The mismatch between morphological and molecular attribution of three Glossogobius species in the Mekong Delta
BMC Zoology volume 7, Article number: 34 (2022)
The Vietnamese Mekong Delta (VMD) is the granary for the whole country, providing animal and plant resources, especially fish. Among the fish species, the genus Glossogobius are the majority. Until now, research for this species has been solely relied on fish morphology for identification. Hence, the present study aimed to describe the morphological variations of the morphologically identified gobies and to validate them at the molecular level through the sequencing of the barcode region, the mitochondrial cytochrome C oxidase subunit I (COI) gene to preliminary provide fundamental information for conservation.
The mitochondrial cytochrome C oxidase subunit I genes were amplified successfully with an approximate size of 650-680 bp. Their morphometries were quite different, and the genetic distance (p-value) among groups and within groups ranged from 0.00 to 0.12. The similarity of the COI gene sequences between the analyzed samples and in the NCBI database was from 87.01 to 100%. The specimens of G. aureus, G. giuris and G. sparsipapillus were interspersed in small branches of the phylogenetic tree with a low genetic distance highlighting that the genetic diversity of COI gene was low among species. Therefore, it is recommended that a combination of morphological method and mtCOI DNA barcoding is required for accurate classification.
This study helps determine three distinct lineages of Glossogobius species, so an appropriate strategy can be proposed for exploitation and conservation.
The Mekong Delta region encompasses a large portion of south-western Vietnam of over 40,500 km2 and is covered by water depending on the season. The wet coastal geography makes the region an essential source of agriculture and aquaculture products for the whole country . Not only famous for being a large granary, the Mekong Delta is also considered a source of genetic diversity, presenting various living organisms, especially gobies, which are among the most common species [2, 3].
The Glossogobius spp. are the primary source of protein for local residents in the Mekong Delta and constitutes the central part of the diet in different cultures, and they also play an essential role in the local economy, specifically providing jobs and investment opportunities for many countries . The morphometrics and meristics of Glossogobius spp. in the Mekong Delta, including G. giuris, G. aureus and G. sparsipapillus, has been found to change with ecoregions along the riverine [5, 6] and coastline regions [7,8,9,10,11,12,13,14]. However, this morphological variation could be due to the environmental adaptation or genetics. Genetic variation is the raw material in a species and population, enabling them to adapt to changes in their environment. This study, therefore, aimed to describe the morphological variations of the identified gobies and to validate them at the molecular level through the sequencing of the barcode region, the mitochondrial cytochrome C oxidase subunit I (COI) gene to preliminary provide fundamental information for conservation.
Study site, fish collection and analysis
This research was carried out at four sites along the riverine to estuarine and coastline ecoregions, including Cai Rang in Can Tho (CRCT), Long Phu in Soc Trang (LPST), Hoa Binh in Bac Lieu (HBBL), and Dam Doi in Ca Mau (DDCM) (Fig. 1). These regions are characterized by a semi-diurnal tidal range of ~ 1.2 m, a temperature of ~ 27 °C, pH of ~ 8, and salinity of ~ 12 ‰ in LPST and 0‰ in CRCT. It rarely rains in the dry season (from January to May) but rains heavily almost every month in the wet season (from June to December), with an average monthly rainfall of 400 mm [1, 16].
Fish specimens were collected monthly from 01/2020 to 12/2020 using trawl nets with a 1.5 cm mesh aperture at the cod-end. After 2–3 h setting at the highest tide in each study site, nets were retrieved to collect fish specimens which were then identified based on their external description . Thereafter, twelve separate samples representing three morphological species at four sampling sites were stored in 96% ethanol for DNA extraction. A total of 742 G. aureus, 1291 G. giuris and 764 G. sparsipapillus used for morphological analysis were observed and the external morphological traits of the three fish species were recorded before fish were euthanized with MS222 and fixed with 5% formalin solution for other experiments (Animal Welfare Assessment BQ2020–02/KSP). The total length (TL), body depth (BD), head length (HL), eye diameter (ED), the distance between eyes (DE), specimen weight and morphometrics traits such as ED/HL, DE/HL, BD/TL, HL/TL were measured at the laboratory.
