Proc. Nati. Acad. Sci. USA Vol. 91, pp. 994-998, February 1994 at the population level: Pupfish HindIll satellite DNA sequences JOHN F. ELDER, JR.* AND BRUCE J. TURNER Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Communicated by Bruce Wallace, October 18, 1993

ABSTRACT The canonical monomers (170 bp) of an organisms. There are very little data on their variation within abundant (1.9 x 10' copies per diploid genome) satellite DNA or divergence among conspecific natural populations. sequence family In the genome of Cyprinodon wriegau, a We report here sequence comparisons ofthe predominant "pph" that ranges along the Atlantic coast fom Cape Cod or "canonical" monomers of a satellite DNA array in sam- to central Mexico, are divergent in base sequence in 10 of 12 ples of 12 natural populations of Cyprinodon variegatus smle collected from natural populations. The divergence (Cyprinodontidae), a coastal killifish species. Ten of these Involves sbsitions, deletis, and insertions, is marked in samples have distinctive and characteristic canonical mono- scoe (mean pairwise sequence slarit = 61.6%; range = mers with high levels of intraindividual and intrapopulation 35-95.9%), Is largely ed to the 3' half of the monomer, homogeneity.t In other words, this satellite DNA has appar- and Is not correlated with the disace among cllg sites. ently undergone concerted evolution at or near the level of Repetitive loning and direct genomic sequencing expriments the local population. failed to detect intrapopulation and intraindividual variation, A preliminary account of some of our early findings has hig levels of sequence homogeneity within popu- appeared in a symposium volume (6). latios. The satellite s has therefore undergone "con- certed evolution," at the level of the local population. Con- certed evolution has previously almost always been dsused in MATERIALS AND METHODS terms of the divergence of species or higher taxa; its intraspe- Organism and Samples. C. variegatus Lacepede, some- cific occurnce apparently has not been reported previously. times called the "Sheepshead minnow," is a common, in- The gnerality of the observation is difficult to evaluate, for shore, euryhaline, "pupfish" species that ranges from Cape altough Esatellite DNAs fom a large number oforganisms have Cod, MA, to the mouth ofthe Rio Tuxpan, Mexico. Related, been su ied In detail, there appear to be little or no other data sometimes morphologically divergent, populations exist in on their sequence variaton in natural populations. The rela- some Floridian freshwater lakes, Yucatan/Belize, Bahamas, tionp (if any) between concerted, population level, satellite several Caribbean islands, and coastal Venezuela (7, 8). Most DNA divergence and the eatent of flow/genetic Iolation specialists would agree that its distribution is "patchy" among conspeiflc natural populations remains to be estab- throughout its range-i.e., it is locally (and sometimes sea- lished. sonally) abundant in appropriate habitats. However, little else is known of its population structure, dynamics, or local Comparisons ofparalogous repetitive DNA sequences in the genetic contiguity. It was selected for further study from an genomes of often reveal striking intraspecific initial restriction enzyme survey of satellite DNAs in 20-30 similarities but marked interspecific divergences. This ap- fish species (B.J.T., unpublished data) because ofthe appar- parent evolutionary nonindependence of the members of a ent simplicity ofits satellite monomers and its abundance and sequence family is termed "concerted evolution" (1). The ease of sampling. Samples are detailed in Fig. 1. Sampling forces that mediate concerted evolution are believed to locations were determined solely by convenience and the include biased , unequal crossing-over, and field activities of colleagues and do not reflect any putative replicative transposition (2). The term "molecular drive" (2) subdivisions within the species. Two to nine pUC19 clones is sometimes used to describe these forces acting together were sequenced from 1-5 specimens per site. HindIII ge- with stochastic population events (, founder nomic bands were directly sequenced (see below) from 5-12 effects, etc.). specimens per site (depending on sample size). The significance ofmolecular drive or its component forces DNA Preparations and Detection/Cloning of Satellite Se- at the population level is currently unknown. Some theoret- quences. Genomic DNA was purified from homogenates of ical studies (e.g., ref. 3) suggest that it could be important in whole fish (less scales, skin, and gut) in 4 M guanidinium promoting the divergence of conspecific populations in a isothiocyanate, followed by phenol/chloroform and chloro- non-Darwinian manner. However, this intriguing notion is form extractions, as described (9). Presumptive satellite currently without empirical support, for there appear to be no sequences were detected as coherent bands upon agarose gel published examples of interpopulation (but intraspecific) electrophoresis of HindIII or Sst I digests of genomic DNA. concerted evolution. Only the HindIll satellite is discussed here. Bands were Highly repetitive, tandemly arrayed, "satellite" DNAs are excised from low-melting agarose; purification and ligation general features of eukaryotic genomes (4). These sequence into the HindIII cloning site of plasmid pUC19 followed families often provide clear-cut examples of interspecific standard methods (10, 11). concerted evolution (e.g., ref. 5). Most satellite DNAs have Genomic Copy Number and Hybridization Experiments. been characterized only from laboratory strains, cell lines, or Satellite copy number was estimated by hybridization of a limited (and frequently pooled) sample of field-caught *To whom reprint requests should be sent at the present address: Department of Biology, University of North Dakota, P.O. Box The publication costs ofthis article were defrayed in part by page charge 8238, University Station, Grand Forks, ND 58202. payment. This article must therefore be hereby marked "advertisement" *The sequences reported in this paper have been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. U02331-U02340). 994 Downloaded by guest on October 2, 2021 Evolution: Elder and Turner Proc. Natl. Acad. Sci. USA 91 (1994) 995

0 100 240 i- i I ---- kilometer 50 150

FIG. 1. C. variegatus population samples and locations. 1A, Cape Cod, MA, site I (north shore, Buzzard's Bay) (3 specimens); 1B, Cape Cod, MA, site II (Oyster Pond, Woods Hole) (7 specimens); 2, Hereford Inlet, NJ (12 specimens); 3, Lusby, MD (Patuxent R. at Drum Point Lake) (15 specimens); 4, Sapelo Island, GA, two proximate stations: site I (west side of island) (16 specimens) and site II (Light House Ditch) (16 specimens); 5, Jacksonville, FL (Forest Street culvert) (12 specimens); 6, Tampa Bay, FL, site I (Pinellas Pt.) (20 specimens); 7, Tampa Bay, site II (Port Manatee) (12 specimens); 8, Key West, FL (8 specimens); 9, New Orleans, LA (Lake Pontchartrain) (8 specimens); 10, High Island, TX (intercoastal waterway bridge) (5 specimens). cloned monomer to a dilution series of slot-blotted genomic monomer (see below). The Vent (exo-) cycle sequencing DNAs. Signal intensities were compared to those generated system (New England Biolabs) was used with the 35S incor- by known amounts of purified satellite monomer applied to poration protocol recommended by the manufacturer, except the same membrane (12). Cross-reaction of C. variegatus that a single initial denaturation period of 5 min at 950C was HindIII satellite monomer with sequences in the genomes of used, followed by 40 cycles of950C for 30 sec, 650C for 30 sec, other fish species was also assessed by slot blot hybridiza- and 750C for 30 sec. The superior thermostability of Vent tion. In all such experiments, probes