4080
Kazuo Umetsu1
Naruki Iwabuchi2
Isao Yuasa3
Naruya Saitou4
Paul F. Clark5
Geoff Boxshall5
Motoki Osawa1
Keiji Igarashi61

Department of Forensic
Medicine, Yamagata University
School of Medicine,
Yamagata, Japan
2
Sendai Science Museum,Sendai, Japan3
Department of Legal Medicine,
Tottori University School of
Medicine, Yonago, Japan
4
Laboratory of Evolutionary
Genetics, National Institute of
Genetics, Mishima, Japan
5Department of Zoology,
The Natural History Museum,London, UK
6Faculty of Agriculture,
Yamagata University,
Tsuruoka, Japan

Electrophoresis 2002, 23, 4080–4084

Complete mitochondrial DNA sequence of atadpole shrimp (Triops cancriformis) and analysis
of museum samples
The complete mitochondrial DNA (mtNDA) of the tadpole shrimp Triops cancriformis
was sequenced. The sequence consisted of 15 101 bp with an A1T content of 69%.
Its gene arrangement was identical with those sequences of the water flea (Daphnia
pulex) and giant tiger prawn (Penaeus monodon), whereas it differed from that of thebrine shrimp (Artemia franciscana) in the arrangement of its genes for tRNAs. Phylogenetic analysis revealed T. cancriformis to be more closely related to the water flea than
to the brine shrimp and giant tiger prawn. We also compared the 16S rRNA sequencesof five formalin-fixed tadpole shrimps that had been collected in five different locations
and stored in a museum. The sequence divergence was in the range of 0–1.51%, suggesting that those samples were closely related to each other.
Keywords: Formalin-fixed sample / Mitochondrial DNA / Molecular phylogeny / Notostraca /Tadpole shrimp
EL 5169

1 Introduction
Fossils of the tadpole shrimp from approximately 200 million years ago are indistinguishable morphologically fromthe present species. Consequently, the tadpole shrimp
has been regarded as a “living fossil” [1, 2]. Three speciesof tadpole shrimps are found in Japan: Triops longicaudatus, Triops granarius, and Triops cancriformis. T. cancriformis is widely distributed in North Africa, Europe, the Middle East, India, and Siberia [2] and has been classified as
an endangered species in some regions. However, it israther common in Japan, where it was reported for the
first time in 1951 [3]. Several habitats have been verified
in Yamagata Prefecture. The origin of the T. cancriformis
in Japan was uncertain, because the species has notbeen recorded to exist in neighboring countries such as
Korea and China. Mitochondrial DNA (mtDNA) is one of
the best markers for revealing phylogenetic relationships
among related groups of animals [4–6]. But information on
mtDNA of the tadpole shrimp is scarce [7, 8]. Because

museum-stored samples collected from various locations are available, such samples would be useful if DNA
information can be extracted from those formalin-fixedsamples. The aim of the present study was to determine
the complete sequence of the T. cancriformis mitochondiral genome and to clarify the usefulness of the formalinfixed samples for examining phylogenetic relationships in
T. cancriformis.

2 Materials and methods
2.1 mtDNA cloning and sequencing

Abbreviations: AA, amino acid; NJ, neighbor-joining; nt, nucleotide

Samples of T. cancriformis were collected from rice fields
in Yamagata Prefecture, Japan. The mtDNA was preparedfrom tissues and eggs according to standard protocols[9]. The first primers for cloning (TaKaRa LA PCR in vitro
cloning kit; TaKaRa, Tokyo, Japan) were designed fromthe nucleotide (nt) sequence of the COI gene of Triops
australiensis mtDNA (DDBJ/ENBL/GenBank accession
number AF218284). Sequencing was performed by primer walking (primers were synthesized on the basis of
the newly clarfied mtDNA sequence of T. cancriformis).
The PCR products were separated on 1% agarose gels,and then purified by using a Gel Extraction Kit (Qiagen,Hilden, Germany). PCR products were subjected to direct
DNA sequencing using an ABI PRISM 310 sequencer and 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

0173-0835/02/2412–4080 $17.501.50/0

Correspondence: Dr. Kazuo Umetsu, Department of Forensic
Medicine, Yamagata University School of Medicine, 2-2-2 IidaNishi, Yamagata 990-9585, Japan
E-mail: kumetsu@med.id.yamagata-u.ac.jp
Fax: +81-23-628-5273

