Studies on the taxonomy of honeybees in the Sudan By Elhadi Adam Omer BSc. (Science) University of El Azhar, Cairo, Egypt. MSc. (Agric.) University of Khartoum. A thesis submitted in fulfillment of the requirements for the degree of Doctor of philosophy Supervisor Professor Mohamed Abd El Halim Siddig Co supervisor Professor Mohamed Saeed Ali El Sarrag Department of Crop Protection Faculty of Agriculture University of Khartoum. September 2007 I ﺑِ ﺴْ ﻢِ اﻟﻠّﻪِ ا ﻟ ﺮﱠ ﺣْ ﻤَ ﻦِ اﻟﱠﺮﺣِﻴﻢ {37} وَﻣَﺎ ﻣِﻦ دَ ﺁ ﺑﱠ ﺔٍ ﻓِﻲ ا ﻷَ رْ ضِ وَ ﻻَ ﻃَ ﺎ ﺋِ ﺮٍ ﻳَ ﻄِ ﻴ ﺮُ ﺑِ ﺠَ ﻨَ ﺎ ﺣَ ﻴْ ﻪِ إِ ﻻﱠ أُ ﻣَ ﻢٌ أَ ﻣْ ﺜَ ﺎ ﻟُ ﻜُ ﻢ ﻣﱠﺎ ﻓَﺮﱠﻃْﻨَﺎ ﻓِﻲ ا ﻟ ﻜِ ﺘَ ﺎ بِ ﻣِﻦ ﺷَ ﻲْ ءٍ ﺛُ ﻢﱠ إِﻟَﻰ رَ ﺑﱢ ﻬِ ﻢْ ﻳُ ﺤْ ﺸَ ﺮُ و نَ {38} ﺳﻮرة اﻷﻧﻌﺎم. II DEDICATION I dedicate this work to those whom I love and admire particularly, to the unassuming person, who lightened and still light many dark corners in this life, to my mother and father for their relentless efforts towards my education in spite of their very meager resources. To my wife to whom I am greatly indebted for her patience towards my kids while, I was outside country during the practical course of the study, I dedicate this work to her with my cordial appreciation. To my kids, brothers and sisters with great gratitude and love. III ACKNOWLEDGEMENT My sincere gratitude is devoted to my supervisor professor Mohamed Abd Elhalim Siddig for his professional guidance and sincere interest in this study. Thanks are extended to my co-supervisor professor Mohamed S. A. Elssarag for his advices and grateful helps. I am greatly indebted to Dr. S. Fuchs for his deep interest, keen supervision, patience and continuous advices and encouragement throughout the practical and analysis part of the morphometric section of this study. My gratitude’s are equally due to professor N. Koeniger (director) for availing me every facilitates in the institute [Institute fuer Bienenkunde (Polytechnische Gesellschaft) Fachbereich Biologie der J. W. Goethe- Universitaet Frankfurt am Main Karl-von-Frisch-Weg 2, D-61440 Oberursel, Germany, where I finished the biometric analysis]. Thus my deep thanks are converted to all the staff members of the institute. I feel pleased to express my deep thanks and sincere gratitude to professor Dr. Moritz and professor Hans (Institut für Zoologie Martin- Luther-Universitut Halle-Wittenberg Hoher Weg 4, D 06099 Halle/Saale, Germany.) for their grateful supervision and criticism during the molecular genetic practical part of this study at their institute. My thanks and wishes are offered to Dr. Marina Meixner (Research Associate Department of Entomology Washington State University Pullman, WA 99164-6382. USA.) for her considerable assistance and advices during the molecular