1 Introduction

Sillaginids are small to moderate sized Indo-West Pacific fishes, commonly inhabiting in inshore waters of sandy substrates or estuarine areas of rivers (McKay, 1992). The family consists of thirty-six species (Fricke et al., 2020), including five new described species during last ten years: S. caudicula Kaga, Imamura & Nakaya, 2010; S. sinica Gao & Xue, 2011; S. suezensis Golani, Fricke & Tikochinski, 2014; S. shaoi Gao & Xiao, 2016; and S. panhwari Panhwar, 2017 (Kaga et al., 2010; Gao et al., 2011; Golani et al., 2014; Xiao et al., 2016; Panhwar et al., 2018). Furthermore, another unnamed species, Sillago sp., was recorded from the southern coast of China (Xiao, 2018).

Sillaginids are easily identified as a family by their similarity of external morphological characters (Sano and Mochizuki, 1984; McKay, 1992), but hard in specific identification. The external morphological similarity has made many true species be concealed in the synonymy of those wide-ranging species. For instance, separate identities of the three trumpeter sillaginids, Sillago maculata, S. aeolus, and S. burrus, were only recognized in 1985 and are elevated to full species by McKay in 1992 (McKay, 1985, 1992). In addition, Sillago sihama, an Indo-West Pacific species, exhibits many cryptic lineages across its distribution range (McKay, 1992). The morphology of the swim bladder has been proved to be effective in distinguishing Sillago species (McKay, 1985, 1992; Xiao, 2018). According to this character, McKay (1985) recognized three subgenera of Sillago: Sillaginopodys Fowler 1933 (swim bladder reduced, no duct-like process); Sillago Cuvier 1817 (swim bladder divided posteriorly into two tapering extensions, duct-like process present); and Parasillago (McKay, 1985) (swim bladder with a single posterior extension and the duct-like process). However, it may be still difficult to distinguish close-related species in this groups based on morphology only. In the past decades, the utility of DNA barcoding on species delimitation and identification has been demonstrated by numerous studies (Hebert et al., 2003; Ward et al., 2005; Burns et al., 2008; Locke et al., 2010). And DNA barcoding is particularly useful in recovering possible new species when morphological differentiation within groups is imperceptible (Gao et al., 2011; Golani et al., 2014; Xiao et al., 2016).

In the present study, we delimited a new Sillago species from two locations of southern coast of China through morphological and genetic comparisons. A phylogenetic tree was reconstructed to help distinguish species. We also provided a distribution map and a key of Sillago species with two posterior extensions on the swim bladder.

2 Materials and Methods 2.1 Materials Examined

Specimens of the new species were collected from Zhanjiang and Beihai, China. Institutional abbreviations follow Fricke and Eschmeyer (2020) except for Fishery Ecology & Marine Biodiversity Laboratory, Fishery College, Zhejiang Ocean University (FEMBL).

Comparative materials (see Table 1 for number of specimens). Sillago caudicula: BPBM 36393, holotype, 148.6 mm SL, Salalah fish market, Oman, photograph. Sillago indica: NMMB-P13995, 174.2 mm SL, Phant Thiet, Vietnam, photograph; USNM FIN34286, 151 mm SL, Salalah, Oman, February, 1993, photograph. Sillago intermedius: BMNH 1976.11.17.1–2, 2, paratypes, 81.0 – 101.0 mm SL, photographs. Sillago panhwari: FAMB-01-1611, holotype, 160 mm TL, northern Arabian Sea coast of Pakistan, photograph. Sillago shaoi: FEMBL 150001, holotype, 182.2 mm SL, Xiamen, China, December 2013; FEMBL150002 – 150016: 15, paratypes, 173.0 – 203.3 mm SL, Xiamen, China, December 2013; FEMBL 150017 – 150019: 3, paratypes, 146.2 – 167.6 mm SL, Changhua County, Taiwan, China, July 2014; FEMBL 150020 – 150039: 20, paratypes, 182.0 – 216.0 mm SL, Dongshan, China, April 2014. Sillago sihama: FEMBL 150101 – 150108: 8, Zhuhai, November 2014; FEMBL 021573 – 021583: 11, Bahrain, October 2014; FEMBL 021220 – 021224: 5, Karachi, January to February 2016; FEMBL020918 – 020927: 10, Phuket Island, March 2015. Sillago sinica: OUC_FEL100348, holotype, 122.79 mm SL, Wenzhou, China, June 2009; OUC_FEL174001 – 174030: 30, 101.8 – 162.5 mm SL, Rushan, Shandong, China, August, 2014; OUC_FEL100362 – 100388, 100403: 28, 94.36 – 134.17 mm SL, near Dongying, Shandong, China, September, 2009. Sillago suezensis: HUJF 7134, holotype, 137 mm SL, Abu Zanima, Egypt, Gulf of Suez, Red Sea, photograph. Sillago sp.: FEMBL 021255 – 021283: 29, 127.8 –155.6 mm SL, Xiamen, China, December, 2015; FEMB L021284, 167.6 mm SL, Zhanghua, Taiwan, China, July 2014.

