A New Species of the Bengal Spined Anchovy Stolephorus from the Eastern Indian Ocean and Redescription of Stolephorus dubiosus Wongratana, 1983, with Comments on the Evolution of Prepelvic Scute Numbers within Stolephorus (Clupeiformes: Engraulidae)

© 2022 Academia Sinica, Taiwan Open Access A New Species of the Bengal Spined Anchovy Stolephorus from the Eastern Indian Ocean and Redescription of Stolephorus dubiosus Wongratana, 1983, with Comments on the Evolution of Prepelvic Scute Numbers within Stolephorus (Clupeiformes: Engraulidae) Harutaka Hata1,* , Sébastien Lavoué2, and Hiroyuki Motomura3 1National Museum of Natural History, Smithsonian Institution, 10th and Constitution Ave, NW Washington, DC 20560, USA. *Correspondence: E-mail: [email protected] (Hata) 2School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia. E-mail: [email protected] (Lavoué) 3The Kagoshima University Museum, 1-21-30 Korimoto, Kagoshima 890-0065, Japan. E-mail: [email protected] (Motomura) Received 19 January 2022 / Accepted 25 July 2022 / Published 25 November 2022 Communicated by Hin-Kiu Mok The Bengal Spined Anchovy, Stolephorus taurus sp. nov. is described from 21 specimens from the northern Bay of Bengal. The new species closely resembles Stolephorus dubiosus Wongratana, 1983, which is redescribed. Both species have a predorsal scute, spine on the pelvic scute, long maxilla posteriorly slightly short of or just reaching the posterior margin of the opercle, 25 or more gill rakers on the lower limb of the first gill arch, and double black lines on the dorsum posterior to the dorsal fin. However, the new species differs from S. dubiosus in having a longer pelvic fin with the posterior tip of the depressed fin beyond vertical through the dorsal-fin origin (vs. usually not reaching to vertical through dorsal-fin origin), longer pectoral fin, second dorsaland third dorsal-fin rays, second analand third anal-fin rays, and greater interorbital width. Stolephorus taurus sp. nov. is closely related to Stolephorus baganensis Delsman, 1931 and S. dubiosus, although at least 2% mean p-distance divergence in the mitochondrial cytochrome c oxidase subunit I (COI) gene separates each of the three species. A phylogenetic reconstruction of the evolution of the number of prepelvic scutes within Stolephorus indicated that having six scutes was the most likely ancestral condition in the genus, and was later reduced in the evolution of Stolephorus to five or four scutes. One such reduction occurred recently in the lineage of Stolephorus taurus sp. nov. Key words: Clupeomorpha, Taxonomy, Bay of Bengal, Stolephorus baganensis, Stolephorus tri. BACKGROUND Stolephorus Lacepède, 1803, comprising 41 valid marine and/or brackish water Indo-Pacific species, is included in the subfamily Engraulinae (family Engraulidae) (Whitehead et al. 1988; Wongratana et al. 1999; Kimura et al. 2009; Hata and Motomura 2018a– e 2021a–c 2022; Hata et al. 2019 2020a b 2021 2022; Gangan et al. 2020). Among them, Stolephorus dubiosus Wongratana, 1983, distributed in the Indo-West Pacific from the Bay of Bengal to Borneo (Wongratana 1983; Whitehead et al. 1988; Wongratana et al. 1999), is Citation: Hata H, Lavoué S, Motomura H. 2022. A new species of the Bengal Spined Anchovy Stolephorus from the eastern Indian Ocean and redescription of Stolephorus dubiosus Wongratana, 1983, with comments on the evolution of prepelvic scute numbers within Stolephorus (Clupeiformes: Engraulidae). Zool Stud 61:58. doi:10.6620/ZS.2022.61-58. Zoological Studies 61:58 (2022) doi:10.6620/ZS.2022.61-58 1 © 2022 Academia Sinica, Taiwan diagnosed by the presence of spines on the pelvic and predorsal scutes (the latter just anterior to the dorsal-fin origin), 25 or more gill rakers on the lower limb of the first gill arch, and a long maxilla with the posterior tip just reaching the posterior margin of the opercle. During a revisionary study of this genus, a species previously identified as S. dubiosus from the Indian and Pacific oceans was found to comprise two distinct species, clearly separated morphologically and genetically. The Indian Ocean species, described herein as new, had longer fins than true S. dubiosus (endemic to the western Pacific Ocean and redescribed herein). In addition, two mitochondrial nucleotide markers, the complete cytochrome b gene and partial cytochrome c oxidase subunit I (COI) gene, were used to evaluate the genetic distinctiveness of the new species compared with 21 other species of Stolephorus (including two morphologically similar species, S. dubiosus and Stolephorus baganensis Delsman, 1931). The phylogenetic tree of Stolephorus was used to reconstruct the evolution of prepelvic scute numbers within the genus. MATERIALS AND METHODS Counts and proportional measurements followed Hata and Motomura (2017). All measurements were made with digital calipers to the nearest 0.01 mm. Standard and head lengths are abbreviated as SL and HL, respectively. Osteological characters, including vertebral counts, were observed on radiographs of 8 specimens of S. baganensis, 13 S. dubiosus and 