Taxonomic triage of the genus Thrissina from Indian waters: a step towards diversity assessment

doi:10.21203/rs.3.rs-4122485/v1

Anchovies of the genus Thrissina (family Engraulidae) are common pelagic fishes that constitute a high-volume fishery resource along the Indian coast. Their accurate identification is essential for the fishery and management of resources. 314 specimens of six species of the genus Thrissina were collected to generate data for exact field identification and species-specific molecular signatures. The identification of the specimens was confirmed using morphometric and meristic parameters, along with COI-based DNA barcodes. This integrative study also aimed to reconstruct a phylogeny to clarify the evolutionary relationships among the species. The morpho-meristic results indicated that maxillary length, number of scutes, number of lower gill rakers, and anal fin ray count were the main characteristics for identifying Thrissina species. The species barcoded included T. setirostris, T. malabarica, T. mystax, T. baelama, T. polybranchialis, and T. vitrirostris. The nucleotide content of each species was examined and the genetic distance was calculated. The interspecific genetic distance (K2P) ranged from 13–22% with the highest distance observed between T. baelama and T. malabarica (22%) and the lowest between T. mystax and T. polybranchialis (13%). The average genetic distance within the genus was 19%. The topology of the phylogenetic tree showed that all the species in this study formed separate clades with a common ancestor. This work provides the first COI-barcode of the least investigated T. polybranchialis from the Indian coast, and establishes the relationship between T. mystax and T. polybranchialis. The integrative data generated here will aid in the correct identification and global studies on the genus. More morphological and molecular studies are recommended to clarify the relationship between all available species within this genus.

Anchovy

barcode

COI

Engraulidae

India

phylogenetic tree

Anchovies are important small-sized marine food fishes of the world. Theyhave a standard-length of 7-17cm, and their fishery is high volume, valuable, and priced. Anchovies belong to the family Engraulidae of the order Clupeiformes. They are widely distributed along the Indian coastline and form a major contribution to the finfish fishery along with Indian mackerel and Bombay duck (CMFRI annual report, 2022). In total 42.9% of pelagic fish catch 3.72% were composed of Anchovies (MPEDA, 2021). There are 28 species of Anchovies reported in Indian waters. However, major contributions are due to Stolephorus, Engraulis, Thrissina, Setipinna, and Coilia genera. There are with 17 valid genera in anchovies, including Thrissina that are distributed entirely in sea residences of the temperate and tropical regions (Eschmeyer et al. 2017).

Grande and Nelson (1985) classified anchovy into two subfamilies, Coilinae and Engraulinae, and the genus Thrissina falling under the latter. Thrissina (Jordan and Seale 1925), previously regarded as the incorrect spelling Thryssa (Cuvier 1829), is found throughout the Indo-Pacific region and forms commercially important resources in India, especially on the southwest and southeast coasts. They are silvery-coloured, highly abundant, marine, and carnivorous fishes that form large schools in near-shore habitats at depths of less than 50 meters. Thrissina contributed 1.3% of the total marine landings in India (CMFRI, 2022). Gill net and mini purse seines are the major gear employed for their fishery. There are 29 species reported worldwide, of which 18 are reported in India. They have a medium-compressed or strongly compressed body with 21-32 keeled scutes on the abdomen that are present from before the pectoral fin base to the anus. The length of their maxilla can vary from short to very long, with 25-45 branched anal fin rays. They possess 6-7 pelvic fin rays, a pre-dorsal scute, and small jaw teeth that are not canine-like. Their upper pectoral fin rays do not extend as a filament (Whitehead et al. 1988).

The identification and classification of fish are mainly based on morphometric and meristic characters (Triantafyllidis et al. 2011). Many authors have reported the taxonomic analysis of various Thrissina species from Indian waters (Lakra et al. 2011; Rajan et al. 2013; Ganga 2015; Hata and Motomura 2020; Hata et al. 2023). In general, species identification of Anchovies is very difficult because of their small size, soft body, presence of ambiguous characters, and rapid spoilage (Whitehead et al. 1988; Khan et al. 2010).

