A Morphometric Analysis of Apis Cerana F and Apis Nigrocincta Smith Populations from Southeast Asia

Original article

A morphometric analysis of Apis cerana F and Apis nigrocincta Smith populations from Southeast Asia

MS Damus GW Otis

Environmental Biology, University of Guelph, Guelph, ON N1G 2W1, Canada

(Received 15 August 1996; accepted 14 August 1997)

Summary — Apis cerana F and Apis nigrocincta Smith, two species of Asian cavity nesting honey bees, were analyzed for interpopulation morphological variation using conventional morphometric techniques. The well-known honey bee subspecies Apis cerana cerana and A c japonica are confirmed as being distinct from the rest of A cerana, even though the analysis removed the confounding influence of ’overall size’ from the data set. There are at least three other morphologically distinct groups: one ranging from Sri Lanka through to Flores and central Sulawesi, a second group so far only found in southern Sulawesi, and a group from Timor. A large phenetic shift has occurred in the second Sulawesi group, indicating that it may have evolved in isolation for a long period of time, or under- gone rapid divergence following a more recent bottleneck event. A nigrocincta is shown to be pre- sent over most of western Sulawesi, on Mindanao and on Sangihe, a small island between Min- danao and Sulawesi. The Mindanao and Sulawesi forms are distinct, but the small sample size did not allow for subspecific recognition. Bees from Luzon, previously identified as A c philippina, are shown to be very distinct from both A cerana and A nigrocincta, and the possibility of their belonging to the species A nigrocincta is discussed.

Apis cerana / Apis nigrocincta / morphometrics / Southeast Asia / honey bees

INTRODUCTION been well studied over most of their range, and have been assigned to many subspecies The honey bees of the world (Apidae: Apis (races or geographical variants) on the basis spp) are widespread over their native range of morphometric analyses. Understanding of Europe, Africa and Asia, and have been of honey bee morphological variation is successfully transplanted to other regions incomplete, however. New species (Apis and continents (Ruttner, 1988). They have nigrocincta, Hadisoesilo and Otis, 1996;

* Correspondence and reprints: Department of Biology, Queen’s University, Kingston, ON K7L 3N6, Canada Tel: (1) 613 545 6000 x4974; fax: (1) 613 545 6617; e-mail: [email protected] Apis nuluensis, Tingek et al, 1996) have Appendix and fig I). For the analysis, one sample recently been discovered from Sulawesi and consisted of ten bees from a single colony. Many of the were collected the authors, but Borneo, respectively, and their ranges have samples by some were collected by others and sent to us (see not yet been adequately determined. No Acknowledgments). The bees were killed with inclusive study of other Southeast Asian ether and preserved in 75% ethanol. The right island bee populations has been carried out fore and hind wings were removed and mounted either, although Ruttner (1988) said that if on glass slides with glycerin. The right hind legs new forms were to be discovered, that is were removed, disarticulated and mounted in the same manner. A second slide was on where they were most likely to be found. glass placed top to ensure that the parts stayed parallel to the Recently, Smith and Hagen (1996) pub- plane of measurement (coverslips were not heavy lished an analysis of mitochondrial DNA enough to prevent the parts rolling and the wings No distortion due to was variation among populations of cavity nest- folding). compression detectable. ing honey bees in Asia, which attempted to phylogenetically link the various popula- The specimens were placed on the stage of an inverted and an of the tions. They were able to resolve four mono- microscope image body parts was onto paper. that are relevant to this projected Magnifications phyletic lineages were x45 for most measurements, or ×21.5 for cerana indica from Sri Lanka study: 1) Apis the wing lengths. Distance measurements were and other parts of India; 2) A c cerana from made with a ruler and were accurate to the near- China and A c