BiochemicalSystematicsand Ecology, Vol. 8, pp. 297 to 304. © PergamonPressLtd. 1980. Printedin England. 0305 1978/80/0901--0297 $02.00/0 Electrophoretic Evidence for Relationships and Differentiation among Members of the Percid Subgenus Microperca DONALD G. BUTH*, BROOKS M. BURR¢§ and JOHN R. SCHENCK:I: *Department of Biology, University of California, Los Angeles, CA 90024, USA t Department of Zoology, Southern Illinois University, Carbondale, IL 62901, USA CEspey, Huston and Associates, 3010 S. Lamar, Austin, TX 78704, USA Key Word Index - Microperca; Etheostoma; Percidae; Perciformes; isozymes; starch gel electrophoresis; phenetics; cladistics; chemosystematics. Abstract - Forty-seven allelic products were electrophoretically resolved at 23 presumptive loci in 10 populations of fishes of the percid subgenus Microperca, genus Etheostoma. Phenetic and cladistic analyses of these genetic data support the recognition of two species-groups within the subgenus: (a) E. fonticola and E. proeliare; (b) E. microperca, confirming previously described morphological interpretations. Additional morphologically based hypotheses receiving genetic support include: (c) recognizing E. proeliare as the most primitive and E. microperca as the most advanced species of the subgenus; and (d) assigning derived status to the differentiation exhibited by the Ozark populations of E. microperca. Etheostoma fonticola is more advanced on the genetic level than had been morphologically ascertained. Introduction Electrophoretic analyses have proved useful in. studies of the relationships and genetic structu re of natural populations of percid fishes especially those of the speciose tribe Etheostomatini [1-7]. Such methods are valuable in providing additional data which may be used to test hypotheses based on morphological criteria. In a recent study of the morphology, relationships, and distribution of the percid subgenus Microperca (genus Etheostoma), Burr [8] concluded that the three species in the subgenus could be placed into two species-groups: (a) Etheostoma fonticola (Jordan and Gilbert) and E. proeliare (Hay); (b) E. microperca Jordan and Gilbert. Burr also discovered a considerable amount of morphological differentiation in the Ozark populations of E. microperca. To test Burr's interpretations regarding the phylogeny of Microperca and intraspecific differentiation in E. microperca, we have conducted an electrophoretic investigation of protein variability within the subgenus. A report of this genetic variation and its phylogenetic significance forms the basis of this study. §Please address reprint requests to B. M. Burr. Results and discussion A total of 47 allelic products were electrophoretically resolved at 23 presumptive loci in the 10 populations examined (Tables 1 and 2). Low levels of intraspecific variability were observed in the subgenus M/croperca with heterozygosity levels approximately equivalent among the three species (Table 3). Populations of E. proeliarewere found to be essentially genetically identical despite sampling geographically distant localities throughout its range. Thus, for future work, a relatively small sample of E. prod~are from a single locality would suffice to genetically characterize this species [9, 10]. A comparable geographic sampling was not achieved in E. microperca, although the genetically similar Ozark populations were separable