Constitutive Heterochromatin Differentiation and Evolutionary Divergence of Karyotype in Oriental Anopheles (Cellia) 1

VISUT BAIMAI 2

ABSTRACT: Analysis of the mitotic karyotype of two clusters of closely re lated species of oriental Anopheles, the A . balabacensis and A. macula/us com plexes, has revealed interspecific differences in the amount and distribution of constitutive heterochromatin, particularly in sex chromosomes. Such a quali tative diagnosis of heterochromatin is useful in identification of these sibling species. The cytological evidence indicates a significant role of heterochromatin in chromosomal evolution of anopheline mosquitoes. The novel heterochro matin differentiation in sex chromosomes suggests an evolutionary role in the process of species divergence. Furthermore, extensive intraspecific variations of sex chromosome heterochromatin have been observed in natural popula tions of A . dirus A and B, while chromosomal rearrangement is very rare, if not absent. The gross heterochromatin variation may be correlated with variability in vectorial capacity, which may reflect its functional significance in coevolu tionary processes.

CYTOLOGICAL VARIATION IS A general phe dipteran insects. Several authors working nomenon of the eukaryotic genome. Chromo with Drosophila (Carson and Yoon 1982, somal polymorphism usually persists in nat Carson et al. 1970, Stalker 1972), Chirono ural popul ations of higher organi sms in two midae (Ma rtin et al. 1974, Newman 1977), forms: chromosomal rearrangements and and Simuliidae (Bedo 1977) have discussed quantitative differences in constitutive hetero phylogenetic relationships among closely re chromatin. The former are well known in lated species groups through detailed investi man y groups of Diptera, and are believed to gation of chromosomal rearrangements. The have some adaptive significance in different application of this technique , based on com microenvironments (Dobzhansky 1970). The paratively fixed chromosome inversion and latter, on the other hand, although not un sex chromosome differences of unique arrays common in karyotypic evolution in many of chromosomal polymorphisms, is also use groups of eukaryotes (White 1973), is a form ful in the recognition of cryptic species of of polymorphism whose functional role and anophelines (Coluzzi and Kitzmiller 1975, implications in species differentiation remain Green 1982, Green and Miles 1980, Lambert unclear (Chang and Carson 1985, review by 1983, Saguna 1982, Subbarao et al. 1983). John and Miklos 1979). Constitutive heterochromatin is a general Detectable cytological differences are gen feature of the chromosome complement in erally useful in studies of taxonomy and eukaryotic karyotypes. The pericentromeric evolutionary relationships ofmany groups of heterochromatin can be readily demonstrated by C-band techniques (Arrighi and Hsu 1971). There have been numerous reports I This investigation received financial support from on the natural occurrence of quantitative the UNDP/World Bank/WHO Special Program for Re heterochromatin variation in both plants search and Training in Tropical Diseases, the U.S. and animals (e.g., Baverstock et al. 1982, Army Component (AF RIMS), and Mahidol University Fund . John 1981, John and King 1977, 1983, 2 Mahidd University, Department of Biology, Faculty Pathak et al. 1973, Patton and Sherwood of Science, Rama VI Road, Bangkok, Thailand 10400. 1982, Schweizer 1980, Weimarck 1975). Dif- 13 14 PACIFIC SCIENCE, Volume 42, January/July 1988 ferent degrees of staining intensity and differ chromosomes (2n = 6) with extensive hetero ent locations of constitutive heterochromatin chromatin variation. Kitzmiller (1976) listed apparently are due to different patterns of a total of only 30 species of Anopheles of C-bands. This has been a useful cytotaxo which metaphase karyotype descriptions or nomic technique (White 1978). Of particular figures had been demonstrated. More re interest, detectable heterochromatin dif cently, there have been some reports dealing ferences may provide a useful criterion for with inter- and intraspecific variation of sex identification of closely related species, for chromosome heterochromatin (Baimai et aI. example, in Drosophila (Baimai and Ahearn 1984a, Bonaccorsi et al. 1980, Gatti et al. 1978, Baimai et al. 1983). Recently, this 1977,Green et al. 1985, Vasantha et al. 1982, C-band method for detecting heterochromatin Wibowo et al. 1984). differences together with new fluorescence Comparative studies on karyotype analy techniques have been used successfully in sis of constitutive heterochromatin and chro separating certain cryptic species of Anopheles mosomal rearrangements thus offer a prom (Baimai et al. 1981,Gatti et al. 1977,Vasantha ising opportunity to explore the implications et al. 1982,Wibowo et al. 1984).Thus, hetero of natural chromosomal variation for a bet chromatin variation in natural populations ter understanding of the microevolutionary is of special interest as a model for examining process of oriental anophelines. This paper its possible role in evolutionary divergence at summarizes the cytological observations in the genomic level. some Anopheles species groups that suggest From the evolutionary genetics point of the evolutionary role of heterochromatin view, anopheline mosquitoes are one of the differentiation and briefly outlines some most interesting groups of the oriental fauna . of its implications in the epidemiology of Some of the Anopheles species groups play human malarial parasites. very important roles in transmission of human pathogens in the tropical region. Of the genus Anopheles, subgenera Anopheles SERIES NEOMYZOMYIA and Cellia are the most predominant groups , . each with some 200 known species (Harrison A focal point of attention is the leucosphy and Scanlon 1975, Rao 1984, Reid 1968). rus species group, belonging to the Neomy Some of these described species have re zomyia series of the subgenus Cellia, since ceived close attention for detailed investiga most of its species members, especially the tion because they serve as important vectors Anopheles balabacensis complex, are primary for human malarial parasites. Naturally, vectors of human malarial parasites in their much of the genetic studies of anopheline particular areas of distribution. This species mosquitoes have been made mainly as applied complex is exclusively a forest and foothills field researches on relatively few species of group, and is widely distributed throughout medical importance (Coluzzi and Kitzmiller the oriental region ranging from India 1975, Kitzmiller 1976, White 1980). Little (Assam) through all countries in Southeast work has been done broadly in terms of evo Asia, Taiwan, and the southern part of the lutionary biology. Like all other dipterans, Republic of China (Figure 1). Early sys information on chromosomal polymorphism tematic studies of this species complex of anophelines is best known from studies of showed a wide range of geographical and chromosomal rearrangements as seen in the morphological variation (Colless 1956, Reid polytene chromosomes prepared from larval 1968). Recently recognized species of the salivary glands or ovarian nurse cells of half A . balabacensis complex include A. balaba gravid females (Coluzzi et al. 1979, Green censis, sensu stricto from Sabah and the 1982, Kitzmiller et al. 1973). In contrast, less Philippines, A. takasagoensis from Taiwan, emphasis has been made with regard to the and A. dirus from Thailand (Peyton and Har analysis of metaphase karyotype of an rison 1979, 1980). The results of cytogenetic ophelines in spite of its small number of studies of these species showed that they Differentiation and Divergence in Oriental Anopheles- BAIMA I 15

