Morphological and genetic evaluation of the thumbprint emperor, Lethrinus harak (Forsskål, 1775) in the Pacific and Indian Oceans

Ethical statement

This research was conducted for three months (November 25, 2019 – February 24, 2020) under the Enhancing International Publication Program conducted by the Directorate for Human Resource Qualifications Directorate General of Resources for Science, Technology & Higher Education Ministry of Research, Technology and Higher Education of the Republic of Indonesia in collaboration with the University of Miyazaki. This study complied with relevant ethical regulations in Japan and Indonesia. In Indonesia, the doctoral research proposal of the first author was assessed and accepted by an academic panel. This assessment included compliance with institutional ethical guidelines in vigour at Universitas Hasanuddin. A letter of acceptance was issued by the Head of the Laboratory of Fisheries Sciences, Faculty of Agriculture, Miyazaki University, permitting the research activities at the University of Miyazaki's Division of Fisheries Science Museum (MUFS). The issuance of this acceptance was based on the research proposal, including compliance with institutional ethical guidelines in vigour at Miyazaki University. The use of the samples in this study did not require specific ethical approval for the following reasons: all fish specimens used were already dead when they were acquired; there was no use of live animal specimens; specimens obtained from fish port auction houses had been captured by legal fisheries and were obtained following all applicable regulations; the IUCN Red List assessment lists Lethrinus harak in the Least Concern category (not considered at risk of extinction); this study was based on field research (surveys) with no experimental component.

Fish specimens

Specimens of L. harak were selected as paratypes at the Marine Biology Laboratory, Faculty of Marine Science and Fisheries, Universitas Hasanuddin (MSFUH), and the Division of Fisheries Science, Miyazaki University. A total of three specimens for morphometric measurements namely MSFUH000591, MSFUH0000592, MSFUH0000593, and only one specimen is taken for DNA barcoding (MSFUH000591), collected from the Makassar Strait (-4°58’N, 119°17’17’’E), were obtained from the Paotere fish auction site, Makassar City, South Sulawesi, Indonesia. The samples were placed in a coolbox with ice for transport to the Marine Biology Laboratory, Universitas Hasanuddin, Makassar. The samples were cleaned under running water to remove any dirt and organic matter attached the exterior of the specimens. Each sample was placed on its right-hand side and sterilised using alcohol. A fin-clipping of around 1.8cm in length was taken from the pectoral fin and inserted into a 2 ml micro tube 2 filled with 90% alcohol. Each sample was numbered and labelled according to the Marine Science and Fisheries Universitas Hasanuddin (MSFUH) catalogue number system, preserved in 10% formalin and stored in the laboratory collection.

The seven specimens measured in Japan formed part of the pre-existing fish collection of the University of Miyazaki Division of Fisheries Science Museum (MUFS). These specimens had been collected from Yonashiro, Uruma, and Okinawa, Japan, with catalogue and sample numbers based on the cataloguing system of Miyazaki University Department of Fisheries Science (MUFS): MUFS 43290, MUFS 4417, MUFS 6025, MUFS 8819, MUFS 12632, MUFS 2098, and MUFS 6014. One specimen had been collected from Meitsu, Nago, Miyazaki, Japan (MUFS 16284) and one from the Philippines (MUFS 6136) (Figure 1).

Figure 1. Specimens of Lethrinus harak.

A. Specimen JN311937, paratype, SL 170mm, collected from Arumbai Fish Market (-3°68’N, 128°18’E), Ambon, Indonesia, 06 January 2016 (photographed by Limmon, reproduced from BOLD under a CC BY-NC-SA 3.0 license). B. Specimen MUFS43290, paratype, SL 131mm, from the MUFS (Miyazaki University, Fisheries Sciences) museum collection (photographed by the authors). C. Specimen MSFUH000591, paratype, SL 217mm, collected in the Makassar Strait (-4°58’N, 119°17’17’’E), 20 January 2019 (photographed by the authors – Muhammad Afrisal). D. Specimen HM423533, paratype, SL 219mm, collected from Lizard Island (-14°66’N, 145°44’E), Queensland, Australia, 07 September 2008 (photographed by the Australian Museum team, Sydney, modified from BOLD under a CC BY-NC-SA 3.0 license). E. Specimen HQ561476, paratype, collected in Mozambique (-22°84’N, 35°55’E), 15 November 2010 (photographed by Connell, modified from BOLD under a CC BY-NC-SA 3.0 license). F.Specimen MH331781, holotype, SL 200mm, collected from Thuwal, Mecca, Saudi Arabia (22°30’N, 39°09’N), 22 March 2017 (photographed by catta, modified from GBIF under a CC BY-NC 4.0 license). G.Specimen JQ350086, paratype, SL 190mm, collected from Nosy Tanikely-West, Quest (-13°48E, 48°24N), Antananarivo, Madagascar, 07 May 2008, (photographed by Planes et al., 2008, modified from BOLD (no rights reserved)).

