The mitochondrial genome of Euphausia superba (Prydz Bay)(Crustacea: Malacostraca: Euphausiacea) reveals a novel genearrangement and potential molecular markers

Xin Shen Æ Haiqing Wang Æ Jianfeng Ren ÆMei Tian Æ Minxiao Wang

Received: 3 December 2008 / Accepted: 24 June 2009 / Published online: 4 July 2009

� Springer Science+Business Media B.V. 2009

Abstract Euphausiid krill are dominant organisms in the

zooplankton population and play a central role in marine

ecosystems. In this paper, we described the gene organiza-

tion, gene rearrangement and codon usage in the mitochon-

drial genome of Euphausia superba Dana 1852 (sampling

from Prydz Bay, PB). The mitochondrial genome of

E. superba is more than 15,498 bp in length (partial non-

coding region was not determined). Translocation of four

tRNAs (trnL1, trnL2, trnW and trnI) and duplication of one

tRNA (trnN) were founded in the mitochondrial genome of

E. superba when comparing its genome with the pancrusta-

cean ground pattern. To investigate the phylogenetic rela-

tionship within Malacostraca, phylogenetic trees based on

currently available malacostracan mitochondrial genomes

were built with the maximum likelihood and the Bayesian

models. All analyses based on nucleotide and amino acid

data strongly support the monophyly of Stomatopoda,

Penaeidae, Caridea, and Brachyura, which is consistent with

previous research. However, the taxonomic position of

Euphausiacea within Malacostraca is unstable. From com-

paring the mitochondrial genome between E. superba (PB)

and E. superba (sampling from Weddell Sea, WS), we found

that nad2 gene contains maximal variation with 61 segre-

gating sites, following by nad5 gene which has 12 segre-

gating sites. Thus, nad2 and nad5 genes may be used as

potential molecular markers to study the inherit diversity

among different E. superba groups, which would be helpful

to the exploitation and management of E. superba resources.

Keywords Malacostraca � Euphausiacea �Mitochondrial genome � Gene rearrangement �Phylogenomics

Abbreviations

atp6, and 8 ATPase subunits 6 and 8

bp Base pair (s)

cox1-3 Cytochrome c oxidase subunits I–III

PCGs Protein-coding genes

nCR Non coding region

cob Cytochrome b

mtDNA Mitochondrial DNA

nad1–6, and 4L NADH dehydrogenase subunits 1–6

and 4L

srRNA, and lrRNA Small and large subunits ribosomal

RNA

tRNA Transfer RNA

L1 tRNALeu(CUN)

L2 tRNALeu(UUR)

S1 RNASer(AGN)

S2 tRNASer(UCN)

BPNn Bootstrap probability of neighbor

joining based on nucleotide data

BPNa Bootstrap probability of neighbor

joining based on amino acid data

BPMn Bootstrap probability of maximum

likelihood based on nucleotide data

Xin Shen and Haiqing Wang contributed equally to this work.

X. Shen � M. Tian

Jiangsu Key Laboratory of Marine Biotechnology/College

of Marine Science, Huaihai Institute of Technology,

222005 Lianyungang, China

X. Shen (&) � H. Wang � J. Ren � M. Wang (&)

Institute of Oceanology, Chinese Academy of Sciences,

266071 Qingdao, China

e-mail: shenthin@163.com

M. Wang

e-mail: wangminxiao@ms.qdio.ac.cn

123

Mol Biol Rep (2010) 37:771–784

DOI 10.1007/s11033-009-9602-7

BPMa Bootstrap probability of maximum

likelihood based on amino acid data

BPPn Bayesian posterior probability based

on nucleotide data

BPPa Bayesian posterior probability based

on amino acid data

Introduction

With a few exceptions, metazoan mitochondrial (mt)

DNAs are circular molecules, 13–20 kb in size, containing

37 genes: 13 for proteins of electron transport (cox1–cox3,

cob, nad1-4-4L-nad6, atp6 and atp8), 2 for ribosomal

RNAs (srRNA and lrRNA), and 22 for transfer RNAs. Over

the past decades, inference of phylogenetic relationship and

population diversity of metazoa species based on mito-

chondrial genome sequences has become popular [1–6].

