Change in a Secondary Sexual Character As Evidence of Incipient Speciation in Drosophila Silvestris* (Evolution/Behavior/Morphology/Sexual Selection/Hawaii) HAMPTON L

Home , Drosophila silvestris

Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1929-1932, April 1979 Evolution Change in a secondary sexual character as evidence of incipient speciation in Drosophila silvestris* (evolution/behavior/morphology/sexual selection/Hawaii) HAMPTON L. CARSONt AND PETER J. BRYANTt tDepartment of Genetics, University of Hawaii, Honolulu, Hawaii 96822; and *Center for Pathobiology and Department of Developmental and Cell Biology, University of California, Irvine, California 92717 Contributed by Hampton L. Carson, January 29, 1979

ABSTRACT Search for genetic changes that are pivotal in In the present paper we report progress in a renewed search species formation has led to intraspecific studies of Drosophila for cases wherein certain intraspecific populations give evidence silvestris, a giant species found only on the geologically new of incipient speciation (5). This approach unfortunately suffers island of Hawaii. Males bear large, curved, modified bristles or cilia on the dorsal surface of the foreleg tibia and tarsus. In from the same difficulty that confronts the comparison of full males from the south and west parts of the island there are two species: How can crucial genetic changes be identified within rows of cilia separated by a naked area, but in males from the segments of what appears to be an incompletely subdivided north and east there is a mean of between 20 and 30 additional gene pool? How can the change be judged as novel and func- cilia between the two major rows on the tibia. These extra cilia tionally important, and how can its age be determined? are absent in closely related species of this subgroup, including present and cytological findings in the sympatric species D. heteroneura and three species from We here morphological adjacent islands. Males use the foreleg tibiae in vibratory the study of populations of the species Drosophila silvestris movements against the female's abdomen during courtship, so from the island of Hawaii. Hawaiian Drosophila provide an this character difference is likely to be important in the repro- extraordinary opportunity to identify incipient species. Recent ductive biology of the species. Inversion polymorphisms are exuberant speciation has occurred; in less than six million years similar in both northeast and southwest populations; they show about 500 good species of the genus Drosophila have evolved large and strikingly parallel altitudinal shifts in frequency dis- tributions involving the same inversions. Populations from in situ on these extremely isolated oceanic islands (6). Evolution various parts of the island cannot be distinguished by routine has, in most cases, progressed stepwise down the archipelago electrophoresis of soluble proteins encoded by 25 loci. Thus the in a line from the older islands and volcanoes in the northwest "extra cilia" character is superimposed on a more ancient ge- to the newest island (Hawaii) in the southeast. netic background of similarity involving both chromosomal and D. silvestris is confined to the high-altitude mesic forests of electrophoretic polymorphisms. We interpret the extra cilia as Hawaii island, where all of the lava flows are younger than a specific new embellishment of a secondary sexual character brought about by altered sexual selection occurring very recently 700,000 years. Populations of this species, furthermore, extend in one part of the species range. This suggests incipient spe- onto the slopes of the two newest volcanoes (Mauna Loa and ciation. Kilauea), which are currently active volcanically. The leg-bristle character dealt with in this study is particu- Identification of the crucial genetic changes that occur as a larly relevant