An Odyssey of Integrative Taxonomy Unveils Marine Fish Diversity, New Records and Cryptic Species in Malaysian Waters

© 2024 Academia Sinica, Taiwan Open Access An Odyssey of Integrative Taxonomy Unveils Marine Fish Diversity, New Records and Cryptic Species in Malaysian Waters Danial Hariz Zainal Abidin1, Siti Azizah Mohd. Nor2,6 , Ying Giat Seah3,4 , Mohd Sharol Ali3, Jamsari Amirul Firdaus Jamaluddin6, Masazurah A Rahim5, Nur Syafiqa Zulkifly3, Min Pau Tan2, Khaironizam Md Zain6, and Tun Nurul Aimi Mat Jaafar3,* 1Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, 43600, Selangor, Malaysia. E-mail: [email protected] (Zainal Abidin) 2Institute of Climate Adaptation and Marine Biotechnology, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia. E-mail: [email protected] (Nor); [email protected] (Tan) 3Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030, Kuala Nerus, Terengganu, Malaysia. *Correspondence: E-mail: [email protected] (Mat Jaafar) E-mail: [email protected] (Ying); [email protected] (Ali); [email protected] (Zulkifly); [email protected] (Mat Jaafar) 4South China Sea Repository & Reference Centre, Institute Oceanography and Environment, Universiti Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia 5Fisheries Research Institute, 11960, Batu Maung, Penang, Malaysia. E-mail: [email protected].my (A Rahim) 6School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, Malaysia. E-mail: [email protected] (Jamaluddin); [email protected] (Md Zain) Received 27 December 2023 / Accepted 26 June 2024 / Published 11 November 2024 Communicated by Ryuji Machida This study elucidates the species diversity of marine fishes in the Exclusive Economic Zone (EEZ) of Peninsular Malaysia (PM) using an integrative approach combining DNA barcoding and morphological identification. Our focus was on demersal surveys conducted on the east coast of PM in the South China Sea. We re-evaluated the diversity of 475 specimens across 93 putative species (92 barcoded morphospecies), from 16 orders and 41 families, including two IUCN vulnerable species. A total of two species - Saurida isarankurai and Oxyurichthys auchenolepis - are presented as new record, and three species - Nemipterus balinensoides, Gymnothorax reevesii and Synodus hoshinonis - as the first specimen-based records in Malaysian waters. Cytochrome c oxidase subunit I (COI) sequence analyses delineated 95 consensus Molecular Operational Taxonomic Units (MOTUs), exceeding morphological diversity. Interestingly, the barcode analysis revealed several MOTUs delimited within one morphologically identified fish species, with both intraspecific and interspecific genetic divergences exceeding 2%, indicating substantial intraspecific genetic divergence within species groups or the existence of morphologically cryptic species within our dataset. These findings highlight the complexity of species delimitation and the value of genetic methods. Our study provides valuable insights into marine fish diversity from the east coast of Peninsular Malaysia and enhances our understanding of genetic diversity, distribution, and conservation needs of ecosystems through DNA barcoding. By integrating DNA barcoding with morphology, we present a comprehensive framework for future research to develop conservation and management strategies for Malaysia’s marine biodiversity. The expansion of the genetic barcode database generated in this study will facilitate future molecular taxonomy research. Key words: Peninsular Malaysia, DNA barcoding, Exclusive Economic Zone (EEZ), South China Sea, Marine fishes, Demersal survey, Cryptic species Citation: Zainal Abidin DH, Nor SAM, Seah YG, Ali MS, Jamaluddin JAF, A Rahim M, Zulkifly NS, Tan MP, Md Zain K, Mat Jaafar TNA. 2024. An odyssey of integrative taxonomy unveils marine fish diversity, new records and cryptic species in Malaysian waters. Zool Stud 63:30. doi:10.6620/ ZS.2024.63-30. Zoological Studies 63:30 (2024) doi:10.6620/ZS.2024.63-30 1 © 2024 Academia Sinica, Taiwan BACKGROUND Understanding the biological diversity within marine ecosystems is of paramount importance for the conservation and sustainable management of these critical habitats. Notably, fish biodiversity plays a crucial role in maintaining ecosystem functionality, economic well-being, and food security (Mora et al. 2011). Situated in the South China Sea, the Exclusive Economic Zone (EEZ) of Peninsular Malaysia (PM), which spans an area of nearly 130,000 km2 (Nadira et al. 2019), is a productive hotspot for marine biodiversity and hosts an impressive diversity of fish species (Allen 2008; Myers et al. 2000). These vibrant fish communities contribute significantly to the region’s ecological equilibrium and form a vital part of Malaysia’s economy and local livelihoods (Teh and Pauly 2018). To date, is no large-scale and comprehensive ichthyofaunal assessment has been done in this area, except for a few smaller scale regional studies (Chong et al. 2010; Du et al. 2019; Matsunuma et al. 2011; Motomura et al. 2021; Seah et al. 2020). Nonetheless, our understanding of this biodiversity remains partial, and is largely hindered by the challenges of morphological identification. The South China Sea and surrounding Southeast Asian waters are home to an incredible diversity of marine fish species. With over 3,300 recorded species from more than 250 families (Allen et al. 2000; Froese and Pauly 2023), this biodiversity hotspot harbours a high diversity of marine ichthyofauna. Malaysia, Indonesia, the Philippines and Vietnam