Advancing DNA Barcoding to Elucidate Elasmobranch Biodiversity in Malaysian Waters

Simple Summary

One-third of shark and ray species are threatened due to overfishing, but a lack of information on each species makes conservation decisions difficult. To address this issue, we conducted a study to identify the different species of sharks and rays in Malaysian waters using DNA barcoding of the CO1 gene, which is akin to DNA fingerprinting for species. We collected 175 individuals between June 2015 and June 2022, randomly selecting up to six specimens from each species. We successfully generated DNA barcodes for 67 species, belonging to 44 genera, 20 families, and 11 orders. Accurate species identification will improve species-specific catch landing data and accelerate the identification of use and illegal trade in Malaysia.

Abstract

The data provided in this article are partial fragments of the Cytochrome c oxidase subunit 1 mitochondrial gene (CO1) sequences of 175 tissues sampled from sharks and batoids collected from Malaysian waters, from June 2015 to June 2022. The barcoding was done randomly for six specimens from each species, so as to authenticate the code. We generated barcodes for 67 different species in 20 families and 11 orders. DNA was extracted from the tissue samples following the Chelex protocols and amplified by polymerase chain reaction (PCR) using the barcoding universal primers FishF2 and FishR2. A total of 654 base pairs (bp) of barcode CO1 gene from 175 samples were sequenced and analysed. The genetic sequences were blasted into the NCBI GenBank and Barcode of Life Data System (BOLD). A review of the blast search confirmed that there were 68 valid species of sharks and batoids that occurred in Malaysian waters. We provided the data of the COI gene mid-point rooting phylogenetic relation trees and analysed the genetic distances among infra-class and order, intra-species, inter-specific, inter-genus, inter-familiar, and inter-order. We confirmed the addition of Squalus edmundsi, Carcharhinus amboinensis, Alopias superciliosus, and Myliobatis hamlyni as new records for Malaysia. The establishment of a comprehensive CO1 database for sharks and batoids will help facilitate the rapid monitoring and assessment of elasmobranch fisheries using environmental DNA methods.

1. Introduction

Elasmobranchs, a subclass of Chondrichthyans that include sharks and batoids (rays, skates, guitarfish, and sawfish), are widely distributed in the Southeast Asian (SEA) region and encompass more than 300 species from freshwater environments to deep seas [1]. Within the SEA region, Malaysia is one of the top countries with rich biodiversity of elasmobranch species. To date, there are at least 70 species (19 families) of sharks and 91 species (11 families) of batoids that have been reported to occur in Malaysian waters (Supplementary Materials Table S1) [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. Although new species are continuously discovered, the populations of sharks and batoids have gradually decreased over the past decades [18]. The reduction in the population sizes of sharks and batoids is mainly due to high bycatch rates within commercial fisheries and recreational fishing activities [19]. Additionally, due to their K-selected life histories, some elasmobranch species are threatened with extinction [20,21]. Malaysia is the world’s ninth largest producer of shark products, especially shark fins, and the second largest importer in terms of volume, as reported by the Food and Agriculture Organisation (FAO) of the United Nations [22]. It is also one of the major countries globally with high annual elasmobranch landings [8]. These sharks and batoids are generally landed whole and are mostly fully utilized. According to the reported annual fish landings in Malaysia (http://www.dof.gov.my, accessed on 5 January 2023), the biomass of sharks and batoids reduced around 15-fold (6487 tonnes to 438 tonnes) and 9-fold (11,993 tonnes to 1372 tonnes), respectively, from 2018 to 2021 [18,23,24]. These data suggested the depletion of shark and batoid populations in Malaysian waters, parallel to the findings on global shark and batoid landings [20,25]. However, as explained by the Department of Fisheries Malaysia (DOFM) [26], sharks are not specifically targeted by fishers in Malaysia, but they are caught as bycatch along with other commercially important species by various fishing gears.

Globally, the estimated species numbers of elasmobranchs threatened with extinction have increased from approximately one-quarter [20] to more than one-third [27] due to targeted and incidental overfishing. This can also be explained by their conservative life history traits, including slow growth, long lifespan, late maturity, and low fecundity, that render elasmobranch populations difficult to recover from anthropogenic pressures [20,21,28,29]. The decrease in elasmobranch populations demonstrated the urgent need for conservation and management plans [20,21,25,30,31]. Due to the lack of species-specific information and variability of population sizes among species, the current landing records fail to provide details as to which species require protection the most. Accurate species identification is one of the most important elements to take into consideration in order to carry out suitable conservation and management programmes. The field identification of several closely related sharks (including carcharhinid, sphyrnid, and triakid sharks) and batoids (Myliobatiformes and skates) is often challenging, which might result in inaccurate species compositions and diversity in catch reports [32,33]. Moreover, only a few detailed studies have been conducted on the taxonomy and diversity of elasmobranchs in Malaysia [9,34,35]. The lack of data in this field is mainly due to large specimen sizes, ethical reasons, lack of experienced taxonomists, and high field survey costs, which, in turn, render accurate identification more challenging [36]. Thus, there is the potential that many undiscovered species remain unknown. A simple and accurate species identification tool is therefore crucial to allow the species-specific development of fishery conservation and management plans, especially those pertaining to the mislabelling and misidentification of shark and batoid products [12].

Over the past two decades, DNA barcoding has been introduced for its efficiency and accuracy in the identification of challenging species from different taxa [37,38]. The idea of this approach is conceptually straightforward—it uses a short DNA fragment of a specific gene of an approximately 650 bp region to be compared against reference sequences for accurate animal species identification [38]. The Cytochrome c oxidase subunit 1 mitochondrial gene (CO1) is suggested as a highly suitable marker for DNA barcoding, as it can discriminate between closely related species across diverse animal phyla [36], including sharks and rays [39,40]. To date, DNA barcoding has been successful in identifying elasmobranch species from dried fins, tissues, and carcasses [41,42,43,44,45,46,47]. This is highly beneficial for non-taxonomists to identify species with reasonable confidence even in the absence of whole specimens. Species can be identified by comparing their DNA barcode sequences against an online repository of barcode references, such as the NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank, accessed on 5 January 2023) and Barcode of Life Data System (BOLD) (http://www.boldsystems.org, accessed on 5 January 2023) [38,48,49]. However, DNA barcoding has limitations, such as the possibility of errors due to poor DNA quality or contamination, the potential for hybridisation or incomplete lineage sorting, and the lack of universality of CO1 primers across different taxonomic groups.

DNA barcoding—more specifically, CO1 analysis—has been used widely to aid in the species identification of elasmobranchs from various regions, such as Australia [36,42,43], China [44], the Philippines [50], Indonesia [51], Singapore [45,47], India [19,52,53], Bangladesh [54], Southern Africa [46,55], the United Kingdom [56], the Mediterranean Sea [57], the North Atlantic Ocean [58], the United States [49], and Brazil [59,60,61]. To date, limited published DNA barcoding studies in Malaysia have focused on rays but not on sharks [9,34]. From the available records in BOLD [62], a total of 783 sequences representing 120 species (primarily from the orders Myliobatiformes and Carcharhiniformes) from Malaysian waters were found. These suggested that elasmobranchs in Malaysian waters are not completely barcoded with potential for undiscovered species. Therefore, the present study used DNA barcoding to elucidate the species diversity of sharks and batoids, as well as to update the online database of DNA sequences of elasmobranchs in Malaysian waters. The results could reveal a greater elasmobranch diversity and help support the implementation of elasmobranch conservation and management programs in Malaysian waters.