14 Stolephorus taurus sp. nov. “Pelvic scute” refers to the scute associated with the pelvic-fin insertion, whereas “prepelvic”, “postpelvic”, and “predorsal scutes” refer to the scutes anterior to the pelvic scute, posterior to the pelvic scute, and just anterior to the dorsalfin origin, respectively. Institutional codes generally followed Sabaj (2020). USMFC indicates Universiti Sains Malaysia Fish Collection, School of Biological Sciences, Penang, Malaysia. The ethical guidelines for experimental use of animals, is not applicable for this study. A phylogenetic tree of the genus Stolephorus based on two molecular markers (the complete [1140 base pairs (bp)] cytochrome b gene and partial [about 650 bp] COI gene) both being newly sequenced for two specimens each of S. baganensis and Stolephorus teguhi Kimura, Hori and Shibukawa 2009, and one specimen of S. dubiosus (Table 1), following Hata et al. (2019). The cytochrome b gene was amplified using the following primers: forward L14740 (5'-CCG TTG TAT TCA ACT ACA GAA-3') and reverse H15913 (5'-TCG ATC TCC GGA TTA CAA GAC CG-3') (all primer references in Hata et al. 2019). The partial COI gene was amplified using the following primers: forward COI_FishF1 (5'-TCA ACC AAC CAC AAA GAC ATT GGC AC-3') and reverse COI_FishR2 (5'- ACT TCA GGG TGA CCG AAG AAT CAG AA-3'). The sequences subsequently generated were deposited into the GenBank database (accession numbers given in Table 1). Data from the five newly-sequenced specimens were combined with the data set of 27 specimens of Stolephorus previously examined by Hata et al. (2019 2020a b 2021), plus data from six specimens available in GenBank and examined by Lavoué et al. (2010), Egan et al. (2018), and Gangan et al. (2020). Cytochrome b and COI sequences were separately aligned by eye, with no indels required. The final alignment combining the two genes comprised 1788 nucleotide positions (38 specimens of Stolephorus) (Table 1). Two specimens of the genus Encrasicholina Fowler, 1938 were collectively used to root the phylogenetic tree. Uncorrected pairwise genetic distances (i.e., p-distances) were calculated for each gene using Mega v10.1.7 (Stecher et al. 2020). An ultrametric phylogenetic tree was inferred using a Bayesian method under a relaxed molecular clock in BEAST v.2.6.5 (Bouckaert et al. 2019). The BEAST input file was built in BEAUti v.2.6.5, and the GTR + G model of sequence evolution selected. The respective monophyly of the ingroup and outgroup (i.e., the two species of Encrasicholina) was a priori enforced to root the tree. The relative age of the tree root was calibrated to ‘‘1”. Two independent runs of 10 million generations each were performed using BEAST v.2.6.5. Estimations of trees and divergence times were sampled once every 1000 generations and the parameters of each run checked for convergence with Tracer v.1.7.2 software (available at https://github.com/ beast-dev/tracer/releases). After removing the burn-in portion of each run (25%), the remaining tree samples from the two runs were pooled into a combined file using LogCombiner v.2.6.5, and the maximum clade credibility tree with posterior probabilities at nodes, calculated from the file using TreeAnnotator v.2.6.5. The evolutionary history of the number of prepelvic scutes was reconstructed onto the ultrametric phylogenetic tree assuming a model of character evolution in which transitions between each state occur at the same rate (model “Mk1”). Character state reconstructions were performed using Mesquite v3.61 (Maddison and Maddison 2019). A discrete category (corresponding to the number of prepelvic scutes 4, 5 or 6) was assigned to each species, with the exception of Stolephorus tamilensis Gangan, Pavanpage 2 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan Kumar, Jahageerdar, and Jaiswar, 2020 (prepelvic scute numbers variable, 5 or 6). Although the latter species was successively categorized, its categorization did not modify the reconstruction hypothesis regarding the ancestral state in Stolephorus (see DISCUSSION). RESULTS Stolephorus dubiosus Wongratana, 1983 (English name: Thai Anchovy) (Figs. 1–6; Tables 2, 3) Table 1. Information on the genetic markers used in the present study. “/” indicates corresponding sequence undetermined; bold accession numbers indicate sequences determined in present study; Cytb cytochrome b, COI cytochrome c oxidase subunit I Museum registration # Accession numbers Species [DNA tissue code] Origin Cytb COI Stolephorus acinaces No voucher [SI17] Fish market, Sibu, Malaysia MH3807433MH3806553 No voucher [SI18] Fish market, Sibu, Malaysia MH3803973MH3805183 Stolephorus andhraensis NTUM 12328 [Bg14] Fish market, Bangkok, Thailand MH3806561MH3807441 NTUM 12328 [Bg18] Fish market, Bangkok, Thailand MH3806571MH3807451 Stolephorus babarani KAUM–I. 62918 off Iloilo, Panay Island, Philippines MH3803652MH3804862 KAUM–I. 62920 off Iloilo, Panay Island, Philippines MH3803662MH3804872 Stolephorus baganensis USMFC (82) 00032 [IK100] Sungai Batu, Penang Isl., Malaysia MT080913 MT080441 USMFC (82) 00035 [IK104] Sungai Batu, Penang