A COMPARATIVE STUDY OF TWO SPECIES OF

CAVITY-NESTING HONEY BEES OF SULAWESI, INDONESIA

A Thesis

Presented to

The Faculty of Graduate studies

of

The University of Guelph

by

SOESILAWATI HADISOESILO

In partial fulfilment of the requirements

for the degree of

Doctor of Philosophy

June, 1997

O Soesilawati Hadisoesilo, 1997 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. me Wellington Ottawa ON KIA ON4 OttawaON K1AON4 Canada Canada

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A COMPARATIVE STUDY OF TWO SPECIES OF CAVITY- NESTING HONEY BEES OF SULAWESI, INDONESIA

Soesilawati Hadisoesilo Advisor: University of Guelph Dr. G-W. Otis

This thesis is a comparative study of two cavity-nesting honey bees of

Sulawesi, Indonesia, Apis cerana and A. nigrocincta. These two species have been wnfirmed to be distinct by the temporal segregation of their drone mating flights at three different observation sites. Drones of A. cerana always took their mating flights about 2 hr earlier than do drones of A. nigrocincta, with little overlap in the distributions of flight times.

The distinction between A. nigrocincta and A. cerana was supported by the results of rnorphometric (Le., cluster, principal cornponent. and discriminant) analyses. These analyses yielded congruent results. In al1 cases two distinct groups were formed that correspond to the a pion identification to A. cerana and A. nigrocincta. No single sample was misclassified. No intermediate forms were found among 126 samples, even in the two zones of sympatry.

The species status of A. nigrocincta is further supported by the failures of the queens of A. cerana and A. nigrocincta to mate when conspecific drones are absent, even though allospecific drones are present. In addition, the drone cells of

A. nigrocincta are soft and lack pores, unlike those of A. cerana which are always hardened and have pores. The distributions of A. cerana and A. nigrocincta are largely allopatric, with narrow zones of overlap. In Central Sulawesi, the abrupt transition corresponds to the restriction of A. nigrocincfa to forests and of A. cerana to disturbed agricultural areas. This is unusual for honey bees which are generally believed to generalist that occupy various habitats. The unusual distribution might be caused by climatic limitations, com petitive exclusion, andior habitat preferences. The possible explanations cannot be distinguished without further studies.

Although it is confirmed that A. cerana and A. nigrocincta are distinct species, the male genitalia of these species are similar. This phenomenon is not expected by many honey bee experts who usually demand differences in male genitalia before accepting a new species. This evidence leads to some indirect evidence that behavioural traits form the basis for speciation in the genus Apis. I would like to express my gratitude to my supervisor, Dr. G.W. Otis, for the guidance and constant support he provided throughout the course of my study. His patience, critical wmments, and suggestions were invaluable for the improvement of my manuscript. Most importantly, I thank him for his friendship. I shall never forget how he jeopardized life and limb to help me to collect my bee samples. 1 also thank the other rnembers of my committee Drs. P.G.Kevan, C. Scott-Dupree, and

A.L. Middleton for their insight and constructive suggestions for the improvement of my thesis.

I am sincerely grateful to many people who, in one way or the other, made my field study a success. Special thanks to Dr. Mappatoba Sila who facilitated the applied field work; to A. Rahim, the late Fitrah Halim, and Sugiyani for their assistance when I did my field work. I am extremely grateful to the following people in South Sulawesi: the former (Ir. Soeparno, M-Sc.) and the present (Dr. D.

Mulyadhi) Kepala Balai Penelitian Kehutanan Ujung Pandang (BPK-UP) who let me use the lab and other office facilities; Bapak Salman and his family at Bontobulaeng and Bapak Arief Arief Tabang and his family at Palangisang who took care of me when I did myfield work in those two villages; and all of my colleagues at the BPK-

UP for their friendship.

My sincere thanks to Drs. Nikolaus and Gudrun Koeniger of the lnstitute für

Bienenkunde, Oberursel, Germany for their support and valuable information

1 provided during the completion of my study. l also thank Dr. S. Marshall for allowing me to use his Iab and the microscopes and for his valuable suggestions; M. Damus for the help with morphometric analyses and M. Nasr for data analyses; Don

Hamilton for his help in making photographs and visual aids; and Dr. G.W. Otis who took excellent photographs for this thesis.

Acknowledgement is due to the Department of Forestry of the Republic of lndonesia for providing me most of the money for my study. Monies were also generously provided by a Beatty-Munro scholarship, the Gordon F. Townsend scholarship, Beenet Asia, and Balai Penelitian Kehutanan Ujung Pandang. I also thank the Agency of Forestry Research and Development for granting me a study leave throughout the duration of my studies.

I thank my Canadians friends, especially Gillian Ferguson, who always showed regard to my well-being and Ann-Marie Cooper who maintairied our friendship since I met her. i value your friendship greatly.

Thanks to Yusman Syaukat and his family for their hospitality while I stayed with them for almost two years - I really enjoyed staying with them; and also for al1 my Indonesian friends who always help me in many ways. Thank you al1 and see you in fndonesia.

Finally, this thesis is dedicated to my parents who have always encouraged me to pursue a higher education. 1 shall never forget the support and encouragement of sister, brothers, and relatives during my long academic struggle. PAGE

ACKNOWLEDGEMENTS ...... i ... TABLE OF CONTENTS ...... III

LIST OF TABLES ...... vii

LIST OF FIGURES ...... ix

CHAPTER 1 GENERAL INTRODUCTION ...... 1

1.1 Historical Overview of the Diversity of Cavity-nesting Honey Bees . 1 1.2 The Biotic Uniqueness of Sulawesi ...... 4 1.3 Honey Bees of Sulawesi ...... 7 1.4 Objective of Studies ...... 9

CHAPTER 2 A COMPARISON OF DRONE MATING FLJGHT TIMES OF TM/O SPECIES OF CAVITY-NESTlNG HONEY BEES IN SULAWESI. INDONESIA ...... 11

2.1 INTRODUCTION ...... II 2.2 MATERIALS AND METHODS ...... 13 2.3 RESULTS ...... 18 2.4 DISCUSSION ...... 24

CHAPTER 3 THE DISTRIBUTION AND MORPHOMETRIC ANALYSES OF Apis cerana AND A . nigrocincta IN SULAWESI. INDONESIA ...... 27

3.1 INTRODUCTION ...... 27 3.2 MATERIALSAND METHODS ...... 31 3.2.1 Distribution ...... 31 3.2.2 Morphometric Analyses ...... 33 3.2.2.1 Sample collections and measurement techniques 33 3.2.2.1.1 South Sulawesi ...... 33 3.2.2.1.2 Central Sulawesi ...... 35

iii

5.2.4 Case4 ...... 125 5.3 RESULTS ...... 126 5.3.1 Case1 ...... 126 5.3.2 Case2 ...... 128 5.3.3 Case3 ...... 130 5.3.4 Case 4 ...... 132 5.4 DISCUSSION ...... 134

CHAPTER 6 DIFFERENCES IN DRONE CAPPING BEHAVIOUR OF Apis cerana AND Apis nigrocincfa ...... 139

6.1 INTRODUCTION ...... 139 6.2 MATERIALS AND METHODS ...... 141 6.3 RESULTS ...... 143 6.4 DISCUSSION ...... 146

CHAPTER 7 VARIOUS SPECIES CONCEPTS AS THEY APPLY TO HONEY BEES ...... 151

lNTRODUCTlON ...... 151 TYPOLOGICAL SPECIES CONCEPT ...... 152 BIOLOGICAL SPECIES CONCEPT ...... 156 7.3.1 Premating Isolation ...... 157 7.3.1.1 Mechanical lsolation - Differences in the male genitalia ...... 157 7.3.1.2 Behavioural (Temporal) lsolation - Drone Mating Flights ...... 158 7.3.1 -3 Behavioural (Spatial) lsolation - Location of Drone Congregation Areas ...... 159 7.3.1.4 Behavioural Isolation - Chernical Signais ..... 161 7.3.2 Postmating Isolation ...... 162 RECOGNITION SPECIES CONCEPT ...... 164 7.4.1 The Timing of the Mating Flights of Drones and Queens 165 7.4.2 Locations of Drone Congregation Areas ...... 166 7.4.3 Sexual Pheromones ...... 166 7.4.4 Contact Stimuli ...... 167 7.4.5 Male Genitalia ...... 168 PHYLOGENETIC SPECIES CONCEPT ...... 169 THE SP EClFlC CASE OF Apis nigrocincta ...... 172 CONCLUSIONS ...... 173 CHAPTER 8 GENERAL CONCLUSIONS ...... 176

REFERENCES ...... 183 LIST OF TABLES

PAGE

Table 2.1 Locations, dates, and colonies observed for drone flights in 1995 . 15

Table 2.2 Means of time of first departure, time of entry to colonies, and last time of entry of drones of different pairs of colonies of A. cerana and "A. nigrocincfa" observed simultaneously at Palangisang and Bontobulaeng (South Sulawesi) and Kamarora and Bobo (Central Sulawesi) ...... 20

Table 3.1 Location of colonies located and analyzed in South Sulawesi. .... 37

Table 3.2 Distribution patterns of A. nigrocincta, and A. cerana colonies at different elevation ranges ...... 42

Table 3.3 Number of colonies located at Kamarora and Rahmat in 1995 and1996 ...... 49

Table 3.4 Correlations of 22 characters with the first three Principal Components for 102 samples...... 52

Table 3.5 Correlations of 18 characters with the first three Principal Component for 24 samples...... 64

Table 4.1 Sites from which drones were collected for morphological comparison of endophalti ...... 89

Table 4.2 The number of nodules on the dorsal cornua (uneverted and everted samples), number of folds on the ventral side of the cervix, and the number of papillae of the fimbriate lobe ...... 94

Table 5.1 The presence or absence of the queen and eggs in the colony of A. cerana allowed to rear a queen cell of A. nigrocincta at Balong, South Sulawesi...... 127

Table 5.2A The presence or absence of queens and eggs of A. nigrocincta in colonies of A. cerana at Jeneponto, South Sulawesi ...... 129

vii Table 5-26 The presence or absence of queens and eggs of A. cerana in colonies of A. cerana, at Jeneponto, South Sulawesi...... 129

Table 5.3 The presence or absence of the queen and eggs in the colony of A. cerana allowed to rear a queen cell of A. nigrocincta at Palangisang, South Sulawesi ...... 131

Table 5.4 The presence and absence of queens and eggs in queenless colonies of A. cerana moved to Manipi, South Sulawesi...... 133

Table 6.1 Number of cell caps with and without pores obsewed on capped drone cells of A. cerana and A. nigrocincta in Sulawesi, Indonesia. .. 144

Table 7.1 Typological species of honey bees as recognized by different authors ...... 155

viii LIST OF FIGURES

PAGE

Figure 1.1 "Total evidence" phylogeny of honey bees ...... 3

Figure 1.2 Map showing the location of Sulawesi relative to surrounding islands ...... 5

Figure 2.1 Map of Sulawesi indicating location of drone flight study sites. . . 14

Figure 2.2 Timing of drone fiights at Palangisang, South Sulawesi ...... 21

Figure 2.3 Timing of drone flights at Bontobulaeng, South Sulawesi . . + . . . . 22

Figure 2.4 Timing of drone ftights at Kamarora and Bobo, Central Sulawesi . 23

Figure 3.1 The hind legs of worker bees of A. cerana and A. nigrocincfa . . . . . 32

Figure 3.2 Characters measured for morphometric analysis of bees collected in Sulawesi ...... 34

Figure 3.3 Seventeen locations surveyed of colonies of A. cerana andlof A. nigrocincta in South Sulawesi...... - . . . 43

Figure 3.4 Locations of colonies in the area of sympatry in South Sulawesi . 44

Figure 3.5 Colonies located at Kamarora ...... 46

Figure 3.6 Colonies tocated at Rahmat ...... 48

Figure 3.7 Scatterplot of Principal Components 1 and 2 for 102 samples predominantly from South Sulawesi ...... 53

Figure 3.8 Scatterplot of Principal Components 1 and 2 for individual bees from the zone of sympatry ...... 54

Figure 3.9 Plot of 95% confidence ellipses for discriminant functions 1 and 2. Samples predominantly from South Sulawesi ...... 56 Figure 3.1 0 Scatterplot of femur lengths versus forewing lengths for samples of A. cerana and A. nigrocincta predorninantly from South Sulawesi...... 58

Figure 3.1 1 Scatterplot of hindwing widths versus hindwing lengths for samples of A. cerana and A. nigrocincta predorninantly from South Sulawesi...... 59

Figure 3.12 Scatterplot of angles 84 and J 1O for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi...... 60

Figure 3.13 Scatterplot of angles D7 and B4 for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi...... 61

Figure 3.1 4 Scatterplot of angles JI0 and D7 for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi...... 62

Figure 3.15 Scatterplot of Principal Components 1 and 2 for 24 samples from Central Sulawesi ...... 65

Figure 3.16 Scatterplot of Principal Components 1 and 2 for individual bees fromRahmat ...... 67

Figure 3.17 Plot of 95% confidence ellipses for discriminant functions 1 and 2. Samples from Rahmat and Kamarora ...... 68

Figure 3.1 8 Scatterplot of femur lengths versus forewing lengths for samples of A. cerana and A. nigrocincta from Central Sulawesi...... 70

Figure 3.19 Scatterplot of hindwing widths and hindwing lengths for samples of A. cerana and A. nigrocincta from Central Sulawesi...... 71

Figure 3.20 Scatterplot of angles 64 and JI0 for samples of A. cerana and A. nigrocincta from Central Sulawesi...... 72

Figure 3.21 Scatterplot of angles D7 and 84 for samples of A. cerana and A. nigrocincta from Central Sulawesi...... 73

Figure 3.22 Scatterplot of angles JI0 and D7 for samples of A. cerana and A. nigrocincta from Central Sulawesi...... 74 Figure 4.1 A dorsal view of the uneverted endophalli of A . nigrocincta andA.cerana ...... 93

Figure 4.2 Variability in the shapes of dorsal cornua of uneverted endophalli of A . cerana and A . nigrocincta ...... 95

Figure 4.3 A ventral view of the uneverted endophalli of A . nigrocincta andA.cerana ...... 97

Figure 4.4 Variability in the shapes and the pattern of the most densely pubescent portion of the vestibulum of uneverted endophalli of Axerana and A . nigrocincta ...... 98

Figure 4.5 Everted endophalli of A . cerana and A . nigrocincta ...... 101

Figure 4.6 Everted endophalli of A . nigrocincta and A . cerana ...... 102

Figure 4.7 Ventral and dorsal cornua of everted endophalli of A . cerana from Kamarora and A . nigrocincta from Rahmat ...... 103

Figure 4.8 Everted firnbriate lobes of two specimens of A . nigrochcta andA.cerana ...... 105

Figure 4.9 Estimated divergence within the genus Apis ...... 112

Figure 5.1 A capped queen cell cut along with a small piece of the comb . . 120

Figure 5.2 A capped queen cell inserted between two frames ...... 121

Figure 5.3 A capped queen cell securely held in place between two frames . 121

Figure 5.4 The opening of a queen cell indicating that the queen had emerged...... 123

Figure 6.1A Drone cells of A . cerana ...... 142

Figure 6.16 Drone cells of A . nigrocincta ...... 142

Figure 6.2 The bottorn board of the hive of A . cerana with an accumulation of drone cell caps ...... 145 CHAPTER 1

GENERAL INTRODUCTION

1.1 Historical Overview of the Diversity of Cavity-nesting Honey Bees

The taxonomy of honey bees (genus Apis) is confusing despite much

research devoted to the genus. Several revisions have attempted to clarify the

classification of the genus (Gerstacker, 1862 reviewed by Alexander, 1991a; Smith,

1865; Ashmead, 1904; Buttel-Reepen, 1906; Maa, 1953). The rnost recent major taxonornic revision was published by Maa (1953) whu split honey bees into three genera (Micrapis, Megapis, and Apis) and 24 species. His description was largely

ignored initially because of his treatment of what is now recognized as Apis mellifera. He recognized several populations as distinct species, even though it was known that several of the populations could be hybridized to yield viable offspring that exhibit no apparent reduction in fitness. Because most bee researchers at that time rejected his classification of European and African honey bees, they also rejected his notions concerning Asian honey bee species. More recently, his typological treatment has failed to meet the increasingiy more stringent requirements of modern systematics which rely upon the application of cladistic and other more objective techniques. Given the general lack of information on honey bees in different parts of Asia and the large amount of variation found within some species, most scientists in this century have adopted a conservative view of Apis species: A. mellifera in Europe and Africa, and A. cerana, A. dorsata, and A. florea in Asia (Ruttner, 1968, 1988; Gould and Gould, 1988). However, recent validation of some of Maa's species, namely A. koschevnikovi Buttel-Reepen, A. labonosa

Smith, and A. andrenifonnis Smith (see Otis, 1991 for review), has eneouraged the reinvestigation of other taxa recognized by Maa (1953). The currently accepted,

"total evidence" phylogenetic tree for the genus Apis is shown in Figure 1.1 (Engel and Schultz, 1997) to which have been added the subgenera as defined by

Ashmead (1904)

In contrast to A. mellifera, which has been the subject of numerous of studies, the cavity-nesting honey bees of Asia have been poorty sstudied. Before the

19801s, the only cavity-nesting honey bee recognized in Asia was Apis cerana. It is a widespread species found from Pakistan northeastward through China to southern

Russia and Japan, and southeastward to the Philippines and through the lndonesian Archipelago to Timor. Over this range, it occupies different climates and shows a high degree of geographic variability (Ruttner, 1988; Verma, 1995). [t is the only species recognized by Ruttner (1988) in his major review of the honey bees, but as a result of recent studies, it has become clear that his view of the diversity of cavity-nesting honey bees in Asia was too restrictive. For exarnple, in 1988 two separate research teams rediscovered A. koschevnikovi in northeastern Borneo

(Mathew and Mathew, 1988; Tingek et al., 1988). An additional species that is being called A. nuluensis was recently confirmed from the mountains of Borneo (Koeniger et al., 1996a; Tingek et al., 1996). In addition, there are several subspecies of A. cerana. Ruttner (1988) recognized A. cerana cerana, A.c. iiidica, A.c. japonica, and Figure 1.l "Total evidence" phylogeny of honey bees (redrawn from Engel and Schultz, 1997, Figure 2e); to which have been added the Iikely branches related to A. laboriosa, A. nuluensis, and A. nigrocincta. A. t7orea

A. andreniformis

A. rnellfem

A. koschevnikovi

A. nuluensis

A. cerana

A. nigrocincta A.c. himalaya. Peng et al. (1 989) sumrnarized results of Chinese authors who have

reported five geographical races in mainland China inctuding the bees from Hainan,

Eastern and Southern Yunan, Aba, and Xizang (Tibet). Vema et al. (1994)

obse~edthat A.c. cerana in north-west lndia comprises two geographic populations

in Kashmir and Himachal regions respectively. From north-east India, three

geographic ecotypes (locally adapted populations) of A.c. himalaya are recognized

from (1) Naga and Mizo Hills, (2) Brahamputra and Khasi Hills, and (3) the foothills

of the Hirnalayas (Singh et al., 1990; Verma, 1995). A detailed morphometric

analysis of cavity-nesting honey bees frorn Malesia, Hong Kong, Japan, and Sri

Lanka by Damus (1995; Damus and Otis, subrnitted) suggested 2-4 additional

subspecies. Against this backdrop of diversity in cavity-nesting honey bees, Otis

and Hadisoesilo (1922) cfiz~veredtwo different bee morphs in South Sulawesi, the taxonomic status of which is uncertain (see section 1.3,).

1.2 The Biotic Uniqueness of Sulawesi

The island of Sulawesi (previously known as the Celebes; see Figure 1.2) occupies a central position in the Malay Archipelago. Irnmediately to the north are the Philippine islands, to the west is Borneo, to the east are the Mollucan islands, and to the south are the Lesser Sunda Islands and Timor. Sulawesi is unique geologically. The western parts appear to be of Sundaic (Asiatic) origin, eastern parts of Sahul (Australian) origin, and certain parts (e.g., northern part on which

Dumoga-Bane Park lies) have arisen more recently as a result of tectonic pressures Figure 1.2 Map showing the location of Sulawesi relative to surrounding islands (map from Whitrnore, 1987). and active vulcanism (Marshall and Collins, 1990). It is an island with cornplex biogeographical relationships, possessing characteristics peculiar to continents as well as to oceanic islands (Wallace, 1860; Cranbrook, 1981). Sulawesi is one of the geologically most ancient parts of the Malay Archipelago (Wallace, 1860, 1869;

Audley-Charles, 1987). Most evidence indicates that it has been completely isolated from neighbouring land masses for at least 15 million years (Audiey-Charles, 1987).

Based on the thickness of Cenozoic sediments and marine sedimentary rocks,

Audley-Charles (1981) suggested that Sulawesi has been separated from Borneo by the strait of Makassar at least since the Eocene epoch. Also, the absence of taxa

(e-g., fresh water fish, frogs) in Sulawesi that are intolerant of salt water, indicates that Sulawesi was probably never connected physically to neighbouring islands

(Mayr, 1944; Cranbrook, 1981; Musser, 1987). Because it has been isolated by water barriers for such a long period of tirne, peculiar faunas have evolved in

Sulawesi as first discovered and described by Wallace (1869).

For taxa shared between Sulawesi and neighbouring regions, the biogeographic affinities Vary among taxa (Whitmore. 1981, 1987: Knight and

Holloway, 1990). It is indisputable that the vertebrate fauna of Sulawesi is anomalous in many respects. The vertebrates of Sulawesi of Asian origin greatly outnumber those of Papuasian affinity (Cranbrook, 1981; Musser, 1987). By contrast. a phenetic analysis of butterfiy distributions indicates that Sulawesi has its strongest overall association with the Philippines (Holloway, 1987), but certain taxonornic groups are linked more closely to other neighbouring biogeographic

regions (Vane-Wright, 1990). Although sorne plant taxa colonized Sulawesi frorn

Sundaland (e.g., Dipterocarpaceae and Magnoliaceae) and Papuasia (e-g.,

Proteacea and Fagaceae) (Whitmore, 1981; Veevers-Carter, 1984), the flora of

Sulawesi appears to be rnost distinctiy allied to the flora of the Philippines, the

Mollucas, the Lesser Sunda Islands, and Java depending on the taxonornic group

(van Balgooy, 1987). The unique flora and fauna of Sulawesi have been at the centre of a large number of studies and have a complex basis (c.f.,Whitmore, 1981,

1987; Knight and Holloway, 1990).

1.3 Honey Bees of Sulawesi

Although much research has been conducted in Sulawesi, surprisingly little attention has been directed towards the honey bees. The first honey bee collected in Sulawesi by A.R. Wallace as reported by Smith (1859), was named Apis zonafa, a large species simifar to Apis dorsata of South East Asia. This name has been changed several times to Megapis zonata (Ashrnead, 1904), A. dorsata binghami

(Cockerell, l9O6), and M. binghami (Maa, 1953). The last two names are both in current use, with Ad. binghami more appropriate at present (Ruttner, 1988; Otis,

1991). It has both morphological and behavioural differences with A.d. dorsata, but probably not in characters that would lead to reproductive isolation.

