Description of a species of Fabaeformiscandona (Ostracoda, Crustacea) from Kushiro Marsh, Hokkaido, Japan, with the nearly complete mitochondrial genomic sequence

Abstract Background So far, 16 species of non-marine ostracods have been reported from Kushiro Marsh, Kushiro Shitsugen National Park, eastern Hokkaido, Japan (Hiruta and Smith 2001, Smith and Hiruta 2004). Nine of these species are in Candonidae, the second-most diverse family of non-marine ostracods. This family contains ca. 550 species, or around 25% of the total number of non-marine ostracod species (Martens et al. 2008). New information We sampled ostracods in Kushiro Marsh on 27 December 2012 and identified an undescribed species in the family Candonidae, herein described as Fabaeformiscandona kushiroensis sp. nov. This species belongs to the F. acuminata species group and is characterized by the shapes of the elongate, dorsally directed medial and outer lobes on the distal end of each hemipenis. We also determined for this species the sequence of the nearly complete mitochondrial genome, the first record from the order Podocopa. The genome (ca. 17 kbp) contains two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes, as also found in other arthropods for which the mitochondrial genome has been sequenced. The gene arrangement is similar to the pancrustacean ground pattern, except that in the control region there is an approximately 2 kbp tandem repeat region composed of 220-bp motif sequences. We describe the genetic features of the mitochondrial genome, including nucleotide composition and the secondary structures of tRNAs and rRNAs, and compare them with the genome of Vargula hilgendorfii (Myodocopa, Ostracoda).


Introduction
Ostracods are small crustaceans, with most species being approximately one millimeter in length, which have a bivalved carapace covering non-mineralized body and appendages. They occur in almost every aquatic environment, including marine, brackish-water, freshwater, and groundwater. Ostracods have the most complete and continuous fossil record of any extant arthropod group, attributable to small body size, the calcified valves, and large population sizes (Moore 1961). The fossil record shows that the extant orderlevel lineages were already established by around 500 Ma, within ca. 50 million years (Maddocks 1982, Martens et al. 1998). Most traditional classifications in the Linnean system have ranked the Ostracoda at the class level (e.g., Davis 2001, Forest 2004), although recent phylogenomic studies (Regier et al. 2010, Oakley et al. 2013) have placed the group in the clade Oligostraca, together with Mystacocarida, Branchiura, and Pentastomida. In these molecular phylogenetic studies, Myodocopa and Podocopa were poorly resolved among arthropods and showed deep divergence, although these groups might be sister taxa, and there is no contradictory evidence for that.
Kushiro Marsh, situated in Kushiro Shitsugen National Park, eastern Hokkaido, is the largest marshland in Japan (Fig. 1). It covers an area of 269 km , which is 60% of the total area of Japan's freshwater wetlands. Most of the shallow waters in the marsh are frozen from December to February. Previous studies have reported 16 species of non-marine ostracods from Kushiro Marsh (Hiruta and Smith 2001, Smith and, nine of which are in the family Candonidae, the second-most diverse family of non-marine ostracods. Candonidae contains around 550 species, or roughly 25% of the total number of non-marine ostracod species (Martens et al. 2008).
Although complete mitochondrial genomic sequences are useful for phylogenetic and population genetic studies (e.g., Kilpert et al. 2012, Omote et al. 2013, only one has been determined from ostracods. Ogoh and Ohmiya (2004) reported the complete mitochondrial genomic sequence for the sea-firefly, Vargula hilgendorfii (Müller 1890) (Myodocopa, Ostracoda), but no complete mitochondrial genomic sequence was available for any species in Podocopa. In this report, we describe a new species in Fabaeformiscandona and report its nearly complete mitochondrial genomic sequence, the first record from the order Podocopa. We also describe the features of the mitochondrial genome and compare them with the genome of V. hilgendorfii.

Specimens
Material was collectednear the Onnenai Visitor Center (43°06′17.6″N 144°19′46.5″E) in Kushiro Marsh, Hokkaido, Japan (Fig. 1) on 27 December 2012, when the ambient temperature was −10°C. The sampling site is located in a spring area beside a hill. Water and bottom sediment were strained through a 0.1-mm-mesh sieve. Specimens were sorted under a stereoscopic microscope and preserved in 99% ethanol.
Selected specimens were dissected, and the appendages were mounted in Hoyer's solution on glass slides and drawn with the aid of a camera lucida. Some carapaces were pasted with a tragacanth gum solution onto microfossil slides. For scanning electron microscope (SEM) observation, carapaces and soft parts were mounted on stub after treatment with hexamethyldisilazane (HMDS) (Nation 1983). Specimens were coated with gold and examined with an S-3000N (Hitachi High Technologies) SEM at 15-20 kV accelerating voltage. The material used in this study has been deposited in the Invertebrate Collection of the Hokkaido University Museum, Sapporo (ICHUM).
The chaetotaxic notation follows that of Broodbakker and Danielopol (1982), as revised for the antennae by Martens (1987) and for the thoracopods by Meisch (1996). Hemipenis terminology follows that of Danielopol (1969). We use the same abbreviations for limbs as Meisch (2000) and Smith and Janz (2008).

