qiagen). Portions of
four mitochondrial genes, cytochrome b (cyt b), cytochrome oxidase subunit I (COI), NADH dehydrogenase subunit II (ND2), and 12S ribosomal RNA (12S),
were amplified by the polymerase chain reaction
(PCR) as described by Yukawa et al. (2003) and purified using the QIAquick PCR Purification Kit
(Qiagen). The sequencing reaction was performed
using PCR primers and the BigDye Terminator Cycle
Sequencing Reaction Kit (Applied Biosystems) and
was electrophoresed on an ABI 3100 sequencer
(Applied Biosystems).
The primers used in the analysis were: CB9 5′-GCC
GAG ACG TGA ATA ATG GAT-3′ (Litzenberger &
Chapco, 2001) and *CBN 5′-TAA CTC CTC CTA ATTT
ATT AGG AAT-3′ (modified from the CBN primer
sensu Simon et al., 1994; following the sequence data
of Locusta migratoria migratorioides identified by
Flook, Rowell & Gellissen, 1995) for the cyt b gene;
LCO1490 5′-GGT CAA CAA ATC ATA AAG ATA TTG
G -3′ and HCO2198 5′-TAA ACT TCA GGG TGA CCA
AA AAT CA-3′ (Folmer et al., 1994) for the COI gene
of S. gregaria, which is used in the DNA barcoding
project of animals (Hebert et al., 2003); and
*LCO1490 5′-TCT CAA CAA ACC ACA AGG ACA
TTG G-3′ and *HCO2198 5′-TAA ACT TCT GGG TGA
CCA AG AAT CA-3′ (modified from the LCO1490 and
HCO2198 primers following the sequence data of L.
migratoria migratorioides identified by Flook et al.,
1995) for the COI gene of L. migratoria, A. thalassinus tamulus, and O. infernalis; *ND2A 5′-CGT TGA
TGA TAG GAA CGT ACC-3′ and *ND2B 5′-GGT GTC
TAA TTG ATG ATT ATG C-3′ (Litzenberger & Chapco,
2001) for ND2; and SR-J-14199 5′-TAC TAT GTT ACG
ACT TAT-3′ and SR-N-14594 5′-AAA CTA GGA TTA
GAT ACC C-3′ (Kambhampati & Smith, 1995) for
12S. These primers, respectively, amplified a 620-bp
fragment of cyt b, a 658-bp fragment of COI, a 467 bp
fragment of ND2, and a 419–432 bp fragment (419–
421 bp for L. migratoria and O. infernalis, 430 bp for
A. thalassinus tamulus, and 432 bp for S. gregaria) of
12S regions. The nucleotide sequence data reported in
the present study are deposited in the DNA Data
Bank of Japan (DDBJ), European Molecular Biology
Laboratory, and GenBank nucleotide sequence databases under the DDBJ accession numbers shown in
Table 1.