This resulted from many advantages offered by mt gen-

omes over other molecular markers. Compared with

sequences of individual gene, mitochondrial genomes

could provide sets of genome-level characteristics, such as

the relative arrangements of gene orders, which are valu-

able characters for studies on evolutionary relationships [1,

2, 7].

Euphausiid krill are significant and dominant organisms

in the zooplankton population and play a central role in

marine ecosystems, which represent important elements in

the transfer of energy from the lower trophic levels through

the planktonic food web upwards to apex predators [8–10].

Euphausia superba Dana 1852, belonging to the order

Euphausiacea, is a dominant krill species and has a huge

biomass in the Antarctic ecosystem. Machida et al. [11]

determined the partial mitochondrial genome sequence of

E. superba sampling from Weddell Sea (Fig. 1). However,

due to the lack of partial srRNA gene and three tRNAs,

many genomic characters and phylogenetic relationship of

Euphausiacea within Malacostraca have not been further

analyzed.

In this paper, we described the gene organization, gene

rearrangement and codon usage of E. superba (sampling

from Prydz Bay, PB) (Crustacea: Malacostraca: Euphausi-

acea). Furthermore, the phylogenetic relationship of Eup-

hausiacea has been analyzed based on 23 malacostracan

mitochondrial genomes. In addition to a better understanding

of the phylogenetic history of the crustaceans, this work

Fig. 1 The sampling location

of E. superba (PB) and

E. superba (WS)

772 Mol Biol Rep (2010) 37:771–784

123

should be useful to the practice of biodiversity management

and conservation strategies of this ecological and commer-

cial significant species.

Materials and methods

Sample collection and DNA extraction

A single specimen of E. superba was obtained from the

Prydz Bay (64�440S, 73�000E; Fig. 1) and was preserved in

99.5% ethanol immediately after collection. Total genomic

DNA was extracted from the muscle tissues using a

DNeasy tissue DNA extraction kit (Promega) following the

manufacturer’s instructions, and was dissolved in TE

buffer.

Long PCR and sequencing by primer walking

The mitochondrial genome of E. superba (PB) was amplified

using a long PCR protocol [12]. Based on partial mito-

chondrial genome sequence of E. superba, five pairs of

primers (Eus-cox1F: GGT GCA TGA GCT GGA ATA GT,

Eus-cox1R: TTA AGT TGT GCA CCG TGA AG; Eus-

cox3F: GCA CAC GGA TTT CAC ACA TA, Eus-cox3R:

GCT GGC TGA AAA GTG ACA AC; Eus-nad5F: TTA

TGA ATT ACA GCC CCA GC, Eus-nad5R: AGG TTG

AGA TGG GTT AGG GT; Eus-cobF: ATC GCA AAT AGA

GCA CTG GT, Eus-cobR: AAA ATA ATG GTG GAA TGG

GA; Eus-srRNAF: TAA GAA TGA GAG CGA CGG G, Eus-

srRNAR: TTT GGC GGT GTC TTA GTC TAG) were

designed to amplify the entire mitochondrial genomes in five

long PCR reactions (Eus-cox1F/Eus-cox3R, Eus-cox3F/Eus-

nad5R, Eus-nad5F/ Eus-cobR, Eus-cobF/Eus-srRNAR and

Eus-srRNAF/Eus-cox1R). PCR reactions were conducted in

a Mastercycler gradient machine (Eppendorf AG Inc.) in a

total volume of 30.0 ll, containing 20.4 ll sterile distilled

H2O, 3.0 ll 109 LA PCR buffer II (Mg2? plus, Takara),

0.6 ll dNTP (10 mM each), 2.0 ll each primer (5 lM),

1.0 ll LA-Taq polymerase (1 unit, Takara), and 1.0 ll DNA

template. The thermal cycling profile was as follows: initial

denaturation at 94�C for 2 min and followed by denaturation

at 94�C for 20 s, annealing at 52�C for 60 s, and extension at

65�C for 16.0 min, for 33 cycles. PCR products were purified

using the Montage PCR Cleanup Kit (Millipore) and

sequenced by primer walking with ABI 373091 DNA

Analyzer.

