Result of the maximum likelihood IQtree and the population structure suggested that the fresh, estuarine and marine water of Bangladesh have a single population of Hilsa shad (Fig. 3; Fig. 5). In-addition both PCA and network on SNP loci analysis also represented the same trend (Fig. S1, Fig. 4). In the phylogenetic tree, samples of all locations were mixed together without making any specific cluster. In the population structure analysis, a single population was present with some admixture individuals bearing small portion of genes from other group. Pairwise FST value between maximum locations were poor with non-significant P value (P< 0.05), that support the deprived local population differences and homogeneity of this fish population throughout our studied locations.
Bangladesh has diversified fresh water habitats for Hilsa shad migration including main river system, coastal and freshwater small rivers, hill stream rivers, haors etc. but anadromous migration of this shad starts from same marine water body, the Bay of Bengal, which is their living ground. Furthermore, this fish has highly migratory nature among marine, estuarine and fresh water bodies. Therefore, it is difficult to draw a conclusion that there is more than one population in this water system. Low variation among groups and among population within groups also did not support more than one population. FST value between most of the locations was poor with non-significant P value, which suggested that the population differences were not significant. Although in some cases, P value was significant but due to their poor FST value did not provide strong support of local population differences. Present findings of this study were supported by the findings of some previous researchers who represented the single gene pool or stock of this species in the Bay of Bengal with a substantial gene flow18,52,53.
All of the spawning grounds of Hilsa shad were identified in the coastal areas of Bangladesh especially at the lower stretches of the Meghna, the Tetulia, the Ander Manik and the Shahabazpur River e.g., Hatia (Moulavir char) Sandwip (Kalir char) and Bhola (Dhal char and Monpura)6,21. However, migratory plan is mainly initiated during the spawning season, which is activated with follow of fresh water runoff from the inland rivers, and naturally it occurs with the commencement of the south-west monsoon and consequent flooding of all the major rivers draining down to the upper Bay of Bengal and there are no considerable differences in any context. Isolation of spawning ground is an important factor for population differentiation11. Due to presence of un-alienated spawning grounds, it is less feasible to draw population differences of Hilsa shad in the upper streams of different rivers and in their living ground, Bay of Bengal. Therefore, the unique spawning grounds and sole major migratory down-stream route strengthen the presence of single population in all over the Bangladesh water without any significant population clusters. Without specify exact spawning grounds for every cluster, it is unrealistic to draw several clusters in this population.
Hilsa population studies in Indian part across the Hoogli, the Bhagirathi, the Ganges and the Brahmaputra Rivers also suggested single and genetically homogeneous population in Indian part10,17,18. Hilsa shad population of the Hoogli-Bhagirathi river system and Hilsa stock of Bangladesh water used same natal habitat, Bay of Bengal. Moreover, the River Ganges is the upstream of the Padma River (Bangladesh) and the Bhagirathi River (India) as well as the Brahmaputra is the upstream of the Jamuna River (Bangladesh). Most of the Hilsa shad of River Ganges comes from the Padma River and as the same way the Brahmaputra river has no other significant source of this fish except the Jamuna River. So genetic homogeneity and unique population across these rivers of Indian part also supported the Hilsa shad’s single population in the Bangladesh water.
Nevertheless, Rahman and Naevdal (2000) based on allozymes and muscle proteins as well as Mazumder and Alam (2009) based on mitochondrial D-loop region figured out more than one Hilsa population in Bangladesh water15,54. Rahman and Naevdal (2000) mentioned two populations: 1. Marine and 2. Estuary and fresh water but they processed without explaining how this highly migratory species was separated into two distinct cohorts. Mazumder and Alam (2009) divided the population into two clusters like previous study but poor pairwise FST value between two groups showed that there were no differences between fresh water and marine-estuarine locations. Recently Asaduzzaman et al. (2020) reported three clusters in the Hilsa population in Bangladesh water, first one was in marine and estuarine waters and another two belonged to north–western riverine (turbid freshwater) and north-eastern riverine (clear freshwater) ecotypes20. Existing of a single population, the most likely assumption from the present research varied with their findings. Our result suggested that as a highly migratory species, Hilsa shad is incapable to belong to more than one population when sampled at different sections of their migration route. Our postulation is the presence of single cluster in the Bangladesh water because all water bodies are almost connected to each other, raising high rate of gene flow and created large population size. Western and eastern river systems of Bangladesh have immaterial dissimilar water quality (e.g., turbidity) but this is not enough to make population differences of Hilsa shad since they migrate and start their life from same spawning grounds and used almost same route across the lower stream and coastal estuaries during their breeding migration. Asaduzzaman et al. (2020) reported that samples of the Meghna river (MR) was included in the north-eastern riverine (clear freshwater) ecotypes by PCA and neighbor-joining tree analysis20. However, their sample collection site (MR) was located in the common migratory route for north–western riverine (turbid freshwater) and north-eastern riverine (clear freshwater) ecotypes. Therefore, this site should be representing the samples of both ecotypes rather than specific one.
