The Southeast Asian Journal of Tropical Biology Vol. 31 No. 2, 2024: 238 - 252 DOI: 10.11598/btb.2024.31.2.2143 ISSN: 0215-6334 | e-ISSN: 1907-770X 238 MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF Donax faba (BIVALVIA: DONACIDAE) OBTAINED FROM KUTANG BEACH, LAMONGAN, INDONESIA Reni Ambarwati1*, Fida Rachmadiarti2, Herlina Fitrihidajati2, Tarzan Purnomo2, Dwi Anggorowati Rahayu1, and Ulfi Faizah3 1Animal Systematics Laboratory, Biology Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, Indonesia. 2Ecology Laboratory, Biology Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, Indonesia. 3Animal Systematics Laboratory, Biology Education Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, Indonesia. ARTICLE HIGLIGHTS • This study revealed 19 morphological variations of edible wedge clams, Donax faba (Bivalvia: Donacidae), from Kutang Beach, Lamongan, Indonesia, which has significant role as part of coastal ecosystem. • This reseach also highlights the DNA barcoding of Donax faba based on COI gene (Cytochrome C Oxidase Subunit I). • The average genetic distance of the research samples was 0.46%, while the value of this parameter between the research samples and the ingroup was found to be 1.51%. Article Information Received 5 December 2023 Revised 23 April 2024 Accepted 2 May 2024 *Corresponding author, e-mail: reniambarwati@unesa.ac.id Research Article ABSTRACT Donax faba is a type of wedge clams with significant ecological and economic roles, as well as variations in color, pattern, and morphometric properties. Recently, a particular population of unidentified D. faba was reported from Kutang Beach, Lamongan, Indonesia. Therefore, this research aimed to assess the morphological variations and molecular characteristics of D. faba obtained from Kutang Beach based on COI gene. A total of 288 samples were collected during the lowest tide for morphological characterization of the color, pattern, and morphometrics of the shells. Additionally, molecular characterization was conducted based on the composition of nucleotide bases and amino acids of COI gene, genetic distance, as well as the relationships. The results showed that D. faba had 19 morphological variations, among which the most dominant type contained a whitish exterior with brown spots and a whitish purple interior. The average genetic distance of the samples was 0.46%, while the value was calculated as 1.51% between the samples and the ingroup. Automatic Barcode Gap Discovery (ABGD) analysis performed using a prior maximal distance of 0.001 showed the separation of these species into distinct categories. Keywords: coastal ecosystem, COI gene, dna barcoding, wedge clams INTRODUCTION Donax faba is popularly known as wedge clams living in intertidal areas (Ambarwati & Faizah 2017; Alyani & Ambarwati 2018) along sandy beaches (Yambem et al. 2017; Rittiboon et al. 2019; Signorelli & Printrakoon 2020) and mangrove forests (Singh et al. 2011; Kassim et al. 2018). These clams are harvested in Vietnam and Thailand for the nutritional value (Krishnan & Tharavathy 2016) and trade purposes (Poutiers 1998), while being consumed in several regions of Indonesia including West Java (Dharma 2005) and Madura (Ambarwati & Faizah 2017; Alyani & Ambarwati 2018; Wasilah et al. 2021). Donax faba is capable of accumulating heavy metals (Singh et al. 2012; Wasilah et al. 2021) and serves as a potential source of anticoagulant compounds (Periyasamy et al. 2013). Moreover, the shells can be processed into flour, serving as a mineral source in feedstuff (Lalopua & Sukisman 2023). https://doi.org/10.11598/btb.2024.31.2.2143 Morphological and molecular characterization of Donax faba - Ambarwati et al. 239 Members of the Donacidae family, including D. faba, have been reported with morphological variations. Tan & Low (2013) detected variations in color, shape, and pattern among D. faba samples in Singapore, while Rittiboon et al. (2019) identified eight color patterns from Bangling Beach, Thailand. Dharma (2005) described five types of D. faba shells collected from West Java. Ambarwati and Faizah (2017) as well as Alyani and Ambarwati (2018) reported 12 and 15 respective variations of patterns and colors from Nepa and Tengket Beaches in Madura. Atlanta et al. (2022) recently found some D. faba at