Sequence analysis

Base calling was performed with PHRED [13, 14] and

sequence reads were assembled in PHRAP with default

parameters. All assembled sequences were manually

checked by using CONSED to remove misassemblies [15].

The locations of thirty PCGs and two ribosomal RNAs were

determined with DOGMA [16] and subsequently aligned

with malacostracan mitochondrial genomes. The majority

of tRNA genes were identified by using tRNAscan-SE 1.21

under the default mode [17]. Remaining tRNA genes were

identified by inspecting sequences for tRNA-like secondary

Fig. 2 A phylogeny of

Malacostraca derived from [35],

indicating species for which

mitochondrial genome

sequences are available so far.

Note: * means the species

displays mt gene

rearrangements in comparison

to the pancrustacean ground

pattern (shared by Penaeus,

Daphnia, Locusta, Drosophila,

and many others)

Mol Biol Rep (2010) 37:771–784 773

123

structures and anticodons. Gene map of E. superba (PB)

mitochondrial genome was drawn by OGDRAW [18].

Codon usage in the 13 PCGs of the E. superba (PB)

mitochondrial genome was estimated with DnaSP 4.10.7

[19].

Phylogenomic analysis

Along with the mitochondrial genome sequence from

E. superba (PB) all currently available malacostracan

mitochondrial genome data were used in phylogenomic

analysis, which included Penaeus monodon, Fennerope-

naeus chinensis, Litopenaeus vannamei, Marsupenaeus

japonicus, Macrobrachium rosenbergii, Halocaridina

rubra, Ligia oceanica, Callinectes sapidus, Cherax

destructor, Eriocheir sinenesis, Geothelphusa dehaani,

Pagurus longicarpus, Shinkaria crosnieri, Panulirus

japonicus, Portunus trituberculatus, Pseudocarcinus gigas,

Gonodactylus chiragra, Harpiosquilla harpax, Lysiosquil-

lina maculate, Squilla empusa, Squilla mantis and Eup-

hausia superba (WS) [5, 11, 20–34]. Their traditional

classification according to Martin and Davis is illustrated in

Fig. 2 [35].

The nucleic acid and amino acid sequences from all 13

PCGs were aligned using ClustalW 1.83 [36] under the

default settings. The concatenated alignment of amino acid

sequences and nucleotide sequences consisted of 11,396

and 3,753 sites for nucleic acid and amino acid sequences,

respectively. Three phylogenetic reconstruction approaches

were applied including Neighbor Joining (NJ) of pairwise

distances using MEGA 4.1 [37], maximum likelihood

(ML) using PHYML 3.0 [38] and Bayesian inference

analyses using MrBayes 3.1 MPI version [39].

To determine the best fitting model of sequence evolu-

tion for the nucleic acid dataset, a nested likelihood ratio

test was performed using MODELTEST 3.8 [40]. After the

evolutionary model (GTR ? I ? G) was determined, phy-

logenetic relationships were inferred by using PHYML 3.0

and MrBayes 3.1 MPI version. The NJ analyses were built

using the maximum composite likelihood model of evolu-

tionary change. In the NJ and ML methods, the assessment

of node reliability was done using 1,000 bootstrap

Fig. 3 Gene map of

mitochondrial genomes of

E. superba (Malacostraca:

Euphausiacea). Note: Protein-

coding genes are transcribed in

a clockwise direction, except for

nad1, nad4, nad4L, and nad5genes. The two ribosomal RNA

genes are encoded on the lightstrands. Transfer RNA genes are

designated by single-letter

amino acid codes. Genes

encoded on the heavy and lightstrands are shown outside and

inside the circular gene map,

respectively. Inner ring shows

GC content graph. The entire

E. supera (PB) mtDNA

sequence has been deposited in

GenBank under accession

number EU583500

774 Mol Biol Rep (2010) 37:771–784

123

replicates. In the case of the Bayesian analyses, the Markov

Chain Monte Carlo analyses were run for 1,000,000 gen-

erations (sampling every 1,000 generations) to allow ade-

quate time for convergence. After approximate 100,000

generations, the log-likelihood values of each sampled tree

had stabilized. After omitting the first 100 ‘‘burn in’’ trees,

the remaining 900 sampled trees were used to estimate the

50% majority rule consensus tree and the Bayesian pos-

terior probabilities (BPPn).