Isl., Malaysia MT080916 MT080444 Stolephorus balinensis KAUM–I. 106302 Ho Chí Minh, Vietnam MH3804351MH3805561 KAUM–I. 106303 Ho Chí Minh, Vietnam MH3804361MH3805571 Stolephorus bataviensis KAUM–I. 110294 off Dong-gang, Pingtung, Taiwan MH3803351MH3804561 KAUM–I. 110295 off Dong-gang, Pingtung, Taiwan MH3803361MH3804571 Stolephorus baweanensis KAUM–I. 94725 Ha Long Bay, northern Vietnam MH3803551MH3804761 KAUM–I. 94779 Ha Long Bay, northern Vietnam MH3803561MH3804771 Stolephorus belaerius ADC10_55.3 #4 Mhlathuze estuary, South Africa / HQ9459377 Stolephorus eldorado KAUM–I. 113149 Donggang, Taiwan MH3803301/ KAUM–I. 94509 Ha Long Bay, northern Vietnam MH3803181/ Stolephorus brachycephalus JFBM 48023 Australia MG9581785/ Stolephorus continentalis KAUM–I. 94540 Ha Long Bay, northern Vietnam MH3803061MH3804501 KAUM–I. 94541 Ha Long Bay, northern Vietnam MH3803071MH3804511 Stolephorus dubiosus NTUM 12329 Ganh Rai Bay, southern Vietnam MH380393 MH380514 No voucher [KU06] Kuching, Malaysia MH3806331MH3807231 Stolephorus holodon ADC55.2-3 Mlalazi Estuary, South Africa / JF4945987 Stolephorus indicus KAUM–I. 59676 off Phuket, Thailand MH3803984MH3805194 KAUM–I. 59677 off Phuket, Thailand MH3803994MH3805204 Stolephorus mercurius KAUM–I. 60731 off Nha Trang, Khánh Hòa, Vietnam MH3803741MH3804951 KAUM–I. 60732 off Nha Trang, Khánh Hòa, Vietnam MH3803751MH3804961 Stolephorus nelsoni JFBM 48063 Australia MG9581775/ Stolephorus oceanicus KAUM–I. 106329 Ho Chí Minh, Vietnam MH3806591/ KAUM–I. 106330 Ho Chí Minh, Vietnam MH3803151/ Stolephorus rex KAUM–I. 80769 off Oton, Panay Island, the Philippines MH3803844MH3805054 Stolephorus tamilensis No voucher [CIFEFGB-SNS-0086] Thoothukudi, Tamil Nadu, India / KX7688926 No voucher [CIFEFGB-SNS-0087] Thoothukudi, Tamil Nadu, India / KX7688936 Stolephorus taurus sp. nov. No voucher [CL14] Fish market, Calcutta, India AP0115678AP0115678 Stolephorus teguhi FRLM 34849 Pintukota, Lembeh Island, Bitung, North Sulawesi, Indonesia /ON791254 FRLM 34852 Pintukota, Lembeh Island, Bitung, North Sulawesi, Indonesia /ON791255 Stolephorus tri NTUM 12578 [SI56] Fish market, Sibu, Malaysia MH3803901MH3805111 NTUM 12578 [SI57] Fish market, Sibu, Malaysia MH3803911MH3805121 Encrasicholina heteroloba No voucher [Ra81] Fish market, Ranong, Thailand MT0809233MT0804513 Encrasicholina punctifer No voucher [Ra79] Fish market, Ranong, Thailand MT0809193MT0804473 Sequence sources: 1Hata et al. (2019), 2Hata et al. (2020a), 3Hata et al. (2020b), 4Hata et al. (2021), 5Egan et al. (2018), 6Gangan et al. (2020); 7D. Steinke and collaborators (unpublished), 8Lavoué et al. (2010). page 3 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan Stolephorus baganensis baganensis (not of Delsman): Hardenberg 1934: 333 [in part: Susang, Labuan, Batavia (currently Jakarta), Cheribon (Cirebon), Kendal, Semarang, Tuban, Surabaja (Surabaya), and Kumai, Indonesia]. Stolephorus dubiosus Wongratana, 1983: 400, fig. 18 (in part: original description; type locality: Songkhla Lake, Thailand; other localities: Paknam, Bangkok, Samutsakorn, Nakornsrithammaraj, and Surajthani, Thailand; Aluhaluh on Barito River, Kalimantan, Indonesia); Wongratana 1987a: 107 (in part: Thailand, Malay Peninsula, and Indonesia); Wongratana 1987b: 8 (in part: Gulf of Thailand and Java Sea); Whitehead et al. 1988: 411, unnumbered fig. (in part: Gulf of Thailand to Java Sea); Kottelat et al. 1993: 31, fig. 94 (in part: Sundaland and Thailand); Wongratana et al. 1999: 1734, unnumbered fig. (in part: Gulf of Thailand, Java Sea to Kalimantan); Nakashima 2005: 72 (Samut Prakan Province, Thailand); Jutagate et al. 2009: 123 (Pak Panang Bay and estuary of Pak Panang River, Thailand); Matsunuma 2013: 33, unnumbered fig. (Bang Pakong, Gulf of Thailand, Thailand); Tran et al. 2013: 43, unnumbered fig. (Mekong Delta, southern Vietnam); Kottelat 2013: 55 (Songkhla Lake, Thailand); Rupawan 2017: 132 (Panjan Strait, Riau Province, Sumatra, Indonesia); Hata et al. 2019: 30, fig. 14 (in part: Kuching, Sarawak, Malaysia; Songkhla Lake and Gulf of Thailand, Thailand); Syafei et al. 2020: 4, fig. 2 (Pabean Bay, Indramayu, West Java, Indonesia); Hata et al. 2020b: table 1 (in part: Bangkok, Thailand); Nagao Natural Environment Foundation 2021: 55, unnumbered fig. (Mekong Basin in Vietnam). Holotype: BMNH 1969.4.22.1826, 70.0 mm SL, Songkhla Lake, Thailand, 2 May 1966, I. A. Ronquillo. Non-type specimens: 22 specimens, 49.9–78.1 mm SL. THAILAND: BMNH 1977.11.30.63, 63.2 mm SL, Nakornsrithammaraj; THNHM-F021231, 68.1 mm SL, THNHM-F021232, 72.1 mm SL, THNHM-F021233, 65.6 mm SL, THNHM-F021234, 60.7 mm SL, THNHM-F021235, 64.4 mm SL, Bang Pakong River estuary, Chachoengsao Province; THNHM-F021236, 58.2 mm SL, Gulf of Thailand; THNHM-F021237, 66.0 mm SL, THNHM-F021238, 64.5 mm SL, THNHM-F021239 (cleared and stained), 63.6 mm SL, Thachin River estuary, Muang, Samut Sakhon Province; NSMT-P. 55363, 3 specimens, 55.1–59.1 mm SL, Songkhla Lake, 1.5 m depth, shrimp trap; NSMT-P 127425, 3 specimens, 49.9–59.4 mm SL, Songkhla Lake; URM-P 12176, 65.1 mm SL, Songkhla; URM-P 27308, 59.4 mm SL, URM-P 