Research Article |
Corresponding author: Mizuho Munakata ( munakata.mizuho.k0@elms.hokudai.ac.jp ) Academic editor: Kay Van Damme
© 2022 Mizuho Munakata, Hayato Tanaka, Keiichi Kakui.
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:
Munakata M, Tanaka H, Kakui K (2022) Taxonomy and natural history of Cavernocypris hokkaiensis sp. nov., the first ostracod reported from alpine streams in Japan. Zoosystematics and Evolution 98(1): 117-127. https://doi.org/10.3897/zse.98.80442
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We describe the cypridoidean ostracod Cavernocypris hokkaiensis sp. nov. from riverbed sediments in an alpine stream at an elevation of ca. 1850 m in the Taisetsu Mountains, Hokkaido, Japan. This species differs from congeners in having (1) the outer surface of the carapace smooth, with sparse, tiny setae, but without shallow pits; (2) the carapace elongate rather than triangular in lateral view; (3) the antennula consisting of seven podomeres; (4) first palpal podomere of maxillula with five dorsodistal and one ventro-subdistal setae; (5) the fifth limb lacking setae b and d; and (6) the fifth limb lacking a vibratory plate. We provided the key to the Cavernocypris species. We determined partial sequences for the cytochrome c oxidase subunit I (COI; cox1) and 18S rRNA (18S) genes in C. hokkaiensis. Our sample contained only females, and we obtained a partial 16S rRNA sequence for the endosymbiotic bacterium Cardinium from C. hokkaiensis, indicating the possibility that this ostracod species reproduces parthenogenetically. Our field survey and observations of captive individuals suggested that C. hokkaiensis may be endemic to the Taisetsu Mountains, with a low population density, narrow distributional range, and slow maturation to sexual maturity.
Crustacea, cryophilic, Cypridoidea, ecology, lotic, Ostracoda
The genus Cavernocypis Hartmann, 1964, one of 20 genera in the subfamily Cypridopsinae Kaufmann, 1900 (
To date, six Cavernocypris species have been described from the Palearctic and Nearctic regions (
Streams in the alpine vegetation zone between the treeline and the permanent snow line are cold and nutrient-poor (
The Taisetsu Mountains in Daisetsuzan National Park are located in the center of Hokkaido, Japan, and consist of several gently sloping volcanic peaks in the 2000 m class. Above the treeline at ca. 1400–1500 m elevation (
Here we describe a new species of Cavernocypris from an alpine stream in the Taisetsu Mountains, the first record of an ostracod from cold alpine waters in Japan. We present nucleotide sequences for this species for parts of the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear 18S rRNA (18S) genes and provide preliminary comments on its phylogenetic position based on 18S data. We also present information on its natural history based on a field survey and preliminary rearing results. Finally, we demonstrate with molecular data (part of the mitochondrial 16S rRNA gene, 16S) the infection of this species by Cardinium, a group of “reproduction-manipulating” endosymbiotic bacteria (
Sampling was conducted at seven sites, including four streams (Stns 1–3, 7) fed by springs, a hot spring, and/or snowmelt, two ponds (Stns 5, 6), and one waterfall basin (Stn. 4) (Table
Station number and name | Environment | Coordinates | Elevation | Mean water temperature | Sampling date |
---|---|---|---|---|---|
1. Hokkai-sawa Stream | Stream fed by spring | 43°41'08"N, 142°55'28"E | 1853 m | 3.5 (2.2–4.8) °C | 25.viii.2020, 26.vii.2021 |
2. Hokkai-sawa Stream | Stream fed by spring and snowmelt | 43°41'17"N, 142°54'33"E | 1837 m | 4.1 (2.4–5.7) °C | 25.viii.2020, 26.vii.2021 |
3. Akaishi Stream | Stream fed by hot spring with high-H2S concentration and snowmelt | 43°41'23"N, 142°54'35"E | 1829 m | 13 °C | 26.vii.2021 |
4. Momizi Fall | Fall basin in Akaishi Stream | 43°43'72"N, 142°57'41"E | 813 m | 13 °C | 27.vii.2021 |
5. Sugatami Pond | Pond | 43°39'41"N, 142°49'58"E | 1665 m | No data | 26.viii.2020 |
6. Unnamed Pond | Pond | 43°39'43"N, 142°49'32"E | 1597 m | No data | 26.viii.2020 |
7. Daisetsu Asahidake Spring | Stream fed by spring | 43°37'59"N, 142°41'31"E | 445 m | 7.0 °C | 26.viii.2020 |
Sampling sites for Cavernocypris hokkaiensis sp. nov. A. Map showing location of Daisetsuzan National Park in Japan; B. Map showing the sampling sites (Stns 1–7) in Daisetsuzan National Park; C. Enlarged map of the area corresponding to the square in Fig.