were radiolabeled (32p) (exo-) tm polymerase allowed the use of very high temper- by random oligonucleotide priming (13). Nylon membranes atures during the sequencing reactions; this resulted in the (Zeta-Probe, Bio-Rad) were prehybridized in 50%o forma- elimination ofsome "stop" sequencing artifacts, presumably = due to template secondary structure, which were visible mide, 1% SDS, 4x SSPE (lx SSPE 0.18 M NaCl/10 mM when Taq polymerase was used in initial experiments. sodium phosphate/i mM EDTA), 0.5 mg of tRNA per ml, Direct sequence comparisons of cloned monomers and and 0.5% Blotto at 42TC. Hybridization was allowed to genomic monomer fractions were made by excising the proceed overnight at the same temperature and in the same cloned monomers from recombinant plasmids, purifying solution but with 10%6 dextran sulfate and 106 cpm of probe them by gel electrophoresis, and then using them as tem- added per ml ofhybridization fluid. Membranes were rinsed, plates in Vent (exo-) tm cycle sequencing reactions. These at room temperature, consecutively for 15-min periods in 2x employed an identical protocol to, and were conducted SSC, 0.5x SSC, and O.lx SSC (all made 0.1% in SDS; lx simultaneously with, genomic sequencing reactions. The SSC = 0.15 M NaCl/15 mM sodium citrate); the final wash sequences of cloned and genomic monomer fractions from was for 30 min at 50TC in O.lx SSC/1% SDS. the same individual or population were then compared side- Sequencing of Cloned Monomers. Satellite monomers by-side on "Long Ranger" modified polyacrylamide gels (AT cloned in pUC19 were sequenced as double-stranded tem- Biochem, Malvern, PA) with 7.8 M urea and a 10%o sucrose plates (14) with T7 DNA polymerase (Sequenase, United gradient to retard migration of smaller fragments. States Biochemical) in the "forward" and "reverse" direc- Sequence alignments and comparisons were done with the tions. "GAP" and "DISTANCES" subroutines of the Sequence Anal- Genomic Sequencing. Monomer bands (=170 bp) were ysis Software Package (version 6.0; Genetics Computer gel-purified (15) after HindIII digestion of C. variegatus Group, Madison, WI). genomic DNA and incorporated directly into a PCR-based, cyclic sequencing protocol. Approximately 200-400 ng of purified monomer was used as double-stranded templates in RESULTS the sequencing reactions. The primer was a relatively invari- Bands of about 170 bp in length, visible in EtBr-stained ant 25-bp segment at the arbitrarily designated 5' end of the HindIII digests of C. variegatus genomic DNA, are the Downloaded by guest on October 2, 2021 996 Evolution: Elder and Turner Proc. Natl. Acad. Sci. USA 91 (1994)

monomers a 1 50 apparent of classic "alpha" satellite (16) array Texas TCAAATCACA AGTTGAGGCC AAAAT.GCTC TTTTCACAGT GAGATAAAGC (Fig. 2). Slot blot dilution experiments indicate that the Louis ------G------G------Tampa I ------.----.------satellite comprises about 5.5% of the diploid genome. The Tampa II ------.------x Jacksonv ------.____. monomer is therefore present at about 1.9 106 copies per Key West ------TA-G- A--GGCCTAA --TCGCTGAT diploid genome (experimental error = 0.6% over three rep- Sapelo ------Capecod ------licates). Lusby ------. T------GCAT------TG Nucleotide sequence data for the monomers characterized Hereford ------TA-G- AAAC-T-TA- T-T-CTGCAT are 51 100 from each population given in Fig. 3 (see also Table 1). Texas TATTTTGG.. CCTGTGGAGT TATAACAG.. ACAAACCATT AATG.ATACA The following are noteworthy: Louis ------Tampa I ------(i) No intraindividual variation in monomer sequence was Tampa II -----G--.. -T-----C------GA C------Jacksonv ------..