to the behavior and evolution of Hawaiian Dro- diploid species diverges into two or more new species has sophila. One of the most striking characteristics of this fauna proved to be very difficult (1, 2). Even with the newer molec- is the extraordinary elaboration of male secondary sexual ular and chromosomal methods for assaying genetic variability characters. Wings, antennae, and mouthparts of males show (3), the study of closely related species still suffers from the old bizarre modifications, but the most elaborately changed organ problem: How can the pivotal changes that occurred when the is the male foreleg (7). This is presumably related to the ex- species were diverging be distinguished from changes that tensive use made of the forelegs by the male in stimulating the follow or are irrelevant to the speciation process? female during courtship (8). Much recent research has been devoted to making detailed We will show that a novel foreleg character has evolved in comparisons between full species that are obviously closely certain populations of D. silvestris and that this change has been related and thus presumed to have speciated recently. From superimposed on an underlying similarity of chromosomal and these data, extrapolations to the formative phase of the species' allozymic polymorphisms. We regard it as a character that has history are frequently made (2). A more difficult approach is been newly acquired by sexual selection and that is intimately to attempt to identify incipient speciation within what appears concerned with the reproductive biology of the species. As such, to be, by most criteria, a single species. Such attempts, for ex- it is relevant to the problem of species incipience. ample, led to the original discovery of morphologically cryptic (sibling) species. Recent research has shown, however, that most such species are widely different in reproductive biology, ad- MATERIALS AND METHODS aptations, and physiology. They are frequently disparate ge- Specimens of D. silvestris were captured by using yeasted ba- netically, as indicated by molecular genetic comparisons (4). nana and mushroom baits at 11 sites on the island of Hawaii Indeed, morphological similarity can be a misleading facade; (Fig. 1). Further information on most of these sites is given in sibling species are usually full species and are far from being ref. 9. New sites with altitudes are: 1, Hualalai, 1400 m, North incipient (3). * This paper is no. 6 in the series "Genetic variation in Hawaiian The publication costs of this article were defrayed in part by page Drosophila." Paper no. 5 is ref. 9. The substance of this paper was charge payment. This article must therefore be hereby marked "ad- presented at a symposium, "The Dynamics of Speciation," sponsored vertisement" in accordance with 18 U. S. C. §1734 solely to indicate by the United States-Japan Cooperative Science Program held at this fact. Tokyo, Japan, October 14-18, 1978. 1929 1930 Evolution: Carson and Bryant Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Number of cilia on the tibia of the male foreleg in various populations of D. silvestris from the island of Hawaii (mean and SEM) Site and sample N* Row 5 Middle rows Row6 1. Hualalai U5B 20 22.5 0.55 0.35 ± 0.15 27.8 ± 0.54 2. Pauahi S89; T90 21 21.0 I 0.44 0.38 + 0.15 26.2 ± 0.44 3. Kahuku T96V 20 23.0 + 0.48 0.20 i 0.12 28.3 ± 0.52 4. Pahip'a U27 45t 19.9 + 0.29 0.31 d 0.09 23.0 ± 0.44 5. Waihaka U52; U69 5 21.6 b 0.93 0.60 k 0.25 28.0 + 1.11

6. Kohala T89B 10 30.1 1.11 23.3 1.66 26.5 + 0.64 7. Mawae U18 20 35.2 + 1.02 32.8 + 1.70 30.7 + 0.76 8. Piihonua U57G 23 28.8 0.42 20.2 + 0.84 28.7 + 0.49 9. Olaa U13B 20 34.4 1.01 26.1 ± 1.71 31.3 ± 0.73 10. Volcano T81 18 33.7 0.80 30.4 ± 1.41 31.1 ± 0.64 11. Kilauea T92 17 33.1 1.01 32.5 + 1.45 30.5 ±0.62 * N, number of wild males examined. t F1 males from several wild-caught females.