DNA extraction and polymerase chain reaction (PCR)
The genomic DNA from the twelve specimens was extracted following the method of Rogers and Bendich . The DNA amplification was carried out at the Laboratory of Molecular Biology of the Biotechnology Research and Development Institute, Can Tho University, based on the research methods of Nguyen and Duong . One primer pair was used to identify the genetic relationship among samples of this genus and to evaluate the effectiveness compared with the morphological classification method. The sequences of the two primers for detecting COI gene  were given as followings:
Fish F: 5′- TCAACCAACCACAAAGACATTGGCAC-3′.
Fish R: 5′ - TAGACTTCTGGGTGGCCAAAGAATCA-3′.
The sequences were used to amplify the COI gene by PCR (Polymerase Chain Reaction) method. PCR reaction was performed in a total volume of 50 μL, consisting of the components of 20 μl My Taq mix buffer 1X; 1 μl of each primer (0.25 × 10− 6 mol/l); 3 μl DNA corresponding to about 100 ng; and distilled water (remaining volume equivalent to 25 μl).
Polymerase chain reaction conditions were one cycle at 95 °C (2 min), 39 cycles at 94 °C (30s), 40s at the annealing temperature of 52 °C, and 1.5 min at 72 °C, and a final extension at 72 °C for 10 min. The PCR products were then sent to Macrogen Ltd. Company, the Republic of Korea, for sequencing using the method of Sanger, Nicklen and Coulson .
The normal distribution of morphometric ratios (ED/HL, DE/HL, BD/TL, HL/TL) was tested by the Kolmogorov-Smirnov test with a sample size greater than 30 . Thereafter, the Kruskal-Wallis Test was applied to analyze them if they were not normally distributed. On the contrary, the one-way ANOVA with Turkey post hoc test was used to test the spatial variation of these meristic parameters.
If morphometric ratios showed differences between species and sampling sites, the principal component analysis (PCA) was applied to determine which environmental factors and morphological characteristics (ED, DE, BD, HL, TL, W, ED/HL, DE/HL, BD/TL, HL/TL) were the main factors affecting these differences. PCA was run by PRIMER v.6 software.
Three gobioid species were identified from collected samples, comprising G. aureus, G. sparsipapillus, and G. giuris. The COI sequences of G. giuris from Australia (MW574775) and India with accession number of MK714087, MK902713, MK348190, whereas G. aureus from the Philippines (KJ013044), all were used as in-group controls. Two mtCOI sequences of Butis koilomatodon in Vietnam (OK076879) and Periophthalmus chrysopilos in Bangladesh (MK572461) were the out-group controls.
The obtained COI sequences with Querry ID (Table 1) were aligned in Bioedit v7.2 . The genetic distances amongst three Glossogobius species were performed following the Kimura 2-parameters method in Mega 7.0. The genetic relationship of the twelve collected gobies specimens was identified by the “Maximum Likelihood method” with a bootstrapped value of 1000 times and performed by Mega 7.0 software . “Maximum likelihood” is a the commonly used method to construct the phylogenetic tree and is used by many molecular biologists [25, 26, 27].
Species identification using morphologies
A total of 742 G. aureus, 1291 G. giuris and 764 G. sparsipapillus collected in the Mekong Delta were classified based on their morphological characteristics described in Table 1. Glossogobius sparsipapillus differed from G. aureus and G. giuris in that it had a vertical transverse of sensory papillae in the middle operculum. In the case of the distinction between G. aureus and G. giuris, the amount of predorsal scale of G. aureus (22–27) was greater than that of G. giuris (22). The values of W, TL, ED, DE, HL and BD were different from site to site (Table 2); thus, the ratios of morphometric were considered site-specific. Specially, as expressed in Table 3 (raw data can be found in supplementary material: Raw data Glossogobius genus), the statistical results of measurement ratios showed that G. giuris was different from the other two congeners. Meanwhile, G. aureus and G. sparsipapillus were statistically similar in all morphometrics traits. Namely, ED/HL and DE/HL of G. giuris were greater than G. aureus and G. sarsipapillus, while the opposite results were found in HL/TL and BD/TL.
