(Family Hydrocharitaceae) Leaf Shape Variation between Halophila ovalis (R. Br.) Hook F. and Halophila minor (Zoll.) den Hartog (Family Hydrocharitaceae) Armin S. Coronado, Christian Joy O. Regala and Michelle O. Casayuran Department of Biology , College of Science, Polytechnic University of the Philippines ABSTRACT In the Philippines, even there were interesting questions related with the diversity and widespread occurrence of seagrasses, still few papers has been published. Recent studies used quantitative leaf descriptors in order to discriminate each taxon, which rely on the basic linear measurement of its length, width and height. This study was conducted to determine the extent of leaf shape variation between H. ovalis and H. minor. Digitized images of the collected leaves were measured using ImageJ (linear measurement) and used to construct the leaf contours using elliptic Fourier at 20 harmonics. The total variation attributed by each component to the overall leaf shape variation was ascertained by performing separate analyses for both symmetric and asymmetric variations. Results showed that H. ovalis have long and narrow leaves as compared with H. minor which has shorter and wider leaves. Leaf geometry showed that 92.71% contributed to overall variation and 86.63% was attributed to its symmetric components while only 6.08% from its asymmetric components. The former has high genetic bases while the latter can be a manifestation of the environmental stressor and structural adaptation exhibited by the Halophila. INTRODUCTION Genus Halophila is a seagrass that belong to order Alismatales under the family Hydrocharitaceae. Halophila is readily distinguishable on the basis of the leaf form. The leaves are variably oval and elliptical to oblong in shape (Lanyon, 1985). At present the delimitation of Halophila species posed a problem such that Halophila showed a synonymous taxonomy in terms of their morphological features (Sachet and Forsberg, 1973). It is then necessary to find characters that would delineate one taxon from another. Thus, quantitative analysis of the leaf outline between Halophila taxa was explored. The laminar shape of H. ovalis ranges from elliptic to ovate. The apex of the leaf lamina has an obtuse to acute angle. The shape margin has also an obtuse to acute base angle. On the other hand, H. minor has a laminar shape that ranges from ovate to oblong. The apex of the leaf blade and the leaf margin has an obtuse to acute angle. Kruskal-Wallis test revealed that leaf length and leaf width between the two Halophila species are significantly different (∝0.05> 0.0001) from each other. The observed difference depends on the organisms. Halophila strongly compete for light with other macrophytes in the seagrass community, which is their adaptation to commensurate the light requirement in their habits (Hedge et al., 2009).  IV. RESULTS AND DISCUSSION Figure 2. Leaf descriptions observed from the two (2) Halophila species. Images on the left are the normalized leaf shape (mean ± 2SD). Images on the right are photographs of the representative samples. II. OBJECTIVES OF THE STUDY This study was conducted to determine the extent of leaf shape variations between Halophila ovalis and Halophila minor. Specifically, it aimed to: characterize the leaf morphological differences exhibited from the collected taxa; quantitatively describe the differences in leaf shape variations in H. ovalis and H. minor; and determine the asymmetrical and symmetrical variations between H. ovalis and H. minor. Leaf Shape Geometry The pattern of leaf shape variation between H. minor and H. ovalis were visualized by reconstructing a closed two-dimensional contour using Elliptic Fourier descriptors. The significant principal components derived from the over all leaf shape variation between the two Halophila species as well as the symmetrical and asymmetrical components is presented in Figure 3. The symmetrical variation was reflected in PC1 and PC3 while the asymmetrical variation in PC2 and PC4. Kruskal-Wallis test (Table 1) showed that PC1 and PC3 were significantly different (PC1= 0.05>0.01; PC2= 0.05>0.0001) while PC2 and PC4 were comparable (PC2= 0.05<0.155; PC4= 0.05<0.335) between the two taxa. A non parametric post hoc test, Mann-Whitney was utilized to identify the principal component that highly contributed for the differences between a paired taxon. Results revealed that PC1 and PC3 were significantly different between H. ovalis and H. minor. Separate analysis for both symmetric and asymmetric variation was performed to determine the total variation contributed by each component to the over all leaf shape variation between the two taxa. The high percentage value of symmetrical components (93.44%) suggests a high genetic variation while the occurrences of low percentage value of assymetrical component (6.56%) suggest two possible reasons: developmental instability and fluctuating asymmetry. Fluctuating asymmetry is defined as random deviations from perfect symmetry in bilateral traits (Coronado, 2009; Hodar, 2002; Palmer and Strobeck, 1992). On the other hand, developmental instability is influence by environmental factor (Coronado, 2009 and Babbit, 2008). III. MATERIALS AND METHODS H. minor and H. ovalis were collected at Bolinao, Pangasinan and San Juan, Batangas. For each species, one hundred (100) leaf samples were collected from at least ten (10) individuals. To asses the leaf shape geometry, the method used by Futura (1985) was adapted to determine the patterns of leaf shape variation. The outline-based morphology of the leaves were initially constructed by assigning chain code (Neto et al., 2006) to the binary images (black background and white objects) of the leaf samples. The assigned codes were then converted into shape variables in the form of Elliptic Fourier Coefficients (EFC) by using the first 20 harmonics and were normalized using 80 elliptic Fourier descriptors (Kuhl and Giardana, 1982) to reconstruct the leaf outline. Figure 3. Variations explained by each principal component shown as ±2 standard deviations from mean leaf shape between the two Halophila species. The numbers correspond to the significant principal components, respectively. Figure 1. Summary of the various analyses used in this study to determine the patterns of leaf shape variations between H. ovalis and H. minor Table 2. Eigenvalues and contributions of each principal component to leaf variation between the two (2) Halophila species. V. CONCLUSIONS H. ovalis has long and narrow leaves while H. minor has shorter and wider leaves; The leaf shape outline enabled to discriminate H. ovalis and H. minor; and The pattern of leaf shape variations between H. ovalis and H. minor can be ascribed with their leaf aspect ratio.