Model selection for the amino acid dataset was done

with ProtTest 1.4 [41]. Due to the Akaike information

criterion MtArt ? C ? I model performed best with our

dataset [42]. NJ and ML analyses of 13 concatenated

mitochondrial PCGs (amino acid data) were built using the

Table 1 Mitochondrial gene

profile of E. Superba(Malacostraca: Euphausiacea)

Note: a Numbers correspond to

the nucleotides separating

different genes. Negative

numbers indicate overlapping

nucleotides between adjacent

genes. ‘‘-’’ Indicates

termination codons completed

via polyadenylation

Feature Position Strand Size (bp) Codon Intergenic

nucleotidesa

From–to Start Stop

nCR 1–316 ? 316

tRNAAsn 317–381 ? 65 0

tRNAGln 379–447 - 69 -3

tRNAMet 447–514 ? 68 -1

nad2 515–1,514 ? 1,000 ATT T- 0

tRNACys 1,515–1,578 - 64 0

tRNATyr 1,590–1,656 - 67 11

tRNATrp 1,672–1,741 ? 70 15

cox1 1,746–3,284 ? 1,539 ACG TAA 4

tRNALeu(CUN) 3,298–3,363 ? 66 13

cox2 3,364–4,051 ? 688 ATA T- 0

tRNALys 4,052–4,120 ? 69 0

tRNAAsp 4,121–4,188 ? 68 0

atp8 4,189–4,347 ? 159 ATC TAA 0

atp6 4,341–5,015 ? 675 ATG TAA -7

cox3 5,015–5,807 ? 793 ATG T- -1

tRNAGly 5,808–5,874 ? 67 0

nad3 5,875–6,228 ? 354 ATT TAA 0

tRNAAla 6,228–6,293 ? 66 -1

tRNAArg 6,295–6,361 ? 67 1

tRNAAsn 6,362–6,426 ? 65 0

tRNASer(AGN) 6,426–6,493 ? 68 -1

tRNAGlu 6,496–6,564 ? 69 2

tRNAPhe 6,664–6,731 - 68 99

nad5 6,731–8,461 - 1,731 ATG TAA -1

tRNAHis 8,462–8,527 - 66 0

nad4 8,528–9,865 - 1,338 ATG TAA 0

nad4L 9,861–10,158 - 298 ATG TAA -5

tRNAThr 10,161–10,226 ? 66 2

tRNAPro 10,228–10,294 - 67 1

nad6 10,298–10,819 ? 522 ATT TAA 3

cob 10,819–11,955 ? 1,137 ATG TAA -1

tRNASer(UCN) 11,976–12,046 ? 71 20

nad1 12,064–13,002 - 939 ATA TAG 17

tRNALeu(UUR) 13,019–13,084 - 66 16

lrRNA 13,085–14,410 - 1,326 0

tRNAVal 14,411–14,482 - 72 0

srRNA 14,483–15,290 - 808 0

tRNAIle 15,291–15,357 ? 67 0

nCR 15,358–15,498 ? 141 0

Mol Biol Rep (2010) 37:771–784 775

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Poisson correction and MtArt model respectively. The

assessment of node reliability was also done using 1,000

bootstrap replicates (BPNa and BPMa stand for NJ and ML

methods, respectively). Given MtArt model could not be

implemented in Bayesian analysis, where we used the best

scoring alternative, MtRev matrix and the gamma ? invar

model of evolutionary change. For a likelihood analysis,

we implemented the MtArt matrix in PHYML 3.0 [38].

