Anaulaciulus koreanus (Verhoeff, 1937)

Hwang, Ui Wook, 2017, The complete mitochondrial genome of the Korean endemic millipede Anaulaciulus koreanus (Verhoeff, 1937), with notes on the gene arrangement of millipede orders, Zootaxa 4329 (6), pp. 574-583 : 576-580

Characterization of the complete mitochondrial genome of A. koreanus .

The complete mitochondrial genome sequence of A. koreanus is 14,916 bp in length. It is within the range of other diplopod mitochondrial genome lengths from 14,747 bp to 15,282 bp ( Table 2 ). The mitochondrial genome of A. koreanus contains 13 proteincoding genes, two ribosomal RNA genes (12S rRNA and 16S rRNA), and 22 transfer RNA genes ( Fig. 1 , Table 3). The mitochondrial genes are arranged in the following 5’ – 3’ order (underlined genes are translated from the opposite strand): tRNA Ile, tRNA Met, ND2, tRNA Trp, tRNA Cy s, COI, COII, tRNA Lys, tRNA Asp, ATP8, ATP6, COIII, tRNA Gly, ND3, tRNA Ala , tRNA Arg, tRNA Asn, tRNA Ser(AGN), tRNA Glu, ND6, Cytb, tRNA Ser(UCN), tRNA Gln, tRNA Tyr, tRNA Phe, ND5, tRNA His, ND4, ND4L, tRNA Thr, tRNA Pro, ND1, tRNA Leu(UUR), tRNA Leu(CUN), 16S rRNA, tRNA Ľal, and 12S rRNA. The base composition of the heavy strand that encodes the majority of genes in the mitochondrial genome of A. koreanus is 36.1% adenine, 39.0% thymine, 14.0% cytosine, and 10.9% guanine, with a total A+T content of 75.1%. The overall A+T content of A. koreanus is the third highest of all diplopod mitochondrial genomes ( Table 2 ). In contrast with that of A. koreanus , also belonging to the same order Julida , A. gracilipes has a relatively lower A+T content (62.1%) ( Woo et al. 2007).

In the A. koreanus mitochondrial genome, G+C rich codons are rarely used for protein-coding genes, and nucleotides of the third codon position in protein coding genes are predominantly A or T due to generally high A+T content. The metazoan mitochondrial protein-coding genes are located principally between single or multiple tRNAs, but some protein-coding genes neighbor other protein-coding genes. There are five pairs of protein-coding genes without any intervening tRNA genes in the mitochondrial genome of A. koreanus : COI-COII, ATP8-ATP6, ATP6-COIII, ND6-Cytb and ND4L-ND4 ( Table 3). At these five gene junctions, there are sequences with the potential for forming stem-and-loop structures with a variable stem and loop ( Fig. 2 ). Of 22 tRNAs, 19 have a typical four-leaf clover shape, but three of tRNA Ser(AGN), tRNA Gln and tRNA Ile have unusual and unstable structures of the DHU arms ( Fig. 2 ). The stem and loop structure has been shown to operate as the regulatory signal that facilitates the precise cleavage of the mature protein-coding gene transcript from the primary multicistronic transcripts ( Ojala et al. 1980, 1981).

The mitochondrial genome of A. koreanus has only one large non-coding region of 590 bp located between 12S rRNA and tRNA Ile. This is different from those of other previously sequenced juliformian species which have two non-coding regions ( Lavrov et al. 2002, Woo et al. 2007). The large non-coding region has a 77.5% A+T content, which is higher than the overall A+T content of the mitochondrial genome (75.1%). The higher A+T content may favor the melting of the DNA strands required to initiate replication and transcription. Thus, the largest non-coding region is considered as a plausible control region for initiating replication and transcription of the mitochondrial encoded genes ( Clayton 1984, Cao et al. 2004).

The large non-coding region has a single stem and loop structure with the conserved 3' flanking "G(A)nT" and the 5' flanking "TATA" sequences ( Fig. 4 ). The motif of the single stem and loop structure has been observed in other arthropods ( Zhang et al. 1995, Zhang & Hewitt 1997, Brehm et al. 2001, Schultheis et al. 2002, Cha et al. 2004); however, this is the first report for Diplopoda.

Gene arrangement among millipede orders, While the gene arrangement patterns of ten millipede mitochondrial genomes are similar to one another (Fig. 5), the sphaerotheriid species appears to be very different from other millipedes, but similar to that of Limulus polyphemus , considered as an arthropod ground pattern ( Dong et al. 2012). The biggest differences between the mitochondrial genome of the order Sphaerotheriida versus the genomes of the other diplopod orders are the translocation of tRNA Tyr into the position between ND2 and COI in association with the translocations of ND6 and Cytb. These translocations are among the characters that separate the mitochondrial genome of the order Sphaerotheriida from other diplopod orders.