account for most of this regional diversity (Allen and Adrim 2003). Recent molecular studies utilising DNA barcoding have identified more than 116 putative fish species from major fishing regions in the South China Sea (Xu et al. 2021). Additionally, a four-year (2015–2018) survey of demersal fish communities of the South China Sea revealed a remarkably diverse assemblage of over 250 fish species inhabiting its northern continental slope waters (Zhang et al. 2022). While there is no definitive current estimate, experts estimate that there are > 3,700 fish species across the entire South China Sea basin, reflecting diverse neritic, oceanic and coral reef habitats (Pauly and Liang 2020). However, our understanding of this exceptional biodiversity, especially of the highly diverse fish community, remains insufficient. Several factors contribute to the exceptional marine fish diversity observed in this region. The wide range of habitats, including over 160,000 km2 of coral reefs, mangroves, and estuaries, provides unique ecological niches that promote population differentiation and ultimately speciation over time (Burke and Selig 2002). This habitat heterogeneity supports and accelerates divergence among fish assemblages (Burke and Selig 2002). Complex circulation patterns and seasonal monsoons also enhance productivity and connectivity between populations within this area (Fang et al. 2010). Nonetheless, major knowledge gaps remain regarding the taxonomy, biogeography, ecology, and population genetics of South China Sea fishes. Many cryptic and undescribed species are likely to exist (Hou et al. 2018; Mat Jaafar et al. 2012; Puckridge et al. 2013). Connectivity patterns between regional fishery stocks are poorly resolved, hampering spatial management (Pauly and Liang 2020). In addition, there is a lack of data on vulnerable and data-deficient species within this important region (Huang et al. 2016). Thus, targeted biodiversity assessments, DNA barcoding, and range-wide ecological studies are urgently needed to better characterise and conserve the marine biodiversity in this region. Over the past two decades, DNA barcoding has emerged as a formidable tool for species identification and biodiversity assessment. By analysing a short sequence from a standardized region of the genome, typically the mitochondrial cytochrome oxidase I gene (COI) in animals, DNA barcoding facilitates rapid, precise, and cost-effective species identification (Hebert et al. 2003b). The generation of DNA barcodes (speciesspecific sequences) provides diagnostic markers that supplement classical morphological taxonomy and accelerate taxonomic identification and discoveries, but they are not intended to replace it (DeSalle et al. 2005). Its efficacy in delimiting marine and freshwater fishes has been demonstrated in regional barcoding studies, including those conducted in the Indian Ocean (Lakra et al. 2011), among Indo-Pacific coral reef fishes (Hubert et al. 2012), Indonesian freshwater species (Hubert et al. 2015), and in the west coast of PM (Zainal Abidin et al. 2021). When coupled with morphological techniques, barcoding offers comprehensive and reliable insights into biodiversity (Ward et al. 2009), unveiling cryptic and potentially novel species (Mat Jaafar et al. 2012; Seah et al. 2017; Zainal Abidin et al. 2021). Leveraging on these advancements, this study aims to elucidate the marine fish diversity within Peninsular Malaysia’s east coast EEZ by integrating DNA barcoding and morphological identification. The findings contribute to building a comprehensive genetic reference database of local fish diversity, serving as a valuable resource for future environmental DNA (eDNA) metabarcoding assessments – an increasingly important method for monitoring marine biodiversity (Alshari et al. 2021; Zainal Abidin et al. 2022). By providing a robust genetic baseline, our study enables effective utilization of eDNA techniques to track changes in fish communities and assess impacts of page 2 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan environmental factors or management strategies. We delve into the genetic diversity of regional fish populations, offering valuable insights to facilitate their preservation and sustainable management. Ultimately, this study underscores the power of amalgamating traditional and contemporary methodologies to decode complex marine biodiversity. It establishes a comprehensive framework, encompassing genetic reference data and morphological records, to guide future research and decision-making processes for conserving and managing Malaysia’s marine biodiversity and fishery resources. MATERIALS AND METHODS Sample collection A total of 475 fish specimens were collected during demersal surveys conducted from May to July 2016 in the Exclusive Economic Zone (EEZ) along the East Coast of PM (ECPM). The surveys were organized by the Department of Fisheries Malaysia with the use of bottom trawls onboard the research vessel MV SEAFDEC II. Sampling was performed at 41 stations distributed within the EEZ (Fig. 1). Samples were collected using a bottom trawl with a 40 mm Fig. 1. Map showing the locations of survey stations where fish samples were collected within Peninsular Malaysia’s Exclusive Economic Zone (EEZ) in the South China Sea. N page 3 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan cod end mesh net. The trawl sampling lasted for 60 minutes at a speed of 3.2 knots, covering 3 nautical miles. Information on sampling locations (geographical