Grande and Nelson (1985) revised the phylogeny of the New World anchovies. Recent studies have revealed new insights into the genus Thrissina. Hata and Motomura (2020) identified specimens of T. cultella from the Bay of Bengal and T. serena from the northwestern Indian Ocean. A new species T. supra with a redescription of T. whiteheadi was identified by Hata et al. (2021). Hata et al. (2022) described another new species T. katana from the Western Pacific Ocean. Hata et al. (2023) also described a new species T. aurora from the Andaman Sea and conducted a reassessment of the taxonomic status of T. cuvierii and T. malabarica.

DNA barcoding is an effective method for identifying the species by using short standardized DNA sequences and helps to determine the phylogenetic relationship within the species (Herbert et al. 2004). Numerous studies have shown that the mitochondrial cytochrome c oxidase subunit I (COI) gene's roughly 650 bp-long sequence is more suited for DNA barcoding than other genes (Lakra et al. 2011; Ma et al. 2015; Zhang et al. 2016). The DNA barcoding of three species of the Anchovies (Stolephorus, Encrasicholina, Thrissina) was reported by Lakra et al. (2011). Lavoue et al. (2007) described the molecular systematics of the Anchovy in the Northwest Pacific. Many authors have hinted at the phylogenetic analysis of various anchovy species (Bloom and Lovejoy 2012; Chairi and Rebordinos 2014; Afrand et al. 2017; Lavoue et al. 2010, 2017). Recently, Mahrus et al. (2022) observed the phylogenetic relationship of Anchovies using COI genetic markers.

Integrative taxonomic studies of the genus Thrissina in Indian water are limited. The present study aims to provide valuable information about the classical taxonomy, molecular identification, phylogenetic relationship, and genetic divergence of Thrissina species from the Indian water. The current work combines morphology, morphometry, meristic, and genetic traits of all species from different locations along the India. All the species of genus Thrissina viz. T. setirostris, T. malabarica, T. mystax, T. baelama, T. polybranchialis, T. vitrirostris have been analysed in this study.

Taxon sampling and morphological identification

A total of 314 specimens of the genus Thrissina were collected from four localities in Indian waters from January 2022- July 2023(Fig.1). These fish were caught by gillnet and mini purse seines, at a depth of 15-20 meters. The collected sample was ice-packed on the site and brought to the laboratory. Morphological characters were observed using a vernier caliper with 0.1cm accuracy and the total weight (TW) of each fish was taken by using an electronic weighing balance with 0.01g accuracy. Meristic characters were observed and counted. Most important taxonomic characters like gill raker count were analysed using a stereo zoom microscope. Species identification was carried out by using Whitehead, Nelson, and Worgratana (1988).

Approximately 100 mg of white muscle tissue samples were extracted from each specimen and preserved in 95% ethanol for genetic analysis. The names and geographic locations of the collected samples are presented in Table 1. The GenBank and accession number, collection location, and type locality of each species were listed in Table 2.

DNA extraction and PCR condition

The genomic DNA of each species was extracted from the muscle tissue using a standard Phenol Chloroform extraction method (Sambrook and Russel. 2006). Amplification of partial sequences of mitochondrial cytochrome oxidase subunit (COI) sequence was done by using primer set LCO1490/ HCO2198 (Folmer et al. 1994) under standard conditions. The PCR products were purified using Qiagen PCR purification kit. The purified PCR product was used as the template for sequencing PCR with the same primer. The sequencing was done at Eurofins Scientific, Bangalore, India. The chromatograms were visually with the aid of ABI sequence editor 3.3 (Applied Biosystems, Waltham, MA).

DNA sequencing and data analysis

Sequencing was carried out with the forward and reverse primer to conform the nucleotide bases. The raw sequences were edited manually and the forward and reverse sequences of the genus were combined using BioEdit sequence alignment editor version 7.0.5.2 (M. J. Research Inc., Waltham, MA) (Hall 1999). The alignments were manually checked and corrected by using the BioEdit assembly program, aligned sequences had a trimmed length of 597 bp. All sequences generated from the study have been deposited in NCBI GenBank ( Table. 2). The analysis of GC, AT content, and sequence divergence was carried out based on the Kimura 2 model in MEGA Version 11 (Tamura et al. 2021).