DNA extraction
Total genomic DNA was extracted from the whole body of one individual by using a DNeasy Blood & Tissue Kit (QIAGEN), with modifications from Johnson et al. (2004). Specimens were incubated in ATL buffer with proteinase K for at least 48 h at 55°C to lyse the tissue. Before the lysis mixture was pipetted into a spin column, the exoskeleton and carapaces of the specimen were retrieved and mounted in Hoyer's solution on a glass slide.

Amplification of partial mitochondrial gene sequences
Initially, universal primer sets were used to amplify parts of the cytochrome c oxidase subunit I gene (COI) and 12S rRNA (srRNA) genes (primers LCO1490 and HCO2198 for COI, Folmer et al. 1994;121Sa and 12Sb for srRNA, Palumbi 1996). PCRs were performed in an ABI 2720 Thermal Cycler (Applied Biosystems) in 10-µl volumes containing 1 µl of template solution, 0.8 µl of 2.5 mM each dNTP, 10 pmol of each primer, and 0.25 U Ex Taq polymerase (Takara) in 1× buffer (Takara). Amplification conditions for COI and srRNA were 95°C for 1 min; 35 cycles of 95°C for 30 sec, 50°C for 30 sec, and 72°C for 1 min; and 72°C for 7 min.

Amplification of the whole mitochondrial genome
The COI and srRNA sequences were used to design new primer sets (Suppl. material 5;Fab_MtF_COIF,Fab_MtF_COIR,Fab_MtF_12S,and Fab_MtF_12R), which were used for long-PCR amplification of the whole mitochondrial genome in two parts. Long-PCRs were carried out in 50-µl reaction volumes containing 1 µl of template solution, 4 µl of 2.5 mM each dNTP, 10 pmol of each primer, and 1.25 U PrimeSTAR GXL DNA polymerase (Takara) in 1× buffer (Takara). Amplification conditions for the two fragments were 30 cycles of 98°C for 10 sec and 68°C for 10 min; and 68°C for 7 min. Primers used for primer walking to sequence the long amplicons obtained are listed in Suppl. material 5. The direction and position of each primer is shown in Suppl. material 1.

Amplification of nuclear rRNA genes
Nuclear rRNA genes were amplified with primer set 18S_F1 and Mallat_R. Long-PCR in 50-µl volumes containing 1 µl of template solution, 4 µl of 2.5 mM each dNTP, 10 pmol of each primer, and 1.25 U PrimeSTAR GXL DNA polymerase (Takara) in 1× buffer (Takara). Amplification conditions were as for the whole mitochondrial genome fragments. Internal primers used for sequencing the nuclear fragments are listed in Suppl. material 5.

Sequencing
Amplification products were purified by the method of Boom et al. (1990). Nucleotide sequences were determined by direct sequencing using a BigDye Terminator Cycle Sequencing Kit ver. 3.1 and an ABI 3730 DNA analyzer (Applied Biosystems). Sequences have been deposited in Genbank under accession numbers AP014656 (mitochondrial DNA) and AB996740 (nuclear rRNA).

Length estimation for the tandem-repeat region
To estimate the length of tandem-repeat region within the control region (CR), primers were designed (Fab_CRF and Fab_CRmF; Suppl. material 5) that bound close to the repeat region on either side. The repeat region was amplified by PCR and its length estimated by electrophoresis.

Sequence analysis
Nucleotide sequences were assembled and edited with MEGA5 (Tamura et al. 2011). Each gene in the mitochondrial genome was identified on the MITOS Web Server (Bernt et al. 2013). Area of ND4 and COIII genes were detected into separated regions by using MITOS Web server, so that we conformed the nucleotides and the translated amino acid sequences to determine each of the coding area. The putative secondary structures of the tRNA genes were also estimated by MITOS Web server.
The boundaries and secondary structures of the mt rRNA genes were determined by using Centroidfold (Sato et al. 2009).
For the nuclear rRNA genes, information on secondary structure from Apis mellifera (Gillespie et al. 2006) and the secondary structure estimated by Centroidfold were used to determine the boundaries of each gene.