~ dirusA

~~APAN \ ~ ~ I I I " . / n m XY " mil takasagoensis . ~ J ' - ' ,.,, '.... INDIA .1! ' ;;;i~~ :~ II :: nHDr HXY ~ nPHILIPPINES IV ~~ TNA M ~ § balabacensis I Dr XY KAMPUCHEA ,'1;,] . '", ~ ';'.Y~::::/ ~ ~ ~ dirus C i F ·~_.~..... ~ i. SABAH ~ .. .~ ~~IBI fd 'to X Y nH m HX y ? "'JAVA ~s~~ , ~ :••.. ~~~ '0" ~ B dirusB ~oo~~-p~ '. ," . INDONESTA ,f .. ~ I HDr XY AUSTRALIA n

F IGURE I. Distributions of the six species of the Anopheles balabacensis compl ex in Southeast Asia, depict ed with diagrammatic representati ons of mitot ic chro moso mes using Giemsa staining. Heterochromatin is indicated in black or shaded. were distinct biological species (Baimai et al. easily by chromosomal analyses. In addition, 1981 , Hii 1982). However, Kanda et al. species members of the A . balabacensis com (1981, 1983) have suggested that the strain s plex have different geographical distributions of " A. balabacensis" from Thailand, pen (Table 1, Figures I, 2). Anopheles balabacensis insular Malaysia, and Sabah represent three is restricted to Sabah , East Kalimantan, and subspecies of this species complex. Detailed Palawan, while A. takasagoensis has been taxonomic studies (Peyton and Harrison, per recorded only from Taiwan. Members within sonal communication) together with cyto the taxon A . dirus have been found on the genetic analysis of natural populations of mainland of Southeast Asia. It is interesting A. dirus (Baimai et al. 1984b, 1987) have re to note that A. dirus D is relatively wide vealed that it is actually a cluster of at least spread in western Thailand, occurring in sym four closely related species provisionally patry with its siblings. On the other hand , designated as dirus A, B, C, and D. These A. dirus A is common in central and northern siblings are only partially morphologically Thailand, while A. dirus B is narrowly dis distinguishable, but they can be separated tributed in the area of the Thailand-Malaysia 16 PACIFIC SCIENCE, Volume 42, January/July 1988

TABLE I COLLECTING DATA FOR POPULATION SAMPLES AND MITOTIC SEX CHROMOSOME CONFIGURATIONS OF THE A . balabacensis AND A . maculatus COMPLEXES

SEX CHROMOSOME

SPECIES LOCALITY (COLLECTION DATE, MONTHjYEAR) X Y

Anopheles balabacensis complex dirus A I-CM(9j84), 2-LP(6j84), 3-PA(8j83), 4-PN(9j84), t* t* 5-L0(7j84), 6-KN(lOj80), 7-NN(9j82), 8-PB(8 j82), 9-TL(8j83), IO-CH(8j83), II-RN(10j84), 16-SK(8 j85) dirus B 12-PG(6j82), 14-PT(2 j85), 15-TG(5j82) a* a* dirus C 6-KN(10j80), I3-SC(12j84), 14-PT(2j85) t* dirus D I-CM(9j84), 4-PN(9j84) , 6-KN(lOj80), 7-NN(9j82), t* 8-PB(8 j82), 9-TL(8j83), IO-CH(8j83), 11-RN (lO j84), 12-PG(6j82) balabacensis SAB (4j84) t* taka sagoensis TAWt t* Anopheles maculatus complex maculatus A I-CM(9j84), 3-PA(8j83), 6-KN(lOj82), 7-NN(9j82), 9-TL(8j83), IO-CH(8j83) maculatus B I-CM(9j84) , 7-NN(9j82), 9-TL(8j83), IO-CH(8j83) sm* a maculatus C I-CM(9j84),6-KN(lOj82) t t maculatus G 9-TL(8j83) sm* sm

