PATTERNS OF MOLECULAR VARIATION. 11. ASSOCIATIONS OF ELECTROPHORETIC MOBILITY AND LARVAL SUBSTRATE WITHIN SPECIES OF THE DROSOPHZLA MULLERZ COMPLEX1

Transcription

1 PATTERNS OF MOLECULAR VARIATION. 11. ASSOCIATIONS OF ELECTROPHORETIC MOBILITY AND LARVAL SUBSTRATE WITHIN SPECIES OF THE DROSOPHZLA MULLERZ COMPLEX1 R. H. RICHARDSON,z PETER E. SMOUSE394 AND MARTHA E. RICHARDSON Department of Zoology, University of Texas, Austin, Texas Manuscript received May 10,1976 Revised copy received July 30, 1976 ABSTRACT Electromorphic variation among populations of Drosophila mojavensis, D. arizonensis and D. longicornis was examined for seven genetic loci. The average electrophoretic mobility for a population was used as the metric. D. mojauensis and D. arizonensis use larval substrates in different parts of their geographic ranges, while D. Zongicornis is more narrowly restricted to different species of the cactus Opuntia in different localities. There is marked electromorphic variation among populations of either D. mojuvensis or D. arizonensis, and the bulk of this variation is accounted for by differences in lava1 substrate. There is somewhat less variation among populations of D. longicornis, and only a moderate portion of this is accounted for by larval substrate differences. There appears to be an association between the taxonomic diversity of the larval substrates and the electromorphic diversity of the Drosophila populations utilizing those substrates. Evidence is reviewed that suggests physiological mechanisms for these possibly adaptive associations. N an earlier paper (RICHARDSON and SMOUSE 1976), we used the average I electrophoretic mobilities of seven multiple-electromorph loci to measure the molecular variation among species of the Drosophila mulleri complex. One of the more tantalizing observations of that paper was a major electrophoretic difference in esterase-c between the giant cactus breeders (D. mojauensis and D. arizonensis) and the Opuntia ( prickly pear cactus) breeders (D. longicornis, D. pachuca, D. propachuca, D. desertorum, D. tira, D. ritae, D. hexastigma, D. aldrichi and D. mulleri). This finding, when coupled with a consideration of possible modes of evolution of electrophoretic differences, suggests that the larval substrates utilized by the flies might bear some causal relationship to the array of electromorphs recovered for certain loci. The purpose of the present paper is to pursue this matter at the intraspecific (interpopulation) level. D. mojavensis and D. arizonensis not only utilize a taxonomically disparate set of larval substrates from those used by other species of the D. mulleri complex, but each exhibits important interpopulation larval substrate variation as well; Supported by ERDA Contract AT-(40-1)4023. NIH Career Development Award GM a Supported by ERDA Contract E(l1-1) Department of Human Genetics, University of Michigan, Ann Arbor, Michigan Genetics 85: January, 1977