RESULTS The basic pattern of bristle rows on Drosophila legs is evolu- tionarily conservative so that we have been able to use for D. silvestris the same nomenclature used for D. melanogaster (11). Accordingly, the bristles in rows 5 and 6 on the dorsal surface FIG. 1. The island of Hawaii, showing the places from which of the tibia, which are posterodorsal and anterodorsal, respec- samples of D. silvestris came. At sites 1-5 (Kona-Ka'd), males have are very few cilia in the middle rows of the foreleg tibia. At sites 6-11 tively, modified into cilia in males of all the populations of (north and east populations) males have 20-30 extra cilia in this region D. silvestris studied here, as well as in males of many other of the tibia. Hawaiian drosophilids. These cilia are distinguished from bristles in being long, recurved, and unbracted. Table 1 gives cilia counts for various populations of D. sil- Kona District, directly up the mountain from the older site at vestris. In males from the west and south sides of the island 1000 m; 3, Kahuku Ranch, 1220 m, Ka'u District; 5, Waihaka (Kona and Ka'u Districts; sites 1-5, Fig. 1; Table 1) there are Gulch, 1310 m, Ka'u District; 8, Piihonua, 1341 m, South Hilo virtually no cilia on the bare dorsal surface between rows 5 and to as District (formerly referred "Kipuka, 4400 ft"); and 9, 6. This is illustrated in Fig. 2A, in which only a single cilium is Volcano Experiment Station, 1250 m, Puna District. Generally, shown; the largest known number is two. On the other hand, the flies occur in small local populations near their principal host tibiae of males from populations elsewhere on the island (sites plants (lobeliads of the genus Clermontia) in rain forests be- 6-11, Fig. 1; Table 1) show means of 20-33 cilia on this same tween 1100 and 1650 m altitude. Except in the Kohala Range, surface (Fig. 2B). These extra cilia are arranged in several (two where it occurs alone, D. silvestris is broadly sympatric with to four) irregular rows ("Middle rows," Table The a second species, D. heteroneura, although only D. silvestris 1). northern reaches the higher altitudes. and eastern flies also appear to show more cilia in the marginal From each locality a series of wild-caught males were ob- rows, especially in row 5. tained. One foreleg of each such male was mounted in Euparol Close re-examination of the data on the inversions of D. sil- between two coverslips, and tibial bristle counts were made vestris reveals an interesting pattern. Populations to the south under the compound microscope. Females captured in the same and west (Table 2) have only five common inversions, whereas areas were placed in separate culture tubes and allowed to most of the populations to the north and east show seven (Table produce F1 larvae. Salivary gland chromosome smears were 3). Four of the five inversions common to the two areas, how- prepared in acetic acid/lactic acid/orcein, and the frequency ever, show striking parallel altitudinal variations (Tables 2 and of inversion polymorphisms was scored according to the usual 3; summarized in Fig. 3). Altitudinal clinal change over a 6-km method (9). New data have been used to supplement earlier transect in the Kilauea area is observed for inversions 3m, 4k2, information (9, 10). 412, and, to some extent, 4t. Very similar changes are observed

Table 2. Inversion frequencies in south and west populations of D. silvestris No. of Altitude, chromosomes Inversion frequencies Site m sampled 3m 3r 4k2 4t 412 4m2 2o Hualalai, low 1100 76 0.8031: 0.066 0.5791 0.776 0.0 0.0 0.0 Hualalai, high 1400 162 0.574J 0.006 0.370J* 0.698 0.037 0.0 0.0 Pauahi 1372 216 0.713 0.009 0.653 0.796 0.0 0.0 0.0 Kahuku, low 1220 216 0.9171 0.0 0.7731 0.806 * 0.0051 * 0.0 0.0 Pahipa, high 1646 58 0.446] * 0.0 0.241] * 0.569: 0.362] * 0.0 0.0 **, P = <0.01; ***, P = <0.001. Evolution: Carson and Bryant Proc. Natl. Acad. Sci. USA 76 (1979) 1931