NOTE : t = telocentric (rod shape); a = acrocentric; sm = submetacentric. See Figure 2 for locality numbers and abbreviations of collecting sites; SAB = Sabah, East Malaysia; TAW = Taiwan. • Heterochromatin variation observed . t Laboratory colony maintained at AFRIMS. border (Figure 2). These two species are allo takasagoensis, each of which shows X and Y patric. Species C has a somewhat limited dis chromosomes of roughly similar size. Never tribution in southern peninsular Thailand. theless, A. dirus A, C, and takasagoensis Analyses of larval mitotic chromosomes of display significantly different fluorescent the six members of the Anopheles balaba banding patterns of major blocks of inter censis complex using conventional Giemsa calary heterochromatin in the X and Y chro staining methods (Baimai et al. 1981) and mosomes, although they cannot be readily Hoechst 33258 fluorescent banding tech distinguishable by the Giemsa staining tech niques (Wibowo et al. 1984) have revealed nique (compare Figures 1 and 3). The sex marked differences in the amount and distri chromosomes of A. dirus B, on the other bution of constitutive heterochromatin both hand, are unique in having distinct acrocent in sex chromosomes and at centromeric posi ric configurations. The short arm of these sex tions of autosome(s) (see Figures 1, 3). The chromosomes is entirely heterochromatic, metaphase karyotype (2n = 6) of all mem which is likely to be due to the addition of bers of the A . balabacensis complex, except extra heterochromatin, although the possi for A . dirus B, exhibits typical telocentric sex bility of pericentric inversion of the centro chromosomes of various sizes, which are meric heterochromatin cannot be ruled out. clearly attributable to different amounts of Additionally, A. dirus B shows a consider heterochromatin in the vicinity of the cen able amount of centromeric heterochromatin tromere as well as qualitative differences of in all autosomes. Thus, the apparent hetero intercalary heterochromatin. Thus, the sex chromatin differentiation is most extensive in chromosomes of A. dirus D are relatively A. dirus B compared with the other members smaller than those of A. dirus A, C, and of the complex. While sex chromosomes of 20 BURMA

18 18"

16 16 "

ANDAMAN 14 SEA 14"

10 0 A dirusA -10 • dirus B 6. dirus C Odirus 0 s 8 °

99" 1050 107 ~

FIGURE 2. Map of Thailand and northern part of peninsular Malaysia showing collection localities of the four siblings within the taxon Anopheles dirus used for chromoso me analyses. Th e localities (with abbreviations) of these sibling species and sample sizes (indicated in parenth eses) are as follows: I, Chiangmai-CM (A = 14, D = 6); 2, Lamp ang-LP (A = 6); 3, Phrae-PA (A = (5); 4, Pitsanulok-PN (A = 133, D = 15); 5, Loei-LO (A = 6); 6, Kanchanaburi-KN(A = 7, C = 9, D = 5); 7, Na khon Nayok-NN (A = 4, D = 3); 8, Prachinburi-PB (A = 7, D = 5); 9, Petchabu ri-TL (A = 17, D = 25); 10, Chantaburi-CH (A = 16, D = 12); II , Ranong-RN (A = 2, D = 59); 12, Phangnga-PG (B = 2, D = 5); 13, Sichol-SC (C = 73); 14, Phatthalung-PT (B = 46, C = 2); 15, Trengganu, Malaysia-TG (B = 5); 16, Sakon Nakh on-SK (A = 52). 18 PACIF IC SCIENCE, Volume 42, January/July 1988 dirus A dirus 8

][ Dr x y n m x y dirus C dirus D , II ill x y II Dl X y balabacensis takasagoensis

i'"" I y n n X y II ill X FIGURE 3. Diagrammatic representations of fluorescent banding patterns of mitoti c karyotypes of the six species of the Anopheles balabacensis complex. Variable heterochromat ic portion is indicated in black or shaded. Differentiation and Divergence in Oriental Anopheles- HAIMAI 19

