Evolution of the Alpha-Esterase Duplication Within the Montana Subphylad of the Virilis Species Group of Drosophila

EVOLUTION OF THE ALPHA-ESTERASE DUPLICATION WITHIN THE MONTANA SUBPHYLAD OF THE VIRILIS SPECIES GROUP OF DROSOPHILA

WILLIAM K. BAKER Department of Biology, University of Utah, Salt Luke City, Utah 84112 Manuscript received May 1, 1979 Revised copy received August 27, 1979

ABSTRACT Previous studies on linkage disequilibrium involving four tightly linked genes that code for the alpha-esterases of Drosophila montana suggest that these loci arose from a primitive esterase gene by gene duplication, Iollowed by tandem duplication (ROBERTSand BAKER 1973). We have examined the esterase variants in the closely related species, Zacicola, fluvomontana and borealis. These studies reveal that borealis has only a single esterase locus, and flmomontana may have only two loci. Cytological studies, using aceto-orcein staining and Hoechst fluorescence of squashes of ganglion chromosomes, reveal acrocentric Y chromosomes for all six species of the montana phylad, with the exception of borealis, which has the primitive rod-shaped Y chromosome. These studies provide evidence ayainst the hypothesis (STONE,GUEST and WILSON1960) that borealis and flavomontana are derived from montana, but support THROCKMORTON’S(1978) conclusion of the early divergence of the former two species. This phylogenetic relationship supports our contention that the difference in the number of esterase genes with active alleles between borealis and montana is based on an increase in the number of genes coding for the alpha-esterases, rather than the retention in borealis of three genes with null alleles.

HERE are four tightly linked genes on chromosome 2 of D. montana that code for esterases and that can utilize alpha-naphthyl acetate as a substrate. They have the following characteristics (ROBERTSand BAKER1973; BAKER1975): (1) Each gene has one active allele (whose protein has a characteristic electro- phoretic migration) and a null allele, both of which are present in the population at high frequencies. Other active alleles are known at each locus, but are only rarely found in natural populations. (2) The gene order on the chromosome is GI,Gs, G,, G,. (3) There is strong linkage disequilibrium in natural populations favoring chromosomes with two active and two null alleles. Chromosomes with 0, 1, 3 and 4 active alleles are found in the populations much less frequently than expected. (4) However, only particular chromosomes with two active alleles are “favored,” namely those with one active allele at gene 1 or 2 and the other active allele at gene 3 or 4.

Genetics 94: 733-748 March, 1980. 734 W. K. B.SKER Data (BAKER1975) support the hypothesis that this specific and unusual linkage disequilibrium is maintained by selection, instead of being caused by histori- cal accident, since it is stable over time and space. On a selective basis, one can propose that G, and G, code for a group of related esterases, Cs, and G, and G, for E,’s; a chromosome with two active loci that code for both types of esterases is favored. Such a system of related and tightly linked genes could have arisen during evolution by either of the processes of gene duplication shown in Figure 1. It should be realized that the proposed scheme of gene order and function in present-day montana is based on a selectionist interpretation of the linkage disequilibrium data from natural populations and that the postulated duplicative events leading to this arrangement are hypothetical. We have studied the alpha-esterase variants in a series of species closely related to montarm and have found substantial support for the duplicative origin of these loci, as proposed in Figure 1. Cytological studies confirm that borealis, with but a single esterase locus, is phylogenetically more primitive than montana: with four loci.