Similarly, the order Polydesmida lacks tRNA Cys between ND2 and COI, whereas this gene occurs in the other diplopod orders. Therefore, the absence of tRNA Cys between ND2 and COI may be a synapomorphy of order Polydesmida .

The close relationship between the order Callipodida and the superorder Juliformia was previously reported ( Sierwald & Bond 2007, Brewer et al. 2013). Sierwald & Bond (2007) suggested that two orders, Callipodida and Chordeumatida, form a clade with the superorder Juliformia ( Sierwald & Bond 2007). Their suggestion may be supported by the similar gene arrangement of CytB and ND5 genes of both Callipodida and Juliformia. Such a gene arrangement pattern is also different from those of other orders, which lack tRNA Tyr (e.g., the order Platydesmida ), or lack tRNA Gln, tRNA Tyr, and tRNA Phe and has translocation of tRNA Thr (e.g., the order Polydesmida ).

In the superorder Juliformia, three hypotheses of phylogenetic relationship among its three orders have been presented so far ( Enghoff 1984, Enghoff et al. 1993, Sierwald et al. 2003, Woo et al. 2007, Brewer et al. 2013). Enghoff (1984), Enghoff et al (1993) and Brewer et al. (2013) reported a close relationship between Julida and Spirostreptida , whereas Sierwald et al. (2003) suggested that the order Julida is closer to the order Spirobolida . Woo et al. (2007) suggested a close relationship between Spirobolida and Spirostreptida based on the mitochondrial gene arrangement pattern inferred from 13 mitochondrial protein-coding genes. In the present study, two julidan species, A. gracilipes and A. koreanus , have the same gene arrangement pattern between ND4L and ND1. The tRNA Thr was re-translocated into the position between ND4L and tRNA Pro. On the contrary, both the species of the orders Spirobolida and Spirostreptida have tRNA Thr to be relocated to after tRNA Ser. This evidence is more likely to support the close relationship between Spirobolida and Spirostreptida as Woo et al. (2007) suggested. Recently, the geographical distribution patterns of three orders Julida , Spirobolida and Spirostreptida were explored ( Shelley & Golovatch, 2011), which coincidently supported the closer relationship of the orders Spirobolida and Spirostreptida separate from the order Julida .

Conclusion

The complete mitochondrial genome of a julid A. koreanus ( Verhoeff, 1937) is 14,916 bp in length, and consists of 13 protein coding genes, 22 transfer RNAs, two ribosomal RNAs (12S and 16S rRNAs) and a large non-coding region (590 bp). Its genome size is within the general size range of other known diplopod species, and the A+T content is 75.1%. In the two julidan species A. gracilipes and A. koreanus , tRNA Thr was translocated into the position between ND4L and tRNA Pro, whereas the spirobolid and spirostreptid species have tRNA Thr to be relocated to after tRNA Ser. The results may support the close relationship between Spirobolida and Spirostreptida as Woo et al. (2007) suggested. Additional millipede mitochondrial genomes will be helpful for assembling a database of mitochondrial gene arrangement patterns to be used for informing millipede phylogeny.

Figures

FIGURE 1. The mitochondrial genome structures of Anaulaciulus koreanus (Verhoeff, 1937). The direction of transcription for each gene is shown by an arrow. The hatch-marked area is a large non-coding region.

FIGURE 2. Potential stem and loop structures of the junctional sequences of (A) COI-COII, (B) ATP 8 - ATP 6, (C) ND 6 - Cytb, (D) ND 4 L-ND 4, and (E) ATP 6 - COIII, and (F) a non-coding region in the mitochondrial genome of Anaulaciulus koreanus (Verhoeff, 1937). The anticodon sequences are boxed, and termination codons or incomplete termination codons are drawn with an underline or side-line. The number of nucleotide sequences in each loop is shown.

FIGURE 4. Comparison diagram of mitochondrial gene arrangement patterns of nine millipede species. One-letter codes corresponding to the amino acids of the tRNAs are used. Diagram is adapted and modified from Brewer et al. (2013).

Tables

TABLE 2. The whole mitochondrial genome lengths and A + T / G + C contents of ten millipede species examined in the present study

Order	Species	Genome length	A+T	G+C
Callipodida	Abacion magnum	15,160	66.6%	33.4%
Julida	Anaulaciulus koreanus	14,916	71.1%	28.9%
Julida	Antrokoreana gracilipes	14,747	62.1%	37.9%
Polydesmida	Appalachioria falcifera	15,282	64.0%	36.0%
	Xystodesmus sp.	15,791	67%	33%
	Asiomorpha coarctata	15,644	67.4%	32.6%
Platydesmida	Brachycybe lecontii	15,115	76.6%	23.4%
Spirobolida	Narceus annularis	14,868	63.7%	36.3%
Sphaerotheriida	Sphaerotheriidae sp.	14,970	71.2%	28.8%
Spirostreptida	Thyropygus sp.	15,133	67.8%	32.2%