Nucleotide sequences of cyt b, COI, and ND2 were
easily aligned because they contained no gaps, and
those of 12S were aligned by CLUSTALX (Thompson
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
141.42°E
140.33°E
141.08°E
141.13°E
139.82°E
138.27°E
139.93°E
129.32°E
130.33°E
43.00°N
42.93°N
40.50°N
39.92°N
39.78°N
38.63°N
37.10°N
36.92°N
36.05°N
34.95°N
34.90°N
34.43°N
34.25°N
33.55°N
33.10°N
32.88°N
32.87°N
2 Hitsujigaoka,
Sapporo, Hokkaido
Japan
3 Jozankei, Sapporo,
Hokkaido, Japan
4 Hirosaki, Aomori,
Honshu, Japan
5 Hachimantai,
Iwate, Honshu,
Japan
6 Morioka, Iwate,
Honshu, Japan
7 Tsuruoka,
Yamagata,
Honshu,
Japan
8 Joetsu, Niigata,
Honshu, Japan
9 Nishinasu,
Tochigi, Honshu,
Japan
10 Tsukuba, Ibaraki,
Honshu, Japan
11 Shizuoka, Honshu,
Japan
12 Sanda, Hyogo,
Honshu, Japan
13 Kansai IAP,
Osaka, Honshu,
Japan
14 Tsushima Is.,
Nagasaki, Japan
15 Fukuoka City,
Fukuoka, Kyushu,
Japan
16 Hachijo Is., Tokyo,
Japan
17 Koshi, Kumamoto,
Kyushu, Japan
18 Isahaya, Nagasaki,
Kyushu, Japan
130.12°E
130.73°E
139.78°E
135.22°E
135.2°E
138.37°E
140.08°E
141.15°E
141.68°E
43.05°N
1 Yubari, Hokkaido,
Japan
Longitude
Latitude
Number and locality
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Adult
Third
instar
Adult
and fifth
instar
Adult
Stage
October
2008
October
2007
September
2006
October 2007
July &
September
2008
August 2007
October 2007
September
2007
October 2007
September
2007
October 2007
August 2008
August 2008
August 2008
September
2007
NGS
KMM
HCJ
3
3
3
3
8
TSM
FKK
3
3
3
3
3
3
3
3
3
3
2
4
3
Number of
individuals
examined
OSK
HYG
SZK
IBR
TCG
NGT
YMG
IWM
IWH
AOM
HKJ
HKS
September
2008
September
2008
HKY
Code
August 2008
Collection
date
Y. Ikesaki
N. Endo
S. Tanaka
M. Tokuda &
D. Yamaguchi
S. Tanaka &
K. Maeno
S. Tanaka & K.
Maeno
H. Nakamine
Y. Hirai
S. Tanaka
S. Tanaka, M.
Tokuda &
K. Maeno
H. Higuchi
K. Tanaka
M. Sakakibara
K. Tanaka
Y. Ando
S. Tanaka
S. Tanaka
Y. Hashimoto
Collectors
AB497139–41
AB497136–38
AB497133–35
AB497130–32
AB497122–29
AB497119–21
AB497116–18
AB497113–15
AB497264–66
AB497261–63
AB497258–60
AB497255–57
AB497247–54
AB497244–46
AB497241–43
AB497238–40
AB497235–37
AB497232–34
AB497107–09
AB497110–12
AB497229–31
AB497226–28
AB497223–25
AB497220–22
AB497217–19
AB497215–16
AB497211–14
AB497208–10
COI
AB497104–06
AB497101–03
AB497098–100
AB497095–97
AB497092–94
AB497090–91
AB497086–89
AB497083–85
cyt b
AB497389–91
AB497386–88
AB497383–85
AB497380–82
AB497372–79
AB497369–70
AB497366–68
AB497363–65
AB497360–62
AB497357–59
AB497354–56
AB497351–53
AB497348–50
AB497345–47
AB497342–44
AB497340–41
AB497336–39
AB497333–35
ND2
Data Bank of Japan accession number
Table 1. Collection information for Locusta migratoria specimens and outgroup taxa used in phylogenetic analysis
AB497514–16
AB497511–13
AB497508–10
AB497505–07
AB497497–504
AB497494–96
AB497491–93
AB497488–90
AB497485–87
AB497482–84
AB497479–81
AB497476–78
AB497473–75
AB497470–72
AB497467–69
AB497465–66
AB497461–64
AB497458–60
12S
Loc128–130
Loc019, 064, 065
Loc143–145
Loc015, 062, 063
Loc107–109, 136–140
Loc013, 058, 059
Loc014, 060, 061
Loc012, 056, 057
Loc011, 054, 055
Loc009, 050, 051
Loc010, 052, 053
Loc125–127
Loc119–121
Loc122–124
Loc008, 048, 049
Loc114–115
Loc110–113
Loc116–118
Specimen voucher
number
572
M. TOKUDA ET AL.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
142.20°E
116.35°E
E 114.35°E
3.17°E
127.90°E
27.07°N
25.82°N
24.78°N
47.26°N
40.45°N
38.50°N
35.25°N
35.02°N
19.14°N
9.85°S
20.65°S
43.16°N
26.66°N
22 Chichijima,
Ogasawara Isls,
Tokyo, Japan
23 Minami-daito Is,
Okinawa, Japan
24 Miyako, Okinawa,
Japan
25 Jiminay, Xinjiang,
China
26 Huludao,
Liaoning, China
27 Dagan, Tianjin,
China
28 Jining, Shandong,
China
29 Fengqui, Henan,
China
30 Baisha, Hainan
Is., China
31 Timor Is., Timor
Leste
32 Queensland,
Australia
33 Narbonne-Plage,
France
34 Okinawa Is.,
Okinawa, Japan
(laboratory
population)
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
Adult
Adult
Aiolopus
thalassinus tamulus
Oedaleus infernalis
October 2006
January
2008
AFR
Adult
FRN
AUS
TML
HND
HNN
SDN
TNJ
LNN
JMN
MYK
MDT
OGS
IHY
AMM
MYZ
OKN
June 2003
March 2007
May 2007
November
2005
August 2004
August 2003
August 2003
August 2003
August 2004
February
2008
January
2008
November
2007
October 2006
September
2007
October
2007
First
instar
Adult
Adult
Adult
Adult
First
instar
First
instar
First
instar
First
instar
First
instar
Adult
Adult
Adult
141.73°E
109.27°E
E 114.25°E
117.30°E
120.51°E
85.53°E
125.28°E
131.23°E
Adult
Adult
Adult
Adult
Schistocerca
gregaria
Outgroup taxa
35 Ethiopia
(laboratory
population)