The second report on honey bees of Sulawesi was published in 1861, also by F. Smith, who described a honey bee collected by A.R. Wallace near Makassar (now known as Ujung Pandang) as Apis nigro-cincfa (In curent nomenclature iules,

names that were hyphenated in original descriptions are correctly used without the

hyphen), The original description is not very clear. It is based primarily on colour of

the antennae, clypeus, labrum, mandibles, legs, and abdomen. This description by

itself does not allow one to differentiate this form from some other geographic

populations of bees in Asia. Therefore, some authors recognized A. nigrocincta as

a distinct species but some did not. Ashmead (19O4), in his revision of honey bee

classification recognized three genera and seven species, one of which was A.

nigrocincta. However, Buttel-Reepen (1906) believed that this form was only a

variety of the taxon he called A. mellifica indica. In recent history, only Maa (1953)

continued to recognize Apis nigrocincta as a distinct species. His work, however, was largely ignored by scientists because he did not prepare a key to Apis species

and he Iacked the biological data to test the species status of his taxa (Ruttner,

1988; Otis, 1991). Most 20th century authors have subsumed A. nigrocincta under the taxon A. cerana which is often depicted as inhabiting the entire island of

Sulawesi (Gould and Goufd, 1988; Ruttner, 1988).

In 1989, Otis and I collected specimens from Mo distinct populations of cavity-nesting honey bees, in a survey in South Sulawesi (Otis and Hadisoesilo,

1990). The distributions of these two morphs, initially referred to as "black" and

"yellow" morphs, were largely allopatric, although one colony of the "black morph and one colony of the "yellow" morph were found within 12 km of each other.

Morphometric analyses by Damus (1995), Hadisoesilo et al. (1995), and Damus and Otis (subrnitted) suggested that the darker, srnaller morph belongs to an endemic

subspecies of the widely distributed A. cerana, which is most similar to populations

inhabiting the Lesser Sunda Islands. Based on extensive rnorphometric analyses,

Damus (1995) conservatively suggested that the "yellow" morph (also found in

Central and North Sulawesi and the north coast of Mindanao) should be referred to

as A. cerana nigrocincta until such time other evidence would warrant elevating it

to species status. Genetic evidence also indicates the existence of a clade of cavity-

nesting bees in common between Sulawesi, Mindanao, and Luzon (Smith and

Hagen, 1996).

1.4 Objective of Studies

The description by Smith (1861) and Maa (1953) of A. nigrocincta as the only

cavity-nesting honey bee species on Sulawesi, is contradicted by the preliminary

recognition of two distinct bee morp hs by Hadisoesilo et a141 995). This latter

situation, unusual for honey bees, stimulated me to conduct the present studies, the

primary objective of which was to clarify the species status of the "yellow" cavity-

nesting honey bee morph in relation to A. cerana. This has been done by examining

the timing of drone flights, delineating the geographic distributions of the two bee

morphs, conducting morphometric analyses of specimens, and comparing male genitalia. These studies also include other aspects of biology of the "yellow" morph.

For convenience, the "yellow" rnorph will be referred to initially as "A. nigrocincta"

(note quotation marks). In the text that follows, Chapter 2 deals with the timing of mating flights of drones of the two morphs in South and Central Sulawesi and discusses the validity of "A. nigmcincfa" as a biological species. Chapter 3 describes the partially sympatric distributions of "A. nigmcincta" and A. cerana and the results of rnorphometric analyses of these two morphs that further confirm the species status of "A. nigrocincta" based on the absence of evidence of hybridization. Chapter 4 compares the male genitalia of the two species and discusses the role of male genitalia in speciation of honey bees. Chapter 5 is a short report on observations of mating of A. cerana and A. nigrocincta in regions inhabited by allospecific or conspecific drones, intended to confirm whether or not hybridization can occur.

Chapter 6 presents a short discussion on the differences in the capping of drone cells of the two species and their biological implications. Chapter 7 summarizes the various species concepts as they apply to honey bees. Finally, Chapter 8 synthesizes the results of my thesis. CHAPTER 2'

A COMPARISON OF DRONE MATING FLIGHT TIMES OF TWO SPEClES OF CAVITY-NESTING HONEY BEES IN SULAWESI, INDONESIA

2.1 INTRODUCTION

Recently two morphs of cavity-nesting honey bees, were discovered on

Sulawesi (Otis and Hadisoesilo, 1990; Otis, 1991). Morphometric and DNA analyses of specimens (Damus, 1995; Hadisoesilo et al., 1995; Smith and Hagen, 1996) indicated that the smaller, darker bees are Apis cerana, the broadly distributed

Asian hive bee. In Sulawesi, this species has been collected only frorn the extreme southern part of South Sulawesi and from a small region of Central Sulawesi. In contrast, the larger, yellower morph is widely distributed on Sulawesi as well as the islands of Sangihe and Mindanao to the north (Damus, 1995; Otis, 1996). It is easily separated from A. cerana of Sulawesi on the basis of size and colour as well as detailed morphometric analyses (Chapter 3).

The obvious question is whether or not the yellow bee morph of Sulawesi constitutes a subspecies of A. cerana or a distinct species, A. nigrocincta, as previously discussed by Hadisoesilo et al. (1995). For convenience it is tentatively referred to as "A. nigrocincta" throughout this chapter. One relevant observation is

'A rnodified version of this chapter has been published as: Drone Flight Times Confirm the Species Status of Apis nigrocincta Smith, 1861 to be a Species Distinct from Apis cerana F, in Sulawesi, lndonesia (1996). Apidologie 27(5):361 -369. that nearly al1 specimens of cavity-nesting honey bees from Sulawesi have been clearly assignable to one morph or the other. No intermediate forms have been observed in South Sulawesi, including those sampled from an area of sympatry

(Bontobulaeng area) (Damus, 1995; Hadisoesilo et al., 1995; Damus and Otis, submitted). A very small percentage of morphologically intemediate bees that may represent hybrids were collected from another area of sympatry at the Palolo Valley in Central Sulawesi (details presented in Chapter 3). That these two populations differ significantly in extemal morphology contrasts with the preliminary obsenration that "there are no substantial differences in the male genitalia of the two morphotypes" (Otis, 1991).

In other studies conducted in Asia where two or more honey bee species coexist, it has been found that the timing of drone rnating flights differs such that there is Iittle or no opportunity for hybridization to occur ((Sri Lanka: A. flores, A. cerana, A. dorsata (Koeniger and Wijayagunasekera, 1976); Borneo: A. nuluensis,

A. andreniformis, A. cerana, A. koschevnikovi, A. dorsata (Koeniger et al., 1988.

1996a); Thailand: A, andreniformis, A. florea, A. cerana, A. dorsata (Rinderer et al.,

1993); Japan: A. mellifera, A. cerana (Yoshida et al., 1994)). This has been interpreted as a good evidence that the populations in question are actually valid species. I assessed the species status of "A. nigrocincta" by comparing the timing of its drone flights with those of sympatric A. cerana in Sulawesi. 2.2 MATERIALS AND METHOOS

Drone flights were quantified in Palangisang and Bontobulaeng (South

Sulawesi) and KamaroraIBobo (Central Sulawesi; Figure 2.1). Palangisang is located I20 km NE of Bulukumba at elevation 200 m. Afternoon drone flights were monitored on one colony of A. cerana and one colony of "A. nigrocincta" for 7 d, over the period from 17-23 August, 1995 (Table 2.1). The black colony originated in Palangisang. Because "A. nigrocincta" does not exist in Palangisang, one colony was brought from Manipi (1800 m elevation, 190 km NW of Palangisang) in

January, 1995. Bontobulaeng is 30 km NW of Bulukumba at 1420 rn elevation and is within a zone of sympatry that extends over approximately 20 km (Chapter 3).

One colony of A. cerana (B-2) was moved from Palangisang in March, 1995; the other colony of A. cerana and three colonies of "A. nigrocincta" were endemic feral colonies transferred to hives in March and April, 1995 (Table 2.1). Drone flights were observed for 15 d during the period of 5-27 September, 1995. Kamarora, at

630 m, is located 155 km SE of Palu in Central Sulawesi; both morphs of bee occur in the village, but A. cerana is more common (Chapter 3). One colony of A. cerana was observed. Because the only hoknown colonies of "A. nigrocincta" in Kamarora lacked adult drones at the tirne of observation, a colony of "A. nigrocincta" was observed in Bobo, a village at 1730 m situated 16 km NW of Kamarora. Kamarora and Bobo were considered a single study site. Observations were made from 17-20

November 1995 (Table 2.1). Figure 2.1 Map of Sulawesi indicating location of drone flight study sites.

Table 2.1 Locations, dates, and colonies observed for drone flights in 1995. C = A. cerana; N = "A. nigmcincta". Each colony from which drone flights were observed is indicated by a number (e.g., colony C-3 was obsewed on 6 d between 18-25 September).

1718 1818 1918 2018 2118 2218 2318 Palangisang C-1 C-1 C-1 C-1 C-1 C-1 C-1

17111 18111 19111 20111 Kamarora and Bobo C-4 C-4 C-4 C-4 Colonies were placed 150-500 m apart. One colony of A. cerana and 1-2 colonies of "A. nigrocincta" were watched simuItaneously by 2-3 people on observation days. Prior to data collection, I observed colonies of both morphs from

12:00 to 18:OO h for 5 d to obtain preliminary information on timing of drone flights.

The earliest observed departures of drones of A. cerana and "A. nigrocincta" occurred at 12:45 h and 14:45 h respectively. Consequently, I initiated data collection at 12:OO h for A. cerana and 14:OOh for "A. nigrocincta" in order to record al1 drone flights; on al1 days of data collection a minimum of 15 min passed before any drones took flight. After recording the time at which the first drones departed,

1 recorded the number of drones that entered each nest during 5 min intervals.

Observations were terminated when 15 min had passed with no drones returning to colonies. Observers were 0.5-0.8 m from the hive entrantes to enable drones to be readily counted without disturbing the colonies. Times were recorded in Central

Indonesian Standard Time (Universal Time + 8 hours). Because al1 locations were within 10-28 km of the meridian 120°, data required no adjustment to solar zenith time prior to statistical comparisons. Daylength was almost the same at al1 three sites because the proximity to the equator (Sunset: Palangisang, 17:57;

Bontobulaeng, l8:Ol; Kamarora and Bobo, l8:O4; List. 1968).

Data on first drone departure, last drone entrance, and mean drone entrance times (after grouping data into 15 min intervals) (f S.E. of rneans) of the two bee morphs were analyzed using one-way analyses of variance (Statistical Analysis

SystemO, SAS Institute Inc., Box 800, Cary, North Carolina). Location effects on these variables were analyzed with a No-way analysis of variance. Durations of the

16 drone Right periods for the two morphs in the three locations were compared with a two-way analysis of variance after log-transformation of the data. The percentages of drones of both morphs that returned during the period of overlap (the period during which bees of both rnorphs were away from colonies on flights) were calculated. General Linear Means Procedure was used for al1 statistical analyses. 2.3 RESULTS

At each of the three sites, the tirnes of the first departures of drones, the last

returns, and rnean tirnes of returns differed significantly (P<0.0001; Table 2.2;

Figure 2.2,2.3,2.4), with drones of A. cerana nearly completing their flights before the first drones of "A. nigrocincta" initiated theirs. At Palangisang there was great overlap in the distributions of the drone flight tirnes of the two rnorphs: 18.9 I8.99%

(Range: 049.8%) of drones of A. cerana flew in the period of overlap; for drones of "A. nigrocincta" 2.3 * 1.2% (0-8.2%). In the other two sites there was very little overlap in the distributions of flight tirnes of drones of A. cemna and "A. nigrocincta"

(Bontobulaeng: drones of A. cerana 0.41 I 0.27% (04.5%); drones of "A. nigrocincta" 0.3 k 0.27% (O - 1.4%); Karnarora and Bobo: drones of A. cerana 0.3

I0.20% (0-0.7%); drones of "A. nigrocincta" 0.5 i 0.31 % (0-1.3%)).

For both morphs, the rnean tirnes of first departure, mean tirnes of drone entrance, and rnean times of last returning drones differed significantly between the three sites (Pc0.05; Table 2.2). The rnean tirnes of first departure of drones in

Karnarora and Bobo occurred earlier than in Palangisang. Drones of both morphs stopped flying later in Palangisang than in Bontobulaeng and Kamarora or Bobo.

The earliest mean entrance tirnes of drones of A. cerana and "A. nigrocincta'' were recorded in Karnarora and Bontobulaeng, respectively.

There was variation in the mean duration of the drone flight periods of the two morphs in the three locations. Cornparing the two morphs, the durations of the flight period were significantly different in Palangisang (P=0.0006) and

Bontobulaeng (P=0.0001), but did not differ statistically in Kamarora and Bobo

(P=0.1654). Considering only the drones of A. cerana the drone flight period was longer in Kamarora (129 i 14.5 min) than at Palangisang (i03 i 10.9 min; P=0.049) or Bontobulaeng (100 I7.2 min; P=0.010). The flight periods at the latter two sites were not significantly different (P=0.62). For the drones of "A. nignicincta" the mating flight period was shorter in Bontobulaeng (127 I5.8 min) than in either

Palangisang (149 I 10.9 min; P=0.048) or Bobo (155 I 14.5 min; P=0.035;.

Duration of the flight periods in the latter two sites did not differ (P=0.64). Table 2.2 Means of time of first departure, time of entry to colonies, and last time of entry of drones of different pairs of colonies of A. cerana (C) and "A. nigrocincta" (N) observed simultaneously at Palangisang and Bontobulaeng (South Sulawesi) and Kamarora and Bobo (Central Sulawesi).

Mean time of Mean drone Mean time of first entrance last drone No. departure f times f SE entrance f Location obs. SE (xhr k min) SE (days) (xhr~min) (xhr i min)

Pafangisang

Bontobulaeng C-2 & N-2

AI1 colonies cornbined

Kamarora & Bobo

* Significant differences (Pe0.05) between values within a column are indicated by different letters. All pairwise cornparisons between A. cerana and "A. nigrocincta" are highly significant (P<0.0001) Figure 2.2 Timing of drone flights at Palangisang, South Sulawesi from 17 to 23 Auçust, 1995. The data are represented as proportion of drone flights ending during each 15-min period relative to the maximal number recorded during any 15 min period. The data are plotted at the starting times of each observation period. N = the total number of drones observed entering hives. Arrows indicate mean drone entrance times.

Figure 2.3 Timing of drone flights at Bontobulaeng, South Sulawesi on 5, 12-21, 24-27 Septernber, 1995. The data are represented as proportion of drone flights ending during each 15-min period relative to the maximal nurnber recorded during any 15 min period. The data are plotted at the starting times of each observation period. N = the total number of drones observed entering hives. Arrows indicate mean drone entrance times. A. cerana "A. nigrocincta"

Time of Day (Central lndonesian Time) Figure 2.4 Timing of drone flights at Kamarora and Bobo, Central Sulawesi on 17- 20 November, 1995. The data are represented as proportion of drone flights ending during each 15-min period relative to the maximal number recorded during any 15 min period. The data are plotted at the starting times of each observation period. N = the total number of drones observed entering hives. Arrows indicate mean drone entrance times.

2.4 DISCUSSION

My results show that there is a highly significant difference in the timing of flights of drones of A. cerana and "A. nigrocincta" in Sulawesi. Because the distribution of queen mating flight times is similar to the flight times of drones within a population (reviewed by Koeniger, 1991; see also Verrna et al., 1990; Yoshida et al. ,1994; Yoshida, 1995), quantification of the timing of drone flights is an easy way to detemine whether or not two populations difler with respect to their mate recognition system (see Chapter 7). In my study, even though the timing varied between sites, the almost complete separation in mating flight times results in almost no opportunity for hybridization between the two morphs. Morphornetn'c and genetic analyses (Damus, 1995; Hadisoesilo et al., 1995; Smith and Hagen, 1996) have demonstrated that "A. nigrocincta" is clearly distinct from A. cerana from al1 the islands surrounding Sulawesi (Damus, 1995) and from A. koschevnikovi of Borneo

(Damus, 1995; Hadisoesilo et al., 1995). The drone flight data not only support the status of A. nigmcincta as a distinct population, but they lead to the conclusion that

A. nigrocincta is a previously overlooked species of Apis. For this reason, further references to A. nigrocincta in my thesis will be made without the use of quotation marks. Specimens I have examined are similar to the type specimen collected by

A. R. Wallace near "Makassar" (=Ujung Pandang) in 1856 that was later described by Frederick Smith (1861) as Apis nigro-cincta (G.W.Otis, pers. comm). The type specimen is housed in the Oxford University Museum Collection, Oxford, United Kingdom.

There are significant differences between sites in the recorded timing of drone flights and the duration of the drone fiight periods of A. cerana and A. nigrocineta. The reasons for these differences are not clear, but may be related in part to afternoon temperatures (Taber, 1964; Rowell et al., 1986): at the higher elevation sites (Bontobulaeng, Kamarora and Bobo), recorded temperatures were cooler (maximum 29°C) and drone flights generally occurred earlier than at

Palangisang (maximum 34%). Unfortunately, because weatherklimate effects were not the main focus of this study, detailed weather records were not taken and detailed analyses could not be performed. Because daylength, latitude, and longitude were almost identical for Palangisang and Bontobulaeng, their effects on the data sets were inconsequential and thus did not aid in understanding differences between the sites.

A comment is warranted conceming the specific timing of drone flights of A. cerana in Sulawesi relative to other locations where drone flights have been monitored. In Sulawesi, the mean time at which flights occurs (returning drones; hereafter referred to as mean flight time) was 13:44 to 14:32 h. This is somewhat earlier than observed in nearby location in Sabah, Borneo (Koeniger et al., 1988;

1996a), where the mean flight time (exiting and returning drones combined) occurred at approximately 14:20 h (data corrected to solar zenith time). In contrast, drones of A. cerana in Thailand have a mean flight time of approximately 1558 h

(corrected to solar zenith time by subtracting 24 min; Rinderer et ai., 1993) which is much later than in the previous two sites. The mean flight (combined exiting and

entering bees) occurs even iater in the day in Japan (exiting bees only), at

approximately l5:l5 h (Yoshida et al., 1994; Sasaki et al., 1995), and in Sri Lanka

(cornbined exiting and entering bees), at approximately I6:S h (corrected to solar tirne; Koeniger and Wijayagunasekera, 1976). This variability between sites is

remarkable. It is presumably infiuenced by the other species of honey bees that CO- occur with A. cerana at the above sites, as pointed out by most authors (Koeniger,

1991; Yoshida et al., 1994; Yoshida, 1995), but that alone does not explain al1 the differences. In Sulawesi, the influence of other species is negligible because in

addition to A. nigrocincta and A. cerana, the only other species present is A. dorsata binghami whose drones fly at dusk. CHAPTER 3

THE DISTRIBUTION AND MORPHOMETRIC ANALYSES OF Apis cerana AND A. nigrocincta IN SULAWESI, INDONESIA

3.1 INTRODUCTION

Nearly al1 species with wide distributions exhibit some geographic variation.

The importance of such variation was identified by Darwin (1859) and Wallace

(1876) although they did not know its source. We now know the basic source of genetic variation lies in relatively rare mutations. Subsequently, gene frequencies are shaped by interactions with the environment through natural selection, as well as by random changes that become particularly important when population sizes are small. Any disruption of gene flow, often as a result of physical barriers, further allows differentiation to occur between isolated populations. Finally, the environment can influence the resultant phenotype. The result is that wide-ranging species that occupy many different habitats and climatic conditions usually exhibit extensive intraspecific variability (Mayr, 1964; 1970).

Several species of honey bees, particularly A. mellifera and A. cerana, demonstrate extensive phenotypic variability. Both have huge distributions that encompass widely different geographic and climatic conditions, from tropical forests to deserts to temperate climates, and many populations are geographically isolated.

Not surprisingly, both species exhibit extensive geographic variation. For example, the continent of Africa harbours no less than eight recognized subspecies, or races, of A. mellifera (Ruttner, 1988). Kenya alone has four races that are distributed between mountain, savannah, coastal, and semi-desert regions of the country

(Mbaya, 1993). These races have been defined largely on the basis of morphological measurements, although behavioural traits are also usually considered. The situation with A. cerana has been less well studied, but numerous races have been recognized (Ruttner, 1988; Peng et al., 1989; Verrna et al., 1994.

Verma, 1995). Ruttner (1988) documented A.c. cerana, Asc.japonica, Asc.indica. and A.c. himalaya. His analysis also suggests that A.c. philippins is a valid race.

Chinese researchers, as surnmarised by Peng et ai. (1989) distinguished three to four additional races. Finally, a recent morphornetric assessrnent of Southeast

Asian and Malesian samples by Damus (1995) confirmed the distinctness of A.c. phiîippina and suggested that populations in South Sulawesi and Timor deserve su bspecific status.

Morphometrics is the measurement and analysis of fomi (Daly, 1985). It is a relatively quick and inexpensive technique that can be applied to specimens preserved and stored in museums (Daly, 1985,1992). Honey bees were among the first organisms to which morphometric techniques were applied because these techniques are particularly appropriate to identifying intraspecific variation. Until now, morphometric characters have proven to be the most effective tool to distinguish races of honey bees; the results are correlated well with biogeography and biology (Ruttner, 1988; Daly, 1992). Unfortunately, although morphometric analyses indicate differences between populations, they usually cannot demonstrate unequivocally the existence of previously unrecognized species (but see Daly, 1985) unless traits differ significantly and consistently between populations that could potentially interbreed. Variation in highly variable, widespread species such as Apis cerana is usually assumed to represent intraspecific variation. The alternative, that a previously unrecognized species exists, is feasible, but usually requires much supporting documentation, including details of distribution and evidence of hybridization or the lack of it where the two populations are in contact.

A relevant situation haç been discovered relating to A. cerana and A. nigrocincta in Sulawesi. It has been confirmed, by documenting the temporal segregation of their mating flights, that A. cerana and A. nigrocincta are distinct species (see Chapter 2; Hadisoesilo and Otis, 1996).These two forms are so similar rnorphologically (Damus, 1995) that no diagnostic character, other than colour of body parts (e-g., femur, clypeus), has yet been identified. Initially the distributions of these species were reported to be parapatric or partially sympatric in South Sulawesi (Hadisoesilo et al., 1995). Subsequently, two different zones of sympatry were discovered in South and Central Sulawesi. However, several questions still need answering to confirm the species status of Apis nigrocincta.

How broad are these zones of sympatry? Do the two forms maintain their distinctiveness in these zones of sympatry, or are there intermediate bees suggestive of hybridization? In this chapter, I report on my detailed surveys of the two zones of sympatry.

Numerous colonies of A. cerana and A. nigrocincta were located and mapped, and sarnples taken for morphometric analyses to determine if intemediate forms indicative of hybridization exist. The results are discussed in relation to the species status of A. nigrocincta. 3.2 MATERIALS AND METHODS

3.2.1 Distribution

From June, 1994 to March, 1995, 1 accessed the knowledge of local

infamants to locate feral as well as hived colonies of cavity-nesting honey bees in

17 localities in the southern part of the Province of South Sulawesi. I selected sites to survey based on the preliminary distribution data of Otis and Hadisoesilo (1990).