Electrophoresis 2002, 23, 4080–4084

2.2 Phylogenetic analysis
The nt and aa sequences were aligned by using the
CLUSTAL-W program [12]. Evolutionary distances werecomputed by Kimura’s two-parameter method [13], andthe phylogenetic analysis was performed by the neighbor-joining (NJ) method [14] and by the use of TreeView[15]. Comparisons were made between the completesequence of tadpole shrimp (T. cancriformis) mtDNA andthose sequences of eight other arthropods. The species
and common names, and DDBJ/ENBL/GenBank accession numbers of the sequences are as follows: Daphnia
pulex (water flea, AF117817) [10], Artemia franciscana(brine shrimp, X69067) [16], Penaeus monodon (giant
tiger prawn, AF217843) [17], Ixodes hexagonus (tick,
AF081828) [18], Limulus polyphemus (horseshoe crab,
AF216203) [19], Drosophila yakuba (fruit fly, X03240) [20],Anopheles gambiae (mosquito, L20934) [21], and Locusta
migratoria (grasshopper, S80245) [22]. Crassostrea gigas(Pacific oyster, AF177226) was included as an outgroup.

2.3 Analysis of mtDNA from formalin-fixedsamples
As listed in Table 1, five specimens of T. cancriformis,
which had been kept at The Natural History Museum,London, were analyzed for the 16S rRNA gene of their
mtDNA. They were most probably fixed initially in formalin
and then transferred into industrial-methylated spirit (90%ethanol, 3–10% methanol, 0–7% water) for long-term storage with the reference collections. DNA was preparedfrom about 10 eggs of the tadpole shrimp. The eggs ofeach sample were crushed in a 1.5 mL tube, and 50 mLof proteinase K solution (25 ng of proteinase K; 20 mM
Tris-HCl, pH 8.0; 20 mM EDTA; 0.1 M 2-mercaptoethanol;
1% sodium N-lauroyl sarcosinate) was added; and the
mixture was then incubated for 1 h at 607C. Subsequently, 150 mL of guanidine-detergent lysing solution (4.5 M
guanidine thiocyanate; 20 mM Tris-HCl, pH 8.0; 20 mM
EDTA; 0.4 M NaCl; 0.1 M 2-mercaptoethanol; 0.5%
sodium N-lauroyl sarcosinate; 20 ng of glycogen) was
added. The supernatant was precipitated with 2.5 volumes of ethanol. Since DNA extracted from formalin-fixedsamples is decomposed into very small molecular sizes[23], small fragments ranging from about 100 to 200 bp

4081

Table 1. List of the T. cancriformis specimens used in thisstudy (The Natural History Museum)
Locality

Collector/present

Date

Doybayazit, Turkey
Vienna, Austria
Dayat, Algeria
Srinagar, India
Kirkcudbrigh, Scotland–
Vienna Museum
Mon L. W. Rothschild
Zool. Survey of India
F. Baljour Brown

March 22, 1961
November 8, 1911
March 16, 1912
October 7, 1954
October 17, 1907

were the target of the PCR. Three pairs of primers (16SF1:
5’-TCCCTAGGGTAACTTGATCTG-3’ and 16SR1: 5’-ACT
CTAAGTTATTGGGTTGGG-3’; 16SF2: 5’-GATCCATAGGG
TCTTATCGTC-3’ and 16SR2: 5’-TTGAGGTATAGCCTGC
TCTATG-3’; 16SF3: 5’-TATATTTGCCGAGTTCCTTTG-3’
and 16’SR3: 5’-AATTAGGGTTAGAGGGATTAGC-3’) wereused to amplify the 16S rRNA gene. The PCR products
were separated on 1.5% agarose gels, and then purified
by using a Gel Extraction Kit (Qiagen). Sequencing of thepurified products and phylogenetic analysis were completed by using the methods described above.

3 Results and discussion
The mitochondrial genome of the tadpole shrimp (T. cancriformis) was found to be a circular one with 15 101 nts. It
encoded information of 22 tRNAs, 13 protein-coding
genes and 2 rRNAs as is characteristic of mtDNAs ofother metazoans. The complete nt sequence of the
mtDNA of T. cancriformis was registered in DDBJ/EMBL/
GenBank nt sequence databases with the accession
number AB084514. The organization of the genome isshown in Fig. 1. The overall base composition of theL-strand was A: 36%; C: 18%; T: 33%; G: 13%. The A1T
content was similar to that of the giant tiger prawn [17],but somewhat lower than that of the water flea [10] or thebrine shrimp [16).
The location and the putative initiation and termination
codons of the protein-coding sequences are shown in
Table 2. The three initiation codons (ATA, ATG, GTG) arecommonly found in other animals. The use of GTG for
the initiation of translation of the ND5 mRNA is also
shared by other curstaceans [10]. A 4 bp initiation codon(ATTA) for ND2 in T. cancriformis has also been proposedfor other arthropod taxa, though this has not been confirmed experimentally [10, 18]. In nine genes (ND2, COI,
COII, ATP8, ATP6, ND4L, ND6, Cytb, and ND1), TAA isused as the stop codon; and TAG in one (ND3). Three
genes (COIII, ND5, and ND4) are characterized by having
an incomplete stop codon TA. The tRNA genes were 64–
71 bp long. The control region comprised 467 nts, andwas the smallest among the crustaceans tested so far.