2 142 R. H. RICHARDSON. P. E. SMOUSE Ah-D M. E. RICHARDSON different genera of cactus are used in different parts of the geographic rangt. In view of the esterase-c difference mentioned above. we wish to determine the relationship between the electrophoretic mobilities of various loci and the larval substrates of different populations within each of these species. We shall also examine D. longicornis, whose larval diet is restricted to several species of the genus Opuntia, and which constitutes a control. Formally, we wish to answer the following questions. (1 ) Do D. mojnriensis and D. nrizonensis. which utilize different genera of cacti in different regions, show more electromorphic variation among pcpulations than D. longicornis, which utilizes different Opuntia species in different parts of its range? (2) To what extent is electromorphic variation among populations correlated with larval substrate variation among these same populations? The answers to these questions lead to some interesting suggestions concerning the source of much electrophoretic variation among populations. Population material MATERIALS.4ND METHODS We shall report here on twenty-nine (29) populations of the three species. The collection localities and principal cactus substrates are listed for all accessions in Tahle 1. The Sonora and Arizona populations of D. ~r~~jai~nsis are found on Larnaireocereus thui-beri (organ pipe), whereas those of Baja California. the Gulf Islands, and Desemboque (Son.) are recovered from gumrnosus (agria). The populations from California were obtained b>- baiting, and their lard substrate is still a mystery. It is worth noting, however. that since neither Lamaireocereus nor Machaerocerius is prcwnt ill this region. a third substrate is implicated. The California collections are morphologically and chi omosomally somewhat unusual. and METTLER (1963) designated this type Race A. METTLER S Race R includes both the and 1.amaireocereus collections. The D. arkonensis populations from western Mexico are recovered from Rnrhbunia alnniocriiis (cina), while those from central and eastern,mexico arc recovered from Myrtillocactus gc7orrzetriznns (garamhullo), The Opuntia species utilized by D. longicornis often occur in sympatry. and we know that more than ono host ma>- he utilizrd in a single locality. All of our D. lonpicornis collectioiis were ohtained from rot pockets. \vhich collections were stronglj- dominated by the substrates listed in Tahle 1. At least for the collections at hand. geography and substrate are totally confounded. Electrophoretic analysis The loci reported here are the same as those of the earlier paper: malate dehydrogenase (Mdh). octanol tlehytlrogenas:. (Odh). alcohol dehydrogenase (A&). pliosplioglucoinutase (Ppri). glutaniic-oxalonc:,tic traiisamiriase (Got). aldehyde oxidase (Ao). a~itl esterase-c (Esl). The electrophor:.tic assay procedures are those of Jorr~son. ei al. (1966). JOHNbON. RICHARDSON and K..\MHYSEI.LIS (1968). arid KO.JIMA. C~ILI.F.SPIE and TORARI (1970). We use the same metric hrre as in the eai.lier paper. nanielj- the weighted avcrt~gc elertrophoretic mobility (or mohilit~- ) of all rlectromorphs of a given locus recovered in a single population. We havtb listed the mohilitirs and average within-population variances for all seven loci and all twenty-nine populations in Table 2. It is worth noting at the outset that Got is segregating onlj- within D. lorzgicornis; and Adh is monomorphic in this same species. L nrintion anal3.sis For a single locus. the variation among a set of I populations is computed as

3 E1,ECTROPHORETIC-SUBSTRATE CORREI.ATION 143 TABLE 1 Collection locali/ies arid principnl crrclus suhsf rules of tuenly-nine populnlions of Drosophila niojavensis, D. ariz.or~ensis arid D. longicornis 33"OO' N 32"50' N 11 6"30' TV 115"OO' W Unknown Unknown Enseriada. Baja N. Sri. E'elipe, Baja N. Desenil)oque. Son. I. Tiburon. Baja N. Sri. Borja, Baja N. I. Sri. Esteban, Baja N. Sn. Ignacio, Baja S. Pt. Concepcion. Baja S. Haricho Cunano, Baja S. Todos Siiritos, Baja S. 31"iO'N 31"lO' N 29" IO' N 29'00' N 28"jO' N 28"40' N 27"30' N 26"JO' N 23"iO' N 23"20' N 116"10'W 1 1 'i"i0' w ' w 112"2O' w 113"iO' W 112"30'W 112"3' w 11l"iO' w 11 (I"4O' 1%' ' w i Machaerorereus 13 Navojoa, Son. 11. Enipalme. Son. 15 Herniosillo, Son. 16 Senita Basin, Ariz. 27"IO' N 28"OO' N 29"OO' N 32"OO' N 109"3O' W 1 1 O"40' W 110"~0'W 11 2Y0' w Lamaireocereus 1,ainaireocereus Laniaiteocereus Larnaireocereus D. arizonensis 17 Sn. Fr. Madrang,: Tam 18 Guayalejo, Tam. 19 Reyes, SLP 20 Venados, Hdg. 21 Venatlos, Hdg. 22 Navojoa, Son. 23 Hermosillo, Son. 23"30'N 23"'O' N 21 "io' N 20"30' N 20"30' N 27"lO' ht 29"00' N 99"30'W 99"OO' w loo"50' w 38"40'W 98"40' W 109" 30' w 110"5o'w My-rtillocactus Myrtillocactus Myrtillocactus Myrtillocactus Myrtillocactus Rathbunia Rathhunia D. longicornis 24 Austiii. Tex J Reyes, SLP 26 Venatlos, IIdg. '7 Pacliura, Htlg 30"10' N 21 "50' N 20"30' N 20" 10' N 97" io' w 1 OO"5O' w 98"10' TI' 98"40' w 0. Iindheinieri 0. rohusia 0. robusfa 0. leucotricha 28 Alarms, soil. 29 Narojoa, Sori '7"OO' N 27"lO' N 108"50' IT 109"30' 11' 0. phaeacaniha 0. phueacaniha where 'j2, and p are the average niobilitirs of the i-th population arid the whole species, respectively, and U? is thc weighted estimate of the within-population variation. The total intraspecific variation may be suhtlivitled to yield AL'(T) = A?(S) + A'(P), (2) where A'(S) is the variation among substrate types. and l'(p) is the variation arnong populatioris writhin substrate types. To ohtain a multiple-locus analysis, we simply add across loci. The motivatiori arid derivatiorr of this arialysis are presented in the Appendix of RICH.ARDSON and SMOUSE (1976). Past rxperience indicates that the mobilitj- metric extracts most of the useful