2. Materials and Methods

2.1. Tissue Sampling and Processing

Shark and batoid tissue samples were collected at various landing sites and markets from four major coastal areas: west coast of Peninsular Malaysia (WP), east coast of Peninsular Malaysia (EP), Sarawak (SR), and Sabah (SB) (Figure 1), from June 2015 to June 2022. These landing sites were chosen based on previous surveys conducted by the Department of Fisheries Malaysia (DOFM), which highlighted prime locations for local elasmobranch fisheries. Moreover, additional samples along EP, SR, and SB were obtained through opportunistic fisheries—independent demersal trawl surveys within the Exclusive Economic Zone (EEZ) organised by the Department of Fisheries Malaysia (July 2015 to July 2016). They were identified in the field and the laboratory by KCL and AJXL using authoritative species identification guides and taxonomic references of the region [4,6,13,14,63,64,65,66]. No live animals were collected or killed during this study. No permission to collect data from EP was needed at the time of the research, and permission to collect data from Sarawak waters was granted by the Fisheries Research Institute Sarawak, Department of Fisheries Malaysia. A Sabah sampling permit was provided by the Sabah Biodiversity Council (SaBC), Ref. No. JKM/MBS.1000-2/2 JLD.9 (22) and JKM/MBS.1000-2/3JLD.4 (18). At least one fin clip of each recorded species was collected and preserved either in absolute ethanol or gently squashed onto a Whatman FTA^® Elute card before the subsequent molecular analysis.

2.2. Extraction, PCR Amplification, and DNA Sequencing

One to six tissue samples for each species were selected for barcoding and molecular analysis (

Table 1. Verification of the identity of barcoded shark and batoids species. Given are the sample code, sample location, NCBI accession number, field identification, best matched species from NCBI GenBank, similarity species and % from BOLD, tree-based identification, and the consensus identification. West coast of Peninsular Malaysia (WP): HM = Hutan Melintang, Pen = Penang, PP = Pasir Penambang, Sb = Sungai Besar, and Tk = Tanjung Karang; East coast of Peninsular Malaysia (EP): En = Endau, EP(D) = East coast of Peninsular Malaysia (DOF survey), Kt = Kuantan, Mer = Mersing, and Trg = Terengganu; Sarawak (SR): Btw = Bintawa, Kc = Kuching, Mk = Mukah, Sib = Sibu, SR(D) = Sarawak (DOF survey), and Srk = Sarikei; Sabah (SB): Kd = Kudat, KK = Kota Kinabalu, San = Sandakan, SB(D) = Sabah (DOF survey), and Tw = Tawau.