------.. ------A----- detected by iterative cloning. Key West CCGGCAC-.. .GC-ACT-CA --AGTG-TTG CGT-TTTTGC -GCTTCG-GC (ii) With a single exception (see legend to Fig. 3), no Sapelo ------Capecod ------.. ------ATAG-. variation in monomer sequence was detected among individ- Lusby ------CAT --G-A-CGAA A--CGAGTAG C--TG-TC-- TCACGTAGT- uals within populations. Hereford AT-GCAA-CA T-G-CAA-AC --C-TAGAAG T-G-TTTTGC G--TT-GGTC (iii) Monomers cloned 10 of a total of 12 101 150 from population Texas GCCATGATCT TTTCTGGCAA TCACCTAGAG CTGAAAACTA ACAATATAAG samples are divergent in sequence; two samples from Cape Louis ------A Tampa I ------A Tampa II ------CTA---C------CTAC- TAT-AG--GA a Jacksonv ------T------A------A Key West TG-TCA---C -C-G--TAGC G-TA--TAC- CA-C-G-GCG CACTCTG-GT Sapelo - .------.------A Capecod .-A---C-T- --GGCAA--T C--TAGGC-T GAAC--TCA- -T-TAGAT-- Lusby --AT--C-GA GAATAC-AC- CT-T-ATCGC A---TCT-GC --GCACATCA Herefor& AG-T-TA--C GC-.A-CTT- G-TGAGTAC- --AC-T-TAG TTG--GCT-C 151 179 Texas TA.GA...... Louis GT.AGATTTG AGCAGATGTT TTGGTTCCA Tampa I CT.AGATTTG ACCAGATGTT TTGGTTA.. Tampa II ...... TTG AGC&GATGTT TTCTCATTA Jacksonv GT.AGATTTG AGCAGATGTT TTGGTTA.. Key West GCGACGACGT CGCTA...... Sapelo GTCAGATTTG AGCAGATGTT TTTGGTTA. Capecod --GCGAAGTG A...... Lusby TGA--GCGTT GCTA...... Hereford GCTC-GACTC AGGACATA ...... FIG. 3. Sequence alignments of the C. variegatus population canonical sequences generated with the GCG "PILEUP" subroutine. Periods ite relative insertions or deletions, dashes indicate identical sites, and substitutions are written. Note: Asingle specimen from sample 2 yielded a canonical monomer with three apparent base substitutions compared to the sequence shown for that population. Cod, MA (locations 1A and 1B) and two from Sapelo Island, 2h 3h 4h 5h 18h GA (location 4) yielded identical monomer sequences. The b Ih divergence involves substitutions, insertions, and deletions and is quite marked in extent: the average pairwise sequence similarity (Table 1) among population samples is 61.6% w:...w...wST:P w (range, 35-95.9%o). The variation is nonrandomly distributed; t the overwhelming bulk occurs in the 3' half ofthe monomer. Age -art (iv) Genomic sequencing experiments yield the same nu- >oS>e ::; cleotide sequences as do the cloned monomers. When com- :.: .: pared on the same gel, cycle sequencing ofcloed monomers *: : .....: :: and genomic Hindm monomer bands yield identical results 1722- (Fig. 4), though background is often higher in the genomic lanes. Direct genomic sequencing therefore independently 984- confirms the results obtained by cloning with respect to 861- monomer sequence homogeneity within genomes and diver- 738- gence among population samples. Mixing experiments (using 615- 492- Table 1. Pairwise proportional similarities of C. variegatus 369 - HindII canonical satellite monomers among 10 population 246 samples (see Fig. 1) as generated by the oA subroutine of the

123 - Genetics Computer Group analysis package (identity = 100) Population sample no. FIG. 2. Documentation of a HindI satellite DNA array in the 2 3 4 5 6 7 8 9 10 genome of C. variegatus. (a) Electrophoresis in a 1% agarose gel of overnight digests of genomic DNA with HindHI; the sample in each 1 35.0 48.9 71.6 71.1 69.4 66.5 41.1 70.3 74.2 lane is from an individual specimen from Jacksonville, FL (standard: 2 42.4 41.8 41.4 36.3 38.5 50.2 36.5 44.6 123-bp ladder). Note the characteristic bright band (presumptive 3 52.5 52.3 53.5 48.0 47.6 54.8 50.2 satellite monomer) at about 170 bp. (b) Electrophoresis (as above) of 4 88.0 92.3 77.5 47.7 94.2 80.5 incremental timed digests of C. variegatus (Jacksonville) genomic 5 90.3 80.1 47.6 90.5 81.3 DNA with HindIl, probed with radiolabeled, cloned 170-bp pre- 6 83.2 48.2 95.9 88.5 sumptive satellite monomer. (The lane immediately to the right of 7 35.8 80.1 73.4 each Hindm ladder is an Sst I digest ofequivalent duration, showing 8 43.9 37.4 no cleavage within the HindIll satellite.) The positions of molecular 9 weight markers are indicated. 84.4 Downloaded by guest on October 2, 2021 Evolution: Elder and Turner Proc. Natl. Acad. Sci. USA 91 (1994) 997 predominant monomer in each genome may therefore ap- proach 70-80%o.