The Markov Chain Monte Carlo analyses were run for

1,000,000 generations (sampling every 1,000 generations)

to allow adequate time for convergence. After approximate

100,000 generations, the log-likelihood values of each

Fig. 4 Comparison of gene arrangements in the mtDNA of Mala-

costraca. Note: Gene segments are not drawn to scale. All genes are

transcribed from left-to-right except those indicated by underlining,

which are transcribed from right to left. Shadows indicate changes

compared to the pancrustacean ground pattern

Fig. 5 Linearized representation of mitochondrial gene rearrange-

ment for E. superba (Malacostraca: Euphausiacea). Note: § means

duplication. Arrows show the rearranged genes or gene blocks. The

circling arrows indicate inversions. Gene segments are not drawn to

scale. All genes are transcribed from left-to-right except those

indicated by underlining, which are transcribed from right to left

776 Mol Biol Rep (2010) 37:771–784

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sampled tree had stabilized. After omitting the first 100

‘‘burn in’’ trees, the remaining 900 sampled trees were used

to estimate the 50% majority rule consensus tree and the

Bayesian posterior probabilities (BPPa). The root of each

tree was determined by using the data from five stomato-

pods as an outgroup.

Results and discussion

Genomic characters

The mitochondrial genome of E. superba (PB) is more than

15,498 bp in length (a partial non-coding region was not

determined) and encodes 38 metazoan genes (13 protein-

coding, 23 transfer RNA, and 2 ribosomal RNA genes),

which has an extra trnN gene when comparing with the

standard set of metazoan mitochondrial genomes (Fig. 3;

Table 1). Though no significant similarity is found between

the sequences of the two trnN, they share an identical

anticodon. Compared with the mitochondrial genome of

E. superba (WS) [11] which lacks partial srRNA gene and

nearly four tRNAs, 892 bp were extended in the mito-

chondrial genome of E. superba (PB). The overall A ? T

content of E. superba (PB) (68.1%) appears to be very

similar to that observed in other malacostracan [5]. The

entire E. superba (PB) mitochondrial genome sequence

was deposited in GenBank with accession number

EU583500 (Fig. 3).

Gene arrangement

Pancrustacea or Tetraconata (uniting Crustacea and Hexa-

poda) share the same ground pattern in mitochondrial gene

order [1, 2, 5, 30]. Among 37 species of Crustacea

with complete mitochondrial genome sequences deposited

in GenBank, the gene order of 13 species is identical to

the pancrustacean ground pattern. Within Malacostraca,

12 of the 22 species retained the pancrustacean ground

pattern, including five mantis shrimps (Stomatopoda), four

penaeid shrimps (Dendrobranchiata) and three members

Table 2 Codon usage in 13 protein-coding genes of E. superba(Malacostraca: Euphausiacea)

Phe UUU 220 Ser UCU 111

UUC 73 UCC 18

Leu UUA 281 UCA 90

UUG 45 UCG 9

Leu CUU 101 Pro CCU 62

CUC 32 CCC 13

CUA 97 CCA 55

CUG 17 CCG 14

Ile AUU 217 Thr ACU 89

AUC 65 ACC 23

Met AUA 170 ACA 85

AUG 46 ACG 11

Val GUU 117 Ala GCU 107

GUC 18 GCC 29

GUA 105 GCA 98

GUG 32 GCG 10

Tyr UAU 106 Cys UGU 34

UAC 43 UGC 8

End UAA 9 Trp UGA 77

UAG 1 UGG 24

His CAU 42 Arg CGU 24

CAC 43 CGC 2

Gln CAA 46 CGA 29

CAG 27 CGG 7

Asn AAU 87 Ser AGU 37

AAC 40 AGC 17

Lys AAA 64 AGA 54

AAG 20 AGG 19

Asp GAU 49 Gly GGU 98

GAC 33 GGC 20

Glu GAA 46 GGA 87

GAG 28 GGG 40

Table 3 Genomic characteristics of E. superba (Malacostraca:

Euphausiacea) mtDNAs

Species E. superb(PB)

E. superb(WS)

GenBank accession no. EU583500 AB084378

Heavy-strand Length (bp) 15,498 14,606

A ? T (%) 68.1 67.7

Protein-coding

genes

No. of amino-acidb 3,711 3,714

A ? T (%)