Scheme 1 Scheme 2

.Primitive gene U Differentiation of function duplication U 1 Gene Any duplication -0- joining the two alleles to the same Differentiation of chromosome is function favored because it preserves polymorphism

Tandem Tandem duplication I -1 duplication

Present-day montana

FIGURE1.-Two schemes for origin of the apparently tandemly duplicated alpha-esterase loci of montana. Scheme 1 proposed by BRUCE WALLACE(personal communication); Scheme 2 proposed by ROBERTSand BAKFR(2973). EVOLUTION OI? A GENE DUPLICATION 735

MATERIALS AND METHODS

The alpha-esterase enzymes were studied in the following species of the montana phylad of the virilis group: monrana (MO);ouiuororum (Ou), a strain of montana; Zacicola (Lc);fluvomontana (Fl) ; and borealis (Bo). Listed in Table 1 are geographic locations of the strains of these species that were examined. Most of these strains were derived from single females inseminated in nature or from pair matings of virgin females to males from the same locale. Thus, barring multiple inseminations, four second chromosomes were originally segregating in each strain. Electrophoresis was performed in 7% acrylamide slab gels, using the Aardvark Instruments apparatus. Individual flies were homogenized in the electrophoresis buffer (0.1 M tris-borate with EDTA, pH 8.9) with 10% sucrose. Electrophoresis was for 2 hr at MO volts, after which the gels were placed in 0.5 M boric acid in the cold for 30 min, then stained for 60 min or more

TABLE 1

Geographical location of strains used

Number of strains examined of each species Location MO Ov Lc RI Bo Bethel, Alaska I Yukon, Alaska I Anchorage, Alaska 1 Mount Hood, Oregon 1 Pompeys Pillar, Montana 1 Shields River, Montana Woods Landing, Wyoming 2 Jackson, Wyoming 4 Chester, Idaho 1 Little Salmon River, Idaho 1 Verdi, Utah 1 Cottonwood Canyon, Utah American Fork Canyon, Utah 29 1 Wadsworth, Nevada 3 Craig, Colorado 19 13 Hamilton, Colorado 1 Walden, Colorado 1 Spencer Heights, Colorado 3 Gothic, Colorado 253 (1012 chromosomes) Horse Ranch Park, Colorado (190 chromosomes) Ohio Creek, Colorado 58 (2.30 chromosomes) Mount Lemon, Arizona 1 Whiteshell Provincial Park, Manitoba 8 31 Beaver Creek Campground, Manitoba 8 Jamestown, North Dakota 5 Lake Itasca, Minnesota 15 Fenske Lake, Minnesota Brule, Wisconsin Sioux Narrows, Ontario Saranac, New York Kent, Connecticut 1 Karesuando, Sweden 1 Inari, Finland 1 Total strains 382 2 20 65 74 Total locations 18 2 6 9 8 736 W. K. BAKER in 100 ml of 0.1 M phosphate buffer, pH 6.5, containing 20 mg each of alpha-naphthyl and beta- naphthyl acetate, plus 50 mg Fast Blue BB. Routinely, in pockets 1, 8, 16 and 24 of these 24-pocket gels, a mixture of montana esterase enzymes with mobilities of 93, 100, 106, 112, and 118 was placed to serve as markers. In order to accommodate the variants with new migration characteristics found in the other species, we have chnnged from an alphabetical system of nomenclature for the variants to a numerical one. Variant F of montana is given a migration value ol100, and the other variants are designated by their migration relative to 100. For the previously reportec! variants of montana, the conversion is as follows: A = 81, B = 87, B' = 90, C = 93, D = 96, E = 98, F = 100, G = 101, H = 103, I = 106, J = 107, K = 108, L = 109, M = 112, N = 114, P = 118 (see Table 2). Even with the use of these five marker enzymes of montuna in pockets distributed throughout the gel, it was occasionally not possible to determine with certainty whether an enzyme in one species co-migrated with one or the other of two close-migrating montana enzymes, or whether its migration was distinct. In these cases, gels were made with the two close-migrating montana enzymes in pockets oi the gel that alternated with the enzyme from the other species being characterized. This usually resolved the question. Of course, co-migration is not proof of protein identity. The cytological preparations in Figure 2 are aceto-orcein squashes of ganglion cells photo- graphed with phase microscopy. The specific strains of flies used in both Figures 2 and 3 are: Ou-Karesuando, Sweden; MeGothic, Colorado, GR7-10-76 Q b; Lc-WSPP, Manitoba, is0 Q g; Bo-WSPP, Manitoba is0 0 ee, and Craig, Colorado, C8-17-74k 0 x C8-17-74i 6' ; FZ- Wadsworth, Nevada, is0 8 a; ezouna Ez-Hokkaido, Japan, 30W.35; Zittorilas Lt-Finland, 480. Fluorescent Hoechst 33258 staining was accomplished according to the method of GATTI, PIMPINELLIand S~NTTNI(1976); however, the fixation was in one part 45% acetic acid to One part 95% ethanol, and no hypotonic treatment was given. The slides were examined in a Zeiss Ultraphot microscope under incident UV illumination from a HBO 200 mercury lamp. Since the UV illumination passes through the objective onto the preparation, only a small area is jlluminated, reducing fading. Scanning and examination were done with a loo><, 1.3 NA oil immersion Ph3 Neofluar Zeiss objective. Examination and photomicrography employed the Zeiss optically matched filters for examination of rhodomine or those matched for fluorescene.