from the sample from northern Illinois. The random association of subunits yielding an expected number of isozyme electromorphs in heterozygous and/or multilocus multimeric situations was observed in all cases except for LDH. Instead of the expected five-electromorph pattern in the tetrameric multilocus LDH system, threeelectromorph patterns were observed in E. fonticola and E. proeliare while E. microperca exhibited only a two-electromorph pattern (Fig. 1). The directional reduction of LDH isozymes in teleosts yields derived states in which not all (Received 14 January 1980) 297 DONALD G. BUTH, BROOKS M. BURR AND JOHN R. SCHENCK 298 TABLE 1. ENZYME-PROTEIN SYSTEMS ELECTROPHORETICALLY EXAMINED Enzyme protein EC No. Acid phosphatase Adenosine deaminase Alcohol dehydrogenase Aspartate aminotransferase Aspartate aminotransferase Calcium-binding protein Calcium-binding protein Creatine kinase Fumarate hydratase Glucosephosphate isomerase Glucosephosphate isomerase Glycerol-3-phosphatedehydrogenase Isocitrate dehyd rogenase Lactate dehydrogenase Lactate dehydrogenase Malate dehydrogenase Malate dehydrogeease Malate dehydrogenase Man nosephosphate isomerase Phosphogrucomutase Sorbitol dehydrogenase Superoxidedismutase Xanthine dehydrogenase 3.1.3.2 3.5.4.4 1.1.1.1 2.6.1.1 2.6.1. I Locus No. of alleles resolved Acp-A Ada-A Adh-A M-Aat-A S Aat-A Cbp-1 Cbp 2 2.7.3.2 Ck-A 4.2.1.2 FumA 5.3.1.9 Gpi-A 5.3.1.9 Gpi-B 1.1.1.8 G 3-pdhA 1.1.1.42 M-Icdh-A 1.1.1.27 Ldh-A 1.1.1.27 Ldh-B 1.1.1.37 M M d h A 1.1.1.37 S-Mdh-A 1.1.1.37 S Mdh-B 5.3.1.8 Mpi-A 2.7.5.1 Pgm-A 1.1.1.14 Sdh-A 1.15.1.1 Sod-A 1.2.1.37 Xdh-A 3 4 1 1 2 2 1 1 1 1 5 2 1 2 2 1 1 2 1 7 2 2 2 expected interlocus heterotetramers are observed [1 1]. Thus, in Microperca, E. microperca exhibits the derived condition expressing LDH products only in the homotetrameric state. Etheostoma fonticola and £. proe/iare retain the plesiomorphic ability to form the Ldh-A2B2 heterotetramer. All 0.5 I , 0.6 I , 0.7 I , three species have lost the ability to form the asymmetrical heterotetramers, Ldh-A1B3 and LdhA3B1, an apomorphic loss event which apparently predated the radiation of the Etheostomatini [2, 3]. Coefficients of genetic similarity and genetic distance [12] calculated between all pairs of populations are given in Table 4. A phenetic treatment of these data is illustrated in Fig. 2 and yields a cluster of E. fonticola plus the populations of E. proel/are and another cluster comprising the populations of E. microperca. A second, cladistic, method of analysis was also employed. To hypothesize the phylogeny of Microperca, a phylogenetic treatment on a character-bycharacter (loci) basis would be most informative. However, due to tissue, specimen and character state distribution limitations, a cladistic analysis employing data for all loci was not possible. Sufficient information exists for allelic character state relationships to be hypothesized for the following six polymorphic loci: A. Ada-A-The Ada-A 87 allele expressed in E. m/croperca is shared with E. fHo/olepis) graci/e, a member of the most closely related subgenus to Microperca [8], and thus is assigned ancestral status. The Ada-A t°° allele must also have been present in the common ancestor of M/croperca and is presently symplesiomorphically shared by E. microperca and E. proe/iare. Etheostoma font/- 0.8 I 0.9 I , ~ 1.O I E. mlcroperca (Kankakee dr~ IL) E. microperca (Gasconade dr. ~(~1, MO) E. microperca (ONse dr. ~1, MOJ E. microperca ~Ot~tgedr. ~ 2 , MOJ E. micropea~a (Gsseonade dr. :N=2,MOJ £. fort.cola ~Guadalupedr, TX) r E. proellare d (Ohio dr. [L) ]L E. proeliare . | iBi8 Sl=ek dr.,MS) IT E. proeliare I~ (Alabams dr., AL) L E. proeUare ~Pearl dr., LA) I 0.5 ' : 0.6 ' : 0.7 ' : 0.8 ' : 0.9 1.0 Genetic Simfim~ty ( ! ) FIG. 2. PHENOGRAM OF GENETIC RELATIONSHIPS BASED ON NEI'S [12] COEFFICIENT OF GENETIC SIMILARITY (/) CLUSTERED USING THE WEIGHTED PAIR-GROUP METHOD WITH ARITHMETIC MEANS (WPGMA). Calculations are based on the analysis of 47 alleles encoded by 23 loci 299 ® + 65 ~ ~ n ~ ~ ~ 5 Origin~ m m 1 2 E. fonticol " 8 3 E. microperca FIG. 1. LACTATE DEHYDROGENASE PHENOTYPES OF ETHEOSTOMA FONTICOLA (3-ELECTROMORPH PATTERN) AND E. MICROPERCA (2-ELECTROMORPH PATTERN) OBTAINED VIA THE ELECTROPHORESIS OF MUSCLE EXTRACTS. Subunit composition of each electromorph is indicated. The loss of the ability to form an Ldh-A2B2 heterotetramer in E. microperca is the derived character state. GENETIC RELATIONSHIPS IN MICROPERCA 301 TABLE 2. ALLELE FREQUENCIES A T 13 P O L Y M O R P H I C LOCI IN 10 P O P U L A T I O N S ( S U B G E N U S MICROPERCA) (POPULATIONS A R E N U M B E R E D IN THE ORDER IN W H I C H THEY A R E LISTED IN THE E X P E R I M E N T A L SECTION) E. microperca E. proeliare Locus 1 Acp-A 2. A d a A Allele ~ fontlcola 1 13 67 100 1.00 0,20 0.80 52 0.10 70 0.90 87 2 3 1.00 1.00 - 4 - 1.00 1.00 1.00 1.00 75 100 1.00 1.00 1.00 11~ 1.00 100 144 0.95 0.05 1.00 . 1 .(30 79 89 100 110 132 1.00 0.10 0.05 0.90 - 0.90 63 100 1 .(30 0.05 0,95 7. L d h - A 100 165 1.00 1.00 - 8. Ldh-B 75 100 9. S - M d h - B I00 5. Gpi B 6. G - 3 - p d h - A - 1.00 . 2 3 4 5 1.00 1.00 - 1.00 1.00 - - - - - 1.00 . 1.00 1.00 1.00 1.00 1.00 1.00 1.00 - 1.00 1.00 - 1.00 1.00 1.00 1 .(30 1.00 0.25 0.75 1.00 - 1.00 1.00 1.00 . 0.05 1.00 - 1.00 1.00 1.00 1.00 1.00 1.00 . 1.00 1.00 1.00 1.00 1 .(30 1.00 1.00 1.00 1.00 1.00 1.00 0.75 0.20 0.05 1.00 0.35 0.65 0,10 0,45 0.45 11. S d h - A 1.00 - 1.00 12. Sod A 1.00 1.00 13 Xdh-A 77 86 94 100 108 123 167 0.25 075 100 217 1.00 100 118 1.00 88 100 - 1.00 1.00 - 1.00 cola has lost both the Ada-A 87and Ada-A ~°°alleles replaci ng these with Ada-A ~2a nd Ada-A m. B. Gpi-B-The Gpi-B 1°° allele expressed by E. microperca and E. proeliare is symplesiomorphically shared with E. (Hololepis) grac#e. Additional species-specific apomorphic alleles have developed in all th ree species of Microperca with G pi-B89 becoming fixed in E. fontico/a. C. L d h - A - The Ldh-Al°°issymplesiomorphically shared by E. fondco/a, E. proe#are and E. (Holo/epis) graci/e. T he Ld h-A 65allele fixed in popu latio ns of E. microperca is assigned derived status. D. Ldh-B - Species of Microperca do not share Ldh-B alleles with E. (Hololepis) gracile [2] but E. fonticola and E. proe#are do share the Ldh-B ~°° 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.40 0.60 1.00 1.00 - 0.90 0.10 1.00 1.00 1.00 1.00 100 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 - 0.05 117 10. P g m - A - 0.95 - 4. Cbp 1 1.00 - 1.00 100 3. S A a t A 1 1.00 1.00 - allele with members of the su bgenus Oligocephalus (e.g.E. spectab#e) [2]. Thus, the Ldh-B 1°° allele is assigned primitive status while the Ldh-B m allele of E. microperca is interpreted as the derived state. E. P g m - A - T h e Pgm-A 1°° allele is assigned primitive status