journal of molecular evolution - beverleybeverleylab.wustl.edu/pdfs/006. beverley.wilson j.mol.evol....

J Mol Evol (1982) 18:251-264 Journal of Molecular Evolution C) Springer-Verlag 1982

Molecular Evolution in Drosophila and Higher Diptera

I. Micro-Complement Fixation Studies of a Larval Hemolymph Protein

Stephen M. Beverley 1,2 and Allan C. Wilson 1

1Department of Biochemistry, University of California, Berkeley, CA 94720, USA 2Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

Summary. LHP is a suitable protein for studying evolution in flies (Diptera). This blood protein, which occurs at high concentration late in larval development, was purified to homogeneity from 5 species of Drosophilidae and one species each of Tephritidae and Calliphoridae. Rabbit antisera to the purified LHPs allowed immunological comparisons to be made with the micro-complement fixation technique. Various indirect tests indicated that immunological distance is a refiable estimator of the degree of amino acid sequence difference between LHPs from different species. An evolutionary tree for the 7 LHPs was constructed from the immunological distances with the method of Fitch and Margoliash (1967) to provide the branching order and the method of Chakraborty (1977) to provide the branch lengths. A modified method of tree construction allowed LHPs from 10 additional species to be attached to this tree. The resulting LHP tree for 17 species agrees approximately in branching order with that based on Throck- morton's study of 60 anatomical traits. However, the ratio of anatomical change to LHP change along branches within the tree varies widely, confirming the independence of organismal and molecular evolution. The LHP tree thus provides a new perspective on evolution within and among the families of higher Diptera.

Key words: Anatomical vs. protein evolution - Cyclor- rhaphan relationships - Molecular evolutionary trees

Introduction

It is important to obtain a quantative picture of evolutionary relationships among lineages within the genus

Offprint requests to: S.M. Beverley

Drosophila. The rapid advances now taking place in our understanding of the Drosophila genome and its role in embryonic development (Ashburner and Novitski 1976; Gehring 1978) promise to make this genus one of the best taxonomic groups with which to examine the molecular basis of evolution.

Proteins provide a moderately reliable way of con- structing an evolutionary tree for the species within such a taxonomic group. This is known from research on vertebrates, where comparative studies of a single protein, such as serum albumin or cytochrome c, have yielded trees whose branching orders agree approximately with those based on anatomical evidence as well as evidence from other proteins (Wilson et al. 1977). A molecular tree is a quantitative temporal framework with which to analyse the molecular and nonmolecular differences among species. The tree is thus a tool for studying the mechanism of evolution. For these reasons, we decided to build an evolutionary tree based on protein comparisons for the main lineages within the genus Drosophila.

Protein methods have, of course, been used before to study evolutionary processes in this genus. Such studies, however, dealt typically with electrophoretic differences among proteins from individuals within a species or species group (Lewontin 1974; Ayala 1975). The electrophoretic methods did not permit assessment of evolutionary relationships among species groups and subgenera in the genus. To estimate the extent of protein sequence divergence among such distantly related taxa, one must use another method, such as amino acid se- quencing (Dayhoff 1972) or quantitative micro-complement fixation (Champion et al. 1974). Except for the pioneering work of Collier and Maclntyre (1977, 1978), little has been done along these lines within Drosophila.

This paper shows how to choose a Drosophila protein suitable for micro-complement fixation studies and pres-

0022-2844/82/0018/0251/$ 02.80

252

ents results al lowing the cons t ruc t ion o f a tree tha t

por t rays genealogical relat ions among lineages in the

genus Drosophila. The pro te in chosen is a major compo-

n e n t in the h e m o l y m p h o f the late larval and early

pupal stages. It is referred to as LHP, which s tands for

larval h e m o l y m p h pro te in 1 .

Materials and Methods

Fly Strains. The sources of species from which LHPs were purified are given in Beverley (1979). The gl 3 mutant of D. melanogaster (containing an electtophoretic variant of LHP-Hubby, 1963) was obtained from the Division of Biology, California Institute of Technology, Pasadena, CA. The National Droso- pbila Species Resource Center, University of Texas, Austin, Texas, provided all remaining species of Drosopbila utilized; the exact strains are given by Beverley (1979). Megaselia scalaris were provided by Bo Slatko, Department of Genetics, Univer- sity of California, Davis, CA. Cbrysops birsuticallus and mosqui- to larvae were coUected in the field. Dacus cucurbitae were provided by the USDA, Hawaii; Cochliomyia hominivorax by the screwworm eradication program, USDA, Texas.

Antisera. Antiserum to chicken lysozyme c (Pool 4B) was provided by Dr. E~i. Prager (Prager and Wilson 1971a,b); antiserum (6F) to human serum albumin was provided by Dr. V. Sarich (Sarich and Wilson 1966).

Purification of LHPs and Criteria of Purity. The purification of LHPs has been described (Beverley 1979). The LHP preparations were shown to contain only LHP by electrophoresis in acrylamide gels under denaturing conditions (sodium dodecyl sulfate; Ames 1974) and in nondenaturing conditions (pH 8.9 system of Maurer 1971) at several acrylamide gel con- centrarions.

The electtophoretic pattern of hemolymph and purified LHP from D. melanogaster is shown in Fig.1. LHP from D. melanogaster was also shown to be homogeneous by sedimenta- tion velocity centrifugation at pH 6 (polymeric form) and pH 8 (monomeric form).

Preparation of Monomeric and Polymeric LHP. Monomeric LHP preparations in this work are obtained by dialysis (or Se- phadex G-25 chromatography) in 0.1M Tris, pH 8.4, 4°C. Poly- meric LHP preparations may be obtained by dialysis against either 0.1M potassium phosphate, pH 7 or 0.01M sodium barbi-

1 A number of hemolymph proteins have been described in insects (Thomson 1975; Wyatt and Pan 1978). One group of proteins seems to function primarily as storage proteins during larval development; LHP is a member of this group. The protein we designate as LHP (Beverley and Wilson 1977) has also been termed Pt-1 (Hubby 1963) and LSP-2 (Roberts et al. 1977) in Drosophila melanogaster, and its probable homologue in CaIliphora stygia is termed Protein B.

The nomenelatural problems evident for LHP also exist for another group or related hemolymph proteins - these include caUiphorin (Munn et aL 1971), Protein C (Kinnear and Thomson 1975), lucitin (Thomson et al. 1976) and LSP-1 (Wolfe et al. 1977). We propose elsewhere a unifying nomen- clature for these proteins, based upon the degree of amino acid compositional and immunological relatedness (Berverley 1979): a LHP class (including LHPs or LSP-2 of Drosophila and Protein B) and a calliphorin class (including calliphorin, Protein C, lucilin and LSP-1). Thus, we now refer to protein B as LHP from Calliphora stygia, in accord with the usual manner of naming homologous proteins from different species.

tal, pH 7.4, 4°C. The monomeric or polymeric nature of these LHP preparations may be rapidly tested by immunodiffusion (Results; see Fig.2). The pH-dependent dissociation of LHPs is reversible, either completely (D. melanogaster, D. mulleri, Scaptomyza and Cocbliomyia) or partially (D. crucigera, D. mimica and Dacus).