27309, 63.4 mm SL, Pak Nam, Samut Prakan Province; USNM 119676, 2 specimens, 58.4–62.3 mm SL, Bangpakong River. VIETNAM: NTUM 12329, 78.1 mm SL, Ganh Rai Bay. Diagnosis: A species of Stolephorus with the following combination of characters: predorsal scute and spine on pelvic scute present; gill rakers on first gill arch 20–24 (modally 21) (upper series), 26–29 (28) (lower series), 46–53 (49) in total; gill rakers on second gill arch 16–19 (16) (upper series), 25–29 (27) (lower series), 41–48 (43) in total; gill rakers on third gill arch 12–14 (13) (upper series), 14–17 (15) (lower series), 27–31 (28) in total; gill rakers on fourth gill arch 8–11 (10) (upper series), 11–14 (12) (lower series), 20–25 (22) in total; prepelvic scutes 5–7 (6); maxilla long, posterior tip just reaching or slightly short of posterior margin of opercle; small teeth on dorsal surface of hyoid bone; posterior border of preopercle convexly rounded; distinct paired dark patches on parietal and occipital regions; no dark lines on dorsum anterior to dorsal-fin origin; double pigmented lines on dorsum from dorsalfin base end to caudal-fin base; no melanophores below eye or on mandibular tip; posterior tip of pelvic fin usually not reaching vertical through dorsal-fin origin (sometimes reaching to vertical through first or second dorsal-fin ray origin) when depressed; body scales not deciduous, with numerous separations of grooves; pectoral fin short, 15.6–17.4% SL; pelvic fin short, 9.5–10.8% SL; second dorsal-fin ray short, 4.9–8.5% SL; third dorsal-fin ray short, 16.8–19.0% SL; second anal-fin ray short, 4.3–5.9% SL; third anal-fin ray short, 13.4–15.1% SL; interorbit narrow, 21.5–24.8% HL. Description: Data for holotype presented first, followed by other specimen data in parentheses (if different). Counts and measurements, expressed as percentages of SL or HL, given in tables 2 and 3. Body laterally compressed, elongate, deepest at dorsal-fin origin. Dorsal profile of head and body gradually elevated from snout tip to dorsal-fin origin, thereafter, nearly linear, gently lowering to uppermost point of caudal-fin base. Ventral profile of head and body gently lowering from snout tip to just below pectoral-fin insertion, thereafter parallel to body axis to anal-fin origin. Ventral contour from anal-fin origin to lowermost point of caudal-fin base gently elevated. Single spine-like scute just anterior to dorsal-fin origin (Fig. 2). Abdomen rounded, covered with five (four to seven) spine-like scutes anterior to pelvic-fin insertion. Pelvic scute attached to pelvic girdle, with a hard backwardly projecting spine (Fig. 3). No spinelike scutes on ventral surface posterior to pelvic fin. Anus just anterior to anal-fin origin. Snout tip rounded, projecting; snout length less than eye diameter. Mouth large, inferior, ventral to body axis, extending backward beyond posterior margin of eye. Maxilla long, posterior tip pointed, reaching to (or just short of) posterior margin of opercle. Premaxilla and first supramaxilla elongate. Second supramaxilla asymmetrical, lower page 4 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan part larger than upper part. Lower jaw slender. Conical teeth in single rows on each jaw and palatines. Several teeth on vomer. Several rows of teeth on inner surface of pterygoids. Small conical teeth on dorsal surface of hyoid bone. Eye large, round, covered with adipose eyelid, positioned laterally on head dorsal to horizontal through pectoral-fin insertion, visible in dorsal view. Pupil round. Orbit elliptical. Nostrils close to each other, anterior to orbit. Posterior margins of preopercle and opercle rounded, smooth. Subopercle with rounded posterior margin. Gill membrane without serrations. Interorbital space flat. Interorbital width less than eye diameter. Pseudobranchial filaments present, length of longest filament less than eye diameter. Gill rakers long, slender, rough, visible from side of head when mouth opened. Isthmus muscle long, reaching anteriorly to posterior margin of gill membranes. Urohyal hidden by isthmus muscle (not visible without dissection). Gill membrane on each side joined distally, most of isthmus muscle exposed (not covered by gill membrane). Head scales absent. Lateral line absent. Fins scaleless, except for broad triangular sheath of scales on caudal fin. Scales on lateral surface cycloid, thin, not deciduous. Numerous separations of grooves on body scales (Fig. 4). Dorsal-fin origin posterior to vertical through base of last pelvic-fin ray, slightly posterior to middle of body. Dorsal and anal fins with three anteriormost rays unbranched. First dorsaland anal-fin rays minute. Anteriormost three rays of both dorsal and anal fins closely spaced. Anal-fin origin just below bases of tenth (eighth to eleventh) dorsal-fin ray. Posterior tip of depressed anal fin not reaching caudal-fin base. Uppermost pectoral-fin ray unbranched, inserted below midline of body. Posterior tip of pectoral fin pointed, Table 2. Meristics of specimens of Stolephorus taurus sp. nov., S. baganensis, and S. dubiosus Stolephorus