Molecular Identification of Mid to Final Stage Slipper Lobster Phyllosoma Larvae of the Genus (Crustacea: Decapoda: Scyllaridae) Collected in the Pacific with Descriptions of Their Larval Morphology. Author Ueda, Kenta Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1 - 1 - 1, Bunkyo, Tokyo 113 - 8657, Japan. * Correspondence: E-mail: ayamakw @ g. ecc. u-tokyo. ac. jp (Yamakawa) E-mail: ueken 2840 @ gmail. com (Ueda); mari @ g. ecc. u-tokyo. ac. jp (Kuroki) & Fisheries Agency, Kasumigaseki 1 - 2 - 1, Chiyoda, Tokyo 100 - 8907, Japan. E-mail: ueken 2840 @ gmail. com (Ueda) ayamakw@g.ecc.u-tokyo.ac.jp Author Yanagimoto, Takashi Fisheries Resources Institute, Fukuura 2 - 12 - 4, Kanazawa, Yokohama, Kanagawa 236 - 8648, Japan. * Correspondence: E-mail: chow @ affrc. go. jp (Chow). E-mail: yanagimo @ affrc. go. jp (Yanagimoto) chow@affrc.go.jp Author Chow, Seinen Fisheries Resources Institute, Fukuura 2 - 12 - 4, Kanazawa, Yokohama, Kanagawa 236 - 8648, Japan. * Correspondence: E-mail: chow @ affrc. go. jp (Chow). E-mail: yanagimo @ affrc. go. jp (Yanagimoto) chow@affrc.go.jp Author Kuroki, Mari Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1 - 1 - 1, Bunkyo, Tokyo 113 - 8657, Japan. * Correspondence: E-mail: ayamakw @ g. ecc. u-tokyo. ac. jp (Yamakawa) E-mail: ueken 2840 @ gmail. com (Ueda); mari @ g. ecc. u-tokyo. ac. jp (Kuroki) ayamakw@g.ecc.u-tokyo.ac.jp Author Yamakawa, Takashi Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1 - 1 - 1, Bunkyo, Tokyo 113 - 8657, Japan. * Correspondence: E-mail: ayamakw @ g. ecc. u-tokyo. ac. jp (Yamakawa) E-mail: ueken 2840 @ gmail. com (Ueda); mari @ g. ecc. u-tokyo. ac. jp (Kuroki) ayamakw@g.ecc.u-tokyo.ac.jp text Zoological Studies 2021 2021-12-07 60 75 1 21 http://dx.doi.org/10.6620/ZS.2021.60-75 journal article 10.6620/ZS.2021.60-75 1810-522X PMC9169114 35774270 12825880 Chelarctus virgosus ( Yang and Chan, 2012 ) Stage V (KY18-1, Fig. 4 ) TL = 8.45 mm ; CL = 5.73 mm ; CW = 6.71 mm ; TW = 3.50 mm ; AL = 1.24 mm ; CW/CL = 1.17; CW/ TW = 1.92. This description was completed for the specimen collected at 27°02'N , 142°05'E (offshore of the Ogasawara Islands) on 31 July 2018 . Cephalic shield ( Fig. 4A ): sub-pentagonal; slightly wider than long, nearly twice as wide as thorax. Antennule ( Fig. 4B ): biramous, 3-segmented; distal segment with two flagella (primary and accessory); primary flagellum unsegmented with 9 ‒ 10 rows of sensory setae, slightly shorter than accessory flagellum. Antenna ( Fig. 4B ): bifurcated, unsegmented, half H-shaped (├), exceeding antennule; short lateral process projecting horizontally. Maxillule 1 ( Fig. 4E ): anterior lobe with denticulate three strong setae; posterior lobe with denticulate two strong setae. Maxillule 2 ( Fig. 4C ): rudimentary flattened. Maxilliped 1 ( Fig. 4C ): rudimentary bud. Maxilliped 2 ( Fig. 4C, D ): 5-segmented, coxal spine absent; three short setae on dactylus; at least five long setae on proximal margin of propodus ( Fig. 4D ). Maxilliped 3 ( Fig. 4A ): 5-segmented, tiny ventral coxal spine; distal parts of propodus and dactylus setosed densely; exopod absent. Pereiopods 1‒4 ( Fig. 4A ): biramous, tiny coxal spine and 5-segmented endopod; ischio-merus fused to basis, one stronger and one smaller distal spines on outer and inner sides of ischio-merus; a tiny distal spine on carpus; a dense of setae covering ischio-merus. Pereiopod 5 ( Fig. 4F, G ): elongated bud, coxal spine absent, much shorter than abdomen. Abdomen ( Fig. 4F, G ): four pairs of undeveloped pleopod buds; slightly bilobed uropod, rounded posterior margin locating far anterior of central margin of telson; prominent posterolateral spines of telson far beyond posterior central margin of telson, more