Samples	Location	Accession Number	Field Identification	NCBI Best Matched	%	BOLD Similarity	%	Tree Match	Consensus Identification
2-073	SB(D)	OQ384975	Heterodontus zebra	H. zebra	99.54	H. zebra	100	H. zebra	H. zebra
2-075	SB(D)	OQ384976	Heterodontus zebra	H. zebra	99.54	H. zebra	100	H. zebra	H. zebra
2-170	HM	OQ384977	Chiloscyllium hasseltii	C. hasseltii	100	C. hasseltii/griseum	100	C. hasseltii	C. hasseltii
2-502	Kt	OQ384978	Chiloscyllium hasseltii	C. hasseltii	100	C. hasseltii griseum	99.85	C. hasseltii	C. hasseltii
3-487	Trg	OQ384979	Chiloscyllium hasseltii	C. hasseltii	100	C. hasseltii griseum	100	C. hasseltii	C. hasseltii
S12	HM	OQ384980	Chiloscyllium hasseltii	C. hasseltii	100	C. hasseltii griseum	100	C. hasseltii	C. hasseltii
S25	HM	OQ384981	Chiloscyllium hasseltii	C. hasseltii	100	C. hasseltii griseum	100	C. hasseltii	C. hasseltii
3-215	Mk	OQ384982	Chiloscyllium indicum	C. indicum	100	C. indicum	100	C. indicum	C. indicum
SLG15	HM	OQ384983	Chiloscyllium indicum	C. indicum	100	C. indicum	100	C. indicum	C. indicum
2-452	SB(D)	OQ384984	Chiloscyllium plagiosum	C. plagiosum	99.85	C. plagiosum	100	C. plagiosum	C. plagiosum
4-357	EP(D)	OQ384985	Chiloscyllium plagiosum	C. plagiosum	99.85	C. plagiosum	100	C. plagiosum	C. plagiosum
J8	En	OQ384986	Chiloscyllium punctatum	C. punctatum	99.69	C. punctatum	99.85	C. punctatum	C. punctatum
J9	En	OQ384987	Chiloscyllium punctatum	C. punctatum	99.69	C. punctatum	100	C. punctatum	C. punctatum
Q11	Mk	OQ384988	Chiloscyllium punctatum	C. punctatum	99.69	C. punctatum	100	C. punctatum	C. punctatum
5-257	KK	OQ384989	Alopias pelagicus	A. pelagicus	100	A. pelagicus	100	A. pelagicus	A. pelagicus
Asup1	HM	OQ384990	Alopias superciliosus	A. superciliosus	100	A. superciliosus	100	A. superciliosus	A. superciliosus
2-842	HM	OQ384991	Atelomycterus marmoratus	A. marmoratus	100	A. marmoratus	100	A. marmoratus	A. marmoratus
4-883	San	OQ384992	Atelomycterus marmoratus	A. marmoratus	99.53	A. marmoratus	99.84	A. marmoratus	A. marmoratus
Q50	Sib	OQ384993	Atelomycterus marmoratus	A. marmoratus	99.37	A. marmoratus	99.85	A. marmoratus	A. marmoratus
2-013	SB(D)	OQ384994	Cephaloscyllium sarawakense	C. umbratile LS	98.93	C. umbratile LS	98.92	-	C. sarawakense I
3-640	SB(D)	OQ384995	Cephaloscyllium sarawakense	C. umbratile DS	99.85	C. umbratile DS	99.85	-	C. sarawakense I
2-011	SB(D)	OQ384996	Halaelurus buergeri	H. buergeri	99.69	H. buergeri	99.85	H. buergeri	H. buergeri
2-109	SB(D)	OQ384997	Halaelurus buergeri	H. buergeri	99.69	H. buergeri	99.85	H. buergeri	H. buergeri
2-536	Kt	OQ384998	Hemigaleus microstoma	H. microstoma	100	H. microstoma	100	H. microstoma	H. microstoma
3-367	Mk	OQ384999	Hemigaleus microstoma	H. microstoma	100	H. microstoma	100	H. microstoma	H. microstoma
4-419	EP(D)	OQ385000	Hemigaleus microstoma	H. microstoma	100	H. microstoma	100	H. microstoma	H. microstoma
TW12	Tw	OQ385001	Hemigaleus microstoma	H. microstoma	99.69	H. microstoma	100	H. microstoma	H. microstoma
2-746	Kt	OQ385002	Hemipristis elongata	H. elongata	100	H. elongata	100	H. elongata	H. elongata
3-423	Mk	OQ385003	Hemipristis elongata	H. elongata	100	H. elongata	100	H. elongata	H. elongata
5-082	San	OQ385004	Hemipristis elongata	H. elongata	99.69	H. elongata	100	H. elongata	H. elongata
3-754	Mk	OQ385005	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
3-850	Mk	OQ385006	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
3-960	Mk	OQ385007	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
3-131	Mk	OQ385008	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
3-133	Mk	OQ385009	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
Q3E	Srk	OQ385010	Carcharhinus amblyrhynchoides	C. amblyrhynchoides MM	100	C. amblyrhynchoides MM	100	C. amblyrhynchoides	C. amblyrhynchoides
3-353	Mk	OQ385011	Carcharhinus brevipinna	C. brevipinna	100	C. brevipinna	99.85	C. brevipinna	C. brevipinna
3-535	HM	OQ385012	Carcharhinus brevipinna	C. brevipinna	100	C. brevipinna	100	C. brevipinna	C. brevipinna
Q19	Srk	OQ385013	Carcharhinus brevipinna	C. brevipinna	100	C. brevipinna	100	C. brevipinna	C. brevipinna
2-246	Pen	OQ385014	Carcharhinus leucas	C. leucas	100	C. leucas	100	C. leucas	C. leucas
2-530	Kt	OQ385015	Carcharhinus leucas	C. leucas	100	C. leucas	100	C. leucas	C. leucas
S1	Tk	OQ385016	Carcharhinus leucas	C. leucas	100	C. leucas	100	C. leucas	C. leucas
3-129	Mk	OQ385017	Carcharhinus leucas	C. amboinensis DS	99.69	C. amboinensis DS	100	C. amboinensis	C. amboinensis
3-038	Kc	OQ385018	Carcharhinus limbatus	C. limbatus MM	100	C. limbatus MM	100	C. limbatus	C. limbatus
2-588	Kt	OQ385019	Carcharhinus limbatus	C. limbatus MM	99.54	C. limbatus MM	99.84	C. limbatus	C. limbatus
3-331	Mk	OQ385020	Carcharhinus melanopterus	C. melanopterus	100	C. melanopterus	100	C. melanopterus	C. melanopterus
2-538	Kt	OQ385021	Carcharhinus sealei	C. sealei	100	C. sealei	100	C. sealei	C. sealei
3-103	Mk	OQ385022	Carcharhinus sealei	C. sealei	99.85	C. sealei	100	C. sealei	C. sealei
5-083	San	OQ385023	Carcharhinus sealei	C. sealei	99.85	C. sealei	100	C. sealei	C. sealei
2-380	HM	OQ385024	Carcharhinus sorrah	C. sorrah	99.85	C. sorrah	99.85	C. sorrah	C. sorrah
2-578	Kt	OQ385025	Carcharhinus sorrah	C. sorrah	99.85	C. sorrah	99.85	C. sorrah	C. sorrah
3-077	Mk	OQ385026	Carcharhinus sorrah	C. sorrah	100	C. sorrah	100	C. sorrah	C. sorrah
S23TC	Tk	OQ385027	Carcharhinus sorrah	C. sorrah	99.85	C. sorrah	99.85	C. sorrah	C. sorrah
3-740	Mk	OQ385028	Lamiopsis tephrodes	L. tephrodes	100	L. tephrodes	100	L. tephrodes	L. tephrodes
3-944	Mk	OQ385029	Lamiopsis tephrodes	L. tephrodes	100	L. tephrodes	100	L. tephrodes	L. tephrodes
3-988	Mk	OQ385030	Lamiopsis tephrodes	L. tephrodes	100	L. tephrodes	100	L. tephrodes	L. tephrodes
Q2	Srk	OQ385031	Lamiopsis tephrodes	L. tephrodes	100	L. tephrodes	100	L. tephrodes	L. tephrodes
Q21	Mk	OQ385032	Lamiopsis tephrodes	L. tephrodes	100	L. tephrodes	100	L. tephrodes	L. tephrodes
3-739	Mk	OQ385033	Loxodon macrorhinus	L. macrorhinus	100	L. macrorhinus	100	L. macrorhinus	L. macrorhinus
3-909	Mk	OQ385034	Loxodon macrorhinus	L. macrorhinus	100	L. macrorhinus	100	L. macrorhinus	L. macrorhinus
3-928	Mk	OQ385035	Loxodon macrorhinus	L. macrorhinus	99.85	L. macrorhinus	99.85	L. macrorhinus	L. macrorhinus
2-438	Kt	OQ385036	Rhizoprionodon acutus	R. acutus	100	R. acutus	100	R. acutus	R. acutus
3-321	Mk	OQ385037	Rhizoprionodon acutus	R. acutus	99.85	R. acutus	99.85	R. acutus	R. acutus
Q42	Mk	OQ385038	Rhizoprionodon acutus	R. acutus	99.85	R. acutus	99.85	R. acutus	R. acutus
Q43	Mk	OQ385039	Rhizoprionodon acutus	R. acutus	99.85	R. acutus	99.85	R. acutus	R. acutus
Q23	Mk	OQ385040	Rhizoprionodon acutus	R. acutus	100	R. acutus	100	R. acutus	R. acutus
3-211	Mk	OQ385041	Rhizoprionodon oligolinx	R. oligolinx	99.69	R. oligolinx	99.85	R. oligolinx	R. oligolinx
4-706	HM	OQ385042	Rhizoprionodon oligolinx	R. oligolinx	99.54	R. oligolinx	100	R. oligolinx	R. oligolinx
J10	Mer	OQ385043	Rhizoprionodon oligolinx	R. oligolinx	99.54	R. oligolinx	100	R. oligolinx	R. oligolinx
J11	Mer	OQ385044	Rhizoprionodon oligolinx	R. oligolinx	99.54	R. oligolinx	100	R. oligolinx	R. oligolinx
S13	HM	OQ385045	Rhizoprionodon oligolinx	R. oligolinx	99.54	R. oligolinx	100	R. oligolinx	R. oligolinx
Q6	Srk	OQ385046	Scoliodon macrorhynchos	S. macrorhynchos	100	S. macrorhynchos	100	S. macrorhynchos	S. macrorhynchos
Q8	Srk	OQ385047	Scoliodon macrorhynchos	S. macrorhynchos	100	S. macrorhynchos	100	S. macrorhynchos	S. macrorhynchos
Q41T	Mk	OQ385048	Scoliodon macrorhynchos	S. macrorhynchos	99.85	S. macrorhynchos	99.85	S. macrorhynchos	S. macrorhynchos
K01	Kd	OQ385049	Triaenodon obesus	T. obesus	99.69	T. obesus	100	T. obesus	T. obesus
2-664	Kt	OQ385050	Galeocerdo cuvier	G. cuvier	100	G. cuvier	100	G. cuvier	G. cuvier
3-115	Mk	OQ385051	Galeocerdo cuvier	G. cuvier	100	G. cuvier	100	G. cuvier	G. cuvier
4-996	San	OQ385052	Galeocerdo cuvier	G. cuvier	100	G. cuvier	100	G. cuvier	G. cuvier
2-226	HM	OQ385053	Sphyrna lewini	S. lewini	100	S. lewini	100	S. lewini	S. lewini
2-604	Kt	OQ385054	Sphyrna lewini	S. lewini	99.69	S. lewini	99.80	S. lewini	S. lewini
3-225	Mk	OQ385055	Sphyrna lewini	S. lewini	100	S. lewini	100	S. lewini	S. lewini
T3	Tw	OQ385056	Sphyrna lewini	S. lewini	100	S. lewini	100	S. lewini	S. lewini
2-041	SB(D)	OQ385057	Squalus altipinnis	S. brevirostris LS	98.78	S. brevirostris LS	98.92	-	S. altipinnisI
2-047	SB(D)	OQ385058	Squalus altipinnis	S. edmundsi DS	99.54	S. edmundsi DS	100	S. edmundsi	S. edmundsi
2-103	SB(D)	OQ385059	Squatina tergocellatoides	S. tergocellatoides	99.85	S. tergocellatoides	99.85	S. tergocellatoides	S. tergocellatoides
2-114	SB(D)	OQ385060	Squatina tergocellatoides	S. tergocellatoides	100	S. tergocellatoides	100	S. tergocellatoides	S. tergocellatoides
4-322	EP(D)	OQ385061	Narcine brevilabiata	N. brevilabiata	99.69	N. brevilabiata	99.85	N. brevilabiata	N. brevilabiata
4-368	EP(D)	OQ385062	Narcine brevilabiata	N. brevilabiata	99.85	N. brevilabiata	100	N. brevilabiata	N. brevilabiata