Nucleic acids

BigDye Terminator Cycle Sequencing FS Kit (Applied Biosystems, Foster City, CA, USA). The 5’- and 3’-ends ofgenes and amino acid (aa) sequences of protein-coding
genes of T. cancriformis mtDNA were inferred from alignments with the Daphnia pulex sequences [10] and by
BLAST searches [11].

mtDNA sequence of tadpole shrimp

4082

K. Umetsu et al.

Electrophoresis 2002, 23, 4080–4084
Table 2. Organization of the T. cancriformis mitochondrial genome
Gene

Figure 1. Genetic map of. T. cancriformis mtDNA. The
tRNAs are identified by the 1-letter aa code.
This region was situated between the 12S rRNA andtRNAIle genes. The aa sequences deduced from the protein-coding genes of tadpole shrimp were subjected topairwise comparisons with the homologues of water flea,
brine shrimp, giant tiger prawn, tick, horseshoe crab, fruit
fly, mosquito, and grasshopper. The results showed that
tadpole shrimp proteins had greater similarity in aa
sequence to those of water fleas than to those of the brine
shrimp and giant tiger prawn.
NJ trees (Fig. 2) were constructed from aa sequences of
two well-conserved genes; cytochrome c oxidase subunit
I (COI) and cytochrome b (Cytb), of one nonconserved
gene, NADH dehydrogenase subunit 5 (ND5), and all
protein-coding genes. Almost identical topologies wereobtained in all four trees. T. cancriformis was more closely
related to the water flea than to the brine shrimp andgiant tiger prawn. However, the branching patterns of
the brine shrimp were unstable in the mitochondrial trees.
The subclass Branchiopoda of the class Crustacea iscurrently classifed into four orders, i.e., Anostraca (brine
shrimps), Notostraca (tadpole shrimps), Conchostraca(clam shrimps) and Cladocera (water fleas). mtDNA genearrangements are generally well-conserved among arthropod groups, and suggest that gene arrangements could
be used to deduce phylogenetic relationships [24]. The
arrangement of T. cancriformis tRNA genes differs fromthat in the brine shrimp [16], but the arrangement of theprotein-coding genes is the same as that in them. These
results indicate that the tadpole shrimp is more closely
related to the water flea than to the brine shrimp.
The use of mtDNA as a maternally inherited marker is now
widespread to elucidate evolution and migrations. thepartial sequence of the 16S rRNA gene of T. cancriformis
has already been reported [7]. However, those data aretRNAIletRNAGlntRNAMetND2tRNATrptRNACystRNATyr
COI
tRNALeu(UUR)
COII
tRNALystRNAAsp
ATP8
ATP6
COIII
tRNAGly
ND3tRNAAlatRNAArgtRNAAsntRNASer(AGN)tRNAGlutRNAPheND5tRNAHisND4
ND4L
tRNAThrtRNAPro
ND6
Cytb
tRNASer(UNC)
ND1tRNALeu/CUN)
16S rRNAtRNAVal
12S rRNA
Control region

From
1
63
128
190
1188
1252
1320
1378
2915
2987
3669
3738
3813
3956
4627
5413
5480
5828
5901
5967
6037
6102
6169
6234
7959
8026
9338
9656
9723
9788
10285
11417
11509
12436
12502
13807
13879
14635

To
65
131
192
1191
1257
1318
1383
2919
2982
3667
3738
3804
3962
4627
5414
5476
5830
5896
5964
6036
6101
6167
6233
7960
8025
9344
9655
9723
9786
10288
11421
11487
12426
12500
13807
13877
14634
15101

Size (bp) Start
65
69 (L)
65
1002
70
67 (L)
64 (L)
1542
68
681
70
67
150
672
788
64
351
69
64
70
65
66
65 (L)
1727 (L)
67 (L)
1319 (L)
318 (L)
68
64 (L)
501
1137
71
918 (L)
65 (L)
1306 (L)
71 (L)
756 (L)467