Figure 2 summarizes the results from the first exploratory multivariate analysis, PCA for 3 species in Glossogobius genus. In general, with 2 principal components extracted, the results explained a 59.5% of the variances (PC1: 37% and PC2: 22.5%). The principal component 1 (PC1) was the most associated with HL/TL, BD/TL, ED/HL, and DE/HL factors, whereas PC2 was the most associated with environmental factors (salinity, temperature, and pH). Figure 2 also expresses that G. aureus and G. sparsipapillus have more morphological similarities than G. giuis because they are superimposed in the graph while most G. giuis separates into two groups.
The principal component analysis of each species were also performed to determine the factors causing the difference between these three species. The results showed that, in all three Glossogobius species, the characteristic indexes of HL/TL, BD/TL, ED/HL, DE/HL and morphological measurement of HL, BD, ED, DE and W played an essential role in causing the differences amongst three species. Besides, environmental factors varied from species to species (Fig. 3). Specifically, in G. aureus, salinity had the most significant influence on morphological characteristics compared to temperature and pH. In the case of G. giuris, all three environmental factors such as salinity, temperature and pH affected the outside features, but the temperature was the strongest influencing factor, followed by pH and salinity. Whereas in the case of G. sparsipapillus, morphology was closely related to temperature and pH but not affected by salinity.
The mitochondrial cytochrome oxidase I (COI) region of all samples was successfully amplified using PCR. The sequences varied from 650 bp to 680 bp, with the composition of nucleotide presented in Table 4. There was not too much difference in the percentage of the base composition of COI sequences in G. aureus and G. sparsipapillus, namely, %T content was the highest, followed by %C, %A and the lowest was %G. A different order was observed in G. giuris in CRCT, LPST,HBBL and G. sparsipapillus in CRCT. The %C and %T were approximately the same, followed by %A and %G presented the lowest. In most cases, % AT content was always higher than %GC.
Species identification using COI sequences
Analyzing the intraspecific alignment results of G. aureus, G. giuris and G. sparsipapillus by the “Align by ClustalW” method showed the variable nucleotides were 83/591; 78/591 and 79/591, respectively. In addition, the most conserved nucleotides were found in G. giuris (513/591), followed by G. sparsipapillus (512/591), and lastly G. aureus (508/591).
The twelve obtained COI sequences were compared to similar sequences in the Genbank by the BLAST program (Table 5) to re-identify the species. Glossogobius giuris - HBBL; G.giuris - LPST and G. sparsipapillus - CRCT were similar to G. giuris (MW574775) in Australia with 100% similarity, while 100% similarity was also seen between G. aureus- LPST and G. aureus (KJ013044) in the Philippines. There were two notable results as presented in Table 4. First, the COI gene sequence of G. aureus from CRCT, HBBL and DDCM showed a relative similarity with G. giuris (MK714087 and MK902713) from India. Second, G. sparsipapillus - CRCT was identical to G. giuris from Australia (100%), while G. sparsipapillus from LPST, HBBL and DDCM were only 87.25–87.48% similar to G. giuris prevailing from India instead of Australia.
The genetic distance analysis of the twelve samples of goby species was quite different. The values are presented in Table 6. Generally, “intra-species” the genetic distances of the three species in Glossogobius genus were similar, and ranged 0.00 to 0.12. Notably, for G. aureus, G. giuris and G. sparsipapillus, the “intra-species” genetic distances were similar to that observed for “inter-specific” pairwise comparisons.
Genetic relationship analysis
The phylogenetic tree of G. aureus, G. giuris and G. sparsipapillus in Fig. 4 showed that species samples had a very distinct division with a bootstrap index in many nodes as high as 100% for the effectiveness and accuracy of the species identification by the COI gene. The phylogenetic tree of the studied fish in Fig. 4 was divided into five main groups. In Group I, two G. aureus individuals in CRCT and HBBL were identical to G. sparsipapillus individuals in LPST, because the number of substitutions per site was 0.00. While G. giuris-DDCM and G. aureus-DDCM exhibited similarities to G. sparsipapillus-LPST and G. sparsipapillus-HBBL, respectively.