japonica from Japan; 3) A c est 0.01 mm, and wing angles were accurate to indica from Malaysia and Indonesia; and 4) the nearest tenth of a degree, measured with a A nigrocincta (see Hadisoesilo and Otis, protractor. The values of the variables for each sample were calculated as the arithmetic aver- 1996) from Sulawesi with A c philippina age over the ten bees from that hive. Consistency (sensu Ruttner, 1988) from the Philippines. was achieved in that one person (MSD) per- Unfortunately, the variation between the formed all of the measurements. In total, 98 sam- samples was too great to root the resulting ples were analyzed. were tree, and consequently they not able The characters measured are listed in table I. to discern phylogenetic relationships The fore wing and leg characters are the same between them (Smith and Hagen, 1996). as described by Ruttner (1988) and Ruttner et al Both Smith and Hagen (1996) and our study (1978), while the hind wing characters are those of Mattu and Verma One new character, used many of the same samples. Therefore (1984). width of the tibia, was introduced. It was defined this can be seen as a to companion study as the width of the distal end of the metatibia. theirs, investigating morphological variation as opposed to genetic variation. It was our intention to the islands of survey major Morphometric analyses Southeast Asia where cavity nesting honey bees are known to and to do a exist, pre- Plots of the raw data, taken two variables at a assessment of their liminary interpopula- time, were made to determine if they could dis- tion morphometric variation. criminate between the samples taken from the different localities. Cluster analysis (using UPGMA and Ward’s MATERIALS AND METHODS minimum variance and Euclidean distance options in SYSTAT; Wilkinson 1990) assigned to of overall and these Collection data and measurement samples groups similarity, groups were entered into a multiple-group prin- techniques cipal components analysis, which determined the characters most correlated with ’size’ [see Honey bees were collected from natural nests Thorpe (1988) and Daly (1992)]. The MG-PCA and semi-managed hives from many sites in was used simply to determine those variables Malaysia, Indonesia and eastern Asia (see correlated with ’size’, and not to compare populations. Variables identified as ’size’ variables localities not studied or reviewed by Ruttner were regressed on hind wing length (the vari- (1988) formed distinct clusters. able with the highest correlation, > 0.95, with PCA 1 in single-group analyses), and those that had high significant correlations with hind wing Smith and Hagen’s (1996) results suggested width (R2 > 0.6, P < 0.05) were classified as size- that the bees known as A c philippina from the related. The overall influence of size was Philippines [both Luzon and Mindanao accord- removed from those characters using the com- ing to Ruttner (1988)] may be more closely puter routine ’Burnaby’ recommended by Rohlf related to A nigrocincta, since they have similar and Bookstein (1987) and available by anony- DNA sequences in the area of the mitochondria mous ftp from life.bio.SUNYSB.edu/morphmet they investigated. A discriminant analysis (DA) as part of the ’Macleod’ package. The new ’size- was designed, assigning those bees known to be free’ variables were rejoined with the original A cerana (from Ruttner, 1988) and A nigrocincta variables that were shown to be uncorrelated (from Hadisoesilo and Otis, 1996) to two groups, with ’size’ into a new data set. This was the size- and testing to which of the two groups the bees free data set used for the rest of the analyses. from the Philippines and one other group from the island of Sangihe (half-way between Sulawesi The size-free data set was subjected to a sim- and Mindanao, see fig 1) belonged. A second ple principal component analysis as an DA was performed in which the Philippine sam- exploratory analysis to determine whether any ples were placed into their own a priori group, to of the recognized subspecies or samples from determine their relative fit to each other. All statistics were performed with SYSTAT version 5.0 for DOS (Wilkinson, 1990).