Stop(A)TTA TAA

ATA

TAA

ATG

TAA

ATA
ATG
ATG

TAA
TAA
TA2

ATA

TAG

GTG

TA2

ATG
ATG

TA2
TAA

ATA
ATA

TAA
TAA

ATG

TAA

L, indicated that the gene is transcribed from the lightstrandprobably erroneous, because there was no counterpart
with the present mtDNA sequence data. Their PCR products would have been derived from the genomic DNA.
To investigate the tadpole shrimp lineage, we examined
the base sequence of 16S rRNA from specimens storedin The Natural Histrory Museum, London (Table 1). The
majority of these specimens had been preserved in formalin to conserve their morphology, with the consequence that the DNA had decomposed into fragments oflow molecular weight. Other studies have shown that the
reduction of PCR product lengths can be used to improveElectrophoresis 2002, 23, 4080–4084

mtDNA sequence of tadpole shrimp

4083

Figure 2. The phylogenetic tree
for nine arthropods constructedby the NJ method, based on
diffent aa sequences (A, COI;
B, Cytb; C, ND5; D, all coding
regions). Bootstrap values werecomputed for 1000 replications.
The oyster was used as an outgroup for rooting trees.

the typing of highly degraded DNA [23]. In spite of the useof formalin-fixed samples, some sequence data (465 bp:
nt 12 771–12 850, nt 12 937–13 096 and nt 13 181–13 405)
revealed of mtDNA common to all samples (Table 3).
Based on the observed sequence divergences of the
16S rRNA gene (0–1.51%), populations of T. cancriforms
from six localities were more closely related to each otherthan to T. granarius (Fig. 3). The 16S rRNA sequence wassuitable for estimating phyogenetic relationships in tadpole shrimps. T. cancriformis from high latitude regions in
the Northern Hemisphere (Scotland, Austria and Japan)
were relatively homogeneous for the nt sequence of 16S
rRNA. These results do not indicate that T. cancriformis
have been living in Japan for a long time, but suggestthat they migrated recently from a high latitude in Eurasia.
Since eggs of the tadpole shrimp are resistant to desication, passive dispersal by human activity, migratory birds
or wind is assumed to be responsible [2].

Figure 3. NJ trees based on 16S rRNA sequences of formalin-fixed samples. Bootstrap values were computedfor 1000 replications. T. granarius (AF200963) was used
as an outgroup for rooting the tree.

4084

K. Umetsu et al.

Electrophoresis 2002, 23, 4080–4084

Table 3. Phylogenetically informative sites of 16S rRNA
sequence data
nt No.

T. cancriformis

T. granariusa)

The authors would like to thank Dr. S. Chiba, Biological
Institute, Tohoku University, Japan, for stimulating discussions.
Received June 17, 2002

12771
12784
12798
12800
12804
12809
12835
13061
13369
13377
13384

Japan Scotland

Austria India

Turkey Algeria Japan

C
T
A
T
T
A
C
T
A
A
A

C
T
A
T
T
A
C
T
A
A
A

C
T
A
T
T
A
T
C
T
G
G

C
T
A
T
T
A
C
T
A
A
A

C
T
A
T
T
A
T
T
A
A
A

T
C
T
C
C
T
T
T
A
A
A

T
C
T
T
C
A
T
C
T
G
T

a) Accession number, AF200963

Detailed elucidation of the phylogenetic relationships between subspecies of T. cancriformis [2] must await further
studies, including morphological diversification. Furthermore, the role of morphological studies will become
more important with the advancement of molecular studies. Thus, a more detailed study of combined morphological and molecular evolution in tadpole shrimps isplanned.

5 References[1] Trusheim, F., Paleont. Z. 1938, 19, 198–216.
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4 Concluding remarks
We determined the complete 15 101 bp sequence of the
mtDNA of the tadpole shrimp (T. cancriformis). Phylogenetic analysis using the NJ method on the mtDNA
revealed that the tadpole shrimp was more closely related
to the water flea (Daphnia pulex) than to the brine shrimp(Artemia franciscana). Formalin-fixed samples, commonly
stored in museums, were useful for the investigation of
DNA. The analysis of mtDNA should become a good
method for investigating both the geographical distribution and lineage relationships of tadpole shrimps. Somesubspecies are morphologically distinguishable [2], and
it is necessary to search for correlations between morphology and mtDNA data.

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667–668.