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J. APIC. SCI. Vol. 57 No. 2 2013 DOI: 10.2478/Jas-2013-0028 J DOI: 10.2478/jas-2013-0028 J. APIC. SCI. Vol. 57 No. 2 2013 J. APIC. SCI. Vol. 57 No. 2 2013 Original article Landmark-Based morphometric study in the Fore and hind Wings oF an iranian race oF european honeyBee (apis melliFera meda) Lotfali Dolati1 Javad Nazemi Rafie2* Hoda Khalesro3 1Assistant Professor of Entomology Department, Faculty of Agriculture, University of Zanjan, Iran 2Assistant Professor of Entomology Department, Faculty of Agriculture, University of Kurdistan, Iran 3MSc Student of Entomology Department, Faculty of Agriculture, University of Zanjan, Iran *corresponding author: [email protected] Received 21 July 2013; accepted 13 November 2013 a b s t r a c t honeybee (Apis mellifera ) populations are usually distinguished using standard morpho- metric methods, mainly based on multivariate analysis of distances and angles. recently, geometric morphometrics, another method of statistical analysis of shape, has been devel- oped. this research was conducted on european honeybees in iran in 2012. multivariate analysis on hind wings identified significant differences between honeybee populations from different areas and significant differences in centroid size of fore wings in different geographical areas (F = 10.6, p = 0.000). populations from nine areas were classified us- ing discriminate function analysis based on shape variables of fore and hind wings. cross- validation tests based on discriminant functions of front wings correctly classified 68.2% of the colonies, and cross-validation tests based on discriminant functions of hind wings correctly classified 43% of the colonies. keywords: apis mellifera meda, fore wing, geometric morphometrics, honeybee, landmark. introduction diversity (Tofilski, 2004). The first comprehen- sive classification and distribution of Iranian The honeybee Apis mellifera L. is widespread honeybees was done in 1985 by Ruttner et al. in Africa, Europe, and parts of Asia with a wide (1985). The name A. m. meda was first given by diversity of subspecies that can be classified Skorikov in 1929 to a honeybee in the Soviet with morphometric tools (Ruttner et al., 1978). Union close to the Iranian border (Ruttner, Honeybees differ in their morphology, behavior, 1988). Multivariate analysis of 63 samples from and physiology according to the environmental across Iran and 142 samples from neighboring conditions to which they have adapted (Ruttner, countries (Turkey, Iraq, Syria, Lebanon, Israel, 1992). According to Ruttner (1988), there are Jordan, and Cyprus) resulted in partly overlap- at least 24 Apis mellifera subspecies grouped ping clusters. Ruttner et al. (1985) discriminated into three or four evolutionary branches, based six local populations for A. m. meda, as follows: on morphometric data. With the help of unvaried West and Central Iran (Azerbaijan-Iranian statistics, the discriminations were improved highlands), the subtropical coast of the Caspian (Alpatov, 1929); additionally, the first attempts Sea (Mazandaran), Northeast Iran (Mashad), to classify bee subspecies were based mainly Southeast Iran (Kerman), Iraq, and Southeast on color and size (Ruttner et al., 1978). Discrimi- Anatolia, from Van Lake to Hatay. nating among honeybee species is important Another modern morphometric method that for beekeeping and preserving honeybee bio- is also very promising for shape studies is 187 Dolati et al. Geometric morphometric study of Apis mellifera meda geometric morphometrics based on the de- Tarom (Emamkandi): 42°95´ N, 48°9´ E; Zanjan scription of shape in Cartesian coordinates (Hajarash): 36°40´ N, 48°30´ E; Khoramdareh: (Bookstein, 1991). Instead of distances and 35°31´ N, 49°17´ E; Ijrood: 36°5´ N, 32°49´ E; angles, it uses the coordinates of points called Khodabandeh: 38°14´ N, 39°16´ E; Mahneshan: landmarks. The landmarks are superimposed 37°10´ N, 48°10´ E; Zanjan (Nikpey): 36°89´ N, by translation, scaling, and rotation. After su- 46°22´ E; Tarom (Dastjerdeh): 42°52´ N, 49°11´ E) perposition, the landmark configurations differ (Tab. 1). Sampling was conducted from hives only in shape and can be analyzed by multivari- that had not migrated for many years. Samples ate statistical methods (Zelditch et al., 2004). In of honeybees were preserved in preservative honeybees, geometric morphometric analyses solution (30 parts distilled water, 15 parts 95% of wing shape have provided many new insights ethanol, 7 parts 38% to 40% formaldehyde into characters and identification of populations and 2 parts acetic acid) to prevent sample de- or lineages (Francoy et al., 2008; 2009; Tofilski, formation. Forty samples of honeybees were 2008; Miguel et al., 2011). collected from each colony. Collected samples The aim of this study was comparison of from each area were mixed and 40 specimens venation variations in the front and hind wings were randomly selected. Then, 40 right front of honeybee populations in