Glossogobius giuris from CRCT, LPST, and HBBL together with G. sparsipapillus were in group II. Compared with the in-group control sequence of G. giuris from Australia, they were also the same in heredity, however they expressed difference from G. giuris in India, due to the identical percentage of 87.01%. This showed that the G. giuris specimens were correctly identified but needed to be reviewed for G. sparsipapillus. Group III consisted two in-group controls of G. giuris from India, while group V was two out-group controls of B. koilomatodon-Vietnam and P. chrysopilos-Bamgladesh. Group IV included G. aureus - LPST and G. aureus from the Philippines (in-group control) with 100% similarity. Overall, the twelve selected sequences did not cluster according to the morphological attribution, being interspersed in the phylogenetic tree, which however, identified three main groups (excluding two control groups III and V), suggesting the existence of three distinct lineages.
This study involved species identification based on the morphology and COI sequences as DNA barcoding. The morphological descriptions of Glossogobius species were similar to the studies on the morphology of G. giuris of Herre  and Tran, Shibukawa, Nguyen, Ha, Tran, Mai and Utsugi ; the study on G. aureus of Phuong and Binh ; and the study on G. sparsipapillus of Tran, Shibukawa, Nguyen, Ha, Tran, Mai and Utsugi . Tran, Shibukawa, Nguyen, Ha, Tran, Mai and Utsugi  reported that the standard length of G. giuris, G. aureus and G. sparsipapillus in the Mekong Delta region could reach different sizes. However, very few morphological characters alone are sufficient to identify G. sparsipapillus, G. aureus and G. giuris, namely the vertical transverse of sensory papillae in the middle operculum and the number of predorsal scales.
Kamboj and Kamboj  and Ujjania, Kumar, Langar and Krishna  noted that the morphometric parameters increased proportionally to the length of the fish. Meanwhile, meristic counts and meristic variables were independent of fish size, but affected by the phylogenetic origin and gender [32, 33]. In the present study, the measurement ratios of G. giuris were different from those of the two other species. However, whether these morphological differences were due to genetics or adaptation of the fish to the environment needed to be determined based on the study of the mtCOI gene.
There were discrepancies in the BLAST results of Glossogobius specimens. For example, G. aureus - DDCM was relatively similar to G. giuris (MK902713) from India, rather than G. aureus, which may be due to an error in the sequence. Furthermore, all G. sparsipapillus specimens were low homologous to G. giuris (MK902713) from India because the genetic data of G. sparsipapillus was unavailable in the Genbank. In addition, G. sparsipapillus - CRCT was identical to G. giuris from Australia (100%), while G. sparsipapillus - DDCM was only 87.48% similar to G. giuris from India instead of Australia. The inconsistency in these results may be due to the interfered nucleotides (errors in sequencing), resulting in a different intra-species genetic distance (0.11) of G. sparsipapillus. Therefore, it was necessary to reclassify this species by both morphology and DNA barcoding methods.
The COI sequence was reported to be informative in analyzing genetic diversity in fish, including Australian fish species , medicinal fish of Culter (Pisces: Cyprinidae) , pufferfish species , transparent gobies , and Sillaginidae fishes (Perciformes) . Within the scope of this study, three Glossogobius species exhibited a lot of similar outside traits as mentioned by Hoese and Allen , G. giuris, G. aureus and G. sparsipapillus had a cylindrical body, with two distinct dorsal fins and fused pelvic fins. Some other features were notable, such as largemouth (10–15% SL), depressed head, long and pointed snout l, projecting lower jaw, at least six lines of longitudinal papilla running longitudinally on the cheek, 27–30 vertebrae, a bilobed tongue, gill opening reaching below a point just before to just behind posterior preopercular margin. However, G. giuris had 22 predorsal fin rays, a unique criterion distinguishing tank goby from two other congeners , and G. sparsipapillus had a vertical transverse of sensory papillae in the middle operculum. The fact that G. sparsipapillus was identical to G. giuris or G. aureus could be due to two main reasons; one was that the COI gene sequence of G. sparsipapillus was not available in the gene bank; and the second was that COI gene sequence of G. sparsipapillus still had many overlapping nucleotides at different peaks. Glossogobius aureus had the same characteristic documented by Hoese and Allen  such as the blackish spots on the 2nd dorsal fin and in the caudal peduncle, as well as the longitudinal black lines on the side of the body which are usually blurred as reported by Phuong and Binh . The phylogram showed that the three species of the Glossogobius genus were interspersed in small clades. Moreover, the genetic distance between the three Glossogobius species (≤ 0.159 or 15.9%) was smaller than the average difference between species of the same genus in the suborder Butidae (22.2%) , but higher than between fish species in Australia (9.93%) . The result showed that the difference in COI sequence of species in the Glossogobius genus was relatively low. As such it is recommended that it is with morphological methods or the other mitochondrial DNA barcodes such as cytochrome b, 12S or 16S rRNA to classify the species more accurately.