Production of Antisera. The strategy employed in the production of antisera for use in complement fixation has been described (Prager and Wilson 1971; Champion et al. 1974). Anrisera were elicited to LHPs from Drosophila melanogaster, D. crucigera, D. mimica, D. mulleri and Scaptomyza adusta (Drosophilidae), Cocbliomyia bominivorax (screw-worm fly; CaUiphoridae) and Dacus cucurbitae (Tephritidae). For each antigen, four rabbits were immunized intradermaUy with 6 6 - 150 ttg of purified monomeric LHP, emulsified with Freund's complete adjuvant (supplemented with 4 mg/ml killed Myco- bacteria). Intravenous booster injections of 20-60 #g were given at periodic intervals, and bleedings were taken 7--10 days later. The final bleedings were taken from 30-38 weeks after the initial immunization. Heat-treated sera were prepared as described (Champion et al. 1974) and stored at -20°C. The sera from individual rabbits were pooled in inverse proportion to their immunological titers (Prager and Wilson 1971). Dur- ing the lengthy course of immunization several rabbits died, as is evident in Table 1, which summarises information about the antisera.

It is possible that under physiological conditions in the rabbit LHP may reassodate to form a polymeric molecule, rather than the desired monomeric form° We have not experi- mentally tested the LHP antigen from rabbits during the course of immunization; however, several observations as suggest that polymerization would not occur in vivo: 1. At concentrations of 1 mg/ml LHP requires approximately

one day at pH 6 to reassociate completely; at pH 7.4 3--4 days are required (Beverley, unpublished observations).

2. The low concentration of LHP in situ would not favor the polymerizatio n . Micro-Complement Fixation. Micro-complement fixation

was performed as described by Champion et al. (1974) but with an important modification: the diluent utilized was Isotris buffer (Champion et alo 1974) supplemented with 1 mg/ml chicken egg albumin (Sigma) and adjusted to pH 8.15 at 4°C° This buffer was made immediately prior to use. We term this

Orlgin 0 pH 8.9

C

Fig. 1. Electrophoretic evidence for the purity of larval hemolymph protein (LHP) from Drosophila melanogaster. Electro- phoresis was performed in polyacrylamide gel in a nondenaturing buffer at pH 8.9 (acrylamide concentration = 7.5%);the protein bands were visualized by staining with Coomassie brilliant blue G-250. Sample a, purified LHP; b, bovine serum albumin; c, hemolymph. LHP was purified as described by Beverley (1979); briefly, hemolymph from late third-instar larvae was fractionated with ammonium sulfate, then subjected to DEAE-cellulose chromatography, Sephadex G-100 chromatography, and preparative electrophoresis using polyacrylamide gels

Table 1. Description of antisera to larval hemolymph proteins

253

Average Average slope "m ' 'a Number of titer of

Species of LHP rabbits antiserum pool Homologous SD Heterologous SD

Drosophila melanogaster 3 7430 335 34 (8) 309 41 (19) Drosophila crucigera 4 1240 326 44 (6) 372 77 (17) Drosophila mulleri 3 1910 324 56 (6) 338 85 (20) Scaptomyzaadusta 3 700 363 56 (6) 354 45 (8) Drosophila mimica 2 1090 357 95 (7) 329 97 (12) Dacus cucurbitae 4 2900 234 53 (4) 259 56 (5) Cochliomyia hominivorax 4 810 335 13 (2) ND ND

a Slopes were calculated according to Equation 1 ; the number in parenthesis refers to the number of determinations (homologous) or heterologous LHPs. ND = not done. SD = standard deviation

the Isovotris buffer. All antisera and antigen preparations were O . ,

centrifuged (35,000 x g, 20 minutes, 4 C) immediately prior to use. All dilutions of antigen and antiserum were made in glass tubes which had been washed in chromic acid cleaning solution and washed extensively in distilled water. These steps help to ensure reproducible results.

Preparation of Antigens for Complement Fixation. Puri- fied LHPs were dialyzed against O.1M Tris, pH 8.4 (4°C) before use in complement fixation tests. Crude preparations of LHP were obtained by placing third instar larvae (either fresh or frozen, stored at -80°C) in approximately 2 volume of 50% saturated ammonium sulfate solution (4°C), and crushing them gently with a glass rod. The mixture was then centrifuged to remove body parts and precipitated protein; this supernatant was collected and re-centrifuged. The final clear supernatant was passed over a 10 ml Sephadex G-25 column in the pH 8.4 Tris buffer described above. Fractions emerging at the ex- cluded volume were collected, pooled, immediately frozen at -80°C, and stored at -20°C.

This method of antigen preparation is efficient and yields a stable, monomeric LHP preparation suitable for use in complement fixation tests (Beverley 1979). It is recommended that such extracts be thawed for the briefest possible period when removing aliquots for testing.

Criteria of Immunochemical Purity. Antisera were tested in three ways for immunochemical purity: 1. immunodiffusion against polymeric LHP at pH 7.4 (O.O1M

sodium barbitol buffer, room temperature) 2. immunodiffusion against monomeric LHP at pH 8.4 (O.1M

Tris CI, room temperature) 3. immunoelectrophoresis against monomeric LHP (O.1M

Tris CI, pH 8.6, 4°C). In every case, both crude and purified LHP preparations were examined and only a single precipitin band developed, corresponding to LHP. (The antigen concentration were monitored to assure that the immunoprecipitates were occuring at concentrations appropriate for this test; Prager et alo 1978).

Antisera to LHPs from D. melanogaster, D. crucigera and D. mulleri were also subjected to micro-complement fixation tests against crude and purified preparations of the homologous LHP. In each case, the maximum percentage of complement fixed was the same for the two preparations within the experimental error, and the maximum reaction occurred at similar LHP concentrations.

Estimation of Immunological Distance. The micro-complement fixation method (Champion et al. 1974) was used to measure immunological distance between pairs of monomeric LHPs from different species. The results were checked quali- tatively by immunodiffusion tests, which evaluated the extent

of spur formation (cf. Prager et al. 1976). In all cases there was qualitative agreement between the results of the two methods. Purified LHPs gave the same results as crude preparations.

The average reproducibility of the immunological distances was within -+2.6 units (standard deviation). In addition, preparations of LHP from larvae, pupae and one-day-old adults from the same species gave immunological distances that were the same, i.e. within the range of experimental error stated above (Beverley 1979).

Results

pH-Dependent Aggregation. LHP undergoes a pH depen-

d e n t d i ssoc ia t ion w h i c h af fec ts i ts an t igen ic p roper t i e s .