The elevations of these sites range from 40m to 1500 m. From this survey, I found one area of sympatry near Bontobulaeng, approximately 30 km NW of the city of

Bulukumba. I subsequently surveyed this area in more detail from March to

September, 1995.

Another zone of sympatry was located in the Palolo Valley of Central

Sulawesi, 55 km SE of Palu, in September, 1994. Subsequently, a detailed survey was made in the area immediately surrounding the villages of Kamarora and

Rahmat, in November, 1995 and September, 1996.

Prior to detailed analysis, I tentatively identified colonies as being either A. cerana or A. nigrocincta based on the colour of the workers' hind femora (Figure

3.1 ). A colony was identified as A. cerana if the hind femora of worker bees were brown to blackish brown ("fuscous" in Smithe, 1975), and as A. nigrocincta if the hind femora were yellow ("cream colour" in Srnithe, 1975). 1 used the colour of workers' hind femora to distinguish these two species because it is more reliable than abdominal colour. Tergite colour is known to Vary with elevation (pers. obs.; Figure 3.1 The hind legs of worker bees of A. cerana (right) and A. nigrocinda (lefi); arrows indicate femora.

Ruttner, 1988) and temperature (Le., season: Spivak et al., 1992; Matsuka et al.,

1995), whereas the colour of hind femora of A. nigrocincta and A. cerana workers was constant over elevations (0-1 500 m) and seasons. Colonies were located with the help of local residents from each location. When a nest was found, its elevation was recorded. The locations of al1 colonies were mapped.

3.2.2 Morphometric Analyses

3.2.2.1 Sample collections and rneasurement techniques

3.2.2.1 '1 South Sulawesi

About 40% (n=96) of located colonies (n=236) in South Sulawesi were sampled. Each colony represented one sample. Honey bee samples were collected directly from colonies in situ. Samples were taken of worker bees from inside the hives or existing colonies. The bees were killed with ether and presenied in 70% ethanol. I also included two samples of A. nigrocincta from North Sulawesi

(elevation < 25 m ) collected in 1994, one from Kamarora (elevation 630 m) collected in 1994, and three colonies of A. cerana sampled in Kamarora in 1994. AI1 sarnples were taken back to the Forest Research Institute in Ujung Pandang for morphometric analyses. In total , 102 samples were analyzed.

The characters I measured differed slightly from those used by Ruttner et al.

(1W8), Mattu and Verma (1984), and Ruttner (1988). Several characters were omitted because they could not be measured accurately. The 22 characters I measured are illustrated in Figure 3.2. Each sample consisted of 15 bees. Figure 3.2 Characters measured for morphornetric analysis of bees collected in Sulawesi. A = hind leg; B = forewing; C = hindwing; D = forewing showing the measured angles. All characters were rneasured for samples from South Sulawesi. For samples from Central Sulawesi characters number 11, 16, 19, and 21 were not included. All characters of forewings, legs, cubital veins A and B, and angles, are those Iisted in Ruttner et al., 1978 and Ruttner, 1988. Hindwing characters are those listed in Mattu and Verma, 1984.

1. Metafernur length (Fe) 2. Metatibia length (TI) 3. Metatarsus length (MI) 4. Metatarsus width (MW) 5. Forewing length (FI) 6. Forewing width (Fw) 7. Hindwing length (HI) 8. Hindwing width (Hw) 9. Cubital vein A (a) 10. Cubital vein B (b) 11. Length of indica vein (IV) 12. Angle A4 13. Angle B4 14. Angle 07 15. Angle €9 16. Angle G18 17. Angle J10 18. Angle JI6 19. Angle KI9 20. Angle LI3 21. Angle N23 22. Angle 026

The right forewings and hindwings were removed. Wet forewings were spread on a pieces of glass (5x5 cm), allowed to dry, covered with a thin piece of clear plastic (overhead projector transparency), and sealed with a piece of clear tape. The hindwings were mounted in the same way on a microscope slide. The right hind legs were removed, arranged on another microscope slide, mounted with a mixture of arabic gum, glycerin, acetic acid, and distilled water, and then covered with another microscope slide. Three slide mounts contained ail the body parts for each sample.

To measure al1 characters, except the angles of the forewing, the specimens were studied with a Wild Heerbrugg dissecting microscope (approximately 9x, 12x,

18x, 25x, and 50x depending on the characters measured) following calibration of exact magnification with a 1 cm micrometer. The results were then converted into actual lengths and widths. The angles of the forewings were rneasured by projecting the image of wings ont0 a wall with a slide projector. The positions of the 18 vein intersections (Figure 3.2D) were marked on a piece of paper at the approximate centres of the vein intersections, after which angIes were rneasured using a protractor.

3.2.2.1.2 Central Sulawesi

About 27% (n=20) of located colonies (n=73) in Kamarora and Palu in 1995 and 1996 were sampled. Six colonies of A. cerana and four colonies of A. nigrocincta were sampled in Kamarora; six colonies of A. nigrocincta and four colonies of A. cerana were collected in Rahmat. In addition, four colonies of A. cerana were collected in Lolu, 20 km southeast of Palu in Central Sulawesi. In total,

14 colonies of A. cerana and 1O colonies of A. nigrocincta from Central Sulawesi were taken to the University of Guelph for morphometric analyses.

The same characters described for the South Sulawesi samples, with the exception of angles (318, KI9, and N23, and the indica vein length, were measured for samples from Central Sulawesi. These characters were not measured because the results from previous analyses revealed that they contributed little to the separation of these taxa. In total, 18 characters were measured. The data for these measurements were analyzed separately from those from South Sulawesi because a different microscope was used and a different person made the measurements.

The measurements of al1 characters except the angles, were measured with a Zeiss stereo microscope (approximately 1Ox, 20x, 40x, and 80x magnification depending on the characters measured). Exact rnagnifications were determined with a 1 cm micrometer. The angles were measured as described previously. For these samples, measurements were made on samples of 1O bees per colony.

3.2.3 Data analyses

The first set of analyses were completed on of 102 sarnples: 96 samples from South Sulawesi (Table 3.1), four from Kamarora, Central Sulawesi, and two from North Sulawesi. Samples with black femora were designated a priori as A. cerana, while those with yellow femora were designated as A. nigmcincfa. Samples Table 3.1 Location of colonies located and analyzed in South Sulawesi. N = A. nigrocincta, C = A. cerana. X signifies locations within the area of sympatry.

Site Location of Elevation Colonies Samples number colonies (m) N C N C

1 Tabo Tabo*

2 Ujung Pandang

3 Jeneponto*

4 Bantaeng

5 Tanaberu

6 Sinjai*

7 Salassae

8 Palangisang

9 Herlang

10 Palampang *X

11 Bulo Bulo X

12 Bontobulaeng'X

13 Tanete X 14 Patongko*

15 Gang king

16 Manipi*

17 Borrongrappoa

Tobl

* Asterisks indicate that sorne sarnples from these locations were measured previously and reported by Hadisoesilo et al., (1995). were divided into four groups: C = 28 samples of A. cerana from South Sulawesi,

N = 70 samples of A. nigrocincta from South (n=68) and North Sulawesi (n=2), K

= three samples of A. cerana from Kamarora, and Y = one sample of A. nigrocincta from Kamarora.

The second set of analyses were completed on 24 samples: 14 A. cerana and 10 A. nigrocincta collected in Central Sulawesi in 1996. Samples were separated into four groups: K = samples of A. cerana from Kamarora (n=6); c = samples of A. cerana from Rahmat (n=4); palu = samples of A. cerana collected from Lob (n=4), and n = samples of A. nigrocincta collected from Kamarora (n=4) and Rahmat (n=6).

Character values for individual bees were entered into LOTUS 1-~-3~~ version 2.0 and averages based on 10 or 15 bees from each sample were calculated. Statistical analyses were performed using SystatTM version 5.0

(Wilkinson, 1990). The analyses were done in three ways:

1. Cluster Analysis using Unweighted Pair-group Method Using Arithmetic

Averages (UPGMA). Before analyzing, the original data were standardized

by converting the original data to new unitless data. Standardizing rnakes

characters (attributes) contribute more equally to the similarities among

samples (Sneath and Sokal, 1973; Romersburg, 1984).

2. Principle Component Analysis (PCA) was performed on the data to

discover the relationships among samples and among variables without a

prion division of the samples into separate groups (Foottit and Sorensen, 1992) and to assign individuals to groups that were created as a function of

the analysis (Pimentel, 1992). The PCAs were done separately for (1) colony

samples and for (2) individual bees in the two areas of sympatry,

Bontobulaeng and Rahmat.

3. Discriminant Analyses, consisting of a Manova and Canonical Analysis of

Discriminance (CAD) (Pimentel, 1979; 1992) were employed. These

analyses involved grouping of samples before analyses (Foottit and

Sorensen, 1992). A Manova was used to check for inequality of group

centroids (confirmation or rejection of distinct group status) and CAD was

used to calculate distances between group members and each group

centroid in discriminant space. The discriminant analyses were carried out

twice. The first discriminant analysis was done by grouping samples into two

a pnongroups, A. cerana and A. nigrocincta, based on the colour of workers'

hind femora. From South Sulawesi (see section 3.2.2.1.1 ), K and C samples

were placed in the group of A. cerana while N and Y samples were placed

in the group of A. nigrocincta. From Central Sulawesi (see section 3.2.2.1.2)

K, c, and palu samples were grouped under A. cerana and N samptes were

placed under A. nigrocincta. ln the second analysis, samples of A. cerana from Kamarora (K) were assigned into a separate a prion group. Because the PCA results for individual bees from Rahmat did not completely separate

A. cerana from A. nigrocincta, a discriminant analysis was carried out for

individual bees. In addition, several bivariafe scatterplots (HI and Hw, FI and Fe, JIO and 04,

JI0 and 07, 84 and D7) were examined to determine if these combinations of two characters were capable of discriminating A. cerana from A. nigrocincta. The choice of these characters was based on the contributions of 22 (18) characters to the first principal component. 3.3 RESULTS

3.3.1 Distribution

3.3.1.2 South Sulawesi

I located 236 colonies: 115 colonies were of A. nigrocincta and 121 colonies

were of A. cerana (Table 3.1). The colonies were arranged into five groups based

on the elevations where samples were collected (Table 3.2). Most colonies of A.

nigrocincta (89.6%) were found above 400 m elevation and most colonies of A.

cerana (86.8%) were found at lower elevation below 400 m. The only colonies of A.

nigrocincta which were found at lower elevation were at the northernmost site, Tabo

Tabo, a forested site at 70 m,and Sinjai at an eastem coastal site. Apis nigrocincta

usually inhabited more forested areas or mixed culture areas, while A. cerana

mostly was found in disturbed areas such as villages or areas with monocultures of coconuts.

Figure 3.3 represents the distribution of colonies located in South Sulawesi.

From Figure 3.3, it is clear that the distribution of the two types of bees is mostly allopatric with a narrow zone of sympatry approximately 10-15 km in width. lt should be noted that because I located all colonies through information provided by villagers, the sampling was not randornized. Each nest represents a naturally occurring colony but the data do not allow for cornparisons of colony density between sites. Figure 3.4 shows the distribution of colonies in the sympatric area in more detail. Sixty five colonies were found in this area (19 A. cerana, Table 3.2 Distribution patterns of A. nigmcjncfa, and A. cerana colonies at different elevation ranges. T = total numbers of colonies Iocated; NT = total numbers of colonies of A. nigrocincta located; Ç = total numbers of colonies of A. cerana located; Np = percentage of colonies of A. nigmcincfa located, Cp = percentage of colonies of A. cerana located.

Elevation (m) T NT C, Np C, 0- 199 60 8 52 13.3 86.7 200 - 399 57 4 53 7.0 93

Total 236 fi5 121 Figure 3.3 Seventeen locations surveyed of colonies of A. cerana andlor A. nigrocincïa in South Sulawesi. Solid portions of the symbols indicate the proportion of colonies of A. cerana the white portions of the symbols signify the proportion of colonies of A. nigrocincta. Nurnbers show the numbers of located colonies at each site. Ellipse indicates the area of sympatry shown in more detail in Figure 3.4.

Figure 3.4 Locations of colonies in the area of sympatry in South Sulawesi. Black syrnbols represent A. cerana, open symbols represent A. nigrocincta. Different symbols are used to indicate the numbers of colonies of each species in each location. KAB. = Kabupaten = District. The village of Tanete is provided as a reference point 0 i Colony alYNETE 0 2 Colonies v 3 Colonies A 4 Colonies O 5 Colonies 010 Colonies KAB, BULUKUMBA 46 A. nigrocincfa). The distances between colonies of A. cerana and A. nigrocincfa

were between 50 to 500 m, showing that they were truly sympatric. Most colonies

of A. nigrocincta were inside natural or man made cavities and most of them were

close to a stream, whereas most A. cerana colonies nested inside hollow trees or

houses.

3.3.1.2 Central Sulawesi

The distributions of A. cerana and A. nigrocincfa in Kamarora and Rahmat

are as follows: A. nigrocincfa typically nested in forested areas of Lore Lindu

National Park but colonies of A. cerana were usually in villages or disturbed areas.

Although this separation was not as clear in Kamarora, al1 colonies of A. cerana

(n=24) in Kamarora were found in the village or other disturbed areas. No colonies

of A. cerana were located inside the park. The numbers of colonies of A. nigrocincfa

(n=9) I found in the village were much lower than colonies of A. cerana.

Unfortunately, I found only two colonies of A. nigrocincta, of which both were inside

the park (Figure 3.5). 1 have plotted the locations of al1 nests whose locations were

known to villagers. Because nesting sites of these two species are similar, there is

not likely any substantial bias in nest location.

In Rahmat, on the other hand, the geographic separation was rernarkable.

All colonies of A. cerana (n=14) were found in the village. In contrast, only 20%

(n=5) of colonies of A. nigrocincfa I found (n=24) were located in the village. The Figure 3.5 Colonies located at Kamarora. Solid symbols indicate colonies of A. cerana open symbols signrfy colonies of A. nigrocincta Squares indicate colonies located in 1995; diamonds signify colonies located in lW6. The Jepang road forms the Lore Lindu National Park boundary and separates the forest frorn disturbed habitats (villages and agricultural areas). Refer to Figure 2.1 for general location. rest of colonies of A. nigrocincta were located inside or at the Lore Lindu

NationalPark boundary (Figure 3.6). The numbers of colonies located at Kamarora and Rahmat in 1995 and 1996 are presented in Table 3.3. Figure 3.6 Colonies located at Rahmat. Solid symbols indicate colonies of A. cerana colonies; open sym bols signify colonies of A. nigmcincta colonies. Squares indicate colonies located in 1995; diamonds signrfy colonies located in 1996. The Jepang road forms the Lore Lindu National Park boundary and separates the forest frorn disturbed habitats (villages and agriculturaI areas).

Table 3.3 Number of colonies located at Kamarora and Rahmat in 1995 and 1996. N = A. nigrocincta; C = A. cerana.

Location 1995 1996 Total N C N C N C

Kamarora 7 22 4 2 11 24

Rahmat 13 7 11 7 24 14

Total 20 29 15 9 35 38 3.3.2 Morphometric Analyses

3.3.2.1 Samples Primariiy from South Sulawesi (1994)

3.3.2.1.1 Cluster Analysis

The cluster analysis generated homain clusters: one comprises al1 colonies of A. cerana and the other al1 colonies of A. nigrocincta. Most of the samples within species collected from relatively nearby locations (cl 0 km apart) were usually more similar than samples collected from more distant areas, but this generalization did not always hold. For example, five out of six samples of A. cerana collected near

Bantaeng clustered together. The sixth sarnple from Bantaeng, however, was more similar to the samples collected in Bontobulaeng and Palangisang than to those from Bantaeng. In another case, only one sample of A. cerana from the area of sympatry failed to cluster with other samples collected in that region. The three samples of A. cerana from Kamarora were very similar to each other and made a separate group, distinct from the samples of A. cerana from South Sulawesi.

As for A. nigrocincta, I found the same situation. Although most sarnples collected from nearby colonies were more similar than those collected from further areas, this generalization did not always hold. Some samples from Manipi, for instance, were more sirnilar to samples collected in Bontobulaeng (- 25 km from

Manipi) than from those collected in Manipi. 3.3.2.1.2 Principal Component Analysis (PCA)

The purpose of the PCA is to discover the relationships among samples without a priori division of the samples into separate groups (Foottit and Sorensen,

1992). The correlations of the 22 characters with the first three principal cornponents are presented in Table 3.4. The first three components described

66.1 % of the total variation of the original variables. The first component accounted for 47.8% of the total variation; it is mostly characterized by variables that have high correlations with general size (lengths and widths except for FI and IV) and some angles. The second component described 10.3% of the total variation. lt had higher correlations with sorne angles but had smaller correlations with characters that differed in size. The third cornponent, accounting for 8.1 % of the total variation, had relatively low correlations with al1 variables except with the indica vein length. To visually detect natural groups among the scatter of sarnple points, their PC1 and

PC2 scores were plotted. This plot shows two discrete clusters, one for A. cerana and one for A. nigrocincta (Figure 3.7). There are no samples with intermediate scores.

The PCA of individual bees from the syrnpatric area gave the same results.

Two separate clusters were fonned. One comprised only A. cerana and the other

A. nigrocincta (Figure 3.8). Notably, bees with intemiediate scores are absent. The separation of these two groups were mainly based on the characters that related to size. Table 3.4 Correlations of 22 characters with the first three Principal Cornponents for 102 samples.

Principal Component Character 1 2 3

1. Metafemur length (Fe) 2. Metatibia length (TI) 3. Metatarsus length (MI) 4. Metatarsus width (MW) 5. Forewing length (FI) 6. Forewing width (Fw) 7. Hindwing length (HI) 8. Hindwing width (Hw) 9. Cubital A (a) 10. Cubital B (b) 1 1. Length of indica vein (IV) 12. Angle A4 13. Angle 84 14. Angle D7 15. Angle E9 16. Angle G18 17. Angle J 10 18. Angle JI6 19. Angle K19 20. Angle LI3 21. Angle N23 22. Angle 026

52 Figure 3.7 Scatterplot of Principal Components 1 and 2 for 102 samples predominantly from South Sulawesi, based on 22 standardized variables. N and Y represent colonies of A. nigrocinda; K and C represent colonies of A. cerana.

Figure 3.8 Scatterplot of Principal Components 1 and 2 for individual bees from the zone of sympatry using 22 standardized variables. Circtes represent A. nigfocincta; small squares represent A. cerana.

3.3.2.1.3 Discriminant Analyses

For the first and the second discriminant analyses, the MANOVA confirmed inequality of the group centroids (Le., the groups were distinct, P<0.001).

When al1 samples were forced to be placed into one of two a priori groups,

A. cerana or A. nigrocincta, no single sample was misclassified. Two distinct groups, A. cerana (C and K) and A. nigrocincta (N and Y)formed. No intermediate samples were found between these groups. The second analysis aIso properly classified aIl samples to the a priori determined group. All A. nigrocincta fell into one group, aIl samplss of A. cerana from South Sulawesi into a second group, and samples of A. cerana from Kamarora (K) classified into a third group. Distances between members of the K group to the group centroid of A. cerana were less than to the group centroid of A. nigrocincfa. No intemediate samples were found. Figure

3.9 shows the biplot of Factor 1 and 2 and the corresponding ellipses of 95% confidence. It is clear that A. cerana and A. nigrocincfa fall into different groups. As for A. mana from Kamarora, its ellipse seerns to overlap the A. cerana ellipse. The

95% ellipse surrounding the A. cerana samples from Kamarora is large because of the srnail sample size (n=3) wmpared to those of A. cerana from South Sulawesi

(n=28) and A. nigrocincta (n=71). Figure 3.9 Plot of 95% confidence ellipses for discriminant functions 1 and 2. N = A. nigrocincta; C = A. cerana; K = A. cerana from Kamarora.

3.3.2.1.4 Bivariate Scatterplots

I selected several characters that had high loadings in the principal component analysis to produce two dimensional graphs. This was done to determine if any simple set of variables would allow for separation of A. cerana and

A. nigrocincta.

Figure 3.10 is a scatterplot of the femur lengths (Fe) against the forewing lengths. Based on these two characters alone, I could separate A. nigrocincta from

A. cerana with 96.8% accuracy .

Figure 3.11 shows a scatterplot of hindwing widths (Hw) and hindwing lengths (HI). Based on these two characters only, one can easily separate A. cerana from South Sulawesi and Kamarora from A. nigrocincta. The hindwings of A. nigrocincta are significantly narrower (Pc0.0005) than A. cerana hindwings. The ratio of sample means of HI to Hw for A. nigrocincta were between 3.6 and 3.9, while for A. cerana they were between 3.2 and 3.4. Figure 3.12 presents a scatterplot of angles B4 against JI0. The results indicate that based on these two characters, misclassification of A. cerana and A. nigrocincta is also unlikely to happen although the separation is not as strong as that in Figure 3.1 1. Scatterplots of angles D7 versus 64 (Figure 3.13) and JI0 against D7 (Figure 3.14) show that misclassification between A. nigrocincta and A. cerana occurs if these combinations of characters are used, although the overlap in the data is slight. Figure 3.10 Scatterplot of femur lengths versus forewing lengths for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi.

Figure 3.1 1 Scatterplot of hindwing widths versus hindwing lengths for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi.

Figure 3.12 Scatterplot of angles B4 and JI0 for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi.

Figure 3.13 Scatterplot of angles 07 and B4 for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi.

Figure 3.14 Scatterplot of angles JI0 and 07 for samples of A. cerana and A. nigrocincta predominantly from South Sulawesi.

3.3.2.2 Samples ,from Central Sulawesi (1996)

3.3.2.2.1 Cluster Analysis

The results of the cluster analysis of samples collected from the zone of

sympatry in Central Sulawesi in 1996 are similar to those collected in 1994

predominantly from South Sulawesi. Two main clusters, A. nigrocincta and A.

cerana were generated. Samptes of A. nigrocincta from Kamarora and Rahmat

clustered together, as did samples of A. cerana from Lolu and Rahmat. For example

one sample of A. nigrocincta from Kamarora was more similar to a sample of A.

nigrocincta from Rahmat than to a sample of A. nigrocincta from Kamarora. Most

samples of A. cerana (n=5) from Kamarora, were similar to each other and made

a distinct cluster separate from the rest of samples of A. cerana. The rernaining

sample of A. cerana from Kamarora clustered with samples collected in Rahmat.

3.3.2.2.2 Principal Component Analysis

The contributions of the 18 characters with the first three principal components are presented in Table 3.5. The first three components describe 72.2% of the variation of the original variables. The first component accounted for 52.1 % of the variation whife the second and the third components described 11.4% and

8.7% of the variation respectively. The first and second principal components from this analysis are presented graphically in Figure 3.15. The first component is dominated by variables related to general size (lengths and widths, but excluding

Hw) and some angles. Hindwing width, on the other hand contributed strongly to the Table 3.5 Correlations of 18 characters with the first three Principal Component for 24 sarnples.