different areas using and 20 hind wings were separated and mounted a geometric morphometric method. Questions in Canada balsam. Digital photos were taken we addressed with this method were whether from mounted wings using a DP12 camera and geographically isolating landforms such as a SZX12 OLYMPUS stereomicroscope. Eighteen mountains, valleys, or other natural obstacles landmarks on fore wings and six on hind wings affect morphological characteristics; whether were digitized on photos by tpsDig software this geometric morphometric method can dis- (Fig. 1). Next, digitized landmark outputs were criminate honeybee populations in nine areas analyzed by tpsRelw, tpsReg (Rohlf, 2010), and based on the front and hind wings; and which NtSys Pc. 2. Finally, samples of nine areas were wing (front or hind wing) better discriminates classified with discriminant function analysis area populations. (DFA) by SPSS ver. 18. All non-shape variations of these landmarks such as orientation (or materiaL and methods rotation), scale, and size were removed. A mul- tivariate analysis of variance (MANOVA) was The colonies were sampled from nine areas (three carried out on the landmark data to compare colonies in each area) in different geographic honeybee populations. regions of Iran (Abhar: 36°8´ N, 49°13´ E; Table 1. List and code of collection sites of Apis mellifera in Iran Fore wing Hind wing Population Sample Sample Code Code number number Abhar A 40 a 20 Tarom (Emamkandi) B 40 b 20 Zanjan (Hajarash) C 40 c 20 Khoramdareh D 40 d 20 Ijrood E 40 e 20 Khodabandeh K 40 k 20 Mahneshan M 40 m 20 Zanjan (Nikpey) N 40 n 20 Tarom (Dastjerdeh) T 40 t 20 188 J. APIC. SCI. Vol. 57 No. 2 2013 resuLts the junction of the 1m-cu and cu veins, had the minimum variation (S2 = 0.00001) because the Variance of coordinates of 18 landmarks on fore eighth landmark is near the centroid of the wing wings and 6 landmarks on hind wings as well as and landmarks 12, 18, and 13 are much further coordinates of landmarks of average shape were from the centroid. At the hind wing, the third evaluated. At the front wing, the fourteenth landmark, the junction of the M and cu veins landmark, the junction of the Rs and 2r-m veins, (S2 = 0.00019), and the fourth landmark, the had the maximum variation (S2 = 0.00005) in junction of the M and r-m veins (S2 = 0.00003), collected populations from nine areas of Iran; had the maximum and minimum variations, re- landmarks 12, 18, and 13 had the next greatest spectively (Fig. 1 and 2). variations. Furthermore, the eighth landmark, a b Fig. 1. Eighteen landmarks and six landmarks on vein junctions of fore wing (a) and hind wing (b) of Apis mellifera. 189 Dolati et al. Geometric morphometric study of Apis mellifera meda Allometry testing on fore wings and hind wings there was allometry in hind wings between was performed. The results identified a sig- centroid sizes and six shape variables (Wilks’ nificant difference between the size (centroid lambda = 5.89, p = 0.0001). size) of the front wings and 32 shape variables Results of MANOVA of fore wings (with (Wilks’ lambda = 0.705, p = 0.0001); therefore, 32 shape variables) showed significant dif- the shape variations of the fore wings were not ferences between populations of the nine uniform. Then, with the increasing of size of the areas (F = 4.97, p = 0.000). Moreover, multi- front wings, wing shape changed. In addition, variate analysis on hind wings (with eight shape a b Fig. 2. Average of schematic variations of 18 landmarks on the fore wing (a) and 6 landmarks on hind wings (b) of Apis mellifera. Table 2. MANOVA for shape variables of fore and hind wings of Apis mellifera in different geographical populations of Iran Worker Wilks’ F df1 df2 p wing lambda Fore wing 0.041 4.97 256 2506.1 0.000 Hind wing 0.281 3.64 64 946.7 0.000 190 J. APIC. SCI. Vol. 57 No. 2 2013 variables) identified significant differences Khodabandeh, and Zanjan (Nikpey). The hind among honeybee populations of the nine areas wings of the honeybee population of Tarom (F = 3.64, p = 0.000) (Tab. 2). (Dastjerdeh) had the minimum centroid size Centroid sizes of fore wings and hind wings (C = 223.8). were compared in different geographical areas Populations of nine zones based on shape of Iran. Results showed a significant difference variables of fore and hind wings were classified in centroid size of fore wings (F = 10.6, by DFA. Cross-validation tests based on dis- p = 0.000). Front wings of honeybee popu- criminant functions of front wings correctly lations in Zanjan (Nikpey area) had maximum classified 68.2% of the colonies (Tab. 4). Sta- centroid size (C = 600.6), but the populations of tistical analysis results for 32 shape variables the Khodabandeh area did not differ. Additional- of the front wings by DFA showed that the ly, the front wings of the Mahneshan population honeybee population of Khodabandeh was had the minimum centroid size (C = 582.9) almost separated (90%) from other areas.
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