2.2 Morphological Analysis

The genus and species classification followed McKay (1985) unless otherwise noted. The terminology of appendages of the swim bladder followed Shao et al. (1986) and Kaga and Ho (2012). In the descriptive section, holotype data were given first, followed by paratypes data in parentheses. Specimen length was standard length, SL. Head length was abbreviated as HL. Measurements were made with dial calipers and the dividers were accurate to 0.1 mm and weights to the nearest 0.1 g. The definition of the modified vertebrae followed McKay (1992). The swim bladder anatomy observations were performed after dissection of 20 paratypes. Specimens of the new species were preserved in alcohol.

After measurements and counts, both sagittaes were taken from each specimen. Each was placed in a 1.5-mL plastic tube containing distilled water and soaked for 1 h. Followed by ultrasonic cleaning for about 30 min, the otoliths were baked in an oven for 24 h at 55℃ until a constant mass to the nearest 0.01 mg was achieved. The otolith's digital images were taken with a Nikon SMZ800 microscope equipped with a Nikon digital sight DS-Fi1 (Tokyo, Japan).

2.3 Genetic Analysis

To analyze genetic differences between new species and other Sillago species, mitochondrial (mt) DNA cytochrome oxidase subunit I (COI) fragments were amplified based on the method of Ward et al. (2005). Genomic DNA extraction and polymerase chain reaction (PCR) followed Gao et al. (2011). The COI fragment amplification primer used in PCR reaction system is L5956-COI (5'-CACAAA GACATTGGCACCCT-3') and H6558-COI (5'-CCTCCT GCAGGGTCAAAGAA-3') (Inoue et al., 2001).

COI sequences produced in the present study were submitted to GenBank with the following accession numbers (see Table 1): S. parasihama (MH 093482 – MH093490), S. panhwari (MF571945, MF571947, KU051787 and KU 051788), and Sillago sp. (MG571458, MG571455, MG 911029, MG911030, KU 051812). The COI sequences of other species were downloaded from GenBank with the following accession numbers (see Table 1): S. indica (KM 350229 – KM350232), S. shaoi (KU051872, KU051 873, KU051879, KU051886 and KU05 1887), S. sihama (KU 051813, KU051819, KU051857, KU051803 and KU051 881), S. sinica (KU052012, KU052017, KU052023, KU 052025 and KU05 2029), S. suezensis (FJ155362 – FJ155 364), and S. parvisquamis (HQ389247 – HQ389249). In total, the presently available nine Sillago species with two posterior extensions of the swim bladder were used for genetic comparison. Sequences were then checked and aligned using the DNASTAR software (DNASTAR Inc., Madison, WI. USA). MEGA 5.0 (Tamura et al., 2011) was used to reckon the genetic distances and reconstruct a Neighbor-joining (NJ) tree under the Kimura 2-parameter (K2P) model using Sillaginodes punctata as the outgroup.

3 Results 3.1 Taxonomy

Family Sillaginidae Richardson, 1846

Genus Sillago Cuvier, 1817

Sillago (Sillago) parasihama sp. n. Gao, Xiao & Guo (Fig.1)

Holotype FEMBL150036, 132.5 mm SL, Zhanjiang, Guangdong Province, China, collected by Jiaguang Xiao, January 2016.

