Metaphase Karyotypes of Fruit Flies of Thailand. V. Cytotaxonomy of Ten Additional New Species of the Bactrocera dorsalis Complex

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1 Cytologia 65: , 2000 Metaphase Karyotypes of Fruit Flies of Thailand. V. Cytotaxonomy of Ten Additional New Species of the Bactrocera dorsalis Complex Visut Baimai1,*, Chalao Sumrandee1, Saen Tigvattananont2 and Wachareeporn Trinachartvanit3 of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut Institute of Technology, Lat Krabang, Bangkok, Thailand of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand Accepted September 12, 2000 Summary Analysis of mitotic chromosomes, with special emphasis on the amount and distribution of constitutive heterochromatin, from larval samples of Thai populations of flies in the Bactrocera dorsalis complex has revealed 10 distinctive forms of metaphase karyotypes. This cytological evidence coupled with morphological observations of adults and host relationships with different host plant species have suggested the existence of 10 new cytotaxonomic species provisionally named species Q-Z belonging to the B. dorsalis complex. Metaphase karyotypes of Thai fruit flies were previously classified into 4 groups based on the quantitative differences in pericentric heterochromatin. Metaphase karyotypes of species Q, R, W, X, Y and Z are characterized by the presence of prominent blocks of pericentric heterochromatin in the X chromosomes and autosomes similar to the mitotic patterns found in B. kanchanaburi and B. raiensis. Hence, they are placed in Group 2 of this classification. Species U and V exhibit chromosome X patterns similar to that of B. carambolae and other related species. They are thus assigned to Group 4. In contrast, species S shows a minimum amount of pericentric heterochromatin in all autosomes, although the X chromosome follows the pattern of species Y. Interestingly, species T exhibits a unique pattern of euchromatin and heterochromatin in the X chromosome and conspicuous pericentric heterochromatin in all autosomes. Thus, species S and T are assigned to 2 new categories. Heterochromatin accumulations in the genome, as demonstrated in this study, appear to have played a significant role in chromosomal evolution. The different degree of variation in pericentric heterochromatin in mitotic chromosomes is, therefore, useful for cytotaxonomically distinguishing members of some cryptic species complexes of the dipteran insects. Key words Bactrocera dorsalis complex, Heterochromatin accumulation, Chromosomal evolution. The tropical forests of Southeast Asia are rich in floral and faunal species diversity. Dipteran insects including fruit flies of the subfamily Dacinae (Diptera : Tephritidae) are especially notable for their species diversity within Southeast Asia. Of the Dacinae, 507 species have been recognized and formally described from the Asian-Pacific-Australasian Regions (Drew 1989, White and Hancock 1997). More recently, White and Evenhuis (1999) have described 24 new species of Dacini from Southeast Asia and Australasia. Among the fruit flies of Southeast Asia and Australia, the species belonging to the Bactrocera dorsalis (Hendel) species group have received close attention because of species complexity, cryptic species problems and the economic importance of some species within the group (White and Elson-Harris 1992). Drew and Hancock (1994) recognized and described 40 new morphological species belonging to the B. dorsalis complex. In our ongoing study of the population cytogenetics of flies in this complex, we have described and characterized * Corresponding author, scvbm@mahidol.ac.th

2 410 Visut Baimai et al. Cytologia 65 Table 1. Wild larval samples of 10 new species (Q-Z) of the Bactrocera dorsalis complex collected from infested fruits of a variety of host plant species in Thailand (Regions: 1= South; 2 =West; 3 = Central) metaphase karyotypes of 10 known species and 7 newly discovered species occurring in Thailand (Baimai et al. 1995, 1999a, b). In this report we present metaphase karyotypes of an additional 10 new species of the fruit flies belonging to the B. dorsalis complex in Thailand. Materials and methods A major survey for fruit flies in Thailand was carried out by collecting larvae-infested fruits from a variety of plants at different localities throughout the country (Table 1). The wild caught larval specimens were reared in the laboratory at Mahidol University until fully developed third instar larvae, pupae and adults were available. Metaphase karyotypes were prepared from the brain ganglia of healthy third instar larvae randomly selected from the samples. Chromosome analysis was performed on a series of photomicrographs of mitotic karyotypes as previously described by Baimai et al. (1995). Adults that emerged from infested fruit were keyed out to the B. dorsalis complex (Hardy and Adachi 1954, Drew and Hancock 1994) but could not be keyed out to any of the known members of the complex. Differences in mitotic karyotypes were then correlated with differences in external morphological characters of the adults (S. Tigvattananont, unpublished) to characterize these new species. Hoechst stained heterochromatin usually showed very bright fluorescent staining but some regions showed dull fluorescent staining. Euchromatin regions showed very light fluorescence. The diagrams of chromosome size, shape and patterning of each metaphase karyotype were generated by a computer software program Visio Technical 4.5 for Microsoft Windows.