Kutang Beach, Lamongan, but morphological variations in this population remain unexplored. High species variations often lead to difficulties in the identification process, which can only be enhanced by the availability of comprehensive information regarding the morphological variations. Additionally, DNA barcoding is among the current valuable tools used to strengthen identification based on morphological data (Moritz & Cicero 2004; Hebert & Gregory 2005; Ferri et al. 2009; Packer et al. 2009). Research across various animal taxa, including Porifera (Cárdenas et al. 2009; Vargas et al. 2012), Echinoderms (Layton et al. 2016), Mollusca (Juniar et al. 2021; Sari et al. 2021), and fish (Rahayu et al. 2019; 2012), had used DNA barcoding markers for identification. COI DNA barcoding was previously applied to successfully identify variations in D. incarnatus from Madura (Wijaya et al. 2023). Additionally, genetic distance analysis based on COI gene sequences of certain samples showed high similarity with comparison species from GenBank and Bold systems. Differences in average genetic distance values can be caused by intragroup genetic diversity. A genetic distance is assumed to be very low when after conversion to less than 2% the values signify the same species. However, values greater than 2% suggest the existence of a species different from other group members (Wong & Hanner 2008; Wong et al. 2009). In an investigation performed by Sari et al. (2021), genetic identification of D. faba from Nepa Beach, Madura showed COI gene sequence of 650 bp with similarity values ranging from 72.01% to 72.12% when compared to sequence data from GenBank. COI gene sequence of D. faba is characterized by a high mutation rate, leading to significant genetic variations and geographic influences on genetic plasticity. Consequently, further research is recommended to determine the molecular characteristics of D. faba. Considering the provided background, this research aimed to assess the morphological variations and molecular characteristics of D. faba samples collected from Kutang Beach based on COI gene in mtDNA, followed by the conduction of phylogenetic relationship analysis. The molecular characterization and phylogenetic analysis data obtained will serve as a foundation for further investigation into the evolutionary history, population dynamics, and adaptation of D. faba in the natural habitat. MATERIALS AND METHODS Field Work Samples of D. faba were collected from Kutang Beach, Lamongan (Fig. 1) during the lowest tide. Five plots measuring 1 m x 1 m were placed horizontally along the beach line with 3 m interval between each plot. Samples for shell morphological analysis were preserved in 70% alcohol, then three from the most dominant shell types were selected and preserved in absolute alcohol for molecular analysis. Laboratory Work Identification and Morphometric Measurements The shells of collected Donax clams were cleaned and identified using the identification books of Poutiers (1998), Dharma (2005), Huber (2010), and Ambarwati & Faizah (2017). Subsequently, morphometric measurements, including shell length (SL), shell height (SH), shell width (SW), dorsal height (DH), dorsal and umbo outline (Fig. 2), were conducted using calipers with an accuracy of 0.01 mm (Ambarwati & Faizah 2017). SL is defined as the perpendicular distance between the anterior and posterior shell, while SH is measured from the highest dorsal to the lowest ventral part of the shell. Furthermore, SW represents the distance between the protruding parts of the lateral sides of two shells. DH is measured from the highest part of the dorsal side to the pseudo line with a perpendicular distance between the anterior and posterior shell. The umbo margin line (UML) is described as the distance from the most dorsal part of the shell to the posterior. BIOTROPIA Vol. 31 No. 2, 2024 240 Figure 1 The map showing the sampling location of D. faba (red dot) in Kutang Beach Figure 2 Morphometric measurements of D. faba Molecular Work (DNA Isolation and Sequencing) Total DNA isolation from muscle tissue was performed using the Isolation Kit (Roche) (Kit catalogue number 05985536190) with several modifications. Initially, 200 µL of Buffer GT1 was pipetted into a 1.5 mL tube and mixed by vortexing. Next, 