DOI: 10.5061/dryad.02v6wwq0m
title: Differentiating three Indian shads by applying shape analysis from digital images

description: Tenualosa ilisha, T. toli and Hilsa kelee are three Indian shad species of
 the family Clupeidae. In past, relationships among these shads have been
 explored in few morphological studies but still remain poorly understood.
 In this study, 120 specimens of three shad species were collected from the
 wild and a truss network system of 13 landmarks was interconnected to
 yield 77 size-adjusted characters. Discriminant function analysis (DFA)
 produced 13 discriminating variables in delineating the three species. The
 landmark based morphometric distance between origin of dorsal fin and
 posterior end of eye was the most important character in discriminating
 the three species. Three prominent characters extracted from DFA (distance
 between posterior end of operculum and posterior end of eye, distance
 between origin of dorsal fin and posterior end of eye and distance between
 insertion of pelvic fin and end of operculum) were used as traditional
 morphometric characters and their ratio to standard length were found
 significant in differentiating the three shad species. The present study
 strongly supports the efficiency of landmark-based morphological
 characters generated from digital images in discriminating the species and
 can be used as a rapid diagnostic tool in recognizing them.
description: <b>Study areas and sample collection</b>             Samples of <i>T. ilisha</i> were collected from Digha and Kakinada on the coast of Bay of Bengal and Bharuch on Narmada River. River Narmada originates as Narmada Kund, located at Amarkantak and flows through the Gulf of Khambhat into the Arabian Sea. Samples of <i>T. toli</i> were collected from Okha, Gulf of Kachchh on the Arabian Sea coast (Singh, 2002). Samples of <i>H. kelee</i> were collected from Rajahmundry on the Godavari River which flows to Southeast coast and join the Bay of Bengal. A total of 120 samples comprising of <i>T. ilisha</i>, <i>T. toli</i> and <i>H. kelee</i> were collected based on taxonomic key characters (Whitehead, 1985). The sample size and related information for three species selected to document morphometric variations are presented in Table 1 and Figure 1. <b>Ethics statement</b> As per IUCN status, <i>T. ilisha</i> is under Least Concern (LC), and <i>T. toli</i> and <i>H. kelee </i>are under Not Evaluated (NE) category. Fish samples were obtained from wild, directly from the commercial catches. Sites from where fishes were collected fell outside Protected Areas (PAs) and therefore no permits were required from State Forest and Wildlife Department. <b>Meristic characters</b> Meristic counts were followed as per Whitehead (1985). Fin rays were counted under transmitted light using a stereoscopic microscope. Results from previous studies (Whitehead, 1985; Quddus <i>et al</i>., 1984a; Narejo <i>et al</i>., 2008; Jawad <i>et al</i>., 2011b; Antony <i>et al</i>., 2005; Losse, 1968) were also included in this study for comparison between species. <b>Landmark based morphometric analysis</b> Digital images of individual fish, labelled with a specific code for identification, placed on laminated graph sheets, body posture and fins were teased into a natural position, was captured using a Cyber shot DSC-W300 digital camera (Sony, Japan). Two–dimensional Cartesian coordinates of 13 landmarks were recorded on the left view of each specimen (Figure 2). Data were generated from digitized images using a combination of three different softwares: tpsUtil was used for converting graphics images in to ‘tps’ format; tpsdig was used for fixing landmarks on the images and also setting scale for the image; PAST (Hammer <i>et al</i>., 2001) was used for generating truss data based on the landmarks (Rohlf, 2006). A total of 78 inter landmark morphometric characters were extracted by measuring distances between landmarks. The data were log-transformed (Strauss, 1985) and size effect was removed by using the following (Elliott <i>et al</i>., 1995): M<sub>adj</sub> = M(L<sub>s</sub>/L<sub>0</sub>)<sup>b</sup> where M is the original measurement, M<sub>adj</sub> the size adjusted measurement, L<sub>0</sub> the standard length of the fish, L<sub>s</sub> the overall mean of standard length for all fish from all samples in each analysis, and b estimated for each character from the observed data as the slope of the regression of log M on log L<sub>0</sub> using all fish from each group.             Standard length (SL, character code 1-6) was not used in the final analysis because SL was used as a basis for transformation (Mamuris <i>et al</i>., 1998) and thus 77 morphometric characters were retained. Univariate analysis of variance (ANOVA) was performed to assess the significant difference among the three species. The significant variables (p&lt;0.01) were subjected to Principal Component Analysis (PCA) and Discriminant Function Analyses (DFA). Box plot was prepared for each discriminant morphometric characters. Range bar of ratios of traditional morphometric data with standard length, extracted from differentiating landmark based morphometric characters were prepared. Canonical Variate (CV) plot was generated from significant variables to visualize the variations among groups. Squared Mahalanobis distance among the three species was computed (Puillandre <i>et al</i>., 2012) and distances between the centroids of the three species were visualized by performing non-metric multidimensional scaling (Legendre & Legendre, 1998). An unweighted per-group method with arithmetic averaging (UPGMA) cluster analysis (Hair <i>et al</i>., 1998) based on the Squared Mahalanobis distance between the group centroids was applied to determine the similarity/dissimilarity between species. Statistical analyses for morphometric data were performed using the software packages viz. SPSS version 16, JMP version 9 and Excel (Microsoft Office 2007).