RESULTS

Several plots of the raw variables were searched for patterns of variation between the groups. Plots of lengths versus widths of wings and leg segments showed significant discrimination between the samples, and separated the two species A cerana and A nigrocincta from one another and the subspecies A c cerana (the samples from Hong Kong) and A c japonica (from Japan) from the rest of A cerana (fig 2). They also highlighted the reason behind the desire to remove the effect of ’size’ from the variables. The smallest bees measured were the samples from Luzon, the largest from Japan, and they occupy opposite ends of the graph (fig 2). It was not desired that populations be classified by their size differences, and the removal of overall size from the variables allowed us to analyse samples by their shape, instead of by size. UPGMA clustering of the data identified 16 groups, mostly arranged by location of collection. One sample from Timor was grouped into the cluster of Malay peninsula samples, another Timorese sample clustered with the samples from Flores, and one Flo- res sample clustered with the south Sulawesi samples. The Javanese samples (western and eastern) were all in one group, and one Balinese sample clustered with them. One sample from Sulawesi A nigrocincta clustered with the Mindanao group, and two from Sulawesi A nigrocincta clustered with the Sangihe group. A discriminant analysis, performed with the 16 groups, reas- signed these samples into their respective islands (with probability > 0.95). The 16 For further of the two A cer- analysis species groups entered into the MGPCA were: Java, ana and A non-metric multidimen- nigrocincta, Bali, Lombok, Flores, Timor, South sional scaling ordinations were performed as recommended by de Queiroz and Good (1997), and Sulawesi (Sulawesi a), central Sulawesi confirmatory analysis was carried out with dis- (Sulawesi b), Sulawesi A nigrocincta, criminant analysis. Sangihe, Malay peninsula, Sabah/Palawan, Luzon, Mindanao, Hong Kong, Japan and nigrocincta, and the samples from Luzon Sri Lanka. Those variables that had their were nearby. The samples of Sulawesi A highest absolute correlations with factor 1 of cerana were isolated into two distinct the MG-PCA and had high significant cor- groups, one (Sulawesi a from southern relations with hind wing length were clas- Sulawesi; clear polygonal shape) was sepa- sified as size related. They are highlighted in rate from but adjacent to the Javanese sam- table I. ples, and one from central Sulawesi (Sulawesi b; light grey polygonal shape) The principal component analysis of the plotted within the Javanese samples. The size-free data set, which compressed 62% samples from Sabah and Timor formed dis- of the variation in the samples into the first tinct groups, and the samples from Sri Lanka two components (fig 3), revealed many of overlapped slightly with the samples from the previously known subspecies of A cer- Java. ana. Both A c cerana from Hong Kong and A c japonica from Japan were well isolated The discriminant function analysis per- from the rest of the samples as was A formed to identify the Philippine and nigrocincta from Sulawesi (dark grey polyg- Sangihe bees placed the Sangihe and Min- onal shape). The Mindanao and Sangihe danao samples and four of the seven Luzon samples plotted with the samples of A samples in with A nigrocincta (probability 1.0), while the other three Luzon samples and A nigrocincta. The morphologically were identified as being more similar to A ambiguous samples from Luzon were cerana (probability 1.0). However, when included in both analyses, to compare their any three of the seven Luzon samples were phenetic relationship with the two species. placed into their own a priori group, all of the others were classified with them (probability 1.0), indicating that they are more A nigrocincta set like each other than either A cerana or A nigrocincta sensu strictu. The Mindanao This data set included all of the Philippine and were