-- -- .- - . Principal Component C haracter 1 2 3

1. Metafemur length (Fe) 2. Metatibia length (TI) 3. Metatarsus length (MI) 4. Metatarsus width (MW) 5. Forewing length (FI) 6. Forewing width (Fw) 7. Hindwing length (HI) 8. Hindwing width (Hw) 9. Cubital A (a) 10. Cubital B (b) 11. Angle A4 12. Angle 84 13. Angle D7 14. Angle E9 15. Angle JI0 16. Angle JI6 17. Angle LI3 18. Angle 026 Figure 3.15 Scatterplot of Principal Components 1 and 2 for 24 samples from Central Sulawesi, based on 18 standardized variables. Solid symbols represent A. cerana; open symbols indicate A. nigrocincta ; (C24) = sarnple # 24 from Rahmat; (NS) = sample # 5 from Rahmat. FACTOR (2) second component. This plot also shows two separate clusters, A. nigrocincta and

A. cerana. However, when I graphed the first and the second principal components computed for individual bees from Rahmat, one bee of A. cerana (24) and four bees of A. nigrocincta (5) had sirnilar scores on the PC1 which was dominated by size variables (Figure 3.1 6).

3.3.2.2.3 Discriminant Analyses

The first discriminant analyses which had two a priori groups and the second analyses which had three a priori groups confirmed the inequality of the group centroids (Le.,the groups were distinct, Pc0.001). When al1 samples were classified into two groups, A. cerana and A. nigrocincta, no single sample was misclassified and no intermediate samples were found. Two distinct clusters: A. cerana (cl K, and palu) and A. nigrocincta (n) formed. The second results also confirmed that no intemediate samples were found and no single sample was misclassified. In this case, al1 samples of A. nigmcincta fell in one cluster, samples of A. cerana from Kamarora (K) formed a second group, and the rest of samples of A. cerana (c and palu) classified into a third group (Figure 3.17). Again, the distances between the members of the K group to the group centroid of A. cerana were Iess than those to group centroid of A. nigrocincta, suggesting a closer relationship between the two groups of A. cerana colonies. Figure 3.16 Scatterplot of Principal Components 1 and 2 for individual bees from Rahmat, using 18 standardized variables. (5) = four individual bees of colony N5 (A. nigrocincfa);(24) = one individual bee of colony C24 (A. cerana). FACTOR (2) Figure 3.17 Plot of 95% confidence ellipses for discriminant functions 1 and 2. n = A. nigtucincta samples from Rahmat and Kamarora; c = A. cerana samples from Rahmat; palu = A. cerana samples from Lolu; K = A. cerana samples from Kamarora.

When discriminant analyses with two a priori groups were run for the individual bees from Rahmat, the results showed the inequality of the group centroids (P<0.001). All individual bees were correctly classified into A. nigrocincta or A. cerana, including al1 10 bees of colony C24 and of colony N5. All bees of colony C24 were classfied into group of A. cerana and al1 bees of colony N5 were included in group of A. nigrocincta none appeared as intermediates as they had in the PCA.

3.3.2.2.4 Bivariate Scatterplots

The results of the five bivariate scatterplots for Central Sulawesi samples were clearer from those of South Sulawesi samples. Ail combinations of characters allowed for clear separation of A. cerana and A. nigrocincta: Fe and FI (Figure

3.18); Hw and HI (Figure 3.1 9); angles JIO and B4 (Figure 3.20);angles 07 and B4

(Figure 3.21); and angles J1O and D7 (Figure 3.22). This was especially true for the combinations of Hw and HI and angles JI0 and 64. The results also show that angle JIO is relatively larger in A. cerana than in A. nigrocincta. The hindwings of

A. nigrocincta are significantly narrower than the hindwings of A. cerana

(P4.0005). The ratio of sample means of HI to Hw for A. cerana are between 3.4 and 3.5, but for A. nigrocincta they are between 3.7 and 4.0. Figure 3.18 Scatferplot of femur lengths versus forewing lengths for samples of A. cerana and A. nigmcincta from Central Sulawesi.

Figure 3.19 Scatterplot of hindwing widths and hindwing lengths for samples of A. cerana and A. nigrocincfa from Central Sulawesi.

Figure 3.20 Scatterplot of angles B4 and JI0 for samples of A. cerana and A. nigrochcta from Central Sulawesi.

Figure 3.21 Scatterplot of angles D7 and 84 for samples of A. cerana and A. nigrocincfa from Central Sulawesi.

Figure 3.22 Scatterplot of angles JI0 and D7 for samples of A. cerana and A. nigrocincta from Central Sulawesi.

3.4 DISCUSSION

Apis cerana and A. nigrocincta are partially sympatric in Sulawesi. 1 located two areas of sympatry, one in the Bontobulaeng area of South Sulawesi and the other in the Palolo Valley of Central Sulawesi. The distributions of these two species differ in these sites, and are contrasted below.

In South Sulawesi, A. cerana appears to be restricted to the extreme southern part of the southwestern peninsula. It is found mostly in disturbed habitats at elevations below 400 m. In Central Sulawesi, it can be found up to 800 ml but alrnost exclusively in disturbed areas (cultivated agricultural areas, monocultures of coconuts, and villages). This distribution is of interest because reports from many other parts of the range of this species indicate that A. cerana can be found at elevations from 0-2000+ m (Ruttner, 1988; Salmah et al., 1990; Singh et al., 1990;

Verrna et al., 1994). For instance, in Central Sumatra, A. cerana is cornmon from sea level to >2000m, but appears to be slightly more comrnon at the higher elevations. It occupies various habitats, ranging from prirnary and secondary forest to orchard plantations and villages (Inoue et al., 1990; Salmah et al., 1990).

The distribution of A. nigrocincta is also unusual. In my survey, in extreme southern Sulawesi where A. cerana occurs at lower elevations, A. nigrocincta is restricted to higher elevations (400-1 500+ m) and is usually found in more forested areas (primary forest, agricultural areas with forest patches). North of the known distribution of A. cerana, however, A. nigrocincta can be found readily at low elevations (e-g., Tabo Tabo Forestry Training Centre, 70 m; Sinjai, 4 0 ml a coastal nce-growing region). A similar situation occurs in Central Sulawesi. Southeast of

Palu, A. nigrocincta is absent at lower elevations and is only encountered at elevations of approximately 550 m and higher. It is abundant in forested areas of

Lofe Lindu National Park and only occasionally found in villages or crop-growing areas. In cuntrast, near Manado, North Sulawesi, an area from which A. cerana is apparently absent, A. nigrocincta is cornmon in coconut-growing areas near sea

Ievel (pers. obs.). In other parts of Sulawesi where A. nigrocincta has been collected, it occurs at nearly al1 elevations (see Otis, 1996, for review).

Several factors may combine to yield the realized distributions of these species, including climatic conditions, habitat preferences, and cornpetitive exclusion between the two species. The climatic zones of Sulawesi have been mapped in a variety of ways. The mapping system that corresponds most closely to distributions of vegetation uses the ratio between dry (c60mm rain) to wet (>IO0 mm rain) months (Whitmore, 1984). Using this system, the most seasonal regions of Sulawesi are the extreme southern parts of the southwestern peninsula and the region near Palu (Figure 1.13 in Whitten et al., 1987). These areas are classified as Zones D and El indicating they have three to four (or fewer) consecutive wet months and two to six consecutive dry months. These same regions correspond closely to those where A. cerana is known to exist. Most areas inhabited by A. nigrocincta are classified into Zones A, B, or Clwhich have shorter, Iess defined dry periods and more pronounced wet periods. If climate does influence the distribution of either species, it could be acting directly on the bees by altering their survival under different climatic conditions, or indirectly through effects on floral communities. However, it cannot be the only factor influencing distributions because both species coexist under identical climatic conditions in the zones of sympatry.

ln the villages of Kamarora and Rahmat. the remarkably abrupt transition from A. nigrocincta in forested areas to A. cerana in villages and cultivated areas is striking and unusual for honey bees which are typically thought of as generalists

(Ruttner, 1988; houe et al., 1990; Salmah et al., 1990). It suggests strong habitat selection by swarms when searching for new nest sites. However, this remains to be tested through direct experimentation in the zones of sympatry. Elsewhere in its range, A. cerana is a common species which is reported from a wide range of habitats (Ruttner, 1988; houe et al., 1990; Salmah et al., 1990). If it is highly habitat-specific in Sulawesi, this would be a departure from the norm for this species. My observations indicate that A. nigrocincta is usually associated with forests, although it occurs commonly in some other habitats (e.g., coconut-growing regions near Manado, North Sulawesi).

Finally, competition between these two species may influence their distributions. It is interesting that near the two known zones of sympatry, A. cerana occurs by itself at Iower elevations, A. nigrocincta at higher elevations. This pattern is suggestive of cornpetitive exclusion, whereby the two species do not coexist except in narrow zones of sympatry because of competition for some limiting resource (Ricklefs, 1973). These two species do differ iri tongue (proboscis) length

(A. cerana: 4.556 + 0.1413 mm; A. nigrocincta: 4.976 + 0.0932 mm; Hadisoesilo et al., 1995). It is well known that proboscis Iength influences the ability of bees to extract nectar from flowers (Brian, 1957; Inouye, 1977, 1978; Pleasants, 1980;

Harder, 1985)+This difference in tongue length probably decreases competition between the two species by allowing them to utilize nectar from different sets of flowering plant species, and should actually adto enhance coexistence of the two species.

At present it is not possible to differentiate between these three explanations to explain the observed distributions: climatic limitations, habitat preferences, and competitive exclusion. Several experiments could be conducted to help to sort out these effects. Cotonies of both A. cerana and A. nigrocincta could be established in sites where only one species exists naturally, and followed over time for evidence of better survival and reproduction by one species over the other in particular habitats. Collections of bees on flowering plants coupled with measurements of corolla lengths would help in providing information on the degree of niche overlap.

Artificial swarms of both species could be established at the ecotone between forest and croplands in Rahmat or Kamarora to determine if bees of the two species that are scouting for new nest sites differ in the places they inspect or occupy (Lindauer,

1951, 1955). Similarly, dances of successful foragers could be interpreted by observing thern in observation hives, to determine if the two species forage in the same habitats on the sarne food resources wsscher and Seeley, 1982). By adding colonies of bees of either one species or the other to this experimental site, continued quantification of recruitment dances would provide evidence of competitive displacement. An analogous situation occurs in Bomeo where A. nuluensis occurs at higher elevations (1700+ m) (Tingek et a1.1996). At lower elevations A. koschevnikovi is common in forested regions (Otis, 1996), and A. cerana is a generalist more common in disturbed habitats (Otis, pers. comm.). No detailed studies of these species have been done.

The morphometric analyses of bees from the two zones of sympatry proved useful in addressing the species status of Apis nigrocinda as distinct from A. cerana. If A. cerana and A. nigrocincta represent interbreeding populations of A. cerana, bees of intermediate form should be evident in the zone of sympatry. For example, in morphometric studies of fish (Neff and Smith, 1979) and green leafhoppers (Claridge and Gilham, 1992), the discriminant scores of hybrids were always intermediate between the scores of the parents. However, in my study the results strongly support the view that A. nigrocincta is a distinct species. This is particularly true for colonies in South Sulawesi for which the Principal Cornponents

Analysis, conducted on 15-bee samples, clearly separated A. nigrocincta and A. cerana on plots of the first two principal wmponents (Figure 3.7). In the chance that almost no hybridization occurred but was obscured by analyzing colony samples, individual bees from the zone of sympatry were also analyzed and plotted (Figure

3.8). They also separated into two distinct, widely separated groups of bees. There is no evidence of hybridization between A. cerana and A. nigrocincta in South

Sulawesi.

In Central Sulawesi, the morphometric analyses also allowed al1 colony samples to be classified into two clusters, but the results are not quite as distinctive and require additional explanation. The Principal Components Analysis of the 24 samples of bees from this region correctly placed al1 colonies into clusters determined a prion on the basis of femur colour to be either A. nigrocincta or A. cerana (Figure 3.1 5). In addition, the cluster analysis created two main clusters of bees that also matched the a prion identification as either A. nigrocincta or A. cerana. However, when the first and second principal component scores of individual bees coilected in Rahmat were plotted, there were four A. nigrocincfa bees from one colony and one A. cerana bee which have sirniIar scores on PC1 which is primarily comprised of size variables (Figure 3-16).This might indicate a small amount of hybridization. However, the discriminant analysis indicated that al1 individual bees from Rahmat were correctly classified as either A. nigrocincta or A. cerana. No bees of interrnediate form were detected in either Kamarora (an area or sympatry) or Lolu (where only A. cerana occurs).

One possible explanation for these similar PCI scores is the condition of the colonies from which the bees were taken. Honey bees rear progeny on a common diet in comb cells of regular dimensions and at closely regulated temperatures.

Once the imago emerges and the exoskeleton is sclerotized, size and shape are fixed for the life of the bee. As a consequence, healthy workers from a colony usually Vary little (Daly, 1992). However, size can Vary seasonally as a function of the nutritional state of the colony (Dietz and Haydak, 1965;Haydak, 1970). In the present case, colony C24 was a large, healthy A. cerana colony with much stored pollen and honey. In contrast, colony NS was a very small A. nigrocincta colony with almost no food reserves. Given the results of the various statistical analyses, I believe the single large A. cerana worker from C24 and the four unusually small worker bees from A. nigrocincta colony N5 reflect these colony conditions and do not provide evidence of hybridization.

If these five bees with similar scores on PC1 were hybrids, I would expect the discriminant analysis to randomly assign them to the two species (e-g., 2-3 assigned to A. cerana, the remainder to A. nigrocincta, and not necessarily to the species to which they were assigned a prbn). However, the discriminant analysis correctly assigned al1 five bees to the species represented by the overall colony sample. Although this sample size is insufficient to test this statistically, it further supports the daim that these bees are not hybrids. Further study, particularly genetic analyses of bees from this region, would help to resolve the question of hybridization.

There were three additional observations of note that emerge from these analyses. First, the colour of the hind femur appears to be sufficient to correctly identify species of A. cerana and A. nigrocincfa from Sulawesi. The hind femora of

A. nigrocincfa workers are always yellow ("cream colour" in Smithe, 1975), but those of A. cerana are blackish brown ("fuscous" in Smithe, 1975). The cluster analyses and discriminant analyses always yielded identifications that matched the

a prion identification based on femur colour. This identification criterion is very

useful when observing bees on flowers or other situations where they must be

identified rapidly. However, preliminary inspection of specimens of A. nigrocincta

from Mindanao, Philippines, indicates that their hind femora are blacker, with the

yellow colour greatly reduced. Therefore, the character may not prove to be

diagnostic for this species over its entire range.

Second, several plots of just two characters result in clear separation of

these two species in bidimensional space. The most useful of these is the plot of

hindwing length against hindwing width, wtiich results in two discrete, widely

separated clusters of points. From Figure 3.1 1 and Figure 3.1 9, it is evident that A.

cerana has shorter, wider hindwings than does A. nigrocincta. Superficially, it

appears that the ratio of hindwing length to hindwing width may be another reliable

character that could be used to distinguish these two species. Whether it will hold

up as a diagnostic character for A. nigrocincta in the southern Philippines, or

against other cavity-nesting species of honey bees (e.g., A. koschevnikovi, A.

nuluensis) has yet to be determined.

Even more remarkable, the samples of A. cerana from Kamarora always formed a cluster distinct (95% confidence ellipses) from those of A. cerana from

Central Sulawesi (Figure 3.1 7). These results accord with those of Darnus (1995)

and Damus and Otis (submitted), who found that A. cerana from Kamarora was

indistinguishable morphometrically and genetically from Javanese A. cerana, but different from the apparently endemic subspecies of A. cerana of South Sulawesi.

The most surprising result from my analyses is the striking difference between bees from Kamarora and Rahmat. These two villages are located only 8 km apart in the

Palolo River Valley. with no obvious climatologie or environmental differences between them. Additional study is needed to determine if there is a cline between these two populations, or if there are barriers to hybridization between them as I have found for A. nigrocincta and A. cerana. It is also not yet clear whether or not the A. cerana in Lolu and Rahrnat represent the same subspecies known from

South Sulawesi, or if they represent yet another population.

In conclusion, my study indicates that A. cerana and A. nigrocincta have overlapping ranges at least in two different parts of Sulawesi. Within these zones of sympatry, there is no conclusive evidence of hybridization between these two species. These results agree with the conclusions from the mating study (Chapter

5) which also suggest that hybridization does not occur between these two species.

In Sulawesi the two species can be easily distinguished by the colour of the hind femur. Finally, the distributional and morphometrical results confirm the status of

A. nigrocincta as a species distinct from A. cerana and reinforce the evidence for species status based on the temporal segregation of the distributions of mating flight times of drones. Further studies are needed to clarify the role of habitat preferences and cornpetition on the distributions of these two similar species of honey bee as well as the nature of the contact zone of the two different populations of A. cerana inhabiting Rahmat and Kamarora, Central Sulawesi. CHAPTER 4

MALE GENITALIA OF Apis cerana AND Apis nigrocincta

4.1 INTRODUCTION

Divergent evolution in male genital structures is a widespread trend in many animals with internal fertilization (Eberhard, 1985). There are often greater differences among genitalia than other morphological characters of related species, which implies that genitalia evolve more rapidly than other structures. According to

Shapiro and Porter (i989), a generalization has emerged that genital form for species with internal fertilization tends to be species specific. Traditionally, taxonomists who work with insects and other arthropods have relied heavily on genital structures because they frequently provide reliable diagnostic characters

(Eberhard, 1985; Shapiro and Porter, 1989).

Although insect taxonomy is based extensively on male genital differences in differentiating species, the value of genitalia for species-level determinations is not universal and varies among taxonomic groups (reviewed by Eberhard, 1985;

Shapiro and Porter, 1989; Goulson, 1993). There are many instances of species being distinct on the basis of fom, behaviour or both, even though the genitalia are indistinguishable (Brown, 1959; Eberhard, 1985; Shapiro and Porter, 1989). For example in Hawaiian Drosophilidae, there is a high degree of similarity in the external genitalia of different species in the drosophiloid group (Kanishero, 1983). For another example, the five known species of Protemides (Hymenoptera:

Megachilidae) show few differences in male genitalia (Griswold, 1983). In fact,

Thornhill and Alcock (1983) have argued that the importance of the structural

"uniqueness" of genitalia has probably been overrated, and have emphasized various aspects of mating behaviour as reproductive barriers.

In honey bees (Apis spp.), the external parts of the male genitalia are poorly developed, but an endophallus, an elaborate structural modification of the interna1 walls of the aedeagus, is everted during copulation (Fyg, 1952; Snodgrass, 1956;

Simpson, 1960). Until 1990, scientists working on the male genitalia of honey bees agreed that the endophallus was a reliable character for separating species

(Ruttner, 1988; Koeniger et al., 1991, Koeniger, 1995). The six species for which genitalia were described all demonstrated clear diagnostic differences in their endophalli: A. florea, A. cemna, A. mellifera (Simpson, 1960), A. dorsata (Simpson,

1970), A. kuschevnikovi (Tingek et al., 1988), and A. andrenifonnis (Wongsiri et al., l99O). Moreover, interspecific differences in male genitalia were considered to be reproductive barriers between species. It was believed that male and female genitalia fd together much like a key fits a specific lock and that crosses were not possible because of the lack of proper fit (Eberhard, 1985). Most researchers came to expect differences in male genitalia before accepting the species status of newly recognized rnorphs of honey bees.

The Iock and key hypothesis is well supported by some taxa such as damselflies (Odonata: Coenagrionidae) (Robertson and Paterson, 1982) and Apamea rnoths (Lepidoptera: Noctuidae) (Mikola, 1992). For rnany other taxa, the evidence for the fit of male and fernale genitalia is weak (Eberhard, 1985). For exarnple, Porter and Shapiro (1990) reported that in the genus Tatochila

(Lepidoptera: Pieridae), the differences in the male genitalia of T. mercedis and T. vanvolxemii are ineffective as mechanical isolating devices. In the case of honey bees, it has been known for some tirne that queens of A. cerana can mate with drones of A. mellifera (Ruttner and Maul, 1983) even though the male genitalia of these two species differ (Simpson, 1960; Koeniger et al., 1991). In addition, the discovery of very similar genitalia between sorne sister species of Apis also calls into question the universal validity of the lock and key hypothesis in honey bees. For exarnple McEvoy and Underwood (1988) found no differences between the male genitalia of A. laboriosa and A. dorsata, although some scientists have yet to agree with this result (Koeniger et al., 1991). Regardless of this disagreement, A. laboriosa

is now generally accepted as a valid species because the drones of A. laboriosa take their rnating flights earlier in the day than do those of A. dorsata (Underwood,

1990) and because there are major structural differences despite the close geography of the two populations (Sakagarni et al., 1980). More recently, Otis and

Hadisoesilo (1990) found two distinct populations of cavity-nesting honey bees in

Sulawesi, Indonesia, that proved ,on initial inspection, to have sirnilar male genitalia

(Otis, 1991). These two populations have been shown subsequently to be reproductively isolated by differences in reproductive behaviour (Chapter 2;

Hadisoesilo and Otis, 1996). The initial report by Otis (1991) on genital structure of drones of A. nigrocincta was observational and lacked details needed to compare it to that of drones of A. cerana. Furthemore, there have been no studies of intraspecific variability in the fomi of drone genitalia in any Apis species. Consequently, I compared the structure of the male genitalia of A. cerana and A. nigrocincta, I then discuss the role of genitalia in distinguishing between species, and review the variability in male genitalia of Apis both intra- and interspecifically. 4.2 MATERIALS AND METHODS

The endophallus of a honey bee is differentiated into three major parts: 1) the basal portion or vestibulum, which bears cornua, 2) the cervix which has a fimbriate lobe, and 3) the distal portion or bulbus (Snodgrass, 1956; Koeniger, 1986b).These al1 consist of soft tissues which are difficult to measure. Therefore, comparisons of the endophalli of A. cerana and A. nigrocincfa using morphometric methods are not possible, and qualitative comparisons of both uneverted and everted endophalli of each species were used.

4.2.1. Uneverted Endophalli

Drones of A. cerana were collected from South, Central, and North

Sulawesi, East and Central Java, Sumatra, Universiti Pertanian Malaysia, Serdang, in Peninsular Malaysia, and Tamagawa University, Tokyo, Japan. Drones of A. nigrocincta were collected only from Sulawesi (Table 4.1). Drones were killed and preserved in 70% ethanol.

From each colony, 5-10 drones were dissected. After remaving the tergites and the digestive tract, the genitalia were separated by cutting the ductus ejaculatorius and lifting the entire genitalia. The last (9th) sternum, mesorneres, and lamina parameralis were separated from the other stemites but remained attached to the endophallus. The ventral cornua were spread with a small smooth brush. The epithelium was cleaned with a brush until the structures of the endophalli could be Table 4.1 Sites from which drones were collected for morphological cornparison of endophalli. U = uneverted; E = everted; EJ = East Java; CJ = Central Java; S = Sumatra; SS = South Sulawesi; CS = Central Sulawesi; NS = North Sulawesi; PM = Peninsular Malaysia; JP = Japan. Numbers outside and inside parentheses indicate the number of colonies from which drones were collected and the number of drones from which the genitalia were analyzed.

