Using DNA Barcodes to Aid the Identification of Larval Fishes in Tropical Estuarine Waters (Malacca Straits, Malaysia)

© 2019 Academia Sinica, Taiwan Open Access Using DNA Barcodes to Aid the Identification of Larval Fishes in Tropical Estuarine Waters (Malacca Straits, Malaysia) Cecilia Chu1, Kar Hoe Loh1, Ching Ching Ng2, Ai Lin Ooi3, Yoshinobu Konishi4, Shih-Pin Huang5, and Ving Ching Chong2,* 1Institute of Ocean and Earth Sciences, University of Malaya, Kuala Lumpur, Malaysia. E-mail: [email protected] (Chu); [email protected] (Loh) 2Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. *Correspondence: E-mail: [email protected]. E-mail: [email protected] (Ng) 3Department of Agricultural and Food Science, Faculty of Science, Universiti Tunku Abdul Rahman, Kampar, Perak. E-mail: [email protected] 4Seikai National Fisheries Research Institute, Nagasaki, Japan. E-mail: [email protected] 5Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan. E-mail: [email protected] Received 3 April 2019 / Accepted 18 August 2019 / Published 18 October 2019 Communicated by Benny K.K. Chan Larval descriptions of tropical marine and coastal fishes are very few, and this taxonomic problem is further exacerbated by the high diversity of fish species in these waters. Nonetheless, accurate larval identification in ecological and early life history studies of larval fishes is crucial for fishery management and habitat protection. The present study aimed to evaluate the usefulness of DNA barcodes to support larval fish identification since conventional dichotomous keys based on morphological traits are not efficient due to the lack of larval traits and the rapid morphological changes during ontogeny. Our molecular analysis uncovered a total of 48 taxa (21 families) from the larval samples collected from the Klang Strait waters encompassing both spawning and nursery grounds of marine and estuarine fishes. Thirty-two (67%) of the larval taxa were identified at the species level, two taxa (4%) at the genus level, and 14 taxa (29%) at family level. The relatively low rate of species-level identification is not necessarily due to the DNA barcoding method per se, but a general lack of reference sequences for speciose and noncommercial fish families such as Gobiidae, Blenniidae, and Callionymidae. Larval morphology remains important in species diagnoses when molecular matches are ambiguous. A lower ethanol percentage (50%) for larva preservation is also useful to keep the body of larvae intact for morphological identification, and to preserve DNA for subsequent molecular analyses. The 10% Chelex resin used to extract DNA is also costeffective for long term monitoring of larval fishes. Hence, the DNA barcoding method is an effective and easy way to aid the identification of estuarine larval fishes at the species level. Key words: Molecular identification, Morphology, Fish larvae, Coastal fishes, Mangrove-associated. Citation: Chu C, Loh KH, Ng CC, Ooi AL, Konishi Y, Huang SP, Chong VC. 2019. Using DNA barcodes to aid the identification of larval fishes in tropical estuarine waters (Malacca Straits, Malaysia). Zool Stud 58:30. doi:10.6620/ZS.2019.58-30. BACKGROUND One major reason why studies of fish larval in the tropics are not progressing as fast as in temperate regions is the problem of larval fish identification. In the Indo-Pacific region, recent publications on fish larvae descriptions are now more accessible since the period prior to the first Indo-Pacific Fish Conference held in 1981 (Leis 2015). Tropical larval fish descriptions for marine and coastal fishes are very few; notable ones include Chayakul (1990) for Gulf of Thailand, Shadrin et al. (2003) for Vietnamese waters, and Leis and CarsonEwart (2004) for the 124 families that occur in the IndoPacific region; this last publication is the compilation Zoological Studies 58: 30 (2019) doi:10.6620/ZS.2019.58-30 1 © 2019 Academia Sinica, Taiwan PENINSULAR MALAYSIA SUMATRA, INDONESIA SOUTH CHINA SEA Klang Strait Matang mangrove forest Buloh River Janggut River Sementa River mangroves 1 3 6 10 Depths in fathom KEY sandbanks exposed at low tide 3 km Kapar power plant of work done by Leis and Rennis (1983) and