ESTERASE PATTERNS In order for the results to be reviewed with some perspective, the closeness of the relationship between the species being discussed should be pointed out. Hybrids can be produced between many species combinations, although some hybrids are sterile and a few survive only to the larval stage (STONE,GUEST and WILSON1960; PATTERSONand STONE 1952). The morphological similarity of the adults is so close that only fiauomontana (because of its lighter coloration) can be told with assurance. Identification is based on internal dissection but, occasionally, crosses must be made for confirmation. For example, two diagnos- tic characteristics are the number of coils in the testes in males and the shape of the spermatheca in females. Against this background of evolutionary closeness, the diversity we are about to describe in the genetic systems for alpha- esterases appears particularly striking, The data on the alpha-esterase variants present in different strains of the species under discussion are presented in Table 2. Most of the data from montana is derived from the Colorado populations studied for linkage diesequilibrium (ROBERTSand BAKER1973; BAKER1975). However, we looked at 23 isofemale strains of this species that have been in culture for a number of years and found EVOLUTlON OF A GENE DUPLICATION 737 10 of these to be monomorphic. The monomorphism seen is probably due to the loss of some variants after long-term laboratory culture because of 21 newly collected females fertilized in nature, none produced a monomorphic strain. The species montana can be characterized by: (1) the high incidence of null alleles and the high incidence of four (93,100,106,112)of the nineteen active alleles, (2) the presence of a large number of different active alleles and (3) the extreme amount of variability. Recall (BAKER1975) that of 1432 chromosomes examined, there were 105 chromosome types with regard to the esterases they had; 40 of these 105 types were unique, indicating no approach to saturation in our collections of the types present in the population. When one combines these chromosome types into diploid combinations according to Hardy-Weinberg frequencies and then determines the probability that a fertilized female would produce a monomorphic strain, the low probability calculated fits the observa- tion of virtual absence of monomorphic strains. Only two isofemale strains of the newly described species, ouiuororum (LAKO- VAARA and HACKMAN1973), were available for study. One of the strains segre- gated for three of the four common alleles of montana (Table 2). We were surprised by this finding because at that time it was believed that species sym- patric with montana had a radically different esterase pattern, and ouiuororum, of course, came from northern Europe. Only in 1978 did we realize their syn- onomy. Evidence will be presented later that ouiuororum is a strain of montana that probably does not warrant subsepecific classification. The data in Table 2 indicate that, although ZacicoZa has a large number of variants and many polymorphic strains just like montana, it differs from its sister species in the fact that there are no null alleles segregating in the isofemale strains and that one of the most common esterase variants (87) is not one of the most common variants in montana, although the other common variant (100) is abundant in both species. One might attribute the lack of null alleles to the fact that all the strains examined have been in laboratory culture for a number of years, and chromosomes with null alleles (at least in montana) are at a selective disadvantage (ROBERTSand BAKER1973). The three taxa discussed so far do not differ greatly in their pattern of esterase variants; this is not surprising because of the closeness of their phylogenic relationship (see below). However, the pattern observed in flauomontana (Table 2) was unexpected. In the first place, 53 of the 64 strains are monomorphic, despite the fact that 46 of these strains were newly collected. Thus, the monomorphism could not be due to loss of variants because of long-term laboratory culture. Furthermorey almost all strains were characterized by variant 90, which has been found in only four of the 1432 montana chromosomes studied from Gothic, Colorado. In fact, none of the five active variants found in flauomontana was among the ones common in montana. The studies with borealis show an extent of polymorphism intermediate between that of montana and flavomontana: 25 out of 75 strains were monomorphic (Table 2). This monomorphism is not due to prolonged laboratory 738 W. K. EAKEP,