3 2000 Cytotaxonomy of the Bactrocera dorsalis Complex 411 Results The discovery of the 10 additional new species of the B. dorsalis complex, temporarily named species Q, R, S, T, U, V, W, X, Y and Z, resulted from extensive field surveys for larvae of Thai fruit fly populations. The recognition of these new cytotaxonomic species was based on analysis of mitotic chromosomes in comparison with those described previously (Baimai et al. 1995, 1999a, b) coupled with differences in external morphological characters of adults (S. Tigvattananont, unpublished). These 10 new species clearly exhibit different patterns of the amount and distribution of pericentric heterochromatin in the sex chromosomes and/or autosomes. Metaphase karyotypes of the 10 new species are briefly described below: Species Q: The X chromosome has a submetacentric shape. The short arm is totally heterochromatic and has a small portion at the tip separated by a constriction (Figs. 1, 2, 21, 22) while the long arm is euchromatic with a conspicuous block of centromeric heterochromatin. The Y chromosome is small and dot-like shape (Figs. 1, 21). All autosomes contain small amounts of pericentric heterochromatin. Species R: The X chromosome has a large subtelocentric shape. The euchromatic portion is located at the distal end of the long arm which also contains 3 blocks of constitutive heterochromatin (Figs. 3, 4, 23, 24). It may be noted that the short arm contains 2 blocks of centromeric heterochromatin. The Y chromosome has a small subtelocentric shape (Figs. 3, 23). All autosomes consist of conspicuous pericentric heterochromatin. Species S: The X is a small subtelocentric chromosome consisting of a very large block of centromeric heterochromatin and an euchromatic portion at the distal region of the long arm while the short arm is entirely heterochromatic (Figs. 5, 6, 25, 26). The Y is a very small metacentric chromosome (Figs. 5, 25). Interestingly, all autosomes contain a minimum amount of pericentric heterochromatin which is similar to the standard metaphase karyotype of B. dorsalis. Species T: The X chromosome of this species has a unique configuration among all species members of the B. dorsalis complex studied thus far with respect to the amount and distribution of euchromatin. The submetacentric X chromosome exhibits 2 small euchromatic portions of approximately equal size each of which is located at the tip of one arm (Figs. 7, 8, 27, 28). The Y is a small submetacentric chromosome (Figs. 7, 27). All autosomes of species T consist of very prominent blocks of pericentric heterochromatin. Species U: The most striking feature of the metaphase karyotype of species U is the enormous size of the X chromosome which has a submetacentric shape. The euchromatic portion is located in the middle of the long arm whereas the opposite arm contains 2 large blocks of heterochromatin (Figs. 9, 10, 29, 30). The Y is a medium submetacentric chromosome (Figs. 9, 29). Like species T, all autosomes of species U show large blocks of pericentric heterochromatin, particularly chromosome nos. 5 and 6. Species V: The X chromosome has a subtelocentric shape and exhibits a similar euchromatic portion to that of species U, although it is clearly smaller in size (Figs. 11, 12, 31, 32). A small portion of lightly staining heterochromatin at the tip of each arm is noticeable. The Y chromosome is a considerably large telocentric chromosome (Figs. 11, 31). All autosomes of species V contain relatively lesser amounts of pericentric heterochromatin than those of species U. However, autosome nos. 5 and 6 seem to have more pericentric heterochromatin than the other autosomes. Species W: The X chromosome has a large metacentric shape. The euchromatic portion is located distally in one arm while the opposite arm is entirely heterochromatic with a secondary constriction toward the distal region (Figs. 13, 14, 33, 34). The Y chromosome is a medium size and has a telocentric configuration (Figs. 13, 33). All autosomes of species W contain considerable amounts of pericentric heterochromatin similar to those of species V Species X: This species has a small telocentric X chromosome showing a fair amount of