on the basis of its symplesiomorphic expression in E. proe#areand E. (Hololepis) gracile. Apparently, the Pgm-A m3 allele was also present in the common ancestor of Microperca with its present distribution being the predominant allele in E. microperca and a minor allele in E. proe#are (i.e. a symplesiomorphy). Species-specific minor alleles assigned apomorphic status include Pgm-A T1, Pgm-A 94, Pgm-A 1°6 and Pgm-AmL The Pgm-A a6allele is shared by E. fonticola and E. proe- DONALD G. BUTH, BROOKS M. BURR AND JOHN R. SCHENCK 302 TABLE 3. ESTIMATES OF GENETIC VARIABILITY IN 10 POPULATIONS (SUBGENUS M/CROPERCA) Proportion of genome heterozygous per individual (observed)* Proportion of genome heterozygous per individual (expected) 1- Proportion of loci polymorphic Effective No of alleles per locus fontico/a Guadalupe drainage 0.017 0.028 0.130 1.040 proetyare Ohio drainage Big Black drainage Alabama drainage Pearl drainage 0.052 0.011 0022 0026 0043 0008 0.020 0.025 0.174 0.043 0.043 0.043 1.063 1.010 1036 1061 0.013 0000 0002 0017 0.012 0.000 0.016 0.021 0.087 0.000 0.043 0.043 1.014 1.000 1.026 1.040 0000 0.000 0.000 1.000 Population mlcroperca Kanakee drainage Osage drainage (1) Osage drainage (2) Gasconade drainage (1) Gasconade drainage (2) Loci were considered polymorphic if more than one allelic preduct was resolved. Given the constraints of sample size, the rarest allele observed in this study was at frequency 0.05 • Based on the actual proportion of observed heterozygotes. 1~Hardy Weinberg heterozygoslty liare and is interpreted as a synapomorphy, although this may eventually be interpreted as a symplesiomorphy if it could be shown that the common ancestor of the subgenus expressed Pgm-A 8~and that E. microperca has subsequently lost it or is expressing it at a low frequency. E Sod-A-Although Page and Whitt [2] reported Sod-A as identical in E. microperca and E. proe/iare, our examination using the same electrophoretic buffer system showed E. microperca to exhibit a slightly faster anodally migrating product. As the Sod-A t°° allele of E. fontico/a and E. proe/iare is common to several other species of the subgenera Ho/o/epis, Oligocephalus and Catonotus [2], it is assigned primitive status. The Sod-A t~8 allele of E. microperca is the derived state. The allelic data for the six polymorphic loci plus the LDH subunit binding data were used to construct a hypothetical phylogeny of M/croperca shown in Fig. 3. While both the phenetic and cladistic treatments of the data cluster E. fonticola and E. proe//are, the latter approach indicates that a sizeable proportion of genetic characteristics shared between E. font/cola and E. prod/are are symplesiomorphically shared. Only the sharing of the Pgm-A seallele, if this characteristic is indeed a synapomorphy, can be considered as evidence for placing these two species on a common evolutionary line within the subgenus. Both E. fontico/a and E. microperca exhibit numerous apomorphic states indicative of evolutionary advancement in their respective lineages. Etheostoma proeliare is E. microperca .on-" g' m n ,mm- .,ii. E. proeliare E.fontico/a I I I I I V I I f l e I d, m -? l--- ____J ___. m ~ m I. 1, 1, i° 1. ~, b, i1' FIG. 3 HYPOTHETICAL PHYLOGENY OF THE SUBGENUS MICROPERCA INFERRED FROM THE DISTRIBUTION OF DERIVED GENETIC CHARACTER STATES, Derived states are indicated by solid symbols whereas hollow