The phylogenetic tree was reconstructed by using MEGA Version 11 (Tamura et al. 2021). Fifteen sequences collected from NCBI GenBank and 12 sequences identified from this study were used for the analysis (Table. 2). Closely related outgroups like Buccaneer anchovy, Encrasicholina punctifer (GenBank accession number: HQ564397) and Hardenberg's anchovy, Stolephorus indicus (GenBank accession no: FJ347956) were included in the sequence analysis. The phylogenetic tree was constructed using Maximum Likelihood (ML) method.

Our results for the morphological identification of specimens from the genus Thrissina represented in Fig. 2-7 and Table. 3. We successfully identified species within this genus including T. setirostris, T. malabarica, T. mystax, T. baelama, T. polybranchialis and T. vitrirostris.

Morphometric characteristics:

Thrissina spp. are characterised by medium-sized compressed or strongly compressed bodies with 8-15cm in standard length. They have a spine-like small scute present in front of the dorsal fin ray. The belly is sharply keeled with pre and post pelvic scutes from the isthmus to the anus. Maxilla may be short, moderate, long or very long. The anal fin is long usually with 26-45 fin rays. Pelvic fin with 6-7 in number. Tip of the snout in the middle of the eye centre or above the level of the upper rim of the eye. The serrae on the gill raker are clumped or even.

Species identification key:

The major distinguishing characteristics of the all the Thrissina species are as follows:

I) Lower jaw with the high coronoid process, maxilla very long reaching beyond the pectoral tip or at the pelvic fin, first lower gill raker 11-12………………………………… T. setirostris (Broussonet, 1782)

II) Lower jaw slender, maxilla tip not beyond pectoral fin tip.

A) Maxilla long, to pectoral base or beyond.

A 29-37, D 12-13; gill raker serrae uneven but not clumped, first lower gill raker 14-16……………………………………………………T. mystax (Bloch & Schneider, 1801).
A 31-40, gill raker serrae in distinctive clumps, first lower gill raker 18-24……………………………………………T. vitrirostris (Gilchrist &Thompson, 1908).

B) Maxilla short, not reaching to pectoral base.

Maxilla short, reaching to just or beyond the front border of pre-operculum……. ……………………………………………………………...T. baelama (Fabricius, 1775).
Maxilla short, reaching just of the gill cover, first lower gill raker 25-27………………………………………… T. polybranchialis (Wongratana, 1983).
Maxilla moderate, little beyond edge of gill cover, first gill raker 17-19 …………………………………………………………….... T. malabarica (Bloch, 1795).

In this study, all species were genetically characterized using COI regions in which no deletions or insertions were observed. The ATGC content of each species was calculated. (Table.4). Comparison of the ATGC content revealed that the AT content is higher than the GC content. The average nucleotide composition was 25.6%, 28.2%, and 19.3%. 26.9% of A, T, G, and C respectively. The pairwise genetic distance is given in Table. 5. The interspecific genetic distance ranged from 13% to 22%. The average was found to be 19%. The highest genetic distance was observed between T. baelama and T. malabarica (22%). The lowest (13%) was between T. polybranchialis and T. mystax. The phylogenetic tree was reconstructed using the best-fit maximum likelihood (ML) method. (Fig.8). The tree topology indicates that each species observed in this study sprang from a common ancestor to create distinct clades.

Morphological characteristics

The present study successfully identified the characteristics of six species within the genus Thrissina viz. T. setirostris, T. malabarica, T. mystax, T. polybranchialis, T. baelama and T. vitrirostris in Indian waters. The latter four species are widely distributed along the south coast of India, as these coastal areas provide suitable habitats for them. The occurrence of T. baelama was observed in the Andaman and Nicobar Islands (Rajan et al. 2013). In our work, the fishes were initially identified as genus Thrissina based on the physical traits. The taxonomic characters of each species were compared with earlier studies (Al-Faisal 2012; Ma et al. 2015; Gangan et al. 2016; Afrand et al. 2020; Hata and Motomura 2020). These previous studies focused on the taxonomic classification of Thrissina species, emphasizing characteristics such as the length of the maxilla, number of lower gill raker, number of scutes (both pre-and post-pelvic), and anal fin ray number.