4-439	EP(D)	OQ385063	Narcine maculata	N. maculata LS	95.25	N. maculata LS	98.52	-	N. cf. maculata sp. 1 I
2-874	HM	OQ385064	Narcine maculata	Narcine sp.MU	99.35	Narcine cf. oculifera MU	99.69	-	N. cf. maculata sp. 2 I
2-762	Kt	OQ385065	Rhina ancylostomus	R. ancylostomus	99.85	R. ancylostomus	99.83	R. ancylostomus	R. ancylostomus
3-584	HM	OQ385066	Rhina ancylostomus	R. ancylostomus	100	R. ancylostomus	99.85	R. ancylostomus	R. ancylostomus
3-890	Mk	OQ385067	Rhina ancylostomus	R. ancylostomus	99.69	R. ancylostomus	99.67	R. ancylostomus	R. ancylostomus
T2	Tw	OQ385068	Rhina ancylostomus	R. ancylostomus	100	R. ancylostomus	100	R. ancylostomus	R. ancylostomus
2-347	Btw	OQ385069	Rhynchobatus australiae	R. australiae	100	R. australiae	100	R. australiae	R. australiae
2-514	Kt	OQ385070	Rhynchobatus australiae	R. australiae	100	R. australiae	100	R. australiae	R. australiae
3-570	HM	OQ385071	Rhynchobatus australiae	R. australiae	99.39	R. australiae	99.62	R. australiae	R. australiae
TW9	Tw	OQ385072	Rhynchobatus australiae	R. australiae	100	R. australiae	100	R. australiae	R. australiae
2-614	Kt	OQ385073	Rhynchobatus australiae	R. australiae	100	R. australiae	100	R. australiae	R. australiae
KK7	KK	OQ385074	Rhinobatos borneensis	R. schlegelii DS	100	R. schlegelii DS	100	-	R. borneensis I
2-070	SB(D)	OQ385075	Rhinobatos borneensis	R. schlegelii DS	99.85	R. schlegelii DS	99.85	-	R. borneensis I
2-678	SB(D)	OQ385076	Rhinobatos borneensis	R. formosensis DS	100	R. formosensis DS	99.85	-	R. borneensis I
S6	HM	OQ385077	Rhinobatos cf borneensis	R. jimbaranensis LS	97.54	R. jimbaranensis LS	97.64	-	R. cf. borneensis I
S16	HM	OQ385078	Rhinobatos cf borneensis	R. jimbaranensis LS	97.54	R. jimbaranensis LS	97.64	-	R. cf. borneensis I
S17	HM	OQ385079	Rhinobatos cf borneensis	R. jimbaranensis LS	97.54	R. jimbaranensis LS	97.64	-	R. cf. borneensis I
2-093	SB(D)	OQ385080	Okamejei boesemani	O. boesemani MM	99.38	O. boesemani MM	99.38	-	O. boesemani I
2-057	SB(D)	OQ385081	Okamejei boesemani	O. boesemani MM	99.38	O. boesemani MM	99.38	-	O. boesemaniI
4-065	SR(D)	OQ385082	Okamejei boesemani	O. boesemani MM	99.54	O. boesemani MM	99.53	-	O. boesemani I
SK1	Trg	OQ385083	Okamejei hollandi	O. hollandi	99.52	O. hollandi	99.51	O. hollandi	O. hollandi
SK2	Trg	OQ385084	Okamejei hollandi	O. hollandi	99.84	O. hollandi	99.84	O. hollandi	O. hollandi
2-784	SR(D)	OQ385085	Okamejei hollandi	O. hollandi	99.52	O. hollandi	99.51	O. hollandi	O. hollandi
2-327	SR(D)	OQ385086	Okamejei hollandi	O. hollandi	99.68	O. hollandi	99.68	O. hollandi	O. hollandi
2-017	SB(D)	OQ385087	Okamejei hollandi	O. hollandi	99.84	O. hollandi	99.84	O. hollandi	O. hollandi
2-019	SB(D)	OQ385088	Bathytoshia lata	B. lata	100	B. lata	100	B. lata	B. lata
Q25TC	Mk	OQ385089	Brevitrygon heterura	B. heterura	99.69	B. heterura	99.69	B. heterura	B. heterura
S20	HM	OQ385090	Brevitrygon heterura	B. heterura	100	B. heterura	100	B. heterura	B. heterura
S21	HM	OQ385091	Brevitrygon heterura	B. heterura	99.85	B. heterura	99.85	B. heterura	B. heterura
Q20	Mk	OQ385092	Hemitrygon bennettii	H. bennettii	100	H. bennettii	100	H. bennettii	H. bennettii
3-862	Mk	OQ385093	Hemitrygon bennettii	H. bennettii	100	H. bennettii	100	H. bennettii	H. bennettii
2-138	Sb	OQ385094	Hemitrygon bennettii	H. bennettii	100	H. bennettii	100	H. bennettii	H. bennettii
2-059	SB(D)	OQ385095	Hemitrygon parvonigra	H. parvonigra LS	94.78	H. fai LS	100	-	H. cf. parvonigra I
2-448	SB(D)	OQ385096	Hemitrygon parvonigra	H. parvonigra LS	94.78	H. fai LS	100	-	H. cf. parvonigra I
2-127	SB(D)	OQ385097	Hemitrygon parvonigra	H. parvonigra LS	94.78	H. fai LS	100	-	H. cf. parvonigra I
T8	Tw	OQ385098	Hemitrygon parvonigra	H. parvonigra LS	94.78	H. fai LS	100	-	H. cf. parvonigra I
TW4	Tw	OQ385099	Himantura leoparda	H. leoparda	99.08	H. leoparda	99.19	H. leoparda	H. leoparda
K1	KK	OQ385100	Himantura uarnak	H. uarnak	100	H. uarnak	100	H. uarnak	H. uarnak
TW6	Tw	OQ385101	Himantura undulata	H. undulata	100	H. undulata	100	H. undulata	H. undulata
TW5	Tw	OQ385102	Maculabatis gerrardi	M. macrura MM	99.85	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
2-160	Sb	OQ385103	Maculabatisgerrardi	M. macrura/H. gerrardi MM	100	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
2-162	Sb	OQ385104	Maculabatis gerrardi	M. macrura/H. gerrardi MM	100	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
2-166	Sb	OQ385105	Maculabatis gerrardi	M. macrura/H. gerrardi MM	100	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
JHR5TC	Mer	OQ385106	Maculabatis gerrardi	M. macrura/H. gerrardi MM	99.69	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
S27E	HM	OQ385107	Maculabatis gerrardi	M. macrura/H. gerrardi MM	100	M. macrura/H. gerrardi MM	100	M. gerrardi	M. gerrardi I
S10	HM	OQ385108	Neotrygon malaccensis	N. kuhlii DS	99.54	N. kuhlii DS	99.84	N. malaccensis	N. malaccensis
S11	HM	OQ385109	Neotrygon malaccensis	N. kuhlii DS	100	N. kuhlii DS	100	N. malaccensis	N. malaccensis
3-619	HM	OQ385110	Neotrygon malaccensis	N. kuhlii DS	99.85	N. kuhlii DS	100	N. malaccensis	N. malaccensis
4-579	HM	OQ385111	Neotrygon malaccensis	N. kuhlii DS	100	N. kuhlii DS	100	N. malaccensis	N. malaccensis
Q12	Mk	OQ385112	Neotrygon orientalis	N. kuhlii DS	100	N. kuhlii DS	100	N. orientalis	N. orientalis
3-459	Trg	OQ385113	Neotrygon varidens	N. kuhlii DS	100	N. kuhlii DS	100	N. varidens	N. varidens
4-421	EP(D)	OQ385114	Neotrygon varidens	N. kuhlii DS	99.85	N. kuhlii DS	100	N. varidens	N. varidens
Q17	Mk	OQ385115	Pastinachus gracilicaudus	P. gracilicaudus	100	P. gracilicaudus/solocirostris	100	P. gracilicaudus	P. gracilicaudus
3-263	Mk	OQ385116	Pastinachus solocirostris	P. solocirostris	100	P. solocirostris	100	P. solocirostris	P. solocirostris
3-746	Mk	OQ385117	Pastinachus solocirostris	P. solocirostris	100	P. solocirostris	100	P. solocirostris	P. solocirostris
3-841	Mk	OQ385118	Pastinachus solocirostris	P. solocirostris	100	P. solocirostris	100	P. solocirostris	P. solocirostris
Q16TC	Mk	OQ385119	Pateobatis uarnacoides	P. uarnacoides	99.85	P. uarnacoides	100	P. uarnacoides	P. uarnacoides
J12	En	OQ385120	Taeniura lymma	T. lymma	100	T. lymma	100	T. lymma	T. lymma
2-069	SB(D)	OQ385121	Taeniurops meyeni	T. meyeni	99.85	T. meyeni	100	T. meyeni	T. meyeni
S3	Tk	OQ385122	Telatrygon biasa	T. biasa	99.69	T. zugei	100	T. biasa	T. biasa
S18	HM	OQ385123	Telatrygon biasa	T. biasa	99.54	T. zugei	99.83	T. biasa	T. biasa
S19	HM	OQ385124	Telatrygon biasa	T. biasa	99.54	T. zugei	99.83	T. biasa	T. biasa
JHR2E	Mer	OQ385125	Urogymnus asperrimus	U. asperrimus	99.69	U. asperrimus	99.85	U. asperrimus	U. asperrimus
3-051	Kc	OQ385126	Urogymnus lobistoma	H. chaophraya	88.01	H. uarnacoides	100	-	U. lobistoma I
3-821	Mk	OQ385127	Urogymnus lobistoma	H. chaophraya	88.01	H. uarmacoides	100	-	U. lobistoma I
3-876	Mk	OQ385128	Urogymnus lobistoma	H. chaophraya	88.01	H. uarmacoides	100	-	U. lobistoma I
3-936	Mk	OQ385129	Gymnura poecilura	G. poecilura	100	G. poecilura	100	G. poecilura	G. poecilura
3-937	Mk	OQ385130	Gymnura poecilura	G. poecilura	100	G. poecilura	100	G. poecilura	G. poecilura
3-987	Mk	OQ385131	Gymnura poecilura	G. poecilura	99.85	G. poecilura	99.84	G. poecilura	G. poecilura
4-705	HM	OQ385132	Gymnura poecilura	G. poecilura	99.69	G. poecilura	99.69	G. poecilura	G. poecilura
G08c	En	OQ385133	Gymnura zonura	G. zonura	99.54	G. zonura	99.69	G. zonura	G. zonura
G09c	En	OQ385134	Gymnura zonura	G. zonura	99.85	G. zonura	100	G. zonura	G. zonura
2-021	SB(D)	OQ385135	Plesiobatis daviesi	P. daviesi	100	P. daviesi	100	P. daviesi	P. daviesi
TW14	Tw	OQ385136	Urolophus cf. aurantiacus	U. expansus DS	99.07	U. expansus DS	99.84	-	U. cf. aurantiacus I
2-656	Kt	OQ385137	Aetobatus ocellatus	A. ocellatus	100	A. ocellatus	100	A. ocellatus	A. ocellatus
2-946	Kc	OQ385138	Aetobatus ocellatus	A. ocellatus	100	A. ocellatus	100	A. ocellatus	A. ocellatus
S9	PP	OQ385139	Aetobatus ocellatus	A. ocellatus	100	A. ocellatus	100	A. ocellatus	A. ocellatus
K2	KK	OQ385140	Aetobatus ocellatus	A. ocellatus	99.85	A. ocellatus	99.85	A. ocellatus	A. ocellatus
Aves1	Kt	OQ385141	Aetomylaeus vespertilio	A vespertilio	99.07	A vespertilio	98.88	A vespertilio	A vespertilio
T7	Tw	OQ385142	Myliobatis hamlyni	M. hamlyni R	100	M. tobijei	100	M. tobijei	M. hamlyni
2-027	SB(D)	OQ385143	Myliobatis hamlyni	M. hamlyni R	100	M. tobijei	100	M. tobijei	M. hamlyni
2-029	SB(D)	OQ385144	Myliobatis hamlyni	M. hamlyni R	100	M. tobijei	100	M. tobijei	M. hamlyni
3-289	Mk	OQ385145	Rhinoptera jayakari	R. jayakari	100	R. jayakari	100	R. jayakari	R. jayakari
2-516	Kt	OQ385146	Mobula thurstoni	M. thurstoni	100	M. thurstoni	100	M. thurstoni	M. thurstoni
3-781	Mk	OQ385147	Mobula kuhlii	M. kuhlii	100	M. kuhlii	100	M. kuhlii	M. kuhlii
2-023	SB(D)	OQ385148	Chimaera phantasma	C. phantasma LS	97.39	C. phantasma LS	97.67	-	C. cf. phantasma I
2-025	SB(D)	OQ385149	Chimaera phantasma	C. phantasma LS	97.39	C. phantasma LS	97.67	-	C. cf. phantasma I