classi- Sangihe samples always bees, as well as A nigrocincta from Sulawesi fied with A from nigrocincta samples as described by Hadisoesilo and Otis (1996). Sulawesi, not into the Luzon a priori group, The multidimensional scaling analysis indicating that they are more similar to (MDS) resolved 96% of the original vari- Sulawesi A than to the Luzon nigrocincta ability into four dimensions (stress = 0.08) samples. and split the samples into three clear clusters For further analyses, the data set was split (fig 4): A nigrocincta from Sulawesi and into the two groups representing A cerana Sangihe, the Mindanao bees, and the samples from Luzon. These were the a priori Sulawesi b from central Sulawesi) and the groups included in a discriminant analysis samples from Luzon. The MDS was able to (DA). Figure 5 shows the graph of the two resolve 96% of the original variation into factors (= discriminant functions) derived four dimensions, with stress 0.09. Dimen- from the analysis. Ellipses are at the 75% sion 1 (fig 6) split the samples into the three confidence level (Cornuet, 1982; Ruttner, well-established subspecies A c cerana 1988), and define ’discrete groups’. The (Hong Kong), A c japonica and A c indica three groups identified by the MDS were (sensu Ruttner, 1988). Dimension 2 subdi- verified as being distinct. vided A c indica, removing Sri Lanka, Timor and Sulawesi a, and clearly isolated the Luzon samples. Dimension 3 (not shown) A cerana set separated Timor from Sri Lanka, and the Malay peninsula together with This data set included all of the A cerana Sabah/Palawan from the rest of A c indica. samples from Japan, Hong Kong, Sri Lanka, These were the groups then entered into the Malay peninsula, Sabah/Palawan, Java, Bali, DA: Sulawesi a, the combined samples from Lombok, Flores, Timor and Sulawesi Java + Bali + Lombok + Flores + Sulawesi (Sulawesi a from southern Sulawesi and b, Timor, Malay + Sabah, Hong Kong, Japan, Sri Lanka, and Luzon. The graphi- DISCUSSION cal results of the DA are presented in fig- ures 7 and 8. The first three factors sepa- This survey was able to document a high rated all seven of the groups. Factor 1 (fig 7) degree of morphological differentiation of A cerana identified A c cerana and A c japonica as among the island populations being distinct. Factor 2 in combination with and A nigrocincta in Southeast Asia. The factor 1 separated Malay+Sabah and Timor previously recognized subspecies A cerana cerana and A (Ruttner 1988) are from the Java et al group, but they were all c japonica upheld, and the tentative subspecies A c overlapped by A c indica from Sri Lanka. philippina is shown to be divergent from The Luzon bees were well separated, and both A cerana and A nigrocincta. In view Sulawesi a was distinct but to the adjacent of Smith and Hagen’s (1996) results, in Java et al group. Factor 3 (fig 8) divided Sri which mitochondrial DNA sequencing and and fur- Lanka, Malay+Sabah Timor, placed A c philippina in a monophyletic ther isolated Sulawesi a. The Timor and branch with A nigrocincta, and not with any Malay+Sabah samples always remained other A cerana groups, we suggest that A c close to the Java et al group. All ellipses are philippina may, in fact, be more closely at the 75% level of confidence. related to A nigrocincta than to A cerana. Further analyses need to be made, particu- Southeast Asian A cerana remains unstud- larly from other islands of the Philippines, ied. and they should combine genetic, behavioural and morphological studies. The southern Sulawesi samples (Sulawesi a) showed an exceptionally high level of morphological divergence (see figs 2, 3, 6 A c indica from Sri Lanka is shown to and 8) from their nearest neighbours be distinct from the Malaysian and Indone- (Malaya+Sabah and the Java et al group), sian samples, but as we lack geographically even though they have mtDNA haplotypes intermediate samples between Sri Lanka identical to those found on Java through and Malaysia, we cannot at this time