coordinates), collection data, taxonomy and details of voucher specimens can be found in the online project dataset implemented in the Barcode of Life Database (BOLD) under the project code ‘DBEEZ’. Sample processing and morphological identification Fresh specimens were immediately photographed and tissue sampled for DNA analysis. Fin clips were preserved in 90% ethanol for DNA extraction. Whole voucher specimens were fixed in 10% formalin for one week before long-term storage in 70% ethanol. All specimens were catalogued and deposited at the South China Sea Repository and Reference Centre, Universiti Malaysia Terengganu. Initial morphological identification of specimens utilised established taxonomic keys (Carpenter and Niem 1999a b 2001a b). An initial species checklist was built based on the morphological identification. Species names were verified based on Eschmeyer’s Catalogue of Fishes (Fricke et al. 2023). Ordinal and familial classifications follow van der Laan et al. (2023). Details on the specimens and species identified in this study are provided in table 1. Where possible, at least three specimens per morphospecies were selected for DNA analysis to capture intraspecific morphological variability. DNA analyses Genomic DNA was isolated from specimens using the standard phenol-chloroform extraction protocol (Sanbrook et al. 2001). DNA purity and concentration were quantified with a microvolume UV spectrophotometer (Quawell Q300, Quawell, CA) and stored at -20°C until further use. A ~650 bp fragment of the mitochondrial COI gene was PCR amplified using universal teleost primers by Ward et al. (2005): FishF1-5'TCAACCAACCACAAAGACATTGGCAC-3', FishF2-5'-TCGACTAATCATAAAGATATCGGCAC-3', FishR1-5'-TAGACTTCTGGGTGGCCAAAGAATCA-3' and FishR2-5'-ACTTCAGGGTGACCGAAGAATCAGAA-3'. Each sample was amplified in a final volume of 25 µL, containing 5.5 µL of 5x MyTaq™ Reaction Buffer Red (Bioline GmbH, Germany), 0.5 µL of each primer (100 ng/µL), 0.25 µL 5U Taq polymerase (iNtRON Biotechnology Inc., Korea), 2.5 µL of genomic DNA (50 ng/µL) and adequate nuclease-free water to complete the final reaction volume. Thermal cycling conditions were: initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 30 sec, 48°C for 50 sec, and 72°C for 1 min, ending with a final extension at 72°C for 10 min. Negative controls lacking DNA template were included. Amplified PCR products were visualized by 2% agarose gel electrophoresis. Successful amplicons were purified and Sanger sequenced bidirectionally by a commercial provider (Apical Scientific Sdn. Bhd.) using the ABI PRISM 3730XL automated sequencer and the ABI PRISM BigDye terminator cycle sequencing kit v3.1 (Applied Biosystems, Foster City, CA). Phylogenetic reconstruction and automatic species delimitation The chromatogram traces from each sequenced sample were visually inspected before alignment in Geneious Prime v2030.1.2 (Biomatters Ltd., Auckland, NZ). The forward and reverse sequences were proofread, aligned, and examined for any deletions, insertions, or stop codons using the same software. A total of 215 cytochrome c oxidase subunit I (COI) barcode sequences were generated in this study. Complete data are accessible under the project ‘DBEEZ: DNA Barcoding – EEZ Offshore Demersal Survey’ in the Barcode of Life Database (BOLD) (Ratnasingham and Hebert 2007). To evaluate taxon discrimination, pairwise genetic distances were calculated within and between species, genera, and families utilizing the Kimura 2-parameter (K2P) substitution model (Kimura 1980) in the BOLD analysis tools. The barcode gap analysis was computed for all sequences excluding singleton species, within the same BOLD analysis platform. Phylogenetic relationships were inferred using Bayesian Inference (BI) in BEAST 2 (Bouckaert et al. 2014) and Maximum Likelihood (ML) in raxmlGUI (Edler et al. 2021). IQ-TREE v2.2.0 (Kalyaanamoorthy et al. 2017) identified the General Time Reversible with empirical base frequencies, invariant sites and gamma distribution with 4 categories (GTR+F+I+G4) as the optimal evolutionary model for our dataset, as implemented in the CIPRES portal (Miller et al. 2011). The BI analysis employed a relaxed molecular clock and birth-death tree with empirical base frequencies and four gamma categories. Two independent Markov Chain Monte Carlo (MCMC) runs of 40 million generations were sampled every 1000 generations, discarding the first 20% as burn-in. Convergence was assessed in Tracer v1.7.2 (Rambaut et al. 2018) (ESS > 200) before combining runs in LogCombiner, as integrated in the BEAST 2 package. The final BI tree was constructed in page 4 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan Table 1. Morphologically identified fish species (OTU) from the survey of demersal fishes in the Exclusive Economic Zone (EEZ) of PM. This table lists the common name, sample/museum ID, Barcode of Life Database (BOLD) ID for COI gene sequences, number of specimens examined (n) and IUCN conservation status for each morphologically defined OTU ORDER, Family, Species Common Name Sample/ Museum ID BOLD ID nIUCN TORPEDINIFORMES Narcinidae Narcine brevilabiata Shortlip electric ray EEZ 253 DBEEZ122-23 1 VU ACANTHURIFORMES Siganidae Siganus canaliculatus White-spotted spinefoot EEZ 384 DBEEZ036-23 1 LC Siganus fuscescens Mottled spinefoot EEZ 382, EEZ 383 DBEEZ034-23, DBEEZ035-23 2 LC Nemipteridae Nemipterus balinensoides4Dwarf threadfin bream EEZ 119, EEZ 121 DBEEZ108-23, DBEEZ109-23 2 LC Nemipterus bathybius Yellowbelly threadfin bream EEZ 112 DBEEZ107-23 1 