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5 2000 Cytotaxonomy of the Bactrocera dorsalis Complex 413 centromeric heterochromatin (Figs. 15, 16, 35, 36). This type of X chromosome is cytologically characteristic for this species. The Y chromosome has a small metacentric shape (Figs. 15, 35). All autosomes of species X exhibit considerable amounts of pericentric heterochromatin similar to those of species V and W. Species Y. The X chromosome pattern of species Y is in some way similar to that of species R but it is obviously smaller due to a lesser amount of heterochromatin. The X chromosome has a submetacentric shape containing an euchromatic portion at the distal end of the long arm while the short arm is totally heterochromatic (Figs. 17, 18, 37, 38). The Y chromosome is very small and dot-like (Figs. 17, 37). All autosomes contain major blocks of pericentric heterochromatin similar to those of species R. Species Z: The X of this species is a large submetacentric chromosome compared with that of species Y. The euchromatic portion is distally located on the long arm whereas the short arm contains 2 conspicuous blocks of heterochromatin (Figs. 19, 20, 39, 40). The Y chromosome has a medium dot-like shape medium in size relative to those of other species (Figs. 19, 39). All autosomes contain considerable amounts of pericentric heterochromatin similar to those of species V, W and X described above. A diagrammatic representation of metaphase karyotypes of the 10 new species is summarized in Fig. 41. Discussion A combination of mitotic chromosome analysis and morphological observations as well as preferences for specific host plant species of the fruit flies belonging to the B. dorsalis complex have revealed 10 additional new genetic species provisionally named species Q-Z. Cytological evidence for these new species was based on quantitative differences in the gross amount and distribution of constitutive heterochromatin in metaphase chromosomes. Our study thus far has proved the usefulness of cytotaxonomy for revealing cryptic species of bisexually reproducing organisms, especially the dipteran insects (Baimai 1988, 1998). Species distribution Our present data and the previous results (Baimai et al. 1995, 1999a, b) suggest that fruit fly populations in Ranong province are rich in species diversity particularly in the B. dorsalis complex. Eight new species (Q, S, T, U, V, W, X, Z) have been found in this area. Our earlier studies showed that 5 new species (E, J, K, L, M) and 4 known species (B. dorsalis, B. propinqua, B. carambolae, B. melastomatos) occurred in Ranong province (Baimai et al. 1995, 1999a, b). This makes a total of 17 species of the B. dorsalis complex occurring in the tropical forest of this province, which may be regarded as one of the richest areas for biological diversity in Thailand. Interestingly, 5 new genetic species (Q, R, W, X, Z) apparently have preferences for specific host plants (Table 1). Species Q is widely distributed in southern peninsular Thailand ranging from Ranong province to Narathiwat province near the Malaysian border. Despite this wide distribution, species Q has been found only in infested fruits of Azadirachta excelsa (Meliaceae), a common medicinal plant in Thailand. Species R has been detected thus far only in the fruits of Artabotrys siamensis in Surat Thani province, about 180 km south of Chumphon province. However, species S and U seem to have a Figs Photomicrographs of metaphase karyotypes using Giemsa staining of 10 new species of the Bactrocera dorsalis complex. 1) sp. Q d, 2) sp. Q Y, 3) sp. Rd, 4) sp. R Y, 5) sp. S d, 6) sp. S Y, 7) sp. Td, 8) sp. T 7,9) sp. Ud, 10) sp. UY, 11) sp. Vd, 12) sp. Vs, 13) sp. Wd, 14) sp. WY, 15) sp. Xd, 16) sp. X Y, 17) sp. Yd, 18) sp. Y Y, 19) sp. Zd, 20) sp. Zd. An extra block of heterochromatin at the tip of one arm of each X chromosome is indicated by an arrow.