200 µL GT2 buffer and 20 µL Proteinase K were added, and the mixture was thoroughly combined through vortexing. The blend was incubated for 10 minutes at 56 °C, with gentle inversion of the tube every 5 minutes. Subsequently, 200 µL absolute ethanol was introduced to the mixture and briefly vortexed. The sample was transferred to Spin Column and centrifuged for 1 minute at 13,000 rpm. The resulting flow-through was discarded, and 500 µL buffer W1 was added, followed by another round of centrifugation for 1 minute at 13,000 rpm. After disposing of the flow-through, 700 µL buffer W2 containing ethanol was introduced and centrifuged for 1 minute at 13,000 rpm. The flow- through was discarded again, and centrifugation was conducted for an additional 2 minutes. DNA retained in Spin Column was transferred to a new 1.5 mL tube, then 50-100 µL Elution Buffer was added and incubated at room temperature for 1 minute before being centrifuged for 1 minute. Finally, Spin Column was removed, the purified DNA was prepared for further steps, and DNA was temporarily stored at -20 °C over a few days. The isolation results were then amplified using Biorad PCR machine in a 30 µL solution consisting of 15 µL PCR Master Mix Nexpro, 3 µL DNA Template samples (100 ng/µL), 6 µL water, and 3 µL primers (10 pmol each of forward and reverse primers). The primers used were LCO1490 Morphological and molecular characterization of Donax faba - Ambarwati et al. 241 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198(5'-TAAACTTCAGGGTGACC AAAAAATCA-3') (Folmer et al. 1994). Amplification was performed with the following temperature settings including pre-denaturation at 94 ºC for 1 minute, followed by 40 cycles of denaturation at 94 ºC for 45 seconds, annealing at 45 ºC for 45 seconds, and extension at 72 ºC for 1 minute 30 seconds. Subsequently, the post- elongation process was carried out at a temperature of 72 ºC for 10 minutes. PCR results were electrophoresed on 1% agarose, then sequenced using 1st BASE Laboratories Sdn Bhd sequencing services. Data Analysis Analysis of Morphological and Morphometric Data Morphological data were analyzed descriptively, while samples were classified based on shell color patterns, with the relative frequency of each type calculated as a percentage. Subsequently, the average and standard deviation of each morphometric parameter was estimated. Types comprising a minimum of 10 individuals (n ≥ 10) were analyzed for shell patterns using the ratio of each shell size and the relationship between patterns was evaluated through linear regression. To determine significant differences between types, analysis of variance (ANOVA) was conducted followed by the Games-Howell test. Molecular Analysis Sequence data from GenBank NCBI (National Center for Biotechnology Information) in addition to DNA sequence data obtained from this research was used for phylogenetic analysis (Table 1). DNA sequence readings were used to determine the genetic variations of COI gene, the composition of the nucleotide bases and amino acids of COI gene, as well as the genetic distance, followed by relationship analysis. Moreover, the chromatogram data from the sequencing results were visualized using FinchTV to assess sequence quality. The K2P substitution model (Saitou & Nei 1987) was applied in calculating the settings for ML phylogenetic tree reconstructions. A bootstrap consensus tree inferred from 1,000 replicates was used to describe the variation rates among sites. Furthermore, adjacent branches showed the percentage of replicate trees in which the related taxa clustered together in the bootstrap test (1,000 repetitions). Grouping analysis was carried out through a web interface (Puillandre et al. 2012) to examine the distribution and size of a potential barcoding gap for the partial sequence of COI gene dataset. The barcode gap generated through Automatic Barcode Gap Discovery (ABGD) was used to strengthen the species identification process. Table 1 Sequences from NCBI GenBank used as reference species Num Species Sample location Acc number of genbank NCBI 1. Donax faba Phuket, Thailand MT334596.1 2. Donax faba Prachubkirikhun, Thailand MT334599.1 3. Donax faba Rayong, Thailand MT334600.1 4. Donax faba Japan AB040845.1 5. Donax incarnatus Chantaburi, Thailand