EVOLUTION OF HAWAIIAN DROSOPHILIDAE. 11. PATTERNS AND RATES OF CHROMOSOME EVOLUTION IN AN ANTOPOCERUS PHYLOGENY1

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1 EVOLUTION OF HAWAIIAN DROSOPHILIDAE. 11. PATTERNS AND RATES OF CHROMOSOME EVOLUTION IN AN ANTOPOCERUS PHYLOGENY1 J. S. YOON AND R.H. RICHARDSON2 Department of Zoology, University of Texas, Austin, Texas Manuscript received August 10, 1975 ABSTRACT The phylogenetic relationships of seven species of the genus Antopocerus (Family Drosophilidae) have been determined by means of a study of the metaphase configurations and polytene chromosomes. Based on biogeographical, behavioral and cytogenetic information, A. longiseta from Molokai is tentatively identified as the primitive species of the genus. The metaphase karyotypes of all Antopocerus species are either five pairs of rod chromosomes and a pair of dots (5RlD), or six rods (6R). Heterochromatin additions converted the dots to rods. Chromosome breakpoints for inversions also are clustered at heterochromatic loci. The chromosome segments between heterochromatic loci may represent sets of functionally related loci, evolving as a unit. The rate of chromosomal inversion substitution is estimated in the origin of the taxon (probably a subgenus of Drosophila rather than a separate genus). It averages no greater than one substitution per 1,000 years, or one per 5,000 generations. The average genetic death rate per generation of one individual per hundred is required to achieve this substitution rate. The rate of inversion substitution during radiation of this taxon may be only 4.4 x l e3 times as fast as that present in forming the taxon. Alternatively, radiation may have required only 250,000 years if rates of substitution are the same as in the origination of the taxon. Average rates of substitution reflect genetic accidents, selection pressures and rates of adaptation to new niches, as well as the rate of encountering new niches. Rate of adaptation probably is much greater in this instance than rate of encountering new niches. Therefore, the average rate of evolution reflects more nearly biogeographic and ecological factors than genetic factors. genus Antopocerus HARDY (1965), a Drosophilidae endemic to the TEwaiian Islands, is characterized primarily by males with highly branched antennae. Individual sizes range from 5 mm to 7 mm long. Their habitat is the rain-forest above about 700 m elevation, where larvae mine leaves of several plant species (HEED 1968). They seem to be restricted to the islands of Oahu, the 4-island Maui-complex (Maui Nui; MACDONALD and ABBOTT 1970) and Hawaii, never having been collected on Kauai, the oldest major island in the Hawaiian archipelago. SPIETH (1968) concluded from his study of the mating behavior 1 Research supported by U S. Public Health Service Research Grant GM from the National Institute of General Medical Sciences Research Career Development Award GM Genetics 83 : August, 1976.