genetic analysis part of this work. At last and not least I would like to express my thanks to my colleges in the Faculty of Natural Resources and Environmental Studies, University of Juba and the department of crop protection, Faculty of Agriculture, University of Khartoum for sparing good atmosphere to complete this study. IV ABSTRACT A thorough morphometrical and some molecular genetic studies (Mitochondrial DNA) were carried out on the most common honeybees in the Sudan. These so far contribute in the identification of the Sudanese honeybees. Nineteen samples of honeybee workers Apis mellifera L. were collected from four different geographical zones of the Sudan. Four samples of the small Asian bee workers Apis florea obtained from Gerry, Khartoum, Madani and El-Dender were also included in the study. Biometric measurements and analysis were performed for all the samples. The 19 colonies were subjected to morphometric measurements plus another 8 different samples of Apis mellifera L. were further subjected to Mitochondrial DNA investigation and analysis. Results were compared with those of the biometric study. The morphometric statistical analysis of the nineteen samples revealed a wide range of differences in most discriminant characters among the samples. In the principal component analysis (PCA), three clusters were graphically formed. Furthermore, the presence of these three clusters was confirmed by some modern discriminant analysis methods, and they were geographically correlated. The cluster with the smallest measurements of some discriminant characters originated from the forest zone. Its average measurements were as follows: forewing length 8.23 mm., width 2.82 mm.; proboscis length 5.55 mm.; hind-leg length 6.83 mm.; body size (T3+ T4) 3.88 mm., and cubital index 1.85 mm. The second cluster with medium measurements of some discriminant characters, originated from the semi-desert zone. Its mean average measurements were as follows: forewing length 8.27 mm., width 2.88 mm.; V proboscis length 5.63 mm.; hind-leg length 7.00 mm.; body size (T3+ T4) 3.88 mm.; and cubital index 2.04 mm. The third cluster, with the highest measurements of some discriminant characters, originated from the savannah zone; mainly towords the border with Ethiopia. Its average measurements were as follows: forewing length 8.45 mm., width 2.95 mm.; proboscis length 5.59 mm.; hind-leg 7.05 mm.; body size (T3+ T4) 4.00 mm., and comparatively the highest cubital index of 2.24 mm. Comparison between the 19 Sudanese honeybees samples and 242 banck samples (data banck, Institute für Bienenkunde, Oberursel, Germany from a neighbouring countries) was done using PCA. The three clusters of the Sudanese bees were like-wise