LIU

2R 8

iU, 3 g ._U e-U v1 .U -UY

U % r, -a a9 54. $2 "8 UB s

.UF:

Uy1 .Er: U 2U U* *pi' 3 2 Fr EVOLUTION OF A GENE DUPLICATION 739

.e2 4- d 8 .ec1 f .5 w E il w c .; .; 6 2 Hri 133.2 3: ma d% g %WO fS-3 w3m .El&E 2s : .$% 5 $LE ;g.36 =WEEQ 05% Eaa pz8 8.h & g s;aLSE E’S 8 i%,2 * +b 740 W. K. BAKER culture because, among eight virgin females pair-mated to males captured at the same location, two produced monomorphic strains. The data in Table 2 provide preliminary information on the question raised previously about the number of esterase genes in the species being discussed. Note that one monomorphic strain of lacicola shows three esterases with different mobilities. This means that this species has at least three alpha-esterase genes. In contrast, no strains of either flauomontam or borealis showing more than one esterase was monomorphic, an indication that all the variants might be alleles of a single gene. In the case of flrauomontana, we found individuals that give three bands upon electrophoresis, indicating that this species has at least two genes. However, an examination of about 2500 individuals of borealis failed to reveal a single one with more than two bands. Also, we have not been able to synthesize in the laboratory a strain oi borealis homozygous for more than one variant. These findings provide strong evidence that this species may have but one alpha-esterase gene. In order to test directly the allelism of the variants of borealis, the crosses outlined in Table 3 were made. Females heterozygous for two variants were crossed to males homozygous for a third variant. If the two protein variants in the female were coded by different genes, then recombination between the loci should produce some eggs with genes for both variants, others with neither (null). When fertilized by sperm from a third variant, these recombinant eggs would produce offspring with three bands or with one. As can be seen from the table, there is no indication of recombination and, thus, no evidence that variants 103, 107, 95 and 109 are not alleles of a single gene. Thus, the strong evidence for a single gene in borealis derived from the strains from natural populations is supported by the results of laboratory crosses. Only three variants of flravomontana were available for tests of allelism. A homozygous strain of variants 90 4- 95 was crossed to a homozygous strain of 70. The heterozygous F, females were crossed to males homozygous for 90. The data (Table 4) show that if the locus specifying 90 was sufficiently distant from the locus specifying 95 to produce recombinants among the 275 off spring studied,

TABLE 3 Tests for allelism in alpha-esterases of D. borealis

Type of offspring observed Cross One 95f 95-k 95f 1034- 103f Three Females X Males band 103 107 109 107 109 bands 103/107 95 0 72 85 ._ .. .. 0 103/95 109 0 72 87 0 103/109 95 0 87 71 0 107/95 103 0 74 83 0 107/109 103 0 85 74 0 109/95 103 0 80 79 0

Total of 94-9 offspring with no evidence of recombination. EVOLUTION OF A GENE DUPLICATION 741 TABLE 4 Test for allelism of alpha-esterasesin D. flavomontana

Type of offspring observed, expected SO+ Cross 904- 904- 704- Males X Females 95___ 70 90 95 90 95 Expected xxxx 70 0 90 0 Observed 119 156 0 0 900 90 95 -- Expected xx- - 0 70. then 70 and 95 are allelic. Further collections of flavomontana are being made to see whether additional variants will be allelic to one or the other of only two genes.

