Mol Biol Rep (2010) 37:771–784 DOI 10.1007/s11033-009-9602-7
The mitochondrial genome of Euphausia superba (Prydz Bay) (Crustacea: Malacostraca: Euphausiacea) reveals a novel gene arrangement and potential molecular markers Xin Shen Æ Haiqing Wang Æ Jianfeng Ren Æ Mei Tian Æ Minxiao Wang
Received: 3 December 2008 / Accepted: 24 June 2009 / Published online: 4 July 2009 Ó 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 organization, gene rearrangement and codon usage in the mitochondrial 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 noncoding 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 pancrustacean ground pattern. To investigate the phylogenetic relationship 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 comparing the mitochondrial genome between E. superba (PB) and E. superba (sampling from Weddell Sea, WS), we found
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:
[email protected] M. Wang e-mail:
[email protected]
that nad2 gene contains maximal variation with 61 segregating sites, following by nad5 gene which has 12 segregating 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 bp cox1-3 PCGs nCR cob mtDNA nad1–6, and 4L srRNA, and lrRNA tRNA L1 L2 S1 S2 BPNn BPNa BPMn
ATPase subunits 6 and 8 Base pair (s) Cytochrome c oxidase subunits I–III Protein-coding genes Non coding region Cytochrome b Mitochondrial DNA NADH dehydrogenase subunits 1–6 and 4L Small and large subunits ribosomal RNA Transfer RNA tRNALeu(CUN) tRNALeu(UUR) RNASer(AGN) tRNASer(UCN) Bootstrap probability of neighbor joining based on nucleotide data Bootstrap probability of neighbor joining based on amino acid data Bootstrap probability of maximum likelihood based on nucleotide data
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BPMa BPPn BPPa
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Bootstrap probability of maximum likelihood based on amino acid data Bayesian posterior probability based on nucleotide data 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 mitochondrial genome sequences has become popular [1–6]. This resulted from many advantages offered by mt genomes over other molecular markers. Compared with sequences of individual gene, mitochondrial genomes could provide sets of genome-level characteristics, such as Fig. 1 The sampling location of E. superba (PB) and E. superba (WS)
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the relative arrangements of gene orders, which are valuable 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: Euphausiacea). Furthermore, the phylogenetic relationship of Euphausiacea has been analyzed based on 23 malacostracan mitochondrial genomes. In addition to a better understanding of the phylogenetic history of the crustaceans, this work
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should be useful to the practice of biodiversity management and conservation strategies of this ecological and commercial significant species.
Materials and methods Sample collection and DNA extraction A single specimen of E. superba was obtained from the Prydz Bay (64°440 S, 73°000 E; 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.
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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/Eusnad5R, 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.
Long PCR and sequencing by primer walking Sequence analysis The mitochondrial genome of E. superba (PB) was amplified using a long PCR protocol [12]. Based on partial mitochondrial 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; Euscox3F: 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-
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)
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structures and mitochondrial Codon usage mitochondrial [19].
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anticodons. Gene map of E. superba (PB) genome was drawn by OGDRAW [18]. in the 13 PCGs of the E. superba (PB) genome was estimated with DnaSP 4.10.7
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, Fenneropenaeus 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, Lysiosquillina maculate, Squilla empusa, Squilla mantis and Euphausia superba (WS) [5, 11, 20–34]. Their traditional Fig. 3 Gene map of mitochondrial genomes of E. superba (Malacostraca: Euphausiacea). Note: Proteincoding genes are transcribed in a clockwise direction, except for nad1, nad4, nad4L, and nad5 genes. The two ribosomal RNA genes are encoded on the light strands. Transfer RNA genes are designated by single-letter amino acid codes. Genes encoded on the heavy and light strands 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
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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 evolution 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, phylogenetic 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 evolutionary change. In the NJ and ML methods, the assessment of node reliability was done using 1,000 bootstrap
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replicates. In the case of the Bayesian analyses, 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 sampled tree had stabilized. After omitting the first 100 ‘‘burn in’’ trees, the remaining 900 sampled trees were used to estimate the Table 1 Mitochondrial gene profile of E. Superba (Malacostraca: Euphausiacea)
Feature
Strand
Size (bp)
From–to nCR
Codon Start
Intergenic nucleotidesa Stop
1–316
?
316
tRNAAsn
317–381
?
65
0
Gln
379–447
-
69
-3
tRNAMet
447–514
?
68
nad2
515–1,514
?
1,000
tRNACys
1,515–1,578
-
64
0
tRNATyr
1,590–1,656
-
67
11
Trp
tRNA
tRNA
1,672–1,741
?
70
cox1
1,746–3,284
?
1,539
tRNALeu(CUN)
3,298–3,363
?
66
cox2
3,364–4,051
?
688
tRNALys
4,052–4,120
?
69
Asp
tRNA
-1 ATT
T-
0
15 ACG
TAA
4 13
ATA
T-
0 0
4,121–4,188
?
