Total DNA was extracted from individual specimens using a TIANamp Micro DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD) following manufacturer protocols. DNA samples were stored at -20 °C until further use.

The Daphnia pulex mitochondrial genome was amplified using five pairs of primers (Table 1). To obtain the complete sequences of Chinese Daphnia pulex, short-PCR and long-PCR methods were used. The primers employed in this study were designed based on the mitochondrial genomes of the North American Daphnia pulex (GenBank accession number AF117817) (Crease 1999) by using an NCBI primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/).

The PCRs were performed by using an Eppendorf Thermal Cycler (5331AH760577, Eppendorf, Germany) with a 25 µL volume reaction mixture containing 2.5 µL 10×LA-Taq Buffer II(Mg²⁺ plus), 4 µL dNTP Mixture (2.5 mM), 2 µL DMSO, 1 µL genomic DNA, 1 µL 10 µM of each primer, 0.5 µL MgCl₂ (25 mM) and 0.25 µL 2.5 units of LA Taq polymerase (TaKaRa Biomedical, Japan), and 12.25 µL distilled water.

The reaction conditions were one cycle of denaturation at 95 °C 5 min, 35 cycles of denaturation at 95 °C 30 s, annealing at 50 °C 30 s, extension at 72 °C for 2 to 8 min and a final extension at 72 °C for 10 min. Each amplicon (5 µL) was examined with agarose gel electrophoresis to validate amplification efficiency. PCR products were sequenced directly by primer walking from both directions after purification.

The raw sequences of mitochondrial genome were edited and assembled by using the program Seqman (DNAStar, Inc.) and then adjusting them manually. Protein-coding genes and rRNA genes were identified by the MITOS WebServer (http://mitos.bioinf.uni-leipzig.de/index.py) and the similarity between Daphnia pulex and that published in NCBI database were distinguished by BLAST search function (http://www.ncbi.nlm.nih.gov/BLAST/). Nucleotide sequences of PCGs were translated using the invertebrate mitochondrial genetic code. The tRNA genes were initially identified by the MITOS WebServer (http://mitos.bioinf.uni-leipzig.de/index.py) and their secondary structures were predicted and modified based on other metazoan’s secondary structure of tRNA genes.

The exact initiation and termination codons were identified by using Clustal X version 2.0 (Larkin et al. 2007) and relied on reference sequences from other invertebrates. Nucleotide composition and codon usage were calculated with MEGA 6.0 software (Tamura et al. 2013). The sequence data has been deposited into GenBank database under the accession number KT003819.

The mitochondrial genomes of the Chinese Daphnia pulex used in this study were similar to that of the Daphnia pulex in North America (Crease 1999). The complete mitochondrial genome of Chinese Daphnia pulex was a circular molecule 15,306 bp in size, containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes for both the small and large subunits (rrnS and rrnL) and a putative control region (Fig. 1). Among all the 37 genes, 23 genes were encoded on the J-strand. The remaining genes were encoded on the N-strand. 8 overlaps were found between adjacent genes (29 bp in total), among which the longest was 10 bp located at trnS2 and ND1. This included 15 intergenic spacers that ranged from 1 to 31 bp (84 bp in total), of which only one spacer was longer than 10 bp. That occurred between ND4L and trnT.

Figure 1. 

Structure of Chinese Daphnia pulex mitochondrial genome. COI, COII, COIII refer to the cytochrome oxidase subunits, Cytb refers to cytochrome b, ND1 - ND6 refer to NADH dehydrogenase components, and rrL and rrnS refer to rRNAs. tRNA genes are denoted by one letter symbol according to the IPUC-IUB single-letter amino acid codes. L1, L2, S1 and S2 denote tRNALeu(CUN), tRNALeu(UUR), tRNASer(AGN) and tRNASer(UCN), respectively. D-loop indicates A+T-rich region. Gene names outside the ring are coded on the majority strand while those inside are on the minority strand.

The mitochondrial genome of the Chinese Daphnia pulex has an A+T content of 64.50%, which is a little higher than that of the North American species (62.26%). Furthermore, it was determined that the AT skew was 0.006, and the GC skew was -0.107. AT skew and GC skew for a given strand were calculated as (G-C)/(G+C) and (A-T)/(A+T), respectively, with negative values in skewness meaning the coding strand is enriched for T or C. In contrast, positive values infer more As and Gs. On the whole, AT skew was slightly negative, or positive in the third codon position of vestimetiferans, and GC skew was more negative than AT skew (Table 2). Nucleotide bias can also be reflected by codon usage. We found that the RSCU (Relative Synonymous Codon Usage) value of NNA and NNU codons were greater than 1, which indicates that codons were biased in favor of codons with A or T in the third position (Table 3).

Amino acids are denoted as one-letter symbol according to the IUPAC-IUB single letter amino acid codes.

The complete mitochondrial DNA of Chinese Daphnia pulex from Chaohu had 13 protein-coding genes. Nine of these genes were located on the J-strand while the others were found on the N-strand; the same as the Daphnia pulex in North America (Table 3). Ten out of these 13 protein-coding genes initiated with typical ATN codons. ND2, COII, ATP6, COIII, ND4, Cytb and ND1 started with ATG, COI initiated with ATA, and moreover ND3 and ND6 used ATC as the initiating codon. The ATP8 and ND5 genes used GTG. The ND4L gene used none of these as initiating codon, but GCT.