MT334591.1 6. Donax incarnatus Phuket, Thailand MT334590.1 7. Donax incarnatus Prachubkirikhun, Thailand MT334593.1 8. Donax incarnatus Rayong, Thailand MT334592.1 9. Donax cuneatus Chantaburi, Thailand MT334594.1 10. Donax cuneatus Phuket, Thailand MT334595.1 11. Donax cuneatus Japan AB040842.1 12 Donax faba type18 Kutang Beach, Lamongan PP593778 (this research) 13 Donax faba type 2 Kutang Beach, Lamongan PP595807 (this research) 14 Donax faba type 11 Kutang Beach, Lamongan PP595808 (this research) BIOTROPIA Vol. 31 No. 2, 2024 242 RESULTS AND DISCUSSION Morphological Characterization During the field trip to Kutang Beach, a population of D. faba clams was observed in the upper intertidal zone at the tidal boundary with a sandy substrate. This habitat type was consistent with previous research, which reported the habitat of D. faba as a sandy substrate (Ambarwati & Faizah 2017; Tenjing 2017; Yambem et al. 2017). The population of D. faba at Kutang Beach reached 57.6 ind./m2 with a total of 288 identified samples. Previous observation (Eshky 1998) along the sandy substrate in the Red Sea showed that the population density of D. faba ranged from 30 to 296 ind./m2, corresponding to the results of this research. The description of D. faba from Kutang Beach, Lamongan is as follows. Shell shape: thick, flat, inequilateral, trigonal oval. Shell length reaches 32.74 mm; shell height reaches 25.64 mm. Shell sculpture: smooth surface with thin concentric lines that become more prominent posteriorly. Umbo: protrude, prosogyrate. Color: white, cream, brown, purple; often with one or more radial bands or broad random patches; the interior of the shell is white, often with yellow shading, there are purplish to purple spots, and/or radial bands. Dentition: heterodont with anterior and posterior lateral teeth. Shell interior: anterior adductor muscle attachment site elongated and posterior adductor muscle attachment site rounded; deep pallial sinus (approximately ½ shell length); pallial line is clear (Figs. 3 & 4). This description corresponds to the reports of Tan and Low (2013), Ambarwati and Faizah (2017), and Signorelli and Printrakoon (2020). The population of D. faba showed high morphological variations, including different interior and exterior shell color patterns, as well as shell morphometry. Samples collected at Kutang Beach had 19 variations in interior and exterior color (Figs. 3 & 4; Table 2). Variations found with the highest relative frequency were types 18, 2, and 11 (Fig. 5). Particularly, type 18 contained a white shell exterior and a whitish purple interior with a frequency of 18.1%. Type 2 showed a cream exterior shell color with brown spots and a yellowish-white interior, as well as a frequency of 13.5%. Type 11 comprised a cream exterior with brown spots and a brown interior with white spots, constituting 11.5% of D. faba population on Kutang Beach. Table 2 Color and morphometric variations of D. faba from Kutang Beach Type n  Color Morphometry (mean±SD) SL (mm) SH (mm) SW (mm) UML (mm) DH (mm) Type 1 13 ext: yellowish white; int: white 13.70±2.73a 7.93±2.12a 1.47±1.06a 8.43±2.25a 4.75±1.54a Type 2 39 ext: cream with brown maculation; int: yellowish white 20.56±4.34b,c 14.68±4.47c,d 8.84±6.66b,c 15.91±4.43c,d 11.7±3.7c,d,e Type 3 2 ext: white; int: white with radial purple 25.56±1.60 19.59±1.68 12.13±0.88 19.86±1.48 16.57±0.98 Type 4 2 ext: white with a radial band at posterior region; int: white with radial band at posterior region 23.06±3.10 19.31±0.45 12.04±0.40 20.42±0.71 16.02±2.37 Type 5 30 ext: white with radial purple band; int: white 20.90±5.51b,c 15.53±4.51c,d 9.23±3.80c 16.75±5.03c,d 12.58±4.15d,e Type 6 27 ext: cream with radial purple band; yellowish white 21.06±3.23b,c 15.32±2.58c,d 7.94±2.03b,c 14.08±2.88b,c 9.97±2.49b,c,d Type 7 1 ext: light brown; light brown 20.7±0.00 12.94±0.00 8.26±0.00 14.2±0.00 11.34±0.00 Type 8 11 ext: cream with brown maculation; int: dark purple 23.21±2.22c 16.35±1.13d 9.01±1.02c 15.04±2.26b,c,d 12.22±2.57d,e Morphological and molecular characterization of Donax faba - Ambarwati et al. 243 Type n  Color Morphometry (mean±SD) SL (mm) SH (mm) SW (mm) UML (mm) DH (mm) Type 9 11 ext: dark brown with light brown maculation; int: brown with white maculation 18.25±3.82a,b 12.69±2.76b,c 