Description
Description of male.
Md (Fig. 11) consists of coxal plate and four-segmented palp. Coxal plate with anterolateral seta and seven stout teeth, latter interspersed with several setae of various lengths. First podomere of palp with exopodal plate (Exo) and one long and one short, stout inner-distal plumose setae, one long antero-distal seta, and alpha simple seta. Second podomere of palp with beta seta, group of four setae, and one plumose seta. Third podomere of palp with three outer-apical setae, one mid-apical gamma seta, and two long apical setae. Fourth podomere of palp with two claws and two apical setae.   L5 (Figs 14, 15) with palp, two filament-like setae (Exo), one antero-proximal seta, one antero-apical seta, and one postero-apical seta. Palp transformed into asymmetrical clasping processes; left clasping process (Fig. 14) slender, somewhat curved and hook-like in shape, with two short mid-setae; right clasping process (Fig. 15) sharply curved and proximally stout, helmet-like in shape, with two short mid-setae. Masticatory process with numerous setae.  L7 (Fig. 17) five-segmented. Penultimate segment subdivided. First podomere with two setae (d1, dp). Fourth podomere with one apical seta (g). Fifth podomere with two long (h2, h3) and one shorter (h1) setae. One third of distal tips of the h3 seta bend.  Uropodal ramus (Fig. 18) with short anterior seta (Sa) and well-developed posterior seta (Sp) longer than terminal claws. Two terminal claws simple, slender.
FRO (Fig. 23) with elongate, somewhat rounded posterior projection with digitiform end. Spiral canal situated anteriorly, with wall thickened near seminal receptacle. Vaginal opening inconspicuous, without rimmed chitinized ring.  In other characters, female similar to male.

Diagnosis
Carapace with dorsal hump one-third of length from posterior end. Male carapace has anterior and posterior margins equally rounded. Female carapace has straight posterodorsal margin, with distinctive collar-like fold in right valve. Male hemipenis very large; medial lobe elongate, rounded distally; outer lobe elongate, with spine-like protrusion; both lobes toward dorsal side. M-process well developed, S-shaped, with rounded distal end. Projection on female reproductive organ elongate, tapering distally.

Etymology
The specific epithet is an adjective derived from Kushiro Marsh, type locality, in combination with the Latin suffix -ensis.

Taxon discussion
Fabaeformiscandona kushiroensis sp. nov. clearly belongs in the acuminata group [medial lobe elongate and directed in the dorsal direction in F. kushiroensis; relatively small and distally digitiform in F. danielopoli; distally flat in F. nishinoae; distally squareshaped in F. akaina; distally large and square-shaped in C. quasiakaina;]; 3) M-process of Hp [distally rounded in F. kushiroensis; distally digitiform in the other four species]; 4) FRO [with elongate, distally digitiform process in F. kushiroensis; with elongate, proximally somewhat broad process in F. danielopoli, F. nishinoae and F. akaina; with relatively short, proximally stout process in C. quasiakaina]. It is generally similar to F. hyalina (Brady and Robertson 1870) and F. levanderi (Hirschmann 1912) in the shapes of the female and male valves, respectively. However, these three species differ in 1) the postero-dorsal margin of the female RV [almost straight with a conspicuous inner fold in F. kushiroensis; almost straight in F. hyalina; broadly rounded in F. levanderi]; 2) the distal lobes of Hp [outer (a) and medial (h) lobes elongate and directed in the dorsal direction in F. kushiroensis; outer lobe similar to F. kushiroensis, medial lobe stout and broad in F. hyalina; outer lobe broad and partly subdivided, medial lobe lacking in F. levanderi]; 3) M-process of Hp [distally rounded in F. kushiroensis; proximally stout and distally digitiform in F. hyalina; proximally stout and distally inflated in F. levanderi]; 4) FRO [with elongate, distally digitiform process in F. kushiroensis; conical in F. hyalina: with elongate, distally rounded process in F. levanderi]. Karanovic and Lee (2012) pointed out that these four species from east Asia and F. hyalina from Europe were closely related species in which having tapering/pointed tip on outer lobe of Hp. The new species form Kushiro Marsh also may belong to the group, because of having similar outer lobe of Hp.

Gene contents and organization
The mitochondrial genome of F. kushiroensis is about 17 kbp in size and includes 13 protein-coding genes, 22 tRNA genes, and two rRNA genes, as typically found in other arthropods (Fig. 24, Table 1). The order of the tRNA, rRNA, and protein-cording genes is that of the putative pancrustacean gene order (Kilpert et al. 2012), with the exception that the position of tRNA has been translocated to between tRNA and tRNA (Fig. 25). There is one major non-coding region that presumably contains the origin of replication and regulatory elements for transcription, as well as tandem repeat sequences. There are small gene overlaps at 12 gene borders (Table 1), with the largest (between COIII and tRNA ) being 50 nucleotides long. Short, non-coding sequences are also present between these genes. The largest spacer sequence (between ND4 and ND4L) is 80 nucleotides long.