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7 2000 Cytotaxonomy of the Bactrocera dorsalis Complex 415 common host plant species (Aglaia sp.). Moreover, species S and U have been encountered in Chumphon province, about 125 km northeast of Ranong province, in addition to the previous records of 3 known species of the B. dorsalis complex, i.e., B. irvingiae, B. arecae and B. melastomatos (Baimai et al. 1999a). Similar to species S and U, species T and V share the same host plant species, Polyalthia sp., and appear to have a distribution wider than species S and U (Table 1). Likewise, species W has a wide distribution ranging from Ranong province to Phangnga province and it has been found infesting fruits of Salacia chinensis. Species X and Z have been encountered only in Ranong province but in different host plant species, i.e., Strychnos sp. and Garcinia sp., respectively. Species Y has only been found in infested fruits of Walsura sp. in Nakon Nayok, central Thailand. Chromosomal evolution Accumulation of constitutive heterochromatin in the genome has played an important role in chromosomal evolution of some species groups of the dipteran insects (Baimai 1998). Our findings in the present study obviously support this notion. Species U exhibits the most extraordinary case of accumulations of constitutive heterochromatin both in the vicinity of the centromeric regions of all autosomes and in the sex chromosomes. Of particular interest, the X chromosome of species U is very unique in having the largest submetacentic shape of all species with a euchromatic portion located in the middle of the long arm. Species V has a large subtelocentric X chromosome with the euchromatic portion located amidst the heterochromatic long arm somewhat similar to that of species U. Nevertheless, species V shows a smaller amount of pericentric heterochromatin than that of species U. From an evolutionary view point, species U could have evolved from species V, or its ancestor, via the gain of extra heterochromatin in the genome. Moreover, species U and V show patterns of pericentric heterochromatin in mitotic chromosomes similar to those of B. carambolae (Baimai et al. 1999a) and, to a lesser extent, species E (Baimai et al. 1995). Baimai et al. (1995) has classified mitotic karyotypes of members of the B. dorsalis complex into 4 groups. Hence, metaphase karyotypes of species U and V may be classified into Group 4. Species U, V, E and B. carambolae are morphologically distinguishable based on differences in dark color patterns in the costal band and in mid tibiae (S. Tigvattananont, unpublished data). Species R shows relatively more prominent blocks of pericentric heterochromatin in all autosomes than those of species W. In contrast, the X chromosome of species R has a submetacentric shape compared with the large metacentric X chromosome of species W Moreover, the Y chromosome of species W is larger than that of species R. Likewise, species Q, Y and Z exhibit patterns of mitotic karyotype similar to those of species R and W, but their X chromosomes are somewhat smaller. Species X also shows a metaphase karyotype similar to those of species Q, Y and Z. Surprisingly, the X chromosome of species X is rather small compared with those of species Q, Y and Z. Moreover, species X exhibits the pericentric heterochromatin patterns similar to that of species K described previously (Baimai et al. 1999b), but the former species apparently contains more heterochromatin than the latter. Although these 2 species occur sympatrically in Ranong province, they seem to infest fruits of different host plant species. Thus species K was found in Payena sp. while species X was detected in Strychnos sp. On the basis of pericentric heterochromatin patterns in the autosomes and the X chromosome, species Q, R, W, X, Y and Z can be placed in Group 2 similar to metaphase karyotypes of B. kanchanaburi and B. raiensis (Baimai et al. 1995). Figs Photomicrographs of metaphase karyotypes using Hoechst staining of 10 new species of the Bactrocera dorsalis complex. 21) sp. Q d, 22) sp. Q Y, 23) sp. Rd, 24) sp. R Y, 25) sp. S d, 26) sp. S Y, 27) sp. Td, 28) sp. T Y, 29) sp. U d, 30) sp. U Y, 31) sp. V d, 32) sp. V Y, 33) sp. W d, 34) sp. W Y, 35) sp. Xd, 36) sp. X Y, 37) sp. Yd, 38) sp. Y Y, 39) sp. Zd, 40) sp. Z Y. An extra block of heterochromatin at the tip of one arm of each X chromosome is indicated by an arrow.

