1907-770XBIOTROPIABIOTROPIA1907-770X0215-6334SEAMEO BIOTROP10.11598/btb.2024.31.2.2143MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF Donax faba (BIVALVIA: DONACIDAE) OBTAINED FROM KUTANG BEACH, LAMONGAN, INDONESIAhttps://orcid.org/0000-0001-8652-1840AmbarwatiReniIndonesiareniambarwati@unesa.ac.idRachmadiartiFidaIndonesiaFitrihidajatiHerlinaIndonesiaPurnomoTarzanIndonesiaRahayuDwi AnggorowatiIndonesiaFaizahUlfiIndonesiaDepartment of Biology, Faculty of Mathematics and Natural SciencesUniversitas Negeri SurabayaEcology Laboratory, Biology Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, IndonesiaAnimal Systematics Laboratory, Biology Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, IndonesiaAnimal Systematics Laboratory, Biology Education Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Surabaya, Ketintang Campus, Surabaya 60231, Indonesia.CahyaHaritzCorresponding author: Reni Ambarwati, Department of Biology, Faculty of Mathematics and Natural SciencesUniversitas Negeri Surabaya .Email:reniambarwati@unesa.ac.id7820243122382525122023252024Copyright (c) 2024 Reni Ambarwati, Fida Rachmadiarti, Herlina Fitrihidajati, Tarzan Purnomo, Rofiza Yolanda, Dwi Anggorowati Rahayu, Ulfi Faizah2024Reni Ambarwati, Fida Rachmadiarti, Herlina Fitrihidajati, Tarzan Purnomo, Rofiza Yolanda, Dwi Anggorowati Rahayu, Ulfi Faizahhttp://creativecommons.org/licenses/by-nc-nd/4.0This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.Authors who publish with this journal agree with the following terms:
Authors retain copyright and grant the journal right of first publication, with the work 1 year after publication simultaneously licensed under a Creative Commons attribution-noncommerical-noderivates 4.0 International License that allows others to share, copy and redistribute the work in any medium or format, but only where the use is for non-commercial purposes and an acknowledgement of the work's authorship and initial publication in this journal is mentioned.
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Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF Donax faba (BIVALVIA: DONACIDAE) OBTAINED FROM KUTANG BEACH, LAMONGAN, INDONESIAARTICLE 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%.
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.
COI geneDNA barcodingwedge clamcoastal ecosystemmarine organismsFile created by JATS EditorJATS EditorINTRODUCTION
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).
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 & 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); (, 2012), Echinoderms (Layton et al., 2016), Mollusca (Juniar et al., 2021); (Sari et al., 2021), and fish ((Rahayu et al., 2019); (Rahayu et al., 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 METHODSField Work
Samples of D. faba were collected from Kutang Beach, Lamongan (Figure 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 WorkIdentification 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 (Figure 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.
The map showing the sampling location of D. faba (red dot) in Kutang Beach
Image
Morphometric measurements of D. faba
ImageMolecular 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 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-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 AnalysisAnalysis 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.
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)
RESULTS AND DISCUSSIONMorphological 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 (Figure 3 & Figure 4). This description corresponds to the reports of (Tan & Low, 2013), (Ambarwati & Faizah, 2017), and (Signorelli & 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 (Figure 3 & 4; Table 2). Variations found with the highest relative frequency were types 18, 2, and 11 (Figure 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.
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
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; 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).
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.
Image
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.
Image
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.
Relative frequency of morphological pattern of D. faba from Kutang Beach
ImageMolecular 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.
The three highest match values from identification performed through the BOLD Systemwith 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
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.
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 (, 2020). Moreover, (, 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 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 (Figure 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.
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
Phylogenetic topology determined using the Neighbor-Joining Method
Image
Phylogenetic topology determined using Maximum Likelihood Method
Image
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.
Image
The reconstruction of D. faba phylogenetic tree using Neighbour Joining (NJ) and Maximum Likelihood (ML) methods showed the existence of three clusters (Figure 8; (Puillandre et al., 2012)). 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 Figure 8A and Figure 8B. The barcode gap distance value of 0.001 corresponded with the results of ABGD grouping which divided the species into five groups (Figure 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%.
Acknowledgments
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.
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