In our study, we observed that in T. setirostris, T. mystax, T. vitrirostris and T. baelama, the tip of the snout was found to be at the level of the eye centre, while in T. malabarica and T. polybranchialis it was found to be at the upper rim of the eye. Every species is distinguished by having a prominent supra-maxilla in both the first and second positions. It is noted that the first is tiny, round, and positioned under the second. The taxonomy of the genus Thrissina was studied by Ganga (2015). According to the work, this genus was characterized with or without the first supra-maxilla, the level of the tip of the snout with a line drawn through mid-eye, and the first supra-maxilla is minute or lost while the second supra-maxilla is prominent. Our results are consistent with these findings.

Knamees et al. (2018) stated the comparative measurements of fin rays and gill raker of five Thrissina species viz. T. whiteheadi, T. vitrirostris, T. baelama, T. hamiltoni, and T. setirostris from the north-western Arabian Gulf. In our study, the anal fin ray count of T. setirostris was found to range from 32-39, whereas in their work, it was reported as 34-37. Upon comparing our findings with that of the three species (T. vitrirostris, T. baelama, and T. setirostris), it was found that the specimens from our study have anal and pectoral fin ray counts that fall within the same range.

Rundall (1995) mentioned that T. setirostris from Taiwan has an anal fin with 32-39 rays, a range that aligns with our research findings. Iwatsucki (2013) reported that the meristic characteristics of fish may vary in different habitats. Hata and Motomura (2020) observed that T. vitrirostris has a long, slender gill raker, and the serrae on the gill raker was clumped and ranged from 19-23. Hata et al. (2023) examined the original description of T. malabarica and revealed that this species was a senior synonym of T. hamiltonii (Gray, 1835). We compared the characteristics of T. malabarica and found that the anal fin ray count fell between 35-39, with a total of 23-25 scutes, and a lower gill raker count ranged from 17-20, which is in agreement with Hata et al. (2023).

The present study includes the most important taxonomic characters of the genus Thrissina. The data presented (Table. 3) aligned with the findings of Whitehead’s (1988) study, indicating that T. mystax and T. vitrirostris have morphological similarities, that make them difficult to differentiate without accurate taxonomic information. In our results, we found that the maxilla length of T. mystax was identical to T. vitrirostris, both have long maxilla that reaches to the base of the pectoral fin. Serrae on the gill raker are clumped in adult fish of T. vitrirostris, while in T. mystax, they are not clumped.

The lower gill raker of T. baelama differs from those T. setirostris and T. mystax. They fall within the same range as T. vitrirostris. T. mystax can be distinguished from T. polybranchialis by its elongated maxilla. In T. polybranchialis, themaxilla extends to the level of the gill opening, and in T. mystax, the maxilla extends to the pectoral fin base. The tip of the snout is in the middle of the eye in T. mystax, whereas it is above the upper rim of the eye in T. polybranchialis. Gill raker count was a useful trait for distinguishing between these species. T. mystax showed a count of 14-16 gill raker on the first lower gill arch, whereas T. polybranchialis has 25-27 gill raker.

Other morpho-meristic characteristics such as anal fin ray number, transverse scales, and scute count do not differentiate these two species. After examining their morphology, we noted there are huge phenotypic uncertainties in the samples, along with observable variations in morphometric features. This study highlights the similarity of physical traits between T. polybranchialis and T. mystax.

Molecular analysis

The identification of the genus Thrissina is extremely challenging due to the overlapping meristic counts. The limitations of morphologically based fish identification systems have been addressed by the application of genetic approaches. The phylogenetic construction of Thrissina was reconstructed. The tree topology indicates that individuals of the same species formed distinct clusters. This is the first attempt that reveal the relationship between the T. mystax and T. polybranchialis from Indian waters. The morphological characteristics of T. mystax were identical to those characteristics of T. vitrirostris, and from this study, we observed that the species T. mystax and T. vitrirostris diverge at the species level.

In the present study, all Thrissina species were characterized using barcodes and the average inter-specific genetic distance of each species was calculated. According to, Hebert et al. (2003) the minimal genetic distance between species is 2%. The genetic distance values in this study lie well above the accepted species threshold value.

Zhang et al. (2016) examined the morphological description and phylogenetic relationship of six species from coastal waters in China which includes three species included in our study. They observed a genetic divergence of 16% between T. setirostris and T. vitrirostris. They constructed the phylogenetic tree, and the tree topology indicated that T. setirostris was the first species derived from the genus. According to their results, T. vitrirostris, and T. mystax showed a closest relationship with each other. They found that the base composition of A and T was higher than that of G and C which is concordant with our results. The separate genetic status T. vitrirostris from T. mystax also corresponded with their investigation.