Ostracods were fixed in 80% ethanol. The methods used for dissection, preparation of slides, light microscopy, scanning electron microscopy (SEM), and drawing were as described by
The following abbreviations are used in the text: Ca, carapace; LV, left valve; RV, right valve; H, height; L, length; W, width; An1, antennula; An2, antenna; Md, mandible; Mx, maxillula; L5–7, fifth, sixth, and seventh limbs, respectively; UR, uropodal ramus. The appendage chaetotaxy follows
An attempt was made to extract total DNA from the soft parts of three individuals by using a NucleoSpin Tissue XS Kit (Macherey-Nagel, Germany) following the manufacturer’s protocol, but only one of the three extracts allowed successful PCR amplification. Primers used for the PCR amplification and sequencing of ostracod COI, ostracod 18S, and Cardinium 16S are listed in
To explore the phylogenetic position of this species, a maximum likelihood (ML) phylogenetic tree was constructed based on the 18S dataset comprised of 66 ostracod sequences (one our sequence, and 64 cypridoidean and one pontocypridoidean (outgroup) sequences from INSD; 1547 positions in the aligned dataset; see Suppl. material
To obtain information on the life cycle, three non-adult individuals collected on 26 July 2021 were maintained singly in wells of a tissue culture plate filled with water collected from sampling site Stn. 1 and placed in a refrigerator at a temperature of 7 °C. Detritus collected from sampling site Stn. 1 was added to each well as a food source. Observations were made twice or more per month.
Among seven sampling sites, Cavernocypris ostracods were collected from only two sites in the Hokkai-sawa Stream (Stns 1 and 2) (Fig.
Three captive non-adult individuals have remained alive and active for more than five months. The body length (LV-L) of each individual was 0.47, 0.48, 0.39 mm. No molts have been observed to date (the latest observation was on 7 January 2022).
Family Cyprididae Baird, 1845
The epithet hokkaiensis is an adjective referring to the type locality, Hokkai-sawa Stream in Japan.
Shibare-doukutsu-kaimijinko, referring to the habitat having low water temperature. Shibare is derived from the Japanese verb shibare-ru (freeze), in a Hokkaido dialect; Doukutsu-kaimijinko is the Japanese name for Cavernocypris (
Hokkai-sawa Stream, Daisetsuzan National Park, Hokkaido, Japan (Stn. 1: 43°41'08"N, 142°55'28"E).
Holotype : female, ICHUM-8247, one slide and one SEM stub, Stn.1, Hokkai-sawa Stream, riverbed sediment, 26 July 2021. Paratypes (five females): ICHUM-8248, 8249, one SEM stub and one slide for each; ICHUM-8250, 8251, one SEM stub; ICHUM-8252, one slide, voucher specimen for LC666823 (COI) and LC666824 (18S). Collection data for ICHUM-8249, 8252 are same as holotype; ICHUM-8248, 8251 were collected from Stn. 2 (43°41'17"N, 142°54'33"E) on 25 August 2020; ICHUM-8250 was collected from Stn. 1 on 25 August 2020. All individuals were collected by Mizuho Munakata.
Measurements (in millimeters, except for ratios) of carapace and valves: LV-L, 0.59–0.61 (0.60, N=3); LV-H, 0.30–0.31 (0.31, N= 3); LV-H/LV-L, 0.50–0.51 (0.51, N=3); RV-L, 0.58–0.61 (0.60, N=3); RV-H, 0.29–0.31 (0.30, N=3); RV-H/RV-L, 0.50–0.51 (0.51, N=3); Ca-W, 0.25–0.26 (0.26, N=2); Ca-W/LV-L, 0.41–0.42 (0.42, N=2).