LC Nemipterus nematophorus Doublewhip threadfin bream EEZ 032, EEZ 033, EEZ 034 DBEEZ104-23, DBEEZ105-23, DBEEZ106-23 3 LC Nemipterus nemurus Redspine threadfin bream EEZ 411 DBEEZ121-23 1 LC Nemipterus thosaporni Palefin threadfin bream EEZ 030, EEZ 126, EEZ 188, EEZ 189 DBEEZ103-23, DBEEZ110-23, DBEEZ111-23, DBEEZ112-23 4 LC Nemipterus virgatus Golden threadfin bream EEZ 281, EEZ 282 DBEEZ113-23, DBEEZ114-23 2 VU Scolopsis taenioptera Lattice monocle bream EEZ 380, EEZ 381 DBEEZ115-23, DBEEZ116-23 2 LC Scolopsis vosmeri Whitecheek monocle bream EEZ 405, EEZ 406, EEZ 409 DBEEZ118-23, DBEEZ119-23, DBEEZ120-23 3 LC Pentapodus setosus Butterfly whiptail EEZ 397 DBEEZ117-23 1 LC Lutjanidae Lutjanus lutjanus Bigeye snapper EEZ 047, EEZ 049, EEZ 259 DBEEZ137-23, DBEEZ139-23, DBEEZ143-23 3 LC Lutjanus xanthopinnis Yellowfin snapper EEZ 048, EEZ 127, EEZ 128 DBEEZ138-23, DBEEZ141-23, DBEEZ142-23 3 DD Lutjanus vitta Brownstripe red snapper EEZ 052 DBEEZ140-23 1 LC Lethrinidae Gymnocranius elongatus Forktail large-eye bream EEZ 387 DBEEZ145-23 1 LC Gymnocranius griseus Grey large-eye bream EEZ 414 DBEEZ149-23 1 LC Lethrinus genivittatus Longspine emperor EEZ 388, EEZ 389, EEZ 390 DBEEZ146-23, DBEEZ147-23, DBEEZ148-23 3 LC Leiognathidae Photopectoralis bindus Orangefin ponyfish EEZ 169, EEZ 170, EEZ 171 DBEEZ150-23, DBEEZ151-23, DBEEZ152-23 3 NE Haemulidae Diagramma pictum Painted sweetlips EEZ 283, EEZ 284, EEZ 323 DBEEZ165-23, DBEEZ166-23, DBEEZ167-23 3 NE Gerreidae Pentaprion longimanus Longfin mojarra EEZ 199, EEZ 200, EEZ 201 DBEEZ174-23, DBEEZ175-23, DBEEZ176-23 3 LC Chaetodontidae Coradion chrysozonus Goldengirdled coralfish EEZ 291, EEZ 292, EEZ 293 DBEEZ187-23, DBEEZ188-23, DBEEZ189-23 3 LC ANGUILLIFORMES Muraenidae Gymnothorax reevesii4Reeve’s moray EEZ 475 DBEEZ123-23 1 LC Gymnothorax longinquus Yellow-gilled reef-eel EEZ 425 DBEEZ217-23 1 LC AULOPIFORMES Synodontidae Saurida isarankurai1Shortjaw saury EEZ 066, EEZ 068, EEZ 069, EEZ 102, EEZ 264 DBEEZ009-23, DBEEZ010-23, DBEEZ011-23, DBEEZ012-23, DBEEZ019-23 5 LC page 5 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan ORDER, Family, Species Common Name Sample/ Museum ID BOLD ID nIUCN Saurida longimanus Longfin lizardfish EEZ 263 DBEEZ018-23 1 LC Saurida undosquamis Brushtooth lizardfish EEZ 130 DBEEZ013-23 1 LC Synodus hoshinonis4Blackear lizardfish EEZ 165, EEZ 167, EEZ 168 DBEEZ014-23, DBEEZ015-23, DBEEZ016-23 3 LC Trachinocephalus myops Snakefish EEZ 345, EEZ 441, EEZ 442 DBEEZ020-23, DBEEZ021-23, DBEEZ022-23 3 LC BLENNIIFORMES Blenniidae Xiphasia setifer Hairtail blenny EEZ 355, EEZ 416, EEZ 454 DBEEZ203-23, DBEEZ204-23, DBEEZ205-23 3 LC CARANGIFORMES Soleidae Liachirus melanospilus Carpet sole EEZ 079, EEZ 080, EEZ 299, EEZ 300 DBEEZ026-23, DBEEZ027-23, DBEEZ029-23, DBEEZ030-23 4 LC Zebrias zebra3Zebra sole EEZ 275, EEZ 276 DBEEZ028-23, DBEEZ093-23 2 NE Zebrias quagga Fringefin zebra sole EEZ 426, EEZ 427, EEZ 428 DBEEZ031-23, DBEEZ032-23, DBEEZ033-23 3 LC Samaridae Samaris cristatus Cockatoo righteye flounder EEZ 471, EEZ 472, EEZ 473 DBEEZ058-23, DBEEZ059-23, DBEEZ060-23 3 LC Paralichthyidae Pseudorhombus javanicus Javan flounder EEZ 297 DBEEZ094-23 1 LC Pseudorhombus dupliciocellatus Ocellated flounder EEZ 474 DBEEZ096-23 1 LC Pseudorhombus pentophthalmus Fivespot flounder EEZ 298 DBEEZ095-23 1 LC Menidae Mene maculata Moonfish EEZ 016, EEZ 017, EEZ 018 DBEEZ134-23, DBEEZ135-23, DBEEZ136-23 3 NE Cynoglossidae Cynoglossus puncticeps3Speckled tonguesole EEZ 431, EEZ 432, EEZ 433 DBEEZ178-23, DBEEZ179-23, DBEEZ180-23 3 LC Cynoglossus kopsii Tonguesole EEZ 296 DBEEZ177-23 1 Citharidae Laiopteryx novaezeelandiae Yellow-dabbled flounder EEZ 104, EEZ 105, EEZ 106 DBEEZ181-23, DBEEZ182-23, DBEEZ183-23 3 LC Carangidae Seriolina nigrofasciata Blackbanded trevally EEZ 228 DBEEZ193-23 1 LC Turrum coeruleopinnatum Coastal trevally EEZ 229, EEZ 238, EEZ 239 DBEEZ194-23, DBEEZ196-23, DBEEZ197-23 3 LC Uraspis helvola Whitetongue jack EEZ 230 DBEEZ195-23 1 LC CLUPEIFORMES Chirocentridae Chirocentrus dorab Dorab wolf-herring EEZ 021, EEZ 022, EEZ 023 DBEEZ184-23, DBEEZ185-23, DBEEZ186-23 3 NE GOBIIFORMES Gobiidae Oxyurichthys auchenolepis1Scaly-nape tentacle goby EEZ 061, EEZ 062, EEZ 063 DBEEZ168-23, DBEEZ169-23, DBEEZ170-23 3 NE Yongeichthys nebulosus Shadow goby EEZ 455, EEZ 456, EEZ 457 DBEEZ171-23, DBEEZ172-23, DBEEZ173-23 3 LC HOLOCENTRIFORMES Holocentridae Sargocentron rubrum Redcoat EEZ 356 DBEEZ164-23 1 LC Table 1. (Continued) page 6 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan ORDER, Family, Species Common Name Sample/ Museum ID BOLD ID nIUCN KURTIFORMES Apogonidae Rhabdamia sp. Cardinalfish EEZ 084, EEZ 085 DBEEZ210-23, DBEEZ211-23 2 Ostorhinchus nigrocincta Blackbelt cardinalfish EEZ 082 DBEEZ209-23 1 NE Ostorhinchus fasciatus Broadbanded cardinalfish EEZ 001, EEZ 154, EEZ 155, EEZ 156 DBEEZ207-23, DBEEZ212-23, DBEEZ213-23, DBEEZ214-23 4 LC Jaydia truncata Flagfin cardinalfish EEZ 013 DBEEZ208-23 1 NE LOPHIIFORMES Ogcocephalidae Halieutaea stellata2Smoky seabat EEZ 247, EEZ 248, EEZ 267 DBEEZ100-23, DBEEZ101-23, DBEEZ102-23 3 LC Lophiidae Lophiomus setigerus5Blackmouth angler EEZ 278 DBEEZ144-23 1 LC PERCIFORMES Uranoscopidae Uranoscopus cognatus Two-spined yellow-tail stargazer EEZ 096, EEZ 097, EEZ 231 DBEEZ001-23, DBEEZ002-23, DBEEZ003-23 3 NE Caesionidae Dipterygonotus balteatus Mottled fusilier EEZ 257 DBEEZ017-23 1 LC Synanceiidae Choridactylus multibarbus Orangebanded stingfish EEZ 334, EEZ 347 DBEEZ023-23, DBEEZ024-23 2 LC Inimicus cuvieri Longsnout stinger EEZ 420 DBEEZ025-23 