Species of Drones Site No. Location Sites Apis nigrocincta Apis cemna

1 Jeneponto (SS)

2 Bantaeng (SS)

3 Palangisang (SS)

4 Bontobulaeng (SS)

5 Manipi (SS)

6 Bobo (CS)

7 Kamaroraf Rahmat (CS)

8 Manado (NS)

9 Tretes (EJ)

10 Pati (CJ)

11 Riau (S)

12 Tokyo (JP)

13 Serdang (PM)

Total seen easily. If the brushing did not work well, the sarnples were soaked in 10% KOH for 3-5 hours at room temperature. After clearing, samples were washed in water and preserved in Kahlets solution (Martin, 1977) for further examination.

Four characters were compared: 1) the shape and the number of the nodules of the dorsal cornu, 2) the number of folds on the ventral side of the cervix, 3) the shape of the ventral side of the vestibulum, and 4) the pattern of dense hairs on the ventral side of the vestibulurn. These characters were chosen because they are distinct for each of the six species of honey bee mentioned above

(Koeniger et al., 1991). The cornparisons were made using a Nikon stereomicroscope with 20 to 40X magnifications. Some sarnples were drawn with the help of a Nikon camera lucida microscope to compare the above structures.

4.2.2. Everted Endophalli

The eversion of the endopallus results from pressure exerted by the haemolymph when it is compressed by the contraction of the abdominal muscles.

For complete reviews on the process of eversion in A. mellifera and A. cerana see

Woyke and Ruttner (1958) and Ruttner et al.(l973).

To cause the eversion, the head and thorax were pinched to achieve the first stage of eversion. Then the abdomen was pressed between the thumb and the forefinger to achieve the full (stage 9) eversion (Woyke and Ruttner, 1958; Ruttner et al., 1973). Photographs were taken irnmediately with a camera equipped with a macro lens, close up attachments, and a ring flash. Later, I projected the photographie slides onto a wall and traced the images of the everted endophalli ont0 paper. The dead drones with the everted endophalli were preserved in 70% ethanol for subsequent counting the number of papillae of the fimbriate lobe and qualitative assessrnent of the extension of the ventral cornua. the extent bending of the bulbus, and the shape and number of nodules of the dorsal cornu. The observations were carried out using a Nikon stereomicroscope. 4.3 RESULTS

4.3.1 Uneverted Endophalli

I compared the uneverted endophalli of 116 drones from 11 colonies of A. nigrocincta and 123 drones from 14 colonies of A. cerana (Table 4.1).

4.3.1.1 Vestibulum, dorsal side

The dorsal side of the vestibulum of A. cerana and A. nigrocincta bear dorsal and ventral cornua. Both A. cerana and A. nigrocincta have one pair of ventral comua, each of which forms a long simple tubular projection. The ventral cornua in situ are extremely wrinkled and folded. When they are pulled, they can extend in length beyond the fimbriate lobes (Figure 4.1).

The dorsal cornua in both species are also paired and much shorter than the ventral comua. A dorsal cornu consists of several nodules/bulges which were found to be the same in number (P>0.5 ) for both A. cerana (y= 2.9) and A. nigrocincta

(y= 2.9) (Table 4.2; Figure 4.1).

In both species these bulges are elongated into three short tubes. The shape of the dorsal cornua could not be easily drawn because they changed with slight changes the orientation of the specimens. It was difficult to view al1 specimens from the same angle because of the thickness of the specimens (Figure. 4.2). For example, Figure 4.2d appears different from the other A. cerana specimens in

Figure 4.2, but rotated under a stereomicroscope it looked similar to Figure 4.2e. In Figure 4.1 A dorsal view of the uneverted endophalli of A. nigrocincta and A. cerana. B = bulbus; L = fimbriate lobe; vC = ventral cornu; dC = dorsal cornu. Sample collection sites: A. nigrocincta: Bontobulaeng, South Sulawesi; A. cerana: Jeneponto, South Sulawesi.

Table 4.2 The number of nodules on the dorsal cornua (uneverted and everted samples), number of folds on the ventral side of the cervix, and the number of papillae of the firnbriate lobe (x I S.E.) of A. cerana and A. nigrocincta drones.

Species of Drones

-- Character Apis cerana Apis nigrocincta

Uneverted Everted Uneverted Everted

No. nodules 2.9 I0.03 3.0 + O 2.9 =t 0.02 2.9 I0.08 of the dorsal (n=l23; (n=54) (n=116; (n= 13; coma range 2-3) range 2-3) range 2-3)

No. folds of 6.2 I0.05 6.1 0.06 the cervix (n=l15; (n=?16; range 5-7) range 5-7)

18.5 i 1.O7 21.4 i 0.57 No. papillae - (n=22; - (n=7; range 14-24) range 20-24)

All painnn'se cornparisons between A. cerana and A. nigrocincta are not significant (P0.05). Figure 4.2 Variability in the shapes of dorsal cornua of uneverted endophalli of A. cerana (a-f) and A. nigrocincta (g-1). Specimen collection sites: a: Kamarora; b: Tokyo; c: Tretes; d: Jeneponto; e: Riau; f: PM; g&h: Bontobulaeng; i&j: Manipi; k&l: Manado. The nodules of the dorsal cornua of A. nigrocincta are more rounded and a little shorter than those of A. cerana. No consistent differences are found in the length and shape of the dorsal cornua of drones within a colony and across colonies for each species. general, the nodules of the dorsal comua of A. nigrocincta are more rounded and a little shorter than those of A. cerana. Differences in the lengths and the shapes of the dorsal cornua within drones of a colony and across the colonies of each species were obsetved but there were no consistent differences found. In each of the species, Wo of the nodules were easily visible but the third was sometimes too short to see (Figures. 4.1 and 4.2).

4.3.1.2 Vestibulurn, ventral side

The ventral side of the vestibulum of both A. cerana and A. nigrocincfa is hairy (Figure 4.3). It could not be drawn at the same perspective because it was impossible to achieve the same angle of specimen orientation (for example see

Figures. 4.4 a,g, and i). In both species the shape of the vestibulum is similar with slight individual variations. In general the vestibulum is almost quadrate in shape.

It expands at the base; the apical parts are slightly rounded and have denser hair than the other parts of the vestibulum. Slight variations of the shapes of the vestibulum are found across the colonies as well as within drones of a colony; no consistent differences between species were observed (Figure 4.4).

4.3.i.3 Cewix

The cervix starts behind the origin of the cornua and the ventral side of the vestibulum. The cetvix in situ is compressed in an irregular shape; when it is pulled it forms a thin tube which is twisted. Figure 4.3 A ventral view of the uneverted endophalli of A. nigrocincta and A. cerana. B = bulbus; L = fimbriate lobe; Cer = cervix; V = vestibulum. Sample collection sites: A. nigrocincta: Bontobulaeng, South Sulawesi; A. cerana: Jeneponto, South Sulawesi.

Figure 4.4 Variability in the shapes and the pattern of the most densely pubescent portion of the vestibulum of uneverted endophalli of A.cerana (a-f) and A. nigrocincfa (g-1). Specimen collection sites: a: Kamarora; b: Tokyo; c: Tretes; d: Jeneponto; e: Riau; f: PM; g&h: Bontobulaeng; i&j: Manipi; k&l: Manado. The shape of the vestibulum is similar in both species with slight individual variations.

The dorsal side of the cervix has a large hairy patch which is similar in shape in both species. In addition, the dorsal side of the cervix bears a fimbriate lobe

(Figure 4.1) which has a rosette shape, both in A. cerana and A. nigrocincta. The fimbriate lobe has papillae, the number of which cannot be counted accurately in uneverted endophalli.

The ventral side of the cervix has approxirnately the same number of folds

(A. cerana, = 6.2; A. nigrocincta, = 6.1; P>0.2) (Table 4.2; Figure 4.3). The cervical folds of both A. cerana and A. nigrocincfa have trapezoidal shapes. The apical part of these folds is relatively flat and slightly rounded at the corner. The apex of some of these folds carries hairs. The shapes of these folds appear slightly different for A. cerana and A. nigrocincta in Figure 4.3, because they were twisted and the orientation of the specimens was not the same when they were drawn.

4.3.2 Everted Endophalli

Everted endophalli of A. nigrocincta (n=13) and A. cerana (n=54) were compared. Since some of them were not fully everted, a comparison of the number of papillae of the fimbriate lobes could not be made for al1 drones.

4.3.2.1 Vestibulum

The tubular projections of the ventral cornua of both species extended posten'orly and then recurved ventrally. The shapes and the manner in whict-i ventral cornua projected are slightly different in A. nigrocincta and A. cerana (Figures 4.5, 4.6, and 4.7). In Apis nigrocincta they tend to be ionger, more slender and straighter with only the tip recurved.

The dorsal cornua of both A. cerana and A. nigrocincta are usually trifid, forming three tubular projections. These projections are much shorter than the ventral cornua. They are about one third and one fifth the length of the ventral cornua in A. cerana and A. nigmcincta respectively (Figures. 4.5,4.6, and 4.7). In some cases, the dorsal cornua are bifid, forming two tubular projections, the basal projection usually with a short Iateral nodule or a shorter projection. The dorsal cornua of A. nigrocincta are shorter and more rounded than those of A. cerana. No differences were found in the way the dorsal cornua evert: one pair was angled fotward, one pair backward, and one pair laterally (Figures. 4.5, 4.6, and 4.7). The number of dorsal cornua of both species were found to be the same (P>0.5): A. cerana = 3.0 and A. nigrocincta = 2.9 (Table 4.2).

4.3.2.2 Cervix

Upon full eversion, the pressure from the haemolymph causes the cewix to assume a consistent shape. Cervical rotation and twisting that influenced depiction the uneverted endophalli are eliminated (Figures 4.5 and 4.6). The ventral folds appear as small bumps. There are fewer of these bumps than there are folds in the uneverted endophalli. These reductions might result from the extension of the cervix.

Figure 4.7 resulted from tracing photographs. In these figures, the first fold Figure 4.5 Everted endophalli of A. cerana (top) and A. nigrocinda (bottom). B = bulbus; L = fimbriate lobe; vC = ventral cornua; dC = dorsal cornua; Cer = cervix; V = vestibulum.

Figure 4.6 Everted endophalli of A. nigrocincta and A. cerana . 8 = bulbus; L = fimbriate lobe; vC = ventral cornu; dC = dorsal cornu; Cer = cervix; V = vestibulum.

Figure 4.7 Ventral (vC) and dorsal cornua (dC) of everted endophalli of A. cerana (a-f) frorn Kamarora and A. nigrocincta (pl) from Rahmat. Cer = cervix; V = vestibulum. The dC of A. nigrocincta appear to be shorter and more rounded than those of A. cerana. The projections of the vC of A. nigrocincfa tend to be longer, more slender and straighter with only the tip recurved.

of the cervix of A. cerana and A. nigrocincta looks different, but observations on specimens of everted endophalli under a microscope failed to confirm any differences. These apparent differences rnay be caused by differences in the viewing angle when the photographs were taken.

No consistent differences were found in the structure of the fimbriate lobes of A. cerana and A. nigrocincta after eversion. Each is rosette-shaped. Each lobe consists of paired structures with the two halves slightly separated. The fimbriate lobes are apically globulose with distal papillae along the lateral border; the medial papillae are longest. The only slight differences may be in the shape and the length of the papillae. In A. nigrocincta the papillae look wider and shorter (Figure 4.8). In this figure, the base of the fimbriate lobe of A. cerana seems wider that those of A. nigrocincta, but when I measured specimens this apparent difference was not consistent, and the base of the fimbriate lobe of A. nigrocincta (n = 5) was sometimes wider than that of A. cerana (n = 16).

Differences in the number of the papillae for A. cerana (x = 18.5) and A. nigmcincta (x = 21.4) (Table 4.2) were not significant (P~0.08);however, sample size for A. nigrocincta was small (n = 5). Figure 4.8 Everted fimbriate lobes of two specimens of A. nigrocincta and of A. cerana (lateral view). A. nigrocincta A. cerana

1 Specimen 4.4 DISCUSSION

There are almost no differences in the structures of the male genitalia of A.

nigmcincta and A. cerana, with the exception of the shapes and the lengths of the

dorsal cornua and the recurvature of the tips of the ventral cornua in everted

endophalli. However, except for the shape of the dorsal cornua which are less

rounded and longer in A. cerana, even these differences were not always consistent

between the species. The suspected function of the dorsal and ventral cornua is to

prevent the endophalli from retracting after they have been everted (Simpson,

1970). Because the observed differences in the genitalia of these two species of

honey bees is very slight, I believe that there are no anatomical barriers to prevent

mating between these two species in Sulawesi. In addition, because these

differences in genital structure are minor and inconsistently expressed, alone they

are inadequate to justify the species status of A. nigrocincta. However, as

dernonstrated previously through observations of the timing of drone matlng flights

(Chapter 2; Hadisoesilo and Otis, 1996), Apis nigrocincta is a valid species.

My study has also shown that the endophalli of A. cerana drones from distant

sites within its vast range differ only slightly. The rninor differences in the shapes

and lengths of the dorsal cornua, for example, are almost as great among drones from a single colony as they are among drones from widely separated sites.

Surprisingly, mine is the first study that has specifically addressed the intraspecific variation in the structure of male genitalia in honey bees. That there were no locality specific differences found is considered in more detail below.

When one examines the male genitalia of the various species of cavity-

nesting honey bees (i.e., A. cerana, A. nigrocincta, A. koschevnikovi, A. mellifera),

one is struck with the remarkable similarity across taxa. The main differences

between species are in the number and shape of the dorsal cornua, the length of the cewix, the nurnber of folds of the cervix, the pattern of the dense hairy fields of the vestibulum, and the shape of the distal margin of the vestibulum (Koeniger et

al., 1991). The functions of these various parts of the endophallus during the

process of mating are not clear, except in the case of the hairy field of the ventral side of the vestibulum, which serves to rernove the mating sign in A. meIlifera

(Koeniger, 1986b), and the dorsal cornua which are believed to prevent the endophallus from disengaging from the queen during copulation (Simpson, 1970). lt is unlikely that these minor differences are sufficient to prevent interspecific mating between drones and queens of any of the cavity-nesting honey bees. In fact, it is already known that A. mellifera drones can mate with A. cerana queens

(Ruttner and Maul, 1983). These two species have the most different genitalia of al1 combinations of cavity-nesting species which further demonstrates that differences in mate genitalia by themselves are not a physical barrier for mating in this group of bee species (Subgenus Apis).

Most authors have tended to stress the differences rather than the similanties in genital structure of species within the genus Apis (Simpson, 1960, 1970; Ruttner

1988). In fact, when the genus was viewed as consisting of only four species, the di8erences between males of the subgenera Megapis, Micrapis, and Apis (Figure

1.1) were striking and suggest the operation of strong selective forces that lead to divergence in male endophalli. The rnost attractive explanation for these differences is the female choice hypothesis which suggests that the variability in male genitalia results from cryptic female choice among males on the basis of minor structural differences that alter the interna1 contact between male and female structures

(Eberhard, 1985). Divergence between non-interbreeding populations may occur rapidly according to this hypothesis. Eberhard (1985) suggested that complex and diverse endophalli should be found in those species in which females copulate more than once and have an opportunity to choose among competing males. A recent detailed analysis of endophallic structures in the Apoidea confirmed this general correlation between complex endophalli and polyandrous mating systems (Roig-

Alsina, 1993). Roig-Alsina concluded that his analysis supported the fernale-choice hypothesis.

A detailed look at the mating biology of honey bees raises difficulties with

Eberhard's (1985) hypothesis as an explanation of their diverse endophallic structures. When a queen encounters drones at a congregation area, she may be able to exercise some choice of mates before coupling with males, although there is presently no evidence that she actually does choose certain males over others.

However, once a male contacts the queen and she opens her sting chamber, the subsequent eversion is explosive and spem are driven into the queen's oviducts

(or directly into the spermatheca in the case of Micrapis; Koeniger et al., 1989). It is difficult to conceive of mechanisms by which the queen could subtly reject a

1O8 particular male during such a rapid and violent process. An alternative explanation, that the complex male structures found in species within this genus are a result of intrasexual selection for males to be more competitive in efficiently transferring spem, is more likely and could not be ruled out by Roig-Alsina (1993). According to the intrasexual selection hypothesis, males compete with each other to most effectively place their spem in the female genital tract, displace sperm from previous matings, or remove mating plugs of other males (Thornhill and Alcock.

1983).

With the rediscovery of several additional species of Apis (e.g., A. (Micrapis) andrenHomis, Wongsiri et al., 1990; A. (Megapis) laboriosa, Sakagami et al., 1980;

Underwood, 1990; A. (Apis) koschevnikovi, Koeniger et al., 1988; Tingek et al. 1988 and A. (Apis) nigrocincta, Hadisoesilo and Otis, 1996; Chapter 2), the similarity of male genitalia between the species within each subgenus becomes of interest, because bee researchers usually expect differences of male genitalia between species. This similarity demands a reassessment of various ideas concerning the evolution of male genitalia in Apis with respect to the rate of change in male genital structures.

Most studies concerning the phylogenetic relationships of the honey bees have obtained similar results, with a few notable exceptions (e.g., Willis et al.,

1992). The available evidence has been reassessed recently by Engel and Schultz

(1997), and a "total evidence" phylogenetic tree that is well supported has been identified (Figure 1.1). lndependent analyses of two DNA sequences also yields this sarne tree (W. S. Sheppard, pers. comm.). This tree has the Micrapis clade (florea,

andrenifomis) diverging first from the cornmon ancestor of the honey bees, followed

by the separation of the Megapis clade (dorsata group) from the cavity-nesting

subgenus Apis. This tree also agrees well with known behavioural evolution within

the genus (Engel and Schultz, 1997).

To estimate the rate of evolution of genital structures in the genus Apis, one

requires estirnates of the age of the nodes on the phylogenetic tree. Unfortunately,

obtaining accurate dates for these nodes is problematic because of the paucity of

fossilized honey bees (Culliney, 1983; Ruttner ,1988; Engel, submitted). There is

no agreement on when the Micrapis clade diverged from the Megapis-Apis clade:

estimates ranging from 27-30 rnya (Ruttner, 1988) to 10-12 rnya (Sheppard, pers.

comrn.), to 5-6mya (M. Engel, pers. comm.). Suggested dates for divergences of

other clades of Apis are either arbitrary (Ruttner, 1988) or based on DNA sequence

divergence, assurning 2% divergence in DNA sequences per million years as has

been estimated for Drosophiia (DeSalle et al., 1987). For example, the estimated

divergence of A. meliifera from other cavity-nesting honey bees ranges from 1-2

mya (Ruttner, 1988; M. Engel. pers. comm.) to 8-9 rnya (W.S. Sheppard, pers.

comm.). Subsequent divergence within the clades represented by each of the three

subgenera is believed to have occurred relatively recently, within 5-6 mya (W.S.

Sheppard, pers. comm.) or more recently (M. Engel, pers. comm.). No estimates are yet available concerning the recent speciation of A. nigrocincta from A. cerana.

Given the mating biology of honey bees, it is not surprising that there has been very little differentiation in genitalia within species such as A. cerana. In al1 species of Apis, drones congregate at specific sites known as drone congregation areas (DCAs) and queens are believed to mate at these sites (Koeniger, 1986a;

Punchihewa et al., 1990; Koeniger et al., 1994). The extensive outbreeding that

results from this mating system Ieads to a high degree of genetic mixing that tends to reduce differences between geographically contiguous populations. Even many populations on islands such as Bomeo, Java, Sumatra, Taiwan, the Japanese

Islands, and Sri Lanka have been only intermittently isolated from each other as a result of fluctuations in sea fevels during Pleistocene epoch (Heaney, 1986;

Whitmore, 1987).

The most surprising result of my review of genitalic diversity in Apis is the remarkable dissimilarity in genitalia between subgenera compared to general similarity within each subgenus, particularly in view of the differences between researchers in the estimated ages of each clade (Figure 4.9). If Ruttner's assessment is correct, the major differences between subgenera may reflect the slow accumulation of differences over the long evolutionary history of these taxa, the relatively minor differences between species result from the more recent divergence that has occurred within each subgenus. However, if Sheppard is correct, then one must hypothesize variable rates of evolutionary change, with more rapid evolution of genitalia initially after divergence of the subgenera than has occurred more recently. Finally, if Engel, is correct, the entire divergence within the genus occurred very quickly, and a mechanism must be sought to explain the exceptionally rapid divergence of genital structures in a group to which the female

111 Figure 4.9 Estimated divergence within the genus Apis based on the fossil records and the ability of queens of A. cerana to mate with drones of A. mellifera (Ruttner, 1988). DNA sequences divergence (W.S.Sheppard, pers. comm.), geological records and the branching pattern of taxa (M. Engel, pers. comm.) choice hypothesis (Eberhard, 1985) does not seem to apply (see above).

Unfortunately, there is no way to resolve these d iscrepancies without additional fossils or better "molecular clocks".

My companson of A. nigrocincta and A. cerana indicates that the genitalia of males are highly similar. Morphometric analyses of workers, however, indicate that

A. nigrocincta of Sulawesi is morphologically distinct from, but similar to, A. cerana

(Damus, 1995; Hadisoesilo et al., 1995; Damus and Otis, submitted). Furthemore.

A. nigrocincta clusters with one (Philippine) of three groups of A. cerana based on nucleotide sequences of the COI-COI1 intergenic region of mitochondrial DNA

(Smith and Hagen, 1996). Collectively, these observations and data sets suggest that A. nigrocincta evolved from A. cerana and attained its species status relatively recently.

The discovery of two Apis species that are behaviourally distinct (see

Chapter 2, timing of mating flights) but othennrise almost indistinguishable offers a rare opportunity to gain insight into some aspects of the speciation process in honey bees. Two important, albeit tentative, conclusions emerge from the comparison of male genitalia of A. cerana and A. nigrocincta. First, it appears that behavioural differences are more important than morphological differences in the speciation of honey bees. Secondly, althoug h distinctiveness of male genitalia frequently provide clear proof of species status in many insects, the converse situation of absence of genital differences does not always demonstrate that two populations are conspecific. My conclusions do not differ from most current views of speciation (Thornhill and Alcock, 1983; Simon, 1987). However, they do demonstrate that the previous emphasis by honey bee researchers on genital differences as species specific characters is not justified and may have obscured the recognition of additional species based on behavioural differences. CHAPTER 5

OBSERVATIONS ON MATING BY QUEENS OF Apis nigrocincta AND Apis cerana IN ZONES WlTH ALLOSPECIFIC DRONES

5.1 INTRODUCTlON

When a single, interbreeding population of organisms becomes split into or more geographically disjunct populations, character divergence often occurs.

Differences between the populations arise as a result of mutations and recombinations, different selective forces, and stochastic factors such as population bottlenecks and founder effects. This process is the basis of Mayr's model for allopatric speciation (Mayr,1 963,1964). If these divergent populations subsequently corne into secondary contact with one another, there are several possible outcomes

(Raven and Johnson, 1986):

1. Cross matings between individuals of these populations are equally as

successful as intrapopulationai matings and normal offspring are produced.

In this case, no mating barriers have evolved during the time of separation

and these two populations are considered as belonging to one species.

Differences tend to disappear over time if selective forces are sirnilar over

the range of the species.

2. No interpopulational matings take place and no intermediate foms are

found. Mating barrÏer(s) have evolved during the time of separation and the two populations have become distinct species.