Carapace (Fig.
SEM images of carapaces and valves of female Cavernocypris hokkaiensis sp. nov. A. Paratype (ICHUM-8250); B. Paratype (ICHUM-8251); C, D. Paratype (ICHUM-8248); E, F. Holotype (ICHUM-8247); G, H. Paratype (ICHUM-8249); A, B. Ventral and dorsal views of whole carapace; C, D. Outer views of left and right valves; E, F. Inner views of left and right valves; G, H. Inner dorsal views of left and right valves; dorsal portion of left valve broken. Arrows indicate anterior direction. Scale bars: 0.2 mm.
LV (Figs
An1 (Fig.
Cavernocypris hokkaiensis sp. nov., female. A–G, I–L. Holotype (ICHUM-8247); H. Paratype (ICHUM-8249); A, B. Inner views of left and right valves; C. Antennula; D. Antenna, outer view; E. Coxa of mandible; F. Mandible, inner view; G. Maxillula (vibratory plate omitted); H–J. Limbs 5–7; setules of distal setae on protopod and palp of limb 5 omitted; K. Uropodal ramus; L. Rake organ. Abbreviations: RO, Rome organ; WO, Wouters organ. Scale bars: 0.2 mm (A, B); 0.5 mm (C–K); 0.25 mm (L).
An2 (Fig.
Md (Fig.
Mx (Fig.
L5 (Fig.
L6 (Fig.
L7 (Fig.
UR (Fig.
Rake organ (Fig.
Genital hooks on female copulatory organ present (not illustrated).
The partial COI sequence (658 bp, encoding 219 amino acids; LC666823), the nearly complete 18S sequence (2053 bp; LC666824), and a Cardinium 16S sequence (907 bp; LC666825) were determined from paratype individual ICHUM-8252.
The sequences in the INSD most similar to our sequences, determined by BLAST searches, were from the ostracod Bennelongia scanloni
Cavernocypris hokkaiensis sp. nov. resembles C. cavernosa and C. danielopoli Smith & Kamiya, 2017 in lacking setae b and d on L5, but differs from them in that (1) the outer surface of the carapace is smooth, with sparse, tiny setae, but without shallow pits (pits present in C. cavernosa and C. danielopoli); (2) the carapace is elongate rather than triangular in lateral view (triangular in C. danielopoli); (3) first palpal podomere of Mx has five dorsodistal and one ventro-subdistal setae (only five dorsodistal setae present in C. danielopoli; not described in C. cavernosa); and (4) L5 lacks the vibratory plate (vibratory plate present in C. cavernosa and C. danielopoli). Character states in all congeners are summarized in Table
Comparison of morphological characteristics between species of Cavernocypris.
C. cavernosa | C. coreana | C. danielopoli | C. reddelli | C. subterranea | C. wardi | C. hokkaiensis sp. nov. | |
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Shape of valves | elongate | elongate | triangular | elongate | elongate | elongate | elongate |
Surface of valves | with numerous shallow pits, most distinct towards anterior and posterior margins, less distinct in central area | with very finely pitted | with elongate shallow pits in central area, and with much smaller rounded pits in posterior region | with small shallow pits | with small shallow pits in a central transverse band; pits may be reduced or even absent | smooth | smooth |
Number of An1 podomeres | 7 | 6 | 7 | 7 | 7 | 7 | 7 |
Number of setae on 1st podomere of Mx-palp | not described | 5 distal and 1 subdistal | 5 distal | 5 distal | 5 distal and 1 subdistal | 4 distal and 1 subdistal | 5 distal and 1 subdistal |
Seta b on L5 | absent | present | absent | absent | present | present | absent |
Seta d on L5 | absent | absent | absent | present | present | present | absent |
Number of rays comprising vibratory plate on L5 | 2 | 4 | 1 | 4 | 2 | 3 | 0 |
Reference |
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this study |
Our sample comprised only females, indicating that C. hokkaiensis may be parthenogenetic. The endosymbiotic bacterium Cardinium has previously been detected (e.g., by means of 16S sequences) in non-marine ostracods engaged in parthenogenetic or mixed reproduction, and infection with Cardinium might be a causative factor in the parthenogenetic reproductive mode (