127.87°E
27.50°N
21 Iyeha is., okinawa,
japan
129.30°E
28.27°N
20 Amami Is.,
Kagoshima, Japan
131.42°E
31.93°N
19 Miyazaki City,
Miyazaki, Kyushu,
Japan
1
1
1
1
1
5
5
5
5
5
5
5
5
5
1
4
5
3
3
3
S. Tanaka
M. Tokuda
A. Foucart &
M. P.
Chapuis
P. Spurgin
P. Spurgin
D.-H. Zhu &
S. Tanaka
D.-H. Zhu &
S. Tanaka
D.-H. Zhu
D.-H. Zhu
D.-H. Zhu
D.-H. Zhu &
S. Tanaka
S. Tanaka
S. Tanaka
S. Sugiura
M. Yamagishi
& S. Tanaka
K. Kanai
Y. Shintani
AB497591
AB497587
AB497583
AB497207
AB497592
AB497588
AB497584
AB497332
AB497331
AB497326–30
AB497201–05
AB497206
AB497321–25
AB497316–20
AB497311–15
AB497306–10
AB497301–05
AB497520–22
AB497517–19
AB497416–20
AB497411–15
AB497410
AB497406–09
AB497401–05
AB497593
AB497589
AB497585
AB497457
AB497456
AB497451–55
AB497446–50
AB497441–45
AB497436–40
AB497431–35
AB497426–30
AB497594
AB497590
AB497586
AB497582
AB497581
AB497576–80
AB497571–75
AB497566–70
AB497561–65
AB497556–60
AB497551–55
AB497546–50
AB497541–45
AB497536–40
AB497535
AB497531–34
AB497526–30
AB497398–400 AB497523–25
AB497395–97
AB497392–94
AB497296–300 AB497421–25
AB497291–95
AB497286–90
AB497285
AB497281–84
AB497276–80
AB497273–75
AB497270–72
AB497267–69
Ab497196–200
AB497191–95
AB497186–90
Ab497181–85
AB497176–80
AB497171–75
AB497166–70
AB497161–65
AB497160
AB497156–59
AB497151–55
AB497148–50
AB497145–47
AB497142–44
Loc080
Loc084
Loc023
Loc085
Loc005
Loc131–135
Loc097–100, 142
Loc102–206
Loc092–096
Loc021, 040–043
Loc002, 028–031
Loc004, 036–039
Loc003, 032–035
Loc001, 024–027
Loc083
Loc068–071
Loc006, 044–047
Loc146–148
Loc017, 072, 073
Loc020, 066, 067
PHYLOGENY OF THE MIGRATORY LOCUST
573
574
M. TOKUDA ET AL.
et al., 1997). The sequence data were analysed with
the Neighbour-joining (NJ) method using MEGA,
version 3.1 (Kumar, Tamura & Nei, 2004), the
maximum parsimony (MP) method using PAUP*
4.0b10 (Swofford, 2002), and the Bayesian analysis
using MrBayes, version 3.1.2 (Ronquist & Huelsenbeck, 2003). Evolutionary distances for the NJ
method were computed by Kimra’s two-parameter
distances (Kimura, 1980). In the MP method, the
most parsimonious trees were determined under the
heuristic search procedure with tree bisection–
reconnection branch swapping algorithm and gaps
are treated as ‘missing data’. Bootstrap analyses
(Efron, 1982; Felsenstein, 1985) of 1000 pseudoreplicates were conducted by NJ and MP methods to
determine internal support for the NJ and MP trees,
respectively. Best-fit models for the Bayesian analysis
were inferred by hierarchical likelihood ratio tests
using MRMODELTEST, version 2.3 (Nylander, 2004)
for respective regions (GTR+G for cyt b and COI,
KKY+G for ND2, and GTR+I+G for 12S). Markov
chain Monte Carlo simulations were run for 5 000 000
generations with trees sampled every 1000 generations. Then Bayesian posterior probabilities were
estimated after omitting the initial 1 000 000
generations.