Leis and Trnski (1989), and an extension of Leis and CarsonEwart (2000). More recently, Konishi et al. (2012) provided an identification guide for 92 families of larval fishes of the important marine capture species that occur in Southeastern Asian. Even so, the taxonomic problem is further exacerbated by the high diversity of fish species in tropical marine waters, which include several unique habitats, making it more difficult to distinguish congenerics within a family. For instance, fish larvae of more than 100 families have been reported in Thai waters (Janekarn and Kiørboe 1991); in the Banda Sea, Indonesia, 78 families of larval fishes were collected (Soewito and Schalk 1990). In Malaysian marine and brackish waters, as many as 1481 species have been recorded, with as many as 250 being considered as marine euryhaline species (Chong et al. 2010). Liew (1992) managed to identify 61 taxa of larval fishes, mostly at the family level, that was collected from the Straits of Malacca and South China Sea. Noticeably, the Malaysian estuarine coastal area has fewer families of larval fishes; 19 families in the Matang mangrove and adjacent waters (Ooi and Chong 2011); 24 families in the seagrass-mangrove area in Gelang Patah, Johor (Ara et al. 2013); and 20 families in the mangrove estuarine of Marudu Bay, Sabah (Rezagholinejad et al. 2016). From the archived ichthyoplankton samples collected in the Klang Strait, 23 families of larval fishes were identified (Chu, unpublished). The Klang Strait on the west coast of Peninsular Malaysia is a narrow, 70-km long shipping channel flanked by coastal mudflats to its right and a large sand-mud shoal on its left (Fig. 1). Several deltaic islands covered by mangrove forests enclose the strait’s southern flank, breaking up its southern approach into several narrow channels. Evidently, the more offshore waters at the northern approach of Klang Strait are spawning areas for fish and prawns, while the coastal mangroves, mudflats, and estuaries serve as their nursery or feeding areas (Chong and Sasekumar 1981; Chong et al. 1990). Sarpedonti and Chong (2008) used a schematic diagram to show that two engraulid species, Stolephorus baganensis and Thryssa kammalensis, use the Klang Strait as a spawning and feeding around; the former species spawn in more offshore waters before moving into the estuaries as postflexion larvae, whereas the latter species spawn closer to the shore but move further upstream until both species reach the juvenile stage and emerge from the estuaries again to feed in the near inshore waters. Since fish movements performed during ontogenetic development are species-specific, the migratory routes used by other larval species may N Fig. 1. Map of sampling location (left) and enlarged inset box (right) showing five sampling stations (black circles) along the Klang Strait. Right arrow indicates offshore direction of transect line from Kapar power plant. page 2 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan be different. So, unless the larval taxonomy is resolved, the migratory routes used by many of the larval fishes in the strait during their larval development will remain unanswered. Previously, Sarpédonti et al. (2000) identified two engraulid species, S. baganensis and T. kammalensis, based on the morphology of their digestive systems. But for most other larval taxa, identification is mostly done at the family, subfamily, or genus level (Leis and Carson-Ewart 2004). This is because some features such as fins and scales are not fully developed, so there are not enough characters to diagnose at the species level. Therefore, conventional dichotomous keys used to identify adult fish are not so useful for fish larvae identification due to too few characters and the often rapid morphological changes during ontogenesis (Leis 2015). With the advent of molecular techniques, DNA barcodes and other gene markers have been used to differentiate or confirm the identity of larval fish species (Hubert et al. 2010; Wibowo et al. 2015; Azmir et al. 2017; Aoyama et al. 2018) and crustacean larvae (e.g., Chen et al. 2013; Wong et al. 2014) after morphological identification. The DNA technique