Research Article |
Corresponding author: Seinen Chow ( chow@affrc.go.jp ) Academic editor: Yves Samyn
© 2016 Seinen Chow, Kooichi Konishi, Miyuki Mekuchi, Yasuji Tamaki, Kenji Nohara, Motohiro Takagi, Kentaro Niwa, Wataru Teramoto, Hisaya Manabe, Hiroaki Kurogi, Shigenori Suzuki, Daisuke Ando, Tadao Jinbo, Masato Kiyomoto, Mamiko Hirose, Michitaka Shimomura, Akira Kurashima, Tatsuya Ishikawa, Setuo Kiyomoto.
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:
Chow S, Konishi K, Mekuchi M, Tamaki Y, Nohara K, Takagi M, Niwa K, Teramoto W, Manabe H, Kurogi H, Suzuki S, Ando D, Jinbo T, Kiyomoto M, Hirose M, Shimomura M, Kurashima A, Ishikawa T, Kiyomoto S (2016) DNA barcoding and morphological analyses revealed validity of Diadema clarki Ikeda, 1939 (Echinodermata, Echinoidea, Diadematidae). ZooKeys 585: 1-16. https://doi.org/10.3897/zookeys.585.8161
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A long-spined sea urchin Diadema-sp reported from Japanese waters was genetically distinct from all known Diadema species, but it remained undescribed. Extensive field surveys in Japan with molecular identification performed in the present study determined five phenotypes (I to V) in Diadema-sp according to the presence and/or shape of a white streak and blue iridophore lines in the naked space of the interambulacral area. All phenotypes were distinct from Diadema setosum (Leske, 1778) and Diadema savignyi (Audouin, 1829), of which a major type (I) corresponded to Diadema clarki Ikeda, 1939 that was questioned and synonymized with D. setosum by
Diadema clarki , Diadematidae , DNA barcoding, Echinoidea
Long-spined sea urchins of the genus Diadema Gray, 1825 are abundant, widespread and ecologically important species in tropical to temperate areas (
Recently,
In this study, molecular and phenotypic evidence are provided that D. clarki is Diadema-sp and hence a valid species, and we report the geographic distribution of D. clarki based on extensive field surveys.
The twenty localities where field observations and/or collecting of Diadema specimens were carried out in Japanese waters are shown in Figure
Localities where field observation and sampling of Diadema were performed. See Table
Locality information for field survey and number of Diadema-sp and D. savignyi observed.
Locality | Prefecture | Figure |
Lat (N) | Long (E) | Date | n† |
---|---|---|---|---|---|---|
Arasaki | Kanagawa | A | 35°11'50" | 139°35'59" | Dec. 2011‡ March-Sep. 2014 |
>400:0 |
Tateyama | Chiba | B | 34°59'26" | 139°49'28" | March to June, 2014 | 3:0 |
Mera | Shizuoka | C | 34°39'39" | 138°47'10" | May 2 and 23, 2015 | 22:0 |
Shikine-jima | Tokyo | D | 34°19'13" | 139°13'11" | Aug. 6, 2015 | 21:0 |
Haida-ura | Mie | E | 33°59'48" | 136°15'39" | March 10, 2012; April 15, 2015 | 70:0 |
Kushimoto | Wakayama | F | 33°28'33" | 135°44'29" | Sep. 29, 2014 | 1:3 |
Hachijo-jima | Tokyo | G |
33°05'53", 33°07'20" |
139°46'30", 139°49'00" |
Feb. 26, 2007 July 30-31, 2015 |
0:1 4:22 |
Uchidomari | Ehime | H | 32°56'31" | 132°29'14" | Oct. 26, 2014 | 9:1 |
Iki-no-shima | Nagasaki | I | 33°44'58" | 129°38'56" | Sep. 2, 2014 | 37:0 |
Ojika | Nagasaki | J | 33°11'05" | 129°04'21" | July 19, 2014 | 102:0 |
Mie | Nagasaki | K | 32°48' | 129°45' | May 30, 2014 | 36:0 |
Shibushi | Kagoshima | L | 31°27'55" | 131°08'13" | May 17, 2014 | 2:0 |
Kaimon | Kagoshima | M | 31°10'39" | 130°33'14" | Oct. 22, 2014 | 19:0 |
Tanega-shima | Kagoshima | N | 30°49' | 131°02' | April 26, 2015 | 0:2 |
Yaku-shima | Kagoshima | O | 30°27' | 130°30' | Feb. 10, 2004 | 0:2 |
Amami Ohshima | Kagoshima | P | 28°24'12" | 129°27'15" | July 29, 2008 | 0:4 |
Ogasawara | Tokyo | Q | 27°05'44" | 142°11'58" | June 21, 2015 | 0:26 |
Motobu | Okinawa | R | 26°39'16" | 127°52'44" | July 16, 2014 | 0:4 |
Sesoko | Okinawa | S | 26°38'09" | 127°51'55" | May, 2013‡ | 0:4 |
Ishigaki-jima | Okinawa | T | 24°27' | 124°12' | Oct., 2013‡ | 0:7 |
Tulamben, Bari | (Indonesia) | not shown | 8°16'29" § | 115°35'40" | March 8, 2015 | 0:2 |
Six individuals of Diadema-sp (designated as AT1 to AT3, AR54, AR59, AR70) showing phenotypic variation were chosen among the specimens collected at Arasaki during March to August 2014 (Table
Information of five phenotypes (I to V) of six Diadema-sp individuals collected in Arasaki area, three museum specimens.
