Corresponding author: Shimpei F. Hiruta (
Academic editor: Viktor Senderov
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
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.,
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
Total genomic DNA was extracted from the whole body of one individual by using a DNeasy Blood & Tissue Kit (QIAGEN), with modifications from
Initially, universal primer sets were used to amplify parts of the cytochrome
The
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
Amplification products were purified by the method of
To estimate the length of tandem-repeat region within the control region (CR), primers were designed (Fab_CRF and Fab_CRmF; Suppl. material
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
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.
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 -
The mitochondrial genome of
The overall A + T content of the
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.
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
The 12S rRNA (srRNA) and 16S rRNA (lrRNA) genes in
Initiation codons in the mitochondrial protein-coding genes in
The nuclear ribosomal RNA genes in
The description of
Our samples collected at the end of December contained mature males and females, but no juveniles, which suggests that
The gene order in the mitochondrial genome of
The CR in
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.
A, map of Hokkaido, Japan, showing the location of Kushiro Marsh; B, diagram of the study area near the Onnenai visitor center in Kushiro Marsh, with the sampling site indicated by star; C, photograph of the sampling site, small spring.
Gene map of the mitochondrial genome of
Comparison of the mitochondrial DNA gene arrangements between
Organization of the tandem repeat region in the control region of
Organization of the
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 | ||
|
+ | 922 | 1923 | 1002 | -2 | ATA | TAA |
tRNA W | + | 1922 | 1985 | 64 | -8 | ||
tRNA C | - | 1978 | 2037 | 60 | 6 | ||
tRNA Y | - | 2044 | 2105 | 62 | -8 | ||
|
+ | 2098 | 3642 | 1545 | -5 | ATT | TAA |
tRNA L2 | + | 3638 | 3700 | 63 | 0 | ||
|
+ | 3701 | 4381 | 681 | -1 | ATC | TAA |
tRNA D | + | 4381 | 4443 | 63 | 0 | ||
|
+ | 4444 | 4605 | 162 | -7 | ATT | TAA |
|
+ | 4599 | 5264 | 666 | 12 | ATG | TAA |
|
+ | 5277 | 6104 | 828 | -50 | ATT | TAA |
tRNA G | + | 6055 | 6118 | 64 | 54 | ||
|
+ | 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 | ||
|
- | 6879 | 8540 | 1662 | 51 | ATA | TAA |
tRNA H | - | 8592 | 8653 | 62 | -17 | ||
|
- | 8637 | 9896 | 1260 | 80 | ATA | TAA |
|
- | 9977 | 10252 | 276 | 31 | ATT | TAA |
tRNA T | + | 10284 | 10347 | 64 | 0 | ||
tRNA P | - | 10348 | 10408 | 61 | -43 | ||
|
+ | 10366 | 10884 | 519 | 15 | ATC | TAA |
|
+ | 10900 | 12027 | 1128 | 1 | ATT | TAA |
tRNA S2 | + | 12029 | 12091 | 63 | 14 | ||
|
- | 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 |
Characteristics of the mitochondrial genome of two ostracods.
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 | |||
|
|
15923 | 61.6 | 3824 | 58.6 | 1214 | 70.4 | 714 | 67.9 | 778 + 855 | 67.3 |
|
|
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 | |
|
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 |
|
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 |
Supplemental Figure 1.
image
Gene map of the mitochondrial genome of
File: oo_59645.gif
Supplemental Figure 2.
image
Putative secondary structures for mitochondrial tRNA genes in
File: oo_59646.gif
Supplemental Figure 3.
image
Putative secondary structures for the mitochondrial rRNA genes in
File: oo_59647.gif
Supplemental Figure 4.
images
File: oo_65602.zip
Supplemental Table 1.
table
List of primers used in this study.
File: oo_59656.xlsx