transcends the limitation posed by morphological diagnoses because the molecular identities of adults are enough to identify all other ontogenetic stages using readily available DNA barcodes (Hubert et al. 2015). As DNA barcodes are increasingly being deposited into accessible databases such as GenBank and BOLD (Barcode of Life Data system), more species of larval fishes can now be identified by matching DNA sequences. Because of this, DNA barcodes have been used to link larvae to their adult species to answer questions concerning their early life stages (Valdez-Moreno et al. 2010). In other cases, DNA barcoding is also able to reveal cryptic species within the family (Mat Jaafar et al. 2012; Dahruddin et al. 2016). However, while the goal of barcoding is to accumulate more DNA barcodes, these barcodes must come from specimens that are accurately identified (Hubert et al. 2008). The availability of accurate DNA barcodes in adults is thus crucial to the accurate identification of their larval counterparts. In light of the new direction that fish larva identification is taking, further collections of fish larvae are necessary for molecular identification using DNA barcoding. The present study thus aimed to evaluate the usefulness of DNA barcodes to support the use of existing morphological traits for larval fish identification. Among the common tropical fish taxa, subsets of the diverse families of Gobiidae and Engraulidae, typical of mangroves and tropical estuaries, were tested. Problems and limitations that arise from using this method are also discussed. Additionally, molecular identification using larval specimens were further refined by comparing methods for larval preservation and DNA extraction used by past researchers. The best and most cost-effective methods for preserving fish larvae, extracting high DNA output, and processing large quantities of larvae were evaluated. MATERIALS AND METHODS Collection and preservation of larval samples Plankton samples were collected at five sampling stations along an 18-km transect line from Kapar power plant toward the offshore direction of the Klang Strait (Fig. 1). The samples were collected by twin bongo nets with mouth openings of 45-cm diameter, and mesh sizes of 180 µm and 363 µm. Day-samplings were carried out in June and July of 2015, and January, March, and June of 2016. At each station, the net was hauled obliquely by a moving trawl boat for 10 minutes at an approximate speed of 1.5 knots. In initial trials, collected plankton samples were immediately preserved in 99%, 80%, and 50% HPLC grade ethanol to determine the best concentration of ethanol that will keep the larval body intact, whilst preserving its DNA for molecular analysis. Since 50% ethanol showed the best results for all larval stages, subsequent samples were preserved in this ethanol concentration. In the laboratory, all preserved samples were kept in a -20°C freezer until being sorted. Sorting and morphological identification of fish larvae Plankton samples were thawed at room temperature. Then, fish larvae and early juveniles were sorted under a stereomicroscope (Leica M125) connected to an imaging system (Leica Application Suite v4.10). For subsequent molecular analysis, all sorted specimens were kept in individual vials containing 50% ethanol, and individually photographed and measured using the imaging system. Preand flexion larvae were measured for notochord length (NL, mm), whereas the postflexion larvae and early juveniles were measured for their total length (TL, mm). The vials were labelled and stored at -20°C. Before DNA extraction, the specimens were identified to the lowest possible taxon by their morphological characters using key descriptions found in Leis and Carson-Ewart (2004) and/or Okiyama (2014). Extraction and amplification of DNA One eye of each specimen was used for DNA extraction. For preflexion larvae, the whole body was page 3 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan used to extract DNA because there was only a small amount of eye tissue. DNA was extracted using 10% Chelex resin following the procedure found in Hyde et al. (2005), with some modification. Either an eye or a small