2 828 J. S. YOON AND R. H. RICHARDSON that the genus arose in Maui Nui. YOON, RESCH and WHEELER (1972 b, c) studied the intergeneric chromosomal homology between Antopocerus and Drosophila, and concluded that Antopocerus was closely related to Drosophila, but did not postulate a place of origin or phylogeny. In this paper we derive the phylogenetic relationships among seven species, and discuss the rates of genetic divergence in the taxon. MATERIALS AND METHODS The flies were collected on three major Hawaiian Islands (Table 1) and iso-female lines were established by Ms. K. RESCH in our laboratory at the University of Texas at Austin. Individual wild females were allowed to oviposit for about six days in a vial (2.5 x 9.5 cm) of a banana-cactus food (RICHARDSON and KAMBYSELLIS 1968). A folded Tomac tissue dampened either with water or a cold water extract of Clermontia leaves was placed against the side of the vial touching the food. A small amount of cornmeal food was smeared on the tissue, a procedure which seemed to maintain female fertility. After ten to twelve days, both the tissue and the entire larval culture were transferred to a fresh vial of Resch medium 184. Contents were placed on folded tissue dampened with the Clermontia extract in the new vial. As larvae developed, small amounts of media 184 were added to maintain vigorous larvae. For confirmation of species identification, msture males from iso-female cultures were examined by DRS. D. E. HARDY and K. KANESHIRO of the University of Hawaii at Manoa. Both metaphase karyotypes and the salivary gland chromosomes were studied by lacto-acetoorcein squash preparations (YOON, RESCH and WHEELER 1973). Cytological maps of the polytene chromosomes were constructed from photographs made with a Zeiss Phase Photomicroscope. Heterochromatic bands are identifiable by their amorphous appearance, and stain darker than euchromatic bands comprising the majority of the polytene banding pattern. The band sequence of A. longiseta (Q84Y10) was selected as the standard (Figure 1). The banding patterns of corresponding chromosomes of the other species were compared with this standard set. The chromosome nomenclature is that used for the modified mouthparts group of Hawaiian Drosophila (YOON and CARSON 1973). Metaphase Chromosome Configurations RESULTS The metaphase karyotype af A. longiseta was found to be five pairs of rods and a pair of dots (5RlD) (Figure 2), which is the same karyaype as for A. tanythrix, cognatus, arcuatus, and villosus (CLAYTON 1969 and 1971; YOON, RESCH and WHEELER 1972~). However, a new species (Antopocerus species # 1 ) has six pairs of rods (6R), the same as for A. aduncus and A. entrichocnemus (CLAY- TON 1968 and 1971). A. diamphidiopodus (S107Y1, Papa, South Kona, Hawaii) has five pairs of rods and one pair of dots (5RlD), while CLAYTON (1968) reported A. diamphidiopodus from Maui to have six pairs of rods. Presumably these two populations represent different subspecies. The 6R karyotype differs from the 5R1D karyotype by the amount of heterochromatin in chromosome 6 (Figure 3). The rod-shaped chromosome 6 of both A. aduncus and species #1 are about times the length of other autosomes. The dot chromosomes of longiseta and arcuatus are intermediate, being a little larger than other dot chromosomes found in the genus. There is some variation

3 INVERSIONS AND RATE OF EVOLUTION 829

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5 INVERSIONS AND RATE OF EVOLUTION 831 FIGURE 2.-Photomicrographs of typical metaphase karyotypes represented by A. species # 1 (A) and A. diamphidiopodus (B). in heterochromatin in the X-chromosomes as well: The longest heterochromatic sections are found in A. diamphidiopodus, arcuatus, and aduncus. Polytene Chromosome Analysis Salivary gland chromosomes appear as five long arms and one short arm. Each euchromatic arm is easily recognized in polytene systems by a characteristic tip and other morphological features (Figure 1 ). The variations in band sequences between species result from paracentric inversions. We found sixteen different inversions in the twenty strains examined (Table 2). Fifteen were homozygous within species (A, B, C, E, F, G, H, I, K, L, M, N, P, Q and R). The one line of A. arcuatus was segregating for a unique inversion (S/+). The X chromosomes of A. diamphidiopodus and A. cognatus differ from the standard (A. longiseta) by one inversion, A. The new A. species #1 has this and an additional overlapping inversion, B, and A. tanythriz has A, B, and still another included inversion, C. The phylogenetic arrangement based on the overlapping inversion must be longisera * (diamphidiopodus, cognatus) * (species # 1, tanythriz). FIGURE 1.-Salivary gland chromosomes of Anlopocerus longiseta (from Molokai). This species has been used for constructing the standard map of the genus Aniopocerus. Other members of the genus are related to this species by fixed or heterozygous inversion sequences whose limits are defined on the photograph by the letters A through R. The distal ends of the chromosome are located to the left. Notice that the eleven distinct inversions were formed by utilizing only seven breakpoints (see text for details).