In the phylogenetic analyses, sequences from the
four regions were concatenated and analysed as a
combined dataset because tree topologies constructed
separately from each of four regions were fundamentally similar.
Previous studies have reported the presence of multiple nuclear copies of some mitochondrial genes in
locusts (Zhang & Hewitt, 1996) and highlighted the
inappropriateness of mitochondrial gene sequences
for phylogenetic studies of L. migratoria (Zhang,
2006). However, using the primers mentioned above,
we did not encounter such problems in the present
study.
RESULTS
PHYLOGENETIC
RELATIONSHIPS
The combined sequences of four mitochondrial regions
comprised 2181 bp, which contains 294 phylogenetically informative characters. The NJ tree and the
majority rule consensus tree of the MP method are
shown in Figures 1, 2. In the MP analysis, 25 559
equally parsimonious trees with tree length of 901,
consistency index of 0.807, retention index of 0.941,
and rescaled consistency index of 0.760 were found by
the heuristic search. Because the Bayesian analysis
reached fundamentally similar tree topologies with
the MP analysis, the Bayesian posterior probabilities
are shown in the MP consensus tree (Fig. 2).
All analyses strongly suggest monophyly of L.
migratoria and the L. migratoria population was
clearly divided into two clades (Figs 1, 2). One clade
consists of individuals from Hokkaido, Honshu, and
Kyushu of Japan, and the Chinese continent (Figs 3,
4; hereafter the ‘northern clade’), and the other clade
comprises individuals originating from subtropical
islands of Japan, Hainan Island in China, Timor
Leste, Australia, Ethiopia, and France, as well as
some individuals collected on Tsushima Island and
Honshu, Japan (Figs 3, 4; hereafter the ‘southern
clade’). Among eight individuals of the Tsushima
population analysed in the present study, six were
members of the northern clade and two were in the
southern clade. In the Honshu population, 27 individuals were members of the northern clade, whereas
six individuals collected in the western areas of northern Honshu, namely Yamagata (all three individuals
analysed), Niigata (two out of three individuals), and
Tochigi (one out of three), belonged to the southern
clade.
In the northern clade, monophyly of the Hokkaido
and of the Xinjiang populations was supported in all
analyses (Figs 1, 2). The individuals from Honshu,
Kyushu, and the Chinese continent, with the exception of Xinjiang, comprised another clade in all analyses, and the monophyly of this clade was supported
both by the NJ bootstrap test and high Bayesian
posterior probabilities. Hokkaido and Xinjiang populations are sister groups in all MP trees and in the
Bayesian analysis, but the relationship was not supported more than 50% of pseudoreplications by the
MP bootstrap test or by the NJ analysis. Although the
Hokkaido lineage was the first to diverge in the
northern clade, and the Xinjiang population became
the sister group of Honshu, Kyushu, and the Chinese
continent populations in the NJ tree (Fig. 1), such
relationships were not supported by the NJ bootstrap
test. In the clade that includes individuals from
Honshu, Kyushu, and the Chinese continent (with the
exception of Xinjiang), a common haplotype was
shared by the Kyushu and Chinese continent populations, and individuals from Japan and China were
mixed in the clade.
In the southern clade, the six individuals from
Honshu were the first to diverge in all trees and the
monophyly of the clade was highly supported in all
analyses. As mentioned earlier, these individuals are
distributed in western areas of northern Honshu
(three individuals in Yamagata, two in Niigata, and
one in Tochigi). Another clade comprising the other
members of the southern clade was also highly supported in all analyses (Figs 1, 2). In this clade, one (in
NJ) or two (in MP and the Bayesian analysis)
individuals from Timor Leste (TML03, or TML03
and TML05) assumed the most basal position(s).