In vivo and be low pH 7.6 in v i t ro , LHP exis ts as a poly-

mer ( A k a m et al. 1978 ; Bever ley 1979) , whi le above

p H 8 i t exists p r imar i ly as a m o n o m e r , w i t h a m o l e c u l a r

weight o f 7 8 , 0 0 0 da l tons . This p h e n o m e n o n depends o n

the bu f f e r c o n d i t i o n s ; the behav io r descr ibed above

applies to Tris and Isotr is buf fe r s , b u t in ba rb i t a l bu f f e r s

a more c o m p l e x b e h a v i o r is observed ( re fe rences c i ted

previous ly) .

I m m u n o d i f f u s i o n provides a rapid and c o n v e n i e n t

tes t for t he aggregat ion s ta te o f LHP, as i l l u s t r a t ed in

Fig. 2. As s h o w n in the lef t p o r t i o n o f the f igure, the

shape o f the i m m u n o p rec ip i t a t i on l ine depends o n t he

relat ive mo lecu l a r weights o f the a n t i b o d y a n d an t igen

(Will iams and Chase 1971) . When t he m o l e c u l a r we igh t

o f LHP is less t h a n t h a t o f t he a n t i b o d y , the i m m u n o -

d i f fus ion l ine curves a b o u t the a n t i b o d y well. By con-

t ras t , w h e n the molecu la r we igh t o f LHP is m o r e t h a n

t h a t o f t he a n t i b o d y , t he l ine curves a b o u t t he an t igen

well. I t is ev iden t in Fig. 2 t h a t m o n o m e r i c a n d poly-

mer ic LHP p r e p a r a t i o n s (5S and 18S, respec t ive ly ,

Bever ley 1979) yie ld i m m u n o p r e c i p i t a t i o n l ines o f the

e x p e c t e d shape w i t h the LHP-specif ic a n t i b o d y (7S).

Complement Fixation at pH 8.15. Since the rela-

t ion b e t w e e n i m m u n o l o g i c a l d i s t a n c e and p e r c e n t

a m i n o acid sequence d i f ference b e t w e e n p ro t e in s has

on ly b e e n es tab l i shed for m o n o m e r i c p ro t e in s (Cham-

p ion e t al. 1975 ; Wilson e t al. 1977 ; see d iscuss ion) , all

254

E x p e c t e d

o)o M o n o m e r i c

L H P

O b s e r v e d

// 0

P o l y m e r i c

L H P

77 ?Li!!ii!!ii!i I ~( ,!i[~iiii!,~ii~i ii i ~¢i ill ~!(~I[:!~ ~!!~!i~([!ii!il !~!

!

Fig. 2. Dependence of the shape of the immunoprecipitin line on the state of aggregation of LHP. In both the expected and observed cases, polymeric LHP gives a precipitin line that curves about the LHP well, whereas monomeric LHP gives a precipitin line that curves about the antiserum well. In all pictures, the antiserum well is on the left of the precipitin line and the LHP well is on the right. The expected patterns are those appropriate for hexameric LHP (MW 480,000) and a monomeric LHP (MW 80,000) in immunodiffusion tests with antibodies of molecular weight 150,000 (Williams & Chase 1977). The observed patterns are those found with the LHP from D. melanogaster and the antiserum to this protein described in Table 1. The monomeric LHP was prepared as described in the methods. The polymeric LHP was prepared by dialyzing the monomeric LHP against 0.1 M KPO 4 buffer, pH 7.0 (Beverley 1979). The immunodiffusion tests were performed in 1% agarose, 0.1 M Tris, pH 8.4 (monomeric LHP) or 0.1 M KPO4, pH 7 (polymeric LHP) at room temperature (overnight incubation)

complement fixation tests were performed on monomeric LHP preparations. To ensure that LHP was monomeric it was necessary to conduct the complement fixation tests at pH 8.15 (4°C). The pH of the Isotris buffer used in conventional complement fixation tests, however, was measured as 7.6 (4°C). Thus, its was necessary to examine whether this change in pH affected the elaborate series of reactions involved in complement activation and fixation. The reactions of human serum albumin (which is roughly similar in size and charge to LHP) and chicken lysozyme c were examined at both pH 7.6 and 8.15. The shift in pH had no effect on the following parameters (Beverley 1979): a) the typical bell shape o f the complement fixation

c u r v e , b) the maximal percentage of complement fixed, c) the amount of antigen required to obtain maximal

fixation, d) the slope of the line relating maximal fixation to anti-

serum concentration, and e) the immunological distances between human albumin

and the albumins of other primate species. We conclude that micro-complement fixation may be used successfully at pH 8.15 in Isovotris buffer.

iZ

g =E

o~

I 00

50

i i i Q

I0 I 00 1000

ng Protein

8 o~ E

.E_ >c o

1/10,000 I / 8 , 0 0 0 I/6,

Ant iserum Concentrot ion

Fig. 3. Example of the titration of an antiserum with larval hemolymph protein. The antiserum used was anti-D, melanogaster LHP (pool A-2), and the antigen was purified LHP from this species. A Complement fixation curves at differing antiserum concentrations. The concentrations were 1/6200 (o), 1/7200 (o), 1/8300 (A), and 1/9600 (z~). B Relationship of the percent complement fLxed at the reaction peak and antiserum concentration. The maximum percent complement fixed (Y) is plot ted against the logarithm of the antiserum concentration (X), in accord with equation 1. The data shown in part A were used, as well as data from other reaction curves using antiserum dilutions which were not shown in part A: 1/6700, 1/7700, 1/8900, and 1/10,000 ( . ) . The line was drawn by linear regression, and has the form Y = 347 log X + 1408; the correlation coefficient o f this relationship was 0.996. The titer o f this antiserum may be calculated by setting Y = 75, and is 6942

Complement Fixation with LHPo The results of a complement fixation experiment with LHP are shown in Fig. 3. A typical series of bell-shaped curves is obtained (Fig. 3a) and the heights of these curves are proportional to antiserum concentration (Fig. 3b). The straight line conforms to the equation

Y = m l o g X + b (1)

where Y is maximal percentage of complement fixed, X is antiserum concentration and m is the slope. The values of m and the antiserum titer for each antiserum pool are shown in Table 1. Also included in the table are the values of m found when each antiserum is tested with heterologous antigens. The values of m obtained with heterologous and homologous antigens are virtually the same for a given antiserum. For most antisera, the values o f m are also similar, but the antiserum to Dacus LHP has a lower slope. Such slope variability has been observed among antisera to serum albumin from various species of vertebrates (Champion et al. 1974).