7.23±2.13b,c 11.54±3.80a,b 8.71±3.06b,c Type 10 1 ext: brown; int: brown 24.02±0.00 17.64±0.00 9.07±0.00 15.94±0.00 13.41±0.00 Type 11 33 Ext: cream with brown maculation; Int: brown with white maculation 19.20±2.16b,c 13.62±1.40b,c,d 7.13±7.13b,c 15.30±2.62b,c,d 10.2±2.65b,c,d Type 12 2 ext: brown with white maculation; int: dark brown 14.03±3.00 9.70±2.07 5.05±1.46 10.30±2.84 6.65±2.75 Type 13 6 ext: black with white maculation; int: deep dark 19.84±2.08 14.36±1.91 7.61±1.29 14.98±2.20 11.35±2.51 Type 14 10 ext: cream with dark brown maculation; int: white with brown maculation 15.09±2.93a 10.49±2.21a,b 5.29±1.21b 11.73±2.12a,b 7.74±2.14a,b Type 15 8 ext: dark brown; int: dark brown 16.34±2.24 10.49±2.21a,b 5.29±1.21b 11.73±2.12a,b 7.74±2.14a,b Type 16 20 ext: black with white spot: int: whitish black 21.96±5.04b,c 15.70±4.52c,d 8.46±3.20b,c 18.02±4.48d 14.17±4.52e Type 17 3 ext: cream with white radial band; int: white 18.33±3.34 13.22±2.57 7.10±2.04 15.58±3.61 10.03±2.88 Type 18 52 ext: white with brown spots; int: whitish purple 19.70±3.43b,c 14.27±2.87c,d 7.42±1.60b,c 15.22±2.92b,c,d 10.06±2.6b,c,d Type 19 17 ext: cream with brown maculation; int: whitish purple 19.52±2.82b,c 14.20±2.37c,d 7.18±1.49b,c 15.37±2.81b,c,d 10.3±2.48b,c,d P value 0.000* 0.000* 0.000* 0.000* 0.000* Notes: n = number of examined shells; SL = shell length; SW = shell width; DH = dorsal height; SH = shell height; UML = umbo margin line; ext = exterior shell; int = interior shell; SD = standard deviation. * = there are differences in average morphometry between types. a,b,c,d,e = the same letter category on each measurement among types shows the absence of significant mean difference between types. The results of morphometric measurements showed that each type of D. faba had different SL, SW, SH, DH, and UML (Table 2). Analysis identified variations in SL between types comprising 3 subsets, each representing a category with no significant difference in average SL value, while differences were observed between several subsets. Based on the morphometric measurements, types 1, 2, 5, 6, 8, 11, 16, 18, and 19 had different SL values. D. faba type 14 was significantly different from 2, 5, 6, 8, 11, 16, 18, and 19, while type 9 differed from 8. Type 1 had the lowest average SL of 13.7 mm, while type 8 had the highest measuring 23.21 mm. Additionally, the average SH of type 1 and 14 was significantly different from 2, 5, 6, 8, 11, 16, 18, and 19. Type 8 varied from 9, 11, and 18, with calculations showing that type 1 had the lowest average SH of 7.93 mm, while type 8 had the highest at 16.35 mm (Table 2). These dimensions were smaller than those observed for D. faba found in Nepa Beach (Ambarwati & Faizah 2017), but larger than the values measured for samples collected from Tengket Beach (Alyani & Ambarwati 2018). The shells had variations in sizes, pattern, and outline, with ratios including SW and SH, SW and SL, as well as SH and SL appearing significantly different among all types (Table 3). The relationship between shell size parameters was described using linear regression equations, with statistical test results showing different regression equations for each type. The shell size ratios of D. faba from Lamongan were similar to those measured in samples collected from Nepa Beach, Madura (Ambarwati & Faizah 2017). BIOTROPIA Vol. 31 No. 2, 2024 244 Figure 3 Morphological variations of D. faba collected from Kutang Beach, Lamongan Indonesia Notes: A = Type 1; B = Type 2; C = Type 3; D = Type 4; E = Type 5; F = Type 6; G = Type 7; H = Type 8; I = Type 9; J = Type 10; K = Type 11; L = Type 12. Scale bars = 10 mm. Morphological and molecular characterization of Donax faba - Ambarwati et al. 245 Figure 4 Morphological variations of D. faba from Kutang Beach, Lamongan, Indonesia Notes: M = Type 13; N = Type 14; O = Type 15; P = Type 16; Q = Type 17; R = Type 18; S = Type 19. Scale bars = 10 mm. BIOTROPIA Vol. 31 No. 2, 2024 246 Table 3 D. faba shell patterns based on measurement ratio and linear regression Measurement n Type Ratio Regression Regression formula P value regression R-square SW and SH 13 Type 1 0.186a SW = -2.095 + 0.45SHa 0.000* 0.811 39 Type 2 0.602b SW = -4.709 + 0.923SHc 0.000* 0.385 30 Type 5 0.595b SW = -3.409 + 0.814SHc 0.000* 0.936 27 Type 6 0.518b