amount of tissue was inserted into a tube containing 150 µl of 10% Chelex resin (Bio-Rad) in distilled water, and the sample was first heated at 60°C for 2 minutes then at 103°C for 25 minutes. The heated tube containing the sample was then left at room temperature to cool down. PCR-amplification of the Chelex-isolated DNA ensued after cooling down, or stored at -20°C pending PCR. Amplification of the partial cytochrome c oxidase subunit I (COI) was done using the barcoding primers and thermal program described in Ward et al. (2005). A 20 µl PCR reaction was prepared using dry Maxime PCR PreMix (iNtRON Biotechnology) by adding 1 µl of template DNA, 0.5 µl of each primer (10 µM), and 18 µl of UV-distilled water. Successful PCR products were outsourced to First Base Laboratory Private Limited (Malaysia) for purification and sequencing. Initially, samples were sequenced in both the forward and reverse direction; but later sequencing was only done in the forward direction because of the robustness of the sequences obtained. DNA sequence analysis Raw DNA sequences were checked and edited using Sequence Scanner v1.0 (Applied Biosystem) to confirm the correct base calling. Then, the trimmed sequences were searched against GenBank (https://blast. ncbi.nlm.nih.gov/) and BOLD (http://www.boldsystems. org/) to find molecular matches. Molecular matches to larval sequences are listed in table S1. Reference sequences nearest to the subject were downloaded from GenBank and aligned to the targeted sequence using MEGA v.6 (Tamura et al. 2013). The genetic distances were then calculated using the Kimura 2-parameter model, and a 3% threshold for species delineation was used, as suggested by Hebert et al. (2003). A species name was only assigned to the target sequence if it was corroborated with the morphological identification i.e., the genus/family of the specimens. The steps for acquiring the final larval identification are shown in figure S1. Adult collection Adult fish specimens were collected from local landing sites and fish markets, and directly from bag net catches from the fishing villages of the Janggut Buloh, and Sementa rivers (Fig. 1). The first collection was made in September 2017, and a second in October 2018. Additional tissue samples from 22 species of Gobiidae and three species of Eleotridae were borrowed from the Biodiversity Research Center, Academia Sinica, Taiwan; the fishes were previously collected by Huang et al. (2013), mostly from the Matang mangrove forest located approximately 250 km north of the main study area. The adults were identified to the species level and photographed, and tissues were collected and preserved in 95% ethanol. DNA extraction, amplification, sequencing, and sequence analysis were done according to the same methods described above. COI sequences of adults were used to match the sequences of larvae through phylogenetic analysis using MEGA v.6 (Tamura et al. 2013). RESULTS Effects of larval preservation in ethanol The fish larvae preserved directly in 99% ethanol just after collection by bongo net were badly distorted in form or damaged, so that their morphological characters were almost impossible to recognize. However, larvae kept in 50% ethanol were intact. Larger larvae at postflexion or the juvenile stage could be kept in 80% ethanol without body distortion. Essentially, samples preserved in 50% ethanol yielded sufficient DNA concentration, i.e., between 7–68 ng/µl for subsequent analysis. Larval composition A total of 671 larval fish were collected, consisting of preflexion, flexion and post-flexion larvae, and early juveniles. Initial identification based on morphology recorded the families of Clupeidae (n = 225), Blenniidae (n = 105), and Ambassidae (n = 98) as the most abundant; together, they accounted for at least 63.8% of the total larval fish collection. Families of Engraulidae (n = 51), Sciaenidae (n = 40), and Gobiidae (n = 34) were moderately abundant and contributed about 18.7% of the total larval collection. The other 8.0% of the larvae consisted of several families, with fewer than 10 individuals collected per family: Sillaginidae (n = 9), Mugilidae (n = 8), Callionymidae (n = 