Biodiversity Data Journal :
Taxonomic Paper
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Corresponding author:
Academic editor: Viktor Senderov
Received: 04 Nov 2015 | Accepted: 07 Dec 2015 | Published: 11 Dec 2015
© 2015 Shimpei Hiruta, Shin-ichi Hiruta
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hiruta S, Hiruta S (2015) Description of a species of Fabaeformiscandona (Ostracoda, Crustacea) from Kushiro Marsh, Hokkaido, Japan, with the nearly complete mitochondrial genomic sequence. Biodiversity Data Journal 3: e7074. https://doi.org/10.3897/BDJ.3.e7074
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So far, 16 species of non-marine ostracods have been reported from Kushiro Marsh, Kushiro Shitsugen National Park, eastern Hokkaido, Japan (
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).
Kushiro Marsh, freshwater, Ostracoda, mitochondrial genome
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 (
Kushiro Marsh, situated in Kushiro Shitsugen National Park, eastern Hokkaido, is the largest marshland in Japan (Fig.
Although complete mitochondrial genomic sequences are useful for phylogenetic and population genetic studies (e.g.,
Specimens
Material was collectednear the Onnenai Visitor Center (43°06′17.6″N 144°19′46.5″E) in Kushiro Marsh, Hokkaido, Japan (Fig.
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) (
The chaetotaxic notation follows that of
List of abbreviations
a, outer lobe of hemipenis; A1, antennule; A2, antenna; b, inner lobe of hemipenis; E, endopod; Exo, exopod; FRO, female reproductive organ; G1–3, apical claws on penultimate segment of antenna; GM, GM, apical claws on terminal segment of antenna; h, medial lobe of hemipenis; Hp, hemipenis; L5, maxilliped; L6, walking leg; L7, cleaning leg; LV, left valve; Md, mandible; M-process, chitinized internal process of hemipenis; Mx, maxillula; Pr, protopod; RV, right valve; Y, y2, y3, aesthetascs on antenna; ya, aesthetasc of antennule.
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
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,
Amplification of the whole mitochondrial genome
The COI and srRNA sequences were used to design new primer sets (Suppl. material
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
Sequencing
Amplification products were purified by the method of
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
Sequence analysis
Nucleotide sequences were assembled and edited with MEGA5 (
The boundaries and secondary structures of the mt rRNA genes were determined by using Centroidfold (
For the nuclear rRNA genes, information on secondary structure from Apis mellifera (
Description of male.
Carapace (Figs
A1 (Fig.
A2 (Fig.
Md (Fig.
Mx (Figs
L5 (Figs
L6 (Fig.
L7 (Fig.
Uropodal ramus (Fig.
Hp (Fig.
Zenker’s organ (Fig.
Description of female.
Carapace (Figs
A2 (Fig.
Palp of L5 (Fig.
FRO (Fig.
In other characters, female similar to male.
Carapace with dorsal hump one-third of length from posterior end. Male carapace has anterior and posterior margins equally rounded. Female carapace has straight postero-dorsal 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.
The specific epithet is an adjective derived from Kushiro Marsh, type locality, in combination with the Latin suffix -ensis.
Fabaeformiscandona kushiroensis sp. nov. clearly belongs in the acuminata group (
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.
Organization of the Fabaeformiscandona kushiroensis mitochondrial genome.