1 NE Serranidae Epinephelus areolatus Areolate grouper EEZ 241, EEZ 242, EEZ 244, EEZ 245 DBEEZ038-23, DBEEZ039-23, DBEEZ040-23, DBEEZ041-23 4 LC Epinephelus sexfasciatus Sixbar grouper EEZ 234, EEZ 367, EEZ 368 DBEEZ037-23, DBEEZ045-23, DBEEZ046-23 3 LC Epinephelus heniochus* Bridled grouper EEZ 245-A No barcode, only voucher specimen Diploprion bifasciatum Barred soapfish EEZ 319, EEZ 320, EEZ 321 DBEEZ042-23, DBEEZ043-23, DBEEZ044-23 3 LC Cephalopholis boenak Chocolate hind EEZ 419 DBEEZ047-23 1 LC Scorpaenidae Brachypterois serrulata Sawcheek scorpionfish EEZ 136, EEZ 137, EEZ 138 DBEEZ048-23, DBEEZ049-23, DBEEZ050-23 3 NE Scorpaenopsis neglecta Yellowfin scorpionfish EEZ 333, EEZ 336, EEZ 338 DBEEZ051-23, DBEEZ052-23, DBEEZ054-23 3 LC Pterois russelli Plaintail turkeyfish EEZ 337, EEZ 339 DBEEZ053-23, DBEEZ055-23 2 LC Neomerinthe procurva Curvedspine scorpionfish EEZ 354 DBEEZ057-23 1 NE Synanceiidae Ablabys sp. Waspfish EEZ 346 DBEEZ056-23 1 NE Platycephalidae Kumococius rodericensis Spiny flathead EEZ 093 DBEEZ064-23 1 LC Kumococius sp. EEZ 225, EEZ 226 DBEEZ068-23, DBEEZ069-23 2 Rogadius pristiger Thorny flathead EEZ 422, EEZ 423 DBEEZ074-23, DBEEZ075-23, 2 LC Rogadius sp. EEZ 094, EEZ 095, EEZ 424 DBEEZ065-23, DBEEZ066-23, DBEEZ076-23 3 Thysanophrys chiltonae Longsnout flathead EEZ 358, EEZ 359, EEZ 360 DBEEZ070-23, DBEEZ071-23, DBEEZ072-23 3 LC Insidiator macracanthus Large-spined flathead EEZ 421 DBEEZ073-23 1 LC Elates ransonnettii Dwarf flathead EEZ 449, EEZ 450, EEZ 451 DBEEZ077-23, DBEEZ078-23, DBEEZ079-23 3 NE Pinguipedidae Parapercis bicoloripes Sandperch EEZ 053, EEZ 054, EEZ 055, EEZ 059, EEZ 060 DBEEZ080-23, DBEEZ081-23, DBEEZ082-23, DBEEZ083-23, DBEEZ084-23 5 NE Table 1. (Continued) page 7 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan ORDER, Family, Species Common Name Sample/ Museum ID BOLD ID nIUCN Parapercis filamentosa Threadfin sandperch EEZ 348, EEZ 436, EEZ 437 DBEEZ087-23, DBEEZ091-23, DBEEZ092-23 3 NE Parapercis displospilus Doublespot grubfish EEZ 349, EEZ 350 DBEEZ088-23, DBEEZ089-23 2 NE Parapercis xanthozona Yellowbar sandperch EEZ 287, EEZ 332, EEZ 415 DBEEZ085-23, DBEEZ086-23, DBEEZ090-23 3 LC Labridae Iniistius evides Blackspot razorfish EEZ 042, EEZ 043, EEZ 044, EEZ 285, EEZ 288, EEZ 290 DBEEZ155-23, DBEEZ156-23, DBEEZ157-23, DBEEZ161-23, DBEEZ162-23, DBEEZ163-23 6 LC Iniistius trivittatus Blue-razor wrasse EEZ 036, EEZ 037 DBEEZ153-23, DBEEZ154-23 2 DD Choerodon typus Blue-banded wrasse EEZ 144, EEZ 145, EEZ 146 DBEEZ158-23, DBEEZ159-23, DBEEZ160-23 3 LC Apistus carinatus Ocellated waspfish EEZ 446, EEZ 447 DBEEZ215-23, DBEEZ216-23 2 LC SCOMBRIFORMES Ariommatidae Ariomma indicum Indian driftfish EEZ 232 DBEEZ206-23 1 NE SILURIFORMES Plotosidae Plotosus lineatus Striped eel catfish EEZ 177, EEZ 178, EEZ 179 DBEEZ061-23, DBEEZ062-23, DBEEZ063-23 3 NE SYNGNATHIFORMES Mullidae Upeneus moluccensis6Goldband goatfish EEZ 269, EEZ 270, EEZ 271 DBEEZ127-23, DBEEZ128-23, DBEEZ129-23 3 LC Upeneus sulphureus Sulphur goatfish EEZ 224, EEZ 215, EEZ 216, EEZ 217 DBEEZ067-23, DBEEZ124-23, DBEEZ125-23, DBEEZ126-23 4 LC Upeneus tragula Freckled goatfish EEZ 444 DBEEZ130-23 1 LC Centriscidae Centriscus scutatus Grooved razor-fish EEZ 304, EEZ 305, EEZ 306 DBEEZ190-23, DBEEZ191-23, DBEEZ192-23 3 LC Callionymidae Dactylopus dactylopus Fingered dragonet EEZ 352, EEZ 353 DBEEZ198-23, DBEEZ199-23 2 LC Callionymus recurvispinnis Belcher’s dragonet EEZ 460, EEZ 461, EEZ 462 DBEEZ200-23, DBEEZ201-23, DBEEZ202-23 3 NE TETRAODONTIFORMES Tetraodontidae Lagocephalus suezensis Pufferfish EEZ 210, EEZ 211, EEZ 212 DBEEZ004-23, DBEEZ005-23, DBEEZ006-23 3 LC Torquigener gloerfelti Pufferfish EEZ 403, EEZ 404 DBEEZ007-23, DBEEZ008-23 2 LC Ostraciidae Ostracion nasus Shortnose boxfish EEZ 398, EEZ 399, EEZ 402 DBEEZ097-23, DBEEZ098-23, DBEEZ099-23 3 NE Monacanthidae Paramonacanthus pusillus Faintstripe filefish EEZ 149, EEZ 150, EEZ 151 DBEEZ131-23, DBEEZ132-23, DBEEZ133-23 3 LC IUCN - LC: Least Concern; VU: Vulnerable; NE: Not Evaluated; DD: Data Deficient. 1 New record in Malaysia. 2 New record in Peninsular Malaysia (PM). 3 New record in East Coast of PM. 4 New specimen-based record in Malaysia. 5 New specimen-based record in PM. 6 New specimen-based record in East Coast of PM. *Morphospecies without COI sequence (barcode). Table 1. (Continued) page 8 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan TreeAnnotator (Rambaut and Drummond 2013). The ML analysis was performed using 1000 rapid bootstrap replicates under the GTR+I+G model. Resulting phylogenies were visualized and edited in FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). Five different sequence-based methods were used to delimit the Molecular Operational Taxonomic Units (MOTUs) (= putative species) from our dataset - (1) Refined Single Linkage (RESL), (2) Automatic Barcode Gap Discovery (ABGD), (3) Assemble Species by Automatic Partitioning (ASAP), (4) Generalized Mixed Yule Coalescent (GMYC), and (5) Bayesian Poisson Tree Process (bPTP). Employing five different automatic molecular species delimitation methods, each with distinct underlying assumptions, enabled the exploration of the reliability of MOTU partitioning in this study (Luo et al. 2018). The first analysis was done within the BOLD platform using the RESL algorithm (Ratnasingham and Hebert 2013) to assign sequences to dedicated Barcode Index Numbers (BIN). Next, the ABGD (Puillandre et al. 2012) analysis was run using the webserver (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb. html) to census