than three times the length of uropod. Stage VI (KY15-1, Fig. 5 ) TL = 12.66 mm ; CL = 7.96 mm ; CW = 9.73 mm ; TW = 4.58 mm ; AL = 2.41 mm ; CW/CL = 1.22; CW/ TW = 2.12. This description was completed for the specimen collected at 27°07'N , 142°05'E (offshore of Ogasawara Islands), on 30 January 2015 . Cephalic shield ( Fig. 5A ): more than twice as wide as thorax. Antennule ( Fig. 5B ): primary flagellum similar in length to accessory flagellum. Antenna ( Fig. 5B ): similar in length to antennule. Maxilliped 1 ( Fig. 5C ): cone-shaped buds. Maxilliped 3 ( Fig. 5A ): propodus more densely setose than stage V (at least 20 setae). Pereiopods 1−4 ( Fig. 5A ): coxal spines more developed than those during stage V. Pereiopod 5 ( Fig. 5F, G ): 2-segmented, shorter than half of abdomen. Abdomen ( Fig. 5F, G ): four pairs of unbilobed pleopods; bilobed uropods; posterolateral spines of telson twice as long as uropod. Fig. 2. Neighbor-joining phylogenetic tree drawn using the Kimura-2-parameter distances for each of the partial COI sequences between the subfamily Scyllarinae species. Scyllarides brasiliensis was used as the outgroup. Closed squares indicate the phyllosoma specimens analyzed in this study. Bootstrap values of> 50% (from 1000 replicates) are shown at each node. Stage VII (SHU09-1, Fig. 6 ) TL = 18.61 mm ; CL = 11.89 mm ; CW = 14.11 mm ; TW = 7.67 mm , AL = 4.66 mm ; CW/CL = 1.18; CW/ TW = 1.84. This description was completed for the specimen collected at 25°30'N , 126°05'E (around the Ryukyu Archipelago) on 8 June 2009 . Fig. 3. Neighbor-joining phylogenetic tree drawn using the Kimura-2-parameter distances for each of the partial 16S rDNA sequences for species belonging to the subfamily Scyllarinae . Scyllarides brasiliensis was used as the outgroup. Closed squares indicate the phyllosoma specimens analyzed in this study. Bootstrap values of> 50% (from 1000 replicates) are shown at each node. Antennule ( Fig. 6B ): accessory flagellum longer than primary flagellum; larger number of setae on primary flagella than during stage VI. Antenna ( Fig. 6B ): lateral process stouter than that of stage VI. Maxillule 2 ( Fig. 6C ): rudimentary bilobed. Maxilliped 1 ( Fig. 6C ): slightly biramous. Maxilliped 2 ( Fig. 6C, D ): four setae on dactylus; two long terminal setae on propodus with four short simple setae. Maxilliped 3 ( Fig. 6A ): exopod bud, at least 19 setae on propodus. Pereiopods 1−4 ( Fig. 6A ): more spine-like setae on basis surface than during stage VI; at least 26 setae scattered over the surface of the P1 propodus; P4 longer than other pereiopods. Pereiopod 5 ( Fig. 6F, G ): 3-segmented, tiny coxal spine, about half of abdomen. Abdomen ( Fig. 6F, G ): pleopod biramous; posterolateral spines slightly longer than uropod. Stage VIII (SHU09-2, Fig. 7 ) TL = 20.66 mm ; CL = 12.30 mm ; CW = 15.97 mm ; Table 2. Uncorrecred K2P distance (lower diagonal) and standard error (upper diagonal) for the partial COI sequences from the nine phyllosoma larvae analyzed in this study and the five reference Chelarctus sequences from the database

Location*	1	2	3	4	5	6	7	8	9	10	11	12	13	14
1: C. aureus	Taiwan	0.021	0.024	0.023	0.026	0.027	0.026	0.026	0.027	0.004	0.022	0.022	0.022	0.023
2: C. crosnieri	Vanuatu	0.187	0.022	0.021	0.024	0.025	0.024	0.023	0.024	0.022	0.020	0.020	0.021	0.012
3: C. cultrifer cultirfer	Indonesia	0.217	0.190	0.014	0.024	0.024	0.024	0.023	0.024	0.024	0.022	0.022	0.023	0.022
4: C. cultrifer meridionalis	Philippines	0.201	0.178	0.086	0.022	0.023	0.022	0.021	0.022	0.023	0.023	0.023	0.023	0.022
5: C. virgosus	Taiwan	0.242	0.213	0.208	0.184	0.006	0.005	0.005	0.005	0.026	0.024	0.024	0.024	0.023
6: KY15-1	NWP	0.245	0.225	0.208	0.192	0.019	0.005	0.006	0.006	0.026	0.024	0.024	0.025	0.023