Paratypes FEMBL150015 – 150019, FEMBL150031 – 150035, FEMBL150037 – 150050, FEMBL150109 – 150123, FEMBL150131 – 150178, 87 individuals, 95.0 – 166.0 mm SL, collection data in consonance with holotype; FEMBL 2705 – 2736, 32 juvenile individuals, Zhanjiang, Guangdong Province, collected by Xiongbo He, December 2015; FEMBL150124 – 150130, 7 individuals, 85.0 – 138.0 mm SL, an aquaculture base in Yinhai District (21˚26´ 27.52´´N, 109˚17´7.72´´E), Beihai, Guangxi Province, collected by Ting Guo, September 2017.

Etymology The name 'parasihama' indicate the close resemblance to Sillago sihama in external morphology.

Diagnosis Dorsal fin rays XI – XII (mostly XI), I + (18 – 21) (mostly 20); anal fin rays II + (19 – 21) (mostly 20); lateral line scales 65 – 70, scales above lateral line 4 – 5; gill rakers (2 – 3) + (5 – 7); vertebra: abdominal 14, modified 4 – 7, caudal 13 – 16, and total 34 (Table 2). Body without dark blotches or mid-lateral stripe; dorsal fins without several rows of dark spots on rays; anal fin without tiny dark spots. The anterolateral extensions of swim bladder are unique with some dendritic or sometimes stunted blind tubule, unilateral and outward, anterolateral extensions extending along the abdominal and about one-third to half of the body of swim bladder in length.

Description General body features are shown in Fig.1. Counts and measurements are given in Table 2. The description is based on holotype, data of paratypes are shown in parentheses.

Body elongate, abdomen wide, body shape curved, anterior slightly pyramidal, posterior cylindrical; anterodorsal profile smooth. Head large, its length 25.9% (18.4% – 29.0%) in SL. Snout long, its length 43.3% (38.9% – 48.0%) of HL. Eye moderate, its margin slightly covered with the adipose eyelid, diameter 20.0% (14.0% – 37.1%) of HL. Interorbital region flat, width 6.2% (3.5% – 11.1%) of HL. The anterior ocular of eye has two nostrils with a one-way anterior flap on posterior border of anterior nostril and a lack of flaps on posterior nostril. Anterior nostril slightly larger than posterior nostril. Mouth small, terminal, tips of jaws almost on samevertical. Jaws with 2 – 3 rows of small canines forming a wide tooth band gradually becoming narrower posteriorly, ending in one row. Palatine and tongue toothless. Vomer with 3 – 4 rows of canine teeth. Posterior margin of preopercle weakly serrated. Gill aperture large, lateral, extending to the pelvic side of the head, stopping at the middle bottom of opercle. Gill rakers on first arch pointed, short, and sparse. Caudal peduncle short, its depth 77.2% (52.7% – 95.7%) of its length.

Scales deciduous, size moderate, ctenoid but cycloid on prenasal area, cheek and the lower part of pre-opercularmandibular canal. Pectoral fin base and pelvic fin base lacking scales. The lateral line begins above gill aperture and anterior portion of the pectoral fin, extending along the curve of dorsal edge to the end of body.

Dorsal-fin rays XI (XI – XII), I+20 (18 – 21), with blurry black spots on membrane. Anal-fin rays II, 19 (19 – 21), not extending to caudal fin origin when placed flat. Pectoral-fin rays 17 (16 – 17), slender. Pelvic fins broad, I, 5, roughly triangular, and shorter than pectoral fin. Caudalfin rays 17 (16–18), tail fork shallow. Comparisons of meristic characters of twelve Sillago species with two posterior extensions of the swim bladder are shown in Table 3.

Color of fresh specimens Head surface bright-brown and trunk light yellowish-brown, grading to silver on abdomen. Dorsal side of snout brownish-gray. Cheek slightly silver, little black dots massed on the anterior inferior part of eyes, the color dark-brown. Body without stripes. The anterior half of ventral side sometimes with some dark pigments. Dorsal fins yellowish-hyaline, with small dark spots on fin membrane. Pectoral, pelvic, and anal fins yellowish-hyaline without dark spots. Caudal fin yellowish dusky with grayish-brown margin posteriorly.

Swim bladder Swim bladder large, covered by a thick layer of fat; with two anterior extensions extending to the anterior part connected with the occipital bone, two dumpy posterior extensions penetrating into caudal region with a large gap between them, one usually longer than the other. Two anterolateral extensions originate from anteriorly, each branch into anterior and posterior sub-extensions: the anterior one comprising a short, simple blind tubule and sometimes absent. The posterior sub-extensions are unique with some dendritic or sometimes stunted blind tubule, unilateral and outward, extending along the abdominal and about one-third to half of the body of swim bladder in length. A single duct-like process originating from the pelvic surface of swim bladder extending to the urogenital opening (Figs. 2a, 3a).