We reconstructed a phylogenetic tree including T. polybranchialis which is very common in coast waters in India. Our work indicated that T. mystax have the closest connection with T. polybranchialis and T. setirostris and the tree topology showed that T. polybranchialis and T. mystax clustered together with a common ancestor. The barcodes in the present study showed that T. polybranchialis is quite different from T. mystax. Ma et al. (2015) recorded the utility of COI sequence as DNA barcoding for the identification of the genus Thrissina from the Zhejiang Sea in China. They found an average of 29.2% T, 26.2% C, 26.2% A, and 18.1% G in their sequences. The genetic distance ranged from 0.003 to 016, with an average of 0.137. The tree topology indicated that four species formed monophyletic clusters. But T. vitrirostris and T. mystax were clustered together. The phylogenetic relationship of the genus Thrissina in their study indicated a closer relationship between T. vitrirostris and T. mystax with a genetic distance of 0.003. However, their morphological analysis revealed that these two might be one species. We observed that our results differed from their conclusions. The life habits of various fish species are related to convergent evolution which may be the the reason for this variable outcome (Ma et al.2010 and Kruck et al.2013).

Afrand et al. (2020) combined the taxonomic traits of Engraulidae in the Persian Gulf and Oman sea. They successfully discriminated the DNA barcode of five species of Thryssina using the mitochondrial COI gene. The tree topology showed that T. hamiltoni and T. vitrirostris were clustered together. In our work, the phylogram shows that T. baelama was the first species to diverge and T. polybranchialis and T. mystax formed a group. The results from the DNA barcoding and identification based on morphological characters in our study agree with Afrand (2020).

This study reveals integrative taxonomic identification of the genus Thrissina. We successfully examined the morphology and the DNA barcode of all species, and 12 sequences were deposited in GenBank. Our study marks the first comprehensive phenotypic and genotypic evaluation of T. mystax, T. polybranchialis, and T. baelama from Indian waters. This work registers the first COI-barcode of the least investigated T. polybranchialis from Indian waters. The current study will be useful as baseline information for evolutionary biologists for future studies.

Supplementary comments

After December 24, 2024, all accession numbers will be made public. The accession number is available through the request via email.

Ethical Approval and Consent to participate

There is no ethical issue concerning this article.

Human and Animal Ethics

Not applicable

Consent for publication

All authors have approved the manuscript and agreed to submit the manuscript for publication.

Availability of supporting data

Not applicable

Competing interests

All authors declare that they have no conflict of interest.

Funding

Union grants commission

Authors' contributions

AG did the collections and the major work, EMA and JNS did the draft preparations corrections. All authors made substantial contributions to the conception or de-sign of the work.

Acknowledgements

The authors would like to thank the Director, CMFRI and CUSAT for support and facilities provided; Union grants commission for financial support and the fishermen for providing samples for the study.

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Table 1. Name and geographic location of the collected fishes.

Sl. No.	Scientific name	Common name	Geographic Location	Code (Lat. and Long.)	Period of collection
1.	T. baelama	Baelama Thrissina	Andaman and Nicobar Islands	Jungalighat (11°39'33.21'' N, 92°43'41.9088''E)	January 2022
2.	T. malabarica	Malabar Thrissina	Karnataka	Mangalore (12°51'28.728''N, 74°49'57.2736''E) Malpe (13°20'50.19'' N, 74°42' 4.842'' E).	February 2022
3.	T. setirostris	Low jaw Thrissina	Tamil Nadu	Tuticorin (08°47' 36.96'' N, 78°9'37.7676''E)	August 2022
4.	T. polybranchialis	Humphead Thrissina	Kerala	Alappuzha (9°29'53.04'' N, 76°20'19.85''E)	September 2022
5.	T. mystax	Middle jawed Thrissina	Kerala	Kalamukku (09°59'24'' N, 76°4'564'' E) Vizhinjam (08°22'52.59''N, 76°59'4.25'E)	June 2023
6.	T. vitrirostris	Orange mouth Anchovy	Tamil Nadu	Kanyakumari (8°5' 5.649''N, 77°32'30.46''E)	July 2023

Table 2. List of sequences used for analysis. The Asterix indicates sequences generated in this study.