6 832 J. S. YOON AND R. H. RICHARDSON ChROMOSOME X Y 6 A. orcuotus A. oduncus A. longiseto A. diomphidiopodus A. cognatus A. species '1 A. tonythrix 0 Euchromotin D Heterochromotin FIGURE 3.-Diagrammatic representation of the metaphase karyotypes of seven species in the genus Antopocerus. Heterochromatin is shown as darkened parts on the diagrammatic chromosome element. Relative lengths within species are shown, and are also approximately true between species. TABLE 2 Paracentric inversions in the genus Antopocerus species Chromosome element Species X A. arcuatus + + S/ A. aduncus M N,P Q3 A. longiseta A. diamphidiopodus A E,F H A. cognatus A E,F H + A. species # 1 A3 E,F,G H K A. tanythrix A,B,C E,F,G H,J + K,L +

7 INVERSIONS AND RATE OF EVOLUTION 833 Chromosome element 2 of A. diamphidiopodus and A. cognatus differs from the standard by two fixed inversions, E and F. Antopocerus tanythrix and species # 1 have these and one additional overlapping inversion, G. This overlapping inversion also gives a phylogenetic arrangement identical to that found for the X-chromosome. Chromosome 3 of A. diamphidiopodus, cognatus, and species #1 has one inversion, H, and A. tanythrix has this and a second inversion, J. The chromosome 4 of A. arcuatus, diamphidiopodus, cognatus, tanythrix, and species #1 appears identical in band sequences to the standard, A. Zongiseta. A. aduncus has two inversions, Q and R. Chromosome 5 of A. arcuatus, diamphidiopodus and cognatus have banding sequences identical to the standard. That of tanythrix and species #1 differ from the standard by one inversion, K, and tanythrix has an additional inversion, L. Chromosome 6, the dot chromosome of all polytene karyotypes, is the same in all seven species of Antopocerus. Although having the same bands, they are in reverse order to those in the dot chromosome of Droisophila. Chromosomes of A. aduncus differ from the standard by a total of five fixed inversions- / in the X chromosome, N and P in chromosome 2, and R and Q in chromosome 4. As can be seen in Table 2, inversions tend to accumulate simultaneolusly in different chromosomes. Excluding A. aduncus and the polymorphic inversion in A. arcuatus (S), inversions accumulate top to bottom in the table for chromosomes X, 2,3 and 5. This pattern forms the basis for the phylogenetic ordering of species shown in Figure 4. A. aduncus falls outside the main series, having five unique inversions among three chrolmosomes, X, 2 and 4. As shown in Table 2, the banding sequences in polytene chromosomes of A. diamphidiopodus and cognatus are identical with each other, as are those of A. longiseta and A. arcuatus. They are anisohomosequential species (YOON, RESCH and WHEELER ) due to the metaphase configuration differences, although they have the same band sequences in pojytene cells. From metaphase and polytene chromosome analyses, six of the seven species fall into three grolups: 1 ) A. longiseta and arcuatus: anisohomosequential species 2) A. dirrmphidopodus and cognatus: anisohomosequential species 3) A. tanythrix and species #1: share three unique inversions (B, G and K). The remaining species, A. aduncus, could have been derived from or given rise to either A. longiseta or A. arcuatus. A. aduncus cannot be considered to be an intermediate of the other two. The general evolutionary pattern from Maui to Hawaii is clear, although details are debatable. From the longiseta-arcuatus pair, the most similar species are diamphidiopodus and cognatus. However, because of the subspecies of diamphidiopdus on Maui, it is considered to be a closer relative of longiseta than cognatus. Since this subspecies was reported to have the 6R karyotype, while longiseta is 5RlD, there may have been a common ancestor with a 5R1D karyotype. Also karyotyping from adults is difficult and no pictures of the prepa-