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
PHYLOGENY OF THE MIGRATORY LOCUST
575
Schistocerca gregaria (outgroup)
HKJ02
HKS02
HKY03
HKS03
HKJ01
HKS01
HKY02
69
HKY01
59
HKS04
61
JMN05
JMN02
JMN03
JMN04
JMN01
57
IWM03
TSM02
IWH02
IBR02
MYZ01
NGS03
NGS02
HYG01
MYZ03
NGT03
IWM01
TSM03
HYG02
AOM01
AOM03
TCG02
IWH03
NGS01
IWH01
HCJ01
IBR03
MYZ02
HCJ02
HCJ03
88
SZK02
SZK03
IBR01
TCG01
IWM02
SZK01
87
AOM02
TSM08
TSM07
TSM05
OSK02
OSK03
OSK01
79
TNJ01
KMM01
TNJ05
TNJ04
TSM04
62
KMM03
KMM02
FKK03
63
FKK01
LNN02
FKK02
LNN01
SDN04
TNJ03
HNN02
SDN01
TNJ02
HNN01
HNN03
HNN05
66
HYG03
LNN03
LNN04
SDN02
65
LNN05
HNN04
72
SDN05
SDN03
52
MDT04
MDT02
56
MYK01
64
AMM03
AMM02
55
MDT01
OKN
62
IHY01
MDT03
71
IHY02
AMM01
IHY03
HND02
HND04
71
HND01
HND05
52
HND03
TML05
TML02
AUS02
70
AUS04
AUS01
74
TML04
91
TML01
AUS05
66
AUS03
FRN04
AFR
FRN03
74
FRN02
FRN05
FRN01
54
TSM06
TSM01
OGS04
OGS02
OGS01
99 OGS05
OGS03
TML03
NGT01
NGT02
YMG03
YMG01
YMG02
TCG03
99
99
Hokkaido
Xinjiang
70
99
63
99
99
92
99
Northern
clade
Honshu,
Kyushu,
and
Chinese
continent
except
Xinjinag
Southwest
Islands of
Japan
Hainan
Island
Timor Leste
& Australia
Ethiopia
& France
Southern
clade
Tsushima
Ogasawara
Timor Leste
Western areas of
northern Honshu
Oedaleus infernalis
Aiolopus thalassinus tamulus
0.02
Figure 1. Neighbour-joining tree for Locusta migratoria based on 2181 bp of combined cytochrome b (cyt b), cytochrome
oxidase subunit I (COI), NADH dehydrogenase subunit II (ND2), and 12S ribosomal RNA (12S) sequence data. Bootstrap
values are indicated for nodes with > 50% support in 1000 pseudoreplicates. Detailed collection data are shown in Table 1.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
576
M. TOKUDA ET AL.
59 (100)
99
(100)
(60)
(60) 50
52
(60)
52
66
99 (100)
100
61 (100)
98
64 (100)
99
62 (100) 65 (100)
99
94
(100)
94
100 (100)
100
59 (100)
95
(100)
92
63 (100)
96
(50)
64 (100)
96
(50)
68
63 (100)
100
80 (100)
100
(100)
87
100 (100)
100
(50)
85 63 (100)
98
(100)
69
100 (100)
100
66 (100)
100
66 (100)
99
(67)
(50)
78
(67)
(67)
82
60 (77)
95
(54)
92 (100)
100
53 (89)
97
60 (100)
99
64 (100)
99
80 (100)
100
90
90 (100)
(100)
100
99
58 (100)
100
(50)
(100)
100
(72)
92
87 (100)
100
81 (100)
96
100 (100)
100
51 (100)
98
Schistocerca gregaria
JMN03
JMN02
JMN05
JMN04
JMN01
HKS02
HKS01
HKS04
HKS03
HKJ01
HKJ02
HKY02
HKY01
HKY03
TCG01
SZK02
IWM02
IBR01
SZK01
SZK03
HYG03
SDN01
LNN01
FKK01
HNN01
SDN02
LNN04
SDN03
SDN05
HNN04
SDN04
LNN02
LNN03
LNN05
TNJ02
TNJ03
TNJ04
TNJ05
HNN02
HNN05
FKK02
FKK03
KMM02
KMM03
TSM04
HNN03
KMM01
TNJ01
AOM01
HYG01
MYZ01
MYZ02
MYZ03
TSM03
IWM01
NGS01
NGS02
NGS03
AOM02
AOM03
TCG02
NGT03
IBR02
IBR03
HYG02
IWM03
IWH03
HCJ01
HCJ02
HCJ03
OSK01
OSK02
OSK03
TSM05
TSM07
TSM08
IWH02
IWM03
TSM02
MDT03
IHY01
OKN
MDT01
IHY02
MYK01
MDT04
MDT02
AMM03
AMM02
AMM01
IHY03
HND02
HND01
HND03
HND05
HND04
AUS02
AUS01
TML02
TML04
TML01
AUS03
AUS05
AUS04
FRN02
FRN03
FRN05
FRN01
FRN04
AFR
OGS02
OGS01
OGS04
OGS03
OGS05
TSM06
TSM01
TML05
TML03
YMG01
YMG02
YMG03
NGT01
TCG03
NGT02
Oedaleus infernalis
Aiolopus thalassinus tamulus
Figure 2. Majority rule consensus tree of 25 559 equally parsimonious trees (tree length 902, consistency index = 0.807,
retention index = 0.941, rescaled consistency index = 0.760) based on 2181 bp of combined cytochrome b (cyt b), cytochrome oxidase subunit I (COI), NADH dehydrogenase subunit II (ND2), and 12S ribosomal RNA (12S) sequence data.