4 144 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON TABLE 2 Average mobilities for twenty-nine populations of Drosophila mojavensis, D. arizonensis and D. longicornis and average within-population mobility variances for each of seven loci Speries - Genetic loci and population Mdh Odh Adh Pgm Got An Est D. mojavensis D. arizonensis D. longicornis Within population variance ooo 1.OO'O ooo 1.ooo OW84 1.o , ,800, MO ,00095.I , ow 1.ooo 1.ow 1.om 1.MKJ 1.(Foe % OW a , , WO WO 1.om information from a multiple-electromorph locus. For a two-electromorph locus, the variation analysis extracts all of this information. RESULTS An examination of Table 2 will show that each lolcus exhibits a different pattern in each of the three species. It is therefore convenient to begin with a

5 ELECTROPHORETIC-SUBSTRATE CORRELATION 145 locus-by-locus description of these patterns. The separate variation analyses are all presented in Table 3, along with a summary across loci. Malate dehydrogenase (Mdh) None of the species exhibits much variation among populations for this locus (Table 3), and all extant variation derives from population differences in the frequency of rare electromorphs (Figure l), 1.23 in D. mojavensis, 0.74 and 1.23 in D. arizonensis, and 1.43 in D. Zongicornis. What little variation there is, does not appear to be substrate-associated in D. mojavensis and D. arizonensis, but does appear to separate the D. Zongicornis populations on the closely related substrates, 0. Zindeheimeri and 0. phaeacantha, from those found on 0. robusta and 0. leucotricha. We are nevertheless describing very minor differences. Octanol dehydrogenase (Odh) All three species are highly variable for this locus, and the impact of substrates is glaringly obvious (Table 3). Races A and B of D. mojavensis are both fixed, but for different electsomorphs (Figure l), thus accounting for all of the variation among populations. The Rathbunia and Myrtillocactus populations of D. TABLE 3 Components of mobility variation for seven loci in Drosophila mojavensis, D. arizonensis and D. longicmis Degrees Genetic loci Souyce. of of - Total variation freedom Mdh Odh Pgm Ao Est Adh/Got* per df D. moiauensis Among substrate types 2 Race A us. Race Bt 1 Mach. us. Lama. 1 Within substrate types 13 Within race A 1 Within Machaeracereus 9 Within Lamaireocereus 3 Total D. arizonensis 15 Among substrate types 1 Within substrate types 6 Within Rathbunia 1 Within Myrtillocactus 5 Total 7 D. longicornis Among substrate types 3 Within substrate types 2 Within 0. robusta 1 Within 0. phaeacantha 1 Total ,5146.om a043,5344, OO~OO ooa5.acm.0005, om oOOo ONO90.OOO C241.oooo.om I BO oo Q I OOOO W ,2649.OOOO.0112, m I H * Adh for D. moiauensis and D. arizonensis, Got for D. longicornis. + The substrate for Race A is unknown; Race B includes both and Lamaireocereus populations. The races are separated on morphological and cytological criteria.

6 146 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON MALATE DEHYDROGENASdOCTANOL DEHYDROGENASEl PHOSPHOGLUCOMUTASE RACE A MACHAEROCEREUS L EMAIREOCEREUS MYRTIL LOCACTUS RdTH0UNIA c! LINDHEIMERI c! ROBUSTA 0 LEUCOlRfCHA c! PHdEACdNTHA (.?) FIGURE 1.-Relative frequencies of Mdh, Odh, and Pgm electromorphs in different substrate races of Drosophila mojauensis, D. arizonensis, and D. longicornis. arizonensis are also very different, the first having a high frequency of the 1.00 electromorph and the second a high frequency of the 1.07 electromorph. The substrate contribution to variation in D. Zongicornis is also apparent, with the 0. lindeheimeri population differing in a major electromorph from the others. (Figure 1). Phosphoglucomutase (Pgm) The variation among populations of D. mojavensis is minimal (Table 3), and is not particularly associated with substrate differences (Figure 1). The same is true for D. longicornis, although the 0. Zindheimeri and 0. phaeacantha populations differ somewhat in the rarer electromorphs. The Rathbunia and Myrtilloc cactus populations of D. arizonensis are quite different, and are dominated by different electromorphs (1.29 and 1.00, respectively). There is minimal variation among populations utilizing the same larval substrate. AZdehyde oxidase ( Ao) A moderate fraction. of the variation in D. mojauensis (Table 3) is accounted for by frequency differences among the three substrate types (Figure 2), but a