divergence within the analysed dataset for species delimitation. The ABGD analysis was run with the following settings: relative gap width X = 1.0, intraspecific divergence (P) values range from 0.001 to 0.0059 for all distance metrics, while all other parameter values were kept as default. The ASAP (Puillandre et al. 2021) analysis was performed on https://bioinfo.mnhn.fr/abi/public/asap/ asapweb.html using the Kimura 2-parameter (K2P) substitution model. ASAP calculates a score for different MOTU partitioning scenarios above a threshold of 3% genetic distance. Lower ASAP scores indicate more optimal MOTU partitioning. Finally, both GMYC (Pons et al. 2006) and bPTP (Zhang et al. 2013) methods were employed with the fully resolved, BI ultrametric tree using only unique haplotypes (see above for the reconstruction method). The haplotype dataset was built in collapsing all 215 individual COI sequences into 181 unique haplotype sequences using FaBox v1.61 (Villesen 2007). A single-threshold GMYC analysis was run in RStudio (Allaire 2012) with the ‘splits’ package (Fujisawa and Barraclough 2013). The bPTP analysis was performed on the GMYC web server (https:// speciesh-its.org/gmyc/). The final species delimitation scheme was determined based on the consensus of the five molecular delimitation methods. Species initially identified by morphological characteristics are referred to as morphospecies (operational taxonomic unit = OTU), while putative species delimited by DNA barcodes are referred to as molecular operational taxonomic units (MOTUs). To further investigate potential cryptic diversity in three OTUs showing deep mitochondrial divergences - Rogadius pristiger, Kumococius rodericensis, and Upeneus sulphureus - we compiled an expanded barcode sequences (COI) dataset incorporating publicly available verified sequences from the Barcode of Life Data System (BOLD). Additional sequences were sourced across the Indo-West Pacific, including Indonesia, Vietnam, Bangladesh, South China, Australia, and the United Arab Emirates (Fig. 5). Figure 5 provides the BOLD IDs and GenBank accession numbers for all utilised sequences. Phylogenetic reconstruction on this dataset was performed using maximum likelihood (ML) analysis with the previously described methods. Incorporating publicly available barcodes provides geographical context and allows comparison to our OTU lineages to delineate species boundaries and evolutionary relationships. RESULTS Fish diversity This study obtained partial (~650 bp) mitochondrial cytochrome c oxidase subunit I (COI) sequences from 217 out of 475 specimens collected during demersal trawl surveys across the East Coast PM Exclusive Economic Zone (EEZ). The successfully sequenced specimens represented 92 morphological species belonging to 68 genera, 41 families, 16 orders, and two classes – Elasmobranchii and Actinopterygii (Table 1). One morphospecies, the bridled grouper (Epinephelus heniochus), failed to be barcoded but was retained in the final species checklist (Table 1). In the initial morphological identification, all species were identified to species level based on morphological taxonomy, except Rhabdamia sp. and Ablabys sp. which could only be identified to the genus level. The order Perciformes displayed the highest species richness with 26 species, accounting for 28.2% of the total species identified. Acanthuriformes and Carangiformes were the next most speciose orders with 21 species (22.8%) and 14 species (15.2%), respectively (Fig. 2; Table 1). Within Perciformes, Platycephalidae and Scorpaenidae exhibited the greatest diversity with five species each, followed by Pinguipedidae and Serranidae with four species per family. The family Nemipteridae of the order Acanthuriformes contained the highest number of species at nine. Based on International Union for Conservation of Nature (IUCN) Red List assessments, two species in our dataset were classified as vulnerable - the shortlip electric ray (Narcine brevilabiata) and the golden threadfin bream page 9 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan all sequences within lineage 4 (L4) are designated as Rogadius sp. (Table 1). In the second case, the expanded dataset of ten K. rodericensis specimens, including three of our barcodes, formed three distinct COI lineages when analyzed with BOLD sequences (Fig. 5b). One of our barcodes was clustered with specimens from the South China Sea and the Vietnamese coast, while the other two interestingly formed a common lineage with an unidentified spiny flathead sample (i.e., Kumococius sp.) from Vietnam. The third lineage (L3; n = 4) included sequences from the coast of Bangladesh and the South China Sea. Despite minimal within-lineage variation (0–1.0%), substantial inter-lineage divergences up to 23.5% were observed (Table S1), indicating cryptic diversity that is morphologically undetectable within this dataset. Such intraspecific polyphyly despite similar morphology likely reflects taxonomic underestimation of species number in hyperdiverse tropical ecosystems (Hubert et al. 2012). Unlike previous barcoding studies on tropical marine fishes (e.g., DiBattista et al. 2016; Steinke et al. 2009), our flathead lineages do not exhibit allopatric distributions. The overlapping distributions of our lineages instead reject the hypothesis that they simply represent geographically isolated populations with independent evolutionary trajectories. Sympatric cryptic taxa likely reflect incomplete taxonomy and limitations of traditional