6), Apogonidae (n = 5), Carangidae (n = 5), Tetrarogidae (n = 5), Polynemidae (n = 3), Platycephalidae (n = 3), Scatophagidae (n = 2), Soleidae (n = 2), Stromateidae (n = 2), Hemiramphidae (n = 1), Gerreidae (n = 1), Cynoglossidae (n = 1), and Triacanthidae (n = 1). Damaged specimens made up the last 9.5% of the total larval collection. page 4 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan Species assignment through DNA barcodes Of the total larvae collected, only 250 individuals that ranged from 2.0 mm to 46.0 mm TL and representing the various families and ontogenetic stages were selected for molecular identification. The COI gene was successfully amplified in 193 individuals (77%) using the barcoding primers. The 57 samples that failed to amplify were from the families Sciaenidae (n = 14), Blenniidae (n = 13), Gobiidae (n = 9), Engraulidae (n = 7), Ambassidae (n = 5), Callionymidae (n = 3), Tetrarogidae (n = 2), Apogonidae (n = 1), and Scatophagidae (n = 1). The COI sequences from larvae ranged from 546–710 bp, and consisted of 48 taxa belonging to 21 families in 7 orders (Table 1). About 67%, or 32 out of the 48 taxa, were identified to the species level, whereas 4% (two taxa) were identified to the genus level, and 29% (14 taxa) were identified to the family level. Despite being the most abundant, the families Clupeidae and Blenniidae contained only two species each. These two families, together with Ambassidae, each consisted of one abundant species. In contrast, the families Engraulidae and Gobiidae contained the highest number of taxa, six and 13, respectively, in spite of being only moderately abundant. Family Sciaenidae, also from the moderately-abundant group, had only two successful amplifications out of the 16 extracted samples; the two successfully-amplified samples came from two species. The intra-species divergence among larval sequences ranged from 0.0% to 2.2%, whereas the inter-species divergence started from 4.8% to 36.8%. For families with two or more species, the intra-family divergence ranged slightly higher at 1.7% to 32.7%. Interestingly, the families Sillaginidae, Engraulidae, and Gobiidae with three, six and 13 taxa, respectively, had smaller intra-family divergence of 14.6%, 15.7%, and 19.8%, respectively, whereas the families Soleidae and Sciaenidae each with two taxa had higher intrafamily divergence at 29.3% and 32.7%, respectively. The smallest inter-family divergences was between Ambassidae and Apogonidae with 19.3%, and the largest was between Clupeidae and Cynoglossidae at 34.6% (phylogenetic trees can be found in figures S2– S6). Clupeidae The larval specimens of Clupeidae could be differentiated into two species based on their monophyletic groupings. The first monophyletic group consisted of three larval specimens that matched the adult of Anodontostoma chacunda with an intraspecies divergence of 0.4%. Sequences of the other 42 larval specimens of Clupeidae matched the reference sequences of the engraulid species Stolephorus indicus by 99% (KX223955) and 98% (FJ238040 and EU595310), and also to a clupeid species Escualosa thoracata by 98% (AP011601 and MH429324). They are clupeid larvae because they have a long gut of which the anus is located posterior to the dorsal-fin base (Fig. 2), so matches to S. indicus were dismissed as wrong identifications; these 42 specimens were identified as E. thoracata. Engraulidae Among the 13 larval sequences of the Table 1. List of the identified larval fish specimens and their GenBank accession number Order Family Species nAccession No. Beloniformes Hemiramphidae Hyporhamphus quoyi 1MH673896 Clupeiformes Clupeidae Anodontostoma chacunda 3 MH673897 –MH673899 Escualosa thoracata 42 MH673906 –MH673947 Engraulidae Coilia dussumieri 2MH673900 –MH673901 Coilia sp. 1 1MH673902 Stolephorus commersonii 1MH673903 Stolephorus dubiosus 2MH673904 –MH673905 Stolephorus insularis 1MH673948 Stolephorus tri 6MH673949 –MH673954 Mugiliformes Mugilidae Paramugil parmatus 7 MH673955 –MH673961 page 5 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan Order Family Species nAccession No. Osteomugil cunnesius 1MH673962 Perciformes Ambassidae Ambassis gymnocephalus 34 MH673963 –MH673996 Apogonidae Ostorhinchus fasciatus 4MH673997 –MH674000 Blenniidae Blenniidae sp. 1 1MH674001 Blenniidae sp. 2 28 MH674002 –MH674029 Callionymidae Callionymidae sp. 1 3 MH674030 –MH674032 Carangidae Alepes djedaba 1MH674033 Alepes kleinii 4MH674034 –MH674037 Gerreidae Gerres limbatus 1MH674038 Gobiidae Acentrogobius cyanomos 6MH674039 –MH674044 Hemigobius hoevenii 1MH674045 Parapocryptes serperaster 1MH674046 Tridentiger barbatus 1MH674047 Trypauchen sp. 1 1MH674048 Gobiidae sp. 1 1MH674049 Gobiidae sp. 2 2MH674050 –MH674051 Gobiidae sp. 3 1MH674052 Gobiidae sp. 4 1MH674053 Gobiidae sp. 5 1MH674054 Gobiidae sp. 6 1MH674055 Gobiidae sp. 7 6MH674056 –MH674061 Gobiidae sp. 8 2MH674062 –MH674063 Polynemidae Eleutheronema tetradactylum 3 MH674064 –MH674066 Scatophagidae Scatophagus argus 1MH674067 Sciaenidae Pennahia anea 1MH674068 Johnius carouna 1MH674069 Sillaginidae Sillago asiatica 1MH674072 Sillago sihama 4MH674073 –MH674076 Sillaginidae sp. 1 2MH674070 –MH674071 Stromateidae Pampus argenteus 1MH674077 Pampus minor 1MH674078 Pleuronectiformes Cynoglossidae Cynoglossus lingua 1MH674079 Soleidae Zebrias zebra 1MH674080 Soleidae sp. 1 1MH674081 Scorpaeniformes Platycephalidae Kumococius rodericensis 2MH674082 –MH674083 Platycephalidae sp. 1 1MH674084 Tetrarogidae Tetraroge barbata 3 MH674085 –MH674087 Tetraodontiformes Triacanthidae Trixiphichthys weberi 1MH674088 TOTAL: 7 Orders, 21 Families, 48 taxa (n = 193) Table 1. (Continued) page 6 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan Engraulidae, 10 matched the adult sequences of Coilia dussumieri (two matches), Stolephorus dubiosus (two matches), and Stolephorus tri (six matches) with an intra-species divergence of 1.1%, 1.6%, and 0.2%, respectively. Two other specimens each belonged to Stolephorus commersonii and Stolephorus insularis based on matches with reference sequences. The last specimen has a long, distinctly tapering tail (Fig. 4c) which is a specific character for the genus Coilia. Since no molecular matches were found on either databases, the genus Coilia was applied based on its morphology and was named Coilia sp. 1. Inter-species divergence between all six species ranged from 13.5% (between Coilia sp. 1 and C. dussumieri) to 24.3% (between S. dubiosus and S. insularis). Ambassidae All 34 sequences of the ambassid larvae matched the adult sequences of Ambassis gymnocephalus collected from Janggut River, with an intra-species divergence of only 0.2%. But morphologically the ambassid larval specimens in this study had at least three morphotypes based on pigmentation on the top of the head (Fig. 3). Blenniidae Specimens recognized as belonging to the Blenniidae could be distinguished as Blenniidae sp. 1 or Blenniidae sp. 2 based on their monophyletic groupings as well as the pigmentation along the analfin base; Blenniidae sp. 2 (Fig. 4b) was less pigmented at all ontogenetic stages compared to Blenniidae sp. 1 (Fig. 4a). Both types of specimens showed similar characteristics to tribe Omobranchini by having pigmentation on the head and pectoral fin, and along the anal-fin base, and a pair of long preopercular spines that decreases as the fish grows (Leis and CarsonEwart 2004). Intra-species divergence of Blenniidae sp. 2 was 0.3%, and inter-species divergence between Blenniidae sp. 1 and Blenniidae sp. 2 was large at 28.1%. Blenniidae sp. 1 initially matched to Cirripectes stigmaticus (KX223895), but the identification was doubtful since the latter did not cluster with other reference sequences of C. stigmaticus (Fig. S3); the sequence divergence between KX223895 and the Cirripectes group was 18.4%. Similarly, the initial matches to Blenniidae sp. 2 with 99% similarity were to Lutjanus apodus (KX223917, KX223918) and Acentrogobius sp. (KX144848), but both were dismissed as wrong identifications. Thus, neither taxa of the Blenniidae were identified, and they were named Blenniidae sp. 1 and Blenniidae sp. 2 following the morphological identification of their family. Gobiidae Out of the 13 taxa recognized for the family Gobiidae, four taxa were identified as Acentrogobius cyanomos, Hemigobius hoevenii, Parapocryptes serperaster, and Tridentiger barbatus based on sequence matches with an intra-species divergence of 0.4%, 0.7%, 0.8% and 0.8%, respectively. The larval Fig. 2. Ontogenetic series of E. thoracata at preflexion (a); flexion (b, c); postflexion (d, e); early juvenile (f). 