Gene | strand | Begins | Ends | Length | 3' spacer | Start | Stop |
Control region | 1 | 710 | 710 | 0 | |||
tRNA I | + | 711 | 772 | 62 | 15 | ||
tRNA Q | - | 788 | 854 | 67 | 0 | ||
tRNA M | + | 855 | 918 | 64 | 3 | ||
ND2 | + | 922 | 1923 | 1002 | -2 | ATA | TAA |
tRNA W | + | 1922 | 1985 | 64 | -8 | ||
tRNA C | - | 1978 | 2037 | 60 | 6 | ||
tRNA Y | - | 2044 | 2105 | 62 | -8 | ||
COI | + | 2098 | 3642 | 1545 | -5 | ATT | TAA |
tRNA L2 | + | 3638 | 3700 | 63 | 0 | ||
COII | + | 3701 | 4381 | 681 | -1 | ATC | TAA |
tRNA D | + | 4381 | 4443 | 63 | 0 | ||
ATP8 | + | 4444 | 4605 | 162 | -7 | ATT | TAA |
ATP6 | + | 4599 | 5264 | 666 | 12 | ATG | TAA |
COIII | + | 5277 | 6104 | 828 | -50 | ATT | TAA |
tRNA G | + | 6055 | 6118 | 64 | 54 | ||
ND3 | + | 6173 | 6472 | 300 | -2 | ATT | TAA |
tRNA A | + | 6471 | 6531 | 61 | 0 | ||
tRNA K | + | 6532 | 6596 | 65 | 2 | ||
tRNA R | + | 6599 | 6661 | 63 | 0 | ||
tRNA N | + | 6662 | 6721 | 60 | 0 | ||
tRNA S1 | + | 6722 | 6776 | 55 | 0 | ||
tRNA E | + | 6777 | 6840 | 64 | 2 | ||
tRNA F | - | 6843 | 6904 | 62 | -26 | ||
ND5 | - | 6879 | 8540 | 1662 | 51 | ATA | TAA |
tRNA H | - | 8592 | 8653 | 62 | -17 | ||
ND4 | - | 8637 | 9896 | 1260 | 80 | ATA | TAA |
ND4L | - | 9977 | 10252 | 276 | 31 | ATT | TAA |
tRNA T | + | 10284 | 10347 | 64 | 0 | ||
tRNA P | - | 10348 | 10408 | 61 | -43 | ||
ND6 | + | 10366 | 10884 | 519 | 15 | ATC | TAA |
Cyt b | + | 10900 | 12027 | 1128 | 1 | ATT | TAA |
tRNA S2 | + | 12029 | 12091 | 63 | 14 | ||
ND1 | - | 12106 | 13035 | 930 | -3 | ATA | TAG |
tRNA L1 | - | 13033 | 13096 | 64 | 0 | ||
lrRNA | - | 13097 | 14217 | 1121 | 0 | ||
tRNA V | - | 14218 | 14282 | 65 | 0 | ||
srRNA | - | 14283 | 14990 | 708 | 24 | ||
Tandem repeated sequence | 15015 | ca. 2000 | 0 |
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
Taxon | Accession number | Total length (bp) | Total | Protein-coding genes | lrRNA | srRNA | Control region | ||||
%A+T | AA | %A+T | bp | %A+T | bp | %A+T | bp | %A+T | |||
Vargula hilgendorfii | AB114300 | 15923 | 61.6 | 3824 | 58.6 | 1214 | 70.4 | 714 | 67.9 | 778 + 855 | 67.3 |
Fabaeformiscondona kushiroensis | AP014656 | 16355 + α | 68.8 | 3640 | 65.5 | 1121 | 77.1 | 708 | 74.0 | 710 + ca. 2000 | 78.7 |
Nucleotide composition at each codon position in the protein-coding genes of mitochondrial genome for two ostracods.
Taxon | 1st codon position | 2nd codon position | 3rd codon position | Overall | ||||||||||||
%A | %T | %G | %C | %A | %T | %G | %C | %A | %T | %G | %C | %A | %T | %G | %C | |
Vargula hilgendorfii | 27.0 | 28.7 | 26.2 | 18.1 | 17.8 | 42.2 | 18.8 | 21.2 | 29.4 | 30.7 | 18.7 | 21.1 | 32.2 | 29.4 | 12.4 | 26.0 |
Fabaeformiscondona kushiroensis | 28.1 | 32.0 | 22.8 | 17.1 | 18.3 | 45.0 | 15.3 | 21.2 | 31.6 | 41.0 | 11.7 | 15.5 | 26.0 | 39.5 | 16.6 | 17.9 |
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.
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
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
Protein-coding genes
Initiation codons in the mitochondrial protein-coding genes in F. kushiroensis are ATT (ATP8, COI, COIII, Cyt b, ND3 and ND4L), ATA (ND1, ND2, ND4 and ND5), ATC (COII, ND6), and ATG (ATP6) (Table
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.
The description of Fabaeformiscandona kushiroensis sp. n. brings the number of non-marine 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 (
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 (
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 (
We thank Dr Matthew H. Dick (Hokkaido University) and Dr Hiroshi Kajihara (Hokkaido University) for critical comments on the manuscript, and our laboratory colleagues for advice and discussion.
Gene map of the mitochondrial genome of Fabaeformiscandona kushiroensis sp. n., showing the direction and position of PCR primers used in this study. The peripheral ring shows the two large fragments amplified with PrimeSTAR GXL and the primer sets used.
Putative secondary structures for mitochondrial tRNA genes in Fabaeformiscandona kushiroensis sp. n., estimated with the MITOS Web server.
Putative secondary structures for the mitochondrial rRNA genes in Fabaeformiscandona kushiroensis sp. n., estimated with Centroidfold.
List of primers used in this study.