CYTOLOGICAL ANALYSIS Since there are usually four alpha-esterase loci in montana and, apparently, only a single one in borealis, an analysis of the salivary gland second chromosomes of hybrids between the two species was undertaken to see if this difference could be seen. A thorough search of both the long and short arm of this acrocentric chromosome failed to reveal any abnormalities in the hybrid (the montana used was from Gothic, Colorado, and the borealis strain from Craig, Colo- rado). The lack of cytological confirmation of the supposed difference in the alpha-esterase gene number between the species does not necessarily mean that the difference is fictitious. Perhaps it cannot be resolved by light microscopy. Note that the recombination frequency within the four loci of montana is 0.37 map units (ROBERTSand BAKER1973) and that the map distances in virilis (a relative of montana) may be from four to 10 times those of melanogaster for comparable chromosome regions (see cytogenetic maps of FUJII 1942). Therefore, the distance across the esterase loci of montana may correspond to a distance of 0.037 map units in melanogaster, a value somewhat lower than the amount of recombination (0.13 units) across the Notch locus, which is associated with a single salivary chromosome band, 3C7, and the interband spaces on either side (WELSHONS1974). "herefore, the duplicated loci in one chromosome of a montana/boreaZis hybrid may make a difference in the width of only a single band, a difference almost impossible to detect. In 1978, L. H. THROCKMORTONalerted me to the fact that ovivororum and montana hybridize. It was verified that both reciprocal crosses produce fertile hybrids between the Gothic, Colorado, montana and the Karesuando, Sweden, strain of ovivororum. An analysis of the salivary gland chromosomes of the hybrids revealed no cytological differences on the X,2 and 3 chromosomes. There is an inversion difference at or near the tip of chromosome 4 and also one near the base of this chromosome. Chromosome 5 differs by a single inversion in the 742 W. K. BAKER distal half. Since such differences are not greater than those found between North American strains of montana (Hsu 1952), not even subspecific rank seems warranted for the Swedish form. In addition, ouivororum and montana both have the unique two-armed, dot-like chromosome (see below) not found in any other member of the virilis species group. This supports the conclusion that they are strains of the same species. When it was learned that oviuororum would easily hybridize with montana, cytological analysis of larval ganglion cells was undertaken to learn if it had an acrocentric Y chromosome like the other Eurasian forms of the montana phlyad (littoralis and ezoanu) , or whether the Y chromosome was rod-shaped as had been reported for all other members of the virilis species group (see STONE, GUESTand WILSON 1960). To our surprise, ouivororum-montana male hybrids had an acrocentric Y no matter which way the cross was made; in fact, when a Gothic Colorado strain of montana was examined, all males showed an acrocentric Y (see Figure 2). This Y chromosome shows a secondary constriction in the long arm producing three heterochromatic regions of about equal length. It was also apparent that the usual dot-like chromosome 6 was a small acrocentric, a condition not previously reported for members of the virilis group. Both chromosomes from the mantana-ouiuororum hybrids are acrocentxic, indicating that this condition is common to both (Figure 2). These findings provide the incentive to examine the ganglion chromosomes of some strains of the other species. Figure 2 shows that Zacicola also has an acrocentric Y chromosome like montana, with a secondary constriction in YL, but the chromosomes 6 are not two-armed in these species. The first strain of borealis examined was from Whiteshell Provincial Park, Manitoba, and it contained no dot-like chromosomes whatsoever, only five pairs of rods in males! The Y chromosome, as well as the X, were rods (Figure 2). Although strains of borealis from Craig, Colorado, and American Fork, Utah, have a pair of dot-like chromosomes 6, the Y is rod-shaped. The Y chromosome of flauomontana was also found to be acrocentric; however, the secondary constriction was in Ys, as it is in ezoana and littoralis (Figure 2). Note the tiny chromosome 6 in ezoana, a characteristic of both strains of this species that we examined. The question now arises as to whether the difference in Y-chromosome shape (acrocentric) that we found and that previously reported (telocentric) is due to strain daerences within a species or to prior errors in cytological determination. The latter seems more likely since acrocentric Y chromosomes were found in flauomontana, montana and lacicola. It seems improbable that three strains of flavomontana (from American Fork, Utah; Pompeys Pillar, Montana; and Wadsworth, Nevada), two of lacicola (Saranac, New York; Whiteshell Provincial Park, Manitoba) and the strain of montana from Gothic, Colorado, would all show only acrocentric Y’s if rod and acrocentric Y’s were segregating. Also note (from Figure 2) that the telocentric chromosomes of these species are not all of equal length as drawn by previous workers (e.g.,STONE, GUEST and WILSON1960). It became apparent, as will be discussed later, that if the evolutionary sequence of the esterase genes is to be understood, the phylogenic relationship between FIGURE&.-Chromosomes from the neural ganglia of males of species indicated. Arrows point to Y chromosomes. (a) Male hybrid from cross of MOO x Oub ; (b) MO;(c) Le; (d) Bo from Whiteshell Provincial Park, Manitoba; (e) Bo from Craig, Colorado; (f) FI; (g) Ez; (h) Lt. Note similarity of Y chromosome in Ou, MO and Lc (compare a, b and c). This acrocentric Y has a secondary constriction in YL. Note ncrocentric chromosome 6 in Ou and MO (a and b). The Y is rod-shaped in Bo, but the strains from Whiteshell Provincial Park, Manitoba, lack the pair of dot-like chromosomes (d); on the other hand, we have found a strain from Craig, Colo- rado segregating for three of these small chromosomes (not pictured). The chromosomes at the right pole of the anaphase pictured in (e) show unambiguously (all chromosomes are in one plane) that the pair of acrocentric chromosomes 2 is the only pair without a near-terminal centromere, confirming that Bo (strain shown is from Craig, Colorado) has a rod-shaped Y. The acrocentric Y chromosomes of FI, Ez and Li are similar and have a secondary constriction in YB (compare f, g and h). The chromosome 34fusion of Li is seen at the top of metaphase (h). 744 W. K. BAKER montana, lacicola, flavomontana and borealis should be known. As previously noted, the Y chromosomes of montana and lacicola are similar, being acrocentric with a secondary constriction in YL.However, the acrocentric Y of flavomontana has a secondary constriction in Ys like the Eurasian species. In order to get further information on this relationship, Hoechst staining of ganglion chromosomes was employed, which revealed some interesting cytological features (Figure 3).