SW = -2.754 + 0.698SHc 0.000* 0.79 11 Type 8 0.551b SW = -1.348 + 0.634SHc 0.017* 0.486 11 Type 9 0.570b SW = -0.948 + 0.64SHb,c 0.001* 0.700 33 Type 11 0.523b SW = 0.379 + 0.495SHd 0.000* 0.585 10 Type 14 0.505b SW = 0.321 + 0.474SHb 0.001* 0.752 20 Type 16 0.539b SW = -1.6 + 0.641SHc,d 0.000* 0.822 52 Type 18 0.520b SW = 1.557 + 0.411SHd 0.000* 0.541 17 Type 19 0.506b SW = -1.205 + 0.59SHd 0.000* 0.887 P value ANOVA 0.000** 0.000** SW and SL 13 Type 1 0.108a SW = - 3.39 + 0.355SLa 0.000* 0.837 39 Type 2 0.430b SW = -9.141 + 0.875SLc,d 0.000* 0.326 30 Type 5 0.442b SW = -4.540 + 0.659SLc,d 0.000* 0.915 27 Type 6 0.377b SW = -3.664 + 0.551SLc,d 0.000* 0.770 11 Type 8 0.388b SW = 2.817 + 0.267SLd 0.062 0.336 11 Type 9 0.396b SW = 0.514 + 0.368SLb,c 0.027* 0.435 33 Type 11 0.371b SW = -0.719 + 0.409SLb,c 0.000* 0.584 10 Type 14 0.351b SW = -0.108 + 0.358SLb 0.001* 0.753 20 Type 16 0.385b SW = -4.04 + 0.569SLd 0.000* 0.805 52 Type 18 0.376b SW = 1.362 + 0.307SLb,c 0.000* 0.432 17 Type 19 0.368b SW = -2.27 + 0.484SLb,c,d 0.000* 0.840 P value ANOVA 0.000** 0.000** SH and SL 13 Type 1 0.579a SH = -1.865 + 0.715SLa 0.000* 0.847 39 Type 2 0.714b SH = -5.487 + 0.981SLc,d 0.000* 0.907 30 Type 5 0.743b SH = -0.815 + 0.782SLc,d 0.000* 0.913 27 Type 6 0.727b SH = -0.805 + 0.766SLc,d 0.000* 0.917 11 Type 8 0.704b SH = 8.568 + 0.335SLd 0.027* 0.438 11 Type 9 0.695b SH = 2.02 + 0.585SLb,c 0.003* 0.651 33 Type 11 0.709b SH = 0.683 + 0.674SLc 0.000* 0.666 10 Type 14 0.695b SH = -0.718 + 0.743SLa,b 0.000* 0.968 20 Type 16 0.715b SH = -2.91 + 0.847SLc,d 0.000* 0.892 52 Type 18 0.724b SH = 0.288 + 0.709SLc 0.000* 0.719 17 Type 19 0.728b SH = -1.27 + 0.793SLc 0.000* 0.884 P value ANOVA 0.000** 0.000** Note: n = number of examined shells; SL = shell length; SW = shell width; SH = shell height. * = There is an influence of the predictor variable on the response variable. ** = ANOVA test showed differences in the average ratio and regression results between types. a,b,c,d = the same letter category in each measure between types represents the absence of significant differences in ratios or average regression results. Morphological and molecular characterization of Donax faba - Ambarwati et al. 247 Figure 5 Relative frequency of morphological pattern of D. faba from Kutang Beach Molecular Characterization Molecular characterization was performed based on the sequence of Cytochrome C Oxidase subunit I (COI) gene using three samples of D. faba which had the highest relative frequency of morphological variations. COI gene barcode sequence data among the three research samples showed an average composition of G+C nucleotide base at 41.7% and A+T nucleotide base at 58.3%. According to the average results, the nucleotide base composition of G+C was lower than A+T. Furthermore, the G+C and A+T content could provide insights into the evolutionary history and relationships of D. faba. Comparing these values with those of related species would clarify genetic divergence, hybridization events, and adaptive evolution processes of D. faba. The average G+C and A+T nucleotide base compositions in COI gene barcode sequence data offered valuable information about the genetic characteristics and evolutionary dynamics of D. faba. Table 4 The three highest match values from identification performed through the BOLD System with representation of similarity values Sample names Three highest BOLD identification Similarity (%) Status D. faba Type 18 D. faba 99.53 Published D. faba 99.53 Published D. faba 99.37 Published D. faba Type 2 D. faba 99.21 Published D. faba 99.21 Published D. faba 99.06 Published D. faba Type 11 D. faba 98.58 Published D. faba 98.58 Published D. faba 98.43 Published BIOTROPIA Vol. 31 No. 2, 2024 248 Table 5 Composition of nucleotide bases Samples A (%) C (%) G (%) T (%) A+T (%) G+C (%) Donax faba Type 18 20.9 19.9 21.6 37.7 58.6 41.4 Donax faba Type 2 21.2 19.9 21.9 37.0 58.2 41.8 Donax faba Type 11 21.2 19.9 21.9 37.0 58.2 41.7 Average 21.1 19.9 21.8 37.2 58.3 41.7 Notes: A = Adenine; C = Cytosine; G = Guanine; T = Thymine. Table 6 Nucleotide Base Variations Num. Species Nucleotide Base Variations 134 184 1. Donax faba MT334596.1 T T 2. Donax faba MT334599.1 ● ● 3. Donax faba MT334600.1 ● ● 4. Donax faba AB040845.1 ● ● 5. Donax faba Type 18 PP593778 (this