As is the case with some other arthropod species, the initiation functions of the COIcodon has not been fully investigated. Atypical initiating codons for the COI gene in mitochondrial genomes have been reported in many studies, examples of these genes are: CGA (Gong et al. 2012), GTG (He et al. 2011), TTG (Hu et al. 2010, Li et al. 2012), ACG (Wilson et al. 2000), CCG (Fenn et al. 2007), ACC (Yamauchi et al. 2004), and TTA (Yamauchi et al. 2002). In Drosophila, Locusta and Daphnia, there are occasionally some uncommon quadruplets like, ATAA or ATTA, that may serve as an initiation codon (Wilson et al. 2000). One example of this is the COI gene in the North American Daphnia pulex initiating with ATTA (Crease 1999). However, the COI gene of Chinese Daphnia pulex started with classical ATA.

Nine of the 13 protein-coding genes used the typical termination codon TAN. ND2 and ATP8 terminated with TAG. COIII, ND3, Cytb, ATP6, ND4L, ND6 and ND1 all terminated with TAA. COI, COII, ND4 and ND5 used the incomplete termination codon T. Both of the complete termination codons TAG and TAA and two additional abbreviated termination codons T and TA were found in the North American Daphnia pulex (Table 4).

The use of incomplete termination codons on these genes might serve the purpose of avoiding overlapping nucleotides between adjacent genes (He et al. 2012). The incomplete termination codons would become functional termination codons after polycistronic transcript cleavage and polyadenylation processes have occured (Ojala et al. 1981). These incomplete codons and this mechanism has been commonly found in metazoan mitochondrial genomes (Wei et al. 2009, Liao et al. 2010). The total length of the 13 protein-coding genes was found to be 11,026 bp for the Chinese Daphnia pulex, which accounts for 63.37% of the total mitogenome length.

Many composition similarities were noted between the two different species compared in this study (Fig. 2).

Figure 2. 

Nucleotide compositions of the two Daphnia pulex from Chinese Chaohu (Ch) and North America (Na). CDS: protein-coding genes; 1st: first codon position; 2nd: second codon position; 3rd: third codon position; tRNA: tRNA genes; rRNA: rRNA genes; D-loop: A+T-rich region. In addition, stop codons were excluded.

All of the 22 typical arthropod tRNAs were found in the Chinese Daphnia pulex mitochondrial genome. They ranged from 63 to 72 bp in size. A schematic drawing of their respective secondary structures is shown in Figure 3. All tRNA genes had a clover-leaf structure except for trnS1, in which its DHU arm simply formed a loop. This loop in trnS is not uncommon in metazoan mitochondrial genomes (Crease and Little 1997). Whether or not the aberrant tRNAs lose their respective functions is still unknown. However, it’s possible this anomaly may be rectified by subsequent RNA-editing mechanisms (Lavrov et al. 2000, Masta and Boore 2004, Li et al. 2012).

Figure 3. 

Inferred secondary structure of 22 tRNA genes in Chinese Daphnia pulex mtDNA genome.

Non-canonical pairs, which possessed non Watson-Crick matches, commonly manifest in mitochondrial tRNA gene secondary structures. There are 30 base pair mismatches present in the tRNA secondary structures of Chinese Daphnia pulex mtDNA, including 15 wobble G-U pairs, 13 U-G pairs , two U-U pairs, one A-A pair and one U-C pair mismatch (Fig. 3). Nevertheless, the post-transcriptional RNA-editing mechanism can rectify these mismatches to maintain tRNA functions (Tomita et al. 2001, Wang et al. 2014).

Both the rrnL and rrnS genes were present in Chinese Daphnia pulex mitochondrial genome. They were located between trnL1 and the non-coding putative control region and separated by trnV, as similarly found in vertebrate mitochondrial genomes (Delisle and Strobeck 2002, Hwang et al. 2008, Chao et al. 2014).

Large and small ribosomal RNA genes (rrnL and rrnS) in Chinese Daphnia pulex were 1,313 bp and 744 bp long, respectively. The lengths of the two rRNAs were almost similar to that of the Daphnia pulex in North America (1,314 bp and 753 bp, respectively).

There are 15 non-coding regions ranging from 1 to 31 bp except for the A+T-rich region in the Chinese Daphnia pulex mitochondrial genome.

A 31 bp intergenic sequence was present between ND4L and trnT, which is also found in the North American Daphnia pulex mitochondrial DNA. The longest intergenic region in Chinese Daphnia pulex was the A+T-rich region. It was between rrnS and trnI with the length of 723 bp. It has an A+T content of 65.42%. It was a little longer than that of the North American Daphnia pulex mitochondrial DNA (689 bp), but lower in A+T content. This region usually contains replication and transcription areas in both vertebrates and invertebrates (Zhang and Hewitt 1997, Boore 1999). The stem-loop structure and the quantity of multiple repeats of AT sequences are notable features of the control region, ranging from 200 bp to 1,300 bp, and determine the difference in arthropod mitochondrial DNA size (Boore 1999).

The phylogenetic relationships among the Daphnia pulex from different areas were reconstructed based on nucleotide sequences of the COI gene by using the maximum likelihood (ML) mothod (Fig. 4). The phylogenetic analyses show that the Chinese and North American Daphnia pulex are recovered as two monophyletic clades with strong bootstrap support values (bs=100). They maybe evolved into two different species.

Figure 4. 

Phylogenetic tree obtained by the maximum-likelihood (ML) method and bootstrap values (1000 repetitions) of the branches were indicated. D. magna and D. carinata were used as outgroups.