taurus sp. nov. Stolephorus baganensis Stolephorus dubiosus Holotype Paratypes Neotype of Stolephorus baganensis Non-types Holotype Non-types OCF-P 10434 n = 19 BMNH 1967.11.13.526 n = 30 BMNH 1969.4.22.1826 n = 22 Standard length (mm) 52.2 39.8–56.4 Modes 67.6 27.3–70.9 Modes 70.0 49.9–78.1 Modes Dorsal-fin rays (unbranched) 3 3 3 3 3 3 3 3 3 Dorsal-fin rays (branched) 11 11–13 12 12 11–12 12 11 11–13 11 Anal-fin rays (unbranched) 3 3 3 3 3 3 3 3 3 Anal-fin rays (branched) 18 17–19 18 18 17–20 18 18 17–19 18 Pectoral-fin rays (unbranched) 1 1 1 1 1 1 1 1 1 Pectoral-fin rays (branched) 12 11–13 12 12 10–12 11 11 10–12 11 Pelvic-fin rays (unbranched) 1 1 1 1 1 1 1 1 1 Pelvic-fin rays (branched) 6 6 6 6 6 6 6 6 6 Gill rakers on 1st gill arch (upper) 21 19–22 21 17 15–19 17 21 20–24 21 Gill rakers on 1st gill arch (lower) 27 25–29 26 22 20–25 22 28 26–29 28 Gill rakers on 1st gill arch (total) 48 45–49 48 39 35–43 40 49 46–53 49 Gill rakers on 2nd gill arch (upper) 15 14–16 15 13 11–14 12 16 16–19 16 Gill rakers on 2nd gill arch (lower) 25 24–26 25 21 19–23 21 27 25–29 27 Gill rakers on 2nd gill arch (total) 40 39–42 40 34 31–37 35 43 41–48 43 Gill rakers on 3rd gill arch (upper) 12 11–12 12 10 9–11 10 13 12–14 13 Gill rakers on 3rd gill arch (lower) 15 13–16 14 12 11–13 12 14 14–17 15 Gill rakers on 3rd gill arch (total) 27 24–28 26 22 20–24 22 27 27–31 28 Gill rakers on 4th gill arch (upper) 9 8–10 9 9 7–9 8 9 8–11 10 Gill rakers on 4th gill arch (lower) 11 11–12 11 10 9–13 10 12 11–14 12 Gill rakers on 4th gill arch (total) 20 19–22 20 19 16–21 18 21 20–25 22 Gill rakers on posterior face of 3rd gill arch 6 4–7 5 6 3–6 5 6 5–7 6 Prepelvic scutes 5 4–6 5 5 4–7 6 5 5–7 6 Scale rows in longitudinal series 34 33–35 34 35 34–37 35 35 34–36 35 Transverse scales 8 8–9 8 8 8 8 8 8–9 8 Pseudobranchial filaments 18 15–19 16 17 16–20 12 17 16–20 19 Abdominal vertebrae 19 18–19 19 - 19 19 18–19 19 Caudal vertebrae 20 20–21 20 - 20 20 19–21 20 Total vertebrae 39 38–39 39 - 39 39 38–40 39 page 5 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan not reaching vertical through pelvic-fin insertion. Upper, posterior, and lower contours of pectoral fin nearly straight. Pelvic fin shorter than pectoral fin. Posterior tip of depressed pelvic fin not reaching to vertical through dorsal-fin origin (rarely reaching to base of first or second dorsal-fin ray). Caudal fin forked, upper and lower margins of both lobes nearly straight. Posterior tips of both lobes pointed. Skeleton of hyoid arch (Fig. 5): All branchiostegal rays paddle-shaped (posteriorly broad). No branchiostegal rays connected to hypohyal, two rays on epihyal. Caudal skeleton (Fig. 6): Each hypural free, except for second and third conjoined. Dorsal margin of first hypural smooth, without distinct projection. Posterior margins of second and third hypurals broadly concave. Dorsal margins of fourth and fifth hypurals projected anteriorly. Sixth hypural elongate. Coloration when fresh (based on color photographs of THNHM-F021231–021235, 021237): Table 3. Morphometrics of specimens of Stolephorus taurus sp. nov., S. baganensis, and S. dubiosus Stolephorus taurus sp. nov. Stolephorus baganensis Stolephorus dubiosus Holotype Paratypes Neotype of Stolephorus baganensis Non-types Holotype Non-types OCF-P 10434 n = 19 BMNH 1967.11.13.526 n = 30 BMNH 1969.4.22.1826 n = 22 Standard length (mm; SL) 52.2 39.8–56.4 Means 67.6 27.3–70.9 Means 70.0 49.9–78.1 Means As % SL Head length (HL) 24.4 23.5–26.5 25.0 25.3 23.5–26.6 24.8 24.1 23.2–26.4 24.7 Body depth 24.1 21.3–24.3 22.9 24.6 20.2–26.1 23.6 23.7 20.4–27.3 22.5 Pre-dorsal-fin length 54.1 50.3–56.2 52.9 53.9 50.6–56.5 53.6 55.5 51.8–55.9 53.9 Snout tip to pectoral-fin insertion 27.1 24.4–27.3 26.1 27.3 24.9–28.6 26.7 26.6 25.2–28.1 26.9 Snout tip to pelvic-fin insertion 44.8 42.2–47.9 44.9 44.5 42.9–47.7 44.9 44.4 43.0–47.8 44.8 Snout tip to anal-fin origin 64.6 62.8–66.3 64.2 64.2 61.3–67.7 64.3 63.2 61.9–66.9 63.9 Dorsal-fin base length 12.4 12.1–14.5 13.3 14.6 12.0–14.6 13.6 14.4 11.9–14.8 13.4 Anal-fin base length 19.2 18.7–20.9 19.6 21.8 18.8–23.2 21.3 22.4 18.5–22.0 20.6 Caudal-peduncle length 19.1 16.1–20.0 17.9 17.6 15.1–19.7 17.3 16.1 15.8–19.5 17.8 Caudal-peduncle depth 11.6 10.2–12.4 11.5 11.1 7.9–12.1 10.6 9.6 9.5–11.6 10.5 D–P1 35.2 34.7–38.5 36.7 36.4 30.9–38.6 35.8 37.2 34.4–38.4 36.3 D–P2 27.2 22.3–26.4 25.2 25.9 21.4–29.0 25.5 26.3 23.1–28.0 24.9 D–A 24.7 23.2–25.6 24.3 25.9 21.6–27.9 25.3 25.5 21.8–27.9 23.7 P1–P2 18.9 17.4–23.3 19.8 18.1 16.1–21.8 19.0 17.9 17.4–23.2 19.3 P2–A 21.3 16.9–22.8 19.6 16.9 16.9–22.9 19.4 20.0 17.1–21.6 19.5 Pectoral-fin length 17.9 17.8–18.7 18.1 broken 16.5–20.0 18.0 16.5 15.6–17.4 16.7 Pelvic-fin length 12.0 11.6–12.7 12.1 broken 8.7–11.6 10.7 9.5 9.5–10.8 10.2 Maxilla length 21.2 20.7–21.9 21.1 broken 19.2–22.2 20.9 20.8 19.2–22.2 20.5 Mandible length 16.7 16.8–18.4 17.5 16.4 15.6–18.0 16.8 17.3 15.4–17.6 16.6 Supramaxilla end to maxilla end 5.2 3.9–5.3 4.8 broken 3.4–6.2 5.1 5.2 4.1–5.7 4.7 1st unbranched dorsal-fin ray length 2.1 1.2–2.5 1.8 1.6 1.4–3.9 1.9 2.3 1.1–2.1 1.4 2nd