Sl. No.	Scientific name	GenBank Accession number	Collection Location	Type locality of the species
1	T. setirostris	MH380700	Sri Lanka	Tanna Island, Southwestern Pacific
2	T. setirostris	MH380701	Sri Lanka	Tanna Island, Southwestern Pacific
3	*T. setirostris	PP038216	Current study
4	*T. setirostris	PP038217	Current study
5	T. malabarica	MH380708	Sri Lanka	Chennai Tamil Nadu, India
6	T. malabarica	MH380709	Sri Lanka	Chennai Tamil Nadu, India
7	*T. malabarica	PP038238	Current study
8	*T. malabarica	PP038239	Current study
9	T. mystax	MW498834	Malaysia	Calicut, India
10	T. mystax	MW498835	Malaysia	Calicut, India
11	*T. mystax	PP038274	Current study
12	*T. mystax	PP038275	Current study
13	T. baelama	GU674112	Indonesia	Sudan
14	T. baelama	HQ564499	Indonesia	Sudan
15	*T. baelama	PP038031	Current study
16	*T. baelama	PP038032	Current study
17	T. polybranchialis	ON166076	India	Mumbai, India
18	T. polybranchialis	MH380703	Sri Lanka	Mumbai, India
19	T. polybranchialis	MK962527	India	Mumbai, India
20	*T. polybranchialis	PP053492	Current study
21	*T. polybranchialis	PP053493	Current study
22	T. vitrirostris	MN512115	Pakistan	Soth Africa
23	T. vitrirostris	MN512116	Pakistan	Soth Africa
24	*T. vitrirostris	PP038226	Current study
25	*T. vitrirostris	PP038227	Current study

Table 3. The main morphological characters to identify six spp. of genus Thrissina. N-number of specimens, LGR-number of lower gill raker, PPS- Pre pelvic Scutes, POPS-Post pelvic Scutes, TS-Total number of Scutes, PF- Pectoral fin rays, PV-Pelvic fin rays, D-Dorsal fin rays, A-Anal fin rays.

Species	N	LGR	PRS	POS	TS	PF	PV	D	A
T. setirostris	60	11-12	16-18	9-10	25-28	12-13	I+7	13-14	32-39
T malabarica	50	17-19	15-16	8-10	23-26	12-13	I+7	13-14	34-38
T. mystax	100	14-16	17-19	8-13	24-32	13-14	I+7	13-14	29-37
T. baelama	4	18-24	5-7	7-10	12-18	13-14	I+7	11-12	29-34
T. polybranchialis	50	25-27	15-17	9-10	25-27	13-14	I+7	13-14	35-39
T. vitrirostris	50	18-24	16-19	8-12	27-30	13-14	I+7	13-14	31-40

Table 4. Average ATGC percentage in COI region (597 bp) of various species in genus Thrissina.

Species name	T(U)	C	A	G	Total
T. setirostris	27.3	27.9	25.7	19.1	597
T. malabarica	27.5	27.8	24.3	20.4	597
T. mystax	28.2	26.6	27	18.2	597
T. baelama	28.6	27.8	24.6	18.9	597
T. polybranchialis	28.9	25.6	25.7	19.8	597
T. vitrirostris	28.6	25.8	26	19.6	597
Avg.	28.2	26.9	25.6	19.3	597

Table 5. Average genetic distance (%) based on Kimura 2-parameter for COI gene in genus Thrissina.

Species name	T. setirostris	T. mystax	T. malabarica	T. vitrirostris	T. polybranchialis	T. baelama
T. setirostris	-	-	-	-	-	-
T. mystax	16.1	-	-	-	-	-
T. malabarica	18.4	21.0	-	-	-	-
T. vitrirostris	16.9	16.8	19.2	-	-	-
T. polybranchialis	15.8	13.1	19.6	15.2	-	-
T. baelama	21.0	20.6	21.6	18.8	20.6	-

No competing interests reported.

Taxonomic triage of the genus Thrissina from Indian waters: a step towards diversity assessment

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Status:

Version 1