The preceding samples were used in this study to represent the Pacific Ocean. The samples used to represent the Indian Ocean were downloaded from the Global Biodiversity Information Facility (GBIF) and The Barcode of Life Data System (BOLD): KS66045, HQ561476, MH331781, dan JN311937. Measurements of these remotely sourced specimens were made using ImageJ version 1.52a software¹⁴, except for the interorbital width character, which could not be measured from the downloaded photographs. The morphological characters were compared with reference to data from three holotypes: MNHN 9087, holotype of L. azureus; RMNH 5758, holotype of L. rhodopterus; and BMNH 1873.3.160, holotype of L. bonhamensis¹⁵. Measurement of morphometric characters using conventional methods by following the holotype character L. harak included standard length, body depth, head length, pectoral length, pelvic length, orbital length, interorbital width, snout length, suborbital width, upper jaw length.

DNA extraction

DNA extraction specimen Makassar was carried out using DNeasy Blood and Tissue Kit (Qiagen, Cat. No. 69504) following the manufacturer’s protocols. Fin clippings (1.8cm) were taken from the pectoral fin of each specimen using surgical scissors or a surgical scalpel. Each sample was preserved in a labelled 2ml tube filled with 95% alcohol.

A sub-sample weighing 0.025mg was taken from each fin clipping sample and placed in a 2mL tube to which 180µL of ATL solution (tissue lysis buffer) and 20µL of proteinase K were added. The tubes were vortexed and incubated at 56°C for 1–3 hours or overnight. Then 200µL AL buffer (lysis buffer) solution and 200µL ethanol were added before vortexing and centrifuging at 8000rpm for 1 minute. The supernatant was transferred to a new tube, 500 µL AW1 (column wash buffer 1) was added, and the tube was centrifuged for 1 minute at 8000rpm. This step was repeated with AW2 (column wash buffer 2) and centrifuged at 14,000rpm for 1 minute. The column was transferred to a new 1.5mL tube and 100μL warm AE buffer was added, then centrifuged for 1 minute, incubated for 10–15 minutes, and centrifuged at 6000×g (8000rpm) for 1 minute. RNAse was added to the extracted DNA solution and incubated for 24 hours. The extracted DNA was prepared for use through dilution, at a ratio of 30µL DNA and 70µL ddH₂O.

Electrophoresis

Agarose gel was prepared by mixing 1.4g of agarose 0.8% in 180mL of 1x TAE buffer solution. A colouring agent (1.5μl GelRed) was added to the gel while still warm and liquid before pouring it into the mould. Aliquots of 2μL DNA template were pipetted and mixed with 1μl of DNA loading dye solution and loaded into the wells in the agarose gel. Electrophoresis was performed at 100 volts for 90 minutes. The results were visualised using an ultraviolet transilluminator (biostep Dunkelhaube, bi000052).

DNA amplification

The DNA barcode analysis used a segment of mtDNA from the cytochrome b oxidase subunit 1 (COI) gene. The target sequences were amplified using the primer pair developed by Ward et al.¹⁶: Fish F1-5’ TCA ACC AAC CAC AAA GCA TTG GCAC 3 (forward) and Fish R1-5’ TAG ACT TCT GGG TGG CCA AGA ATCA 3 (reverse). Each reaction tube contained 5µL of PCR mix Hotstart (Cat. No. 203645, QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany), 3µL DNA template, 3µL ddH2O, and 0.675µL each of the forward and reverse primers. The PCR amplification procedure (Labcycler Gradient, 012-102, Sensoquest) comprised the following stages: initial denaturation at 95°C for 5 minutes; 35 cycles with denaturation at 94°C for 1 minute, annealing at a primer-specific temperature for 1 minute, elongation at 72°C for 1 minute; and final elongation at 72°C for 10 minutes. The amplified PCR products were stored in a freezer (-20°C) until the separation process. The annealing temperature followed a gradient of ±5°C compared to the reference temperature for the primer pair, in order to determine the optimal annealing temperature, resulting in a bright and sharp band under electrophoresis. PCR products that have been tested positive for a length of 682 bp, then proceed with the sequencing of nucleotides to obtain DNA sequences. The sequencing was carried out at the company 1st Base Asia, Singapore using BigDye® Terminator ver.3.1 cycle sequencing KIT and an automated HiSeq-X sequencing machine. The primers used for the sequencing process are the same as the primers used for the PCR amplification process.