7 ELECTROPHORETIC-SUBSTRATE CORRELATION 147 ALDEHYDE OXIDASE I ESTERASE -C \ALCOHOL DEHYDROGENASE FIGURE 2.-Relative frequencies of Ao, Est, and Adh electromorphs in different substrate races of Drosophila mojauensis, D. arizonensis, and D. longicornis. large fraction of the variation is found among populations which share the same substrate. There is not much variation among populations of D. urizonensis, and most of what there is separates populations within the Myrtillocactus type. There are large differences among populations of D. Zongicornis; a moderate fraction of the variation is accounted for by substrate differences and involves substantial changes in electromorph frequencies. Esterase-C (Est) Races A and B of D. mojauensis are very well separated by this locus (Table 3) ; the former is dominated by the 0.55 and the latter by the 0.45 electromorph (Figure 2). There is essentially no difference between the Machaemcereus and Lamaireocereus populations, and only slight variation among populations within either substrate type. The pattern is similar in D. urizonensis, where the Myl-tillocactus and Rathbunia populations are dominated by different electromorphs. There is not much variation within D. Zongicornis, but what there is can be largely attributed to substrate differences, reflecting a small shift of the 0. phaeocantha populations to the right (the flatter electromorph distribution has no impact on average mobility).

8 148 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON Alcohol dehydrogenase (Adh) The populations of D. mojauensis are most notable for a high frequency olf the 2.63 electromorph, whereas Race A and Lamaireocereus populations have high frequencies of the 0.80 electromorph (Figure 2). These differences contribute a large component of variation for substrates (Table 3). In addition, the populations exhibit considerable variation among themselves, some having high frequencies of 0.80 and some of The variation among populations of D. arizonensis is minimal, but is mostly attributable to subtle differences in electromorph frequencies between the two substrate types. D. longicornis is fixed for the 4.02 electromorph and hence exhibits no variation among populations. Glutamic-oxaloacetic transaminase (Got) There is no variation within either D. mojauensis or D. arizonensis, both of which are fixed for the 2.90 electromorph. The variation within D. longicornis is negligible and unrelated to substrates. We have not graphed the frequencies of this nearly monomorphic locus. Species comparisons Those loci which exhibit large amounts of variation (Odh, Ao, Est, and Adh) within D. mojauensis also show large substrate-associated differences in electrophoretic mobility. Two of the loci (Ao and Adh) also show considerable variation within the type. Those loci exhibiting minimal variation (Mdh and Pgm) show no particular substrate associations. The final column of Table 3 is the sum of all the single-locus components, which are expressed on a per-degree-of-freedom basis. The among-substrates component (31.80) is about six times the size of the within-substrates component (5.22), indicating rather large substrate-associated differences. The Race A us. Race B component (36.97) is not much larger than the variation between the and Lamaireocereus types (26.63), showing that host race formation need not be accompanied by morphological divergence. The results for D. arizonensis are even more clearcut. The loci with large amounts of variation (Odh, Pgm, and Est) are notable for large substrate components, whereas those with less variation (Mdh, Ao, Adh) show no substrate pattern. The among-substrates component (54.00) is about ninety times the size of the within-substrates component (0.64). The results for D. Zongicornis are more subtle. The two loci with large amounts of variation (Odh and Ao) show moderate substrate differences, but the other loci (Mdh, Pgm, Est, and Got) show nothing of particular interest. The amongsubstrates component (4.60) is only about two and one half times larger than the within-substrates component (1.95). We are now in a position to answer the questions posed at the outset. (1) Both D. mojauensis and D. arizonensis exhibit large amounts of electromorphk variation among populations (8.77 and 8.26 per degree of freedom, respectively), while D. Zongicornis is only about half as variable (3.54 per degree of freedom). (2) the first two species utilize alternate larval substrates in different cactus