The results of our field survey suggest that C. hokkaiensis is distributed in an extremely narrow area, only in Hokkai-sawa Stream. It was not found at three sites distant from Hokkai-sawa Stream (Stns 5, 6, and 7). Its absence at two sites in Akaishi Stream (Stns 3 and 4), which Hokkai-sawa Stream joins, may be related to environmental differences between two streams. Hokkai-sawa Stream is fed by spring water and snowmelt, and is thus cold (1.4–6.8 °C measured in the summer season; Table
Our rearing experiment, though we could prepare only three live individuals, provided preliminary data about the life history of C. hokkaiensis. We observed no molting by captive C. hokkaiensis individuals for more than five months at 7 °C. Ostracod life cycles typically comprise eight non-adult and one adult instars, i.e., ostracods molt eight times before becoming sexually mature adults. Instars are not uniform in duration, but tend to become longer with successive instars (e.g.,
Our field survey and observation of captive individuals may indicate that C. hokkaiensis is an endemic species adapted to the harsh alpine environment of the Taisetsu Mountains, with a low population density, narrow distributional range, and potentially slow maturation. If this is the case, then habitat loss and fragmentation due to anthropogenic activities, or a decrease in snowfall and snowfields due to climate change, could lead to a rapid population decline of this species. Additional ecological and biological information is necessary to confirm whether C. hokkaiensis is a narrow endemic, and to design an informed conservation strategy.
1 | An1 with 7 podomeres | 2 |
– | An1 with 6 podomeres | C. coreana (McKenzie, 1972) |
2 | 6th swimming seta of An2 longer than the other 5 setae, second podomere of Md-palp with β+4 setae | 3 |
– | 6th swimming seta of An2 shorter than others, second podomere of Md-palp with β+3 setae | C. reddelli Külköylüoğlu, 2020 |
3 | Seta b present on L5 | 4 |
– | Seta b absent from L5 | 5 |
4 | First palpal podomere of Mx with 5 distal and 1 subdistal setae, vibratory plate of L5 with 2 rays | C. subterranea (Wolf, 1920) |
– | First palpal podomere of Mx with 4 distal and 1 subdistal setae, vibratory plate of L5 with 3 rays | C. wardi Marmonier, Meisch & Danielopol, 1989 |
5 | Carapace triangular in lateral view, with distinctive hump on LV | C. danielopoli Smith & Kamiya, 2017 |
– | Carapace elongate in lateral view, without hump on LV | 6 |
6 | Surface of valves covered with numerous shallow pits, vibratory plate of L5 with 2 rays | C. cavernosa Smith, 2011 |
– | Surface of valves smooth, L5 without vibratory plate | C. hokkaiensis sp. nov. |
We thank Akane Saito and Sota Matsuno in the Ministry of the Environment for support in obtaining a sampling permit; Yuki Kita at Hokkaido University (HU) for supporting the field work; Akira Tsukagoshi at Shizuoka University for literature; Yuki Oya at HU for helping with molecular analyses; and Matthew H. Dick at HU for reviewing the manuscript and editing the English. Permit numbers 1910241 and 2104201 allowed field sampling of animals in Daisetsuzan National Park. This study was funded in part by a research grant from the Research Institute of Marine Invertebrates Foundation to MM.
Table S1
Data type: table (Excel format)
Explanation note: List of species included in the molecular phylogenetic analysis and respective GenBank accession numbers.
Alignment S1
Data type: molecular dataset (fasta format)
Explanation note: Aligned 18S sequences used for the maximum-likelihood analysis, trimmed in MEGA7 to the shortest length among the sequences.
Alignment S2
Data type: molecular dataset (fasta format)
Explanation note:Aligned 18S sequences used for the maximum-likelihood analysis, reduced to 1547 positions by removing alignment-ambiguous sites with Gblocks ver. 0.91b in NGPhylogeny.fr under “relaxed” parameters.
File S1
Data type: text with one figure (docx format)
Explanation note: Phylogenetic analysis of cypridoidean ostracods based on 18S rRNA sequences.