^R = Revised identity based on White et al. [66]—reference sequence EU398924 M. tobijei was revised as M. hamlyni; ^DS = best match differed from field-identified species; ^LS = best match showed low similarity; ^MM = match with multiple species; ^MU = match with uncertain species; and ^I = inconclusive identity.

). Particular attention was given to specimens showing morphological ambiguities during field identification and potential cryptic species, as well as species that have not been barcoded to date; molecular identification was used to clarify species identities in these cases. These includes samples of Cephaloscyllium sarawakense (2-013 and 3-640); Carcharhinus leucas (3-129); Squalus altipinnis (2-041 and 2-047); Narcine maculata (2-874 and 4-439); Rhinobatos borneensis (KK7, 2-070, 2-678, S6, S16, and S17); Okamejei boesemani (2-057, 2-093, and 4-065); Hemitrygon parvonigra (2-059, 2-127, 2-448, and T8); Maculabatis gerrardi (TW5, 2-160, 2-162, 2-166, JHR5TC, and S27E); Urogymnus lobistoma (3-051, 3-821, and 3-876); Myliobatis hamlyni (T7, 2-027, and 2-029); and Chimaera phantasma (2-023 and 2-025). For samples stored in absolute ethanol, the total DNA was extracted using 10% Chelex resin incubated for two minutes at 60 °C, followed by 25 min at 103 °C, following a modified protocol of Hyde et al. [67]. For samples stored using Whatman FTA^® Elute cards, the total DNA was extracted using the modified protocol in Rigby et al. [68].