8 416 Visut Baimai et al. Cytologia 65 Fig. 41. Diagrammatic representation of haploid idiograms of metaphase karyotypes of 10 new species (Q-Z) of the Bactrocera dorsalis complex. Black and shade areas represent heterochromatic portions. The centromeres are indicated by constrictions of each chromosome. Chromosome lengths, arm ratios, and heterochromatic portions are shown in proportion. Species S shows a pattern of heterochromatin in the subtelocentric X chromosome similar to that of species Y. Nonetheless, all autosomes of species S strikingly exhibit very small amounts of pericentric heterochromatin compared with those of species Y. Such a cytological difference is a good diagnostic character for separation of these 2 species. Unlike other species studied thus far, species S exhibits a minimal amount of pericentric heterochromatin in all autosomes and has small X and Y chromosomes. Species T, on the other hand, shows a unique submetacentric X chromosome in that it has a small portion of euchromatin at the tip of each arm. This unique type of X chromosome, only encountered once thus far in the B. dorsalis complex, could have arisen from an event of pericentric inversion of the ancestral X chromosome. In addition, all autosomes of species T contain prominent blocks of pericentric heterochromatin. These features of the mitotic karyotype of species S and T are hence assigned to 2 new categories, in addition to those described in Baimai et al. (1995, 1999a, b). Constitutive heterochromatin is generally thought to be genetically inactive. Yet gain of heterochromatin in the genomes of eukaryotes is a common phenomenon in the process of chromosomal evolution (John and Miklos 1979, John 1988). Although the functional role of heterochromatin is still largely unknown, its widespread occurrence in plants and animals (White 1973, John 1988) suggests an important contribution to biological properties and a genetically significant function in higher organisms. In this regard, it has been recently demonstrated in Drosophila melanogaster that constitutive heterochromatin is rich in transposable elements (TEs) of various families (Dimitri 1997). It is further suggested that the accumulation of transposable elements is also a general phe-

9 2000 Cytotaxonomy of the Bactrocera dorsalis Complex 417 nomenon among higher eukaryotes. Thus some of these parts of constitutive heterochromatin may serve as active domains of the genome (Lohe and Hilliker 1995, Gatti and Pimpinelli 1992). Further detailed study on chromosomal evolution among the species members of the B. dorsalis complex may shed some light on the functional role of constitutive heterochromatin in the speciation processes of these fruit fly groups of Southeast Asia. Acknowledgements We wish to thank John Milne, Faculty of Science, Mahidol University, for valuable comments on the draft manuscript. We also thank Somsak Tiengtrong and Wanpen Seukam for technical assistance. This work was partly supported by the Thailand Research Fund (Grant nos. RTA and RTA/01/2541) and the TRF/BIOTEC Special Program for Biodiversity Research and Training (Grant no. BRT ). References Baimai, V Constitutive heterochromatin differentiation and evolutionary divergence of karyotype in Oriental Anopheles (Cellia). Pac. Sci. 42: Heterochromatin accumulation and karyotypic evolution in some dipteran insects. Zool. Stud. 37: Trinachartvanit, W, Tigvattananont, S., Grote, P. J., Poramacom, R. and Kijchalao, U Metaphase karyotypes of fruit flies of Thailand. I. Five sibling species of the Bactrocera dorsalis complex. Genome 38: Phinchongsakuldit, J. and Trinachartvanit, W 1999a. Metaphase karyotypes of fruit flies of Thailand (III): Six members of the Bactrocera dorsalis complex. Zool. Stud. 38: , - and Tigvattananont, S. 1999b. Metaphase karyotypes of fruit flies of Thailand. IV Evidence for six new species of the Bactrocera dorsalis complex. Cytologia 64: Dimitri, P Constitutive heterochromatin and transposable elements in Drosophila melanogaster. Genetica 100: Drew, R. A. I The tropical fruit flies (Diptera: Tephritidae: Dacinae) of the Australasian and Oceanian regions. Mem. Queensl. Mus. 26: and Hancock, D. L The Bactrocera dorsalis complex of fruit flies (Diptera : Tephritidae) in Asia. Bull. Entomol. Res. (Suppl. Ser.) Suppl. 2: Gatti, M. and Pimpinelli, S Functional elements in Drosophila melanogaster heterochromatin. Ann. Rev. Genet. 26: Hardy, D. E. and Adachi, M. S Studies in the fruit flies of the Philippine Islands, Indonesia, and Malaya. Part I. Dacini (Tephritidae-Diptera). Pac. Sci. 8: John, B The Biology of Heterochromatin. In: Verma, R. S. (ed.). Heterochromatin: Molecular and Structural Aspects. Cambridge University Press, Cambridge. pp and Miklos, G. L. G Functional aspects of satellite DNA and heterochromatin. Int. Rev. Cytol. 58: Lohe, A. R. and Hilliker, H. J Return of the H-word (heterochromatin). Current Opin. Genet. Devel. 5: White, M. J. D Animal Cytology and Evolution. 3rd ed. Cambridge University Press, London. White, I. M. and Elson-Harris, M. M Fruit Flies of Economic Significance: Their Identification and Bionomics. Centre for Agriculture and Biosciences International, Wallingford. - and Evenhuis, N. L New species and records of Indo-Australasian Dacini (Diptera: Tephritidae). The Raffles Bulletin of Zoology 47: and Hancock, D. L CABIKEY to the Dacini (Diptera, Tephritidae) of the Asia-Pacific-Australasian Regions. Centre for Agriculture and Biosciences International, Wallingford. Windows CD-Rom.