A. balabacensis appear to be similar to tho se almost identical (Hii 1982). In addition, of A. dirus D, these two species members do A. dirus D shows a fixed paracentric inver show significant differences in centromeric sion in the X chromosome that differs from heterochromatin of autosome III (Figure 3). the banding sequence of other species mem These two are, of course, allop atric species. bers (Baimai et al. 1987). The remaining four members of this species It may be noted that Anopheles dirus A complex do not exhibit any detectable differ and C are virtually homosequential species, ences of centromeric heterochromatin of the showing almost identical banding patterns autosomes. Such a gross chromosome differ and nearly complete synapsis in F1 hybrid ence with respect to constitutive heterochro polytene chromosome complement, except matin is quite useful in species identification for the density of bands and sequences at of this species complex and is routinely em the very tips of chromosome arms 2R, 2L, ployed in our laboratory. Furthermore, nat and the X chromosome (Baimai et al. 1987). ural population samples of all siblings within Such a remarkable cytological difference is the taxon A . dirus in Thailand exhibit quan extremely useful for the unambiguous iden titative variation of centromeric heterochro tification of these two genetic species, since matin of the X chromosome. To complicate they cannot be distinguished by mitotic sex matters further, A . dirus A and B show even chromosomes using conventional Giemsa more extensive heterochromatin variation in methods, as mentioned above . This phe the vicinity of the centromere of both X and nomenon of banding differences at the free Y chromosomes (Baimai and Traipakvasin ends of polytene chromosome arms has been 1987, Baimai et al. 1984a). The evolutionary observed in some closely related species of role and epidemiological significance of such Drosophila (Hagele and Ranganath 1982), an extensive heterochromatin variation re especially certain homosequential species of main unknown. Hawaiian Drosophila (Ahearn and Baimai Data on polytene chromosome anal ysis 1987). of the Anopheles balabacensis complex is Even though these observations tend to somewhat less extensive due to the difficulty limit the use of mitotic karyotype analysis as of making reasonably good salivary gland a reliable guide to phylogenetic relation chromosome preparations. The limited data ships, the cytological evidence presented here available indicate that A. dirus D is relatively seems to suggest a significant role of hetero highly polymorphic for chromosomal rear chromatin in evolutionary divergence of the rangements. At least six chromosome inver Anopheles balabacensis species complex. In sions have been observed distributed in five the case of sympatric species, for instance, chromosome arms (two in arm 2R and one A. dirus A and D or A . dirus Band D, there each in other arms) in Ranong populations. has been no record of interspecific hybridiza Species A shows a simple inversion in the tion in nature. However, laboratory cross X chromosome in some populations. On the mating experiments among these members contrary, no chromosomal inversion is rec were possible by artificial insemination, and orded in A. dirus Band C from the Thailand produced variable degrees of reproductive population samples examined so far (Baimai, isolation. Data from these hybridization ex unpublished). Chromosomal rearrangement periments between A. dirus A and C indicate of A. balabacensis from Sabah is also rare , if that they are the least reproductively isolated not absent (Hii, personal communication), species compared to other member species while chromosomal polymorphism in nat (Baimai et al. 1984b, 1987). Cross-mating ural populations of A. takasagoensis from involving A. dirus A 0 x C ~ produced all Taiwan is not known. Striking differences fertile F1 hybrids, while the reciprocal cross have been found in the free ends of X chro yielded fertile F1 hybrid females but sterile mosomes of A. dirus A, B, and balabacensis, F1 males. All other combinations of inter even though the general banding patterns of specific crosses gave either sterile F1 hybrid the other polytene chromosome arms were males or even sterile eggs, which reflected 20 PACIFIC SCIENCE, Volume 42, January/July 1988

high degrees of genetic incompatibility. control elements for mating behavior holds These hybridization data seem to support true in general for insects, it is easy to visu the chromosomal observations mentioned alize the effects of heterochromatin differen earlier. tiation in the Y chromosome in association Usually, the general trend ofchromosomal with sexual isolation giving rise to the specia evolution of eukaryotes tends toward gain of tion process of this sibling species complex. heterochromatin (John and Miklos 1979). These findings seem parallel to the situation In this view, Anopheles dirus A may conceiv encountered in the D. grimshawi-bostrycha ably be considered a common ancestor of disjuncta complex of Hawaiian Drosophila this cryptic species, since it has maintained (Ahearn and Baimai 1987, Baimai and the supposedly original types of X, and Y I Ahearn 1978). Further in-depth investigation chromosomes. Moreover, the extraordinary may help elucidate the evolutionary signifi condition of gross DNA content at the free cance of heterochromatin differentiation en ends of chromosome X, arm 2R, and arm 2L countered in sibling species within the taxon of A. dirus C makes it possible to conceive A. dirus. that this species was directly derived from Cytogenetic evidence suggests that An A . dirus A. If such is the case, A. dirus C opheles dirus Band D are remote from A. might be more recently evolved from A. dirus dirus A. There is also some suggestion that A than the other two sibling species. The A. dirus Band D are independently derived process of speciation may be accompanied from a common ancestor, A. dirus A. This by change and/or amplification of DNA involves accumulation of heterochromatin in sequences at the free ends of the three chro sex chromosomes and autosomes in the case mosome arms, probably facilitated by dif of A . dirus B and fixing of a paracentric ferentiation of (intercalary) heterochromatin inversion in the X chromosome and hetero of the sex chromosomes. These kinds of chromatin differentiation in A. dirus D. The chromosomal differentiation may be as present evidence is not conclusive. Since sociated with feeding and mating behavior. A. balabacensis and A. takasagoensis are geo -Indeed, field observations indicate that out graphically isolated from each other and door biting activity of A. dirus C occurs at from all other members within the taxon a high level in the early hours of the night A. dirus, it is not difficult to envisage that (6: 30- 7:30 PM), then decreases sharply, and these two distinct biological species arose by is maintained at a very low level through allopatric speciation via genetic divergence the remainder of the night. On the contrary, expressed, perhaps, by the accompanying outdoor biting activity of A. dirus A starts heterochromatin differentiation. relatively later in the first half of the night, with a peak period around 8:00-9:00 PM, and gradually decreases toward the second half SERIES NEOCELLIA of the night. Such differences in feeding and perhaps mating behavior may significantly The Anopheles maculatus species group of reflect an underlying mechanism for species the Neocellia series of the subgenus Cellia differentiation of these very closely related is of special interest because it exhibits geo species. Concerning this aspect, it has been graphically morphological variation in cor demonstrated that some control factors in relation with variable degrees of vectorial mating behavior of Anopheles mosquitoes are capacity in different areas of its distribution associated with the Y chromosome (Fraccaro (Reid 1968). Detailed cytogenetic studies of et al. 1977). Moreover, data from detailed " A. maculatus" samples of Thailand popula cytological and genetic analyses of the Y tions revealed that this taxon is in fact a cryp chromosome of Drosophila melanogaster tic species comprised of at least four closely suggest that fertility factors are located in the related species that occur in sympatry at some Y chromosome (Gatti and Pimpinelli 1983). localities. These members of the A. maculatus If this phenomenon of fertility factors and complex are provisionally designated macu- Differentiation and Divergence in Oriental Anopheles- HAIMAI 21