FIGURE3.-Hocchest staining of neural ganglion cells from larvae of the six species in the montana phyled. Arrows point to the Y chromosomes. (a) Fl, (b) Bo, (c) MO, (d) Lc, (e) Er, (f) Li. EVOLUTION OF A GENE DUPLIC-4TION 745 A region of strong fluorescence is observed in the proximal region of the long arm of chromosome 2, the chromosome with the pericentric inversion (see Figure 3d). The dot-like chromosome of Lt (Figure 3f) fluoresces strongly, as do those of MO (in particular the long arm) (Figure 3c) ; on the other hand, the dot of Lc has little fluorescence (Figure 3d). In Lt, the 3-4 fusion chromosome shows a fluorescent region in the long arm. Another interesting feature revealed by Hoechst staining is the drawn-out end with a fluorescent dot at one tip of all the telocentric chromosomes. Presumably, this is the centromere end and is a feature not observed in aceto-orcein squashes. (See Figure 3c.) The Y chromosomes seem to be the only ones in which the fluorescence occurs in sharp, narrow bands (see Figure 3f). An examination of the Y chromosomes of the six species shows that each one is different. Even though the Y chromosomes were indistinguishable in aceto-orcein squashes of Lt, Ez and FZ, the Hoechst fluorescence makes each distinctive. This is also true of the acrocentric Y chromosome of MOand Lc. It appears that there is a close resemblance between the Y chromosomes of the three species pairs: Lt and Ez, Bo and FZ and Lc and MO, as one might expect from their phylogenetic relationship. Notice, in the FZ Y chromosome, the small regions of fluorescence and constrictions, which I presume to be the tip of the long arm, and see how closely they resemble one of the tips of the Y in Bo. None of the other species shows this pattern. Therefore, it seems clear that the similarity of the Y chromosomes of FZ, Ez and Lt in aceto- orcein squashes probably does not indicate a direct phylogenetic link of FZ with the Eurasian forms.