unbranched dorsal-fin ray length 10.0 8.7–10.0 9.2 7.0 6.8–10.8 8.1 7.2 4.9–8.5 7.3 3rd dorsal-fin ray length broken 19.5–20.2 19.8 broken 17.8–19.9 18.9 broken 16.8–19.0 18.0 1st unbranched anal-fin ray length 2.3 1.3–2.9 2.1 1.7 1.2–3.5 2.0 1.5 0.7–2.4 1.6 2nd unbranched anal-fin ray length 6.6 6.0–7.3 6.4 broken 3.6–6.9 5.3 4.6 4.3–5.9 5.2 3rd anal-fin ray length 15.8 15.8–17.5 16.4 broken 13.4–17.3 15.7 broken 13.4–15.1 14.4 As % HL Orbit diameter 34.0 30.3–34.5 32.2 29.5 30.1–37.2 32.7 30.4 28.8–33.5 31.3 Eye diameter 27.9 24.2–30.9 27.3 24.4 21.7–29.3 25.9 26.3 23.2–28.8 26.4 Snout length 13.1 11.9–14.8 13.2 13.1 11.6–14.7 13.0 16.0 13.6–16.7 15.1 Interorbital width 25.6 25.2–28.3 26.2 23.0 20.6–26.0 24.0 24.3 21.5–24.8 23.3 Postorbital length 54.8 51.1–56.2 53.9 58.1 51.7–59.0 55.1 57.7 52.4–58.0 55.4 Abbreviations: D–P1 (distance between dorsal-fin origin and pectoral-fin insertion); D–P2 (distance between dorsal-fin origin and pelvic-fin insertion); D–A (distance between origins of dorsal and anal fins); P1–P2 (distance between insertions of pectoral and pelvic fins); P2–A (distance between pelvic-fin insertion and anal-fin origin). page 6 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan Fig. 1. Stolephorus dubiosus: (a) Lateral view of holotype (BMNH 1969.4.22.1826, 70.0 mm SL, Thailand); (b) lateral view in fresh condition; (c) dorsal and (d) ventral views in preserved condition of non-type specimen (THMHM-F021237, 66.0 mm SL, Samut Sakhon Province, Thailand). Fig. 2. (a) Lateral and (b) dorsal views of dorsal-fin origin of Stolephorus dubiosus, NSMT-P 127425, 55.7 mm SL, Songkhla Lake, Thailand (stained with Alizarine Red). Arrows indicate predorsal scute. Fig. 3. Ventral view of pelvic fin of Stolephorus dubiosus. NSMT-P 127425, 49.9 mm SL, Songkhla Lake, Thailand (stained with Alizarine Red). Triangles and arrow indicate prepelvic scutes and hard spine on pelvic scute, respectively. page 7 of 16 Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan Body milky-white, a silver longitudinal band of width subequal to pupil diameter extending from just behind upper opercular margin to caudal-fin base. Lateral surface of head uniformly silver. Snout yellowish-white, semi-translucent. Melanophores scattered on snout tip. Scale pockets on upper part of body yellowish-black. Dorsal fin whitish, semi-translucent, melanophores scattered along fin rays. Pectoral, pelvic, and anal fins uniformly whitish, transparent, without melanophores. Caudal fin yellow, melanophores scattered along fin rays. Posterior margin of caudal fin black. Iris and pupil silver and black, respectively. Coloration of preserved specimens: Body uniformly pale; silver longitudinal band usually lost. Pairs of distinct dark patches on parietal and occipital regions. Scale pockets on dorsum to upper lateral surface of body margined black. Double pigmented lines dorsally posterior to dorsal fin (Fig. 1c). A few melanophores scattered anteriorly on snout. All fins whitish. Melanophores scattered along fin rays of dorsal and caudal fins. Pectoral, pelvic, and anal fins semitransparent, without melanophores. Caudal fin margined black. Distribution: Stolephorus dubiosus is distributed in the western Pacific Ocean from southern Vietnam (Mekong Delta) to Songkhla, Thailand (based on specimens examined in this study), although molecular evidence and some literature records indicate that it is also distributed in Sumatra, Java, and Borneo (Wongratana 1983; Rupawan 2017; Syafei et al. 2020; Fig. 7). The species mainly inhabits estuarine or brackish waters of large-scale rivers (Whitehead et al. 1988; Wongratana et al. 1999; this study). Morphological comparisons: Stolephorus dubiosus is easily distinguished from all other congeners, except for S. baganensis, Stolephorus tri Bleeker, 1852, and S. taurus sp. nov., in having a spine-like scute located just anterior to the dorsal-fin origin and a small spine on the pelvic scute. Stolephorus bengalensis Dutt and Babu Rao, 1959, Stolephorus diabolus Hata, Lavoué and Motomura, 2022, Stolephorus eclipsis Hata, Lavoué and Motomura, 2022, and Stolephorus eldorado Hata, Lavoué and Motomura, 2022 also have a spine-like scute just anterior to the dorsal-fin origin, but lack a spine on the pelvic scute (Whitehead et al. 1988; Fig. 5. Left side of left hyoid arch of Stolephorus dubiosus (THNHM-F021239, 63.6 mm SL, cleared and stained). hypo lo, lower hypohyal; hypo up, upper hypohyal; chy, ceratohyal; gha, groove for hyoidean artery; eph, epihyal; inh, interhyal (broken); br, branchiostegal rays (seventh branchiostegal ray detached). Fig. 6. Left side of caudal-fin complex of Stolephorus dubiosus (THNHM-F021239, 63.6 mm SL, cleared and stained. Caudal-fin rays removed). preu, preural centrum; hpap, hypurapophysis; urn, uroneural bone; epu, epural; hyu, hypural; parth, parhypural bone. Fig. 4. Stained scale removed from right side of midbody (just below dorsal fin) of Stolephorus dubiosus. NSMT-P 127425, 49.9 mm SL, Songkhla Lake, Thailand (left-right inverted). Grooves on posterior part forming numerous separations. page 