Chow S et al. (2016), DNA barcoding and morphological analyses reveal...

ECB-ART-44702

Zookeys 2016 Apr 27;585:1-16. doi: 10.3897/zookeys.585.8161.

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DNA barcoding and morphological analyses revealed validity of Diadema clarki Ikeda, 1939 (Echinodermata, Echinoidea, Diadematidae).

Chow S , Konishi K , Mekuchi M , Tamaki Y , Nohara K , Takagi M , Niwa K , Teramoto W , Manabe H , Kurogi H , Suzuki S , Ando D , Tadao Jinbo , Kiyomoto M , Hirose M , Shimomura M , Kurashima A , Ishikawa T , Kiyomoto S .

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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 Diadema setosum by Mortensen (1940). The holotype of Diadema clarki has not been found, but three unlabeled dried tests of Diadema were found among Ikeda''s original collection held in the Kitakyushu Museum of Natural History and Human History, Fukuoka, Japan. A short mtDNA COI fragment (ca. 350bp) was amplified from one of the tests, and the nucleotide sequence determined (275bp) was nearly identical with that of Diadema-sp. Arrangements of the primary tubercles on the coronal plates in Diadema-sp and the museum specimen also conformed with Diadema clarki, indicating that Diadema-sp is identical to Diadema clarki and a valid species. Narrow latitudinal distribution (31°N to 35°N) of Diadema clarki in Japan was observed, where it co-existed with abundant Diadema setosum and rare Diadema savignyi. No Diadema clarki was found in the southern islands in Japan, such as Satsunan Islands to Ryukyu Islands and Ogasawara Island, where Diadema setosum and Diadema savignyi were commonly observed.

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Genes referenced: LOC100887844 LOC100893907 LOC115919132 LOC580968

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	Figure 1. Localities where field observation and sampling of Diadema were performed. See Table 1 for detailed information. A Arasaki (Kanagawa Prefecture) B Tateyama (Chiba) C Mera (Shizuoka) D Shikine-jima (Tokyo) E Haida-ura (Mie) F Kushimoto (Wakayama) G Hachijo-jima (Tokyo) H Uchidomari (Ehime) I Iki-no-shima (Nagasaki) J Ojika (Nagasaki) K Mie (Nagasaki) L Shibushi (Kagoshima) M Kaimon (Kagoshima) N Tanega-shima (Kagoshima) O Yaku-shima (Kagoshima) P Amami Ohshima (Kagoshima) Q Ogasawara (Tokyo) R Motobu (Okinawa) S Sesoko (Okinawa) T Ishigaki-jima (Okinawa). Diadema setosum was observed in all areas surveyed. No Diadema savignyi but Diadema-sp were observed at localities with yellow circle, Both Diadema savignyi and Diadema-sp were observed at localities with white circle, No Diadema-sp but Diadema savignyi were observed at localities with red circle.
	Figure 2. Underwater aboral view of phenotype I of Diadema-sp, KPM-NJL000035, original specimen number is AT1.
	Figure 3. 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 KPM-NJL000036, original specimen number is AT2 designated as phenotype II B KPM-NJL000037, original specimen number is AT3 designated as phenotype III C KPM-NJL000039, original specimen number is AR59 designated as phenotype IV D KPM-NJL000038, original specimen No. is AR54 designated as phenotype V E KPM-NJL000040, original specimen No. is AR70 designated as phenotype V F color variant of phenotype I observed at Haida-ura in Mie Prefecture (Figure 1E) photographed by T. Ishikawa G Diadema setosum (DST2 in Chow et al. (2014)) H Diadema savignyi (DSV23 in Chow et al. (2014)).
	Figure 4. Preserved specimen of phenotypes I (A), II (B), III (C) and V (D) of Diadema-sp, corresponding to AT1 (KPM-NJL000035) in Figure 2, and AT2, AT3 and AR54 (KPM-NJL000036‒KPM-NJL000038) in Figures 3A, B and D, respectively. All scale bars = 10 mm.
	Figure 5. 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. KMNH IvR 500788, original specimen No. is IK3. White streak-like remnant can be seen (A, B), and the outer and inner series of primary tubercles initiated on the 3rd and 5th coronal plates, respectively (B). Scales bars = 20 mm (A) and 10 mm (B).
	Figure 6. 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 Lessios et al. (2001) and red accession numbers with asterisk (AB900024, AB909927, AB909929–AB909931, AB909933–AB909935, AB909942, AB909945, AB909947, AB909949, AB909953, AB909955, AB909957) are from Chow et al. (2014).

References [+] :

Addison, Multiple gene genealogies reveal asymmetrical hybridization and introgression among strongylocentrotid sea urchins. 2009, Pubmed, Echinobase