research) ● ● 6. Donax faba Type 2 PP595807 (this research) G A 7. Donax faba Type 11 PP595808 (this research) G A 8. Donax cuneatus MT334594.1 ● ● 9. Donax cuneatus MT334595.1 ● ● 10. Donax cuneatus AB040842.1 ● ● 11. Donax incarnatus MT334590.1 ● ● 12. Donax incarnatus MT334591.1 ● ● 13. Donax incarnatus MT334592.1 ● ● 14. Donax incarnatus MT334593.1 ● ● Note: ● = conserved sequence. Table 5 shows transition and transversion mutations of COI gene nucleotide sequence of the samples when compared to related species. Transversion substitution of nucleotide base number 134 presented a change in base T (Thymine) to base G (Guanine). Additionally, the transition substitution of nucleotide base number 184 included a change in base T (Thymine) to A (Adenine). The results showed that two unique nucleotide base patterns, known as automorphic nucleotide bases, were exclusively present in D. faba samples. Automorphic nucleotide bases were specific to D. faba from Kutang Beach, distinguishing this population from other species (Table 6). Jannah & Rahayu (2019) and Priyono et al. (2018) reported that certain species had automorphic nucleotide bases as distinctive markers or features used for differentiation from other species under comparison. Zhang & Zhao (2004) stated that the transversion substitution would elevate with increasing AT base composition in the sequence. The exploration of collected D. faba samples and the ingroup showed an average genetic distance of 0.46%, representing the average genetic divergence among the samples investigated. Genetic distance is a measure of the genetic divergence between populations or individuals, often quantified based on genetic markers such as DNA sequences (Priyono et al. 2018). Furthermore, the average genetic distance between the research samples and the ingroup was determined to be 1.51%. This value showed the average genetic differentiation between D. faba and the ingroup, which could consist of related species or other reference samples. The differences in the composition of the nucleotide bases in each sequence signified the existence of genetic variation between species (Saleky et al. 2020). Moreover, Cai et al. (2016) stated that a genetic distance value of < 2% showed the group comprised the same species, and > 2% suggested the group was a different species from other members. This signified that the samples collected from Kutang Beach were identified Morphological and molecular characterization of Donax faba - Ambarwati et al. 249 as one species with D. faba. Chiu et al. (2013) reported various factors such as environmental conditions, overexploitation, and geographical location to be capable of influencing the diversity of genetic distances in a species. Additionally, certain environmental factors could impact the morphology and phylogenetic characteristics of populations in a species. The reconstruction of the phylogenetic tree of D. faba showed the existence of three clusters (Fig. 6). Cluster 1 consisted of two clades, namely D. faba research samples (featuring a bootstrap value of 70-88) along with the close relative originating from Thailand and Japan. Cluster 2 consisted of D. cuneatus originating from Thailand and Japan, while Cluster 3 comprised D. incarnatus originating from Thailand. Table 7 Genetic distances Samples 1 2 3 4 5 6 7 8 9 10 11 12 13 Donax faba MT334596.1 Donax faba MT334599.1 1.39 Donax faba MT334600.1 1.74 0.34 Donax faba AB040845.1 3.16 2.44 2.80 Donax faba Type 18 PP593778 (this research) 1.39 0.00 0.34 2.44 Donax faba Type 2 PP595807 (this research) 2.09 0.69 1.03 3.15 0.69 Donax faba Type 11 PP595808 (this research) 2.09 0.69 1.03 3.15 0.69 0.00 Donax cuneatus MT334594.1 18.76 19.22 18.76 19.19 19.22 20.09 20.09 Donax cuneatus MT334595.1 17.30 17.75 17.30 16.47 17.75 18.61 18.61 14.96 Donax cuneatus AB040842.1 17.81 18.27 17.81 17.84 18.27 19.13 19.13 5.37 13.24 Donax incarnatus MT334590.1 21.81 21.33 20.86 22.31 21.33 22.23 22.23 19.94 20.09 20.42 Donax incarnatus MT334591.1 23.19 22.71 22.23 23.71 22.71 23.63 23.63 20.36 19.16 21.78 4.63 Donax incarnatus MT334592.1 22.23 21.76 21.29 22.74 21.76 22.67 22.67 20.36 19.16 20.84 3.89 0.69 Donax incarnatus MT334593.1 22.23 21.76 21.29 22.74 21.76 22.67 22.67 20.36 19.16 20.84 3.89 0.69 0.00 Figure 6 Phylogenetic topology