DISCUSSION The discovery that one of the species in the montana phylad, borealis, has but a single active gene for the alpha-esterases and that another species, flavomontana, may have only two active loci is in accord with the hypothetical origin of the arrangement of the four genes in montana. This difference in the esterase loci between borealis and montana has been discussed as if it were a physical increase in the amount of DNA present in the genome that codes for the alpha- esterases. We have no evidence at present that would rule out with certainty the possibility that the two species have the same number of loci, but that in borealis three of the loci are represented only by null alleles. Although this possibility may seem unlikely on theoretical grounds, recall that a fifth locus in same montana was discovered only when its rare active allele was found on the same chromosome with active alleles of each of the other four loci. The null allele-or absence of this fifth locus-is present in 97% of the chromosomes (BAKER1975). In view of the exceptionally high frequency of the “null allele” of this fifth locus, one might well consider that the rare chromosomes with this locus arose by asymmetrical crossing over within the quadruplicated esterase loci. Thus, there may be no true null allele at this fifth locus. Recombination studies place locus 5 to the “left” of loci 2 and 4, but tightly linked to loci 1 and 3. However, an allele is classified as “null” if it has no activity on the artificial substrate alpha- naphthyl acetate. This substrate is rather poor when compared with possible natural substrates (NARISE1973), but the utility of various substrates by homo- 746 W. K. BAKER zygous null individuals has not been checked. The final resolution of the problem of physical duplication ugrsus differential activity will have to await studies on the proteins involved. If one assumes that the duplicative nature of the loci in montana, as compared with borealis, represents an increase in DNA, then borealis might well be thought of as the more primitive. On the other hand, if the “duplicative” nature of the esterases is really a matter of more loci being inactive in borealis, then montana might be thought of as the more primitive. This deduction is based on the premise that, in order for a gene to be maintained in the genome, it must have had some activity for which it was selected at least some time in the past (montana),and that, as evolution proceeded, a loss of activity of all but one gene occurred (borealis). The phylogenetic relationship of the species in the montana phylad is of some importance in interpreting the physical nature of the duplication. The evolution of the virilis group has proceeded along two phylads: the virilis phylad contains the species virilis, americana americana, americana texana and lummei; and the montana phylad contains the species discussed in this paper (see THROCKMORTON1978). One distinguishing character of the montana phylad is the presence of a pericentric inversion in chromosome 2, producing an acrocentric chromosome. The montana pliylad is divided into two subphylads; one includes ezoana and littoralis and the other montana, lacicola, flauomontana and borealis. This subdivision is based primarily 011 the common inversions shared by members of each o€ the two subphylads. Also, it was previously believed that ezoana and littoralis were the only species of the group with an acrocentric Y chromosome, a finding that we believe to be incorrect. Although borealis has a rod-shaped Y chromosome (a characteristic one would expect from a primitive member of the montana phylad) , the inversions in this species must have arisen after the splitting of the phylad into the exoana and montana subphylads, since many of them are shared with other members of the montana subphylad (see STONE,GUEST and WILSON1960). One could view the phylogeny of the montana phylad as that depicted in Figure 4. Primitive ZZZ (see STONE,GUEST and WILSON1960), with its pericentric inversion in chromosome 2 and segregating for a telocentric and an acrocentric Y chromosome, became split into two: one with the two-armed Y modified by a secondary constriction of the short arm, Primitive ZIT, and the other, Primi- tiue ZIP, retaining both Y chromosomes, but with the acrocentric Y modified by a secondary constriction in the long arm. The latter hypothetical species could have differentiated into two species groups: flazjomontana and borealis. The former evolved a new acrocentric Y, the latter retained the original telocentric Y, and the lacicola-montana group, which established the acrocentric Y chromosome with the secondary constriction in the long arm. This phylogeny is hypothetical and awaits confirmation or rejection through use of other characters. In either event, however, it seems highly probable that borealis has not been derived from montana, as inferred from the phylogeny published by STONE,GUEST and WILSON(1960). Therefore, there is some solid basis for the statement that the EVOLUTION OF A GENE DUPLICATION 747