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FIG. 2. (A) Dorsal surface ofthe foreleg tibia of a male D. silvestris from Hualalai (locality 1). The area between the two rows ofcilia is mostly bare; a single extra cilium is indicated (arrow). (B) Similar view of a foreleg tibia of a male from Kilauea Forest Reserve (locality 11). Arrows indicate the presence of extra cilia in the irregular middle rows. Row 6 (anterodorsal) is to the left in each picture; articulation with the tarsus is shown at the lower left-hand side of each figure. in two areas on the west slope of Mauna Loa (Kona) (Fig. 3 and manner as the male performs his head-under-wing movements Table 2). The transects of these latter two areas (Kahuku and orienting to the female. During a crucial stage of courtship, the Hualalai) are over comparable or even shorter linear distances male of D. silvestris folds his forelegs and places the dorsal (6 and 2 km, respectively ). surfaces of the tibiae against the dorsum of the female's abdomen and vibrates them (8). Indeed, a similar movement is a DISCUSSION characteristic and important aspect of courtship behavior of The evidence suggests that, during the last 700,000 years on the all members of the D. planitibia subgroup. Accordingly, the island of Hawaii, sexual selection within some populations of addition to the tibiae of north and east populations of D. sil- D. silvestris has led to the embellishment of a secondary sexual vestris of 20-30 cilia may represent an active new morpho- character on the male foreleg. This divergence has occurred logically expressed behavioral element which reflects an in- with virtually no detectable divergence at the level of allozymic tensification of the action of epigamic sexual selection. or chromosomal polymorphism. That this selection is novel and is directed toward a very re- The dorsal tibial bristles of the foreleg in the D. planitibia cent, perhaps incipient, character is made clear by the fact that subgroup of species, to which D. silkestris belongs, are modified the other closely related species of the subgroup lack the extra into long cilia in males only. These cilia are used in a complex cilia. This includes not only D. heteroneura of Hawaii but, more

Table 3. Inversion frequencies in north and east populations ofD. silvestris No. of Altitude, chromosomes Inversion frequencies Site m sampled 3m 3r 4k2 4t 412 4m2 2o Kohala 1220 44 0.909 0.545 0.705 0.773 0.0 0.182 0.186 Mawae no. 9 1554 172 0.552 0.128 0.395 0.709 0.256 0.087 0.046 Mawae no. 14 1524 166 0.566 0.048 0.404 0.843 0.018 0.066 0.121 Piihonua 1341 77 0.961 0.455 0.818 0.597 0.052 0.208 0.0 Volcano, low 1250 194 FO.954 0.0 * rO.825 0.974 0.0 0.0 0.0 Keahou, medium 1433 86 * LO.6161* 0.023 L0.6981 0.9531 0.0121 0.012 0.0 Kilauea, high 1615 436 0.324J* 0.0 0.511J 0.782J 0.179J 0.016 0.028 *,P = <0.05; **, P <0.01; ***,P = <0.001. 1932 Evolution: Carson and Bryant Proc. Natl. Acad. Sci. USA 76 (1979) WEST SLOPE (KONA) D. silvestris populations is not unexpected in view of the fact INVERSION INVERSION INVERSION INVERSION that pairwise comparisons between D. silvestris and its 3m 4k2 4t 412 sym- LOCALITY patric relative D. heteroneura are so high (0.94) that the two LOH S _ ' HUALALAI :4 I species cannot be diagnosed biochemically (14). Nevertheless, HIGH_._ I D. heteroneura is morphologically and behaviorally distinct from D. silvestris and shows strong ethological isolation from KAHUKU% LOW I that species (17). The great biochemical similarity of east and PAHIPAZ HIGH It --I I I west D. silvestris and D. heteroneura has been ascribed to the extreme recency of the evolution of these forms (18). SLOPE OF MAUNA LOA Mating choice experiments are currently in progress with VOLCANO " LOW laboratory strains derived from many of the sites mentioned KEAHOU Z MEDIUV KILAUEA H;GH here (K. Y. Kaneshiro, personal communication). Investigation of both the mode of inheritance and the geographical variability 0 25 50 75 0 25 50 75 0 25 50 75 0 25 50 of the extra cilia is also proceeding. Nevertheless, the data POPULATION FREQUENCY (PER CENT) presented in this paper support the view that behavioral dif- FIG. 3. Parallel altitudinal dines of inversion frequencies in ferentiation is pivotal in speciation of forms such as these, which D. silvestris from both the east and west sides of the island of Ha- display complex epigamic selection (10). Indeed, the genetic waii. response appears to be to the intraspecific sexual environment significantly, closely related species that are confined