7: KY18-1	NWP	0.242	0.214	0.211	0.184	0.012	0.014	0.004	0.002	0.026	0.023	0.023	0.023	0.022
8: SHU09-1	NWP	0.236	0.211	0.200	0.178	0.013	0.019	0.010	0.005	0.025	0.023	0.023	0.023	0.022
9: SHU09-2	NWP	0.245	0.217	0.213	0.181	0.014	0.016	0.002	0.012	0.026	0.023	0.023	0.024	0.023
10: SHU09-5	NWP	0.010	0.195	0.212	0.196	0.233	0.236	0.233	0.227	0.236	0.022	0.022	0.022	0.022
11: SHU09-7	NWP	0.190	0.150	0.190	0.196	0.213	0.216	0.197	0.199	0.199	0.193	0.006	0.005	0.020
12: SHU09-8	NWP	0.188	0.155	0.188	0.199	0.207	0.208	0.194	0.196	0.197	0.190	0.016	0.005	0.019
13: SHU09-9	NWP	0.190	0.158	0.198	0.199	0.216	0.219	0.199	0.202	0.202	0.193	0.014	0.014	0.020
14: KH16-4-1	CSP	0.208	0.069	0.194	0.196	0.210	0.210	0.202	0.199	0.205	0.205	0.154	0.149	0.159

Accession numbers: JF411065 (1), JX486086 (2), JX486082 (3), JX486085 (4), JX262266 (5), LC632949 (6), LC632948 (7), LC632950−LC632955 (8-13), LC632947 (14). *NWP: Northwest Pacific, CSP: central South Pacific. Table 3. Uncorrecred K2P distance (lower diagonal) and standard error (upper diagonal) for the partial 16S rDNA sequences from the 11 phyllosoma larvae analyzed in this study and the two Chelarctus reference sequences from the database

Location*	1	2	3	4	5	6	7	8	9	10	11	12	13
1: Chelarctus aureus	Taiwan	0.022	0.019	0.020	0.019	0.019	0.003	0.003	0.027	0.027	0.027	0.027	0.023
2: Chelarctus cultrifer	Philippines	0.117	0.016	0.017	0.016	0.016	0.023	0.023	0.017	0.018	0.017	0.017	0.016
3: KY15-1	NWP	0.102	0.071	0.003	0.000	0.000	0.020	0.020	0.021	0.021	0.021	0.021	0.018
4: KY18-1	NWP	0.098	0.068	0.003	0.003	0.003	0.020	0.020	0.021	0.021	0.022	0.022	0.018
5: SHU09-1	NWP	0.098	0.068	0.003	0.000	0.000	0.020	0.020	0.021	0.021	0.021	0.021	0.018
6: SHU09-2	NWP	0.098	0.068	0.003	0.000	0.000	0.020	0.020	0.021	0.021	0.021	0.021	0.018
7: SHU09-3	NWP	0.003	0.121	0.106	0.102	0.102	0.102	0.000	0.027	0.026	0.027	0.027	0.023
8: SHU09-4	NWP	0.003	0.121	0.106	0.102	0.102	0.102	0.000	0.027	0.026	0.027	0.027	0.023
9: SHU09-6	NWP	0.169	0.085	0.115	0.111	0.111	0.111	0.164	0.164	0.004	0.003	0.003	0.018
10: SHU09-7	NWP	0.165	0.088	0.111	0.108	0.108	0.108	0.160	0.160	0.006	0.003	0.003	0.017
11: SHU09-8	NWP	0.169	0.085	0.115	0.111	0.111	0.111	0.165	0.165	0.003	0.003	0.000	0.018
12: SHU09-9	NWP	0.169	0.085	0.115	0.111	0.111	0.111	0.165	0.165	0.003	0.003	0.000	0.018
13: KH16-4-1	CSP	0.132	0.074	0.089	0.085	0.085	0.085	0.136	0.136	0.085	0.078	0.082	0.082

Accession numbers: JN701711 (1), JN701712 (2), LC632673 (3), LC632672 (4), LC632674−LC632682 (5−13). *NWP: Northwest Pacific, CSP: central South Pacific. TW = 7.76 mm ; AL = 5.92 mm ; CW/CL = 1.25; CW/ TW = 2.06. This description was completed for the specimen collected at 23°19'N , 126°09'E (around the Ryukyu Archipelago) on 9 June 2009 . Antenna ( Fig. 7B ): exceeding antennule. Maxillule 1 ( Fig. 7E ): six short terminal setae, two long denticulate setae and two normal terminal setae on the posterior lobe and two sub-terminal setae on the outer side of the posterior lobe. Maxillule 2 ( Fig. 7C ): apparently biramous, more developed posterior lobe than during stage VII. Maxilliped 1 ( Fig. 7C ): unsegmented and bilobed, outer lobe flattened and rounded. Maxilliped 2 ( Fig. 7C, D ): an exopod bud. Maxilliped 3: coxal spine absent; propodus more densely setose than stage VII. Pereiopods 1−4 ( Fig. 7A ): gill buds on base of coxa; at least 33 setae scattered over the surface of the propodus in P1. Pereiopod 5 ( Fig. 7F, G ): 4-segmented, much longer than half of abdomen. Abdomen ( Fig. 7F, G ): biramous pleopods larger than stage VII; round uropod margin near reaching but never exceeding posterior central margin of telson; posterolateral spines of telson similar in length of uropod.