Factors Affecting the Estimation of Immunological Distance. The length o f the immunization period and variability among rabbits affect the immunological distance among LHPs. Fig. 4 shows the immunological distances obtained with individual and pooled antisera that were elicited against LHP from D. crucigera and tested with LHP from D. mulleri. Considerable variation is evident among individual rabbits and over time. Interestingly, one of the antisera (2082) shows an in- crease in immunological distance with increasing length

Table 2. Immunological distances among purified larval hemolymph proteins a

255

Antiserum

Source of antigen mel cruc mull Sc. mim Dac SWF

Drosophila melanogaster (mel) Drosophila crucigera (cruc) Drosophila rnulleri (mull) Scaptomyza adusta (Sc.) Drosophila mimica (mira) Dacus cucurbitae (Dac) Cochliomyia hominivorax (SWF)

0 76 b 74 b 80 b 90 b 185 139 84 0 58 53 32 139 170 78 50 0 56 b 59 195 157 70 42 57 0 57 175 144 79 17 b 52 54 0 180 138

170 135 119 129 133 0 139 95 97 85 116 98 153 0

aThe degree of antigenic difference is expressed in terms of immunological distance units (Champion et al. 1974). The abbreviations utilized for each antiserum are listed in the first column

bThe values listed are the average of 2 or 3 determinations

I

c 60 o

E E

20

80

/

I , I

20 50

Length of Immunization Period

(weeks)

Fig. 4. Dependence of immunological distance on length of immunization. The immunological distance between two purified LHPs (from D. crucigera and D. mulleri) was measured with antisera to one of these LHPs. The antisera were from 4 rabbits immunized for various periods with LHP from D. crucigera. Both individual and pooled antisera were employed. The resultant immunological distances were then graphed against the length of the immunization period involved in the production of each antiserum. The individual antisera utilized were those of rabbit numbers 2048 (o), 2082 (A), 2083 (o) and 2096 (z~); the antiserum pools were B-l, BB-1, and BC-1 (V). See Beverley (1979) for further details

of immunization, in contrast to the other 3 rabbits which exhibit the more common pattern of decreasing immunological distance with increasing length of immunization (Prager and Wilson 1971). Several other rabbits also exhibited this profile. By pooling antisera, the variation in immunological distance is minimized as shown in Fig. 4 (heavy line).

In tests with antisera to other species' LHP we find that at least 30 weeks are required for the antigenic specificity of the pooled antisera to stabilize. When estimating immunological distances in this work we have therefore used only pooled antisera from immunization periods of longer than 30 weeks.

The effect of LHP polymorphism was also examined. The mutant gl 3 of Drosophila melanogaster contains an allele of LHP which differs electrophoretically from the "wild type" allele found in the Oregon-R strain (Hubby 1963). In complement fixation tests the gl 3 allele was indistinguishable from the Oregon-R allele. This observation may be compared to the expected immunological distance for LHPs differing by a single amino acid substitution. LHP contains nearly 700 amino acid sites (Bev- erley 1979; Akam et al. 1978); one substitution would constitute 0.14% sequence difference between LHPs, or 0.7 immunological distance units (Ibrahimi et al. 1979; Champion et al. 1974; Discussion). This value is less than the experimental error (+2.6 units; Methods). Thus, LHP polymorphism may have little effect upon the interspecific immunological distances.

Immunological Distances Among LHPs. We consider first the immunological distances among the 7 LHPs used as reference antigens. These LHPs were from 5 drosophilid and two nondrosophilid species. To obtain the immunological distances shown in Table 2, antiserum to each LHP was tested against every other LHP. The immunological distances range from 17 to 195. The smallest distances were between the LHPs ofD. crucigera and D. mimica, which belong to closely related species groups. Intermediate distances were found among other pairs of drosophilid LHPs and large distances were found between drosophilid and nondrosophilid LHPs.

The 7 x 7 matrix of immunological distances (Table 2) allows a test to be made of their reliability as estima- tors of the degree of sequence difference between LHPs. Consider any two types of LHP, designated A and B. If immunological distance were a perfect estimator, antiserum to A when tested with B should give the same immunological distance as antiserum to B when tested with A. For the data in Table 2, the deviation from perfect reciprocity, as defined by Maxson and Wilson (1975), is 14.2 percent. The corresponding values for 10 other immune systems range from 13.8 to 26 percent (Beverley 1979). Thus, LHP shows satisfactory reciprocity.

256

Table 3. Immunological distances to the larval hemolymph proteins of twenty-four drosophilid species from five reference species

Source of LHP Immunological distance b

Taxonomic group a Species mel cruc mull Sca mim

Genus Drosophila

Subgenus Sophophora

Melanogaster group

Other groups

Subgenus Drosophila

Subgenus Hirtodrosophila

Subgenus Scaptodrosophila

Subgenus Dorsilopha

Genus Chymomyza

Genus Scaptomyza

D. melanogaster 0 76 74 80 90 D. simulans 0 D. takahashi 19 D. yaku ba 21 D. mimetica 29 D. parabipectinata 32 D. pseudoobscura 57 57 109 82 59 D. prosaltans 58 64 89 84 D. willistoni 74 67 83 78 84

D. cardini 74 D. crucigera 84 0 58 53 32 D. colorata 71 51 44 D. flavopinicola 69 60 D. funebris 69 D. immigrans 88 36 92 68 D. melanica 40 39 D. mimica 79 17 52 54 0 D. mulleri 78 50 0 56 59 D. pachae 76 64 D. pinicola 69 31 41 48 D. robusta 44 D. sordidula 67 40 38 60 63 D. virilis 66 40 53 66 59

D. duncani 68 51 68 58 67

D. dimorpha 62 D. lebanonensis 86 60 81 50 63

D. busc kii 67

C. procnemis 105 102 86 112

S. adusta 70 42 57 0 57

aThe classification of the species into species groups, subgenera and genera is based on morphological traits (Throckmorton 1975) blf no value is given the test was not done. The abbreviations stand for the following reference species: mel = Drosophila melanogaster; cru = D. crucigera; mul = D. mulleri; Sca = Scaptomyza adusta; mim = D. mimica

Next, we consider the immunological distances obtained with crude preparations of LHP from 24 additional species of drosophilids. The results with the antisera to 5 drosophilid LHPs appear in Table 3 and the 24 species are listed in accordance with the traditional taxonomy. The antiserum to D. melanogaster LHP reacted best with LHP from the sibling species D. simu-

lans, next best with LHP from other species within the melanogaster species group, less well with LHP from other species groups and subgenera of Drosophila, and least well with LHP from Chymomyza. The remaining 4 antisera reacted moderately well with LHP from various subgenera of Drosophila and less well with LHP from Chymomyza.

Molecular Tree Construction. The relationships among LHPs inherent in the data of Table 2 may be summarised and visualized by the construction of a

molecular tree. As shown by Prager and Wilson (1978) the method of Fitch and Margoliash (1967) is suitable for immunological data. To apply this method t o the data of Table 2, i t was necessary, first, to average the immunological distances obtained in reciprocal tests. The average distance yields the best estimate of the degree of sequence difference among proteins. These averaged values appear in the upper half of Table 4.