68
atp8
4,189–4,347
?
159
ATC
TAA
0
atp6 cox3
4,341–5,015 5,015–5,807
? ?
675 793
ATG ATG
TAA T-
-7 -1
tRNAGly
5,808–5,874
?
67
nad3
5,875–6,228
?
354
ATT
TAA
Ala
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
Phe
tRNA
tRNA
6,664–6,731
-
68
nad5
6,731–8,461
-
1,731
tRNAHis
8,462–8,527
-
66
nad4
8,528–9,865
-
nad4L
0
0 0
99 ATG
TAA
-1
1,338
ATG
TAA
0
ATG
TAA
-5
0
9,861–10,158
-
298
Thr
10,161–10,226
?
66
tRNAPro nad6
10,228–10,294 10,298–10,819
?
67 522
ATT
TAA
1 3
cob
10,819–11,955
?
1,137
ATG
TAA
-1
tRNASer(UCN)
11,976–12,046
?
71
nad1
12,064–13,002
-
939
tRNALeu(UUR)
13,019–13,084
-
66
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
tRNA
Note: a Numbers correspond to the nucleotides separating different genes. Negative numbers indicate overlapping nucleotides between adjacent genes. ‘‘-’’ Indicates termination codons completed via polyadenylation
Position
50% majority rule consensus tree and the Bayesian posterior 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
2
20 ATA
TAG
17 16
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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 Malacostraca. 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 rearrangement 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
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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 stomatopods 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 proteincoding, 23 transfer RNA, and 2 ribosomal RNA genes), 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
Leu
UUG CUU
45 101
UCG CCU
9 62
CUC
32
CCC
13
CUA
97
CCA
55
CUG
17
CCG
14
AUU
217
ACU
89
AUC
65
ACC
23
AUA
170
ACA
85
AUG
46
ACG
11
Ile Met Val
Tyr End His Gln Asn Lys Asp Glu
GUU
117
Pro
Thr
Ala
GCU
107
GUC
18
GCC
29
GUA
105
GCA
98
GUG
32
GCG
10
UAU
106
UGU
34
Cys
77 24
CGU
24
CGC
2
Pancrustacea or Tetraconata (uniting Crustacea and Hexapoda) 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 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
No. of amino-acidb
3,711
3,714
Protein-coding genes
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
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
Length (bp) A ? T (%)
456a 73.2
– –
43
UAA UAG
9 1
Trp
CAU
42
Arg
CAC
43
CAA
46
CGA
29
CAG
27
CGG
7
AAU
87
AGU
37
AAC
40
AGC
17
AAA
64
AGA
54
AAG
20
AGG
19
GAU
49
GGU
98
GAC
33
GGC
20
Putative control region
GAA
46
GGA
87
a
GAG
28
GGG
40
Incomplete
b
Not include stop codon
Gly
8
UGA UGG
Gene arrangement
UAC
Ser
UGC
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 mitochondrial 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).
lrRNA
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778
of Pleocyemata (Figs. 2, 4). Among Decapoda, rearranged 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 Geothelphusa 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
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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 euphausiids and other malacostracans when more mitochondrial genome data from Euphausiacea are available. Protein-coding genes Protein-coding genes were identified with DOGMA [16] and subsequently aligned with malacostracan mitochondrial 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
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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 pancrustacean 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
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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 polyadenylation [43]. The pattern of codon usage in E. superba (PB) mtDNA was also studied (Table 2). Excluding incomplete termination 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 frequently 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)
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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).
and the A ? T contents are 75.7/75.0%, which are similar to other malacostracans (Table 3).
Transfer and ribosomal RNA genes
Malacostracans have been the subject of a huge number of taxonomic and phylogenetic studies employing morphological characters and molecular characters. Though Malacostraca itself is widely regarded as a monophyletic group, the relationship among various groups of Malacostraca 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, Penaeidae, 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
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), ranging 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 putative 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, 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)
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Phylogenomic relationship
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(BPMa \ 50, BPPa = 100) (Fig. 10), which is in coincidence 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 Penaeidae, which destroys the monophyly of Decapoda and disagrees 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 Euphausiacea 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 mitochondrial genomic data, whether the Pleocyemata and Decapoda are monophyletic or not appear ambiguous. Although many questions in the phylogeny of Malacostraca remain unanswered, it is desirable to increase the resolution by adding more molecular information. Further taxon sampling, especially from Leptostraca, Syncarida
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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 significant 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: 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 (BPNa)
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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 Table 4 Mitochondrial gene variant sites and identities among E. superba (PB) and E. superba (WS)
Gene
Including mutations, insertions and deletions
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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
a
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)
351
1
99.715
nad4 nad4L
1,335 297
8 2
99.401 99.327
nad5
1,728
12
99.306
nad6
519
4
99.229
srRNA
618a
2
99.676
lrRNA
1,326
1
99.925
Mol Biol Rep (2010) 37:771–784
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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