First record of the dotted grouper Epinephelusepistictus (Temminck & Schlegel, 1843) (Perciformes, Serranidae) in Malaysia

Abstract Five specimens of Epinephelusepistictus (Temminck & Schlegel, 1843) were collected from a major landing site located on the west coast of Peninsula Malaysia during a fish faunal survey on 23 August 2017. The present study extends the distribution range of E.epistictus southwards from Andaman Sea to the Strait of Malacca. Species identification was confirmed by colour pattern and DNA barcoding (567 bp of cytochrome C oxidase I) of all E.epistictus specimens and nine closely related Epinephelus species. The interspecies genetic distance ranged from 0.002–0.245. This study also presents, for the first time for Malaysia, data on length-weight relationships and otolith measurements. It contributes to a better understanding of taxonomy, and phylogenetic and genetic diversity of E.epistictus.


Introduction
Groupers (subfamily Epinephelinae of the family Serranidae) are a commercially valuable taxa globally and in Malaysia, in particular (Craig et al. 2011;Bray 2018;Froese and Pauly 2018). To date, a total of approximately 65 species in 15 genera have been reported in Malaysia (Department of Fisheries Malaysia 2009; Chong et al. 2010;Matsunuma et al. 2011;Ambak et al. 2012;Yusri et al. 2015).
The dotted grouper Epinephelus epistictus (Temminck & Schlegel, 1843) is in the Least Concern (LC) category (Leung and Sadovy 2018). It is a demersal species, which inhabits the continental shelf over soft or rocky bottom at a considerable depth (71-800 m); however little else is known about this species (Paxton et al. 1989;Heemstra and Randall 1993;Goldshmidt et al. 1996;Sommer et al. 1996;To and Pollard 2008). This is a widely distributed species in the Indo-West Pacific, occurring off South Africa, in the Red Sea (Randall and Adam 1983), off Iraq (Mustafa et al. 2011), Oman (Krupp et al. 2000), on the west coast of India, Korea, Japan, in the South China Sea (Randall and Lim 2000), off Taiwan, Hong Kong, Indonesia (Limmon et al. 2017;Mous and Pet 2018), Papua New Guinea (Froese and Pauly 2018), and northern Australia (Bray 2018;Dianne 2019). It can be identified by a pale brown body with irregular rows of small dark spots on the back and sides of the body. Some specimens have a broad dark band from the eye to the gill cover, and two narrower bands running diagonally across the cheek (Bray 2018).
Epinephelus epistictus is a medium-sized grouper, with a maximum of 80 cm total length, and may be misidentified as Epinephelus magniscuttis Postel, Fourmanoir & Guézé, 1963or Epinephelus heniochus Fowler, 1904(To and Pollard 2008Froese and Pauly 2018;Leung and Sadovy 2018). E. epistictus and E. heniochus differ from E. magniscuttis by having fewer and smaller dark spots on head and body and dark spots arranged in three longitudinal rows on body of juveniles (Heemstra and Randall 1993). However, E. heniochus and E. epistictus share the following characters: distinctly enlarged serrae at the corner of the preopercle, 14 or 15 dorsal-fin rays, interspinous dorsal-fin membranes distinctly incised, midlateral part of lower jaw with two rows of teeth, similar morphometric features and colour pattern (Heemstra and Randall 1993). These similarities might have contributed to E. epistictus misidentifications in the past. Though a number of barcoding (CO1) studies of groupers from Malaysia have been conducted (Chu et al. 2011;Nurnadia et al. 2016;Rahim et al. 2016), the species occurrence remained undetected. A recent ichthyofaunal survey found specimens of E. epistictus in a commonly surveyed major landing site on the west coast of Peninsula Malaysia and the present study reports on size and genetic data that confirm its identification as well as some aspects of its biology and phylogeny.

Materials and methods
Five specimens of E. epistictus were collected from a major fish landing site in Hutan Melintang, northeastern Peninsula Malaysia during a fish faunal survey on 23 August 2017 (Fig. 1). These specimens were caught using trawl nets operating in the Straits of Malacca. The tissue samples were preserved in 95% ethanol solution and deposited in the Institute of Ocean and Earth Sciences (IOES), University of Malaya (UM), Kuala Lumpur. Preliminary species identification was made based on the morphology of the whole fish specimens using the species identification keys and diagnostic features reported in Heemstra and Randall (1993). Morphological measurements taken included total length (TL, mm), standard length (SL, mm), and total weight (Wt, g). Otoliths were extracted and various measurements were made, namely the sagitta otolith length (O L , mm), the longest distance between the most anterior and posterior points, otolith width (O w , mm), the longest distance between the ventral and dorsal edges, and the weight of the sagittal (O wt , g). Otolith aspect ratio (O AS ) was calculated by dividing O L by O W for the left otolith (Table 1).
Molecular DNA sequencing was used to confirm the species identification. Total DNA extractions were performed on the collected tissue samples using the G-spin TM Total DNA Extraction Kit (iNtRON Biotechnology, Inc., Korea) following the manufacturer's instructions. The primers, including combinations of the forward (FishF1 or FishF2) and the reverse (FishR1 or FishR2) primer pairs, followed Ward et al. (2005). A 20 μl PCR reaction mixture was prepared in a 1.5 ml tube containing 13.25 μl double distilled water (ddH 2 O), 2 μl 10x i-Taq plus PCR buffer, 1 μl of deoxynucleotide triphosphate (dNTP), 1 μl of each primer used, 0.25 μl i-Taq plus DNA polymerase and 1.5 μl of total genomic DNA. The Eppendorf thermal cycler was used to run the following thermal cycle profile: initial denaturation at 94 °C for 5 minutes; 35-40 cycles of denaturation at 94 °C for 30s, annealing at 44-50 °C for 30s, extension at 72 °C for 1 minute; followed by a final extension at 72 °C for 5 minutes. The PCR products were stained with loading dye, and loaded on to the wells of 1.0% agarose gel before conducting gel electrophoresis. Successfully amplified PCR products were sent to 1 st BASE Laboratories (Malaysia) with the same primers used for PCR reactions for sequencing.
For systematic relationships with congeners, the raw sequences were first assembled and edited via ChromasPro ver 1.42 (Technelysium Pty Ltd), subsequently aligned using Clustal X v. 2.0.8 (Larkin et al. 2007) and then manually adjusted with Bioedit v. 7.0.9.0 (Hall 1999). The COI gene of the five E. epistictus specimens, and nine   Table 2). The maximum likelihood (ML) tree was reconstructed based on the best evolutionary model, namely the General Time Reversible model (GTR) with the Gamma distributed (G) distance and invariable sites (I), which was selected using the lowest bias-corrected Akaike Information Criterion (AICc) value in model test, with 1000 replications for bootstrap analysis. Both tree construction and model test were completed in MEGA (Molecular Evolution Genetic Analysis) version 7.0 (Kumar et al. 2016). Similarly, a neighbor joining (NJ) tree was constructed based on the pairwise genetic distance using the Kimura 2-parameter (K2P) model with 1000 bootstrap resampling. Genetic distances of the sequences were calculated with the K2P model (Kimura 1980) using MEGA 7.0.