Lt Ez Bo FI I I I

Prim 111 FIGURE4.-Phylogeny of the montana phylad of the virilis species group. The original basis of the phylogeny is the cytogenetic studies of J. T. PATTERSONand colleagues (see STONE,GUEST and WILSON1960). The genealogy within the montana subphylad (to the right) is more hypothetical, but based on cytological studies reported herein. The cytological appearance of the Y chromosomes with aceto-orcein and Hoechst staining is shown, as well as that of chromosome 6. The Hoechst pattern is based on examination of a number of cells. duplicative nature of the alpha-esterase gene complex-when one compares borealis with montana-is founded on an actual duplication of genetic material, rather than on gene inactivation of three of the four loci.

This research is supported by a grant from the National Science Foundation. GLORIA ENRIQUEZassisted in the initial stages of the inv-estigation, and BETHKAEDING provided expert technical help with the later stages. LYNNH. THROCKMORTONprovided many of the isofemale strains utilized. He also willingly gave sage advice and kept me informed of the status of his research on the evolution of the virilis group. This project could not have been accomplished without him. MARTINC. RECHSTEINERtaught me fluorescence microscopy.

LITERATURE CITED

BAKER,W. K., 1975 Linkage disequilibrium over space and time in natural populations of Drosophila montana. Proc. Natl. Acad. Sci. U.S. 72: 4095-4099. FUJII, S., 194.2 Further studies on the salivary gland chromosomes of Drosophila uirilis. Cytologia 12 : 435-459. 748 W. K. BAKER GATTI,M., S. PIMPINELLIand G. SANTINI,1976 Characterization of Drosophila heterochro- matin. I. Staining and decondensation with Hoechst 33258 and quinacrine. Chromosoma 57: 351-375. Hsu, T. C., 1952 Chromosomal variation and evolution in the vjrilis group of Drosophila. Univ. Texas Publ. 5204: 35-72. LAKOVAARA,S. and W. HACKMAN,1973 Drosophila ouiuororum sp. n., a new species of the Drosophila virilis group (Diptera, Drosophilidae) . Notulae Entomologicae 53 : 167-1 69. NARISE,S., 1973 Substrate specificity of a: and p-esterase enzymes of D. uirilis. Drosophila Inform. Serv. 50: 91. PATTERSON,J. T. and W. S. STONE,1x2 Evolution in the genus Drosophila. Macmillan CO., N.Y. ROBERTS,R. M. and W. K. BAKER,1973 Frequency distribution and linkage disequilibrium of active and null esterase isozymes in natural populations of Drosophila montana. Amer. Nat. 107: 709-726. STONE,W. S., W. C. GUESTand F. D. WILSON,1960 The evolutionary implications of the cytological polymorphism and phylogeny of the virilis group of Drosophila. Proc. Natl. Acad. Sci. U.S. 46: 350-361. THROCKMORTON,L. H., 1978 Molecular phylogenetics. pp. 221-239. In: Beltsuille Symposia in Agriculture Research. 2. Biosystematics in Agriculture. Edited by ROMBERGER,FOOTE, KNUTSONand LENTZ. WELSHONS,W. J., 1974 The cytogenetic analysis of a fractured gene in Drosophila. Genetics 76: 775-7944. Corresponding editor: D. HARTL