latus A, B, C, and G, all of which show dif tions of "ma culatus B" may reflect a different ferent banding patterns in ovarian polytene form, or even subspecies, which is provision chromosomes as well as quantitative differ ally designated form E. Thus, form E is the ences of sex chromosome heterochromatin only representative form of maculatus B in (Green and Baimai 1984, Green et al. 1985). southern Thailand, where it is known as one Like the A . balabacensis complex, Giemsa of the vectors of human malarial parasites. C-banding of the metaphase karyotype of Other members of the A. maculatus complex four members of the A. maculatus complex are not considered as vectors in Thailand. It displays distinctive X and Y chromosome is not known whether such cytological differ configurations. The telocentric Y chromo ences are correlated with variation in vecto some of A. maculatus A is longer than that rial capacities among members ofthis species of A. maculatus C. On the other hand, the complex. entirely heterochromatic Y chromosome of Field data are being gathered on the mitotic A. maculatus B is obviously acrocentric com karyotype of inter- and intraspecific vari pared with the extraordinarily large subrneta ation of sex chromosome heterochromatin centric Y configuration of A. maculatus G of other species groups within the series (see Green et al. 1985). The X chromosome Anopheles annularis and others of the ori of both A. maculatus A and C shows telocen ental Anopheles (Baimai, unpublished). The tric shape and similar size, with about two dramatic evidence available so far seems to fifths of the chromosome length at the distal suggest that heterochromatin differentiation end being euchromatic. In contrast, the X often plays an important role in karyotypic chromosome of A. maculatus Band G is evolution in anopheline mosquitoes, at least clearly submetacentric, of which approxi in the oriental region. mately two-thirds of the short arm at the distal region is euchromatic while the long arm is totally heterochromatic. Intraspecific EVOLUTIONARY ROLE OF CONSTITUTIVE heterochromatin variation in the X chro HETEROCHROMATIN mosome of A. maculatus Band G has been observed in natural samples (Baimai, unpub Although the formulation of models for lished). Therefore, apart from interspecific speciation in relation to heterochromatin differences in chromosomal rearrangements, differentiation is inevitably in the realm of distinctive sex chromosome heterochromatin speculation, the foregoing cytogenetic data is also a species-specific cytological char indicate some implications of heterochro acter of these members within the taxon matin in the phylogenetic affinity and con A. maculatus. Like the A. balabacensis com sequently the evolutionary divergence of plex, trends of karyotypic evolution by mem these and other cryptic species of Anopheles bers of the A. maculatus complex apparently in Southeast Asia. are due to accumulation of sex chromosome Cytological differences may not exist in heterochromatin. Furthermore, members of some closely related species, since cytological these four genetic species show morphologi change does not appear to be an absolute cal differences in certain stages of develop prerequisite for speciation (Carson 1982a, ment including egg raft, pupal skin, and Chang and Carson 1985). For example, adult morphology. A formal taxonomic study several members of homosequential species of the A . maculatus group has been made by groups of Hawaiian Drosophila are primarily Rattanarithikul and Green (1986). Interest recognized by morphological and behavioral ingly enough, populations of "maculatus B" differences (Carson 1982b, Carson and Kane in southern peninsular Thailand show sig shiro 1976, Val 1977). In contrast, in many nificantly different frequencies of some chro taxa, cytological differences by virtue ofeither mosomal rearrangements from the central chromosomal changes (e.g., gene rearrange and northern populations. Such unique poly ments , fusions, translocations) or hetero tene banding patterns of southern popula- chromatin variation, or both, occur between 22 PACIFIC SCIENCE, Volume 42, January/July 1988 closely related species or sibling species complex. In contrast, all species members of groups that are virtually similar in gross the A . balabacensis complex are generally morphology. Such chromosomal changes are homosequential, but they are strikingly dif often thought to be involved, to some degree, ferent in the amount and distribution of con in species formation (White 1978). A con stitutive heterochromatin in sex chromosomes siderable number of animals, particularly and the centromeric autosomes. These meta insects, are assigned to certain phylogenetic phase karyotype differences may be accoun relationships of species groups on the basis ted for by gain of heterochromatin, which of chromosome differences and interspecific is a common phenomenon in chromosome mating ability (Dobzhansky 1970, White evolution (John and Miklos 1979). This kind 1973). The existing knowledge in karyotypic of cytological difference is comparable to the evolution comes mainly from detailed inves situation of some homo sequential species of tigation of natural populations of animals, the picture-winged Hawaiian Drosophila, for instance, in Drosophila (e.g., Baimai et al. (Ahearn and Baimai 1987, Baimai and Ahearn 1983, Clayton 1968, 1969, Patterson and 1978, Clayton 1969). Cytogenetic evidence in Stone 1952, Yoon and Richardson 1978), these species groups and others of Anopheles stick insects (Craddock 1974), ants (Imai et examined so far suggests that sex chromo al. 1977), grasshoppers (White 1974), and somes are particularly prone to the accu some groups of mammals (Baverstock et al. mulation of extra heterochromatin, ranging 1977, Mascarello and Hsu 1976, Pathak et al. from noticeably small, to moderately and ex 1973, Patton and Sherwood 1982). Data on tremely large amounts. The mechanism for karyotype analysis of different taxonomic the increased amount of heterochromatin is groups of animals suggest a possible role of not fully understood. It may be the corollary constitutive heterochromatin in the process of different phenomena, e.g., unequal cross of evolutionary divergence. ing over and chromosomal breakage (review Increasing cytogenetic evidence has led by John and Miklos 1979). Thus, heterochro to some new insights into the evolutionary matin differentiation may be generally con biology of Anopheles that may afford answers sidered as a recurrent feature of chromosome to the possible relationships among closely changes, which presumably serve as a medium related species groups and probable modes for genetic divergence and subsequent evolu of speciation (Kitzmiller 1976). Further, re tionary process. cent recognition of cryptic species in some Earlier, it was thought that heterochromatin taxa of the oriental Anopheles provides a was geneticallyinert and hence had no effecton better understanding of mechanisms under the biology and viability of higher organisms lying the speciation processes of these medi (review by Brown 1966). However, biochemi cally important mosquitoes (Baimai et al. cal evidence suggests that heterochromatin 1981, Green and Baimai 1984, Green et al. is correlated with highly repetitive DNA se 1985, Hii 1982, Subbarao et al. 1983). Cyto quences or satellite DNA arranged tandemly logical differences in the karyotype and sali in the vast majority of the genome (Britten vary gland polytene chromosomes of some and Kohne 1968, Peacock et al. 1977). The species of Culicidae have been described by repeated DNA sequence of chromatin repre several workers (for a review, see Kitzmiller sents noncoding DNA, and consequently has 1967, 1976). As exemplified in this paper, a no direct function or immediate phenotypic striking feature of cytological variation of benefit. The presence or absence of a highly the oriental Anopheles is the wide range of repetitive DNA sequence or heterochro chromosomal rearrangements and sex chro matin does not seem to exert direct genetic mosome evolution via variable degrees of effects similar to the unique DNA sequence heterochromatin differentiation. Thus, cyto associated with the addition or deficiency logical differences both in polytene chromo of euchromatin. Nonetheless, some repetitive some inversions and in mitotic sex chromo DNA sequences may serve a regulatory func some heterochromatin are conspicuous among tion upon the unique DNA sequences (Britten the four species members of the A. maculatus and Davidson 1969, Hilliker and Appels Differentiation and Divergence in Oriental Anopheles-HAIMAI 23