All positions 66.2 66.3

First codon

positions

58.8 58.8

Second codon

positions

62.2 62.3

Third codon

positions

77.7 77.9

lrRNA Length (bp) 1,326 1,326

A ? T (%) 75.7 75.8

srRNA Length (bp) 808 618a

A ? T (%) 75.0 74.6

tRNA Length (bp) 1,551 1,234

A ? T (%) 68.5 67.7

Putative control

region

Length (bp) 456a –

A ? T (%) 73.2 –

a Incompleteb Not include stop codon

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of Pleocyemata (Figs. 2, 4). Among Decapoda, rear-

ranged mitochondrial genomes have been found only in

the suborder Pleocyemata. Five species of the infraorder

Brachyura (Callinectes sapidus, Portunus trituberculatus,

Pseudocarcinus gigas, Eriocheir sinenesis, and Geothel-

phusa dehaani), share a translocation of the trnH gene

compared to the pancrustacean ground pattern [24, 25, 27–

29] (Fig. 4). The trnH translocation shared by these five

taxa is regarded as a synapomorphic character and this

pattern of gene rearrangement supports the monophyly of

Brachyura.

Translocation of four tRNAs (trnL1, trnL2, trnW, and trnI)

and the duplication of trnN were founded in the

mitochondrial genome of E. superba when comparing the

genome with the pancrustacean ground pattern (Figs. 4, 5).

These data indicate that gene order is not conserved in

euphausiids mitochondrial genome. Gene order may be

useful for inferring phylogenetic relationship among eup-

hausiids and other malacostracans when more mitochon-

drial genome data from Euphausiacea are available.

Protein-coding genes

Protein-coding genes were identified with DOGMA [16]

and subsequently aligned with malacostracan mitochon-

drial genomes. There are in total nine genes (atp6, atp8,

Fig. 6 Putative secondary structures for 23 tRNA genes in the mitochondrial genome of E. superba (Malacostraca: Euphausiacea). Note:

Watson–Crick and GT bonds are denoted by ‘‘-’’ and ‘‘?’’, respectively

778 Mol Biol Rep (2010) 37:771–784

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cox1, cox2, cox3, cob, nad2, nad3, and nad6) are encoded

on the heavy-strand, while the remaining four (nad1, nad4,

nad4L, and nad5) are encoded on the light-strand (Table 1).

These transcriptional polarities are identical to the pan-

crustacean ground pattern (Figs. 4, 5) [5, 30].

Among the 13 protein-coding genes of E. superba (PB),

there are two reading-frame overlaps on the same strand

(atp6/atp8 and nad4/nad4L, both share seven nucleotides)

(Table 1), and this is common among the crustaceans.

Mitochondrial genes commonly use several alternatives to

ATG as start codons. Six of the thirteen PCGs (atp6, cob,

cox3, nad4, nad4L, and nad5) of E. superba (PB) start with

the ATG start codon, cox2 and nad1 genes start with ATA.

Nad2, nad3, and nad6 genes start with ATT, while atp8

and cox1 genes start with ATC and ACG, respectively

(Table 1).

Ten open reading frames of the E. superba (PB) mtDNA

end with the TAA or TAG stop codon (atp6, atp8, cob,

cox1, nad1, nad3, nad4, nad4L, nad5, and nad6), and the

remaining ones (cox1, cox3, and nad1) have incomplete

stop codons (TA- or T-) (Table 1). Compared with the

mitochondrial genome of E. superba (PB), there are six

alterations in the mitochondrial genome of E. superba

(WS), including atp6, atp8, nad2, nad3, nad4L, nad5, and

nad6. Those variations partially result from the gene

annotation in the mitochondrial genome of E. superba

(WS). Such immature stop codons are common among

animal mitochondrial genomes, and it has been shown that

TAA stop codons are created via posttranscriptional poly-

adenylation [43].