8 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan Wongratana et al. 1999; Kimura et al. 2009; Hata and Motomura 2018a–e 2020 2021a b; Hata et al. 2019 2020a b 2021 2022; Gangan et al. 2020; this study). Stolephorus dubiosus differs from S. baganensis and S. tri in having higher counts of lower gill rakers on the first gill arch [26–29 in S. dubiosus vs. 21–24 (rarely 20 or 25) in S. baganensis and 18–22 in S. tri]. Stolephorus dubiosus is also distinguished from S. baganensis in having the pelvic fin posteriorly short of the dorsalfin origin (rarely reaching to vertical through first or second dorsal-fin ray origin in S. dubiosus vs. reaching to vertical through third to fifth dorsal-fin ray origin in S. baganensis). Dorsal pigmentation from the occipital area to the dorsal-fin origin also separates S. dubiosus (no dark lines) and S. tri (usually paired dark lines; fig. 19 in Hata et al. 2019). Comparisons of S. dubiosus with S. taurus are given under Remarks for the latter. Remarks: Stolephorus dubiosus was originally described by Wongratana (1983) from specimens collected from the Pacific coast of Thailand, Kalimantan, and northeastern India. Subsequently, the species has been considered distributed in both the eastern Indian and western Pacific oceans (Whitehead et al. 1988; Wongratana et al. 1999). However, specimens from the Indian Ocean previously identified as S. dubiosus are newly recognised herein as a new species (described below), the distributional range of true S. dubiosus therefore being restricted to the western Pacific Ocean (Fig. 7). Before S. dubiosus was described by Wongratana (1983), the species had been frequently confused with S. baganensis. However, because specimens collected from Indonesia, reported by Hardenberg (1934) as S. b. baganensis, had 25–29 lower gill rakers on the first gill arch, they probably included S. dubiosus. Stolephorus taurus sp. nov. (English name: Bengal Spined Anchovy) (Fig. 8; Tables 2, 3) urn:lsid:zoobank.org:act:457E9BDD-B565-4CCD-80C883F92055D3B7 Anchoviella baganensis baganensis (not of Delsman): Dutt and Babu Rao, 1959: 160 (Kakinada, Andhra Pradesh, India). Stolephorus dubiosus (not of Wongratana): Wongratana 1983: 400 (in part: Orissa, India); Wongratana 1987a: 107 (in part: Bay of Bengal); Wongratana 1987b: 8 (in part: Bay of Bengal); Whitehead et al. 1988: 411 (in part: northern part of Bay of Bengal); Kottelat et al. 1993: 31 (in part: India); Wongratana et al. 1999: 1734 (in part: northern part of Bay of Bengal); Hata et al. 2019: 30 (in part: India); Gangan et al. 2020: 566, fig. 5 (Digha, Kolkata, India); Hata et al. 2020b: table 1 (in part: India). Holotype: OCF-P 10434, 52.2 mm SL, estuary of Hooghly River, West Bengal, India (purchased in a fish market in Kolkata, West Bengal, India), 9 June 1985. Paratypes: 19 specimens, 39.8–57.7 mm SL (all purchased with the holotype): KAUM–I. 157580, 46.5 mm SL, KAUM–I. 157581, 53.2 mm SL, NSMT-P 141123, 49.0 mm SL, NSMT-P 141124, 48.6 mm SL, URM-P 10942, 5 specimens, 44.9–53.2 mm SL, URM-P 10943, 10 specimens, 39.8–46.7 mm SL. Diagnosis: A species of Stolephorus with the following combination of characters: predorsal scute and spine on pelvic scute present; gill rakers on first gill arch 19–22 (modally 21) (upper series), 25–29 (26) (lower series), 45–49 (48) in total; gill rakers on second gill arch 14–16 (15) (upper series), 24–26 (25) (lower series), 39–42 (40) in total; gill rakers on third gill arch 11 or 12 (12) (upper series), 13–16 (14) (lower series), 24–28 (26) in total; gill rakers on fourth gill arch 8–10 (9) (upper series), 11 or 12 (11) (lower series), 19–22 (20) in total; prepelvic scutes 4–6 (5); maxilla long, posteriorly just reaching or slightly short of posterior margin of opercle; small teeth on dorsal surface of hyoid bone; posterior border of preopercle convexly rounded; distinct paired dark patches on parietal and occipital regions; no dark lines on dorsum anterior to dorsalfin origin; double pigmented lines on dorsum from end of dorsal-fin base to caudal-fin base; no melanophores below eye or on tips of snout and mandible (sometimes a few on snout); posterior tip of pelvic fin reaching to third to sixth dorsal-fin ray base when depressed; body scales not deciduous, with relatively a few separations Fig. 7. Distributional records of Stolephorus dubiosus (circles) and S. taurus sp. nov. (triangles). Closed symbols, based on specimens examined in this study; open symbols, based on literature records or molecular evidence. page 9 of 16Zoological Studies 61:58 (2022) © 2022 Academia Sinica, Taiwan apiensis (Jordan and Seale 1906) (Clupeiformes: Engraulidae). Ichthyol Res 66:280–288. doi:10.1007/s10228-018-00675-5. Hata H, Motomura H. 2020. First Northern Hemisphere records of the Samoan Anchovy, Stolephorus apiensis (Actinopterygii: Clupeiformes: Engraulidae). Acta Ichthyol Piscat 50:367–372. doi:10.3750/AIEP/03015. Hata H, Motomura H. 2021a. Two new species of Stolephorus (Teleostei: Clupeiformes: Engraulidae) from the western Pacific. Raffles Bull Zool 69:109–117. doi:10.26107/RBZ-2021-0009. Hata H, Motomura H. 2021b. Stolephorus grandis, a new anchovy (Teleostei: Clupeiformes: Engraulidae) from New Guinea and Australia. Zootaxa 5004(3):481–489. doi:10.11646/zootaxa. 