1980). Repetitive noncoding DNA also may preeminence of sex chromosome heterochro show much faster sequence divergence than matin in the evolutionary divergence of these the unique DNA sequence of euchromatin closely related species groups, although they (Macgregor et al. 1976).It has been suggested do not deal explicitly with the postulated that species differentiation may involve some correlation. Further investigation into the changes in the gene regulatory system be dynamic element of heterochromatin and its sides mutation of the coding DNA (Britten evolutionary significance remains intriguing and Davidson 1969). From this point of and challenging. view, heterochromatin may be envisaged as a potential source of evolutionary change in the regulatory context. Hence, instead of using the term "genetically inert" for a HETEROCHROMATIN VARIAnON AND highly repetitive DNA sequence or hetero EPIDEMIOLOGICAL IMPLICAnONS chromatin in the sense of selfish, symbiotic, It seems clear that heterochromatin is or ignorant parasitic DNA (e.g., Doolittle involved in karyotypic evolution in many and Sapienza 1980, Orgel and Crick 1980), groups of organisms , but the functional role we may consider the term "dynamic element" of heterochromatin has not been established. of the genome. Several investigators have By and large, heterochromatin does not inter evoked an evolutionary role of heterochro fere with the normal function and devel matin in attempting to explain the common opment of the organisms containing it, but phenomenon of heterochromatin differentia whether it attributes some advantages to the tion (Dover et al. 1981 , Nagl and Capesius organism is still obscure. There is an increas 1977). As yet, there has been no compelling ing awareness of the possibility that hetero evidence supporting these views. chromatin may exert some functional role in The karyotypic differences among mem the internal environment, where it behaves as bers of the Anopheles dirus and A. maculatus a not-too-harmful "parasite" within the host species complexes are due mainly to the cell. In this view, one may speculate that the changes in quantity and/or quality of sex extra ("parasitic") heterochromatin of the chromosome heterochromatin. The direction insect vectors might exercise its dynamic role of chromosome changes tends, in most cases, in interacting favorably with the true para toward the acquisition of extra heterochro sites, e.g., Plasmodium transmitted by the matin , which can be comparatively deter mosquito hosts. Thus, heterochromatin dif mined by Giemsa banding techniques . Thus , ferentiation may play an important role in almost all Thailand populations of these coevolutionary processes. Such speculation species subgroups harbor large amounts of can be tested. In the Anopheles gambiae com heterochromatin variation in sex chromo plex, differences in constitutive heterochro somes, which may reflect, to some degree, the matin in the sex chromosomes may be related different interspecific nature of regulatory to different responses in vectorial capacity of gene systems. The critical problems revolve two closely related species, A . gambiae and around how consistent this kind of hetero A . arabiensis (Bonaccorsi et al. 1980). From chromatin differentiation is, and whether this viewpoint, it is suggested that a serious any differentiation is based on persistent ge effort should be made to determine whether netic divergence. If this type of heterochro there is any correlation between hetero matin differentiation is to play any significant chromatin differences and variable degrees role in influencing levels of genetic divergence of vectorial capacity, both within a species within populations of these mosquitoes, it and between sibling species of potentially should be a repeatable phenomenon both effective vectors, e.g., within the A. dirus within populations of a species over several and A. maculatus complexes. Both variable seasons and between populations of different effects, susceptibility to malarial parasites species that have experienced the same set of and heterochromatin, of the vectors may be selective pressures of the genome. In any acting in concert in nature. In this respect, event, the present data seem to suggest the preliminary laboratory tests show that dif- 24 PACIFIC SCIENCE, Volume 42, January/Jul y 1988 ferent members of these two Anopheles genetic relationships of Drosophila affini species groups disp lay significant differences disjuncta H ardy. Amer. Midl. N at. 99(2) : in susceptibility (R. G . Andre, personal com 352-360. munication). If heterochromatin variations BAIMAI, V., and A. TRAIPAKVASIN. 