dis- Timor (Smith and Hagen, 1996). Although pute Ruttner’s (1988) identification of the not shown graphically, this population is Malaysian and Indonesian A cerana as distinct from others at the 95% level of con- belonging to A c indica. Smith and Hagen’s fidence (95% confidence ellipse). This sug- (1996) data showed A c indica from Sri gests that this is a population that has under- Lanka to have a very different mtDNA hap- gone significant selection and/or drift in lotype from Malaysian or Indonesian A cer- isolation from the other populations. Con- ana. The transition between Indian and versely, a bottleneck experienced during past migration to Sulawesi may have accel- 6-8). Like the Sulawesi a samples, they may erated phenetic diversification, as Bryant represent a divergent form, clearly derived and Meffert ( 1996) have shown to occur in from the Javanese group (and with identical experimentally bottlenecked house flies. mtDNA; Smith and Hagen, 1996), but iso- Such a migration may have been recent, ie, lated for a period of time. Again due to the perhaps aided by humans. Unfortunately, paucity of samples, we refrain from mak- we do not know of a way to test these alter- ing subspecies judgments until more sam- natives with the material we have at pre- ples from a wider area of the island are anal- sent. ysed. The second A cerana population on We have shown that the samples of cav- Sulawesi seems to be indistin- Sulawesi, b, bee from the from Javanese A and it ity nesting honey Philippine guishable cerana, island of Mindanao and from the small be a recent as it seems to may very import, island of Sangihe between Mindanao and be confined to a few sites in central only Sulawesi are most similar to A Sulawesi and phenetically (Hadisoesilo Otis, 1996). nigrocincta (fig 3), although the Mindanao The five samples from Timor were iden- population represents a morphologically dif- tified as being morphologically different ferent group (figs 4 and 5). Smith and Hagen from other A cerana samples, although they (1996) place them within the A nigrocincta are close to the samples from Java et al (figs clade as well, based on molecular evidence. We refrain from judging the Mindanao sam- the Philippines and neighbouring islands ples to be a separate subspecies owing to are sure to result in exciting discoveries con- inadequate samples (four) but believe that cerning the variability and evolutionary his- more sampling will confirm them as being tory of the honey bees of this region. Such significantly different from Sulawesi A studies will hopefully clarify the species sta- nigrocincta. This report and that of Damus tus (A cerana, A nigrocincta or A philip- (1995) are the basis for the assertion by Otis pina) of the bees from Luzon. (1996) that A nigrocincta inhabits Mindanao and Sangihe. ACKNOWLEDGMENTS The congruence of our phenetic results with Smith and Hagen’s (1996) genetic The authors would like to thank those who helped results is encouraging, and it illustrates that collect or donated samples: C Cervancia, M continued use of both genetic and morpho- Chandra, MJ Crosland, A Lao, A Massulukang, logical studies of the cavity-nesting honey RWK Punchihewa, M Sasaki, M Sila, D Smith, L and the Indonesian bees of Southeast Asia should clear up many Wijaya many beekeepers who offered their hives. Thanks to W Matthes- while at the same time questions, leading Sears and HV Daly for help with the morpho- to additional, more ones. Further complex metrics. This research was funded by an NSERC collections and studies of bees from the PGSA scholarship, a University of Guelph Field islands of Sulawesi, Sumatra, the islands of Studies Travel Award, the 1993 Townsend Award, the Thompson fund and a Graduate Stud- monophylétique qu’A nigrocincta de Sula- ies Award to MSD; a grant from the Office of wesi et que les abeilles de Mindanao et de and funds from Research, University of Guelph, et Nos ana- the of Environmental Uni- Sangihe (Smith Hagen, 1996). Department Biology, discriminantes