morphological diagnoses rather than just biogeographic variance (Steinke et al. 2009). Consequently, after careful consideration, all sequences within lineage 2 (L2) are designated as Kumococius sp. (Table 1). The commercially exploited platycephalid flatheads (Perciformes: Platycephalidae) of the IndoPacific exemplify the challenges of coping with long-standing fishing pressure in tropical ecosystem due to obscured cryptic lineages (Imamura and Knapp 2009; Puckridge et al. 2013). The substantial mitochondrial divergences within the flathead OTUs likely signify genuine overlooked diversity rather than anomalous variation, congruent to cryptic diversity patterns reported in other studies. For instance, DNA barcoding uncovered approximately 10% divergence between western and eastern Australian forms of the morphospecies Platycephalus marmoratus (Puckridge 2006), providing robust evidence of an unrecognized species that was subsequently described as P. orbitalis (Imamura & Knapp, 2009). Another comprehensive assessment of P. indicus across the Indo-West Pacific revealed eight remarkably divergent lineages separated by up to 16.37% genetic distance, unveiling extensive cryptic diversity (Puckridge et al. 2013). The large intraspecific divergences correspond to geographically isolated lineages, reflecting recognised biogeographic barriers in the studied region (Puckridge et al. 2013). Moreover, multiple taxonomic work on platycephalids has focused on Australian waters, delineating new flathead species in the region (Imamura 2007; Imamura and Gomon 2010; Knapp and Imamura 2004). This further supports the likelihood that the distinct genetic lineages uncovered in our study may represent undescribed cryptic species within the currently recognized taxa. Expanded analysis of the Upeneus sulphureus OTU included 11 COI sequences, with four from our dataset (Fig. 5c). Three phylogenetic lineages were observed, with three of our barcodes clustering with South China Sea samples in lineage 2 (L2), reflecting a shared genetic pool on a regional scale. Intriguingly, this lineage only diverged by 0.7% from the Australian and Indonesian sequences in lineage 1 (L1) (Table S1, Fig. 5c), despite broad geographical separation. However, one barcode formed a divergent lineage, clustering with an uncertain Upeneus cf. sulphureus specimen from Indonesia at 3.0% distance from other lineages. Similar slightly elevated within species divergence (2.02%) was also documented in U. sulphureus from China (Zhang et 2011). Although intraspecific divergence was moderate in this OTU (2.67%, Table 3), comparison against publicly available data provided a biogeographical context to infer probable cryptic diversity within our specimens. OTU – MOTU discordance Several factors contribute to the discordances between morphospecies (OTUs) and molecular operational taxonomic units (MOTUs) observed in this study. The taxonomic impediment disproportionately impacts the megadiverse tropical ecosystems, where extreme diversity coupled with limited taxonomists results in incomplete alpha taxonomy (Bini et al. 2006). Furthermore, the slower turnover and older age of tropical marine species increase the manifestation of ancestral polymorphisms, leading to deep mitochondrial divergence despite taxonomic integrity (Hubert and Hanner 2015; Rabosky et al. 2018). As seen here, such polyphyly has been attributed to cryptic speciation in fishes, specifically within the tropical South China Sea, validating the barcoding method in identifying known species and highlighting potential new ones (Landi et al. 2014; Lara et al. 2010; Ward et al. 2008). Our integrated taxonomic framework, combining morphology, DNA barcoding, and phylogeography, elucidates evolutionary lineages and biogeography to uncover overlooked diversity. Incorporating verified data from public sources such as the Barcode of Life Data System (BOLD) and GenBank facilitates insight into tropical page 16 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan biodiversity across spatial scales (Sholihah et al. 2020; Zainal Abidin et al. 2022). Although DNA barcoding is not a substitute for morphological taxonomy, it is a powerful supplementary tool for identifying species and guiding future taxonomic research. Our study lays the groundwork for such investigations into the drivers of cryptic diversity patterns in Malaysian demersal fishes. Towards the establishment of a comprehensive DNA barcoding library of the fish community in the EEZ of Peninsular Malaysia Accurate identification of organisms is essential for assessing ecosystem status which is now well acknowledged to require the integration of morphological and molecular techniques (Bourlat et al. 2013). DNA barcoding enables rapid biodiversity assessment, although it is contingent upon availability of comprehensive libraries for comparative analysis (Ward et al. 2005). Hebert et al. (2003a) highlighted the efficacy of the mitochondrial COI gene for delineating species boundaries, suggesting its discriminatory power could enable species identification from sequences alone. Our study highlights the ability of barcoding to reveal potential cryptic diversity, although a formal downstream taxonomic analysis is still required. This study provides the first comprehensive DNA barcoding assessment of demersal fishes in the waters of Peninsular Malaysia’s Exclusive Economic Zone (EEZ) in the South China Sea. Characterising biodiversity in this vast but under-researched marine region is critical for national monitoring