to rather than to environmental factors impinging from outside geologically older islands (D. planitibia of Maui, D. differens the organism. These Hawaiian species may indeed be referred of Molokai, and D. hemipeza of Oahu). Most striking for the to as ethospecies (10), a term which emphasizes that the pri- present case, however, is the fact that D. silvestris populations mary genetic change in speciation is the incipience of a new from the south and west areas of Hawaii also lack this character. repertoire of mating behavior having a genetic basis. When and It may be concluded that these populations have retained the where this occurred in the present case is now under investi- ancestral character in this regard or, conversely and more im- gation. portantly, the character obaerved in the north and east is newly We acknowledge the close cooperation of Drs. D. E. Hardy and K. acquired. Y. Kaneshiro of the Department of Entomology, University of Hawaii. Populations of D. silestris that differ in the bristle character Linden Teramoto and Joyce Kurihara provided valuable technical just described show strikingly similar altitudinal changes in the assistance. This work has been supported by Grants GB 29288, BMS frequencies of certain inversions. These are interpreted as al- 74-22532, and GB 23250 from the National Science Foundation and titudinal shifts of karyotype polymorphisms which have oc- HD 06082 from the National Institutes of Health. curred in response to the demands of very similar selective 1. Ayala, F. J. (1975) Evol. Biol. 8, 1-78. pressures in quite separate populations. Similar altitudinal chines 2. White, M. J. D. (1978) Modes of Speciation (Freeman, San on continental mountains in both eastern (12) and western (13) Francisco). North America have been explained in a similar manner. 3. Carson, H. L. (1976) BioScience 26,700-701. We suggest that both the inversions themselves and their 4. Dobzhansky, Th. (1976) in Molecular Evolution, ed. Ayala, F. capacity to respond altitudinally are relatively ancient features J. (Sinauer, Sunderland, MA), pp. 95-105. of D. silvestris populations and evolved prior to divergence 5. Dobzhansky, Th. & Spassky, B. (1959) Proc. Natl. Acad. Sci. USA within the species into the two morphological types. The idea 45,419-428. that the altitudinally shifting inversions are ancient is reinforced 6. Carson, H. L. & Kaneshiro, K. Y. (1976) Annu. Rev. Ecol. Syst. by the fact that one of them (inversion 3m) is also found in D. 7,311-345. 7. Hardy, D. E. (1965) Insects of Hawaii (Univ. of Hawaii Press, heteroneura and is present as a polymorphism in all known Honolulu, HI), Vol. 12. populations (9, 10). Thus we regard the altitudinal response of 8. Spieth, H. T. (1978) Evolution 32,435-451. D. silvestris inversions on mountainsides as a polyphyletic 9. Craddock, E. M. & Johnson, W. E. (1979) Evolution 33, 137- adaptational change. The facts, however, appear to exclude the 155. interpretation that this inversion variability is leading in the 10. Carson, H. L. (1978) in Ecological Genetics: The Interface, ed. diretimz of incipient, altitudinally differentiated species. Brussard, P. F. (Springer, Heidelberg, West Germany), pp. In addition to the morphological and chromosomal data just 93-107. discussed, information on electrophoretic enzyme variability 11. Hannah-Alava, A. (1958) J. Morphol. 103,281-310. in a number of local populations of D. silvestris is available (9, 12. Stalker, H. D. & Carson, H. L. (1948) Evolution 2, 295-305. 13. Dobzhansky, Th. (1948) Genetics 33,158-176. 14). Although the species is quite polymorphic, is very there 14. Sene, F. M. & Carson, H. L. (1977) Genetics 86, 187-198. little differentiation between populations on any part of the 15. Nei, M. (1972) Am. Nat. 106,283-292. island. Based on 25 loci encoding for soluble proteins, the mean 16. Ayala, F. J., Tracey, M. L., Hedgecock, D. & Richmond, R. C. similarity coefficient (15) for pairwise comparisons of popu- (1974) Evolution 28,576-592. lations is 0.96 (14). This is very close to that found intraspeci- 17. Kaneshiro, K. Y. (1976) Evolution 30, 740-745. fically in the D. uwillistoni group (16). Such similarity between 18. Carson, H. L. (1976) Nature (London) 259,395-396.