ont attribué tous les versity of Guelph. This manuscript has been lyses échantillons de Mindanao et de improved by the comments of three anonymous Sangihe, reviewers. ainsi que quatre échantillons de Luzon, directement à A nigrocincta, les trois autres échantillons de Luzon étant attribués à A cerana (probabilité de 1,0 pour tous les Résumé — Analyse morphologique des échantillons). Lorsqu’on a formé a priori un populations d’Apis cerana F et d’Apis groupe à partir de trois échantillons de nigrocincta Smith d’Asie du Sud-Est. Luzon quels qu’ils soient, tous les autres Depuis l’article de Ruttner et al (1978) échantillons ont été attribués à ce groupe, détaillant les méthodes et les caractères alors que les échantillons de Mindanao et utiles, la classification des abeilles melli- de Sangihe restaient avec A nigrocincta (pro- fères repose sur les techniques morphomé- babilité 1,0). Ce résultat, ainsi que ceux de triques. Toutes les sous-espèces reconnues Smith et Hagen (1996), suggèrent que les d’A mellifera et d’A cerana ont été décrites échantillons de Mindanao et Sangihe sont à l’aide des techniques de morphométrie. phénétiquement différents d’A nigrocincta Seules la Malaisie, l’Indonésie et les Phi- (figs 4 et 5) et d’A cerana (figs 6 et 8) et lippines n’ont pas été étudiées de façon représentent peut-être une sous-espèce dis- appropriée. Il est encore possible d’y trouver tincte qui pourrait être, non pas A c philip- des sous-espèces et même des espèces non pina comme l’avait suggéré Ruttner (1988), décrites à ce jour (Ruttner, 1988). Notre mais A n philippina ou une espèce distincte étude donne un aperçu de la variabilité mor- propre. D’autres études sont nécessaires. phométrique qui existe en Asie du sud est. Les sous-espèces bien connues A c cerana Nous avons utilisé une combinaison des (de Hong Kong) et A c japonica (du Japon) caractères de l’aile antérieure et de la patte ont été séparées dans toutes les analyses et de Ruttner ( 1988) et des caractères de l’aile sur tous les graphiques (figs 2, 3, 6-8). postérieure de Mattu et Verma ( 1984), mais A c indica est composée d’au moins trois la principale différence avec les études pré- groupes : l’un comporte les populations du cédentes est d’avoir exclu l’influence de la Sri Lanka, de Malaisie et des îles indoné- taille sur les variables en utilisant la tech- siennes de Java, Bali, Lombok, Flores, nique de Burnaby, telle qu’elle a été décrite Timor et une petite population dans le centre par Rohlf et Bookstein (1987). Cela nous a de Sulawesi (Sulawesi b), le second n’a été permis d’analyser les populations d’abeilles trouvé jusqu’à présent que dans le sud de en fonction de leur forme et non de leur Sulawesi (Sulawesi a) et le troisième est taille. celui de Timor (figs 2, 3, 6-8). Le groupe De nombreux échantillons ont été utilisés à Sulawesi a a subi une dérive phénétique qui la fois par nous et par Smith et Hagen pour l’a éloigné du reste d’ A c indica. Cela leur étude (1996) portant sur l’analyse géné- indique soit qu’il a été isolé pendant une tique de la variation de l’ADN mitochon- longue période, soit qu’il a subi un goulot drial, si bien que nos résultats peuvent être d’étranglement (« bottleneck ») important, dans les considérés comme le pendant morphomé- peut-être temps récents, peut-être trique de leur étude génétique. Cette der- aussi sous l’influence de l’Homme. nière a montré que les abeilles connues cou- A nigrocincta peut être divisé en deux ramment sous le nom d’A c philippina groupes : l’un de Mindanao, l’autre de Sula- (Ruttner, 1988) sont sur la même branche wesi et de Sangihe (figs 4 et 5). Nous n’avions pas suffisamment d’échantillons stein (1987). Das ermöglichte uns, die Bie- de Mindanao pour être certains de couvrir nenpopulationen nach ihrer Form und nicht correctement la variation qui existe sur l’île; ihrer Größe zu analysieren. Neben unseren aussi nous nous abstenons de considérer ce morphometrischen Studien wurde eine gene- groupe comme une sous-espèce. tische