symbols depict the ancestral states, Character states are as follows: (a) absence of Pgm-A 86, la') development of Pgm A86; Ib) absence of Ada-A 52 and Ada-A 70, (b') development of Ada A 52 and Ada-A70; (c) absence of Gpi B89, (c') development o1 Gpi-B89; (d) ability to form Ldh-A2B2 heterotetramer, (d') loss of ability to form Ldh-A2B2 heterotetramer (e) retention of Ldh-A 10° (e') development of Ldh-A 65; (f) retention of Ldh-B 00, (f,) development of Ldh B/~ (g) retention of Sod-A [uu (g,) develoPlmoent of Sod Al18"; (h) absence of Gpi-B 110, (h' ) development of Gpi B GENETIC RELATIONSHIPS IN MICROPERCA 303 TABLE 4. NEI'S [12] COEFFICIENTS OF GENETIC SIMILARITY (/) ABOVE DIAGONAL AND GENETIC DISTANCE (D) BELOW DIAGONAL CALCULATED BETWEEN POPULATIONS (CALCULATIONS ARE BASED ON 23 LOCI) Population 1 3 4 0.89 0.88 0.89 0.89 0.12 0.12 0.12 0.11 0.00 0.01 0,01 1.00 0.01 0.01 0.99 0.99 0.00 0.55 0,56 0.55 0.60 0.56 0.54 0.47 0.45 0.51 0.47 0.56 0.49 0.47 0.53 0.49 0.55 0.49 0.46 0.52 0.49 E fonticola 1. Guadalupedrainage E. proehare 2. 3. 4 5. Ohio drainage Big Black drainage Alabama drainage Pearldrainage E. microperca 6 Kankakeedrainage 7. Osagedrainage (1) 8. Osagedrainage (2) 9. Gasconadedrainage (1) 10. Gasconadedrainage (2) Population 5 6 2 almost identical on the genetic level to the hypothetical common ancestor of the subgenus Microperca. In view of this high level of genetic plesiomorphy in E. proe/iare, we highly recommend the use of this species to ascertain the evolutionary status of allelic expression in studies of related subgenera, e.g. Holo/epis and Catonotus. In summary, virtually all of Burr's [8] morphologically based hypotheses regarding M/croperca are supported by the electrophoretic data. Etheostoma fonticola and E. proe/iare are shown to be very similar relative to E. microperca, although this similarity is due to a high degree of symplesiomorphy. The recognition of E. proe/iare as the most primitive species and E. microperca as the most advanced species of the subgenus Microperca [8] has considerable genetic support both on the allelic level and on the level of LDH subunit binding. Morphological differentiation between the Ozark and more northern populations of E. microperca is paralleled on the genetic level by allelic differentiation at the adenosine deaminase locus (Ada-A; Table 2). The retention of the Ada-A 87and Ada-A 1°° alleles by the Illinois population may be interpreted as the primitive condition while the loss of Ada-A 87 by the Ozark populations may be interpreted as an evolutionary advancement that is paralleled by E. proe/iare. Further population-level genetic studies of E. microperca are obviously desirable and the Ada-A locus exhibits variability that should be especially informative. Experimen'ml Specimens were collected 13yminnow seine and dip net at the 10 localities listed below. Specimens were placed on dry ice upon capture and kept frozen until dissected. Electrophoretic examinations of individual specimens were completed within 1 yr of their collection. Voucher specimens are deposited at the 7 8 9 10 0.57 0.57 0.56 0.55 0.57 0.99 0.99 1.00 0.58 0.57 0.56 0.58 0.62 0.61 0.61 0.62 0.64 0.63 0.63 063 0.60 0.59 0.59 0.60 0.62 0,61 0.61 0.62 0.55 0.48 0.46 0.52 0.48 0.04 0.04 0.06 0.04 0.96 0.00 0.02 0.00 0.96 100 0.94 0.98 0.98 0.02 0.96 1.00 1.00 0.98 0.02 0.00 Illinois Natural History Survey (INHS). Collection