Three trees were found by this technique to have nearly identical "goodness of f i t" values. Tree I is shown in Fig. 5A. Tree I has an F value (as defined by Prager and Wilson 1978) of 5.6 percent. Tree II differs by reversing the relative positions of the D. mulleri and Scaptomyza lineages, while Tree III unites the lineages leading to these two species prior to attaching them to the tree. The goodness of fit values for these trees are F - 5.3 and F = 5.1, respectively. However, both of these

Table 4. Average of immunological distances obtained in reciprocal tests of larval hemolymph proteins a

257

Species compared mel cruc mull Sc mim Dac SWF

Drosophila melanogaster (reel) Drosophila crucigera (cruc) 76 Drosophila mulleri (mull) 78 Scaptomyza adusta (Sc) 79 Drosophila mimica (mim) 80 Dacus cucurbitae (Dac) 163 Cochliomyia hominivorax (SWF) 131

80 76 75 84 178 117 54 48 24 137 133

52 - - 56 56 157 121 49 55 56 152 130 24 56 53 - - 156 118

151 153 154 155 146 119 121 122 123 146 - -

aThe averages of reciprocal measurements listed in Table 2 are given in the upper right-hand section of the Table; the lower left-hand section contains the reconstructed immunological distances from the phylogenetic tree shown in Figure 5B. The symbols at the column heads correspond to the species indicated in the first column. The standard deviation of the input reciprocal data from perfect reciprocity is 14.2%

.~A (15) }a

13 F I ~ D. crucigera

E 14 D. mirnica 7 D 28 l Scaptomyza

adusta 28 ~ Dmul/eri

- D melanogaster

51 Cochliomyia

(80) Dacus

O. cruc~gero I°

z ~4 D mimica

~ odusto 30 Z7 - - D mulleri

I (iz) I D melanogaster I S7 Cochhomyia I

(77) Oacus

Fig. 5. Evolutionary trees based on LHP immunological distances. A This tree was constructed by the method of Fitch and Margoliash (1967) from the averages of the immunological distances observed in reciprocal tests (see Table 4). The numbers above each branch represent the inferred length in immunological distance units, and each branch was drawn proportional to this length. The position of the ancestral node A, leading to Dacus On the one hand and the remaining species on the other, was estimated by assuming the distance from the node to Dacus was equal to the average distance from the node to the remaining six species. B This tree has the same topology as the tree shown in A; however, the branch lengths were calculated by a modification of the method of Chakraborty (1977). This modification involves considering the lineage B-A-Dacus as a single unit, rather than separately as two lineages B-A and A-Dacus. The position of node A was then calculated as described above

trees require negative branch lengths o f -1 a n d - 2 unit, respectively, and for this reason, were discarded in favor of Tree I.

As noted by many workers, the Fitch-Margoliash

method introduces a systematic bias into the tree: distances involving proteins added late in the procedure

are weighted more heaviliy than those added earlier. Chakraborty (1977) presented a matrix least-squares method for calculating relative divergence times. This method can be modified to apportion distance data on trees of known topology (S.M. Beverley and L.M. Cherry, unpublished). Application of this approach to Trees I, II and III yields lower F values than those obtained with the Fitch-Margoliash method (4.6, 4.7 and 4.5, compared to 5.6, 5.3 and 5.1). Trees II and III still require negative branch lengths, as before. Tree I is shown in Fig. 5B.

Thus, Tree I is considered to be the best tree for describing the evolutionary relationships of LHPs. More- over, the LHP relationships shown among the 5 species of Drosophilidae agree exactly with the branching order obtained by quantitative phylogenetic analysis of 60 morphological traits (Throckmorton 1968, 1975). Be- sides associating the D. crucigera and D. mimica lineages closely, the tree associates these two lineages with D. mulleri, which is classified in the same subgenus (Droso- phila). It is notable that Scaptomyza LHP lineage stems

from within this subgenus. Although Scaptomyza is a distinct genus morphologically, its LHP is more closely related to those of the subgenus Drosophila than is the LHP of D. melanogaster, which is classified in the subgenus Sophophora of the genus Drosophila. The tree shows further that the 5 aforementioned lineages of drosophilid LHPs are related more closely to each other than any of them is to the LHPs of the two nondroso- philids (Cochliomyia and Dacus).

Branching information may also be extracted from the "unidirectional" data of Table 3. For those LHPs which have been tested with several antisera, one may attempt to attach the protein to any branch of the molecular tree; a system of equations is then developed which allows the appropriate branch lengths to be calculated for this attachment site. Several sites are evaluated in this manner, and the "goodness of fit" may be calculated for each using the F parameter. One then chooses among these on the basis of the F parameter. A formal

description of this procedure is given in the appendix and also by Beverley (1979).

258

I

5 / I0

O. crucigero

O. re~mica

26 SCAPTOMYZA

O. plnicolo 30

Hir todrosophl la

D. Immlgrons

24 D. v i r i l l s 21

B ~ D, mul le r i

17 D, sordldula

27 D. me/onogoster

O. pseudoobscura

- D. wlll istoni

O. pro$ol/ans

~ p t o d r o s o p h l l a

58 CHYMOMYZA

Fig. 6. An expanded tree for larval hemolymph proteins of the Drosophilidae. This tree was obtained by adding 10 LHP species (thin branches) to the tree in Figure 5a (thick branches) by the unidirectional attachment method described in the appendix. The lengths of each branch are drawn proportional to the LHP immunological distance, listed above each lineages. The subgenera Scaptodrosophila and Hirtodrosophila refer to the species Do lebanonensis and D. duncani, respectively. "Unidirectional" LHPs descending from the same nodes may share more common ancestry than the diagram indicates; this uncertainty is inherent in the unidirectional attachment method

The results of applying this tree-building procedure to 10 of the LHPs listed in Table 3 appear in Fig. 6. For 7 of these the cladistic position shown is that of lowest F (Chymomyza, D. duncani, D. pinicola, D. prosaltans, D. pseudoobscura, D. sordidula, D. willistoni). For the remaining three species' LHPs (D. immigrans, D. lebanonensis, D. virilis) the tree of lowest F differs by an average of 6 percent (in F value; 3.2-9.5%) from the tree anticipated from Throckmorton's (1975) organismal phylogeny. As this difference is small relative to the uncertainty introduced by the imperfect reciprocity of immunological distances (see above), we decided to put these 3 species on the tree at the positions recommended by Throckmorton (1962, 1968, 1975).

The tree associates three of the 10 lineages with the melanogaster lineage; these three (pseudoobscura, willistoni and prosaltans) are classfied with the melanogaster group on organismal grounds in the subgenus Sophopho- ra. Five of the 10 lineages associate with lineages stemm- ing from the subgenus Drosophila; the latter were referred to when presenting Fig. 6. The ninth lineage, that of D. lebanonensis, which belongs to the subgenus Scaptodrosophila, is as distantly related to the subgenus Drosophila as to the subgenus Sophophora. Thus, we have identified three major lineages within the genus Drosophila, namely the Sophophora, Drosophila and

Scaptodrosophila lineages. The tenth lineage, that of Chymomyza, as expected from the organismal evidence, lies outside the three major lineages leading to species of Drosophila and Scaptomyza.

Discussion

Suitability of LHP for Micro-Complement Fixation

The micro-complement fixation test has been rigorously applied to the study of protein evolution in a wide variety of vertebrates (Wilson et al. 1977), bacteria (Champion et al. 1980) and plants (Wallace and Boulter 1976). The primary advantage of this approach is that one may rapidly obtain estimates of the extent of amino acid sequence difference among proteins without direct knowledge of the individual sequences. When dealing with a new protein antigen, however, one must take careful steps to test whether immunological distance is a reliable estimator of sequence difference. We briefly review these steps in this section and their application in the LHP antigen system.