and management of fisheries resources, as over 50% of the species examined are commercially exploited (DoF 2000 2018). Under the United Nations Convention on the Law of the Sea (UNCLOS), EEZs which extend 200 nautical miles define the marine areas and sovereign rights of the coastal states over the resources (Poling 2013). Safeguarding EEZ biodiversity and ecosystems is therefore crucial for Malaysia’s national food security, economy, and heritage. DNA barcoding relies on constructing comprehensive reference libraries to enable sequence-based species identification (Ratnasingham and Hebert 2007). While public databases like BOLD and GenBank accelerate insights, localised curated repositories provide more practical foundations tailored to regional biota (Bemis et al. 2023; Zainal Abidin et al. 2021). For instance, our Malaysia-focused library better elucidates biogeographic patterns in these demersal fishes. Beyond species discovery, such resources have diverse applications from food authentication to conservation (Chin et al. 2016; Zainal Abidin et al. 2022). Ongoing barcoding efforts should engage taxonomists to integrate multiple lines of evidence for robust species delimitation, especially in understudied tropical ecosystems. Our study establishes an integrative taxonomic framework combining morphology, DNA barcoding, and phylogenetics to elucidate cryptic diversity, thus facilitating species discovery, and provide insights into evolutionary lineages among Malaysian demersal fishes. The 92 morphospecies and distinct mitochondrial lineages uncovered highlight underestimated diversity and represent candidate species for description. The curated DNA barcode library provides a foundation for conservation and sustainable use of these commercially valuable fish stocks (Knebelsberger et al. 2014). As climate change, overfishing, and other stressors rapidly impact tropical marine biodiversity, continuous assessments are imperative (Pecl et al. 2014). Comprehensive barcoding surveys like this research are crucial for monitoring, managing, and protecting Malaysia’s invaluable marine living resources. CONCLUSIONS This study uses an integrative taxonomic approach that combines DNA barcoding and morphological identification to elucidate fish diversity in the Exclusive Economic Zone (EEZ) of Peninsular Malaysia’s east coast. We reassessed 475 demersal fishes comprising 92 putative species and 16 orders, including two vulnerable IUCN species. The DNA barcoding cytochrome c oxidase subunit I (COI) gene revealed 95 consensus Molecular Operational Taxonomic Units (MOTUs) for all automated delimitation methods. Interestingly, several MOTUs within a morphospecies had over 2% intraand interspecific genetic divergence, indicating either deep intraspecific variation or cryptic species. These results highlight the complexity of species delimitation and the value of genetic methods. Our study provides important insights into east coast fish diversity, improves understanding of genetic distribution and conservation needs, and creates a comprehensive framework combining barcoding and morphology to inform future research and management strategies for Malaysia’s marine biodiversity. The expanded genetic barcode database will facilitate ongoing and future molecular taxonomic studies of Malaysian ichthyofauna. Overall, this first large-scale analysis of east coast PM demersal fishes demonstrates the ability of barcoding to illuminate diversity and reveal hidden divergences indicative of reproductive isolation and cryptic speciation. Our contribution to public databases enables species identification by experts and laypeople alike, and offers a wide range of potential applications. page 17 of 21Zoological Studies 63:30 (2024) © 2024 Academia Sinica, Taiwan Further taxonomic research is warranted, as evidenced by the knowledge gaps highlighted in this study. Acknowledgments: We thank the Department of Fisheries Malaysia for the opportunity to participate in the 2016 National Demersal Trawl Survey and for facilitating the sample collection. We also thank the SEAFDEC Training Department in Bangkok for their assistance with sample collection during the survey. Finally, we wish to extend our appreciation to Mrs. Salwani Abdullah and Mr. Muhammad Wan Hanafi, whose assistance in sample collection and processing made this work possible. Danial Hariz Z.A. was supported by a postdoctoral fellowship (JPNP. AUPE002) from Universiti Sains Malaysia (August 2023–March 2024). Availability of data and materials: Voucher specimens are available as described in the text. All the COI sequences determined in this study have been uploaded in BOLD under the public project – DBEEZ: DNA Barcoding EEZ Offshore Demersal Survey and deposited in GenBank (Accession nos. OR918571– OR918786; Table S2). Authors’ contributions: DHZA designed experiments, conducted analyses, data visualisation, and wrote the manuscript. SAMN and TNAMJ provided funding, participated in data collection and analysis. TNAMJ, JAFJ, MSA, YGS, MAR, NSZ, MPT, and KMZ collected specimens, performed morphological identification and laboratory analysis. 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Comparison of K2P intraspecific (bold values) and interspecific genetic divergences of OTUs separated by lineages. The italicised values are the standard error. (download) Table S2. List of GenBank accession numbers with associated information including sample/museum ID, Barcode of Life. (download) page 21 of 21Zoological Studies 63:30 (2024)