Amplification of the partial cytochrome c oxidase subunit I (CO1) gene (650 base pairs, nucleotide position 51-701) was done using universal primers FishF2– 5′ TCG ACT AAT CAT AAA GAT ATC GGC AC 3′ and FishR2—5′ ACT TCA GGG TGA CCG AAG AAT CAG AA 3′ [38]. PCR reactions were performed in 25 μL volumes containing 12.5 μL of 2X PCR Master Mix (1st Base), 1 μL of 10 mM of both primers, 1 μL DNA templates, and 9.5 μL sterilized distilled water. The thermal conditions consisted of the initial preheating at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 44–54 °C for 30 s, and extension at 72 °C for 1 min, then repeated for 36–40 cycles, followed by a final extension at 72 °C of 5 min. The PCR products were checked using 1% agarose in TAE buffer. Only PCR products that showed good PCR amplification were sent for sequencing service at Apical Scientific Sdn Bhd (Selangor, Malaysia). The sequencing results were checked for confirmation of the species using the BLAST tool of the NCBI and compared with field identification. The species identification matching was based on the query cover (%) in the BLAST tool as the query cover that describes how similar the sample sequence is to the reference sequence in GenBank and the similarity in the BOLD system.

2.3. Data Analysis

For the data analysis, the obtained sequences were edited and aligned using BioEdit version 7.2.5 [69]. Molecular species identification was achieved using two approaches: Basic Local Alignment Search Tool (BLAST^®) and phylogenetic tree reconstruction. For the first approach, reviewed sequences were uploaded to BLAST^® (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 5 January 2023) and BOLDSYSTEMS (http://www.boldsystems.org/index.php/IDS_OpenIdEngine, accessed on 5 January 2023) in search for local similarities with the available sequences in the National Center for Biotechnology Information (NCBI) GenBank and Barcode of Life Database (BOLD), respectively. The results from the searches were in the form of scores, query cover, and percentage identity of the 200 most similar sequences available in the databases. The list of sequences that produced significant alignments or best matches with each of our sequence was reviewed manually for validity of the suggested identity. This included their percentage similarity, query coverage, reliability of the sequence sources (from respectable authors in the field), congruency among sequences from different sources, etc. Suspicious sequences in the database were excluded in the process.

For the second approach, a phylogenetic tree was constructed using sequences from the current study and reference sequences available in the NCBI GenBank (Supplementary Materials Table S2). The selection of the reference sequences included the best matched sequences in the BLAST^® search, as well as available sequences of elasmobranchs that are recorded in Malaysian waters. Reference sequences from Malaysian waters were prioritized over sequences from neighbouring waters. The list of elasmobranchs (70 sharks and 91 batoid species) and chimaera species that had been recorded in Malaysia (include some unverified records) is shown in Supplementary Materials Table S1. The number of reference sequences used for each species was limited to one or two sequences, except for the species complex of Maculabatis gerardi and M. macrura.

A midpoint rooted tree maximum likelihood (ML) based on Kimura-2-Parameter (K2P) distances was created using MEGA X [70] with 1000 bootstrap replicates. The general time-reversible model plus gamma distribution rate plus evolutionarily invariable model (GTR + G + I) was selected by MEGA X as the best-fitting substitution model based on the Akaike Information Criterion (AIC). Sequences of closely related cartilaginous fish (Chimaera phantasma) were used as the outgroup. In this approach, morphological identification was verified based on the clustering of the samples in the phylogenetic tree in comparison to the reference sequences. Furthermore, the genetic distance was calculated to evaluate the usefulness of COI across taxonomic rank. We also examined genetic distances (p-distance) within and between species and subspecies. The interindividual distances calculated in MEGA X were sorted into eight inter-rank categories (intraspecific, interspecific, inter-genus, inter-subfamily, inter-family, inter-order, inter-infraclass, and inter-class) based on the species classification in the Eschmeyer’s Catalog of Fishes (CAS) [71] and the International Union for Conservation of Nature’s (IUCN) [72] Red List of Threatened Species. The mean and ranges of p-distance for these categories were calculated and boxplot was generated using Statistica 7 [73]. In addition, the analysis of variance (ANOVA) supplemented with Tukey’s HSD pairwise comparison was performed to determine the variations among inter-rank categories, between infraclass, and among orders of elasmobranchs.

3. Results

A total of 175 individuals belonging to 29 shark and 38 batoid species based on field identification were barcoded in this study. In addition, two individuals of chimaera Chimaera phantasma (closely related cartilaginous fishes) were also barcoded. All sequences were uploaded to GenBank, accession numbers: OQ384975–OQ385149 (n = 175). Another 231 sequences from NCBI GenBank were retrieved for phylogenetic tree reconstruction (Supplementary Materials Table S2). These sequences covered 82% of the total elasmobranch species recorded in Malaysia, i.e., there are 29 remaining elasmobranch species in Malaysian waters that have not been barcoded to date.

3.1. Matches with BLAST

Table 1 provides, for each barcoded individual, the percent matches to the best matched species in the NCBI GenBank and the similarity matched in the BOLD database. The results showed that 137 of the 175 samples strongly matched (>99%) with sequences of the same identity only (average 99.86%). The remaining individuals with uncertainty were noted as being matched with different species (13 individuals), low similarity (88.01–98.93%) with the best matched species (15 individuals), matched with multiple species at average genetic distance of 99.7% (17 individuals), or matched with unknown species (1 individual) (see Table 1).