numbers of voucher samples follow each listed locality in brackets. Numbers in parentheses are specimens electrophoretically examined. Complete collection data are available from the second author. Etheostoma fonticola. Guadalupe drainage: San Marcos R., Hays Co.,TX [INHS 75562] (20). Etheostoma proeliare. Ohio drainage: Max Cr., Johnson Co., IL [INHS 26918] (10). Big Black drainage: Hays Cr., Attala Co., MS (4). Alabama drainage: Taylor Cr., Greene Co., AL [INHS 76250] (10). Pearl drainage: Lee's Cr., Washington Par., LA [INHS 75844] (10). Etheostoma microperca. Kankakee drainage: Tributary of Iroquois R., Iroquois Co., IL [INHS 7235] (10). Osage drainage: Sac-Osage R., Greene Co., MO [INHS 75822] (10); Hahatonka Spring, Camden Co., MO [INHS 75817] (10). Gasconade drainage: Wood Fk., Gasconade R., Wright Co., MO [INHS 75819] (10); Osage Fk., Gasconade R., Webster Co., MO [INHS 75828] (10). In addition to these samples of the subgenus Microperca, specimens of Etheostoma (Hololepis) gracile (Girard) (Ohio drainage: Cypress Cr., Union Co., I L - I N H S 17994) were electrophoretically examined to ascertain allelic products that are symplesiomorphically shared between the subgenera Microperca and Hololepis. The preparation of liver and skeletal muscle extracts followed Buth and Burr [13]. ACP, ADH, SDH, SOD and XDH isozymes were electrophoretically separated [14, 15] from liver extracts whereas all other enzymatic and non-enzymatic proteins were resolved from skeletal muscle extracts. Electrophoretic buffers and conditions include sodium borate (for ADH, SDH and XDH), sodium citrate (for ACP, AAT, G-3-PDH, LDH, MDH and PGM), EBT (for CBP, CK, GPI and SOD), phosphate-citrate (for ADA, FUM and ICDH) and discontinuous Tris-citrate (for MPI). The sodium borate, sodium citrate and EBT electrophoretic buffers are those discussed by Buth and Burr [13]; whereas the phosphatecitrate and discontinuous Tris-citrate (="Poulik") electrophoretic buffers are those of Selander eta/. [15]. The staining procedures for visualizing enzymatic activity and the general protein stain for calcium-binding proteins plus creatine kinase are those previously described or involve slight modifications ot these methods: ACP, G-3-PDH, ICDH, LDH arid XDH [16]; 304 ADA [17]; AAT, ADH, FUM and MDH [14]; CBP and CK [18]; GPI [19], MPI [20], PGM [21]; SDH [22]; SOD [23]. Allelic terminology utilizing the relative differences in the electrophoretic mobility of the respective gene products employed as the reference allele ( = 100) the most common allele at each locus in the Illinois sample of E. proeliare. Acknowledgements-Field work for this study was supported in part by a grant to B. M. Burr (NSF DEB 76-22387). Further support was provided by the Department of Zoology, Southern Illinois University at Carbondale (B.M.B.) and the UCLA Department of Biology Fisheries Grant (D.GB.). A permit to collect the federally endangered Etheostoma fonticola was granted through the cooperation of the U.S. Fish and Wildlife Service, Department of the Interior. We appreciate the field assistance of P. A. Burr, R. L. Mayden, M. A. Morris, L. M. Page and R. D. Wrisberg. We thank G. C. Gorman for the use of his laboratory facilities during a portion of this study. R. W. Murphy, R. D. Orton and T. L. Vance provided valuable laboratory assistance, and R. W. 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