Previously, morphological characters were mainly used to identify fish species and phylogenetic relationships to understand their speciation and evolution [41, 42]. On the contrary, gobiid species were hard to differentiate species because of their similarity in external morphology [4, 43]. Therefore, the reconstructed phylogenetic trees based on morphology were limited, and at times controversial due to the complex evolutionary changes in either morphological or physiological characters [44, 45]. Based on the development of molecular biology techniques, this situation has changed, especially with the application of mtDNA’s genetic analysis to resolve controversial taxonomic problems [46,47,48,49]. This technique is a helpful tool for the determination of molecular markers that can facilitate the discrimination of morphologically similar species. Many previous researchers studied the gobiid fishes and reported that they are monophyletic [42, 44, 45, 50]. In the present research, the COI part of the mitochondrial DNA was sequenced to identify gobies from twelve samples collected from different provinces in the Vietnamese Mekong Delta. Nevertheless, the COI sequence of gobies displayed a similarity to available sequences of the gene bank. As taxonomic ambiguities, successful molecular identification was helpful. The findings showed that the COI gene enabled accurate fish species identification where adequate sequence data exists.
There was an incongruence between morphological and molecular species attribution between morphological and molecular species attribution of three species: G. aureus, G. giuris and G. sparsipapillus collected in brackish and freshwater in the Mekong Delta based on the COI gene sequences. Morphological characteristics and fish body size of G. aureus and G. sparsipapillus had many similarities in the present study such as ED/HL, DE/HL, HL/TL and BD/TL, while G. giuris showed more differences. Their COI sequences were similar up to100% to species in the Glossogobius genus on NCBI. Despite the differences in their morphometric characteristics, G. aureus, G. giuris and G. sparsipapillus were nearly genetically identical up 99–100%. Thus, further research was needed to reclassify Glossogobius species in VDM to contribute to developing a conservation strategy for these economically valued species.
Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information file, namely Raw data_Glossogobius genus]. The sequences of the COI gene were submitted to NCBI and got the ID: OK043694-OK043700 (please find the GenBank_OK043694-OK043700 file submitted to the Journal system].
- COI :
Cytochrome c oxidase subunit I
The Mekong Delta, Vietnam
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The authors would like to thank the local fishers for their assistance with specimen collection. The authors would like to thank Nigel K. Downes, Researcher for Integrated Water Resource Management (IWRM), for proofreading the article.
This research was funded by the Ministry of Education and Training of Vietnam under grant number B2020-TCT-13. Phan Hoang Gieo was funded by Vingroup JSC and supported by the Master, PhD Scholarship Programme of Vingroup Innovation Foundation (VINIF), Institute of Big Data, code VINIF.2021.TS.146.
Ethics approval and consent to participate
The study was reported in accordance with ARRIVE guidelines.
All methods were performed in accordance with the relevant guidelines and regulations.
This research was conducted with approval from The Scientific Committee of School of Education, Can Tho University (No. BQ2019–02/KSP) after animal welfare assessment (please find the Animal welfare assessment_BQ2019–02KSP file submitted to the Journal system).
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The original online version of this article was revised: Table 1 has been updated.
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Truong, N.T., Phan, G.H., Lam, T.T.H. et al. The mismatch between morphological and molecular attribution of three Glossogobius species in the Mekong Delta. BMC Zool 7, 34 (2022). https://doi.org/10.1186/s40850-022-00137-6
- Glossogobius genus
- Genetic distance
- Phylogenetic tree