International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(3), 37-44, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 37 Leaf Architectural Study of Hoya coriacea, Hoya halconensis and Hoya buotii(Apocynaceae)Salvaña F.R.P. and Buot Jr. I.E.Plant Biology Division, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños, Laguna, PHILIPPINES Available online at: www.isca.in, www.isca.me Received 22nd October 2013, revised 8th December 2013, accepted 2nd February 2014Abstract Leaf architecture characters of the three seemingly similar and hence, controversial species of Hoya, namely H. coriacea, H. halconensis and H. buotii were examined. The use of leaf architecture has been demonstrated in paleobotanical studies and other systematic studies to resolve some taxonomic controversies. These characters were used to analyze the similarities and differences of the three Hoya species. The seven characters, leaf base, margin, form, blade class, primary vein size, variation of angle of divergence and areole development, showed to be distinct from either one or two species. Unweighted Pair Group Method with Arithmetic Averages (UPGMA) and Single Linkage were used for cluster analysis. Both results showed that H. coriacea and H. buotii shared more similar characters than H. halconensis. Elliptic to narrow elliptic form, convex leaf base and stout primary vein size were characteristics common to H. coriacea and H. buotii whereas both H. halconensis and H. buotii had leaves with nearly uniform angle of divergence. Moreover, H. coriacea and H. halconensis have moderately developed areoles while H. buotii had well-developed ones. The study showed that variations in leaf architecture characters can be used to illustrate the relationship among the three species of Hoya. Keywords: leaf architecture, Hoya coriacea, Hoya halconensis, Hoya buotiiIntroduction Genus Hoya is considered taxonomically complex brought about by the large number of species that have been published. Approximately, a hundred of species names were used in horticulture and there is an increasing number of individuals collecting and studying this group of plants. Traditionally, morphological parameters have been used for several taxonomic and ecological investigations not only in animals4-6 but also in plants7-10. In the identification and classification of plant taxa, floral characters are given emphasis11. This is also true in Hoya wherein the bases of delineation of the genus are more focused in the qualitative and quantitative features of the corolla, corona and pollinarium12. The use of these characters is also done in separating H. coriacea, H. halconensis and H. buotii. Based on the description of Aurigue13, there is a slight difference on the flower morphology of the three species. The flowers of Hoya buotii are more hairy compared to H. halconensis and H. coriacea. Aurigue13 also noted three color forms existing in the flower of H. buotii, but the one that is considered in this study is the purplish green with light red markings on the corona. The corona of H. halconensis has moderate reddish brown inner lobe and greenish outer lobe whereas H.coriacea has silky-sericeous-velvety inner surface and papillate outer surface as observed by Aurigue13. In this paper we are trying to use leaf architecture data to hopefully supplement the floral information. The use of leaf architecture data has never been optimized despite that they are genetically-fixed and stable especially venation patterns14. In fact, several taxonomic investigations and controversies had been conducted and resolved using these characters including: the determination of the differences in the three leaf types of Sorbus through the examination of the form and venation15; the fundamental differences in the leaf shape of Oak leaves which is suggested by relationship among particular sets of landmark16; the determination of infrageneric and interfamilial relationships of Chloranthaceae17; the determination of the species of Salicaceae18; the relationship of the three subgenera of Ficusincluding Ficus, Sycomorus and Urostigma19; distinguishing some species of family Malvaceae20; and the classification of the species of Camelia (Theaceae)21. There are instances wherein leaf characters are of great importance in taxonomic studies particularly in sterile plants and fossils without reproductive parts22-23. However, even though there are already works suggesting the effectiveness of using leaf characters, full taxonomic potential of these remains relatively unexplored24. The purpose of this study is to document the leaf architectural characteristics of Hoya coriacea, Hoya halconensis and Hoya buotii and to determine whether these leaf architecture characters strongly support the present recognition of these three distinct species. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 37-44, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 38 Material and Methods A total of ten mature leaf samples for each of Hoya coriacea, H. halconensis and H. buotii were collected from the garden of Dr. Inocencio E. Buot Jr. The source of the collection was from the Institute of Plant Breeding: Hoya Accessions, University of the Philippines Los Baños, Laguna, Philippines. All leaf samples were subjected to pressing and drying. All dried leaves were examined using stereomicroscope. The leaf architecture of the three species was described based on established leaf architecture characters and terminologies of Hickey25 and Leaf Architecture Working Group26. Large measurements such as leaf length, leaf width etc. were measured using dial calliper. Angle of Divergence was measured using a protractor. A dichotomous key to the 3 species of Hoya studied was constructed. Descriptions were based on the characteristic of leaf architecture examined in this study. Cluster analysis using PAST- PAleontological STatistics ver. 1.6427 was done to analyze the significant differences among the species. A number of characters were selected and each of its character states was assigned as values corresponding to a legend as follows: F (form)- 1-2; M (Margin)- 1-3; BC (Blade Classs) – 1-2; PVS (primary vein size)- 1-2; ARD (areole development)- 1-2; VAD (variation of angle of divergence- 1-2; LB (leaf base)- 1-2. A dendogram was constructed using Euclidean as distance measure and Unweighted Pair Group Method with Arithmetic Averages or UPGMA Method and Single Linkage or Nearest Neighbor as a linkage method28. Results and Discussion Leaf Architectural Characteristics of H. coriacea, H. halconensis and H. Buotii: The examined leaves of the three species are shown in figure – 1, 2, and 3. In table – 1, leaf architecture characteristics of H. coriaceae, H. halconensis and H.buotii are listed The three species of Hoya examined in this study exhibited several unifying features including the unlobed leaf margin; pinnate, camptodromous, straight primary vein; festooned brochidodromous, sinous, secondary veins; moderately acute angle of divergence; inter-secondaries composite; random reticulate tertiary veins; alternate percurrent quaternaries; looping branches are enclosed by 3° and 4° arches; branched veinlets and random areole arrangement. On the other hand, differences in the three species were also noted in some leaf architectural characters as shown in table - 2. The three species greatly differ in leaf margin, L: W ratio and leaf form. There were also characters common to the two species which differ from the other one. Convex leaf base is a characteristic of Hoya coriacea and H. buotii whereas H. halconensis exhibited rounded leaf base. Moderate primary vein size is exhibited by H. halconensis and stout primary vein size is of H. coriacea and H. buotii. Furthermore, H. buotii has a well developed areoles whereas H. coriacea and H. halconensis has areoles which are moderately developed. In terms of the blade class, both H. halconensis and H. buotii are under notophyll while H. coriacea is under mesophyll. Nearly uniform angle of divergence is recorded in H. halconensis and H. buotii whereas lower secondaries of H. coriacea are more acute than the upper ones. Figure – 1 Examined leaf samples of Hoya coriacea Figure – 2 Examined leaf samples of Hoya halconensis International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 37-44, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 39 Table – 1 Leaf architecture characters of the three species of Hoya Characters Hoya coriacea Blume Hoya halconensis Kloppenburg Hoya buotii Kloppenburg Blade Length (mm) 113- 166 99-136.5 83-137 Blade Width (mm) 34-56 46-60.5 34-49 Blade Area (mm 2 ) 2561-5674 3102-5220 2130-3990 L:W Ratio 2.6:1- 3.5:1 1.7:1-2.4:1 2:1-3.4:1 Blade Class Mesophyll Notophyll Notophyll Form Elliptic to narrow elliptic Ovate to oblong Elliptic Symmetry Symmetrical Symmetrical Symmetrical Apex Acuminate Acuminate Acuminate Base Convex Rounded Convex Margin Smooth Entire Slightly revolute Lobation Unlobed Unlobed Unlobed 1° vein category Pinnate Pinnate Pinnate 1° vein size Stout Moderate Stout Course Straight Straight Straight 2° vein category Festooned Brochidodromous Festooned Brochidodromous Festooned Brochidodromous Relative Thickness Moderate Moderate Moderate Angle of Divergence Moderate acute Moderate acute Moderate acute Variation of Angle of Divergence Lower secondaries more acute than upper ones Nearly uniform Nearly uniform Course Sinous Sinous Sinous Behavior of Looping Branches Enclosed by 3° & 4° arches Enclosed by 3° & 4° arches Enclosed by 3° & 4° arches Inter-2° veins Composite Composite Composite 3° vein category Random reticulate Random reticulate Random reticulate 4° vein category Alternate percurrent Alternate percurrent Alternate percurrent Veinlets Branched Branched Branched Areole Development Moderately-developed Moderately-developed Well-developed Areole Arrangement Random Random Random Table - 2 Characters separating Hoya coriacea, H. halconensis and H. buotii Character Hoya coriacea Blume Hoya halconensis Kloppenberg Hoya buotii Margin Smooth Entire Slightly revolute LWR 2.6:1-3.5:1 1.7:1- 2.4:1 2.4:1-3.4:1 Leaf Base Convex Rounded Convex Form Elliptic to narrow elliptic Ovate to Oblong Elliptic Blade Class Mesophyll Notophyll Notophyll 1° vein size Stout Moderate Stout Variation of Angle of Divergence Lower secondaries more acute than upper ones Nearly uniform Nearly uniform Areole Development Moderately- developed Moderate-developed Well-developed There were recorded variations in the size and shape of the three species of Hoya in the study. These variations could be physiological in nature as in the study of Zhang et al.29. McLellan30 and Sylvester et al.31 observed that there are instances that leaves produced on the shoots at the beginning of a season display gradual leaf shape transition that could spell out difference when they mature. For such reasons, the size and the shape of the leaf were considered to yield poor results. This is true in plotting populations wherein there were significant variations in the area and shape of the leaves due to segregation. However, leaf physiognomy characters can work well within single species32. Most of the leaves examined in the study were symmetrical. However, there were recorded leaf samples which seemed asymmetrical. This conforms to the result of Torres et al.33where there were minor asymmetrical variations in the leaves of 16 species of Hoya. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 37-44, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 40 Dichotomous Key to Three Species of Hoya: 1. Blade ovate to oblong; 1° vein size moderate, H. halconensis, 1. Blade elliptic to narrow elliptic; 1° vein size stout 2. 2. Well-developed areoles; leaf base convex H. buotii, 2. Moderately-developed areoles; leaf base convex H. coriacea. Hoya halconensis Kloppenburg: Blade ovate to oblong with acuminate apex and rounded base, symmetrical, 99-136 mm long, 46- 60.5 mm wide, 1.7- 2.4: 1 ratio, notophyll, entire, unlobed. Venation camptodromous, pinnate. 1° stout; straight. 2° festooned brochidodromous; moderate; sinous; angle of divergence moderately acute and nearly uniform; loop-forming branches enclosed by 3° and 4° arches. Inter-2° vein composite. 3° random reticulate. Higher vein order distinct; 4° alternately percurrent ; highest vein order 5°; moderately-developed and random areoles. Marginal ultimate venation looped. Veinlets branched (figure – 4) Figure – 3 Examined leaf samples of Hoya buotii Hoya buotii Kloppenburg : Blade elliptic with acuminate apex and convex base, symmetrical, 83-137 mm long, 34-49 mm wide, 2-3.4: 1 ratio, notophyll, slightly revolute, unlobed. Venation camptodromous, pinnate. 1° stout; straight. 2°festooned brochidodromous; moderate; sinous; angle of divergence moderately acute and nearly uniform; loop-forming branches enclosed by 3° and 4° arches. Inter-2° vein composite. 3° random reticulate. Higher vein order distinct; 4° alternately percurrent ; highest vein order 5°; 4° and 5° anastomosing to form well-developed, 5 or more- sided and random areoles. Marginal ultimate venation looped. Veinlets branched (figure – 5) Figure - 4 Hoya halconensis Kloppenberg flower (Photo by: Edward Agdeppa) Figure - 5Hoya buotii Kloppenberg flower (Photo by: I.E Buot Jr.) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 37-44, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 41 Hoya coriacea Blume: Blade elliptic to narrow elliptic with acuminate apex and convex base, symmetrical, 113-166 mm long, 34-56 mm wide, 2.6- 3.5: 1 ratio, mesophyll, smooth, unlobed. Venation camptodromous, pinnate. 1° stout; straight. 2° festooned brochidodromous; moderate; sinous; angle of divergence moderately acute, lower 2° more acute than upper 2°; loop-forming branches enclosed by 3° and 4° arches Inter-2° veins composite. 3°random reticulate. Higher vein order distinct; 4° alternately percurrent ; highest vein order 5°; moderately-developed and random areoles. Marginal ultimate venation looped. Veinlets branched (figure – 6) Figure - 6 Hoya coriacea Blume flower (Photo by: I.E. Buot Jr.) There is no doubt that Hoya halconensis, H. buotii and H. coriacea can be delineated into three distinct species through leaf architecture, though the differences are not that striking. Cluster Analysis: Qualitative morphological data including leaf base, form, primary vein size, blade class, variation of angle of divergence, areole development of the three species of Hoyawere subjected to cluster analysis Figure – 7 shows the dendrograms constructed using Unweighted Pair Group Method using Arithmetic Averages and Single Linkage or Nearest Neighbour Clustering. Both dendrograms clearly illustrated that H. coriacea and H. buotii shared more similar characters than H. halconensis at the similarity level of 0.84. This is also depicted in table - 2. Cluster analysis is a statistical tool which produces a hierarchical classification of taxa based on similarity matrix34. Unweighted Pair Group Method with Arithmetic Averages (UPGMA) and Single Linkage or Nearest Neighbour Clustering are clustering approaches commonly used in the analysis. UPGMA algorithm constructs a dendrogram that reflects the structure present in a pairwise similarity matrix and clusters are joined based on the average distance between the members of a group35. Moreover, single linkage clustering constructs a dendrogram which shows the sequence of cluster fusion and the distance at which each fusion takes place and clusters are joined based on the smallest distance between two groups36. Apparently, both dendrograms produced by the two approaches depicts the same pattern of clustering of the three species in question. Hammer et al.27 has stated that if the groupings do not change when tried another algorithm, that grouping should perhaps be trusted. Thus, in terms of leaf architectural characters, H.coriacea and H. buotii are more related to each other than with H. halconensis. The results of the study indicate that leaf architectural characters are necessary and should be combined with floral characters of the three Hoya species to support and strengthen their current status as three distinct and separate species. Conclusion Leaf architectural characters of three species of Hoya namely H. coriacea Blume, H. halconensis Kloppenberg and H. buotii Kloppenberg, were examined. Description of the leaf architecture characters were given for each species. There were characters common in the three species and only seven were found to be different from any one or two of the species. Leaf characters, especially the detailed characteristics of the higher vein orders as well as the areoles, can be considered as good taxonomic markers in classifying plant taxa. These can be used in combining or separating several species. 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