Complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus (Gasterosteiformes, Gasterosteidae)

Abstract

The complete mitochondrial genome of the Greek nine-spined stickleback Pungitius hellenicus was obtained using Illumina high-throughput sequencing of genomic DNA. The genome was 16 713 bp long, and contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. The arrangement of the genes was identical to that of other Gasterosteidae fishes. However, the control region of P. hellenicus contained three copies of imperfect repeated sequences (72–78 bp in single motifs), while P. pungitius and P. tymensis have one or two copies. Nucleotide identity between P. hellenicus and three other Pungitius species across all the 37 genic regions was 93.0% to 95.5%.

The number of valid species in the circumpolarly distributed teleost fish genus Pungitius remains uncertain, but morphological evidence suggests that the Greek nine-spined stickleback (Pungitius hellenicus) is likely to be a valid species (Keivany et al. 1997; Keivany & Nelson 2000). However, little genetic information is available on this species (Geiger et al. 2014). Hence, access to the complete mitochondrial genome sequence of P. hellenicus would allow evaluation of its validity as a distinct species, as well as its phylogenetic positioning within the genus Pungitius (cf., Takahashi & Goto 2001; Keivany & Nelson 2004; Mattern 2004). As the known distribution of P. hellenicus is confined to only a few localities in the Sperchios River basin in Central Greece (Keivany et al. 1999; Keivany & Nelson 2000), and the species is listed as critically endangered (Martins & Wiswedel 2015), the genomic information is likely to support conservation and management planning of this species.

Pungitius hellenicus was collected from the Sperchios River basin in Greece (38°51′N, 22°26′E), and genomic DNA was sequenced using the Illumina HiSeq2000 platform with 100 paired-end strategy. In total, 16.6 million reads were aligned against the reference P. sinensis mitogenome (Hwang et al. 2012a) using bwa-0.5.10 (Li & Durbin 2009). Mean sequence coverage across the mitogenome was 43-fold, and with the exception of a 265 bp gap in the control region, all regions had at least one-fold coverage (83.7% with ≥20-fold coverage). Sanger sequencing was used to fill the 265 bp gap. PCR products were obtained using a primer set (PCR-F: 5′-CAAGGTTGAACATCTTTCGC-3′ and PCR-R: 5′-CTGATACCAGCTCCTTGTTCC-3′) following the protocols and procedures described in Teacher et al. (2011). The consensus sequence of the P. hellenicus mitogenome was generated using SAMtools 1.2 (Li et al. 2009) and manually checked.

The complete mitochondrial genome of P. hellenicus was 16 713 bp long (GenBank Accession No. KU236383), containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a control region. The arrangement of the genes was identical to that of other Gasterosteidae fishes (Miya et al. 2001; Kawahara et al. 2009; Hwang et al. 2012a, 2012b). In the 13 protein-coding genes, an incomplete stop codon was found for four genes (ND2, COII, ND4 and Cytb). Three copies of imperfect repeated sequences (72–78 bp in single motifs) were identified in the control region, whereas one or two copies have been observed in P. pungitius and P. tymensis (Takahashi & Goto 2001). The overall base composition of the entire mitogenome was 27.4% for A, 26.4% for T, 17.6% for G and 28.6% for C.

To investigate the phylogenetic position of P. hellenicus in Gasterosteidae fishes, a maximum-likelihood tree was constructed with the 37 genes (15 581 bp in total) using RAxML v.8.0 under the GTR + GAMMA model, 37 gene partitions and 100 thorough bootstrap replicates (Stamatakis 2014). Pungitius hellenicus was phylogenetically positioned with other Pungitius species (i.e. P. pungitius, P. sinensis and P. kaibarae), showing clear divergence from them (Figure 1). Nucleotide identity in the 37 gene regions was 93.0% to 95.5% between P. hellenicus and the other Pungitius species.

Figure 1.

A maximum likelihood tree inferred from 37 mitochondrial genes among nine Gasterosteidae and an outgroup (Aulorhynchus flavidus) species. Bootstrap support is indicated at nodes. GenBank accession numbers are indicated in brackets.