9 ELECTROPHORETIC-SUBSTRATE CORRELATION 149 genera in different portions of their geographic ranges, and differences among these host races account for most of the electromorphic variation. The third species employs a narrower range of larval substrates (several species of Opuntia), and shows only moderate electromorphic variation among populations utilizing different substrates. These results suggest that the greater the taxonomic diversity of the larval substrates, the greater the electromorphic diversity of the corresponding geographic host races of Drosophila. It is worth noting that all of these electromorph distributions are unimodal, and that population differences are achieved by shifting the whole mobility distribution up or down the electrophoretic ladder. This pattern is a smallscale equivalent of the cascading effect across species so notable in the earlier paper (RICHARDSON and SMOUSE 1976). It is this very regular feature of the electromorphic distribution which permits us to capture the bulk of the useful information in a multiple-electromorph array in the form of two parameters: the average mobility for a given population and the within population mobility variance. DISCUSSION We mentioned earlier that geographic and substrate separation are almost totally confounded for these collections. Inasmuch as geographic separation has concomitants other than substrate differences, how do we know that the patterns observed are due to substrate differences per se? In particular, a number of workers have reported striking associations between climatic variables and electromorph frequencies in several different organisms (KOEHN and RASMUSSEN 1967; JOHNSON et al. 1969; KOJIMA, GILLESPIE and TOBARI 1972; VIGUE and JOHNSON 1973). Although it is possible that the observed variation patterns might be attributable to climatic differences associated with geography, certain auxiliary observations suggest to us that climate is not a strong proximal factor. The populations of D. mojavensis extend over 8 of latitude and 1100 km in Baja California, encompassing a wide range of climatic conditions. Two of the substrate variable loci (Adh and Ao) show large amounts of variation among popula tiom within the type. Geographic variation of Adh electromorph frequencies in D. mlanogaster has been explained in terms of temperature effects on molecular stability and kinetic activity (VIGUE and JOHNSON 1973; PIPKIN, RHODES and WILLIAMS 1973; DAY, HILLIER and CLARKE 1974; AINSLEY and KITTO 1975). A similar explanation might apply here for either Adh or Ao. An alternative explanatioln is possible, however, since Lamaireocereus and are sympatric over much of the southern two-thirds of Baja California. Although D. mojavensis seems to prefer the latter, given a choice (FELLOWS and HEED 1972), it is quite possible that local concentrations of Lamaireocereus-eclosed flies are the source of the extra variation, rather thalz climatic variation. Within single substrate-types of D. arizonensis we see no similar variation patterns. Since the widest range of conditions is experienced by the Myrtillocactus type, which extends in our collections over only 3 of latitude and 300 km,

10 150 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON this is hardly compelling evidence, one way or another. The strongest evidence against climate, as an important proximal factor of geographic variation, is provided by D. longicornis. Our collections extend from Austin to Pachuca (10 of latitude, 1000 km) and from Pachuca to Navojoa (7 of latitude, 1300 km), but there is only moderate variation among populations, much of which is associated with substrate changes. A similar situation is known for D. pachea, which ranges from Senita Basin (Arizona) to Zaragosa (Sinaloa). This species is strictly confined to Lophocereus schottii (senita), and shows virtually no geographic variation among populations (ROCKWOOD-SLUSS, JOHNSTON and HEED 1973), although several of the loci studied are quite polymorphic within single populations. These observations suggest that it is substrate per se, rather than climate or some other correlated factor, which accounts for the frequencies of the various electromorphs. It may well be that climatic variables have a secondary role, in terms of the availability of different substrates. An important test of this substrate hypothesis would be to compare samples of the same species from alternate hosts in the same location. For D. mojauensis, a careful search of Lemaireocereus rot pockets in mixed cactus areas should yield such natural experiments. We have already mentioned the mixed cactus area of Baja California, but should also point out the transition zone from to Lemaireocereus near Desemboque (Sonora). For D. arizonensis, the shift from Myrtillocactus to Rathbunia cannot be accomplished sympatrically, since the two substrates are strictly allopatric. This species is more flexible in its habits than D. mojauensis, however, and with a bit of work can usually be recovered from other cacti in either zone. The natural experiment should be easiest to pursue in D. Iongicornis, where we already suspect that sympatric Opuntia substrates of different species are sometimes utilized. The differences expected here are, of course, somewhat smaller than for the other two species. In any case, it should be possible to supplement the field studies with laboratory experiments of a cross-rearing sort. The results should be enlightening. Irrespective of whether substrate shifts are the proximal factor in the genetic differences observed, there is no guarantee that the loci under observation are the ones of interest. We might be observing a remnant of piggy-backing (KOJIMA and SCHAFFER 1967), due to close linkage of observed markers with others under selective pressure. This possibility is particularly important where inversion polymorphisms must be considered. ZOUROS (1976) has shown that Est, Ao and Odh are located on the second chromosome for these species, while Adh and Mdh are on the third chromosome. METTLER (1963) has shown that D. mojauensis populations differ for second and third chromosome inversions. The Race A and Lamaireocereus populations are essentially fixed for alternative second chromosome inversions. while the populations are polymorphic and rather variable (JOHNSON 1973). Third chromosome inversions are segregating in both substrate types of Race B, and the standard arrangement is fixed in Race A. Inasmuch as both Ao and the second chromosome inversion are rather variable in the populations, one wonders whether the patterns are cor-