3. The two populations have diverged, but the speciation process is

incomplete and interpopulational matings are still possible because mating

barriers are weak or absent. When such mating does occur, there may be

barriers that prevent fertilization or the development of zygotes into normal,

functional, and fertile individuals. For exarnple in insects, successful matings

do not guarantee that the female lays eggs. If she does lay eggs, the eggs

may not hatch or larvae may die before they reach the adult stage. If they do

reach the adult stage, the adults may either be sten'le, or fertile but with

reduced fitness.

Two similar honey bee species, Apis nigrocincta and A. cerana, inhabit the island of Sulawesi. Apis nigrocincta is distributed throughout most of Sulawesi (Otis,

1996), although A. cerana so far is only known to occur in the most ex-treme southern part of Sulawesi and in part of central Sulawesi (Chapter 3). It is likely that

A. nigrocincta and A. cerana evolved allopatrically and only relatively recently became sympatric, possibly through natural dispersa1 of A. cerana to Sulawesi or recent importations of bees for beekeeping projects (from Java to Sulawesi)

(Damus, 1995).

Apis nigrocincta and A. cerana appear to be two distinct species as reflected by the segregation of the timing of drone mating flights (Chapter 2). If hybridization does occur, it is severely limited as evidenced by the small number of intermediate forms collected in one zone of sympatry at the Palolo Valley, Central Sulawesi (Chapter 3). However, no diagnostic morphological characters have been observed

(M. Engel, pers comm.) and the male genitalia are almost identical (see Chapter 4).

This general overall structural similarity suggests that A. nigrocincta and A. cerana are closely related and the divergence into two distinct species happened only recently.

The objective of my study was to attempt to get queens of A. nigrocincta to mate naturally with drones of A. cemna and vice-versa. Because instrumental inseminations could not be perfomed, the study was done by allowing queens of

A. nigrocincta and A. cerana the opportunity to mate in zones with only allospecific drones. 5.2 MATERIALS AND METHODS

This observational study consisted of four cases as described below.

5.2.1 Case 1

One comb of A. nigrocincta from a colony maintained in Palangisang containing a lot of worker brood of al1 ages was brought to Balong, -3 km from

Palangisang, Kecamatan Ujung Bulu, Kabupaten Bulukumba, South Sulawesi

Province on 11 May, 1995. The colony of A. nigrocincta from which the brood was obtained did not have any adult drones or drone brood. No other hived or feral colonies of A. nigrocincta were known to occur in the vicinity of Palangisang and

Balong. There were many mature drones of A. cerana present in these villages that took mating flights during their specific mating tirnes (see Chapter 2). The comb of

A. nigrocincta was placed inside a hive body above the single brood chamber of a populous colony of A. cerana which also had many mature drones. The queen was removed from this colony of A. cerana before having the comb of A. nigrocincta added to it. When the colony was checked on 14 May, one queen cell of A. nigrocincta was left intac while additional queen cells were removed. On 18 May, the single queen cell was capped. The colony was checked again on 27and 31 May and 03, 13, 20 , and 27 June 1995, for the presence or absence of a queen and eggs-

In this case and the other cases that follow, the specific identity of the queens was determined by checking the colour of their hind femora. The queen of

A. cerana had yellowish hind femora, while the queens of A. nigrocincta were blackish. The presence of either queen or worker laid eggs were detemined by the position of the eggs in cells and by the number of eggs in a cell. Eggs produced by a queen bee are laid neatly at the bottom of cells with only one egg in each cell. On the other hand , workers' eggs are placed on the sidewalls of the cells or at one side of the cell base and they are usually many eggs in each cell (Morse and Hoper,

1985; Seeley, 1 985; Gary, 1989).

5.2.2 Case 2

Five queenless colonies of A. cerana were given queen cells of A. nigrocincta as foliows. A colony of A. nigrocincta maintained in Palangisang was divided into two units using a wooden division board. These two units were checked every day, and when the cap of one of the queen cells in the queenless unit had changed to dark brown indicating that the virgin queen was nearly ready to emerge, l removed it along with a small piece of the comb (Figure 5.1) and put it in a colony of A. cerana which had been queenless for 1-2 d. The queen cell was inserted between two frames in the centre of the hive, and the frames were moved together slowly so that the queen ceIl was securely held in place (Figures 5.2 and 5.3). The colony was checked carefully every day for the presence of naturally built queen cups/cells which, if present, were destroyed. When the virgin queen had emerged 5-6 d after Figure 5.1 A capped queen cell cut along with a small piece of the comb. Note the colour of the cap, dark brown, indicating that the virgin queen was ready to emerge.

Figure 5.2 A capped queen cell inserted between two frames

Figure 5.3 A capped queen cell securely held in place between two frames. transplanting (Figure 5.4), 1 checked for the presence of the queen of A. nigrocincta and again removed any queen cells/cups in the original colony of A. cerana. If the queen could not be found, another queen cell was introduced immediately. For controls, four capped queen cells of A. cerana at the same state of development were transplanted into four dequeened colonies of A. cerana. In total, nine colonies of A. cerana were made queenless; five were given queen cells of A. nigrocincta and four received queen cells of A. cerana. All nine colonies were moved to

Jeneponto, a zone containing only A. cerana, about 70 km WSW of Palangisang

(see Chapter 3 for detailed location) on 24 August 1995. Before moving the colonies, three colonies had virg in queens of A. nigrocincta, two colonies were queenless with sealed queen cells of A. nigrocincta, two colonies had young adult queens of A. cerana, and two colonies were queenless with sealed queen cells of

A. cerana.

Upon arriva1 in Jeneponto, the hive entrances were opened to allow free flight and the colonies were checked for the presence of the virgin queens. Only two queens of A. nigrocincta and two queens of A. cerana were found. On 25 August. colonies were checked again. No additional queens had emerged. All colonies were checked again on 26 August. The two queens of A. nigrocincta and two queens of

A. cerana which had already emerged by 25 August were found but no eggs were yet present. The other queens of A. cerana had not emerged yet. Figure 5.4 The opening of a queen cell indicating that the queen had emerged.

Capped queen cells of A. nigrocincta, about 5-8 d after capping, were transported from Palangisang and introduced to the three queenless colonies on 27

August 1995. Before introducing the capped queen cells of A. nigrocincta, Ichecked colonies for the presence of queen cellslcups of A. cerana. Because the two colonies lacking virgin queens of A. cerana still had capped queen cells, no further manipulations of queen cells were undertaken. All colonies were checked again on

2 and 14 September 1995 for the presence of queens and eggs.

5.2.3 Case 3

This case was carried out in Palangisang. Only one colony of A. nigrocincta which had many mature drones was present in this village, and no wild colonies of this species were found. Ten hived colonies plus many wild colonies of A. cerana with numerous mature drones were present in this village.

On 23 August 1995, one capped queen cell of A. nigrocincta was cut and transplanted into a queenless division of a colony of A. cerana. Before transplanting, al1 queen cups/cells of A. cerana were destroyed, as explained in detail for case 2. Two days after transplanting the queen cell, the colony was checked for the presence of the queen, and on subsequent dates ( 03 September;

14 and 22 October) for the development of the colony. Between 03 September and

14 October I was unable to check this colony. It was planned that the colony would be checked again on 29 October 1995 but the colony absconded before that date apparently because it had became heavily infested by wax moths.

5.3 RESULTS

5.3.1 Case 1

Table 5.1 summarizes the observations relating to the queen of A. nigrocincta in an area with only drones of A. cerana present. The queen was still present but had not laid eggs by 24 d after her ernergence. The workers of A. cerana bees began to lay eggs, as determined by the presence of more than one egg in each cell and the position of the eggs in the cells (see section 2.1),between day 17-24. Unfortunately, this queen disappeared before I could check her spermatheca for the presence of sperm. Table 5.1 The presence or absence of the queen and eggs in the colony of A. cerana allowed to rear a queen cell of A. nigrocincta at Balong, South Sulawesi. + means present; - signifies absent. Days were counted starting on the date when the emerged virgin queen was first observed.

Date Day Queen Queen's eggs Workers' eggs (+/-) (+N (+Q 5.3.2 Case 2

The fates of several virgin queens of A. cerana and A. nigmcincta in colonies of A. cerana were determined. The complete results for this set of observations are presented in Table 5.2A and 5.28. Up to 10-11 d after their emergence, tvvo queens of A. nigrocincta (BPK-08 and -10) still had not laid eggs. By 12 d later they had disappeared and no eggs had been laid. The other three queens also disappeared about 9-13 d after their emergence without laying eggs (Table 5.2A).

One colony of A. cerana (Black-2) into which a queen cell of A. cerana was transplanted absconded before the queen laid eggs. One queen of A. cerana

(Black-3) had begun to lay eggs between 12-24 d after she emerged and another queen of A. cerana (Black-4) had laid eggs between 11-12 d after emerging.

Unfortunately the fourth queen (Black-1) disappeared and the workers laid eggs

(Table 28). Table 5.2A The presence or absence of queens and eggs of A. nigrocincta in colonies of A. cerana at Jeneponto, South Sulawesi. + means present; - signifies absent. Q = queen; W = workers.

Date Queen 02109/1 995 14 10911 995 Col. # emewd Q Q W Days after Q W Days after eggs eggs Queen Q eggs eggs Queen emerged emerged BPK-10 22/08/1995 + - - 11 - + 23

BPK-08 23/08/1995 + - 10 - - 4 22 BPK-O1 Between + - - 1-5 - - + 9-13

BPK-05 28/08 and + - - 1-5 - - + 9 - 13 1-5 9-13 BPK-09 01/09/1995 + - - - - +

Table 5.2B The presence or absence of queens and eggs of A. cerana in colonies of A. cerana, at Jeneponto, South Sulawesi. + means present; - signifies absent. Q = queen; W = workers.

Col. # Date Queen Q Q W Days after Q Q W Days after emerged eggs eggs Queen eggs eggs Queen emerged emerged Black-2 21/O811 995 + - - 12 - - - absconded

Black-1 Between + - - 1-3 - - + 13- 15 30108 and Ol/O9/I995 Between Black-4 02 afid - 03/09/1995 5.3.3 Case 3

A capped queen cell of A. nigrocincta was introduced to a division of a colony of A. cerana in a region with drones of both species on 23 August, 1995. One colony of A. nigrocincta with numerous drones was present at this site at this time.

Nine days after the queen of A. nigrocilcta emerged she had already mated and laid eggs (Table 5.3). Fifty three days after the queen emerged, I found both workers of A. nigrocincta and A. cerana mixed in this colony. The species identity of the workers was checked by looking at the hind femora (see Chapter 3). Table 5.3 The presence or absence of the queen and eggs in the colony of A. cerana allowed to rear a queen cell of A. nigrocinda at Palangisang, South Sulawesi. + means present; - signifies absent. Days were counted starting on the date when the emerged virgin queen was first observed. C = A. cerana; N = A. nigro cinefa.

Date Day Queen Queen's eggs Workefs eggs Worker (+4 (+Q (+4 (Cor N) 25/08/1995 O + - - C

14110/'l 995 50 + + - C and N

22/10/1995 58 i- + - C and N 5.3.4 Case 4

Queens of A. cerana were allowed the opportunity to mate in a site where only colonies of A. nigrocincta exist. Obsenrations are summarized in Table 5.3.

Two colonies absconded before the queens of A. cerana laid eggs. Two other queens disappeared before laying eggs. The last time they were observed they were 11-14 d old yet they had not laid eggs. The fifth queen still had not laid eggs by the time she was at least 24 d old. Because I was away to locate and sample colonies at Central Sulawesi, I could not check the spermatheca for the presence of spem. Therefore, I could not confirm if she remained unmated. Table 5.4 The presence and absence of queens and eggs in queenless colonies of A. cerana moved to Manipi, South Sulawesi. + signifies present; - means absence. Q = queen; W = workers.

Colony # Date OU1 111995 1211lM995 queen emerged Q W Days after Q Q W Days afl eggs eggs queen eg9s eggs queen emerged emerge Absconded

Between - - - - i - - 1-4 08 and 1111 11 - - - + d. - 1-4 1995 - - - - + - - 1-4

-- -- Colony # 2Z1if1 995 0511Zig95 . . .- Q Q W eggs Days after Q Q W Days afti eggs queen Wgs e9W queen emerged emergei 5.4 DISCUSSION

Under normal conditions (i-e., conspecific drones present, weather conditions

favourable for mating) a queen of A. cerana indica will usually lay eggs shortly after

emergence (6-10 dl Sharma, 4960; 5-8 dl Adlakha, 1971; 6-8 d, Shah and Shah,

1980; 8-12 dl Verma et al., 1990). A queen of A.c. cerana in Thailand, usually lays

eggs 2-18 d after emergence (Wongsiri, 1995). In my study, when conspecific

drones were present, two queens of A. cerana (Case 2; Black-3 and Black-4)

started laying eggs at ages of 11-12 d and 12-24 d, postemergence, respectively.

In Case 3, a queen of A. nigrocincta initiated oviposition only 9 d after she emerged.

Even though this queen was in a colony of A. cerana, mature drones of A.

nigrocincta were availabte for mating in the area.

My study, although not conclusive, suggests that if conspecific drones are not

present, queens do not mate and lay eggs (Cases 1,2,4) even in the presence of

numerous allospecific drones. One queen of A. cerana (Case 4) and one queen of

A. nigrocincta (Case 1) persisted in colonies for at least 24 d after they emerged with no evidence that they had started to oviposit; it is Iikely they failed to mate.

One possible explanation for these results is that these queens could not mate because they failed to encounter drones from the other species because they take their mating flights at different times (see Chapter 2). The timing of mating flights of the queens is probably independent of the colony in which she resides, as suggested by data on crossfostered queens of A. koschevnikovi(in colonies of A. cerana) which took their mating flights at the same tirne of day as in their

conspecific colonies (Koeniger et al., 1996b). Crossfostered queens (i.e., queen of

A. nigrocincta in a colony of A. cerana) are capable of flying and mating (Case 3).

In addition to differences in timing of mating, there may be species specific mating

locations (Le., DCAs) that contribute to the ethological separation of these species.

Another possibility is that these queens may have mated successfully but when they laid eggs, the workers may have eaten them. This could explain results obtained in Cases 1 and 2, in which the queens of A. nigrocincta were present up to 24 and 10-1 1 d after they ernerged in colonies of A. cerana but no queen laid eggs. Moreover, in case 4, two queens of A. cerana in an area with only drones of

A. nigrocincta were still present at least 10 and 24 d after they emerged but. no queen eggs were found.

In a study in Punjab, India, rejection of uncapped worker brood of A. cerana by workers of A. mellifera and vice-versa was observed by Dhaliwal and Atwal (

1970). All eggs and larvae of A. cerana in the comb were removed by workers of

A.mellifera within 2 d. The workers of A. cerana removed al1 eggs and larvae of

Amellifera on the third day of the comb insertion. This was unlikely to have happened in my study because as seen in Case 3, the brood and resulting workers of A. nigrocincta that emerged in a colony of A. cerana were accepted by workers of A. cerana for over a period of 41 d.

A third possibility is that crossfostered queens actually did fly and encountered drones of the other species. mated interspecifically, but failed to lay eggs. Because I did not observe the crossfostered queens taking their rnating flights and returning with mating signs (Woyke and Ruttner, 1958; Ruttner et al.

1972,1973), 1 could not prove this possibility .

Given the evidence, I believe the first explanatbn, that interspecific mating did not occur, to be the most Iikely, although 1 could not prove this since I did not check the sperrnathecae of the queens for the presence of sperm. The results of these cases are not conclusive but are consistent with the idea that there are barriers (probably premating, such as time and location of rnating, but possibly inviability of hybrids as well) which prevent the interspecific matings between A. cerana and A. nigrocincta.

In A. cerana, the queen usually mates when she is quite young (4-8 d,

Shamia, 1960; 3-5 cf, Adlakha, 1971; 2-6 d, Shah and Shah, 1980; 5-8 d, Vemia et ai., 7990). She is usually eliminated from the colony by the workers when more than

25 d old if she has not mated (Sharma, 1960). In A. rneliifera, workers act aggressively toward their queen untii she mates (Hamman, 1957). According to

Koeniger et ai. (1996b), aggressive behaviours of workers of A. cerana and koschevnikovi toward allospecific queens increased with the age of the virgin queen.

At 3 or 4 d old, the queens were balled, rnutilated, and finally expelted from the colony. The loss of most of the older virgin queens in rny study resulted from worker aggression. However, the possibility remains that these queens disap peared during mating flights.

In this study, although a virgin queen of A. nigmcincta was present for a relatively long period in a colony of Axerana (Case 1) and similarly a virgin queen

of A. cerana persisted in a colony of A. cerana (Case 4), they did not inhibit workers from laying eggs. In contrast, when a crossfostered queen of A. nigrocincta in a

colony of Axerana did successfully mate and worker brood was reared, laying workers did not appear. This phenomenon is likely to be explained by the absence of brood as suggested by Jay (1970,1972). He demonstrated that a volatile brood pheromone produced by worker tarvae or pupae inhibits ovarian development of worker bees. His cases are supported by Willis and Winston (1990) and Winston and Slessor (1992) who mentioned that the five component blend of the queen mandibular pheromone of A. mellifera (9-ODA, 9-HDA, 10-HDA, HOB, and HVA) by itself does not inhibit the ovarian development of workers. Plettner et al. (in press) found that in A. cerana the amounts of 9-ODA and other components of mandibular glands were similar in virgin and mated queens. In fact, Crewe (1982) rnentioned that in three subspecies of A. mellifera (capensis, adansonii, and mellifera), laying workers developed most rapidly in the subspecies in which the queen mandibular glands contained the greatest quantity of 9-ODA. The results of my study fit the other research that indicates that queen mandibular pheromone by itself cannot prevent workers from laying eggs and that other pheromone(s), probably substances produced by larval and pupal bees, are involved in the suppression of ovarian development as suggested by Winston and Slessor (1992).

My study also suggests that the queen of A. nigrocincta would be accepted by workers of A. cerana as long as no conspecific queen cells are present; otherwise she would be balledlkilled by the alien workers (pers. obs.). Because

these two species are closely related, the complex of queen pherornones is

probably rather similar but remains to be determined. The rejection of queens of A.

nigrocincta when queen cells of A. cerana were present was probably because the workers of A. cerana prefer their own queen rather than an alien queen (Breed,

1985). This was also observed by Koeniger et al. (1996b) for A. cerana and A. koschevniko vi.

In summary, my study demonstrated that queen cells of A. cerana and A. nigrocincta and the virgin queens that emerge frorn them are accepted by colonies of the other species, but alien queens are not accepted if conspecific queen cells are present. When conspecific drones are available for mating, surviving queens mate and begin egg laying in a relatively short period of time. However, when only allospecific drones are present, there is no evidence that queens mated. These cases further support the species status of Apis nigrocincta as distinct from A. cerana. CHAPTER 6

DIFFERENCES IN DRONE CAPPING BEHAVIOUR OF Apis cerana AND Apis nigrocincta

6.1 INTRODUCTION

In al1 honey bee species, the drone celt containing a fifth instar lama is capped by worker bees with wax, folIowing which the drone spins a pupal cocoon.

In A. cerana, this cocoon includes a hard yellawish conical "cap". One to 3 d after the cell is sealed, the worker bees remove the waxy cell cover and the hard cap becomes visible (Sakagami, 1960; Hanel and Ruttner, 1985). At the centre of the hard cap lies a pore. This pore is funnel-like, measuring 0.4 mm in depth and 0.25-

0.5 mm in diameter, and is proposed to result from the local dissolution of the wcoon from inside, as in Bombyx mon or other insects, while the wax cover is still intact (Hanel and Ruttner, 1985). This characteristic of the caps of drone cells is evident throughout the range of A. cerana (reviewed by Sakagami, 1960, and

Ruttner, 1988). The pore is absent in A. mellfera, but is found in the drone cell caps of A. koschevniicovi, a species more closely related to A. cerana (Alexander,

1991a,b). So far, there is no published information on the presence or absence of this pore in the recently described species A. nigrocincfa or A. nuluensis (Koeniger et al., 1996a; Tingek et al., 1996).

In my studies of the previously unrecognized cavity-nesting honey bee, A. nigrocincfa,1 casually observed that drone brood cells lacked the pore typical of the cappings of drones of A. cerana. To confirm this observation, I studied the nature

of drone cell caps of A. nigrocincfa and A. cerana in Sulawesi. I also review the

proposed function of the pore. 6.2 MATERIALS AND METHODS

This study was conducted in Mo locations in South Sulawesi (Palangisang and Bontobulaeng) and in three locations in Central Sulawesi (Kamarora, Rahmat, and Bobo). To quantify the absence or presence of pores in capped drone cells, I counted the number of capped drone cells which had visible pores. If the pore was not visible, I removed the wax cap from the cell gently untit the brood inside the cell or the underlying cocoon pore was visible. To make sure that 1 counted drone brood that had developed to the stage that the pore, if present, would be visible, I included only cells in which the compound eyes of the drone pupa were cleariy visible when the cell was completely opened (Figure 6.7 €3). Figure 6.IA Drone cells of A. cerana; sealed cells have distinctive pores in the outrnost portion of the cocoon cell caps.

Figure 6.1B Drone cells of A. nigrocincfa ; sealed cells lack pores in the cet1 caps. The wax caps have been rernoved from three cells to expose the drone pupae and demonstrate the absence of the hard cocoon with pore.

6.3 RESULTS

In total, 515 drone cells from five colonies of A. cerana and 198 drone cells from five colonies of A. nigrocincta were examined. Sealed drone cells of these two species are shown in Figures 6.1A and B. All drone cell caps of A. nigrocincta lacked pores, whereas al1 drone cell caps of A. cerana had pores (Table 6.1). The drone brood of A. nigrocincta lacked a hard cocoon structure. Only the fragile wax capping was present, even in cells with mature drones ready to emerge as adults.

In contrast, the drone pupal cap of A. cerana was always hardened with a pore in its centre. In addition, on the bottom board of A. cerana hives I always found an accumulation of drone cell caps in colonies actively rearing drone brood (Figure

6.2). Hard cell caps were absent on the bottom board of the hives of A. nigrocincta. Table 6.1 Number of cell caps with and without pores observed on capped drone cells of A. cemna and A. nigmcincta in Sulawesi, Indonesia.

Species Location Date # With pores Without pores

A. cerana Col. # 1 Kamarora Col. # 2 Kamarora Col. # 3 Kamarora Col. # 4 Kamarora Col. # 5 Rahmat Total

A. nigrocincta Col. # 1 Bobo Col. # 2 Palangisang Col. # 3 Bontobulaeng Col. # 4 Rahmat Col. # 5 Bobo Total Figure 6.2 The bottom board of the hive of A. cerana with an accumulation of drone cell caps.

6.4 DISCUSSION

This study demonstrated that the caps of drone brood cells of A. nigrocincta are strikingly different from those of A. cerana. In A. cerana, a thin wax cap is added by workers, then removed a few days later to expose the hard cap of the pupal cocoon with a pore in the centre. Apis nigrocincta also seals its drone cells with wax. However, this thin wax capping remains intact until the drone emerges as an adult, and no hard cocoon structure or pore is present.