distinguishable as subclusters. The same results were also confirmed by the discriminant analysis. Therefore, the smallest bees of Sudan were identified as Apis mellifera sudanesis instead of Apis mellifera yemenitica which represent the bees of the forest zone. The medium sized bees were identified as Apis mellifera yemenitica instead of sudanesis., representing the semi-desert zone bees, while the bigest bees retained the name Apis mellifera bandasii., representing the Savannah zone bees. The measurement of genetic variation in the Sudanese honeybees Apis mellifera L., at the mitochondrial DNA level of the 27 samples revealed the present of sex different haplotypes. The cluster with the smallest measurements (forest zone colonies) had only haplotype A1 representing 100% of the whole measured colonies; the medium cluster (semi-desert zone colonies) posses two different haplotypes O1 and Y2 with percentages 75% and 25% respectively from the whole measured colonies of the zone, while the cluster of highest measurements (savannah zone) showed four different haplotypes, O1, O1`, A2 and A4, representing 54%, 13%, 13% and 20% VI respectively. These results partially confirmed the biometric measurements of the PCA and discriminant analysis. The current study represent the first record on the classification of the Sudanese honeybees according to mitochondrial DNA variability. The present study suggest that, the presence of the gene flow among the Sudanese bees in the southern part of the semi-desert zone and almost all the savannah zone of the Sudan is a result of heterogeneous blood mixture between the Sudanese bees and the Ethiopian bees in the border between the two countries and the gene flow direction might be from the low land of the savannah zone of Ethiopia towards the western part of the Sudan in the area between latitudes 9º N and 15º N. Also this study suggests that the origin haplotype of the Sudanese bees is A1 and the pure Sudanese bees might be the south Sudan race (A. m. sudanesis). The four Apis florea samples were also treated by PCA and discriminant analysis, the results obtained so far revealed that, colonies are not very distinct indicating that, all of these colonies were similar and originally they were descendent of the first recorded colony of Apis florea in Khartoum in 1985. Treatment of the four Sudanese Florea samples together with 6 Florea colonies of different origins [2 from Sudan “Moggas ones” and 4 from the data bank, Institute fur Bienenkunde-Oberursel-Germany (Mogga 1988)], by cluster column analysis (which compare values across categories); revealed that, the four target Florea samples of Sudan might be brought from Pakistan or South Iran. VII ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ ﺃﺠﺭﻴﺕ ﺩﺭﺍﺴﺔ ﺸﺎﻤﻠﺔ ﻓﻲ ﺍﻟﻘﻴﺎﺴـﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴـ ﺔ (Biomorphometrics) ﻭﺍﻟﻭﺭﺍﺜـﺔ ﺍﻟﺠﺯﻴﺌﻴﺔ (Molecular Biology) ﻷﻜﺜﺭ ﺃﻨﻭﺍﻉ ﻨﺤل ﺍﻟﻌﺴل ﺇﻨﺘﺸﺎﺭﺍﹰ ﻓﻰ ﺍﻟﺴﻭﺩﺍﻥ. ﻤﻤﺎ ﻴـﺴﺎﻋﺩ ﻓﻲ ﺘﻌﺭﻴﻑ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ. ﺠﻤﻌﺕ ﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﻤﻥ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ Apis mellifera l ﻤﻥ ﺃﺭﺒﻊ ﻤﻨـﺎﻁﻕ ﺠﻐﺭﺍﻓﻴﺔ ﻤﺨﺘﻠﻔﺔ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ . ﻜﻤﺎ ﺍﺸﺘﻤﻠﺕ ﺃﻴﻀﺎ ﺍﻟﺩﺭﺍﺴﺔ ﺃﺭﺒﻊ ﻋﻴﻨـﺎﺕ ﻤـﻥ ﺍﻟﻨﺤـل ﺍﻵﺴـﻴﻭﻱ ﺍﻟﺼﻐﻴﺭApis florea ﺍﻟﻤﻭﺠﻭﺩﺓ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ ﺠﻤﻌﺕ ﻤﻥ ﻜ لٍ ﻤﻥ ﻗـﺭﻯ ، ﺍﻟﺨﺭﻁـﻭﻡ ، ﻤـﺩﻨﻲ ﻭ ﺍﻟﺩﻨﺩﺭ. ﻜل ﻫﺫﻩ ﺍﻟﻌﻴﻨﺎﺕ ﺃﺨﻀﻌﺕ ﻟﻠﻘﻴﺎﺴﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ ﻜﻤﺎ ﺤﻠﻠﺕ ﻨﺘﺎﺌﺠﻬﺎ. ﺴﺒﻌﺔ ﻭﻋﺸﺭﻭﻥ ﻋﻴﻨﺔ ﻤﻥ ﻨﺤل ﺍﻟﻌﺴل ﺍﻟﺴﻭﺩﺍﻨﻲ ﺃﺠﺭﻴﺕ ﻟﻬﺎ ﺩﺭﺍﺴﺎﺕ ﻓﻲ ﺍﻟﻭﺭﺍﺜﺔ ﺍﻟﺠﺯﻴﺌﻴـﺔ Mitochondrial DNA (ﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻤﻘﺎﺴﺔ ﺒﻴﻭﻟﻭﺠﻴﺎ ﺒﺎﻹﻀﺎﻓﺔ ﺁﻟﻲ ﺜﻤـﺎﻨﻲ ﻋﻴﻨـﺎﺕ ﺃﺨﺭﻯ ). ﻫﺫﺍ ﻭﻗﺩ ﻗﻭﺭﻨﺕ ﻨﺘﺎﺌﺞ ﺩﺭﺍﺴﺔ ﺍﻟﻭﺭﺍﺜﺔ ﺍﻟﺠﺯﻴﺌﻴﺔ ﺒﻨﺘﺎﺌﺞ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺒﻴﻭﻟﻭﺠﻴﺔ. ﺃﻅﻬﺭﺕ ﺍﻟﺘﺤﺎﻟﻴل ﺍﻹﺤﺼﺎﺌﻴﺔ ﻟﻠﺘﺴﻌﺔ ﻋﺸﺭ ﻋﻴﻨﺔ ﺍ ﺨ ﺘ ﻼ ﻓ ﺎﹰ ﻭ ﺍ ﻀ ﺤ ﺎﹰ ﻓﻲ ﻤﻌﻅﻡ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﺒﻴﻥ ﺍﻟﻌﻴﻨﺎﺕ . ﻭ ﺒﺘﺤﻠﻴل ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ (PCA) ﺘﻜﻭﻨﺕ ﺒﻴﺎﻨﺎﺕ ﺜﻼﺜﺔ ﺘﺠ ﻤﻌﺎﺕ ﻤﺘﻨﺎﺴﺒﺔ ﺠﻐﺭﺍﻓﻴﺎ . ﻭﻜﺫﻟﻙ ﺒﺘﺤﻠﻴل ﻨﺘﻴﺠﺔ ﺍﻟﻤﻜﻭﻨﺎﺕ ﺍﻷﺴﺎﺴﻴﺔ ﺒﻭﺍﺴـﻁﺔ ﺍﻟﺘﺤﻠﻴـل ﺍﻟﻤﻤﻴـﺯ ﺍﻟﺤـﺩﻴﺙ Discriminant analysis ﺘﻡ ﺍﻟﺘﺄﻜﺩ ﻤﻥ ﻭﺠﻭﺩ ﺘﻠﻙ ﺍﻟﺘﺠﻤﻌﺎﺕ ﺍﻟﺜﻼﺜﺔ ﻭﻫﻰ ﻜﺎﻻﺘﻰ : ﺃ- ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﺼﻐﺭﻯ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﻭﻗﺩ ﻨﺸﺄﺕ ﻫـﺫﻩ ﺍﻟﻤﺠﻤﻭﻋـﺔ ﻤـﻥ ﻤﻨﺎﻁﻕ ﺍﻟﻐﺎﺒﺎﺕ ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻗﻴﺎﺴﺎﺘﻬﺎ ﻜﺂﻻﺘﻲ :- (ﺃ) ﻁﻭل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ 8.23 ﻤﻠﻡ (ﺏ) ﻋﺭﺽ ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ 2.82 ﻤﻠﻡ (ﺝ) ﻁﻭل ﺍﻟﺨﺭﻁﻭﻡ 5.55 ﻤﻠﻡ (ﺩ) ﻁﻭل ﺍﻟﺭﺠل ﺍﻟﺨﻠﻔﻴﺔ 6.83 ﻤﻠﻡ (ﻩ) ﻁﻭل ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﻅﻬﺭﻴﺔ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ (ﺤﺠﻡ ﺠﺴﻡ) 3.88 ﻤﻠﻡ (ﻯ) ﻤﻌﺎﻤل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ (Cubital index) 1.85 ﻤﻠﻡ ﺏ- ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﺘﻭﺴﻁﺔ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ ﻭﻗﺩ ﻨﺸﺄﺕ ﻫﺫﻩ ﺍﻟﻤﺠﻤﻭﻋـﺔ ﻤـﻥ ﻤﻨﻁﻘﺔ ﺸﺒﻪ ﺍﻟﺼﺤﺭﺍﺀ. ﻭﻜﺎﻥ ﻤﺘﻭﺴﻁ ﻗﻴﺎﺴﺎﺘﻬﺎ ﻜﺂﻻﺘﻲ :- (ﺃ) ﻁﻭل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ 8.27 ﻤﻠﻡ (ﺏ) ﻋﺭﺽ ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ 2.88 ﻤﻠﻡ (ﺝ) ﻁﻭل ﺍﻟﺨﺭﻁﻭﻡ 5.63 ﻤﻠﻡ VIII (ﺩ) ﻁﻭل ﺍﻟﺭﺠل ﺍﻟﺨﻠﻔﻴﺔ 7.00 ﻤﻠﻡ (ﻩ) ﻁﻭل ﺍﻟﺼﻔﺎﺌﺢ ﺍﻟﻅﻬﺭﻴﺔ ﺍﻟﺜﺎﻟﺜﺔ ﻭﺍﻟﺭﺍﺒﻌﺔ (ﺤﺠﻡ ﺠﺴﻡ) 3.88 ﻤﻠﻡ (ﻯ) ﻤﻌﺎﻤل ﺍﻟﺠﻨﺎﺡ ﺍﻷﻤﺎﻤﻲ (Cubital index) 2.04 ﻤﻠﻡ ﺝ- ﺃﻤﺎ ﺍﻟﻤﺠﻤﻭﻋﺔ ﺫﺍﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻜﺒﺭﻯ ﻟﺒﻌﺽ ﺍﻟﺼﻔﺎﺕ ﺍﻟﻤﻤﻴﺯﺓ .