If we draw several specific populations or clusters in the upper streams of Bangladesh that means we had the scope to find this shad in the freshwater all over the year round. However, in the freshwater of Bangladesh, this fish was available in the summer (July-October) and winter season (January-March) only; these were related to their summer and winter migration respectably55. If one or two groups of this fish, continue their complete lifecycle in the freshwater (Western/Eastern part of Bangladesh) that states the assurance of continuous supply of this fish almost year round. However, the original scenario does not support this hypothesis. Finally we can conclude that, only one population of this fish inhabit in the Bangladesh water without any instance of different populations and clusters (2-4) but in some specific locations, they had some particular characteristics. The Bay of Bengal is their main living ground, at the time of their breeding they come to the freshwater upper streams, spawn in the estuaries and finally return to the sea. Therefore, using all the same ecosystems (sea, estuary and freshwater rivers) in a cyclic fashion is essential to support their life cycle, which certainly pushes all the individuals to belong a unique population.
In the population structure analysis, only one population of Hilsa shad was identified with some admixture individuals (32%) containing partial genes from other population in the water bodies of Bangladesh. The mentioned other population might not represent the Hilsa population of the Hoogly and Bhagirathi river system, India because, the Hilsa shads of both migratory routes of Bangladesh and India showed genetic homogeneity10,17. The Ganges and Brahmaputra rivers of Indian part are the upstream of the Padma and the Jamuna river of Bangladesh and might be belonged to the same population. However, Hilsa population of the Arabian Sea was genetically heterogeneous from the Bay of Bengal18 and those different population genes of admixture individuals might come from the Arabian Sea by oceanographic dispersion. Once (almost 18,000 years ago) the Arabian Sea had a close connection with the Bay of Bengal through the Laccadive Sea, the Gulf of Mannar and the Palk Bay. Therefore, this likely was an easy way for oceanographic dispersion of Hilsa shad between these two water bodies. After that period, a bridge of limestone shoals, coral reefs and tombolo called as ‘Ram Bridge’ or ‘Adam’s Bridge’ (about 48 km) originated between Pamban Island off the south-eastern coast of Tamil Nadu, India, and Mannar Island, off the north-western coast of Sri Lanka 56,57. Sarker et al. (2020) also mentioned that type of oceanographic dispersion between these two water bodies for another Clupeid fish species, Hilsa kelee49. The Irrawaddy, the Naaf and the Sittang River of Myanmar were also regarded as another important route for Hilsa migration6,58. There is also a possibility of inflowing of these different genes of other population from such population. Still there is no population structure study was conducted in the Myanmar part. Therefore, it is no scope to compare those admixture individuals with the Hilsa population of Myanmar. However, for completing the full scenario, the Hilsa population of Myanmar also claims research attention in population genomics field.
Hill stream river and haor were two important and unique ecosystems for fish diversity in Bangladesh, they belong to the unique characteristics in the ecological factors as well as fish diversity 62,63. Infrequently Hilsa shad use these two water bodies as their migratory routes. Samples were collected from the Shomeswari River and the Dingapota Haor, Mohanganj as the representatives of hill stream river and haor population respectively. However, Hilsa shad of these two exclusive water bodies were similar to the samples of the some other fresh water bodies (i.e., CM, CN and MG) as they were belonging to the Hilsa population without any admixture individual. Samples of SS do not have any significant P value with other locations whereas MO samples had significant P value with five other locations but having poor FST value with three locations (i.e., BL, PP, MG). MO samples had only mentionable FST value with MP (estuarine) and MK (marine), which might be the result of differences in water quality of these two water bodies. In PCA, phylogenetic tree and in network, the samples of hill stream river and haor failed to make any unique cluster or monophyletic clade that represent they are also the part of single unique Hilsa population of Bangladesh water.
Main migration was occurred through the Meghna river estuary, which is connected to the Padma, Meghna and Jamuna river system. However, there are some other alternative routes through some small coastal rivers e.g., the Pashur, the Bishkhali, the Balaswar, the Kocha river, which are connected to the Padma river through the Modhumati and the Gorai river. These coastal rivers passed through or beside the world largest mangrove forest Sundarban. Thus, these two routes are ecologically different from each other. Samples of these two routes have some genetic differences, because most of the locations (MK, CF and BL with PP and KN) of these two estuarine routes had significant P value, but their FST value was not satisfactorily high to make population differences. Ecological differences of these two routes might be played an important role to create this type of slight differences among them. Therefore, these scenarios were not significant enough to describe noteworthy differences in the population level, but may make a sign of upcoming population differences.