The pattern of codon usage in E. superba (PB) mtDNA

was also studied (Table 2). Excluding incomplete termi-

nation codons, there are a total of 3,711 codons in all

thirteen mitochondrial PCGs of E. superba. In the 13 PCGs

of E. superba (PB) mitochondrial genome, the most fre-

quently used amino acids were Leu (15.44%), followed by

Ser (9.57%), Phe (7.90%), Ile (7.60%) and Val (7.33%).

A common feature in most metazoan genomes is a bias

towards a higher representation of nucleotides A and T

which leads to a subsequent bias in the corresponding

Fig. 7 Topology derived from

NJ analysis of 13 concatenated

mitochondrial PCGs (nucleic

acid data) from 23

mitochondrial genomes, which

was built using the maximum

composite likelihood model of

evolutionary change. Note:

Black branches indicate the taxa

whose mitochondrial gene

arrangements are consistent

with the pancrustacean ground

pattern, and gray ones indicate

the taxa whose mitochondrial

genes encountered

rearrangement. Nodal support

indicated by Bootstrap value(BPNn)

Mol Biol Rep (2010) 37:771–784 779

123

encoded amino acids. The overall AT composition of

protein-coding regions is 69.8 and 66.2%, but at the third

codon positions the AT composition elevates to 77.7% in

E. superba (PB) (Table 3).

Transfer and ribosomal RNA genes

The E. superba (PB) mitochondrial genome encodes 23

tRNA genes, and has an extra trnN gene compared with the

standard set of metazoan mitochondrial genomes. Each

folds into a clover-leaf secondary structure (Fig. 6), rang-

ing from 64 to 72 nucleotides (Table 1), and the total

length is 1,551 bp with 68.5% AT (Table 3). Gene sizes

and anticodon usage are congruent to those described for

other malacostracan species (Fig. 6).

DOGMA and BLAST analyses indicate that the lrRNA

gene lies between the tRNALeu(UUR) and tRNAVal genes,

while the srRNA gene lies between tRNAVal and the puta-

tive control region, and both rRNA genes are encoded on

the light-strand. The location and orientation of rRNA

genes is typical to the pancrustacean ground pattern

(Fig. 5). The lengths of lrRNA/srRNA are 1,326/808 bp,

and the A ? T contents are 75.7/75.0%, which are similar

to other malacostracans (Table 3).

Phylogenomic relationship

Malacostracans have been the subject of a huge number of

taxonomic and phylogenetic studies employing morpho-

logical characters and molecular characters. Though Mal-

acostraca itself is widely regarded as a monophyletic

group, the relationship among various groups of Mala-

costraca is still controversial [35]. Here we explored the

phylogenetic relationship among major groups within

Malacostraca and the taxonomic position of Euphausiacea

based on their mitochondrial genomes.

All analyses based on nucleotide and amino acid data

strongly support the monophyly of Stomatopoda, Penaei-

dae, Caridea and Brachyura (Figs. 7, 8, 9, 10), which

is consistent with previous research [5, 32]. However,

the taxonomic position and phylogenetic relationship of

Euphausiacea within Malacostraca is unstable. The ML

and Bayesian analyses based on amino acid data support

the sister-group between Euphausiacea and Decapoda

Fig. 8 Topologies derived from

ML and Bayesian analyses of 13

concatenated mitochondrial

PCGs (nucleic acid data) from

23 mitochondrial genomes,

which was built using the

GTR ? I?G model of

evolutionary change. Note:

Black branches indicate the taxa

whose mitochondrial gene

arrangements are consistent

with the pancrustacean ground

pattern, and gray ones indicate

the taxa whose mitochondrial

genes encountered

rearrangement. Nodal support

indicated by Bootstrap value(BPMn and BPPn stand for ML

and Bayesian methods,

respectively)

780 Mol Biol Rep (2010) 37:771–784

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(BPMa \ 50, BPPa = 100) (Fig. 10), which is in coinci-

dence with traditional views (Fig. 2) [35] and supports the

monophyly of Eucarida. However, the ML and Bayesian

analyses based on nucleotide acid data and the NJ approach

based on nucleotide and amino acid data strongly support

the close relationship between Euphausiacea and Penaei-

dae, which destroys the monophyly of Decapoda and dis-

agrees with traditional classification (Fig. 2) [35]. In

addition, the bootstrap value is very high (BPNn = 100,

BPNa = 99, BPMn = 64, BPPn = 100), so the further

analyses with more mitochondrial genomes from Eup-

hausiacea and closer groups are needed. The NJ approach

based on nucleotide and amino acid data affiliated Isodopa

into Decapoda (Figs. 7, 9). Therefore, according to mito-

chondrial genomic data, whether the Pleocyemata and

Decapoda are monophyletic or not appear ambiguous.