8 834 J. S. YOON AND R. H. RICHARDSON Hawaiian Islands OKAUA Km I I /- I I A. \ \._ I I I \ I FIGURE 4.-Phylogenetic relationships of the seven species within the genus Aniopocerus. The symbols (* and 0) represent actual collection sites; the asterix represents those from which we have studied collections and the open circles are from the published records (see Appendix). rations from the Maui subspecies are published. Confirmation of the earlier report is needed. A. tanythrix and species #1 were prolbably derived from diamphidiopodus. However, at the present, it is not clear if these species evolved linearly, or divergently from a common ancestor. The new species #1 has the 6R metaphase karyotype, which may not be the ancestral karyotype. Furthermore, species #1 was collected on the western slopes of Mauna Loa, opposite from the range of A. tanythriz. There may have been an unknown common ancestor similar to diamphidiopodus. Alternatively, CARSON et al. (1970) have found two major Drosophila colonization routes from Maui-either to the Kohala Mountains, or to South Kona. Since only tanythrix has been collected in the Kohala Mountains, while diamphidiopodus has been collected only at South Kona and the volcano area, the latter route seems the more likely of the two. The number of inversions so far described from the Antopocerus species is summarized in Table 3. The band order for chromosomes X and 2 is the most variable, while it is relatively stable for chromosomes 3 and 5. An average of 2.1 inversions per species have been fixed during their evolutionary history. This is essentially the same as that found in the modified mouthparts group species

9 INVERSIONS AND RATE OF EVOLUTION 835 TABLE 3 Number of inversions and break points in seven species of antopocerus Chromosome X Total No. No. No. points common to invenions fixed points observed more than one inversion (ca. 2 per species), but somewhat more than that found in the picture-winged group species (ca. 1.5 per species) (YOON, et al b). However, if the subspecies of A. diamphidiopadus are actually species, the inversions per species for Antopocerus fall to 1.9. The polytene chromosomes for species of Antopocerus are similar to those of many Hawaiian Drosophila species, including those of the modified mouthparts, the picture-winged and Engiscaptomyza species groups ( YOON, RESCH and WHEELER 1972c and YOON, et al. 1975). At least 30% of their total polytene chromosomes not only have readily identifiable homologous banding patterns, but have remained in approximately the same relative position in the corresponding chromosome element. The remaining 70% appears to be blocks of homologous bands which have been disrupted and their positions changed by included and overlapping inversions. Identification of homologous bands is further restricted by morphological modifications, often a result of position effects. The chromosomal banding patterns of Antopocerus have been homologized in a preliminary fashion with Scaptolmyza. An hojmology of at least 15 % is indicated. As can be seen in Table 3 and Figure 1, there are several breakpoints shared by different inversions. On three chromosomes (X, 2, and 4), seven breakpoints were utilized in the formation of eleven distinct inversions (A, B, C, E, F, G, M, N, P, Q and R). This is accounted for by each inversion sharing a breakpoint with at least one other, and in two instances this point was shared by three inversions. If there are about 1,000 bands per major polytene arm (ignoring chromosome 6), and inversions involving adjacent bands are identifiable, the expected frequency of a common breakpoint for two independent inversions is of the order of Therefore, it is apparent that locations of breakpoints are not independent. There are characteristic morphological features associated with breakpoints. The most common features are deposits of heterochromatin, usually with a constriction (Table 4). All breakpoints of fixed inversions in the Antopocerus genome have heterochromatic deposits associated with them, although not universally found in all species. Furthermore, the thirty heterochromatic regions associated with breakpoints shown in Table 4 comprise over 90% of all those

