Philippine Journal of Science 150 (4): 743-752, August 2021 ISSN 0031 - 7683 Date Received: 25 Nov 2020 Determining Shell Shape Differences in the Horse Mussels Modiolus philippinarum (Hanley 1843) and Modiolus modulaides (Röding 1798) by Morphometric Analysis Kaent Immanuel N. Uba* Department of Fisheries Science and Technology School of Marine Fisheries and Technology Mindanao State University at Naawan Naawan, Misamis Oriental 9023 Philippines In the present study, shell morphological variations of the horse mussel species Modiolus philippinarum and Modiolus modulaides have been explored by the means of linear morphometrics for size and landmark-based geometric morphometrics for shape. Linear morphometrics revealed significant differences in shell length [t(298) = –6.29, p = 1.08 x 10–9), shell height [t(298) = 10.60, p = 1.74 x 10–22], shell width [t(298) = 2.13, p = 0.034], PAMS or posterior adductor muscle scar length [t(298) = 2.16, p = 0.032], hinge length [t(298) = 2.26, p = 0.025], umbo length [t(298) = –5.54, p = 6.73 x 10–8], and anterior length [t(298) = –5.59, p = 5.16 x 10–8] between species. However, upon the use of these morphometric characters to develop an index that will easily discriminate the species, only the relationships of shell length vs. shell width and hinge length were significant (ANCOVA, width/ length F = 18.45, p = 0.0001; hinge/length F = 7.76, p = 0.005) but an invariably high overlap between species was observed resulting in a 23.3% misclassification rate. Contrastingly, the analysis of shape variables through landmark-based geometric morphometrics revealed significant differences in shell shape between the two species [MANOVA, Wilk’s λ = 0.01, F(24,335) = 941.4, p = 3.47 x 10–291] with a 0.0% misclassification rate. Generally, M. modulaides were found to have an elongated shell while M. philippinarum was compressed and convex. Also, visualization through thin-plate spline expansion factor plots revealed that variability in shell shape between the species occurred mostly in the posterior and ventral region of the shell and was attributed to the species ecology. The findings of the present study encourage the use of geometric morphometric methods in species delineation, especially when ecotypes or sibling species are present. Keywords: horse mussels, morphometrics, relative warp analysis, thin-plate spline INTRODUCTION bottoms of sheltered bays while M. philippinarum is found on muddy and gravely mudflats and seagrass The horse mussels Modiolus modulaides (Röding beds (Poutiers 1998; Ozawa 2001; Uba and Monteclaro 1798) and Modiolus philippinarum (Hanley 1843) are 2020). In the Philippines, these species are commercially widespread in the Indo-West Pacific region. Both species exploited for food and aquaculture feed (i.e. freshly fed to inhabit the littoral and sublittoral up to a depth of 40 shrimps and crabs) (Napata and Andalecio 2011; Uba et al. m. However, M. modulaides are often found on muddy 2020). Specimens of M. modulaides measure up to 8 cm shell length whereas M. philippinarum is usually bigger, *Corresponding Author: [email protected] measuring up to 13.5 cm shell length (Poutiers 1998). 743 Philippine Journal of Science Uba: Determining Shell Shape Differences between Vol. 150 No. 4, August 2021 M. philippinarum and modulaides There has been confusion on the identification of these morphometrics to investigate shell morphological horse mussel species, as demonstrated by conflicting variations between species and the feasibility of these reports (Laureta 2008; Napata and Andalecio 2011; del techniques in distinguishing these species. Norte-Campos et al. 2020; Uba and Monteclaro 2020). Most morphological differences between species are only identifiable by experienced observers (e.g. a careful comparison of the anterior margin, posterodorsal margin, MATERIALS AND METHODS presence of periostracum hairs, etc.). Accurate species identification is the first and necessary step when one Collection of Specimens studies species biology and population and community A total of 300 individual horse mussels were randomly ecology. Unfortunately, such identification is not always collected from January–March 2018 at Looc, Romblon easy to perform. Obviously, molecular methods may be (M. philippinarum, n = 150) and Dumangas, Iloilo (M. of great help, but they are costly and may not be always modulaides, n = 150), respectively (Figure 1). The horse on hand. In many cases, researchers have to rely heavily mussel species were identified carefully by their gross on morphological traits. Traditionally, shell morphometric morphology, as described by Poutiers (1998). characters have been used to distinguish bivalve species. However, recent analyses showed that the use of indices (i.e. the arc of maximal convexity of the valve’s outline Linear Morphometric Analysis and the index of convexity ratio) for species delimitation For the linear morphometric analysis, seven inflates the number of species as different ecophenotypes morphometric measurements were recorded for each are identified as separate species (Bolotov et al. individual, which generally followed the description 2015; Klishko et al. 2014, 2016, 2017a and b, 2018). of Maas et al. (1999) (Table 1). Linear morphometric Furthermore, traditional morphometric measurements measurements were taken from the left valve of the (length, width, height) are rarely sufficient because of the shell and measured to the nearest 0.01 cm using a high morphological plasticity of bivalve shells and linear digital caliper. measurements tend to be autocorrelated with the size of Descriptive statistics including mean (x̄ ), range animals (Zelditch et al. 2012). (minimum and maximum value), standard deviation At present, the detection of subtle differences in shape (SD), and coefficient of variation (CV = SD/x̄ ) of each can be done through geometric morphometric analysis. morphological character were calculated using EXCEL Because no deformation of shape occurs during 2019 (Microsoft Corporation, Redmond, WA, USA). manipulation, bivalves are excellent candidates for shape The relationships between these variables were analysis using geometric morphometrics which can be obtained by regression analysis fitting the linearized easily done using landmarks. A landmark is a homologous form [log10Y = log10(a) + blog10(X)], where X is shell point in the shell, which can be identified in all specimens. length in cm and Y is the shell height, shell width, It can be further classified into anatomical landmarks PAMS length, hinge length, umbo length, or anterior (points that correspond between organisms in some length in cm of the power function (Y = aXb). In each biologically meaningful way), mathematical landmarks relationship, differences between the estimated slopes (located on an object according to some mathematical or for each species were compared through ANCOVA geometrical property, e.g. high curvature or an extreme (analysis of covariance). Whenever the results point), and pseudo-landmarks (constructed points on indicated significant differences between the slopes the an object either on the outline or between landmarks) regressions were estimated separately for each species. (Dryden and Mardia 1998). Geometric morphometrics provides statistically robust and visual methods for To test the effectiveness of these characters in predicting shape analysis as it can remove the factor of size and different group locations, which clarify the relative symmetry which enables the study of its variation and importance of such traits as discriminators between relationships (Zelditch et al. 2012). Thus, it has been a priori for groups, a linear discriminant analysis successfully used to discriminate species of aquatic was performed using morphometric measurements organisms (Kennedy and Haag 2005; Rufino et al. 2006; previously transformed with Burnaby’s method for Torres and Santos 2009; Leyva-Valencia et al. 2012; size correction. The classification success rate was Fang et al. 2018; Santos et al. 2019). evaluated based on the percentage of individuals correctly assigned into the original sample by a The present work aimed to determine differences in shape confusion matrix (Kong et al. 2007; Konan et al. 2010; between M. philippinarum and M. modulaides based Gou et al. 2017). on linear measurements and landmark-based geometric 744 Philippine Journal of Science Uba: Determining Shell Shape Differences between Vol. 150 No. 4, August 2021 M. philippinarum and modulaides Figure 1. Location of the horse mussel collection sites. Table 1. Definitions of linear morphometric characters used in a measurement reference. The images were compiled, the study. scaled, and digitized using the tpsDig version 2.12 (Rohlf Morphometric 2008a) and tpsUtil version 1.44 (Rohlf 2009) software. Description characters Twelve homologous anatomical landmarks in the inner left Shell length Widest part across the shell at 90° to the height valves were landmarked, of which seven are anatomical Shell height Distance from the hinge line to the shell margin and five are mathematical landmarks (Bookstein 1991) Shell width The thickest part of the two shell valves as illustrated in Figure 2. Landmark positions were selected according to Valladares et al. (2010) with some PAMS length Length of the posterior adductor muscle scar modifications. Landmarking per specimen was done in Hinge length Distance between the anterior and the posterior triplicates on different days to minimize measurement end