determined using the Neighbor-Joining Method Morphological and molecular characterization of Donax faba 2 BIOTROPIA Vol. 31 No. 2, 2024 250 Morphological and molecular characterization of Donax faba 2 Figure 7 Phylogenetic topology determined using Maximum Likelihood Method Morphological and molecular characterization of Donax faba 3 Figure 8 Barcode Gap Analysis of COI sequences performed through ABGD (Puillandre et al. 2012) Notes: Histograms show the distribution of pairwise genetic distances between each pair of samples: (A) Histogram of distance; (B) Ranked distance; and (C) Number of Primary Species Hypotheses (PSHs) obtained, for each prior intraspecific divergence. Regular red: number of hypothesis species delimitation. Morphological and molecular characterization of Donax faba - Ambarwati et al. 251 The reconstruction of D. faba phylogenetic tree using Neighbour Joining (NJ) and Maximum Likelihood (ML) methods showed the existence of three clusters (Fig. 7). Cluster 1 consisted of two clades, namely D. faba research samples (featuring a bootstrap value of 100) along with the close relative originating from Thailand and Japan. Cluster 2 comprised D. cuneatus which originated from Thailand and Japan, while Cluster 3 consisted of D. incarnatus originating from Thailand. The phylogenetic trees showed that D. faba and the close relative species formed distinct monophyletic branches. However, the proximity at the same node presented genetic relatedness and the positioning of these branches corresponded with a calculated genetic distance of 0.46%, signifying the greatest divergence between each species. NJ, ML, and genetic distance data collectively provided strong evidence that D. faba and the close relatives were genetically distant from each other. ABGD was used to identify five distinct groups for D. faba and the relatives based on the initial method and the bar-code gap threshold calculated by COI dataset, as shown in Figures 8A and 8B. The barcode gap distance value of 0.001 corresponded with the results of ABGD grouping which divided the species into five groups (Fig. 8C). These were categorized as Group [1] (Donax faba 1, D. faba 2, and D. faba 3); Group [2] (D. faba MT334596.1 and D. faba MT334599.1); Group [3] (D. faba MT334600.1 and D. faba AB040845.1); Group [4] (D. cuneatus MT334594.1 and D. cuneatus MT334595.1 and D. cuneatus AB040842.1); and Group [5] (D. incarnatus MT334590.1, D. incarnatus MT334591.1, and D. incarnatus MT334592.1. The application of ABGD analysis, with a prior maximal distance set at 0.001, further strengthened the separation of D. faba and other species in the ingroup into distinct categories. Therefore, D. faba was successfully identified by the combined use of genetic distance, phylogenetic analysis, and ABGD analysis. Based on the comprehensive data obtained through DNA barcoding combined with morphological characteristics, it could be inferred that the focused application of these tools provided a reliable and effective means of identifying D. faba at the species level. CONCLUSION D. faba collected from Kutang Beach had 19 morphological variations. The average genetic distance of the research samples was 0.46%, while the value of this parameter between the research samples and the ingroup was found to be 1.51%. ACKNOWLEDGMENT The authors are grateful to the Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya for providing funding assistance (Rector Decree Number 1117/UN38/HK/ PP/2023). The authors are also grateful to Rofiza Yolanda, Ph.D., for helping with manuscript improvement, as well as the reviewers and editors who thoroughly and constructively reviewed the manuscript. REFERENCES Ambarwati R, Faizah U. 2017. Colour and morphometric variation of donacid bivalves from Nepa Beach, Madura Island, Indonesia. Biosaintifika J Biol Biol Educ 9(3):466- 73. Atlanta V, Ambarwati R, Rahayu DA, Mujiono N. 2022. Diversity of bivalves on the north coast of Lamongan, East Java, Indonesia. Biodiversitas 23(8):4263-71. Cárdenas P, Menegola C, Rapp HT, Díaz MC. 2009. Morphological description and DNA barcodes of shallow- water Tetractinellida (Porifera: Demospongiae) from Bocas del Toro, Panama, with description of a new species. Zootaxa (2276):1-39. 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