10 836 J. S. YOON AND R. H. RICHARDSON i i I rilr, I 1

11 INVERSIONS AND RATE OF EVOLUTION 837 found in the Antopocerus genome. Of the breaks shown in Table 4, 20% are associated with the first appearance of heterochromatin in the derived species (three cases in longisetae -+ aduncus, two in longiseta -+ diamphidiopodus, one species #1 -+ tanythrix). Only one, cognatus 4 species #I, shows loss of heterochromatin. Of the remaining 70% involving heterochromatin both in the primitive and derived species, more than half involve constrictions before and after the appearance of the break. The high degree of clustering of breakpoints resulted in considerable correlations in chromosomal regions involved in different inversions. In two cases, one long inversion affects the same chromosome segment as the combination of two non-overlapping smaller inversions. That is, A=C+M and G=F+N. Such a pattern also was described for species in the mimica subgroup (YOON, RESCH and WHEELER 1972b). Clustered breakpoints were first observed in some mainland Drosophila species by MATHER (1963) and GOLDSCHMIDT (1956). Several additional cases have been recorded subsequently with a variety of explanations of causes and implications (see BICUDO 1973). DISCUSSION Polytene chromosomes have been used extensively in Drosophila to determine phylogenetic relations among closely related species. However, from these phylogenies it is not possible to determine the primitive species. In some instances a relative outside the phylogeny known to be primitive may be related to the species in the phylogeny. Often the comparisons are on morphological or behavioral bases, rather than genetic comparisons. In other cases, biogeographic relations of species in the phylogeny may be used to infer the direction of evolution in a phylogeny. For example, in an earlier study of the D. crassifemur complex, the species seemed most likely to have evolved from the inhabitants of the oldest island to those of the youngest island (YOON et a1 1975). In the case of Antopocerus, the bases for identifying the primitive type are biogeographical, cytological and behavioral. The identification of the primitive karyotype and mating behavior are based upon evidence from other groups of species of Hawaiian Drosophilidae, so that we reference simultaneously several primitive relatives. From the biogeographical point of view, CARSON et al. (1970) have found several cases of colonization from Maui Nue to Oahu and Kauai, but never from Hawaii to other islands. The 6R metaphase karyotype we have found to generally represent phylogenetic termini, and therefore is a derived trait in many instances. We follow SPIETH (1968) and consider tanythrix and cognatus to be derived species from their location on Hawaii and from their having a derived mating behavior. Our species # 1, phylogenetically between cognatus and tanythrix, also has a derived 6R metaphase karyotype. Among the Maui Nui species, diamphidiopodus has both a derived mating behavior and metaphase karyotype, while aduncus has a derived karyotype. Comparing longiseta to arcuatus, the Oahu species, they are morphologically similar (HARDY 1965), but arcuatus was considered to have a primitive mating

12 838 J. S. YOON AND R. H. RICHARDSON behavior by SPIETH (1968) after a study of male fore tarsi. Only longiseta is primitive in both mating behavior and metaphase karyotype. We therefore consider longiseta to be the best representative of a primitive species in our phylogeny. Antopocerus is similar to Drosophila in morphology (THROCKMORTON 1966; KANESHIRO 1974). metaphase karyotype (CLAYTON 1971; YOON et al. 1975), and pdytefie chromosome homology (YOON, et al. 1972c and unpublished). KANESHIRO ( 1974) proposed that certain extreme morphological anomalies should receive less weight, and proposed that Antopocerus should be synonymized with Drosophila. Although at odds with taxonomic criteria for mainland Drosophila, such a change would be in line with the degree of cytogenetic divergence between Drosophila ani! Antopocerus. Furthermore, in agreement with the suggestion of SPIETH ( 1968), Antopocerus arose no more than about 1.8 million years ago, since that is the age of Molokai, the oldest portion of Maui Nui (MACDONALD and ABBOTT 1970). Conventionally we expect generic distinction to be attained more slowly. Regardless of whether this taxon should be given a generic or subgeneric distinction, the rates of chromosome evolution during the origin of the taxon may be compared to radiation of species from the primitive form of the taxon. The average of about two fixed inversion differences per species will not likely increase with additional data, but to the contrary, new homosequential species may be found. We can estimate some limits for the number of inversions between Antopocerus and Drosophila. The maximum number of inversions separating species within the picture-wing group is 14 (ignoring primeua and antigua). and, within the modified mouthparts group, it is 11. We have counted 33 inversions between these two groups and a few more probably exist. Thus, the 50% homology between these groups represents about 35 inversions. The 30% homology between Antopocerus and Drosophila suggests there are somewhat more than 35 inversions involved. Since we observed a range of 3 to 5 heterochromatic sites with inversion breakpoints per chromosome (Table 4), we assume for simplicity five potential heterochromatic sites for each of the five major chromosome elements. (Of course, this is far less than the possible number of rearrangements, since overlapping inversions produce different arrangements when they occur in different orders.) Therefore, based on our observations, we might expect more than 35, but less than 50 inversions. We may take about 40 as a convenient lower limit of inversions separating Antopocerus from Drosophila. The upper limit is arbitrary. If there are 1,000 bands per major chromosome element, then there are 5 (l,y") 2.5 x lo6 possible inversions. Resolution of most of these is impossible, and, in addition, it requires bands in a segment to establish an homologous sequence. If a breakpoint could occur every 20 bands, there would be 5 (","), the same as the previous calculations based on heterochromatic breakpoints. It is clear that lo6 is several orders of magnitude too large. We consider 200 as a reasonable upper limit.