Analyse der Variation mitochondria- Il est certain que d’autres récoltes d’abeilles ]er DNA von Smith und Hagen (1996) et d’autres études faites sur les îles de Sula- durchgeführt, die weitgehend dieselben Pro- wesi, de Sumatra, des Philippines et des îles ben benutzten, sodaß unsere Ergebnisse als voisines fourniront des découvertes pas- morphologische Gegenseite der genetischen sionnantes concernant la variabilité et l’his- Untersuchung angesehen werden können. toire évolutive des abeilles mellifères de Nach den genetischen Befunden gehören cette région. De telles études devraient cla- die bisher als A c philippina (Ruttner, 1988) rifier, nous l’espérons, le statut d’espèce des beschriebenen Bienen zu dem gleichen abeilles de Luzon (A cerana, A nigrocincta monophyletischen Zweig wie Apis nigro- ou A philippina). cincta aus Sulawesi und die Bienen von Mindanao und Sangihe (Smith und Hagen, Apis cerana / Apis nigrocincta / morpho- 1996). In unserer Diskriminanzanalyse wur- métrie / Asie du sud est / abeilles melli- den alle Proben aus Mindanao und Sangihe, fères sowie vier der Proben aus Luzon der Art A nigrocincta aus Sulawesi, die anderen drei Proben aus Luzon wurden A cerana zuge- Zusammenfassung — Eine morphome- ordnet (alle mit einer Wahrscheinlichkeit trische Analyse von südostasiatischen von 1.0). Wenn "a priori" Gruppen aus drei der Luzon-Proben wurden Populationen von Apis cerana F und Apis gebildet wurden, nigrocincta Smith. Morphometrische Tech- die restlichen Proben aus Luzon dieser niken waren seit der detaillierten Darlegung Gruppe zugeordnet, die Proben aus Minda- der Methoden und verwendbaren Merkmale nao und Sangihe blieben aber weiterhin bei durch Ruttner et al (1978) die Hauptstütze A nigrocincta (Wahrscheinlichkeit 1.0). Die der Klassifierung der Honigbienen. Alle Proben aus Luzon dagegen sind phenetisch anerkannten Rassen von Apis mellifera und sowohl von A nigrocincta als auch von A Apis cerana wurden mit morphometrischen cerana (Abb 6-8) verschieden und stellen Techniken beschrieben. Die Gebiete von vielleicht eine eigene Unterart dar, die mög- Malaysia, Indonesien und den Philippinen licherweise nicht die von Ruttner (1988) wurden bislang nicht angemessen unter- vorgeschlagenen A c philippina ist, sondern sucht. Es ist immer noch nicht ausge- eher A n philippina oder sogar einer anderen schlossen, daß bisher nicht beschriebene Art entspricht. Dazu sind aber weitere Stu- Rassen oder sogar Arten in diesem Bereich dien erforderlich. Die gut bekannten Unter- gefunden werden (Ruttner 1988). Unsere arten A c cerana (aus Hong Kong) und A c Studie soll einen Einblick über die mor- japonica (aus Japan) waren in allen Analy- phometrischen Variabilität in Südostasien sen und Graphen gut getrennt (Abb 2, 3, geben. Wir kombinierten eine Reihe von 6-8). Für A c indica konnte gezeigt werden, Ruttner’s (1988) Merkmalen an Vorderflü- daß sie sich aus drei Gruppen zusammen- geln und Beinen mit den Hinterflügelmerk- setzt: eine wird von Sri Lanka, Malaysia, malen von Mattu und Verma (1984). Ein den indonesichen Inseln Java, Bali, Lom- Hauptunterschied unserer Methode ist der bok, Flores und Timor und einer kleinen Ausschluß des Einflusses von Größe auf die Population im zentralen Sulawesi (Sulawesi Merkmale mit Burnaby’s Technik, entspre- b) gebildet, eine 2. Gruppe wurde bislang chend der Beschreibung in Rohlf und Book- nur im südlichen Sulawesi gefunden (Sula- wesi a) und die 3. Gruppe auf Timor (Abb 2, Hadisoesilo S, Otis GW (1996) Drone flight times confirm the species status of Apis nigrocincta Smith, 3, 6-8). Die Gruppe Sulawesi a zeigte eine 1861 to be a species distinct from Apis cerana F phenetische Merkmalsverschiebung von den 1793, in Sulawesi. Apidologie 27, 361-369 restlichen A c indica, was einen Hinweis Mattu VK, Verma LR (1984) Comparative morpho- entweder auf eine lange Isolation gibt oder metric studies on the Indian honeybee of the north- auf eine Population, die einen Engpaß (bottle west Himalayas. 