Sample | Voucher | Phenotype | Test size (mm) | Collection | GenBank | |||
---|---|---|---|---|---|---|---|---|
No. | diameter | height | locality | date | depth (m) | Accession No. | ||
AT1 |
|
I | 42.0 | 22.4 | Arasaki | Mar. 24, 2014 | 2 | LC037355 |
AT2 |
|
II | 46.5 | 19.6 | Arasaki | Mar. 24, 2014 | 3 | LC037356 |
AT3 |
|
III | 54.0 | 22.2 | Arasaki | Mar. 24, 2014 | 2 | LC037357 |
AR59 |
|
IV | 44.0 | 19.2 | Arasaki | July 28, 2014 | 2 | LC037358 |
AR54 |
|
V | 67.0 | 32.6 | Arasaki | June 25, 2014 | 3 | LC037359 |
AR70 |
|
V | 53.8 | 20.8 | Arasaki | Aug. 29, 2014 | 2 | LC037360 |
IK1 |
|
- | 88.5 | 38.6 | Kyushu | 1933 or 1934 | - | - |
IK2 |
|
- | 65.4 | 36.9 | Kyushu | 1933 or 1934 | - | - |
IK3 |
|
- | 64.2 | 31.6 | Kyushu | 1933 or 1934 | - | LC037361 |
OK2 |
|
- | 91.7 | 38.6 | Motobu | July 16, 2014 | 2 | LC037362 |
OK3 |
|
- | 64.0 | 31.5 | Motobu | July 16, 2014 | 2 | LC037363 |
OK4 |
|
- | 40.0 | 19.5 | Motobu | July 16, 2014 | 2 | LC037364 |
All tissues fixed in ethanol and extracted DNA are kept in the National Research Institute of Fisheries Science, Kanagawa, Japan.
Crude DNAs were extracted from the ethanol tissues preserved in ethanol and used for PCR amplification. In addition to the primers (COI120F and COI1300R) previously described (
Underwater images of several phenotypes in D. setosum, D. savignyi and Diadema-sp were presented in
Underwater aboral views of four phenotypes (A–E) and color variants of phenotype I (F) of Diadema-sp, Diadema setosum (G), and Diadema savignyi (H). A
These characteristics in living specimen conspicuous underwater were not well preserved after fixation (Figure
Aboral views of the dried test of IK3 are presented in Figure
Aboral view (A) and enlarged view of a naked space of the interambulacral area (B) in a reference dried specimen of Diadema found in Ikeda’s collection.
Approximately 1,100 bp fragments could be amplified in three D. savignyi (OK) and six Diadema-sp (AT and AR) individuals using a primer pair (COI120FxCOI1300R). All possible primer combinations were tested in three museum specimens (IK1 to IK3), but successful amplification (c.a. 350 bp fragment) was obtained only in IK3 by one primer pair (COI531FxCOI874R). Nested PCR was also attempted for the other museum specimens, but no amplification was observed. IK3 was therefore designated as a reference specimen of the Ikeda’s collection and deposited to the Kitakyushu Museum of Natural History and Human History (voucher:
Neighbor-joining phylogenetic tree drawn using from COI sequence data. Bootstrap support (>50%) after 1,000 replications is shown at each node. Italic accession numbers with dagger (AY012732, AY012733, AY012742–AY012747) are from
Mean percent nucleotide sequence divergence (K2P±SE) within (on the diagonal) and between (below diagonal) species. Number of individuals within brackets.