13 INVERSIONS AND RATE OF EVOLUTION 839 Within the limits of inversions between Antopocerus and Drosophila, and with no known intermediate types, the origin of the taxon would seem to be a saltatory event of massive chromosomal rearrangements, and presumably reflect a genetic revolution. This interpretation deserves closer analysis, however. Can it be interpreted by more conventional genetic theory? The conditions whereby a new taxon may arise are unknown, and at best, speculation can only indicate directions for further work. However, there are some clues. We may assume that the highly modified antennae of Antopocerus are adaptations to at least some of the conditions related to the origin of the taxon. SPIETH (1968) suggested the specialized aristae were an adaptation of the mating behavior, where males lunge forward on the female, the aristae forcing her wings upward and forward. This interpretation is logical and certainly true to a large extent. However, the aristae of females also are modified. Relative to Drosophila, the first segment is larger and the rays are shorter and more numerous. These modifications suggest initially there was specialization in navigation or olfaction, presumably traits present in both sexes. Thus a change in habitat selection in order to exploit a new habitat may have been a factor in the initial divergence of this taxon. As suggested by studies of RICHARDSON (1974) and collleagues (RICHARDSON and JOHNSTON 1975; RICHARDSON and SMOUSE 1975), habitat selection may play a key role in reproductive isolation and genetic divergence, particularily when functionally (pleiotropically?) connected with mating behavior. Although such an initial event may not involve massive genetic change, the cytological modification between Antopocerus and Drosophila suggests that this shift, plus subsequent adaptation, reflected a rather drastic modification during the origin of the taxon. Absence of intermediate chromosomal types (relics) suggests that radiation was delayed until considerable gene substitution had occurred. Our hypothesis, then, is that after an original speciation, the primitive pro- Antopocerus species (taken as represented by A. longiseta for present discussion) evolved until some large degree of differentiation and specialization was reached, whereby radiation began. Since A. longiseta is found only on Molokai, the oldest island of Maui Nui, the time for divergence from Drosophila to the primitive Antopocerus level could be approximately the time Molokai predates Maui, or about 200,000 years ( IO6 generations). The average inversion substitution rate thereby could be as fast as one per 1,000 years, or about one per 5,000 generations. Even at this rate, the origin of the taxon may have been anything but a saltation. Assuming as many as 200 inversion substitutions, a population size of 5 x IO8 and no dominance, an average of only about one per hundred individuals would need to be selectively eliminated each generation in order to achieve these substitutions (from KIMURA and CROW 1969; L, = -2 log,(p,) = 41.5). Fewer inversion substitutions. dominance of adaptive inversions, a smaller population or longer times for substitution would require an even lower selection intensity. Subsequent speciations of an average of two inversion differences per species could have as much as 1.6 million years to evolve. In the phylogeny we have derived, at least four species evolved from longiseta, with an accumulation of only