The pore on the drone cell cap of A. cerana was first noticed by E. Jacobson

(reviewed by Hanel and Ruttner, 1985). The development of this pore was discussed by Hanel and Ruttner (1985), but its biological significance was not clear to them. However, they did not agree with the idea that the pore enhances the ventilation of the pupa necessitated by the hot temperatures experienced by tropical

A. cerana, because populations of this species in regions with cold, temperate climates also exhibit pores in drone cappings.

More recently, Rath (1992a, b) hypothesized that the dense microstructure of the thickened drone cell cap prevents the exchange of CO, and Q gases between the ceIl and the hive environment. According to Rath, the pore of the cap consequently serves as a device to improve gas exchange to the interior of the drone cell. His argument is based on the results of his study in which he experimentally blocked the pores with wax. Most drones did not emerge from the cells when the pores were plugged. Metamorphosis ceased or was retarded in 84% of the treated cells.

Rath's findings (1992a,b) fail to address the associated question: Why does

only the drone brood of A. cerana and A. koschevnikovi (and possibly A. nuluensis)

secrete such hard pupal caps but al1 other species of Apis, including the closely

related A. nigrocincta, lack this structure? Rath (1 992a,b) has further suggested that the hard drone cell cap plays an important role in protecting drone brood from worker bees. His hypothesis is that drone brood of A. cerana (and probably A. koschevnikovias well) emits insufficient q uantities of brood pheromones. This drone brood is consequently not accepted by the workers which would kill it if it was not protected by the hard, thick cell caps. The hard cap protects the brood because the worker bees are not inclined to remove the caps (Rath and Drescher, 1990). There is a major problem with this suggestion. It is diffult to imagine a way in which the hard cell caps could evolve because by this scenario, drone brood that produces insufficient amounts of pheromone would have to precede the appearance of the hard pupal caps. However, because this same drone brood would be detected by workers and probably removed (= killed), drones carrying the genes for the hard cap would only very infrequently survive and mate successfully. The trait would have little or no opportunity to increase in frequency. If Rath's hypothesis is valid, it is curious that drone brood of A. nigrocincta, a species very similar in most respects and closely related to A. cerana, exhibits such a striking difference with respect to this structure, especially because A. nigrocincta likely evolved from A. cerana in the

Philippines (Damus, 1995; Smith and Hagen, 1986). Another possible explanation for the hardened drone cell cap may relate to

parasitism by mites. Varna jacobsoni is found virtually throughout the range of its

host A. cerana and is believed to have coevolved with it over a long period of time.

A similar species, V. rindereri, infests drone cells of A. koschevnikovi (de Guzman

and Delfinado-Baker, 1996). In both bee species, the mites reproduce exclusively on the drone brood (Koeniger ef al., 1981, 1983; de Guzman and Delfinado-Baker,

1996; but see De Jong, 1988). In A. cerana, sealed worker brood that is experimentally infested with mites is removed by worker bees, a process that results in the death of the pupae (Rath and Drescher, 1990). Presumably similar behaviour rnay have occurred with infested drone brood at one point. In that case, drones that could better protect themselves from removal by workers would have had a selective advantage, even if that same trait resulted in the persistence of

Varroa mites. The thickened, hard cap of the drone ceII may have been such a trait.

Drone brood that produced this pupal structure would have been at a selective advantage with respect to worker removal behaviour, while being at a lesser selective disadvantage from the minor effects of mite parasitism. The net balance between these two opposing forces would have favoured the evolution of the hard cap. Drones, being haploid, would have transrnitted that trait upon mating and it would have increased in frequency in the population. The pore, being too narrow for the entry of mites to the cell, would then have had a clear function in enhancing gas exchange (Rath 1992a,b). This hypothesis for the evolution of the hard cell cap is similar to Rath's except for the underlying reasons for drone brood removal. Why does the drone brood of A. nigrocincta lack the hard ceIl cap with the pore? Both V. jacobsoni and V. underwoodi have been found inside the capped drone cells of A. nigrocincta (pers. obs.). One possibility is that the mites may have been introduced recently to Sulawesi with colonies of A. cerana from Java or elsewhere. This seems unlikely because the population of A. cerana in South

Sulawesi represents a unique endemic subspecies (Damus, 1995; Damus and Otis, submitted), suggesting that it has existed in Sulawesi for a long time. Moreover, the host-parasite relationship between A. nigrocincta and its Vanoa parasites seems to be in balance: the mites usually occur at low frequencies and worker brood is not infested. This contrasts with situations in which mites have only recently corne into contact with novel bee hosts, such as occur between Van-oa and A. meliifera, and the mites rapidly kill the bee colony (De Jong, 1990).

Another possibility is that the Varna colonizes drone brood of A. nigrocincta. but there are physiological factors that limit or prevent the reproduction of the mites on this bee species. I have not yet confirmed the reproduction of either V. jacobsoni or V. underwoodi on drone pupae of A. nigrocincta. In this scenario, these mites may be reproducing only on A. cerana and their appearance in colonies of A. nigmcincfa may resuit from contact between adults of the two bee species or they rnay be distributed by other flower-visiting insects such as wasps (Kevan et al.,

1990). The only observation that suggests this explanation is not tenable is my collection of V. jacobsoni in Manipi, South Sulawesi, a site well removed (e.g., >25 km) from any known colonies of A. cerana. Verification of Van-oa reproduction on A. nigrocincta drone brood will eliminate this as a potential explanation.

Whatever the underlying biological basis of the hard pupal cap in A. cerana, the curious absence of this structure in A. nigrocincta presently Jacks an adequate explanation. Studies to confimi the reproduction of Varroa mites on A. nigrocincta drone brood and of the behaviour of A. nigrocincta workers toward experimentally mite-infested drone and worker brood will eliminate some of the hypotheses discussed here. CHAPTER 7

VARIOUS SPECIES CONCEPTS AS THEY APPLY TO HONEY BEES (GENUS Apis)

7.1 INTRODUCTION

The diversity of life is a spectacular attribute of the planet Earth.

Generalizations about diversity are possible only when we have a good taxonomic foundation on which to speculate (Blackwelder, 1967; Mayr, 1976). Classification forms the framework of this work by grouping organisms on the basis of their similarities in an effort to determine their true relationships, but also provides a system by which we can keep track of the of facts discovered about each of the kinds (Blackwelder, 1967).

it is almost universally accepted by biologist that the species is a fundamental and natural unit of classification. lnspite of this general acceptance, there is still no agreement on exactly what a species is or how species should be defined (Ridley, 1993). The term species is frequently used to define a group of similar organisms to which a name has been attached (Mayr, 1976). It is generally taken to be the primary taxonomic unit of biology. Buried within the term, however, are basic aspects of evolutionary theory (Cracraft, 1989). Different taxa, with their widely differing attributes and evolutionary histories, and different evolutionary questions require different species concepts (Endler, 1989); no species concept applies universally to al1 organisms (Cracraft, 1987, 1989). Consequently, it has been stated that, "a species concept can be evaluated only in terms of a particular goal or purpose" (Templeton, 1989). One definition is preferable to another onIy if it allows us to understand with precision what sets species apart (Cracraft, 1987;

1989).

The purpose of this chapter is to summarize several major species concepts as they apply to honey bees (genus Apis). Distinct forms, several of which are now recognized as distinct species, are evaluated with respect to different species concepts.

7.2 TYPOLOGICAL SPECIES CONCEPT

The Typological Species Concept (TSC) is the simplest of the species concepts. A species according this concept is "a different thing" (typological means

"kind of') (Mayr, 1976). A species is defined on the basis of rnorphological characters provided by the type specimen(s). A type specimen is the one designated to represent the species.

The basic concept of the TSC is that "every natural group of organisms, hence every species in classification, is believed to have an invariant or generalized or idealized pattern shared by al1 members of the group" (Simpson,

1961). Numerous terms have been given to these idealized patterns, including "archetype", "bauplan", and "morphotype" (Simpson, 1951). It is believed that al1 mernbers of a species share the same essential nature; they conform to the same type. Variation under this concept is considered a trivial and irrelevant phenornenon

(Mayr, 1976).

The TSC was accepted by taxonornists almost unanimously including the acceptance of four postulates (Mayr and Ashlock, 1991):

1. Species consist of similar individuals sharing the same essences.

2. Each species is separated frorn al1 others by sharp discontinuities.

3. Each species is constant through time.

4. There are strict limits to the possible variation within any one species.

This concept led to arbitrariness in taxonomy because there were no objective methods of determining the essential nature of each species. From a theoretical perspective, the TSC is now almost abandoned by taxonomists except those who believe that species arise as a result of abrupt morphological change from one morphotype to another (Simpson, 1951). However, operationally many systematists today recognize species and develop keys for identification in much the same way their predecessors did 100-200 years ago.

In the early history of honey bee systematics, the TSC (as we know it today) was applied liberally to honey bees based on morphological characters and colour variations of pinned museum specimens. The descriptions of species were usually short and vague. With insufficient diagnostic morphological characters, frequently specimens from one locality were described independently under several names. With no adequate means of dealing with variation within interbreeding populations, newly collected variants were described as new species, resulting multiplicity of descriptions was unavoidable and resulted in superabundant names (Ruttner,

1988). After 200 years of research in honey bee taxonomy, more than 600 names have been used for members of the genus Apis (Maa, 1953) as it is defined by

Ruttner (1988)+

According to Alexander (1991a), Gerstacker (1862) presented a careful and comprehensive survey of variation in a number of morphological traits over the entire known geographic range of honey bees. This was the first attempt to bring order to this confusing mass of species names. Since then, severai other attempts have been made to revise the taxonomy of honey bees using morphological characters (e-g., Smith, 1865; Ashmead, 1904; Buttel-Reepen, 1906; Maa, 1953).

Because these entomologists analyzed different sets of diagnostic characters and weighted charaders differently when they attempted to define their morphospecies, different numbers of species resulted (Table 1). More recently, the widespread application of the Biological Species Concept to a growing body of information on the life history of geographical variants of honey bees made it clear that the application of the TSC in honey bees resulted in a large number of geographic variants being described and categorized as species even though they freely hybridize and produce viable offspring (DuPraw, 1965). Consequently, the TSC has been abandoned by honey bee researchers. Table 7.1 Typological species of honey bees as recognized by different authors. Not al1 Maa's species are given. Dash means the authors did not recognize the species.

-Gerstiiker (1862) Smith (1865) Ashmead (1904) Buttel-Reepen (1906) Maa 11 953) Apis dorsata Apis dorsata Megapis dorsata Apis dorsata var. dorsata Megapis dorsata A. dorsata var. zonata M. laboriosa M. breviligula A. zonata M. zonata A. dorsata var. zonata M. binghami A. indica Apis indica Apis indica A. mellifica indica var. indica A. indica - A. peroni A. nigro-cincta A. nigrocincta A.m. indica var. nigrocincta A. nigrocincta A. sinensis A. cerana A.m. indica var sinensis + japonica A. cerana - A.m. indica var koschevnikovi A. vechti A. m. indica var, koschevnikovi A. koschevnikovi A. mellifca Apis adansonii A. nigritarum A.m. unicolor var. adansoni A. adansonii A. adansonii A. unicolor Amunicolor var. unicolor A. unicolor A. m. unicolor var. intemissa A. intermissa A. m. unicolor var. fasciata A. lamarckii A. mellifera A. mellifera A. mellifica A. mellifera - A. m. mellifica var. remipes A. remipes A. florea Apis florea Micrapis florea A. florea var. florea Micmpis florea A. florea var. andreniformis M. andreniformis 7.3 BIOLOGJCAL SPECIES CONCEPT

The Biological Species Concept (BSC) defines species in terms of one population being distinct from another in part because of a lack of gene flow between them. According to this concept, "a species is a group of interbreeding natural populations that is reproductively isolated from other such groups" (Mayr and Ashlock, 1991). tnterbreeding between species is prevented by isolating mechanisms (Dobzhansky, 1970).

According to Mayr (1970), isolating mechanisms are any biological properties of sympatric or "potentially interbreeding" populations which prevent them from interbreeding. There are two main types of isolating mechanisms: rnechanisms that prevent interspecific mating (premating isolating mechanisrns) and rnechanisms that reduce the full success of interspecific mating (postmating isolating mechanisms). For full classification of isolating mechanisms see Mayr

(1970) for review.

In honey bee mating systems there are four factors that could act as reproductive premating isolating mechanisms: mating flight times of dronestqueens, locations of drone congregation areas, specific sexual pheromones, and the anatomy of genitalia. The BSC has been adopted in honey bee species primarily by viewing male genitalia as a form of mechanical isolation, followed by drone mating fi ight times as a form of behavioural (temporal) isolation, and more recently by the location of drone congregation areas as a secondary form of behavioural (spatial) isolation.

7.3.1 Premating Isolation

7.3.1.1 Mechanical Isolation - Differences in the male genitalia

Barrnann in 1956 (reviewed by Ruttner, 1968) published the first report on diagnostic differences in the uneverted male genitalia of A. mellifera, A. cerana, A. florea, and A. dorsata. Subsequently, several reports on the differences in everted

male genitalia of the honey bee were published: A. mellifera, A. cerana, and A. florea (Simpson, 1960); A. dorsata (Simpson, 7970); A. koschevnikovi (Tingek et al., 1988); and A. andreniformis (Wongsiri et al., 1990). Koeniger et al. (1991 ) compared everted and uneverted male genitalia of six honey bee species: A. andreniformis, A. flores, A. cerana, A. koschevnikovi, A. meIlifera and A. dorsata.

Based on the morphological differences between species, most biologists agree that the anatomy of male genitalia in honey bees is an important criterion for distinguishing species of honey bees. The species status of a population is sometimes wnfimied on the basis of male genitalia only (e.g., Tingek et al., 1988;

Wongsiri et al., 1990). In fact, some experts hesitate to accept the species status of newly discovered morphs of honey bee without distinctive genitalic differences

(e.g., Ruttner, 1988; Koeniger et al., 1991 ; Verma, 1995).

Several recent studies have challenged this view. McEvoy and Underwood

(1988) showed that A. dorsata and A. labonosa have similar genitalia; however there is disagreement over this finding (Koeniger et al., 1991). Despite this controversy, A. laboriosa is now generally accepted as a valid species based on temporal differences of mating flights by drones of A. labonosa and A. dorsata

(Underwood, 1990), as well as extensive differences in extemal anatomy

(Sakagami et al., 1980). In another example, it has been shown that two morphs of cavity-nesting honey bees in Sulawesi, Indonesia, A. cerana and A. nigrocincta, are separate species based on the temporal segregation of their drone mating flights

(Chapter 2, Hadisoesilo and Otis, 1996). However, no obvious differences in the anatomy of their male genitalia have been found yet (Chapter 4). In their preliminary investigations, G. and N. Koeniger (pers. comm.) could not find any discrete genital features to distinguish A. nuluensis from A. cerana, even though it has been proven to be a valid species based on the timing of the drone mating flights (Koeniger el al., 1996a). In addition, it is already known that differences in male genitalia alone are insufficient to prevent mating of A. mellifera drones with A. cerana queens

(Ruttner and Maul, 1983).

7.3.1.2 Behavioural (Temporal) Isolation - Drone Mating Flights

Witi-iin a population of honey bees, mating flight times of drones coincide with mating flight times of queens (reviewed by Koeniger, 1991; see also Verma et al., 1990; Yoshida et al., 1994; Yoshida, 1995). Therefore, timing of mating flights has been accepted as a form of behavioural isolation. It is known that if more than one species of honey bees inhabit the same location, each has a specific distribution of drone mating flight times that is usually distinct from other species: A. florea, Axerana, and A. dorsata in Sri Lanka (Koeniger and Wijayagunasekera,

1976); A. dorsata and A. labonosa in Himalayan valleys (Underwood, 1990); A. fforea, A. andrenfimmis, A. cerana, and A. dorsata in Thailand (Rinderer et al.,

1993); A. andreniformis, A. cerana, A. koschevnikovi, A. nuluensis, and A. dorsata in Bomeo (Koeniger et al., 1988, 1996a); A. cerana and A. nigrocincta in Sulawesi

(Chapter 2; Hadisoesilo and Otis, 1996).

From these reports, it is clear that timing of mating flights is an effective reproductive barrier in honey bees. This fype of reproductive isolation applies to al1 nine recognized honey bee species, with the exeption A. florea and A. cerana in

Thailand which have overlapping distributions of drone mating flight times (Rinderer et al., 1993) . However, this type of behavioural isolation cannot be confirmed in populations that are allopatrically distributed, such as A. dorsata binghami, A.d. breviligula, and A. d. dorsata.

7.3.1.3 Behavioural (Spatial) Isolation - Location of Drone Congregation Areas

It is believed that drones and queens of the genus Apis mate at drone mngregation areas (DCAs) (Zmarlicki and Morse, 1963; Punchihewa et al., 1990;

Koeniger, 1991; Yoshida, 1994a; Yoshida; 1995). Unfortunately, not many studies have been carried out conceming the location of the DCAs of different species from different localities. In Sri Lanka, Punchihewa et al. (1990) found that drones of A.c. indica congregated in clearings below the canopy of trees. In Japan, drones of A.c. japonica congregate above prominent trees at 17.2 to 22.5 m (Yoshida, 1994a;

Fujiwara et al., 1994). In contrast, the DCAs of A. mellifera in Japan are located in

open spaces surrounded by low trees (Yoshida, 1994a). Despite considerable

overlap in drone mating flight times (Yoshida et al., i 994) few drones of A. cerana

were trapped in the DCAs of A. mellifera (Yoshida, 1994a). He concfuded that A.

cerana and A. mellifera do not share the same DCAs in Japan. Drones of A. dorsata

congregate below the crowns of tall emergent trees (Koeniger et al., 1994) in

Borneo. Drones of A. koschevnikovihave been observed flying under the canopy

of trees near ground level and, although they have not yet been found, it is

predicted that their DCAs are also located below the forest canopy (N. Koeniger,

pers. comm.). No other information is available concerning the DCAs of the other

honey bee species in Asia (e.g., A. florea and A. andreniformis, A. nigrocincta and

A. nuluensis).

From the reports that are available so far, there is an indication that DCA

location might serve as a reproductive barrier because honey bees appear to have

well defined conventions for encountering mates in specific locations. However,

because the data are few, more information on DCAs from areas where two or more

species live sympatrically is needed. Also it has not been possible to directly

confirm that mating occurs at the DCAs. Recent studies with radar suggest that

drones of A. mellifera fly along pathways, and where the pathways branch, and

drones reorient, local aggregations ("DCAs") form (Loper et al., 1987, 1992). There

are also some hints that drones can be lured away from these pathways by queen mandibular pheromone or the presenœ of actual queens (O.R. Taylor, pers. comrn.

to G.W. Otis). As discussed for timing of mating flights, this spatial isolation is

impossible to demonstrate in allopatric populations.

7.3.1.4 Behavioural Isolation - Chemical Signals

Sex attraciant pheromones produced by females are widespread in bees and

are used for close-range discrimination (Eickwort and Ginsberg, 1980). These

pheromones are usually species specific, as shown for bumble bees (Free, 1971)

and halictine bees (Barrows, 1975). Therefore, differences in mating attractants

usually are wnsidered to be reproductive isolating mechanisms. However, several

studies on the queen pheromones of honey bees suggest that they do not act as

isolating mechanisms in honey bees. Despite qualititive and quantitive differences

in the mandibular gland pheromone blends of different species of honey bees

(Plettner et al., 1997), a single component, 9-keto-(E)-2-decenoic acid (referred to

as 9-ODA) may attract drones of al1 species. Attraction of A. mellifera (Butler and

Fairey, 1964; Yoshida, 1994a), A. cerana (Punchihewa et al., 1990; Yoshida,

1994a), and A. dorsafa (Koeniger et al., 1994) to synthetic 9-ODA has been demonstrated. Queen mandibular gland extracts also attract drones of several species. Apis mellifera drones were attracted to mandibular gland extracts of A. cefana (Butler et al., 1967; Ruttner and Kaisslhg, 1968) and A. florea (Butler et al.,

1967). A. cerana, A. florea, and A. dorsata drones were attracted to dead queens of A. rnellifera in lndia (Sannasi and Rajulu, 1971). Bornean drones of A. dorsata were also attracted to dead queens of A. meIlifera (Koeniger et al., 1994). Because the sexual attractant in honey bees, queen mandibular pherornone, appears to function interspecifically, I conclude that it should not be considered as a reproductive isolating mechanism.

1.3.2 Postmating Isolation

Premating isolation is usually effective at preventing interbreeding between allospecific populations. However, if these sets of mechanisms fail to prevent interspecific mating, other sets of mechanisms, postmating barriers, may prevent successful hybridization. These mechanisms result in gametic mortality, zygotic mortality, hybrid inviability, or hybrid sterility (Mayr, 1970).

Only two studies demonstrate the existence of postmating rnechanisms in honey bees. Ruttner and Maul (1983) inseminated queens of A.c. indica with semen of A. meIlifera and vice-versa. In both cases the queens laid eggs but the eggs never hatched because the formation of blastoderm was blocked. More recently, Yoshida (1994b) inseminated queens of A. mellifera with semen of A.c. japonica. The results showed that among 682 eggs laid by the queen, 676 (99.1 % failed to hatch and only 6 (0.9%) eggs developed into larvae. However, the sex of these Iarvae couid not be determined because they died immediately after hatching.

In contrast G. and N. Koeniger (pers. comm.) successfully obtained hybrid workers from A. cerana queens inseminated with semen from A. koschevnikovi. These studies indicate that in some instances postmating barriers operate between honey bee species, in this case by causing zygotic mortality, but they can be absent between closely related species.

In summary, the temporal distribution of mating flights and, in some cases, spatial location of mating, both behavioural traits, operate to prevent gene flow between different species of Apis. Under the BSC, they are interpreted to be isolating mechanisms. Genital differences may prevent interspecific mating in some cases, but not al1 (e.g., A. cerana and A. mellifera). Moreover, there are no direct observations of genital differences preventing interspecific mating. Consequently, genital structures may contribute to reproductive isolating mechanisms, but by themselves are inconclusive evidence of species status according to the BSC. Sex pheromones do not appear to operate as isolating mechanisms in Apis because they lack species specificity. According to the BSC, postmating isolating mechanisms may occur also; this has only been observed in crosses of A. mellifera and A. cerana in which egg and larval development is arrested.

If the BSC is applied to the genus Apis, nine species of honey bees are recognized: A. andmiformis, A. florea, A. cerana, A. nigrocincta, A. nuluensis, A. koschevnikovi, A. mellifera, A. dorsafa, and A. laboriosa. The BSC is frequently applied to allopatric populations with the assumption that the members of these populations represent the same species if they are morphologically similar and there is no evidence of reproductive isolating mechanisms that could operate if they were sympatric. Conversely, if their mating systerns differ such that it appears that they wouid be reproductively isolated, then it would be concluded that they represent different species. In the absence of direct observations, conclusions conceming species status of allopatric populations is a matter of personal judgernent.

7.4 RECOGNITION SPECIES CONCEPT

The Recognition Species Concept (RSC), like the BSC, applies only to biparental organisms. In the BSC, a species is defined by its relation to populations of other species. In wntrast, in the RSC a species is defined by the relation of conspecific individuals to each other. Species are not defined relationally but independently. The RSC defines "a species as the most inclusive set of populations of individual biparental organisms which share a common fertilization system"

(Paterson, 1985, and references therein). This concept stresses the importance of how fertilization is brought about between individuals of the same species in their natural habitat.