11 ELECTROPHORETIC-SUBSTRATE CORRELATION 151 related. ZOUROS ( 1976), however, found no such associations, and while we cannot rule this possibility out absolutely, we see no reason to invoke it. WASSERMAN (personal communication) has found three second chromosome inversions in D. longicornis. Most of his samples, however, were from more southern portions of the species range than we have sampled, and the one small sample from the nothern portion of the range was segregating for only one of these inversions. If these northern populations are almost monomorphic, then our observations should not be due to inversion-generated piggy-backing. On the other hand, it is worth noting that Ao and Est are the two most variable loci within D. longicornis. There are no known inversion polymorphisms within D. arizonensis, so that strong linkage-disequilibrium from this source is unlikely for this species. We cannot, of course, rule out piggy-backing per se, which does not require polymorphisms. The only way to demonstrate convincingly that the loci in question are the ones of interest (relative to substrates) is to examine the biochemical behavior of the different electromorphs. Although the relationship between electrophoretic mobility and biochemical properties remains obscure. there are several clues from the literature which suggest that an examination of biochemical behavior for these electromorphs would prove fruitful. AINSLEY and KITTO (1975) and CLARKE (1975) point out that the different electromorphs of Adh exhibit different alcohol substrate affinities and play different roles in alcohol detoxification in D. melanogaster. WILLS, PHELPS and FERGUSON (1975) have shown that D. pseudoobscura strains fixed for different electromorphs of Odh and Est-5 survive differentially under octanol, ethanol, tributyrin and triacetin loading, and they have suggested that these two loci interact at the physiological level. We also know that alcohols affect the in vitro activity of different esterases. For example, different species and strains vary widely in their esterase activities under n-propanol loading (JOHNSON et al. 1966). We have also observed (unpublished data) that methanol and ethanol often enhance in vitro esterase activity, while longer chain alcohols and aldehydes inhibit activity. Numerous paths of interaction among loci, as well as direct evidence for selection. are suggested by extensive research on alcohol metabolism ( LIEBER 1976; GEER et al. 1976), insect toxicology and insecticide resistance (CORBETT 1974; PLAPP 1976). Although there have been several surveys of the Cactaceae ( HEGNAUER 1962), more comprehensive and sophisticated biochemical analyses are badly needed. Toxic alkaloids and similar compounds occur in the Cactaceae, and there is at least one well understood case of Drosophila adapting to a cactus alkaloid (KIRCHER et al. 1967). Alkaloids are not, however, recovered in apprecible quantities from the cacti utilized by these particular Drosophila species. Saponins are found in Lamaireocereus, and Myrtillocactus, but Rathbunia and Optunia have not been examined. A number of other potentially active compounds found in these genera include polyphenols, organic acids, and esterified triterpenes. Furthermore, different cacti are decomposed by different arrays of