1987. In in the sex chromosomes affect the genetic traspecific variation in sex heterochro basis of susceptibility, then the development matin of species B of the Anopheles dirus of strategies for vector control measures be complex in Thailand. Genome 29 :40 1 comes critically important. It is not difficult 404. to visualize how the sex chromosome hetero BAIMAI , V., B. A. H ARRISON, and L. SOMCHIT. chromatin might play an important rol e in 1981. K aryotype differentiation of 3 An reproductive isolation and, subsequently, opheline taxa in the balabacensis complex species differentiation in some anopheline of Southeast Asia (Diptera: Culicidae ). mo squitoes (Bonaccorsi et al. 1980, Fraccaro Genetica 57: 81- 86. et al. 1977). Yet such a deductive interpreta BAIMAI , V., F . M. SENE, and M . A. Q. R . tion is merely circumstantial and aw aits suit PEREIRA. 1983. Heterochromatin and karyo able experimental tests. typic differentiation of some neotropical Genetic changes ranging from point muta cactus-breeding species of the Drosophila tions through chromosomal rearrangement repleta species gro up. Genetica 60: 81-92. and including the load of highly repetitive BAIMAI, V., R. G . ANDRE, and B. A. HARRISON. DNA sequences are principally ra w materials 1984a. Heterochromatin variation in the sex for evolutionary divergence. Studies of the chromosomes in Thailand populations of latter should offer some insights pertaining Anopheles dirus A (D iptera : Culicidae). to the evolutionary role of sex chromosome Can. J . Genet. Cytoi. 26 :633-636. heterochromatin in speciation processes. The BAIMAI , V., C. A. GREEN, R . G . ANDRE, B. A. heterochromatin differentiation in sex chro H ARRISON, and E. L. PEYTON. 1984b. mo somes of the Anopheles species groups is Cytogenetic studies of some species com an appropriate candidate for thi s approach. plexes ofAnopheles in Thailand and South Although it may seem naive, this is an excel ea st Asia. Southea st Asian J . Trop. Med. len t opportunity to put evolutionary genetics Pub. Hlth. 15: 536-546. into the field ofapplied research. BAIMAI , V., R. G . ANDRE, B. A. HARRISON, U . KUCHALAO, and L. PANTHUSIRI. 1987. Crossing and chromosomal evide nce for ACKNOWLEDGMENTS two additional sibling species within the taxon Anopheles dirus Peyton and Harrison I wish to thank H. L. Carson and W. W . M. (Di ptera: Culicidae) in Thailand. Proc. Steiner for their useful comments on the Entomol. Soc . Wash. 89 : 157-166. manuscript. I also thank J. Hii for provid BAVERSTOCK, P. R ., C. H . S. WATTS, and J. T . ing mosquito samples from Sabah. The ex HOGARTH. 1977. Chromosome evolution pert technical assistance of A. Traipakvasin, in Australian rodents. II . The Rattus U. Kijchalao, and K . Vejsanit is greatly group. Chromosoma (Berl.) 61 :227-241. appreciated. BAVERSTOCK, P. R ., M . G ELDER, and A. JAHNKE. 1982. Cytogenetic studies of the Austral ian Rodent, Uromys caudimaculatus, LITERATURE CITED a species showing extensive heterochro AHEARN, J. N ., and V. BAIMA!. 1987. Cyto matin variation. Chromosoma (Berl.) 84 : genetic study of three closely related 517-533. species of Hawaiian Drosophila. Genome BEDO, D . G . 1977. Cytogenetics and evolu 29: 47- 57. tion of Simulium ornatipes Skuse (D iptera: ARRIGHI, F. E., and T . C. Hsu. 1971. Locali Simuliidae). 1. Sibling speciation. Chro za tion of heterochromatin in human chro mo soma (Berl.) 64 :37- 65. mo somes. Cytogenetics 10 :81-86. BONACCORSI, S., G . SANTINI, M. G ATTI, BAIMAI , V., and J . N . AHEARN. 1978. Cyto- S. PIMPINELLI, and M . COLUZZI. 1980. Differentiation and Divergence in Oriental Anopheles-HAIMAI 25