Otolith Otoliths shape fusiform; most of the edge is smooth, with only slight protuberances at the posterior end. There is a main groove throughout the otolith, with an opening at both ends. The surface of the no-groove side is bright, with radial stripes and tumor-like rimes in the middle. There are distinct protuberances on the pelvic edge. There is no significant difference between the left and right otoliths (Fig.4).

Distribution and habitat Sillago parasihama is presently known only from the southern coast of China (Beihai and Zhanjiang). It may distributes along the beaches, sandbars, and the mangrove creeks of sandy substrate. It is generally mixed cultured with other fish or shrimp in openair ponds (usually 1.6 m in deep). The species prefer to dive into the soft sand bottom of ponds.

Comparison According to the subgenera-grading system in genus Sillago proposed by McKay (1985), S. parasihama belongs to subgenus Sillago (Sillago). We confirmed the validity of S. parasihama by comparing it with all species in this group. Among the eleven Sillago species with two posterior extensions, S. parasihama can be easily distinguished from S. intermedius and S. caudicula by the body coloration (dusky black blotches are present on the body of S. intermedius and S. caudicula); from S. parvisquamis and S. sinica by the dusky spots on the second dorsal fin membranes (five or six rows in S. parvisquamis and three or four rows in S. sinica). We could also distinguish S. parasihama from S. indica, S. shaoi, and Sillago sp. by the coloration of anal fin (the anal fin of S. parasihama is hyaline without black spots, but the anal fins of S. indica, S. shaoi and Sillago sp. yellowish, interradial membranes with black dots). Moreover, we could also distinguish S. parasihama from S. indica, S. suezensis, S. panhwari and Sillago sp. by the faint midlateral stripe on the body of them.

Moreover, we could also distinguish most of these species based on the meristic and morphometric characters in Table 3. S. parasihama is easily distinguishable from others by the following characteristics: S. megacephalus by having a smaller head (18.4% – 29.0% SL in S. parasihama vs. 33.0% in S. megacephalus) and fewer soft rays in anal fin (19 – 21 in S. parasihama vs. 23 in S. megacephalus); from S. intermedius by having fewer scales above lateral line (4 – 5 in S. parasihama vs. 6 – 7 in S. intermedius) and a smaller head (18.4% – 29.0% SL in S. parasihama vs. 30.0% – 31.0% in intermedius); from S. shaoi, S. parvisquamis, S. caudicula and S. sinica by having 34 total vertebrae (35 in S. shaoi, 39 – 40 in S. parvisquamis, 35 – 36 in S. caudicula and 37 – 39 in S. sinica); from S. parvisquamis and S. sinica by having 65 – 70 scales on lateral line (79 – 84 in S. parvisquamis and 75 – 79 in S. sinica), from S. parvisquamis by having fewer scales above/below lateral line (4 – 5/8 – 10 in S. parasihama vs. 7/(11 – 12) in S. parvisquamis); from S. sihama by having fewer gill rakers (2 – 3/5 – 7 in S. parasihama vs. 3/8 – 9 in S. sihama); from S. caudicula by having fewer soft rays in dorsal fin (18 – 21 in S. parasihama vs. 22 – 23 in S. caudicula) and soft rays in anal fin (19 – 21 in S. parasihama vs. 23 – 24 in S. caudicula); from S. caudicula and S. suezensis by having fewer gill rakers (2 – 3/5 – 7 in S. parasihama vs. 4/11 in S. caudicula and 3 – 4/8 – 10 in S. suezensis).

S. parasihama is most similar with S. sihama in the countable characters and color pattern. However, their swim bladders are obviously different (Figs.2, 3). The anterolateral extensions of swim bladder of S. parasihama are unique with some dendritic or sometimes stunted blind tubule, unilateral and outward, anterolateral extensions extending along the abdominal and about one-third to half of the body of swim bladder in length; but the anterolateral extensions of swim bladder of S. sihama are kinky, long and complicated, and then extending along the abdominal wall below the peritoneum to the base of posterior extensions. By contrast, S. parasihama is more similar to S. sinica on swim bladder whose anterolateral extensions of swim bladder also with some stunted blind tubule. But this structure is usually more complicated in S. parasihama, and the posterior sub-extensions of swim bladder of S. parasihama extending along the abdominal and about onethird to half of the body of swim bladder in length.