2. Wings. JApic Res 23, 3-10 Otis GW Distributions durchlaufen hat, vor (1996) of recently recognized neck) möglicherweise of bees kürzerer Zeit und durch species honey (Hymenoptera: Apidae; Apis) möglicherweise in Asia. J Kans Entomol Soc 69 suppl, 311-333. menschlichen Einfluß. Bei A nigrocincta Queiroz K de, Good DA (1997) Phenetic clustering in können zwei Gruppen unterschieden wer- biology: a critique. Quart Rev Biol 72, 3-30 den: eine von Mindanao und eine von Sula- Rohlf FJ, Bookstein FL (1987) A comment on shearing wesi und (Abb 4 und 5). Wir hatten as a method for ’size correction’. Syst Zool 36, 356- Sangihe 367 nicht genügend Proben von Mindanao um Ruttner F (1988) Biogeography and Taxonomy of Hon- sicher zu daß die Variation auf der sein, eybees. Springer Verlag, Berlin Insel ausreichend erfaßt und ver- wurde Ruttner F, Tassencourt L , Louveaux J (1978) Bio- zichteten daher darauf, diese Gruppe als metrical-statistical analysis of the geographic vari- eigene Rasse anzusehen. Weitere Proben- ability of Apis mellifera L. 1 Material and methods. Apidologie 9, 363-381 sammlungen von den Inseln Sulawesi, Sumatra, den und den Smith DR, Hagen RH (1996) The biogeography of philippinischen Apis cerana as revealed by mitochondrial sequence benachbarten Inseln werden mit Sicherheit data. J Kans Entomol Soc 69 Suppl, 294-310 zu weiteren interessanten Entdeckungen Thorpe RS (1988) Multiple group principal compo- über die Variabilität und Evolutionsge- nent analysis and population differentiation. J Zool schichte der Honigbienen in dieser Region Lond 216, 37-40 führen. Solche Studien werden dann hof- Tingek S, Koeniger G, Koeniger N (1996) Descrip- tion of a new cavity nesting species of Apis (Apis fentlich auch die Artzugehörigkeit der Bie- nuluensis n.sp.) from Sabah, Borneo, with notes nen von Luzon (A cerana, A nigrocincta on its occurrence and reproductive biology (Insect: oder A philippina) klären. Hymenoptera: Apoidea: Apini). Senckenbergiana Biol 76, 115-119 Wilkinson L (1990) SYSTAT for DOS. Systat, Inc Apis cerana / Apis nigrocincta / Morpho- Evanston, Illinois metrie / Südostasien / Honigbienen

APPENDIX REFERENCES Samples were collected from the following Bryant EH, Meffert LM (1996) Morphometric differ- sites: Philippines: Luzon, Laguna Province, entiation in serially bottlenecked populations of the housefly. Evolution 50, 935-940 College (2), Quezon Province, Quezon City (3), Zambales Province, Santa Cruz (2); Cornuet JM (1982) Représentation graphique de populations multinormales par des ellipses de confi- Palawan, Babuyan (1); Mindanao, Laguna ance. Apidologie 13, 15-20 del Norte, Kauswagan (4); Indonesia: Daly HV (1992) A statistical and empirical evaluation Sangihe, 5 km E of Tahuna (3); Sulawesi of some morphometric variables of honey bee clas- North, Teeb (near Amurang) (4), Sonder sification. In: Ordination in the Study of Morphol- (1); Sulawesi Central, Kamarora (70 km SE ogy, Evolution and Systematics of Insects: Appli- cations and Quantitative Genetic Rationales (JT Palu) (5), Bobo (1); Sulawesi South, Tabo Sorenson, R Foottit, eds), Elsevier, Amsterdam Tabo (60 km N Ujung Pandang) (1), Damus MS (1995) A morphometric and genetic anal- Jeneponto (5), Manipi (4), Bontobulaeng ysis of honey bee (Apis cerana F.) Samples from (4); Java West, Parungpanjang (5), Cianjur Malasia: population discrimination and relationships. Master’s Thesis, University of Guelph, (3); Java East, Tretes (5); Bali, Tunjungan Guelph, Ontario, Canada (2 km N Ubud) (6); Lombok, Senggigi (1), Gunungsari (4 km W of Senggigi) (6); Flo- referred to as one from Hong Kong, for sim- res, Ende and surrounds (4); Timor, 3 km plicity and because of its geographic prox- SW Penin- Kupang (5); Malaysia: Malay imity to and phenetic similarity with those sula, Universiti Pertanian Selangor, Machida Shi Malaysia (5), Sabah, Tenom (5); China: samples; Japan: Honshu, (near Hong Kong (4); Guangdong, Guangzhou Tokyo) (4); Sri Lanka: Colombo (4). Total: (1) — in the body of the paper this sample is 98, all workers.