Data analysis

Principal component analysis (PCA) was used to analyse the morphometric data, and was implemented in MINITAB version 17¹⁷. The quality of the DNA sequences obtained was evaluated using the software Sequence Scanner 2.0 (Applied Biosystems, USA). The sequence data obtained from this study were complemented by additional sequences downloaded from the National Center for Biotechnology Information (NCBI) and DNA Data Bank of Japan (DDJB) databases (accession numbers: HQ561476 Mozambique; KF489627 South Africa; JQ350086 Madagascar; MH331781 Saudi Arabia; JF952781 Japan; JN311937 Indonesia; HM423533 Australia). The forward and reverse sequence data were combined using the software Bioedit version 7.2.6.1¹⁸. The sequences were trimmed and aligned using the ClustalX¹⁹ routine in MEGA 6²⁰. The phylogenetic topology reconstruction was implemented in MEGA 6 with the neighbour-joining method and genetic distance was analysed using the compute within groups mean distance option.

Although PCA in this research was implemented in MINITAB, the free software JASP²¹ could be used as an alternative, although we could not guarantee total equivalence between the two software packages. Genetic analyses in this study were implemented using Sequence Scanner 2.0, ClustalX 2.1 and MEGA 6.

Morphological characters

The PCA analysis shows that the distribution of morphological characters of individual L. harak specimens (see Table 1) indicates a general pattern of close relationships within two population groups: those in the Pacific Ocean and those in the Indian Ocean (Figure 2). Holotype specimens used in this research (Bonham, New Ireland, Singapore) tend to be grouped to the right above the X axis (Figure 2).

Figure 2. Distribution of morphological characters of Lethrinus harak populations with respect to the two principal components (axes) of the principle component analysis.

In general, individuals from Pacific Ocean populations, i.e. from Japan (Okinawa, Miyazaki, and Ishigaki), Indonesia (Makassar and Ambon), the Philippines, and Australia, are predominantly grouped in the upper quadrants of the PCA graph (Figure 2).

Populations from the Indian Ocean were mostly spread across the lower left quadrant, i.e. South Africa, Mozambique, and Saudi Arabia, with the Seychelles being the only population placed (very low) in the bottom right quadrant. However, the specimens from Tanzania and Madagascar grouped together with the Pacific Ocean populations in the upper right quadrant and had morphometric characters similar to the holotype of L. harak.

Genetic distance and phylogenetic topology reconstruction for L. harak

The genetic distance analysis using the compute within groups mean distance method indicates that, in general, L. harak populations in the Pacific Ocean and the Indian Ocean have a close kinship relationship (Table 2). The molecular analysis in this study shows that the smallest genetic distance between populations in the Indian Ocean (South Africa and Mozambique) is 0.000, while the lowest genetic distance in the Pacific Ocean (between Japan and Indonesia) is 0.004. The lowest genetic distance between populations in the two oceans (Saudi Arabia and Indonesia) is 0.031. Meanwhile the largest genetic distance is 0.042 (between Madagascar and Japan, Makassar, and Australia), and is still included in the low category.

Phylogenetic topology reconstruction was made to confirm the morphological grouping and genetic distance between the Pacific and Indian Oceans. The phylogenetic reconstruction (Figure 3) used the neighbour-joining method with outgroup sequence data obtained from GenBank for species in the genus Lethrinus found in eight populations in the Indian and Pacific Oceans.

Figure 3. Phylogenetic reconstruction for eight Pacific and Indian Ocean L. harak populations, nested within the genus Lethrinus, using the neighbour-joining method with the Kimura 2-parameter model and bootstrapping with 1000 replicates.

The topology of this neighbour-joining phylogenetic reconstruction shows two L. harak population clusters. The fish from Mozambique, South Africa, Madagascar, and Saudi Arabia formed a clade with a bootstrap value of 100%. The second clade comprised populations from Indonesia (Makassar and one other location), Japan, and Australia with a bootstrap value of 99%.

Underlying data

Partial cytochrome C oxidase subunit I (COX1) gene mitochondrial DNA sequence of L. harak (specimen voucher MSFUH0000591) from Makassar waters on GenBank, Accession number MT551656.