DISCUSSION The data clearly suggest that intraspecific concerted evolu- tion of HindIII satellite monomers has occurred in C. var- iegatus at or near the level of the local population: divergent canonical monomers, each with high levels of intraindividual and intrapopulation homogeneity, could readily be charac- terized from 10 of 12 population samples. The level of sequence divergence is not correlated with the distances between collecting sites. For example, the two most similar canonical monomers (95.9o similarity) are from New Or- leans, LA, and Pinellas Pt., Tampa Bay, FL, whereas the two most divergent (35% similarity) are from Cape Cod, MA, and Hereford Inlet, NJ. The two pairs of localities are separated by roughly equivalent coastwise distances. Monomers from the two most proximate population samples (Pinellas Pt., and Port Manatee, both in Tampa Bay) are only slightly more similar (83.2% vs. 74.2%) than are those from the two most widely separated samples (Cape Cod, MA, and High Island, TX). Likewise, the satellite variation is not obviously related to previously reported minor morphological or allozyme variation (17, 18) in the species. Although its distribution is patchy, different populations of C. variegatus generally cross readily in the laboratory and produce fertile progeny of both sexes; even partial F1 sterility has been encountered only in a few crosses between mainland and some Caribbean island populations (ref. 17; B.J.T., unpublished data). There is no reason to suggest that our samples are from more than a single species. Our observation of population-specific concerted evolu- tion of a satellite DNA sequence appears to be novel. However, the generality of the phenomenon itself is difficult to assess, for there have been very few studies ofintraspecific variation of satellite DNA monomers (or of repetitive se- quences of any sort) and apparently none that attempted to partition variation into intraindividual, intrapopulation, and interpopulation components. Consequently, at present it is not clear if this type of divergence occurs widely or if it is -F3 7%. limited to species with particular histories, population struc- tures, or other properties. The high levels of intraindividual sequence homogeneity of the C. variegatus HindIII satellite monomer appear to be unusual. Several well-characterized satellites from mamma- lian genomes (e.g., refs. 19-21) are apparently more variable within individuals, though the nature of the samples makes this difficult to evaluate. The intragenomic distribution of ..- --I--; satellite sequence variants in C. variegatus is apparently 11 .1~~ more similar to that of the beetle Tenebrio molitor, in which a single sequence variant can comprise about 50o of all monomers (22), than it is to the satellites thus far character- ized from other vertebrates. It should be noted that we have ACGTACGT studied only monomeric sequences in C. variegatus, and much of the intragenomic variation reported in the satellites c loned uncloned of other species is in modified oligomers (e.g., variant or truncated dimers, etc.). In the discussion that follows, we tentatively accept the emerging consensus that satellite DNAs generally lack se- FIG. 4. Comparison of sequence patterns obtained from a cloned quence-specific biological function and thus that their vari- C. variegatus HindIll monomer from a Key West sample and from ation tends to be phenotypically inconsequential (e.g., ref. equivalent genomic monomer fragments from the same sample 23; see refs. 24 and 25 for reviews). In other words, we ("uncloned") with the direct cycle sequencing protocol. Note iden- assume that we deal with a "selfish" or "ignorant" DNA tity of patterns. sequence. While this assumption appears to be the general view, there are clear-cut exceptions (e.g., transcribed am- varying proportions of recombinant plasmids with different phibian satellite sequences; see ref. 26 for review). HindIII satellite monomer variants) suggest that the genomic In the absence of detailed knowledge of the population sequencing could have detected a minor variant sequence structure and genetics of C. variegatus, attempts to assess the present at as little as 20% ofthe total. The homogeneity of the biological significance of its interpopulation satellite DNA Downloaded by guest on October 2, 2021 cmw 7.70 Evolution: Elder and Turner Proc. NatL Acad. Sci. USA 91 (1994) divergence are necessarily speculative. There appear to be versity research monies. During manuscript preparation, J.F.E. was two general possibilities: supported by National Science Foundation Grant STIA-R11- (i) The divergence is related to the genetic isolation of EPSCOR (to the State of North Dakota). populations. Satellite sequence variants in a given population become new canonical monomers through the action of 1. Arnheim, N. (1983) in Evolution of and Proteins, eds. to Nei, M. & Koehn, R. K. (Sinauer, Sunderland, MA), pp. molecular and population-level stochastic forces, but, due 38-61. limited , they are transmitted to other populations 2. Dover, G. A. (1982) Nature (London) 29, 111-116. only infrequently. The existence of divergent canonical sat- 3. Ohta, T. & Dover, G. A. (1983) Proc. NatI. Acad. Sci. USA 80, ellite monomer variants in two or more populations would 4079-4083. thus be indicative oflimited genetic interchange and therefore 4. Beridze, T. (1986) Satellite DNA (Springer, Berlin). would likely be correlated with the divergence of other 5. Karger, J. & Vogel, F. (1975) J. Mol. Evol. 4, 201-247. genetic features. 