3.2. Tree Matches

The ML tree (Figure 2a–e) matched most of the present sequences to reference sequences forming distinct clades by species (Supplementary Materials Table S3). Some of the exceptions in shark species were listed as follows. Field-identified S. altipinnis (samples 2-041 and 2-047) was found to be paraphyletic based on the ML tree; sample 2-041 formed a potential unique clade on its own (1.3–5.2% genetic difference with other highly similar Squalus species), while sample 2-047 clustered with the reference sequence of S. edmundsi (0.2% genetic distance). Sphyrna lewini (samples 2-226, 2-604, 3-225, and T3) formed two distinct clades, with sample 2-604 and T3 branching out from the other sequences, forming a unique clade (3.7–3.8% genetic distance). Cephaloscyllium sarawakense (samples 2-013 and 3-640) formed a clade with a reference sequence named C. umbratile, with genetic distance ranges from 0.6 to 1.3%. Carcharhinus leucas showed two separate clades: samples S1, 2-246, and 2-530 formed a clade with a reference sequence of C. leucas, while sample 3-129 formed a clade with a reference sequence of C. amboinensis. Sequences of Carcharhinus amblyrhynchoides and C. limbatus from the present study and the reference sequences used formed two separate clades; however, the genetic distance between the clades was low (0.6–0.8%).

The exceptions among batoids were listed as follows. Hemitrygon parvonigra (samples 2-059, 2-127, 2-448, and T8) was separated from the reference sequence of the species at 5.2% inter-clade genetic distance. The species identity of the Neotrygon kuhlii reference sequences found in Malaysian waters were updated to N. malaccensis, N. orientalis, and N. varidens, according to the assigned species names in Borsa et al. [74]. After renaming the references sequences, the Neotrygon sequences from the present study (samples S10, S11, Q12, 3-459, 3-619, 4-421, and 4-457) were found to cluster with the reference sequence of their respective species. Urogymnus lobistoma recorded in the present study showed a unique clade on its own as separate from other Urogymnus reference sequences (genetic distances ranged from 12.1 to 13.7%).

Sequences of M. gerrardi and M. macrura were paraphyletic showing multiple overlapping clades. The inter-individual genetic distances ranged from 0 to 5.1%. The sequence of O. boesemani formed a clade with reference sequences of O. boesemani from China (0.9–1.1% genetic distance with present sequences) and O. cairae from Vietnam (0.5–0.6% genetic distance with present sequences). Two N. maculata barcoded in this study were found to be different genetically and did not form clades with any of the best matched species; the genetic distance between samples 4-439 and N. maculata from India was 4.3% while that between samples 2-874 and N. cf. oculifera was 1.6%. Two or possibly three separate clades of field identified Rhinobatos borneensis were found with samples from Sabah (KK7, 2-070, and 2-678) forming clades with reference sequences named R. formosensis and R. schlegelii (0 to 0.3% within clade genetic distance), while samples from WP (S6, S16, and S17) were likely a unique clade separated from the aforementioned clade at 3.2 to 4.0% genetic distance and from R. jimbaranensis at 2.2% genetic distance. Myliobatis hamlyni (samples 2-027, 2-029, and T7) formed a clade with the reference sequence of the species (species name of sequence EU398924 was revised to M. hamlyni by White et al. [62]), as well as with M. tobijei sequence from South China Sea (0–0.3% genetic distance).

Although the sequences of C. phantasma were not the focus of this study, it was worth noting that the sequences obtained were separated from the reference sequences of C. phantasma at a genetic distance of 2.7 to 3.8%.

3.3. Final Taxonomic Identification

Considering the results of the BLAST matches and the ML tree, taxonomic identities of 121 individuals that showed consensus using both approaches were clearly verified. Seven samples of Neotrygon species were also confirmed after updated species identity of the reference sequences according to Borsa et al. [74]. Six samples of C. amblyrhynchoides (3-754, 3-850, 3-960, 3-131, 3-133, and Q3E) and two samples of C. limbatus (3-038 and 2-588) were cautiously treated as correctly identified due to the presence of misidentified sequences in NCBI GenBank. This suggests the need to review the identity of all submitted sequences in NCBI GenBank to prevent future confusion. Four samples of S. lewini (2-226, 2-604, 3-225, and T3) were also cautiously treated as correctly identified. However, the presence of two distinct clades suggested cryptic species of S. lewini that need further taxonomic evaluation. Three samples of M. hamlyni (T7, 2-027, and 2-029) were treated as correctly identified, backed by the reference sequence (EU398924) and specimens that were used in M. hamlyni redescription [66]. Two individuals, i.e., samples 2-047 and 3-129, were determined to be field misidentifications and were assigned as S. edmundsi and C. amboinensis, respectively, based on the phylogenetic tree information.

The identities of the remaining 30 individuals remained inconclusive based on the two molecular approaches. Field-identified S. altipinnis (2-041) and U. lobistoma (3-051, 3-821, and 3-876) were treated as the final species assignment as sequences of these species were not available in NCBI GenBank. The species identity of the two C. sarawakense samples was retained, as the morphological characteristics of the specimens did not match those of C. umbratile, suggesting a possible error in the submitted C. umbratile sequences in NCBI GenBank. Similarly, the identity of the U. cf. aurantiacus sample was retained, as the morphological characteristics of the sample were found to be different from the best matched species of U. expansus. Species identities of the sequences of three R. borneensis (from Sabah), three O. boesemani, and six M. gerrardi were also retained, as they belong to species complexes that need taxonomic clarification. The three other sequences of R. borneensis from WP were treated as R. cf. borneensis considering the low similarity with the available reference sequences of the genus. Eight sequences that matched at low similarity levels were each treated as N. cf. maculata sp1 (sample 4-439); N. cf. maculata sp2 (sample 2-874); H. cf. parvonigra (sample 2-059, 2-127, 2-448, and T8); and C. cf. phantasma (2-023 and 2-025).

3.4. Genetic Distance

Pairwise genetic distances were progressively increased as the inter-rank categories increased (Figure 3). These increases were significant (p < 0.001)-based overall and pairwise comparisons in the ANOVA and Tukey’s HSD tests. However, the range for among inter-rank genetic distances were considered large and overlapped especially between the neighbouring categories.

Comparison across class/infraclass for each of the inter-rank categories is shown in Figure 4. Within intraspecific categories, the genetic distance of Holocephali was significantly higher than the elasmobranchs (p < 0.001). The genetic distance within batoids, on the other hand, was significantly higher than sharks for the interspecific, inter-genus, inter-family, and inter-order categories (p < 0.001). Comparisons across orders provide further details for the inter-individual distances (Figure 4). Within the intraspecific categories, it showed that most of the genetic distances were in the range from 0 to 1%, except in Hexanchiformes and Chimaeriformes. For interspecific distance, the range for most batoids was found above 8%, except Rhinopristiformes, while below 8% in most of the sharks, except Lamniformes and part of the Orectolobiformes. For the inter-genus category, the genetic distance within Myliobatiformes was significantly higher than the other comparable orders (p < 0.001). For inter-family, the genetic distances within Myliobatiformes and Squaliformes were each highest and lowest among the comparable orders (p < 0.001). Other significant comparisons can be found between Orectolobiformes and Lamniformes, Orectolobiformes and Rhinopristiformes, and Carcharhiniformes with the others, except Orectolobiformes (p < 0.05 or p < 0.001).