According to this concept, each fertilization system comprises a number of components. Motile organisms have a requirement that a subset of al1 the charaders of their fertilization system serve the function of bringing motile partners together, thereby enabling fertilization and syngarny. This subset of possible fertilization systems constitutes the Specific-Mate Recognition System (SMRS)

(Paterson, 1985 and references therein). According to Scoble (1985), the RSC can be cautiously applied to allopatric populations by comparing the sequence of courtship. If no differences are found in the sequence of courtship (Le., MRSs), one would conclude that the populations are part of the same species.

In the genus Apis, matings between drones and queens occur in the air, for a short period time of day, and in certain areas (DCAs) (see Koeniger, 1991 for review). Therefore, in the mating system of honey bees, there are five sequential elements that bring drones and queens together:

(1) the timing of the mating flights of drones and queens,

(2) locations of drone congregation areas,

(3) sexual pherornones,

(4) contact stimuli

(5) male genitalia.

Note that this set of characteristics is nearly the same as the premating isolating mechanisms discussed above under the BSC.

7.4.1 The Timing of the Mating Flights of Drones and Queens

Because the mating flight times of drones coincide with the mating flight tirnes of drones (Koeniger, 1991), the timing of the mating flights is considered a component of the MRS of each species. If drones of two different populations fly at different times of day and queen flights are synchronized with drone flights, then the queens of one population do not have an opportunity to meet drones from the other population (see section 7.3.1 -2).Consequently, if this element of the SMRS is not fulfilled, natural mating does not ocair, and the two popufationsunder consideration must be considered as different species. This criterion is met by al1 recognized species of Apis except A. florea and A. cerana in Thailand which have overlapping distributions of drone mating flights (Rinderer et al., 1993).

7.4.2 Locations of Drone Congregation Areas

Drones and queens of honey bees must meet at a certain place to mate

(section 7.3.1.3). The evidence obtained to date suggests that each species of honey bee has a characteristic site where drones congregate (see section 7.3.1-3).

Therefore, the locations of DCAs may be the second element of the MRS of honey bees. As pointed out previously, there is no direct evidence that queens mate with drones in DCAs. Direct quantification of where mating of different species occurs is needed before DCA location can be unequivocally accepted as a cornponent of the SMRS. If it is shown that mating actually occurs in DCAs, then it follows that if several populations of honey bees have DCAs in different locations, then the queens (drones) of one species do not physically encounter drones (queens) of others. Consequently, natural mating between these two populations does not occur even though they may have the same mating flight times. According to the

RSC, these populations would be considered distinct species.

7.3.3 Sexual Pheromones

Queen substance, the pherornone blend produced by the queen mandibular glands, also serves as a sexual attractant (Gary, 1962; Butler and Fairey, 1964).

A review of queen substance and its apparent role as a general attractant of drones of many species is presented in section 7.3.1 -4. Although the queen mandibular pheromone blend differs between species (Plettner et a1.,1997), there is no evidence of species specific drone attraction.

I still consider mating attractants as a component of the MRS of honey bees that operate in the close range orientation of drones to queens. Without queen substance, the attraction of drones by queens would be based on vision alone and probably would operate less efficiently. However, queen mandibular pheromone does not appear to function as a component of the SMRS of individual species because of interspecific attraction.

7.4.4 Contact Stimuli

A survey of copulation behaviour in insects reveais that in one third of 302 species, the male behaves so as to stimulate the female (Eberhard, 1990; 1991).

His behaviours include stroking, rubbing, or biting the fernale's body (Borror et al.,

1989). Because this sort of behaviour influences the acceptance of the male by the female, it is also considered an element of the SMRS.

In the mating of honey bees (A. mellifera and probably the other species), it has been observed that after a drone approaches the queen, he touches her dorsally with the distal parts of the tarsi of the first and the second pair of legs.

Shortly thereafter, al1 the six legs clasp the queen. In this final position, the hind metatarsi always grasp the fourth and fah tergites where they overlap the sternites.

These metatarsi have special adhering hairs that touch the queen at a consistent place. After holding the queen in the final position he everts his endophallus into her opened sting chamber (Koeniger et al., 1979).

When the drone grasps the queen and contacts her with the modified hairs of the hind metatarsi, the queen may be able to accept or reject the drone based on the contact stimuli she receives. The queen must open her sting chamber to allow copulation and it is possible that she may reject some (e.g., allospecific) drones. However, there is no documented evidence of rejection of drones by queens in honey bees. If it happens it would have to occur quickly because the whole procedure from the final position to the evertion of the endophalli is completed in 1.5 sec (Koeniger et al., 1979).

7.4.5 Male Genitalia

In many insect species, the genitalia are a component of the SMRS. For instance, the genitalia of many organisrns exhibit a "lock-and-key" complementary nature (White et ai., 4995) such as in Enallagrna damselflies (Robertson and

Paterson, 1982) and Apomea moth (Mikola, 1992). However, because of the nature of the mating systern in honey bees, the role of the genitalia as a component of the

SMRS is probably insignificant. After grasping the queen, the drone everts his endophallus into the opened sting chamber of the queen. At this time he becomes paralyzed and the distal part of his genitalia breaks off from the rest of the body. It is difficult to imagine mechanisms by which the queen could terminate copulation

before sperrn tranfer on the basis of genital stimuli (Eberhard, 1990) during such

a rapid violent process (for review see Koeniger et al., 1979; Koeniger, 1986b). It

is known that drones of A. mellifera can physically mate with queens of A. cerana

(Ruttner and Maul, 1983), supporting the idea that the queen exercises little choice

of mates once appropriate physical stimuli from the male have been received.

In conclusion, when the RSC is applied to honey bees, the two most

important components that bring the two sexes together are the timing of mating

flights and the locations of DCAs. The sexual pheromone also acts as a

nonspecific, short-range attractant of drones to queens. Physical stimuli at the time

of contact may serve as the final component of the SMRS. It is unlikely that the

queen can exercise any further choice once mating has occured. Because the

application of the RSC to honey bees relies on the same major characteristics as

reviewed for the BSC, the same nine species of honey bees are recognized.

7.5 PHYLOGENETIC SPEClES CONCEPT

According to the Phylogenetic Species Concept (PSC), "a species is the smallest diagnosable cluster of individuai organisms with a distinct pattern of ancestry and descent" (Cracraft, 1983). The PSC recognizes groups as species even if they are diagnosable by only minute genetic differences (Vrba, 1995). Using this concept one tries to infer the phylogenetic relationships among the clusters.

Unlike the BSC and the RSC, the PSC can be applied to both sexual and asexual groups of organisms.

One requirement under the PSC is that species must be strictly monophyletic, which requires that phylogenetic analyses be conducted before conclusions regarding species are made (the PSC differs from the BSC and RSC in this regard). This concept emphasizes diagnostic character states for each species (Cracraft, 1983,1989). Diagnostic characters can be any intrinsic attribute, they can be morphological, biochemical, physiological or behavioural (Cracraft,

1983). These diagnostic characters must be heritable. In principle, populations should be 100% diagnosable within a species. Even if two sister-taxa can hybridize, both can still be considered to be species if each is diagnosable as a discrete taxon

(Cracraft, 1983). Under the PSC, standard cladistics analyses (Hennig, 1966) that employ appropriate outgroups are made (Eldrege and Cracraft, 1980; Wiley, 1981 ) in order to identify the most likely phylogeny for the taxa being evaluated.

If the PSC is applied to honey bees, our current view of several species remains the same: A. andreniformis, A. florea, A. koschenikovi and A. labonosa.

These are taxa for which no distinctive subgroups have yet been described that would allow hem to be more finely subdivided into additional phylogenetic species.

However, several other species as defined by the BSC and RSC would require changes under the PSC. For example, within A. mellifera and A. cerana there are many geographical races. In A. mellifera, some of these are distinctive and easily identified (e-g., A.m. unicolor, A.m. monficola). It may be possible to define these particular populations on the basis of clear diagnostic traits, in which case they would constitute monophyletic units that would therefore qualify for species status under the PSC. This is also true of the three subspecies of A. dorsata (treated in more detail below) which would almost certainly qualify for the status of phylogenetic species. In fact, because of their clear morphological differences and distinct allopatric ranges, one recognized authority on bee systematics S.F.

Sakagami (Sakagami et al., 1980) has suggested they deserve to be elevated to species status: A. dorsata, A. binghami, and A. breviligula.

The situation surrounding A. nuluensis of Borneo (Koeniger et al, 1996a;

Tingek et al., 1996) points out ways in which this concept and its requirement of monophyly of al1 taxa rnay result in nomenclatural difficulties. Apis nuluensis is morphologically similar to A. cerana, but has temporally separated mating flights

(Koeniger et al., 1996a). Both species also inhabit different habitats (Koeniger et al., 1996a). Arias et al., 1996 used a mitochondrial DNA region (ND2) and a nuclear gene intron (EF-1 a) to construct phylogenies of the cavity-nesting honey bees. The results from ND2 indicate that A. nuluensis is more closely related to A. cerana from

Borneo than to A. cerana from Sri Lanka. In this analysis, the discovery of A. nuluensis makes A. cerana paraphyletic. Under the requirements of monophyly of the PSC, the sister group to A. nuluensis, in this case A. cerana of Borneo, would have to be given a new name if this phylogeny receives further support. Paraphyly was not created in the analysis of the EF-1 a sequence in which A. cerana and A. nuluensis emerged as sister species. A similar situation is likely to be found for A. nigmhcta, wtiich appears to have evolved from one Iineage within A. cerana that colonized the Philippines (Smith and Hagen, 1996).

If al1 populations of Apis are reassessed with the PSC, the number of species recognized would be greater than what are currently recognized by most researchers. They include A. andreniformis, A. florea, A. koschevnikovi, and A. laboriosa; the highly variable species A. meIlifera and A. cerana which have several populations that rnight be sufficiently distinct to receive species status; A. dorsata,

A. binghami, and A. breviligula; and A. nigrocincta, A. nuluensis, and their sister groups (currently recognized as A. ceana) which would be required to be renamed to maintained the strict monophyly of each species. This results in a minimum of 13 species and possibly several more! Clearly the species concept that is adopted strongly affects the nomenclature and estimates of diversity of Apis.

7.6 THE SPECIFIC CASE OF Apis nigrocincta

This species was first classified typologically by Smith (1861 j. Until the mid

1990's only two additionat authors applying TSC methodology (Ashmead, 1904 and

Maa, 1953) recognized A. nigrocincta as a distinct species. Others believed this form was only a variety of the widespread Asian hive bee A. cerana (Enderlein,

1906; Buttell-Reepen, 1906 as A. mellifica indica). In addition, specimens of bees tom Sulawesi were preserved in the Rijks museum of Natural History, Leiden, the

Netherlands but are absent from almost al1 other museum collections (see Otis,

1996 for review). In fact, several recent books fail to indicate that any cavity-nesting honey bees inhabit Sulawesi (Crane, 1990, d993). As a result, until my work, this species was generally overlooked and subsumed under the taxon A. cerana.

I was the first to recognize (1995) that there were two distinct morphs of cavity-nesting honey bees in Sulawesi. Morphometric analyses indicated that one of them is A. cerana (Damus, 1995; Hadisoesilo et al., 1995; Damus and Otis, submitted). The other, which matches the type specimen of A. nigrocinda, appeared to be a distinct form. Although no diagnostic morphological characters have been found between the two forms, behavioural traits have proven that A. nigrocincta is a valid species under the BSC and RSC. This verification was based on the segregation of the drone flight times of these two species (Chapter 2; Hadisoesilo and Otis, 1996). Another trait, the nature of the drone cell caps (Chapter 6), differs between these two forms, thereby also allowing the conclusion that they are different species under both the TSC and the PSC.

7.7 CONCLUSIONS

The application of different species concepts to honey bees results in different numbers of species. The Typological Species Concept, now largely abandoned by honey bee taxonomists, is incapable of dealing with taxa that, like honey bee species, exhibit extensive variability.Early descriptive taxonomy resulted in hundreds of named honey bee species, few of which are still recognized.

At the other extreme, the Biological Species Concept and Rrcognition

Species Concept provide the most conservative view of honey bees, with nine currently recognized species. These concepts emphasize ecological and behavioural traits that enable the union of drones and queens and subsequent fertilization of eggs. Differences in these traits between populations can be interpreted as evidence that successful natural hybridization between members of those populations does occur; in other words those populations represent different species. Unfortunately, no direct evidence can be confirmed if these two species concepts are applied to populations that have non-overlapping ranges, such asthe members of the A. dorsata complex. My analyses (Chapter 2) emphasize the importance of behaviourat isolation (e.g., where and when reproductives fly to mate). Endophallic structure, viewed as a possible isolating mechanism by the BSC, is probably not important under the RSC because of the way in which mating and sperm transfer occur in honey bees. In fact, male genitalia need not differ between species, as has been found to be true for A. nigrocincta and A. cerana and has been suggested by McEvoy and Undewood (1988) for A. laboriosa and A. dorsata, by G. and N. Koeniger( pers. comm.) for A. cerana and A. nuluensis .

Application of the Phylogenetic Species Concept to honey beeç increases the number of species to a minimum of 13 and possibly many more. The recognition of the island populations A. bjnghami and A. brevilgula as species distinct from A.d. dorsata is a relatively arbitrary decision. Several people (e-g., Sakagami et al.,

1980; C.K. Starr, pers. comm. to G.W.Otis) already support their designation as species because of their clear morphological distinctiveness and behavioural difFerences from mainland populations. However, recognition of A. nuluensis as a species may create a paraphyletic situation within the A. cerana clade which can only be resolved by recognizing the sister group to A. nuluensis (at present

Bornean A. cerana) as a separate species. Because it is almost certain that

Bomean A. cerana are not distinct from other A. cerana populations under the BSC and RSC, taxonomie view of A. cerana would not be recognized by many biologists studying honey bees. A similar paraphytetic situation will almost certainly be created by recognition of A. nigmincfa which probably evolved from the Philippine branch of A. cerana. For these reasons, the use of the Phylogenetic Species

Concept to classify honey bees creates more confusion than it resolves and therefore is not recommended. CHAPTER 8

GENERAL CONCLUSIONS

Apis is probably the most widely studied of al1 insect genera (Maa, 1953;

Seeley, 1985, 1995; Winston, 1987; Gould and Gould, 1988; Ruttner, 1988).

However, among the four species of honey bees recognized before the 1980's (A. florea, mellifera, cerana, dorsata), A. mellifera is very well known biologicall y. In contrast, the cavity-nesting honey bees of Asia are relatively poorly known. For most of this century only one cavity-nesting honey bee species, A. cerana with 4-8 subspecies and several ecotypes, was recognized in Asia, (Ruttner, 1988; Peng et al., 1989; Verma, 1995). The recent recognition of A. koschevnikovi (Koeniger et al., 1988; Tingek et al., 1988; Ruttner et al., 1989), A. nigrocincfa (Chapter 2;

Hadisoesilo and Otis, 1996), and A. nuluensis (Koeniger et al., 1996a; Tingek et al.,

1996) shows how poorly we understand this group of bees. I hope the results of my study on two cavity-nesting honey bees of Asia, A. cerana and A. nigrocincta of

Sulawesi, Indonesia, will help fiIl this void.

My study has confirmed that Apis nigrocincta Smith is a valid species. The validity of A. nigrocincta as a species distinct from A. cerana is evident frorn the segregation of the drone mating fiights of these two species. Drones of Apis cerana fly two hours earlier than drones of A. nigrocincta at al1 three observation sites (Chapter 2; Hadisoesilo and Otis, 1996). The species status of A. nigrocincfa is also

supported by the results of morphometric analyses (Chapter 3). Cluster analyses

classify A. cerana and A. nigrocincfa into two well defined groups. No single sample

of A. cerana or A. nigrocincta is misclassified. The principal component analysis

and discriminant analysis give the same results. There are no samples with

intermediate scores and these two species fon two distinct groups without

misclassification even in the two areas of sympatry. In addition, observations on the

mating by queens of A. cerana and A. nigrocincta in zones with allospecific drones

suggest that no interspecific mating occurs (Chapter 5). Collectively these results

reveal that there is at least one premating barrier (i.e., behavioural isolation by

temporal differences) between these two species. In the terminology of Ruttner and

Maul(1983) the process of çpeciation of A. nigrocincta and A. cerana is considered

to be "completed" as indicated by the existence of premating barriers that "prevent

the wastage of gametes".

The morphology of adult workers of these two taxa is sirnilar. No easily

assessed morphological character has been identified that allows identification of

A. nigrocincta (M. Engel, pers. comm.). A possible exception is the relative lengths

and widths of hindwings which do differ among the specimens checked (Chapter

3); this needs to be verified with larger samples of bees from more widely

distributed of the ranges of the two species. The anatomy of the endophalli of these two species are also similar (Chapter 4). This similarity contradicts the general situation in honey bees in which different species exhibit differences in genital

anatomy.

The similarity in the worker morphology and the male genitalia between A. cerana and A. nigmcincta suggests that the divergence between these two species occurred relatively recently. A review of current ideas about the phylogenetic history of the honey bees has pointed out major discrepancies in the estimated ages of various taxa. For that reason, estimation of the age of the speciation event for A. nigrocinda range from less than 54mya (M.S. Sheppard, pers. comm) to less than

1 mya (M. Engel, pers. comm.).

If one examines the male genitalia of the three subgenera of honey bees

(Le., Megapis, Micrapis, and Apis), one finds that among the subgenera, the differences in the male genitalia are pronounced. In contrasi, within each subgenus the male genitalia of al1 species are similar. The subgeneric differences in male genitalia among species are unlikely sufficient to prevent interspecific matings. In fact, in the cavity-nesting bees, it has been observed that differences in the structure of the endophalli by themselves cannot prevent interspecific matings

(Ruttner and Maul, 1983). In the case of A. nigrocinda and A. cerana, because of their almost identical endophallus structures, there is no doubt that interspecific matings are physically possible.

The existence of distinctive male genitalia, as for most insects, is taken as clear evidence of species status in the honey bees (Koeniger et al., 1991 ). My study demonstrates that the converse situation is not always true: the absence of genitalic differences does not always demonstrate that two populations are conspecific. This conclusion differs from what many honey bee experts expect or demand before accepting new species.

The distributions of A. cerana and A. nigrocincta in Sulawesi are unusual in several respects for honey bees. There are two known areas of sympatry, one in

South Sulawesi (Bontobulaeng area) and the other in the Palolo Valley of Central

Sulawesi. In these regions, only A. cerana has been observed at lower elevations

(c 400 m) with pronounced dry seasons (e.g.,the extreme southem parts of the southwestern peninsula and the region near Palu, Central Sulawesi), in the Palolo

Valley it inhabits higher elevations (up to 800 rn) with less pronounced dry periods and it has only been found in disturbed habitats. Apis nigrocincta usually inhabits higher elevations (>400 m) that are more forested areas and have more pronounced wet periods. However, where A. cerana is apparently absent (e-g.,

Manado, North Sulawesi; Tabo Tabo and Sinjai, South Sulawesi) it is also common near sea level in forest and coconut growing areas. Several factors are discussed that may influence these distributions, including climatic conditions, habitat preferences, and cornpetitive exclusion between the two species. Climatic conditions are undoubtly important but cannot be the only factor influencing distributions because both species wexist under identical climatic conditions in the zones of sympatry.

In the villages of Kamarora and Rahrnat, I found an abrupt transition from A. nigrocincta in forested areas to A. cerana in villages and cultivated areas. This is

striking and unusual for honey bees which are typically thought of as generalists

and exhibit little habitat specificity (Ruttner, 1988; Salmah et al., 1990). The

distribution data suggest strong habitat selection by swarms when searching for

new nest sites. Alternatively, cornpetition between these two species could be

influencing their distributions. Near the two known zones of sympatry, the

replacement of A. cerana at lower more disturbed habitats by A. nigrocincta at

higher more forested habitats is suggestive of cornpetitive exclusion, whereby the

two species do not coexist excep: in narrow zones of sympatry because of

cornpetition for some Iimiting resource. Without further experimentation it is not

possible to differentiate between these three explanations: climatic limitations,

habitat preferences, and cornpetitive exclusion.

The discovery of two Apis species that are reproductively distinct (Chapter

2; Hadisoesilo and Otis, 1996) but have morphologically indistinguishabie genitalia provides some indirect evidence on the speciation process in honey bees. There are a number of SMRS components in honey bees that could allow speciation in this genus (Le., timing of mating flights, location of DCAs, sex pheromones, contact stimuli, and morphological compatibility of male and fernale genitalia). It should be relatively easy for differences in the timing of mating flights of queens and drones of Apis population to evolve so as to avoid overlap with sympatric Apis species. Few genetic changes would probably be required to shift mating flights sufficiently to maximize intraspecific encounter rates between queens and drones within a population Mile maximizing interspecific encounters. A similar argument could be made conceming the locations of mating. Unfortunately, there is no information on where DCAs of A. nigrocincta are found. It is likely that behavioural traits, not morphological ones, form the biological basis for speciation in the genus Apis.

My study ais0 reveals differences in the drone capping behaviour of A. cerana and A. nigrocincta. In two other cavity-nesting honey bees of Asia, A. cerana and A. koschevnikovi, the caps of drone cells are always hard cocoon structures with pores. In contrast, no pores are found in the soft beeswax caps of A. nigmcincfa's drone cells. This phenomenon leads to several interesting questions.

Given their close phyiogenetic relationship, why does A. cerana have drone cells with hard caps with pores but A. nigrocincta does not? What is the purpose of the pore in the cell cap? One possible factor could be the present of parasitic mites, but both A. cerana and A. nigrocinda appear to share the same parasites, V. jacobsoni and V. underwoodi. Reproduction of these mites remains to be verified in A. nigmcincta. The pore in the drone cap of A. cerana and A. koschevnikoviremains an unexplained phenomenon.

Apis nigrocincfa and A. cerana in Sulawesi present an interesting situation: what affects the distribution and abundance of these species that are so sirnilar in size and morphology? To understand the unusual distribution of A. cerana and A. nigrocincta one must know what factors influence the distribution (e-g., climatic, habitat preferences, or competition or combinations between thern). One rnay gain insight by placing colonies of both species in different sites and following their subsequent survival and reproduction. Further studies are required to clarify the nature of the current zones of sympatry. Are the current zones of sympatry static. or is one species displacing the other? When searching for new nest sites, do workers exhibit habitat preference that may result in the observed abrupt transition in the distribution of these two species? Do worker bees also exhibit habitat preference during foraging? Given their similarity, how do A. cerana and A. nigrocincta share the pollen and nectar resources in areas of sympatry?

The mating flights of these two species are shown to be temporally segregated. Do differences in drone flight times evotve to prevent overlapping distributions of flight times, as suggested by data? Do drones also segregate spatially by flying to different DCAs? As a recently recognized species, A. nigrocincta warrants further study to better understand its basic behaviour, its management for beekeeping purposes, its role as a pollinator in managed and natural ecosystems, and its pests and diseases. REFERENCES

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