Several statistical methods have been employed to study morphological divergences among wild populations of S. sarana collected from different geographic regimes. This is the first study on the population discrimination of S. sarana using truss network analysis with geometric morphometrics. The results revealed that heterogeneity exists among examined populations of S. sarana procured from the specific sites of geographically isolated rivers (Ganga, Godavari, Narmada, and Mahanadi). Significant variations were detected for most of the analyses. The PCA loadings (truss analysis) of principal components revealed distinctness between populations. Though, there was a slight overlap found in the characters which were examined among the four groups. This separation was corroborated by DFA (truss analysis), showed significant morphological heterogeneity among populations, the level of differentiation between most of them as evidenced by a slight overlap of statistical data on derived plots.
Using geometric morphometrics, the CVA plot obtained, has shown a slight level of overlaps among groups with a high percentage of correct classification suggesting differentiation among the examined populations. The PCA (geometric analysis) and DFA (geometric analysis) further confirmed the morphological heterogeneity among populations of S. sarana. The higher misclassification (DFA) observed for the Ganga with Mahanadi River and least with the Narmada. The biological variations in morphometric characters based on DFA are majorly associated with head morphology, covering lateral body lengths and caudal peduncle regions. Shape differences have been visualized with the deformation grids using geometric morphometrics. Geometric morphometry-based deformation grids (wireframes and relative warps) of average shapes between populations correspond to the high values of statistical distance between them and confirm the distinctness of populations in their immediate anatomical context.
Overall, the variations among the four groups in this study were largely owing to the dissimilarities of morphometric characters broadly associated to head, and body characteristics. However, the shape differences observed in this study presents little practical use in terms of discriminating fish populations in the field. The visualization of the body shape differences, associated with other groups of correlated morphological traits, allowed to obtain a clear diagnosis of fish morphology for each population [43, 44]. Visualization tools might help in further study of the putative underlying mechanisms involved [45]. The result of the present study is in line with other studies based on truss analysis [4, 46-49] and geometric morphometrics [50, 51] which have shown the fish species to have a distinctive morphology.
Lotic ecosystems such as rivers exhibit distinctive spatial and temporal characteristics [52]. The biogeographical patterns of rivers have been determined by specific spatial features which are enclosed in geomorphology of drainage, the structure, and directional connectivity of the dendritic network, elevation area, the flow of water, flow duration, physicochemical milieu, and other abiotic/biotic factors and their interaction [53, 54]. These factors possibly have a role in controlling morphologies of the fish fauna thriving in a particular aquatic environment [55, 56]. The environmental characteristics of the present rivers (Ganga, Godavari, Narmada and Mahanadi) are quite remarkable. The snow-fed Ganga River is young, longer and deeper, perennial in nature, having a good amount of flow throughout the year, exhibiting a vigorous tendency of meandering has its origin from Himalaya while Godavari, Mahanadi and Narmada are rain-fed older Peninsular Rivers, originates from a small plateau, flows straight path, shallower basins, forming broad largely graded shallow valleys, and are characterized by fixed course and absence of meanders. The Narmada River can be further distinguished as being the west-flowing rivers of the peninsular plateau [57-60]. Further peninsular rivers, Godavari, Mahanadi and Narmada are also geographically isolated from each other. Godavari basin is disconnected from the Mahanadi basin by the Bastar hills and the Parasgaon plateau towards the basin’s northern regions. Narmada basin is detached by the Maikala range from the Mahanadi basin in the east and Mahadeo hills in the south isolates the Narmada river basin from the Godavari basin [61].
The different environments often considered as a basis of strong divergent selection in morphological traits in the fish population. There is a relationship between morphological traits and their functions in connection with the environment [62, 63]. Spatial isolation has led to high levels of morphological variability between populations. The highest percentage of correct classification for the Narmada River population indicates greater distinction from the other populations. These morphological changes correspond to variations in spatial and/or temporal features. As mentioned above, the selected populations were geographically isolated from each other which could have hindered the movement of fish from intermingling with populations in other rivers. Therefore, the variability in morphological characters among populations possibly accredited due to separate geographical locations, the microclimate of rivers, the distance between the rivers, as well as the environmental variability of the river experienced by each population which leads to the local adaptations [64, 65]. The morphological variation could probably be coupled with the variation of feeding regimes and above-mentioned habitat circumstances [46, 55, 56, 66-68]. Additionally, different reports indicate variations of the whole fish body are mainly due to fish inhibiting in different flow regimes [56, 69].
Successful efforts have been made to differentiate S. sarana populations using traditional morphometrics [42] and genetics [41] from a neighbor country, Bangladesh which is far smaller in geography when compared to India. Therefore, in a geographically vast country like India having a number of isolated rivers, there is more possibility of variations among fish populations, which can be clearly depicted in the present study. The present study is in agreement with previous studies conducted with the same objective of detecting intraspecies morphological differences in other cyprinids [69-72] and non-cyprinids [49, 73, 74] Though, there are also reports on low morphometric divergences despite of geographically isolated populations [75]. Hence, patterns of morphological structure in fishes differed between river basins/rivers/river stretches. Variations in the degree and pattern of morphological traits correspond to numerical values may depends on the species of fish examined or/and the method/methods utilized for examine the morphological variations thus the reasons of low divergence could be many which is subject of detailed investigation.
Morphological differentiation can enable individuals to survive with existing environmental variability [76]. Fishes are excellent model systems for studies on inter as well as intraspecific divergences to understand ecological correlates of morphological diversifications. Some factors were assumed to be controlling the differences observed such as plasticity owing to habitat dissimilarities or could be due to environment and genotype interactions. Earlier, it was assumed that the variation of morphometric characters was exclusively genetic but recent studies have established its relation with environmental factors [77-79] and the role of epigenetics cannot be ruled out as suggested by many scientists, population differentiation associated with ecological factors have the main element as epigenetic [80].
As mentioned above, intraspecific variability can have huge ecological effects [81-83]. Charles Darwin indicated that variations among individuals of species offer the raw materials for natural selection. All hereditary characters in the genotype are not expressed in the phenotype. Further, variation not attributable to genetic factors not necessarily is environmental. Interestingly, the environment is often made responsible for non-genetic variations in phenotypes but it could be because of meta-stable epigenetic regulation [84]. Considering that morphological variations are raw materials, truss and geometric analysis techniques are the best approaches to discriminate populations on the basis of morphological characters. Results from the present study show that geometric morphometrics can provide additional information for shape delineation between populations that might otherwise be unnoticed. Further, the use of both truss and geometric morphometrics can provide deeper insight into the pattern of shape variations. This study could not answer whether are the results of morphological plasticity, genetic difference, or interaction of either mechanisms or epigenetic related hence, to resolve this, additional studies such as common garden experiments and epigenetic and/or genetics studies can be performed. More precise results might be obtained if larger sample sizes with a greater geographical extent were available. Geometric morphometrics analyses that include other aspects of fish morphology could enhance the precision of results.