14 840 J. S. YOON AND R. H. RICHARDSON seven inversions. Thus the inversions substituted during radiation may have been 7/200 as many over possible a period of 8 times longer length. Th' IS amounts to a selective elimination less than 1 % of that which could have occurred during formation of the taxon. Alternatively, one may take the lower limit of inversion substitutions giving rise to Antopocerus (i.e., 40), and also assume the phylogenic sequence is unbranched in the accumulation of the seven inversions from Longiseta to tanythrix. If the evolutionary rates are equal. it would take almost six times longer to form the primitive species as to achieve the present radiation. Thereby the radiation would have begun only about 250,000 years ago. At this time all islands of the archipelago were present, but Hawaii was much smaller than today. Maui Nui formed one continuous land mass periodically as ocean levels fell during ice ages. If radiation occurred this recently, the primitive species might be expected to be more widely distributed. It therefore seems more likely that rates of evolution were considerably slower during radiation than during the development of primitive Antopocerus. It is apparent from these data that, even for inversions, selection intensities on the average would be very small. However, each inversion substitution presumably represents a set of interacting genes undergoing simultaneous allelic substitution. The tendency for chromosomal segments to be common to sets of inversions suggests that the breakpoints mark the boundaries of genetic complexes, and several loci evolving as a functional set would be expected to exhibit large selection pressures. Rates of fixation would be rapid, but followed by long periods of relative evolutionary stability. Nevertheless, whether functionally related or not, several gene fixations per inversion are expected due to chance inclusion in an inversion of alleles with neutral or even small negative effects. It remains for later studies to relate the evolving chromosomal segments to the average number of nucleotide substitutions. In any case, slow average evolutionary rates should not be taken to imply a gradual accumulation of genetic differences. For example, the genes involved in speciation, sinsu strictu, may have very rapidly approached fixation along with or precedivg the inversions, once a niche became available. The rate of change measured between species or superspecies reflects both rate of encountering new niches, as well as rate of exploiting the niches. The former reflects isolation and development of the plant community in this instance. Therefore, for an additional nongenetic factor, it remains to be determined to what degree inter-island colonization retards radiation. In Antopocerus, the radiation may have been slow on Maui Nui with periodic isolation of volcanoes by water, only to accelerate greatly once Hawaii was colonized. and the volcanoes are connected by continuous land masses. Although selection forces are a pervasive element throughout this evolutionary scheme, the relevant questions of the evolutionary processes seem not to involve differentiation between selective uersus neutral modes of divergence. More critical questions which remain even more elusive are those such as what particular ecological conditions and which particular genetic loci are involved in the origi-