Intraspecific polymorphism of sex chro R. C. King, ed. Handbook of genetics. mosome heterochromatin in two species of Vol. 3. Plenum, New York. the Anopheles gambiae complex. Chromo COLUZZI, M., A. SABATINI, V. PETRARCA , soma (Berl.) 76 :57-64. and M. A. DI DECO. 1979. Chromosomal BRITTEN, R. J., and E. H . DAVIDSON. 1969. differentiation and ad aptation to human Gene regulation for higher cells: A theory. environments in the Anopheles gambiae Science 165: 349-357. complex. Trans. R. Soc. Trop. Med. Hyg. BRITTEN, R. J., and D. E. KOHNE. 1968. Re 73:483-497. peated sequences in DNA. Science 161: CRADDOCK, E. M. 1974. Chromosomal evo 529-540. lution and speciation in Didymuria. Pages BROWN, S. W. 1966. Heterochromatin. Sci 24-42 in M. J. D. White, ed. Genetic mecha ence 151:417-425. nisms of speciation in insects. Australia CARSON, H. L. 1982a. Speciation as a major and New Zealand Book Co ., Sydney. reorganization of polygenic balances. DOBZHANSKY, T. 1970. Genetics of evolu Pages 411-433 in C. Barigozzi, ed. Mech tionary process. Columbia Univ. Press , anisms of speciation. Alan R . Liss, New New York. York. DOOLITTLE, W. F., and C. SAPIENZA. 1980. --- . 1982b. Evolution of Drosophila on Selfish genes, the phenotype paradigm and the newer Hawaiian volcanoes. Heredity genome evolution. Nature 284 :601-603. 48 :3-25. DOVER, G . A., T. STRACHAN, and S. D. M . CARSON, H. L., and K. Y. KANESHIRO. 1976. BROWN. 1981. The evolution of genomes Drosophila of Hawaii: Systematics and in closely related species. Pages 337-349 in ecological genetics. Ann. Rev . Ecol. Syst. G. G. E. Scudder and J. L. Reveal, eds. 7:311 -345. Evolution today. Proc. 2nd Internat. CARSON, H . L., and J. S. YOON. 1982. Ge Congr. Syst. Evol. Biol, Carnegie Mellon netics and evolution of Hawaiian Droso Univ., Pittsburgh, Pa. phila. Pages 297-344 in M. Ashburner, FRACCARO, M., L. TIEPOLo, U . LOUDANI, H . L. Carson, and J. N . Thompson, Jr., A. MARCHI, and D. S. JAYAKAR . 1977. eds. The genetics and biology of Droso Y chromosome controls mating behavior phila. Vol. 3b. Academic Press, New York. in Anopheles mosquitoes. Nature 265 : CARSON, H. L., D . E. HARDY, H. T. SPIETH, 326-327. and W. S. STONE. 1970. The evolutionary GATTI, M., and S. PIMPINELLI. 1983. Cyto biology of the Hawaiian Drosophilidae. logical and genetic analysis of the Y chro Pages 437-543 in M. K. Hecht and W. C. mosome of Drosophila melanogaster. 1. Steere, eds. Essays in evolution and ge Organization of the fertility factors. Chro netics in honor ofTheodosius Dobzhansky. mosoma (Berl.) 88 :349-373. Appleton-Century-Crofts, New York. GATTI, M., G . SANTINI, S. PIMPINELLI, and CHANG, L. S., and H. L. CARSON. 1985. Meta M. COLUZZI. 1977. Fluorescence banding phase karyotype identity in four homo techniques in the identification of sibling sequential Drosophila species from Hawaii. species of the Anopheles gambiae complex. Can. J. Genet. Cytol. 27 :308-311 . Heredity 38: 105-108. CLAYTON, F. E. 1968. Metaphase configura GREEN, C. A. 1982. Population genetical tions in species of the Hawaiian Droso studies in the genus Anopheles. Ph .D . The philidae. Univ. Texas Pub. 6818:263-278. sis. Univ. of the Witwatersrand, Johannes - - -. 1969. Variations in metaphase chro burg, South Africa. mosomes of Hawaiian Drosophilidae. GREEN, C. A., and V. BAIMAI. 1984. Polytene Univ. Texas Pub. 6918 :95-110. chromosomes and their use in species COLLESS, D. H. 1956. The Anopheles leuco studies of malaria vectors as exemplified sphyrus group. Trans. R. Entomol. Soc. by the Anopheles maculatus complex. Lond.108:37-ll6. Pages 89-97 in V. L. Chopra, B. C. Joshi, COLUZZI, M ., and J. B. KITZMILLER . 1975. R. P. Sharma, and H . C. Bansal, eds. Ge Anopheline mosquitoes. Pages 285-309 in netics : New frontiers. Proc. 15th Internat. 26 PACIFIC SCIENCE, Volume 42, January/Jul y 1988

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