Bootstrap values in the maximum parsimony method (> 50% support in 1000 pseudoreplicates) and majority rule
percentages (in parentheses) are indicated above nodes. Bayesian posterior probabilities (> 50%) are shown below nodes
in italics. Detailed collection data are shown in Table 1.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
PHYLOGENY OF THE MIGRATORY LOCUST
Hokkaido
Honshu
577
Monophyly of south-west islands of Japan (including
Amami, Iheya, Okinawa, Miyako, and Minami-Daito
populations), Ogasawara, and Tsushima was supported in all analyses. Individuals collected on each
south-west island of Japan did not form a monophyletic group but were mixed in the clade. The Hainan
population of China formed a paraphyletic group of
the Japanese south-west islands population. Populations distributed in Timor Leste, Australia, and
France became polyphyletic groups in all analyses.
Among them, some individuals from Timor Leste and
Australia were closely related and the French population had a rather close relationship with the African
strain.
Kyushu
SEQUENCE
DIVERGENCE BETWEEN MONOPHYLETIC
CLADES
Ogasawara
Mimami-Daito
Figure 3. Distribution map of Locusta migratoria haplotypes in Japan. Localities where haplotypes belong to the
northern clade are shown in blue, localities where haplotypes belong to southern clade (other than a possible relict
haplotype derived from Honshu) are shown in red, and the
possible relict haplotype is shown in green circles. Localities where both northern and southern haplotypes were
detected are indicated by yellow or light blue circles. The
numerals on the map indicate the locality shown in
Table 1.
In the amino acid-coding regions, pairwise sequence
divergences of L. migratoria from S. gregaria, A.
thalassinus tamulus, and Oedaleus infernalis ranged
between 20.91–21.94%, 18.24–18.85%, and 12.13–
12.89%, respectively. In the L. migratoria clade, pairwise sequence divergences were 1.98–3.79% between
the northern and southern clades, and 3.18–3.48%
and 1.81–2.64% from the Honshu populations in the
southern clade to the populations in the northern
clade and to the other populations in the southern
clade, respectively.
In the northern clade, pairwise sequence divergences were 0.46–0.63% between Hokkaido and
Xinjiang populations, 0.46–0.75% between Hokkaido
populations and Honshu, Kyushu, and the Chinese
continent (with the exception of Xinjiang) populations, and 0.28–0.52% between the Xinjiang population and the Honshu, Kyushu, and Chinese continent
populations.
In the southern clade excluding the Honshu populations, pairwise sequence divergences were 0.40–
0.87% between Ogasawara and the other populations,
0.40–0.92% between Tsushima and the other populations, 0.17–0.52% between Japanese south-west
islands and Hainan populations, and 0.34–0.81%
between Hainan + Japanese south-west insular populations and the other populations.
DISCUSSION
Figure 4. Distribution map of Locusta migratoria haplotypes in the areas other than Japan. Localities where
haplotypes belong to the northern clade are shown in blue
and localities where haplotypes belong to the southern
clade are shown in red circles. The numerals on the map
indicate the localities as shown in Table 1.
The migratory locust is divided into eight to ten
subspecies based on morphological analysis (Uvarov,
1966; COPR, 1982; Chen, 2000), although some recent
genetic studies suggest that this treatment is not
appropriate (Zhang & Kang, 2005; Chapuis et al.,
2008). In the present study, L. migratoria populations
were clearly separated into two mitochondrial haplotype clades (Figs 1–4). The divergence time between
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
578
M. TOKUDA ET AL.
the two clades is estimated to be 0.86–1.89 Mya when
the mitochondrial molecular clock, 2.0–2.3% pairwise
sequence divergence per one million years is assumed
(Desalle et al., 1987; Brower, 1994). This suggests
that temperate East Asian populations and the other
populations have been isolated for one million years
or more.