Desirability of Monomers. The correlation between immunological distance and the extent of sequence difference has been established for a variety of monomeric proteins, including lysozyme, ribonuclease, azu- rin, cytochrome c and plant plastocyanins (White et al. 1978). A similar empirical correlation has not been demonstrated for polymeric protein antigens. Indeed, there are several observations which suggest that polymeric protein antigens may be less satisfactory than monomers in this regard.

Firstly, several studies have shown that polymeric antigens fix complement more efficiently than the constituent monomer, often as much as five-fold higher (Reichlin et al. 1970; Nakanishi 1971; Beverley 1979). Secondly, polymeric proteins often dissociate into smaller oligomers or monomers at concentrations similar to those used in complement fixation tests (approximately 20 nanograms/ml at the peak of reaction); thus one may be dealing with a complex mixture rather than a defined molecular state. Thirdly, antigenic determinants are repeated on the surface of polymeric antigens; therefore, the presence of a single reactive antigenic site may suffice to allow such a molecule to fix complement. In contrast, monomeric antigens require the reactivity of several different antigenic determinants for complement fixation (Rapp and Borsos 1970). For this reason, a larger proportion of the surface of the monomeric antigen is probed. This is of particular importance when one recalls that all portions of the surface of proteins do not evolve at the same rate, and some regions of proteins may evolve slowly (Dickerson 1971). Thus, estimates concerning the reactivity of polymers may be more susceptible to variation from this source.

A study of acid phosphatase evolution in Drosophila deserves mention in this connection (Macintyre et al.

259

1978). The immunological method they used detects univalent interactions between antigen and antibody and for reasons analogous to those stated above is unlikely to be as reliable as micro-complement fixation for estimating extent of sequence homology in the protein.

In summary, we do not assert that immunological reactions of polymeric proteins are unreliable for evolutionary studies. Rather, we point out that at present the assumption of a reliable correlation between the extent of sequence difference and immunological distance for such proteins is premature. For this reason, we have studied the complement fixation reactions of LHP exclusively in the monomeric state.

Desirability of Minimal Post-Translational Modifica- tion. As carbohydrate and lipids bound to proteins may elicit an immune response and react in complement fixation tests (Mayer 1961), the protein should prefer- ably contain little non-protein material. LHP contains from 0.8% (D. melanogaster) to 5.7% (Scaptomyza adusta) carbohydrate by weight, and no lipid (Beverley 1979). The presence of such low levels of carbohydrate on protein antigens has been shown not to have a significant effect on the micro-complement fixation reactions of RNAse (Prager et al. 1978), transferrin (Putnam 1975; Prager et al. 1974) or ovalbumin (Prager and Wilson 1976).

lmmunochemical Purity. Once the target protein is selected, one must establish the immunochemical purity of the antisera. The anti-LHP antisera utilized have been shown to be satisfactory in this regard, as described in the Methods section.

Factors Affecting Antiserum Variability. The specificity of antisera is known to change during the immune response, eventually stabilizing at a "plateau" level after lengthy immunization (Prager and Wilson 1971; Cham- pion et al. 1974). Similarly, variability in specificity exists among individual rabbits; this can be minimized by pooling antisera. We find that a 30-week immunization period is sufficient to allow stabilization of anti- LHP antisera (Fig. 4), and only antiserum pools have been utilized in obtaining immunological distances.

We note that in a previous micro-complement fixation study of a-glycerophosphate dehydrogenase evolution in Drosophila, unusually early bleedings were used (Collier and Macintyre 1977).

Amino Acid Compositional Data. Comparison of indices of amino acid compositional divergence can give a rough idea of the extent o f sequence difference between proteins (Metzger et al. 1968; Marchalonis and Weltman 1971; Davidson and Flynn 1980). Similarly, comparison of compositional indices with immunological distances can provide an indication of the likely correlation between immunological distance and sequence difference (Wallace and Wilson 1972; Prager and Wilson 1971a). Analysis of the amino acid compositions of four LHPs (D. melanogaster, D. mullerL D. mimica

I M M U N O L O G I C A L DISTANCE

5 0 IO0 150 2 0 0

15 ̧

w

I0

51

©

Immn~

• ON~ i ~

/ e

• e / • *

. l / • I l l • • •

/ .e u /

• u n n n • • • ~

ib 26 3b 40 % SEQUENCE DIFFERENCE

Fig. 7. Amino acid compositional difference as a function of percent of difference in amino acid sequence. The extent of amino acid compositional relatedness was calculated using the difference index of Metzger et al. (1968) for 50 pairs of random- ly selected cytochrome c (e), lysozyme (a), and alpha hemoglobin chains (m); Dayhoff (1972) and Prager and Wilson (1971a) were the sources of these data. The difference indices were then plotted against the observed percent amino acid sequence difference for each pair. The dashed line suggests the general trend of the relationship. The difference index was also calculated for all six possible pairs of LHPs whose amino acid compositions are known (Beverly 1979); for these comparisons the percent sequence differences was estimated by assuming that the immunological distance (average of reciprocal comparisons, Table 4) is equal to 5 times the true percent sequence difference. These values were then plotted according- ly (o). It is evident that the LHP points fall in a range similar to that of the proteins of known sequence

and D. crucigera; Beverley 1979) indicate that the immunological distance between LHPs is related to the extent of sequence difference (Fig. 7). This relationship may be similar to that observed for other monomeric proteins, for which

y = 5 x (2)

where y = the immunological distance and x = the percent amino acid sequence difference (Champion et al. 1975; Ibrahimi et al. 1979).

In summary, the criteria applied above indicate that the immunological distances among LHPs are likely to yield satisfactory estimates of the actual degree of amino acid sequence difference. Thus, we find the LHP system suitable for studies of molecular evolution.

Agreement of Molecular and Organismal Trees

Our results demonstrate substantial agreement in branching order between the LHP and organismal trees for the Drosophilidae. This confirms for insects the

260

value of comparative studies on a single protein I . Comparable results have been obtained for vertebrates with serum albumin, cytochrome c, hemoglobin and many other proteins (Wilson et al. 1977 and reference cited therein).

The finding that organismal and LHP trees are nearly congruent allows an important inference to be made concerning the amount of phylogenetic information contained in proteins. Throckmorton's (1962, 1966, 1968, 1975) organismal tree of the Drosophilidae is the result o f a thorough quantitative study of 60 anatomical traits. Hence, a protein such as LHP may contain about as much cladistic information as do 60 anatomical traits. This empirical observation is inconsistent with the expectations of those who have raised theoretical objec- tion to the use of proteins as phylogenetic tools (Throckmorton 1978; see Farris (1979) for additional discussion of the theoretical basis for tree construction).