Nucleotide composition
The overall A + T content of the F. kushiroensis mitochondrial genome is 68.8% (A = 35.1%; T = 33.7%; G = 11.8%; C = 19.4%), which is relatively low among arthropods. Table 2 gives the A + T content for each type of gene and the CR, and Table 3 gives the nucleotide composition of each codon position for the coding genes. Long tandem repeat sequences in the CR The CR contains a long tandem repeat region composed of replicated 220-bp units, three of which were sequenced at each end of the region (Fig. 26). However the sequence of central part of the region is missing due to the technical reasons by Sanger sequencing method. The length of this region is about 2 kbp, estimated from electrophoresis. Table 2.
Characteristics of the mitochondrial genome of two ostracods. Table 3.
Nucleotide composition at each codon position in the protein-coding genes of mitochondrial genome for two ostracods.

Transfer RNA genes
The A + T content of the 22 tRNA genes is 72.2%, which is higher than the overall A + T composition of the mtDNA. The secondary structures of the 22 tRNA genes were determined from the MITOS Web Server to be complete cloverleaves (Suppl. material 2). The anticodon nucleotides for the corresponding tRNA genes are identical to those in V. hilgendorfii. The tRNA and tRNA genes encode UUU and UCU instead of the more common CUU and GCU, respectively, typical of arthropod mtDNA. Furthermore, the tRNA gene has lost the D-arm, which is also the condition in V. hilgendorfii. Several overlaps associated are with tRNA genes (Table 1).

Ribosomal RNA genes
The 12S rRNA (srRNA) and 16S rRNA (lrRNA) genes in F. kushiroensis are 708 and 1121 nucleotides long, respectively. Their locations and lengths were determined from an analysis of similarity to the V. hilgendorfii homologs and by analysis of their secondary structures reconstructed with Centroidfold (Suppl. material 3).

Nuclear ribosomal RNA genes
The nuclear ribosomal RNA genes in F. kushiroensis are determined by a single sequence in which total 7805 nucleotides long, including internal transcribed spacer 1 and 2. Partial 18S rRNA (srRNA), complete 5.8S (lrRNA) and partial 28S rRNA (lrRNA) genes are 1792, 155 and 4007 nucleotides long, respectively. Their locations and lengths were determined from an analysis of similarity to the other sequences of Podocopid ostracods on GenBank and by analysis of their secondary structures reconstructed with Centroidfold.
Organization of the tandem repeat region in the control region of Fabaeformiscandona kushiroensis sp. n.

Discussion
The description of Fabaeformiscandona kushiroensis sp. n. brings the number of nonmarine ostracods species reported from Kushiro Marsh, eastern Hokkaido, to 17 species. The specimens used in this study were collected at the end of December, from a spring area, which is a relatively temperature-stable environment.
Our samples collected at the end of December contained mature males and females, but no juveniles, which suggests that F. kushiroensis does not begin breeding at least before January. If F. kushiroensis starts breeding in early spring, its breeding strategy would be similar to that of congeners (Meisch 2000, Karanovic 2012). In Sarobetsu Marsh, northern Hokkaido, populations of Cryptocandona sp. in the subfamily Candoninae contain juveniles from early spring, and an increasing number of mature individuals from summer to autumn (Hiruta and Mawatari 2013).
The gene order in the mitochondrial genome of F. kushiroensis (subclass Podocarpa) is similar to the pancrustacean ground pattern, with just one difference. In contrast, the mitochondrial genome of V. hilgendorfii (subclass Myodocopa) has a quite different gene order and a duplicated CR, and its evolution from the pancrustacean ground pattern is difficult to explain through a few simple events (Ogoh and Ohmiya 2004; Fig. 25). These differences indicate a deep divergence between the subclasses Podocopa and Myodocopa, as fossil information has also suggested.
The CR in Fabaeformiscandona kushiroensis contains a long tandem-repeat region composed of 220-bp motif sequences. These kind of repeat region are rare not only in Crustaceans but also in other metazoans (Kilpert and Podsiadlowski 2006). While tandem repeats have been found in the CR in various organisms, they are highly variable in the length of the motifs and the number of copies. For example, the tandem repeat regions in rabbit and the fish owl contain 153 bp and 77-78 bp motifs, respectively (Casane et al. 1994, Omote et al. 2013); among arthropods, tandem-repeat sequences have previously been reported in insects and isopods (Zhang and Hewitt 1997, Kilpert and Podsiadlowski 2006).