5004.3.5. Hata H, Motomura H. 2021c. A new species of the anchovy genus Stolephorus Lacepède 1803 from North Sumatra, Indonesia, and redescriptions of Stolephorus pacificus Baldwin 1984 and Stolephorus teguhi Kimura, Hori and Shibukawa 2009 (Teleostei: Clupeiformes: Engraulidae). Zool Stud 60:65. doi:10.6620/ ZS.2021.60-65. Hata H, Motomura H. 2022. Redescription of Stolephorus ronquilloi Wongratana, 1983 and description of Stolephorus hindustanensis, new anchovy from the western coast of India (Teleostei: Clupeiformes: Engraulidae). Taxonomy 2:124–135. doi:10.3390/ taxonomy.201001. Jordan DS, Seale A. 1926. Review of the Engraulidae, with descriptions of new and rare species. Bull Mus Comp Zool Harvard 67:355‒418. Jutagate T, Sawusdee A, Chaidee TT, Thongkhoa S, Chotipuntu P. 2009. Fish in the Pak Panang Bay and river in relation to the anti-salt dam operation, part I: assemblage patterns of the marine and brackish water fish. Kasetsart J (Nat Sci) 43:120–131. Kimura K, Hori K, Shibukawa K. 2009. A new anchovy, Stolephorus teguhi (Clupeiformes: Engraulidae) from North Sulawesi, Indonesia. Ichthyol Res 56:292–295. doi:10.1007/s10228-0090103-4. Kottelat M. 2013. The fishes of the inland waters of Southeast Asia: a catalogue and core bibliography of the fishes known to occur in freshwaters, mangroves and estuaries. Raffles Bull Zool Suppl 27:1–663. Kottelat MA, Whitten AJ, Kartikasari SN, Wirjoatmodjo S. 1993. Freshwater fishes of western Indonesia and Sulawesi. Periplus Editions, Jakarta, Indonesia. Lacepède BGE. 1803. Histoire naturelle des poisons. v. 5. Chez Plassan, Paris, France. Lavoué S, Miya M, Nishida M. 2010. Mitochondrial phylogenomics of anchovies (family Engraulidae) and recurrent origins of pronounced miniaturization in the order Clupeiformes. Mol Phylogenet Evol 56:480–485. doi:10.1016/j.ympev.2009.11.022. Lavoué S, Bertrand JAM, Wang H-Y, Chen W-J, Ho H-C, Motomura H, Hata H, Sado T, Miya M. 2017. Molecular systematics of the anchovy genus Encrasicholina in the northwestern Pacific. PLoS ONE 12:e081329–e081345. doi:10.1371/journal.pone.0181329. Maddison WP, Maddison DR. 2019. Mesquite: a modular system for evolutionary analysis. Version 3.61. Available at: http://www. mesquiteproject.org. Matsunuma M. 2013. Stolephorus dubiosus Wongratana, 1983. In: Yoshida T, Motomura H, Musikashinthorn P, Matsuura K (eds) Fishes of northern Gulf of Thailand. National Museum of Nature and Science, Tsukuba, Research Institute for Humanity and Nature, Kyoto, and Kagoshima: Kagoshima University Museum, Kagoshima, p. 33. Nagao Natural Environment Foundation. 2021. Fishes of the Indochinese Mekong. Nagao Natural Environment Foundation, Tokyo, Japan. Nakashima T. 2005. Composition of trash fish caught by push net in Thailand. Mem Fac Fish Kagoshima Univ 54:69–75. Rupawan. 2017. Catch composition of modified gumbang net in Panjang Strait estuary Riau Province. Maspari J 9:131–138. Sabaj MH. 2020. Codes for natural history collections in ichthyology and herpetology. Copeia 108:593–669. doi:10.1643/ ASIHCODONS2020. Stecher G, Tamura K, Kumar S. 2020. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol 37:1237– 1239. doi:10.1093/molbev/msz312. Syafei LS, Siregar RS, Rahardjo MF, Simanjuntak CPH. 2020. Diet composition and trophic niche similarities of engraulid fishes in Pabean Bay, Indramayu, Indonesia. IOP Conf Ser: Earth Environment Sci 404:1–10. doi:10.1088/1755-1315/404/1/ 012056. Tran DD, Shibukawa K, Nguyen PT, Ha HP, Tran LX, Mai HV, Utsugi K. 2013. Fishes of Mekong Delta, Vietnam. Can Tho University Publishing House, Can Tho, Vietnam. van Hasselt JC. 1823. Uittreksel uit een' brief van Dr. J. C. van Hasselt, aan den Heer C. J. Temminck. Algeme Konst en Letterbode Deel 21:329–331. Whitehead PJP, Nelson GJ, Wongratana T. 1988. FAO species catalogue vol 7. Clupeoid fishes of the world (suborder Clupeoidei). An annotated and illustrated catalogue of the herrings, sardines, pilchards, sprats, shads, anchovies and wolfherrings. Pt 2 – Engraulididae. FAO Fish Synop, no 125 7(2):i– viii + 305–579. Wongratana T. 1983. Diagnoses of 24 new species and proposal of a new name for a species of Indo-Pacific clupeoid fishes. Japan J Ichthyol 29:385–407. Wongratana T. 1987a. Four new species of clupeoid fishes (Clupeidae and Engraulidae) from Australian waters. Proc Biol Soc Washington 100:104–111. Wongratana T. 1987b. Two new species of anchovies of the genus Stolephorus (Engraulidae), with a key to species of Engraulis, Encrasicholina, and Stolephorus. Am Mus Novit 2876:1–8. Wongratana T, Munroe TA, Nizinski MS. 1999. Order Clupeiformes. Engraulidae, Anchovies. In: Carpenter KE, Niem VH (eds) FAO species identification guide for fishery purposes. The living marine resources of the western central Pacific. Vol 3. Batoid fishes, chimaeras and bony fishes pt 1 (Elopidae to Linophrynidae). FAO, Rome, pp. 1698–1753. page 16 of 16Zoological Studies 61:58 (2022)