The agreement between the LHP and organismal trees is not exact. For three of the 15 drosophilid LHPs considered, the branching order differs from that in the organismal tree. The reasons for such discrepancies are discussed by Wilson et al. (1977). Briefly, either the molecular or organismal trees may be in error; alterna- tively, the data sets may not have sufficient resolving power. For the 3 LHPs mentioned above, the discrepancies are smaller than the resolving power of the immunological method.

Future protein work should enable the correct branching order to be ascertained for these three species. Such work should be done with antisera to the LHPs of these species. In addition, a molecular tree could be constructed for another protein (cf. Prager and Wilson 1976). If the latter tree were congruent in branching order with the LHP tree of best fit, one would be justi- fied in preferring it to the organismal tree. It is particu- larly important to conduct such studies with Scapto- drosophila because, if Throckmorton (1968) is correct about its phylogenetic position, this subgenus presents an amazing contrast with the subgenus Drosophila in tempo and mode of evolution at the supramolecular level.

Independence of Organismal and Molecular Change

Although the organismal and LHP trees agree approximately in branching order, they differ spectacularly in branch lengths, as illustrated in Fig. 8. This figure contains a simplified tree, showing the branches leading to

1 Earlier indications that immunological comparisons of insect proteins would prove to be phylogenetically useful have been presented by several authors (Duke and Glassman 1968; Fink et al. 1970; Kunkel and Lawler 1974; Collier and Maclntyre 1977; Maclntyre et al. 1978).

4

subgenus

Drosophilo

Sophophora

Scaptodrosophila

Fig. 8. The relationship between morphological and LHP evolution in the genus Drosophila. The figure presents a simplified phylogeny for the subgenera Scaptodrosophila, Sophophora and Drosophila, in accord with organismal and molecular relationships. The numbers along each lineage (M/P) refer to the magnitude of the inferred morphological change (M) and LHP evolution (P). M is estimated by the number of derived char- acter states assigned to each lineage by Throckmorton (1968, Table 15; the values for the two major subqineages within the subgenus Drosophila have been averaged). P is estimated by the immunological distance associated with each lineage in Fig. 6; the values for the Sophophora and Drosophila are averages (4 and 7 species, respectively)

the three major subgenera, viz Drosophila, Sophopho- ra and Scaptodrosophila. On each branch there are two numbers: the first is an estimate of the number of "derived" anatomical changes (Throckmorton 1968), and the second is the amount of LHP change in immunological distance units (from Fig. 6).

Fig. 8 implies that there is enormous variation in the ratio of anatomical change to LHP change. While the branch leading to Scaptodrosophila underwent no derived changes in anatomy, 37 units of LHP change accumulated. In contrast, during the same time interval, the branch leading to the subgenus Drosophila experienced 64 derived anatomical changes and 32 units of change in LHP.

The contrast between these two branches of the fruit fly tree appears to be even more striking than that between frogs and mammals. The latter two groups have experienced about the same rate of protein evolution but frogs have undergone much slower anatomical change than have mammals (Wilson et al. 1977). Plants (Prager et al. 1976) and bacteria (Champion et al. 1980) provide similar examples of phenotypically conservative lineages that have experienced as much protein evolution as lineages that have undergone rapid phenotypic evolution. Evidently, in insects as well as other creatures, the quantitative processes of organismal and protein change are uncoupled.

It will be of great interest to conduct further studies of evolution in the Scaptodrosophila and Drosophila subgenera. Such studies may give biologists insight into the basis for the large difference in rates of organismal evolution in these two branches of the fruit-fly tree.

Future Studies o f LHP Evolution

LHP evolution can probably be studied in many additional families, of the order Diptera. Every family tested in the dipteran section Schizophora contains an LHP that reacts with antisera to drosophilid LHP; these families include the Agromyzidae, Calliphoridae, Gaste- rophilidae, Muscidae, Ottitidae, and Tephritidae (S.M. Beverley, present and unpublished work). No cross- reaction has been observed with representatives of other sections or suborders in the order Diptera (e.g. Megaselia scalaris, section Aschiza; Chrysops hirsuticallus, suborder Brachycera; mosquitos, suborder Nematocera). This suggests that LHP evolution may be examined through-

out the section Schizophora, which includes over 10,000 species.

The potential of LHP as a phytogenetic probe for the higher diptera (section Schizophora) is illustrated by considering the tree in Fig. 5. The relationships of the LHPs of the Drosophilidae, Calliphoridae (Cochliomyia, the screw-worm fly) and Tephritidae (Dacus) shown in the figure are not in agreement with that anticipated from the usual textbook classification of these families (see for example Borror et al. 1976), which associates the Tephritidae and Drosophilidae together. However, studies of a-glycerophosphate dehydrogenase (Collier and Maclntyre 1977) and several other proteins (W.S. Davidson and S.M Beverley, unpublished) yield results

which agree with the LHP tree. In contrast to the agreement of the protein trees, there is considerable disagree- ment among entomologists concerning the relationships of these three families (Oldroyd 1964; Griffiths 1972; Hennig 1973; Rohdendorf 1974; Steyskal 1974) 1. Therefore, serious consideration should be given to the possibility that the tree shown in Fig. 5 represents the correct phylogeny of these families.

It is notable also that our preliminary studies of relationships among LHPs within the Tephritidae gives results in accord with the organismal classification for this group (Beverley 1979). Thus, it is anticipated that LHP will prove to be as valuable for elucidating phylogenetic relationships within and among families of higher diptera as serum albumin has been in the study

of vertebrate evolution (Wilson et al. 1977) 2.

Acknowledgments. We thank G.L. Bush, S.S. Carlson, G. Cassa- vetes, W.S. Davidson, D.E. Dobson, O. Eugene, G.B. Kitto, J.G. Kunkel, E.M. Prager, M. Revesley, R. Richardson, V.M. Sarich, D. Schultz, T. Shermoen, A.R. Taylor, M.R. Wheeler and H. White for discussions, advice and assistance. We especially

1 The tree proposed by Hennig (1973) is most consistent with the LHP data.

2 Finally, we point out that LHP could be present throughout the class Insecta. This is suggested by evidence for the presence of hemolymph proteins resembling LHP in amino acid compo- sition, size and charge in diverse orders (Kunkel and Lawler 1974; Wyatt and Pan 1978; Beverley 1979).

261

thank the National Drosophila Species Resource Center for supplying Drosophila species, R. Richardson for providing facilities and preparative quantities of several drosophilid species, E.J. Harris of the USDA for providing Dacus, and the screw- worm eradication program of the USDA for providing Cochlio- myia. NIH provided funds for this research through grant GM21509 and training grant 5T01GM31-18.

Appendix: The Method of Unidirectional Attachment

As the average immunological distance obtained in reciprocal tests provides the best estimate of amino acid sequence difference (Prager and Wilson 1971; Champion et al. 1974), it is preferable to construct molecular trees from such data. This requires that antisera be available to every protein on the tree. In practice, however, one usually has antisera to only a few proteins, termed reference proteins. With each antiserum one can measure immunological distances from the reference protein to many other proteins, for which there are no antisera. These immunological distances are termed "unidirectional", We present a five step method of attaching such proteins to a molecular tree. The method takes molecular trees constructed for reference proteins as a fixed framework, and thus minimizes the inaccura- cies inherent in the use of unidirectional distances. The calcula- tions utilized are similar to those of other tree-building methods, such as that of Fitch and Margoliash (1967).