IK3 | Diadema-sp | D. savignyi | D. setosum-a | D. setosum-b | |
---|---|---|---|---|---|
IK3 (1) | - | ||||
Diadema-sp (14) | 0.16±0.09 | 0.26±0.09 | |||
D. savignyi (9) | 12.06±2.39 | 12.14±1.56 | 1.11±0.25 | ||
D. setosum-a (7) | 13.13±2.81 | 16.96±1.93 | 18.50±2.21 | 0.84±0.19 | |
D. setosum-b (2) | 12.72±2.79 | 13.89±2.07 | 19.30±2.61 | 7.22±1.24 | 0.00±0.00 |
Diadema-sp was seen in the subtidal zone, ranging to depths of 8 m but no further attempt was performed to investigate their distribution in deeper zones. Both D. setosum and Diadema-sp were observed in the same habitats and depths, but the former had tendency to aggregate and the later was usually seen as solitary specimens; in consequence both usually did not occur side by side. Although relative abundances of D. setosum and Diadema-sp were not quantitatively investigated, the former species was relatively abundant and ubiquitously observed throughout all the areas examined. However, after the severe winters of 2014 and 2015 (January to February), Diadema-sp were observed to be predominated at Arasaki area, suggesting that it may be more tolerant to cold water than D. setosum. In addition, D. savignyi may be less tolerant to lower temperatures than D. setosum, since D. savignyi was never found at Arasaki area and is not common around Japan mainland.
In contrast to the ubiquitous distribution of D. setosum throughout the survey areas, Diadema-sp was only observed in a narrow latitudinal range around Japan mainland, from Kanagawa (35°11’ N) to Kagoshima (31°10’ N) (see Figure
Frequency of the five phenotypes observed in Kanagawa, Mie, Nagasaki and Kagoshima Prefectures is presented in Table
Relative abundance (percentage) of the five phenotypes of Diadema-sp observed in Kanagawa, Mie, Nagasaki and Kagoshima Prefectures.
Phenotype | Kanagawa | Mie | Nagasaki | Kagoshima | ||||
---|---|---|---|---|---|---|---|---|
Arasaki | Haida-ura | Iki Isl. | Ojika | Mie | Kaimon | |||
June 2014 | July 2014 | Sep. 2014 | April 2015 | Sep. 2014 | July 2014 | May 2014 | Oct. 2014 | |
I | 64.1 | 58.8 | 71.4 | 73.1 | 64.6 | 52.6 | 38.9 | 52.6 |
II | 14.8 | 4.1 | 7.7 | 20.9 | 17.7 | 21.1 | 30.5 | 21.1 |
III | 12.0 | 27.8 | 15.4 | 3.0 | 17.7 | 10.5 | 16.7 | 15.8 |
IV | 6.3 | 6.2 | 3.3 | 3.0 | 0.0 | 5.3 | 11.1 | 10.5 |
V | 2.8 | 3.1 | 2.2 | 0.0 | 0.0 | 10.5 | 2.8 | 0.0 |
total (n) | 142 | 97 | 91 | 67 | 34 | 57 | 36 | 19 |
The present investigation together with previous studies (
Among the several characters of D. clarki described by
Since experimental hybridization between D. setosum and D. savignyi produced viable hybrids (
So far as published data, D. clarki is not observed in the Ryukyu Archipelago (
We are grateful to Yoshikazu Kajigaya, Mayumi Sato, Kyoko Asano, Chie Takahashi, Kimie Aoki and Tomoko Kawashima, National Research Institute of Aquaculture, for their devoted help in the field survey and sample preparation. We are indebted to Yoshikatsu Nakano, the University of Ryukyus, for kindly allowing us to use the Sesoko laboratory. We would like to thank to Takehiro Sato, the Kanagawa Prefectural Museum of Natural History, for aiding voucher specimens, and to Andrew Jeffs, the University of Auckland, for providing us an invaluable reference. We wish to express our special thanks to John S. Pearse, the University of California, Santa Cruz, for improving the manuscript and providing the unpublished data.