The northern clade is subdivided into three clades,
which include (1) Hokkaido populations; (2) Xinjiang
population; and (3) Honshu, Kyushu and Chinese
continent populations, except Xinjiang. The divergence time among the three clades is estimated
as 0.20–0.35 Mya. The phylogenetic relationship
strongly suggests that Honshu and Kyushu populations originated from the eastern region of the
Chinese continent. On the other hand, the origin of
the Hokkaido populations appears to differ from that
of the Honshu and Kyushu populations. The origin of
the Hokkaido population is unknown at present;
however, in light of geography, they might have originated from a Russian population. Because no common
haplotypes were detected between Hokkaido and
Honshu populations, the Tsugaru Straits between
Hokkaido and Honshu may have acted as a geographical barrier for the L. migratoria populations.
However, there is no sign of reproductive isolation
between a Hokkaido and a Honshu population (Hakomori & Tanaka, 1992). On the Chinese continent, the
presence of desert areas may have served as a geographic barrier between the cool-temperate Xinjiang
populations and the temperate populations in the
eastern regions of China, although there is no evidence for reproductive isolation between populations
of the two areas (S. Tanaka & D.-H. Zhu. unpubl.
observ.).
In the southern clade, six individuals derived from
northern Honshu comprised a single subclade and
were the first to diverge from other individuals
(Figs 1, 2). The divergence time between this clade
and other individuals in the southern clade is estimated to be 0.78–1.32 MYA (the Pleistocene glacial
period). Because individuals similar to the northern
Honshu haplotype were not found from the distribution area of other southern haplotypes (i.e. Asian
subtropical and tropical regions as well as from Australia, Africa, and Europe), the northern Honshu haplotype is quite unlikely to have invaded there recently
from tropical and subtropical regions. One possible
explanation is that the common ancestor of the southern clade had been distributed widely from tropical
regions to East Asia in the glacial period and that the
Honshu haplotype in the southern clade is a relict
descendent of the common ancestor. Individuals of
this haplotype have been found in areas of Japan
surrounded by relatively high mountains. Alternatively, the haplotype might have been pushed to the
north-western margins of Honshu after recent invasions by the northern haplotypes from the Chinese
continent.
In the southern clade, individuals from the Southwest Islands of Japan comprised a monophyletic
group with individuals from Hainan Island as a paraphyletic group. This suggests that the south-west
populations originated from Hainan or an adjacent
area. Because no clear geographical variation was
found among the South-west Islands populations,
each island population does not appear to have been
isolated for a long period of time. Occasional outbreaks of the locust in South-east Asia and the frequent occurrence of strong northward air current
(seasonal trade wind) or typhoons might explain the
distribution of the southern clade in Japan. Adults of
this locust exhibited outbreaks in the Philippines in
1922–1923 and some swarms reached Taiwan where
this locust is normally absent (Koidsumi & Ogasahara, 1940). On the other hand, the results obtained
in the present study suggest that the Tokara straits
located north of Amami Island have acted as a geographical barrier, isolating L. migratoria populations
for a few thousand years. Because viable offspring are
produced in crosses between South-west Islands
populations and Honshu or Hokkaido populations of
L. migratoria (Tanaka, 1994a), Japanese populations
in the southern clade appear to show no reproductive
isolation from those in the northern clade. It has been
suggested that populations of the eastern part of
China and Southeast Asia may belong to a subspecies
Locusta migratoria manilensis (Meyen) (Tsou, 1935;
Uvarov, 1936; Chen, 2000). The results obtained in
the present study indicate that the populations in the
eastern part of China belong to the northern clade
and are apparently different from those in the southern part of China (Hainan Island) as well as those of
Southeast Asia (Timor Leste), which are classified
into the southern clade.
Locusta migratoria collected on the Ogasawara
Islands exhibited an endemic haplotype and formed
a single clade. The monophyly was highly supported
in all phylogenetic analyses. This indicates that the
Ogasawara population has a different origin from
the Honshu and Japanese south-west insular populations. The Ogasawara haplotype belongs to the
southern clade and is rather closely related to Timor
Lestre and Australian populations, although the
origin of this population is unknown. It may have
originated somewhere from the Micronesian islands,
which are located south of the Ogasawara Islands.