12 152 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON yeast and bacteria (STARMER et al. 1976; HEED et al. 1976). The different species of this microflora have very different metabolic capabilities, particularly with respect to alcohol production. This rot-pocket microflora represents a crucial portion of the diet for Drosophila, and largely determines the state and chemical composition of the larval milieu. Together, these disparate observations suggest a hypothesis. We postulate that EST, found at high concentration in the gut (KAMBYSELLIS, JOHNSON and RICHARDSON 1968), ADH and ODH are involved in the detoxification of cactus secondary and degradation products, and that the electrommphic variation encountered in the Drosophila utilizing these cacti reflects the diversity of these cactus products found in alternate substrates. We also hypothesize that EST activity is differentially affected by various alcohols produced by the digestion of these cactus products by the microflora of different cacti. Furthermore, the EST pattern may reflect secondary effects of microsomal oxidases, insoluble detoxification enzymes to which EST and (AO) are coupled. There are several known cases in which a reaction product in the detoxification system itself may be highly toxic. Thus, adaptation may sometimes involve reducing the enzyme activity. It also seems reasonable to speculate that the ODH and ADH changes which accompany the substrate shifts are direct responses to these different nutritional challenges. They may play regulatory roles through the inhibitorystimulatory effects of their substrates on EST or other enzymes. The probable relationship of substrate shifts and electromorphic changes at the Ao locus is not at all obvious at this juncture, but it is worth noting that A0 is involved in the metabolism of alcohols and fatty acids, which are important degradation products of cactus sterols. A0 is also coupled to the microsomal oxidase system, and may be secondarily affected. Glycolytic inhibitors are known for PGM (COBBETT 1974; MAHLER and CORDES 1971), and Pgm electromorphic clifferences may relate to differences among cacti, with respect to the sugar and slime which the flies consume and digest. It is obvious that before any of these suggestions may be treated as established fact. a considerable amount of detailed biochemical investigation is needed at the interface between insects, microflora and cactus substrates. The adaptive significance of these electromorphic patterns can only be revealed by combining population genetics and biochemical ecology. The taxonomic assistance and instruction in Drosophila field biology by DR. W. B. HEED is especially acknowledged. We also thank DR. HEED and his students, and DRS. SPENCER JOHN- STON, TOM STARMER, BERNARD WARD and IRE and JEAN RUSSELL for numerous collections and helpful discussions. The collections and cytological assistance of DR. MARVIN WASSERMAN have also been most helpful. His successful recollection of Race A of D. moiavemis deserves special note. The technical assistance of Ms. ANN CAPPS, MR. WILLIAM SIEVERT, MR. WILMER AVERHOFF, Ms. ANDREA LASSETER, and DRS. JONG SIK YOON, RICHARD AINSLEY and LARRY SPRECHMAN has been most helpful. The many cactus identifications and the distribution information given SO freely by PROF. E HERNANDEZ XOLOCOTZI, Chapingo, Mexico, have been of great importance in planning and executing much of the field work. DRS. LYNN THROCKMORTON and MARSHALL WHEELER have freely provided helpful suggestions and encouragement. We also appreciate the helpful criticism of several colleagues during the preparation of this manuscript.