Although many questions in the phylogeny of Mala-

costraca remain unanswered, it is desirable to increase the

resolution by adding more molecular information. Further

taxon sampling, especially from Leptostraca, Syncarida

and Peracarida, will be very useful for determining the

phylogenetic relationship among the major lineages within

Malacostraca.

Genetic variations among euphausiids

Euphausiids are dominant organisms in the zooplankton

population and play a central role in marine ecosystems

[8–10]. Population genetics and molecular ecological

research of euphausiids would be one of the most signifi-

cant areas in marine ecosystem studies. The goal of this

study is to reveal the genetic variation in euphausiids

mitochondrial genomes, which will shed light on the

population genetics and molecular ecology of euphausiids.

From comparing the mitochondrial genome of E. superba

(PB) and that of E. superba (WS) (Table 4), we found that

the variation of atp8 gene is zero, which is different from

previous thought that atp8 gene has the highest variation.

Cox1 gene has been used in barcoding and population

genetics analyses [44, 45]. However, the cox1 gene

Fig. 9 Topology derived from

NJ analysis of 13 concatenated

mitochondrial PCGs (amino

acid data) from 23

mitochondrial genomes, which

were built using the Poisson

correction. Note: Blackbranches indicate the taxa

whose mitochondrial gene

arrangements are consistent

with the pancrustacean ground

pattern, and gray ones indicate

the taxa whose mitochondrial

genes encountered

rearrangement. Nodal support

indicated by Bootstrap value(BPNa)

Mol Biol Rep (2010) 37:771–784 781

123

Fig. 10 Topologies derived from ML and Bayesian analyses of 13

concatenated mitochondrial PCGs (amino acid data) from 23

mitochondrial genomes; which were built using the MtArt and

MtRev matrix, respectively. Note: Black branches indicate the taxa

whose mitochondrial gene arrangements are consistent with the

pancrustacean ground pattern, and gray ones indicate the taxa whose

mitochondrial genes encountered rearrangement. Nodal support

indicated by Bayesian posterior probabilities (BPMa and BPPa stand

for ML and Bayesian methods, respectively)

Table 4 Mitochondrial gene

variant sites and identities

among E. superba (PB) and

E. superba (WS)

a Including mutations,

insertions and deletions

Gene Length (bp) Variant sitesa Identities (%)

atp6 672 2 99.702

atp8 156 0 100.000

cob 1,134 3 99.735

cox1 1,536 3 99.805

cox2 687 4 99.418

cox3 792 3 99.621

nad1 936 5 99.466

nad2 990 61 93.838

nad3 351 1 99.715

nad4 1,335 8 99.401

nad4L 297 2 99.327

nad5 1,728 12 99.306

nad6 519 4 99.229

srRNA 618a 2 99.676

lrRNA 1,326 1 99.925

782 Mol Biol Rep (2010) 37:771–784

123

contains only three alterations within 1,536 variable sites,

which is not enough in population genetic research. Among

all major mitochondrial PCGs, nad2 gene contains a

maximal variation with 61 alterations within 990 variable

sites, following by nad5 gene which contains 12 alterations

within 1,728 variable sites. Therefore, nad2 and nad5

genes were suggested as potential molecular markers.

Those markers may be used to study the inherit diversity

among different E. superba groups, which may be helpful

to the exploitation and management of E. superba biotic

resources reasonably.

Acknowledgments This study was supported by Jiangsu Natural

Science Funds (BK2007066), Lianyungang Natural Science Funds

(ZH200805) and Huaihai Institute of Technology Natural Science

Funds (Z2008044).

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