6.7 mm NL (a) 8.2 mm NL (b) 9.4 mm NL (c) 15.3 mm TL (d) 18.5 mm TL (e) 19.0 mm TL (f) page 7 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan sequence that was embedded between Trypauchen pelaeos and Trypauchen vagina with an inter-species divergence of 7.0% and 10.0%, respectively, could be another species of Trypauchen, and was thus named Trypauchen sp. 1. Morphologically, the larval specimen showed the characteristics of the subfamily Amblyopinae as described by Leis and Carson-Ewart (2004) which contains the genus Trypauchen. The eight other taxa of gobiid larvae were unidentified because no close matches (98–99% similarity) were found, but they formed monophyletic groupings with less than 3% intra-species divergence. Gobiidae sp. 1, Gobiidae sp. 2, and Gobiidae sp. 3 seemed to be embedded within the species complex of Mahidolia mystacina; the nearest inter-species divergence of 4.8% was between Gobiidae sp. 2 and Gobiidae sp. 3, and the largest was between Gobiidae sp. 1 and Gobiidae sp. 3 with 21.0% divergence. Gobiidae sp. 4 was nearest to another gobiid species Odontamblyopus rubicundus, with an interspecies divergence of 17.1%. Gobiidae sp. 5 showed 13.9% divergence from Gobiidae sp. 6, whereas the latter was very close to Parachaeturichthys polynema (4.2% divergence). Gobiidae sp. 7 had the largest intraspecies divergence of 2.2% among all recognized taxa, although they seemed to consist of two very closely related taxa; the distance between these two branches was 3.4%, but since they had similar morphology and pigmentation (Fig. 4o, 4p), they were both classified as Gobiidae sp. 7. Lastly, Gobiidae sp. 8 was only distantly related to Scartelaos gigas (12.4% divergence). Other families One larval sequence of the Sillaginidae matched the adult sequence of Sillago asiatica, with an intraspecies divergence of 0.3%, whereas four other larval sequences were identified as Sillago sihama, with 0.4% divergence. The remaining two sillaginid sequences were only distantly related to Sillago ingenuua, with 13.4% divergence. All specimens of the families Mugilidae, Apogonidae, Sciaenidae, and Stromateidae were identified based on matches with their adult sequences, i.e., the mugilid Paramugil parmatus and Osteomugil cunnesius, the apogonid Ostorhinchus fasciatus, the sciaenid Pennahia anea and Johnius carouna, and the stromateid Pampus argenteus and Pampus minor with an intra-species divergence of 0.4%, 0.2%, 0.2%, 0.4%, 1.0%, 0.2%, and 0.5%, respectively. Specimens of the families Hemiramphidae (Hyporhamphus quoyi), Carangidae (Alepes djedaba and A. kleinii), Gerreidae (Gerres limbatus), Polynemidae (Eleutheronema tetradactylum), Scatophagidae (Scatophagus argus), Cynoglossidae (Cynoglossus lingua), Soleidae (Zebrias zebra), Platycephalidae (Kumococius rodericensis), Tetrarogidae (Tetraroge barbata) and Triacanthidae (Trixiphichthys weberi) were identified based on matches with their reference sequences. The last three unidentified taxa, which were identified solely on morphology, were Callionymidae sp. 1 (three specimens), Soleidae sp. 1 (one specimen), and Platycephalidae sp. 1 (one specimen). DISCUSSION Sample preservation Plankton samples with fish larvae are normally preserved in buffered 4% formalin in seawater immediately after collection. But for molecular Fig. 3. Three degrees of pigmentation on the top of head of A. gymnocephalus larvae; heavy pigment (a); moderate pigment (b); sparse pigment (c, d). 4.8 mm TL (a) 5.2 mm TL (c) 7.5 mm TL (b) 7.5 mm TL (d) page 8 of 15Zoological Studies 58: 30 (2019) © 2019 Academia Sinica, Taiwan Fig. 4. Unidentified taxa. Blenniidae sp. 1 (a); Blenniidae sp. 2 (b); Coilia sp. 1 (c); Callionymidae sp. 1 (d); Sillaginidae sp. 1 (e); Soleidae sp. 1 (f); Platycephalidae sp. 1 (g). 5.2 mm NL (a) 4.0 mm NL 5.7 mm NL 5.3 mm NL 8.6 mm TL 9.0 mm TL 14.7 mm TL (b) 4.0 mm NL (g) 5.5 mm NL (f) 8.1 mm TL (e) 5.7 mm TL (d) 19.2 mm TL (c) page 9 of 15Zoological Studies 58: 30 (2019)