13 ELECTROPHORETIC-SUBSTRATE CORRELATION 153 LITERATURE CITED AINSLEY, R. and G. B. KITTO, 1975 Selection mechanisms maintaining alcohol dehydrogenase polymorphisms in Drosophila melanogaster. In: Isozymes, Vol. 2, Physiological Function. Edited by C. L. MARKERT. Academic Press, New York. CLARKE, B., 1975 The contribution of ecological genetics to evolutionary theory: Detecting the direct effects of natural selection on particular polymorphic loci. Genetics 79: CORBETT, J. R., 1974 The Biochemical Mode of Action of Pesticides. Academic Press, New York. DAY, T. H., P. C. HILLIER and B. CLARKE, 1974 Properties of gemetically polymorphic isozymes of alcohol dehydrogenase in Drosophila melanogaster. Biochem. Genet. 11 : FELLOWS, D. P. and W. B. HEED, 1972 Factors affecting host plant selection in desert-adapted cactiphilic Drosophila. Ecology 53 : GEER, B. W., S. N. KAMIAR, K. R. KIDD, R. A. NISHIMURA and S. J. YEMM, 1976 Regulation of the oxidative NADP-enzyme tissue levels in Drosophila melanogaster. J. Exp. Zool. 195: HEED, W. B., W. T. STARMER, M. MIRANDA, M. W. MILLER and H. J. PHAFF, 1976 An analysis of yeast flora associated with cactiphilic Drosophila and their host plants in the Sonoran Desert and its relation to temperate and tropical associations. Ecology 57: HEGNAUER, R., 1962 In: Chemotaxonomie der Pflanzen, Vol. 3. pp JOHNSON, F. M., H. E. SCHAFFER, J. E. GILLASPY and E. S. ROCKWOOD, 1969 Isozyme genotypeenvironment relationships in natural populations of the harvester ant, Pogonomyrmex barbatus, from Texas. Biochem. Genet. 3: JOHNSON, F. M., C. G. KANAPI, R. H. RICHARDSON, M. R. WHEELER and W. S. STONE, 1966 An operational classification of Drosophila esterases for species comparisons. In: Studies in Genetics Ill. Edited by M. R. WHEELER. University of Texas Publication No. 6615: , Austin. JOHNSON, F. M., R. H. RICHARDSON and M. P. KAMBYSELLIS, 1968 Isozyme variability in species of the genus Drosophila Qualitative comparison of the esterases of D. aldrichi and D. mulleri adults. Biochem. Genet. 1 : JOHNSON, W. R., JR., 1973 Chromosome variation in natural populations of Drosophih moiauensis. Unpublished MS Thesis, Department of Biological Sciences, University of Arizona, Tucson. KAMBYSELLIS, M. P., F. M. JOHNSON and R. H. RICHARDSON, 1968 Isozyme variability in species of the genus Drosophila. N. Distribution of the esterases in body tissue of D. aldrichi and D. mulleri adults. Biochem. Genet. 1 : KIRCHER, H. W., W. B. HEED, J. S. RUSSELL and J. GROVE, 1967 Senita cactus alkaloids: Their significance to Sonoran Desert Drosophila ecology. J. Insect Physiol. 13: KOEHN, R. K. and D. J. RASMUSSEN, 1967 Polymorphic and monomorphic serum esterase heterogeneity in Catostomid fish populations. Biochem. Genet. 1 : KOJIMA, K., J. GILLESPIE and Y. N. TOBARI, 1970 A profile of Drosophila species enzymes assayed by electrophoresis. I. Number of alleles, heterozygosities, and linkage disequilibrium in glucose-metabolizing systems and some other systems. Biochem. Genet. 4: KOJIMA, K. and H. E. SCHAFFER, Survival process of linked mutant genes. Evolution 21: KOJIMA, P. SMOUSE, S. YANG, P. S. NAIR and D. BRNCIC, 1972 Isozyme frequency patterns in Drosophila pauani associated with geographical and seasonal variables. Genetics 72 :

14 154 R. H. RICHARDSON, P. E. SMOUSE AND M. E. RICHARDSON LIEBER, C. S., 1976 MAHLER, H. R, and E. H. CORDES, 1971 York. The metabolism of alcohol. Sci. Amer. 234: Biological Chemistry. Harper and Row Publishers, New METTLER, L. E., 1963 Drosophila mojavensis baja, a new form of the mulleri complex. Drosophila Inform. Sew. 38: PIPKIN, S. B., C. RHODES and N. WILLIAMS, 1973 Influence of temperature on Drosophila alcohol dehydrogenase polymorphism. J. Heredity 64: PLAPP, F. W., 1976 Biochemical genetics of insecticide resistance. Ann. Rev. Entomol. 21: RICHARDSON, R. H. and P. E. SMOUSE, 1976 Patterns of molecular variation. I. Interspecific comparisons of electromorphs in the Drosophila mulleri complex. Biochem. Genet. 14: ee ROCKWOOD-SLUSS, E. S., J. S. JOHNSTON and W. B. HEED, 1973 Allozyme genotypeenvironment relationships. I. Variation in natural populations of Drosophila pachea. Genetics 73 : STARMER, W. T., W. B. HEED, M. MIRANDA, M. W. MILLER and H. J. PHAFF, 1376 The ecology of yeast flora associated with cactiphilic Drosophila and their host plants in the Sonoran Desert. Microbial Ecology 3: VIGUE, C. L. and F. M. JOHNSON, 1973 Isozyme variability in species of the genus Drosophila. VI. Frequency-propertyenvironment relationships of allelic alcohol dehydrogenases in D. melanogaster. Biochem. Genet. 9: WILLS, C., J. PHELPS and R. FERGUSON, 1975 Further evidence for selective differences between isoalleles in Drosophila. Genetics 79: ZOUROS, E., 1976 The distribution of enzyme and inversion polymorphism over the genome of Drosophila: Evidence against balancing selection. Genetics 83: Corresponding editor: R. W. ALLARD