Genetic analysis of the COI gene Forty-three sequences of 9 Sillago species were used in the genetic analysis. The genetic distances among Sillago species ranged from 0.084 to 0.231, and the intraspecific distances ranged from 0.000 to 0.004 (Table 4). The NJ tree based on COI gene sequences (Fig.5) revealed that all the 9 species formed exclusive lineages.

4 Discussion

The new species would be identified as S. sihama according to the countable characters and color pattern (McKay, 1992). However, S. parasihama is clearly different from S. sihama in the shape of the swim bladder (see Comparison part). The swim bladder of S. parasihama is similar to S. sinica. But S. parasihama is more complex, and can be distinguished from S. sinica by the differences on counts of lateral line scales and vertebrae, as well as the color of the second dorsal fin. In addition, S. parasihama is allopatric with S. sinica which occurs from the Bohai Sea to the Taiwan Strait (Gao et al., 2011). S. megacephalus was described based on holotype only. Unfortunately, the holotype may have been lost (McKay, 1985), and its swimbladder structure and vertebral count are unknown. However, S. megacephalus still can be clearly distinguished from S. parasihama by obviously larger head (Table 2). S. parvisquamis is another species we did not examine any specimens. But this species is different with S. parasihama in color pattern and scale counts above/below lateral line (see Comparison part), and in genetics.

The genetic distances between S. parasihama and other Sillago species ranged from 0.159 to 0.231, much larger than intraspecific genetic distances, strongly supporting the validity of the new species. The phylogenetic analysis recovered nine exclusive lineages representing nine valid Sillago species. S. parasihama was grouped with the clade including S. shaoi, S. sinica, and S. parvisquamis. Among genus Sillago, S. sihama exhibits high genetic diversity in Indo-West Pacific. A number of recently described Sillago species across Indo-West Pacific – S. caudicula (from Oman and Madagascar), S. sinica (from China), S. suezensis (from the northern Red Sea and Mediterranean), S. shaoi (from Taiwan Strait) and S. panhwari (from the northern Arabian Sea) – have been ever misidentified as S. sihama (Kaga et al., 2010; Gao et al., 2011; Kaga and Heemstra, 2013; Golani et al., 2014; Xiao et al., 2016; Panhwar et al., 2018). S. parasihama is a new one isolated from S. sihama. Thus, a thorough taxonomic revision of S. sihama species from Indo-West Pacific is necessary.

A synopsis of Sillaginids from China is presented based on this study and previous records (McKay, 1985, 1992; Gao et al., 2011; Xiao et al., 2016). Presently, a total of 37 valid species of Sillaginidae are recognized. Fourteen of them have been recorded from China, including S. aeolus, S. ingenuua, S. sihama, S. chondropus, S. parvisquamis, S. sinica, S. microps, S. japonica, S. boutani, S. asiatica, S. megacephalus, S. shaoi, Sillago sp. and S. parasihama (McKay, 1985, 1992; Gao et al., 2011; Xiao et al., 2016; Xiao, 2018). Among them, S. aeolus, S. ingenuua, S. sihama, and Sillago sp. are identified from the Taiwan Strait to the South China Sea; S. japonica is widely distributed from the south of Bohai Sea to the South China Sea; S. sinica distributes from the Bohai Sea to the Taiwan Strait; S. shaoi is currently sampled from the Taiwan Strait; S. asiatica and S. parasihama are found from the northern coast of China; S. chondropus, S. parvisquamis, S. microps have only been recorded from Taiwan; S. megacephalus and S. boutani have been described in Hainan and the Beibu Gulf, respectively (Xiao, 2018). We also provided a distribution map (Fig.6) and a key characteristic of the 11 Sillago species with two posterior extensions on swim bladder, which should be particularly helpful for species identification and further taxonomic study of Sillaginids.

Acknowledgements

We are very grateful to Mr. Jianwei Zou for help in sample collection. This work is supported by the National Key Research and Development Program of China (No. 2019 YFD0901301), and the National Natural Science Foundation of China (Nos. 41976083, 41776171).