6. Turner, B. J., Elder, J. F., Jr., & auin, T. F. (1991)J. Fish (ii) The divergence is related solely to the molecular Biol. 39, Suppl. A, 131-142. biology of particular sequence variants. New sequence vari- 7. Johnson, W. E. (1980) in Atlas ofNorth American Freshwater ants arise by in some populations, and those that are Fishes, eds. Lee, D. S., Gilbert, C. R.,Hocutt, C. H., Jenkins, replicated (e.g., by biased gene conversion) most efficiently R. E., McAllister, D. E. & Stauffer, J. R., Jr. (North Carolina eventually spread to adjacent populations, even if gene flow State Museum Nat. Hist., Raleigh), p. 504. in them as well. Their spread 8. Hubbs, C. L. (1937) Carnegie Inst. Wash. Publ. 457, 157-287. is limited, and become common 9. Turner, B. J., Elder, J. F., Jr., & Laughlin, T. F. (1989) continues until they encounter other sequences ofequivalent Fingerprint News 1, 15-16. or greater replicatory efficiency. In this interpretation, the 10. Sambrook, J., Fritsch, E. F. & Manitis, T. (1989) Molecular satellite sequence variants might be considered as rough Cloning: A Laboratory Manual (Cold Spring Harbor Lab. analogs ofhypothetical, newly arisen, supernumerary ("B") Press, Plainview, NY), 2nd Ed. , which mediate their own meiotic accumula- 11. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., tion (27) but which otherwise presumably have little effect on Seidman, J. G., Smith, J. A. & Struhl, K., eds. (1987) Current the genomes of their "hosts." The existence of divergent Protocols in Molecular Biology (Wiley Interscience, New satellite monomers would thus be largely unrelated to the York). divergence of other genetic features. 12. Krane, D. E., Clark, A. G., Cheng, J. F. & Hardison, R. C. The first possibility, if supported, would have potentially (1991) Mol. Biol. Evol. 8, 1-30. important theoretical and practical implications: Concerted 13. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132, feature 6-13. divergence ofrepetitive sequences might be a general 14. Gatermann, K. B., Rosenberg, G. H. & Kaufer, N. F. (1988) of genetically isolated populations, one associated with the BioTechniques 6, 951-952. process of allopatric divergence. Moreover, the genetic iso- 15. Qian, L. & Wilkinson, M. (1991) BioTechniques 10, 736-738. lation of populations, including those of commercially ex- 16. Horz, W. & Zachau, H. G. (1977) Eur. J. Biochem. 73, 383- ploited species with unknown or unresolved stock structure, 392. might be conveniently assessed by comparing canonical 17. Darling, J. D. (1976) Ph.D. dissertation (Yale Univ., New satellite monomers. (These could then be considered "ge- Haven, CT). netic tags" ofhigh specificity.) The second alternative offers 18. Duggins, C. F., Jr., Karlin, A. A. & Relyea, K. G. (1983) novel insight into the population genetics of selfish DNA Northeast GulfSci. 6, 99-107. 19. Gaillard, C., Doly, J.. Cortadas, J. & Bernardi, G. (1981) sequences. It would suggest that the molecular drive-related Nucleic Acids Res. 9, 6069-6082. properties of a sequence might be more important than 20. Manuelidis, L. & Ward, D. C. (1984) Chromosoma 91, 28-38. population structure in determining its distribution. At pres- 21. Maresca, A., Singer, M. F. & Lee, T. N. H. (1984) J. Mol. ent, no data exist by which the two interpretations can be Biol. 179, 629-649. distinguished. 22. Plohl, M., Borstnik, B., Lucijanic-Justic, V. & Ugarkovic, D. (1992) Genet. Res. 60, 7-13. We thank W. P. Davis, K. A. Goddard, J. J. deGreef, D. Huie, A. 23. Ohta, T. & Dover, G. A. (1984) Genetics 108, 501-521. Morales, B. Nelson, J. Pitcairn, K. Reich, R. Socolof, C. H. StQwell, 24. Miklos, G. L. G. (1985) in Molecular Evolutionary Genetics, M. Szesze, J. Trexler, R. Wildekamp, M. C. Wooten, and their field ed. MacIntyre, R. J. (Plenum, New York), pp. 241-321. associates for collecting specimens; T. F. Laughlin for assistance in 25. John, B. & Miklos, G. L. G. (1988) The Eukaryotic Genome in the field and laboratory; and J. Cranford and M. Lederman for Development and Evolution (Allen & Unwin, London). helpful discussions and editorial advice. This research was supported 26. Green, B., Pabon-Pena, L. M., Graham, T. A., Peach, S. E., in part by the Lerner-Grey fund ofthe American Museum ofNatural Coats, S. R. & Epstein, L. M. (1993) Mol. Biol. Evol. 10, History (J.F.E.), National Science Foundation Grant BSR 8506417 732-750. (B.J.T.), and various Virginia Polytechnic Institute and State Uni- 27. Jones, B. (1991) Am. Nat. 137, 430-442. Downloaded by guest on October 2, 2021