The divergence time between Ogasawara and the
other southern populations is estimated to be 0.17–
0.44 Mya and the former population is likely to have
been isolated for this period after its colonization of
the islands.
© 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 570–581
PHYLOGENY OF THE MIGRATORY LOCUST
Two out of eight individuals collected from
Tsushima Island were included in the southern clade.
The haplotype was closely related to Ogasawara,
Timor Leste, and Australian populations rather than
the Hainan and Japanese south-west insular populations. This result is rather surprising from a geographic standpoint and the origin of this haplotype is
presently unknown. It may have arisen from an accidental invasion of the island as a result of recent
human activities. Further studies will be needed to
clarify the distribution of northern and southern haplotypes on Tsushima Island.
No tight relationship has been found between haplotypes and life-history traits such as voltinism and
diapause in L. migratoria (Hakomori & Tanaka, 1992;
Tanaka, 1994a, b; Yamagishi & Tanaka, 2009; S.
Tanaka & M. Tokuda, unpubl. observ.). Populations in
Africa and Australia have no diapause (Farrow &
Colless, 1980). In Japan, the life cycle of L. migratoria
is univoltine in Hokkaido and northern Honshu,
bivotine in central and southern Honshu and northern Kyushu, and multivoltine in southern Kyushu
and in the South-west Islands. Embryonic diapause is
found in all Japanese populations including subtropical populations, although the incidence and the environmental factors controlling the diapause vary with
the climatic zone, with the pattern of latitudinal
variation being similar to that in the Chinese Continent where the tropical populations on Hainan Island
also show embryonic diapause (Tanaka & Zhu, 2008).
According to a microsatellite analysis conducted by
Chapuis et al. (2008), a Japanese Honshu population
was phylogenetically distant from Chinese continental populations but rather close to Madagascar and
Réunion populations. However, Honshu and Madagascar are geographically quite distant from each
other and no close relationships have been reported
for any other organisms. In their analysis, a close
relationship between Australian and South-east
Asian populations was suggested, but the relationships of these populations with other local populations are unclear.
Phylogenetic relationships among Chinese L.
migratoria populations were recently analysed by
Zhang & Kang (2005) by the random amplified polymorphic DNA technique and Zhang et al. (2009)
employing a multilocus microsatellite genotyping
analysis. In Zhang & Kang (2005), populations in
north-eastern China were close to the Hainan population, and Xinjiang populations became the paraphyletic group of them. By contrast, Zhang et al.
(2009) claimed the presence of a close relationship
between East China and Xinjiang populations.
Although Xinjiang populations were not clearly separated from East Chinese populations in Zhang et al.
(2009), our phylogenetic analyses showed that the
579
Xinjiang population has been isolated from the East
Chinese populations for 0.20–0.35 Mya. Zhang et al.
(2009) further claimed that the populations in East
China are different from either the Hainan Island
or Tibetan populations and should be regarded as
Locusta migratoria migratoria instead of L. m.
manilensis. However, their samples from the southern China were collected only at one location (i.e.
Hainan Island) and the relationships of the Hainan
Island populations with the southern populations
such as South-east Asia and Oceania remained to be
clarified. The present study has provided evidence
indicating that the Hainan populations belong to the
southern clade that we defined. Although the present
study did not contain Northwestern populations such
as Tibetan populations, the results reported both by
Zhang & Kang (2005) and Zhang et al. (2009) appear
to indicate that the Tibetan populations are distant
from East China and Hainan Island populations.
ACKNOWLEDGEMENTS
We thank the following individuals for offering L.
migratoria specimens: Y. Ando, M. P. Chapuis, N.
Endo, A. Foucart, Y. Hashimoto, H. Higuchi, Y. Hirai,
J. Huybrechts, Y. Ikezaki, K. Ito, K. Kanai, K. Maeno,
H. Nakamine, M. Sakakibara, Y. Shintani, P. Spurgin,
S. Sugiura, M. Takeda, K. Tanaka, M. Yamagishi, and
D. Yamaguchi. We also thank N. Hinomoto, T. Higaki,
K. Maeno, and H. Noda for their support with the
molecular experiments; N. Utsuki for his support in
the phylogenetic analyses; and S. Masaki and Y. Ito
for information about the literature. This study is
partly supported by a Kakenhi grant of Japan
(No.19380039) to S.T.
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