4. Discussion

A comprehensive DNA barcode reference library is one of the fundamental building blocks allowing application of the increasingly popular environmental DNA (eDNA) metabarcoding approach for the assessment and environmental monitoring of biodiversity [75]. While gaps remain in the current elasmobranch biodiversity reference library, collective efforts, including ours, have advanced barcoding data for both common and rarely occurring freshwater and marine species of sharks, batoids, and skate in Malaysia. Additionally, the field identification and molecular verification efforts added four new records for Malaysia, namely Squalus edmundsi, Carcharhinus amboinensis, Alopias superciliosus, and Myliobatis hamlyni. With the exception of A. superciliosus that was sampled from WP, the other three species were sampled from Borneo (Sabah and Sarawak). These findings further affirm that the waters of Malaysian Borneo remain understudied in terms of the full biodiversity characterization of local elasmobranchs.

From the collation of available studies, there are 70 species of sharks (19 families) and 91 species of batoids (11 families) recorded in Malaysian waters (Supplementary Materials Table S1). The combined records from various studies had excluded species that were unaccepted in World Register of Marine Species (WORMS) (Cephaloscyllium circulopullum and Narcine indica) and species that had been verified as absent from Malaysian waters (Brevitrygon walga and B. imbricata in Last et al. [13]). These records have been improved in terms of classification within the family Dasyatidae [13]; incorporation of the revision of Telatrygon species in Malaysian waters [14]; delimitation of cryptic species in Neotrygon kuhlii complexes [15,74,76]; new recorded species in Malaysian water, including Carcharhinus tjutjot [10], Fluvitrygon kittipongi, and F. oxyrhyncha [16,17]); and Scoliodon laticaudus [35] (previously revised as S. macrorhynchos in Last et al. [6]).

Among the new species records, there are two species that are field-misidentified based on their morphological characteristics, namely S. edmundsi and C. amboinensis. These misidentifications are unsurprising, given how uncommon these two species are worldwide; the former was only described in 2007 in specimens from Australia [77], while the latter is likely often misidentified as highly similar-looking bull shark C. leucas, resulting in poor distribution records [78]. The usefulness of the molecular approach is particularly evident in separating these two morphologically similar carcharhinid species that are genetically distinctive. The discovery of spurdog S. edmundsi was possible due to the Department of Fisheries Malaysia trawl survey within the deeper EEZ waters off the coast of Borneo. This relatively newly described species is currently known to occur only off the continental waters of Australia and Indonesia [79].

The new finding of the bigeye thresher A. superciliosus in a major fisheries landing site in Hutan Melintang, along the central-west coast of Peninsula Malaysia, was rather surprising. This thresher species is thought to occur in deeper waters and generally less catchable than the tropical congener A. pelagicus [80], which had only been reported off the open waters of Sabah. The trawl fisheries in the landing site where A. supercilious was found typically operates in the relatively shallow Strait of Malacca. Further investigations into this finding will help shed light on the ecology of the thresher.

For the eagle ray M. hamlyni, existing molecular evidence suggested a best match with M. tobijei from South China Sea (0% genetic difference) and M. hamlyni from Indonesia (0.3% genetic distance). The latest distributional assessment on both species by White et al. [66] showed that M. tobijei is restricted to the western North Pacific, while M. hamlyni occurs in neighbouring Indonesia and the Philippines. Moreover, the morphological characteristic of the present specimen best matches the species description of M. hamlyni in White et al. [66], especially the purplish brown dorsal coloration (compared to yellowish brown in M. tobijei), as well as the slightly convex cranial fontanelle that is visible in the dorsal view (nearly straight and broader in M. tobijei). More importantly, the M. hamlyni specimen corresponded to the reference sequence used in the present analysis (EU398924), i.e., one of the type of specimens in White et al. [66]. This suggests that the reference sequence for M. tobijei KP267630 (published in December 2015) was likely an outdated species identity. Prior to the White et al. [66] redescription study, M. tobijei was thought to be distributed from Japan down south to Indonesia and the Philippines [66]. The field-identified U. cf. aurantiacus showed the highest genetic similarity to U. expansus found only in Southwestern Australia, but the known distribution of U. aurantiacus in the Philippines makes the latter species identity more feasible. The U. cf. aurantiacus specimen may therefore be an undescribed species, and the significance of this finding is that no urolophids are known to occur within Malaysia to date.

One of the most fundamental requirements in conservation and fishery management of sharks and batoids is correct species identification. While some advances have been made from our study, improvement in the records is still needed, including verification of the 40 recorded species of concern (recorded by single/multiple reference/s by the same research team or absent in Malaysia according to the IUCN assessment). The use of available genetic sequences without careful morphological examination and corroboration from existing distributional records would result in erroneous species assignment. The BOLD species assignment of Narcine cf. oculifera and Cephaloscyllium umbratile are inconclusive, as the percentage match with the present sequences (except sample 3-640) was less than 99%, and they had not been previously recorded in Malaysian waters. Despite the use of multiple approaches, our work highlights the need to focus on select taxonomic groups that may be species complexes or cryptic species. Sphyrna lewini (which was also pointed out by Naylor et al. [81]), the R. borneensis-schlegelli group, and the M. gerrardi-macrura group are among those requiring further investigations that are feasible due to the relatively common occurrence locally from our past surveys. On the other hand, taxonomic work to resolve unclear identities of uncommon species of S. altipinnis (2-041); C. sarawakense (2-013 and 3-064); N. maculata (4-439 and 2-874); and H. parvonigra (2-059, 2-448, 2-127, and T8) may be hampered without extensive field samplings.

The use of barcoding approaches is also fundamental in describing species that are still unknown to the scientific communities. This is important to prevent the loss of species before even being discovered, as seen in Carcharhinus obsolerus [82]. However, to advance the use of barcoding for accurate biodiversity monitoring, there remains the urgent need for the scientific names in the NCBI database to be updated regularly. One example is the revision of the family Dasyatidae that has resulted in the renaming of Himantura walga to Brevitrygon heterura (the revised B. walga is found to be confined to the Indian Ocean) [13]. Another example is the identity of reference sequence EU398924 as M. tobijei in NCBI GenBank, which likely should be M. hamlyni after the updated study of White et al. [66]. It is imperative to review the identity of all submitted sequences in NCBI GenBank to prevent future confusion, preferably through collaboration with reputable taxonomist with molecular knowledge to further enhance the validity of the submitted sequences. Moving forward, it is also essential to increase the amount of localized data on individual species, and the correct identification of species through traditional morphology and the uploading of correct sequences for the right species are highly essential to allow full functionality of barcoding technology for species identification.

5. Conclusions

The barcoding of animal tissues has proven to be an important technique to identify species from animal remains that cannot be identified by conventional morphological means and therefore cannot be adequately traced. Accurate species identification will improve species-specific catch landing data and ease and accelerate the identification of their illegal trade and use in Malaysia. Overall, this will provide baseline and monitoring data for conservation and fisheries management. The sharks and batoids CO1 database establishment and improvement will also help facilitate the rapid monitoring and assessment of elasmobranch fish through environmental DNA methods. In the future, rapid and large-scale surveys can be conducted to obtain the distribution points of cartilaginous fishes and predict their potential habitat areas, thereby providing a scientific basis and basis for the identification of elasmobranch conservation priority areas.