1. Select the starting "framework" tree, which has been constructed from bidirectional distances (i.e., obtained in reciprocal tests); for example, the molecular trees shown in Fig. 5 are appropriate.

2. Correct the unidirectional immunological distances to unattached LHPs. The need for correction is evident from Table 2, where the antiserum to LHP from 1). crucigera always yields immunological distances which are less than the distance observed in the reciprocal direction. If unidirectional distances obtained with this antiserum were compared directly to bidirectional distances they would probably appear to be less. To compensate for this, unidirectional immunological distances are multiplied by the correction factor Rp. For antiserum to protein P, this factor is calculated as

Rp= 1 ~ 2Pi/(p i+ Qi) (1) n i=l

4 9

I0 ~ O, crucigera

D. mimica

| 24 S. adusta

D. mulleri 27-b " ~ b

~ O. sordidula

D. rnelanogaster

Fig. AI. Application of the method of unidirectional attachment to the LHP from Drosophila sordidula. The framework tree (heavy lines) was constructed from reciprocal data involving the five drosophilid antisera, using the Fitch-Margoliash method; the topology is identical to that shown in Figure 5, and the branch lengths are very similar. The LHP of Drosophila sordidula is shown attached (broken line) to the branch leading from node D to D. mulleri

262

Table AI , Values needed for unidirectional attachment of Dro- sophila sordidula LHP to an evolutionary tree

Reference Unidirectional distance Patristic LHP to D. sordidula LHP distance*

observed corrected

D. mulleri 38 38 a + b D. melanogaster 67 67 76 + a - b D. crucigera 40 47 55 + a - b Scaptomyza 60 60 54 + a - b D. mimica 63 56 60 + a - b

*From Figure A1

where Pi represents the immunological distance obtained using antiserum to protein P tested with protein Q and Qi the immunological distance obtained using antiserum to protein Q tested with protein P, the summation is over all (n) reciprocal comparisons involving antisera which were used in construction of the framework tree. If Rp is not significantly different from 1 it is set equal to 1. A similar correction has been proposed by Sarich and Cronin (1976). As an example, table A1 shows the corrected distances from LHPs on the framework tree to the LHP of D. sordidula.

3. Attach each LHP to the framework tree at any given node or branch as illustrated in Fig. A1, where the LHP ofD. sordidula is attached to the D. mulleri branch. To calculate the branch lengths a and b, one considers both the corrected unidirectional distances to D. sordidula LHP and the patristic distances (taken

Table A2. Evaluation of alternative attachment sites for larval hemolymph proteins

Number of Species comparisons Site a

Calculated limb Goodness of lengths b fit

a b r F c

Drosophila duncani 5

Drosophila immigrans 4

Drosophila lebanonensis 5

Drosophila pinicola 4

Drosophila prosaltans 4

Drosophila pseudoobscura 5

Drosophila sordidula 5

Drosophila virilis 5

Drosophila willistoni 5

Chymomyza procnemis 5

D-E 30 3 0 8.6 node E 30 - - 8.6*+ E-F 35 3 12 9.8 node E 41 - 19.3"+ node D 41 - - 19.3 D-mel 40 48 1 18.9 E-Sc 32 18 6 10.5 D-E 37 -5 8 10.8 node F 36 - 13.4 node D 36 - - 14.9 D-mel 36 50 -1 15.2 node C 37 - 16.0"+ E-F 13 14 1 4.2 D-E 13 -1 4 4.2 node E 13 - 4.2"+ D-mull 13 27 0 4.2 D-mel 29 29 20 8.3+ D-mel (a = 33) 33 33 16 10.5+* E-C 40 22 -7 19.7 D-E 40 8 -5 19.7 D-mel 30 27 22 21.5"+ E-C 39 7 8 28 D-mull 17 21 6 7.1 *+ node D 21 - - 10.8 D-mull 25 28 -1 11.3 D-mel 21 45 4 8.8 D-E 24 5 -2 12.7 E-F 24 14 1 11.3 node D 24 - - 12.0"+ F-mim 28 25 -10 19.7 D-mel 37 37 12 4.4+ D-mel 35 33 16 5.4+* (a = 33) B-C 58 10 21 8.9*+ node C 64 - - 11.8

aThe site refers to the branch or node used in the examination of goodness-of-fit; the abbreviations are those found in Table 2 br, a, and b are calculated as described in the text and depicted in Figure A1; r represents the branch corresponding to 27-b in

the figure CF is defined by Prager and Wilson (1978) *Denotes the attachment site selected for Figure 6 +Denotes the attachment site most similar to that proposed in conventional organismal studies (Throckmorton 1962, 1968, 1975)

263

from the framework tree) appropriate for the selected attachment site. For the example in Fig. AI, it is evident that the patristic distance between sordidula and a typical reference LHP (e.g., D. crucigera) is

1 0 + 1 5 + 3 + r + a (2)

where a is the immunological distance along the sordidula branch and r is the immunological distance from node D to the sordidula-mulleri branch. It is evident that r = 27 - b, where b is the immunological distance along the mulleri branch, and therefore the patristic distance from sordidula to crucigera reduces to

55 + a - b (3)

The patristic distances for all five reference LHPs from D. sordidula are shown in Table A1.

By equating the patristic distances to the corrected unidirectional distances a system of equations in two unknowns is obtained: the (a + b) equation

a + b = 38 (4)

and the (a - b) equations

a - b + 55 = 47 (5)

a - b + 76 = 67 (6)

a - b + 54 = 60 (7)

a - b + 60 = 56 (8)

By averaging the (a -b) equations (5 - 8) we obtain equation

a - h = -4 (9)

From equation 4 and 9 we solve for a and b, obtaining

a= 17, b = 2 1 , r = 2 7 - b =6

4. Calculate a goodness-of-fit parameter for the tree with this attachment site; i.e., compare the starting corrected immunological distances with the reconstructed immunological distances calculated from the results of step 3. We recommend the F parameter (prager and Wilson 1978) for this purpose.

5. Steps 3 and 4 are then repeated for the other possible attachment sites one wishes to examine. Suitable equations may be developed for any site, including directly to nodes, or with any of the values a, b, or r specified to be a given value. One then selects the preferred attachment site on the basis of lowest F, or other criteria (if applicable).

The application of this method to the unidirectional immunological distances among LHPs (Table 3) is summarized in Table A2. The attachment sites whose resultant trees were presented in Fig. 6 are indicated. We stress that this method cannot ascertain relationships among proteins attached by the unidirectional method (due to the lack of an immunological distance between the proteins), but can only examine relationships of each such protein to the proteins of the framework tree.

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Received September 17, 1981/Revised December 7, 1981

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