15 INVERSIONS AND RATE OF EVOLUTION 841 nation of a new evolutionary lineage. In the long run, the average selection forces might be small and difficult to measure, as the populations approach fixation quickly. In such cases, the average rates of evolution are more highly correlated to rates of exploitation of new niches, rather than rates of genetic change once the exploitation begins. We are indebted to all participants in the Hawaiian Drosophilidae Project for their cooperation in this study. In particular, the assistance of DRS. D. E. HARDY, H. L. CARSON, and K. Y. KANESHIRO of the University of Hawaii at Manoa is appreciated. We appreciate the continued encouragement in our work froin DR. M. R. WHEELER. Ms. K. RESCH cultured the material we studied, and much insight was gained in our discussions with her. We thank DRS. M. MAGUIRE and A. TEMPLETON for critically reading the manuscript. ADDENDUM: DRS. D. E. HARDY and K. KANESHIRO of the University of Hawaii at Manoa stated in recent personal communications that they are taxonomically recategorizing the genus Antopocerus to be cogeneric with the genus Drosophila, based on new data including our recent cytogenetic studies. LITERATURE CITED BICUDO, H. E. M. DE CAMPOS, 1973 Chromosomal polymorphism in the saltans group of Drosophila. I. The saltans subgroup. Genetica 44: CARSON, H. L., D. E. HARDY, H. T. SPIETH and W. S. STONE, 1970 The evolutionary biology of the Hawaiian Drosophilidae. In: Essays in Evolution and Genetics in Honor of Th. Dobzhansky. Edited by M. K. HECHT and W. C. STEERF.. Appleton-Century-Crofts, New York. CLAYTON, F. E., 1968 Metaphase configurations in species of the Hawaiian Drosophilidae. Univ. Texas Publ. 6818: , 1969 Variations in metaphase chromosomes of Hawaiian Drosophilidae. Univ. Texas Publ. 6818: , 1971 Additional karyotypes of Hawaiian Drosophilidae. Univ. Texas Publ. 7103: GOLDSCHMIDT, E., 1956 Chromosomal polymorphism in a population of Drosophila subobscura from Israel. J. Genet. 54: HARDY, D. E, 1965 Znsects of Hawaii, Vol.le. Diptera: Cyclorrapha 11, Series Schizophora Section Acalypterae I. Family Drosophilidae. University of Hawaii Press, Honolulu. HEED, W. B., 1368 Ecology of the Hawaiian Drosophilidae. Univ. Texas Publ. 6818: KANESHIRO, K. Y., 1974 Phylogenetic relationships of Hawaiian Drosophilidae based on morphology. In: Genetic Mechanisms of Speciation in Insects. Edited by M. J. D. WHITE. AUStralia and New Zealand Book Co., Sydney. KIMURA M. and J. F. CROW, : MACDONALD, G. A., and A. T. ABBOTT, 1970 lulu. Natural selection and gene substitution. Genet. Res., Camb. Volcanoes in the Sea. Univ. Hawaii Press, Hono- MATHER, W. B., 1963 Patterns of chromosomal polymorphism in Drosophila rubida. Am. Nat. 97: RICHARDSON, R. H., 1974 Effects of dispersal, habitat selection, and competition on a speciation pattern of Drosophila endemic to Hawaii. In: Genetic Mechanisms of Speciation in Znsects. Edited by M. J. D. WHITE. Australia and New Zealand Book Co., Sydney. RICHARDSON, R. H. and J. S. JOHNSTON, 1975 Ecological specialization of Hawaiian Drosophila Habitat selection in Kipuka Puaulu. Oecologia 21 : RICHARDSON, R. H. and M. P. KAMBYSELLIS, 1968 A cactus-supplemented banana food for cultures of Repleta group Drosophila. Drosophila Information Service 43 : 187.

16 842 J. S. YOON AND R. H. RICHARDSON RICHARDSON, R. H. and P. E. SMOUSE, 1975 Ecological specialization of Hawaiian Drosophila: 11. The community matrix, ecological complementation, and phyletic species packing. Oecologia 22: SPIETH, H. T., 1966 Courtship behavior of Hawaiian Drosophilidae. Univ. Texas Publ. 6615: , 1968 Evolutionary implications of the mating behavior of Antopocerus (Drosophilidae) in Hawaii. Univ. Texas Publ. 6818: THROCKMORTON, L. H., 1966 The relationships of the endemic Hawaiian Drosophilidae. Univ. Texas Publ. 6615: YOON, J. S. and H. L. CARSON, 1973 Codification of polytene chromosome designations for Hawaiian Drosophilidae. Proc. XI11 Int. Congr. Genetics 74: s YOON, J. S., K. RESCH and M. R. WHEELER. 1972a Cytogenetic relationships in Hawaiian species of Drosophila. I. The Drosophila hyrtricosa subgroup of the modified mouthparts species group. Univ. Texas Publ. 7213: , -, 1972b Cytogenetic relationships in Hawaiian Drosophila. 11. The Drosophila mimica subgroup of the modified mouthparts species group. Univ. Texas Publ. 7213: , 1972c Intergeneric chromosomal homology in the family Drosophilidae. Genetics 71 : , 1972d Cytogenetic evidence for one-ancestral relationship within Hawaiian Drosophilids. Genetics (Abstracts) 71: S YOON, J. S., R. H. RICHARDSON and M. R. WHEELER, 1973 A technique for improving salivary chromosome preparations. Experientia 29 (5): YOON, J. S., K. RESCH, M. R. WHEELER and R. H. RICHARDSON, 1975 Evolution in Hawaiian Drosophilidae: Chromosomal phylogeny of the Drosophila crassifemur complex. Evolution 29: Corresponding editor: J. F. CROW

17 INVERSIONS AND RATE OF EVOLUTION 843 h a a