Results
The colour pattern of the Malaysian specimens identified as E. epistictus was similar to the species' description in Bray (2018) (Fig. 2). The five specimens were 122-154 mm SL (145-180 mm TL) and 36.44-70.75 g total weight. Estimates of the length-weight parameters were -2.0542 for log a (95% CI = -2.1378, -2.0353). The length-weight relationship based on total length and total weight showed positive allometric growth (b   = 3.1245) and the coefficient of determination (r 2 ) of the regression was 0.9725. Otolith measurements were presented using mean (standard deviation): O L 7.98 (0.46) mm; O w 3.82 (0.11) mm; and O W t 0.0257 (0.0029) g (Table 1). The otolith aspect ratio (O AS ) averaged 2.09 (0.12) for the left otolith (Fig. 3).
The 19 grouper specimens were classified as ten species based on external morphology, which was consistent with the species names in both the GenBank BLAST and BOLD-IDS. DNA sequences from the above said ten species were submitted to Gen-Bank (PubMed) and their accession number were given in Table 2. There was a total of 181/567 bp of variable sites and 174/567 bp of parsimony-informative sites after aligning the sequences. The average base composition obtained was 25.0% A bases, 27.7% of C bases, 16.4% of G bases and 30.9% of T bases.
Both ML and NJ trees showed two major groups: one group containing of E. epistictus, E. heniochus and the other comprising other Epinephelus species. All species are monophyletic with bootstrap values range of 63-100%. All five E. epistictus samples were clustered together with the E. epistictus reference sequences from GenBank (KM226255, KU499627) with high bootstrap values (63-99% in ML tree, 62-100% in NJ tree) (Fig. 4).
Genetic distances between grouper species based on the 567 bp COI consensus sequences and the respective reference sequences with the K2P model were given in Table 3. The intraspecific nucleotide distances for E. epistictus were low, ranging from 0.000-0.005. The interspecific differences between other grouper species of the genus Epinephelus ranged from 0.002-0.245. The largest difference was found between E. heniochus and E. sexfasciatus (0.245).

Discussion
Our study confirmed a new record of E. epistictus in the waters of Malaysia (in the Strait of Malacca). Although the strait is one of the busiest shipping channels in the world, our discovery of this commercially important grouper species suggests that much work remains to be done with documenting the local fish diversity. While the strait itself is considerably shallow in the south (close to Singapore) with average minimum depth of 25 m, the northern part of the Strait connecting to the Andaman Sea is up to 200 m deep; this depth profile is consistent with the depth range at which the species reportedly occurs. An undergraduate thesis reported the use of this species at an aquaculture farm in the Sabahan Borneo (Chen 2015), however, examination of the photos included in the thesis revealed that the species was erroneously identified as E. epistictus. A morphometric comparison and characters distinguishing E. epistictus from E. bleekeri, E. heniochus and E. latifasciatus were compared with existing literature (Sommer et al. 1996;Krupp et al. 2000;Froese and Pauly 2018) in Appendix 1.
Our study demonstrated evidence to support fine-scale monophyly for the subset of Epinephelus species examined in this region based on COI sequence data. Despite    comparison of specimens from various distant locations, namely India, Saudi Arabia and Malaysia, the intraspecific nucleotide distances of E. epistictus was relatively low (0.000-0.005). The interspecific differences between ten grouper species examined ranged from 0.002 to 0.245, and 0.213 from the closest outgroup Anyperodon leucogrammicus. This species was included by some Hastings 2007, Ma andCraig 2018) in the genus Epinephelus but others have kept it in the monotypic genus (Rhodes 2018) until further phylogenetic study is done. A recent study placed groupers in the family Epinephelidae sensu Smith and Craig (2007) and supports the idea of assigning both E. epistictus and E. heniochus to Mycteroperca (Ma and Craig 2018). At the species level, the status of E. epistictus has not been questioned.
The findings of this study contribute to better understanding on the taxonomy, biology, phylogenetic and genetic diversity of E. epistictus, which is important for sustainable management of the species in Malaysia.