Brief Communications

Brief Communications

Genetic Variation Detected by Microsatellites in Five Spanish Dog Breeds L. Morera, C. J. Barba, J. J. Garrido, M. Barbancho, and D. F. de Andre´s Estimates of genetic variation detected by microsatellite loci were obtained at the intra- and interbreeds levels for five Spanish dog breeds: Alano, Galgo, Podenco Andaluz, Perro de agua, and Maneto. Four microsatellites described in the literature were used. All were polymorphic in the five breeds. Average heterozygosities within breeds ranged from 0.70 to 0.77 and the mean number of alleles per breed ranged from five to seven. Wright’s F statistics were calculated to analyze the differences between observed and expected heterozygosities. Our results showed an average deficit of heterozygotes at all loci for each breed (FIS 5 0.085 6 0.034). The genetic differentiation among breeds was estimated as FST 5 0.108 6 0.015. Both estimates were significant at the 99% level. FST was also used to obtain genetic distances between different breeds. Our estimates are of similar magnitude to those reported in Spanish dog breeds using allozyme markers. The lowest distance was obtained for the Galgo–Podenco Andaluz pair. The greatest distances were obtained between Alano and the other breeds. This can be due, at least in part, to a bottleneck effect caused by the drastic reduction in population size for this breed at the end of the last century. In the last few years microsatellites have become the preferred type of genetic markers because of their abundance, random distribution, codominant inheritance, high variability, possibility of automated detection (Goldstein and Pollock 1997), and ability to show genetic polymorphism

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even in species in which classical genetic markers have proven to be unsuccessful (Petit et al. 1997). Several groups have published results on the application of microsatellites to the study of genetic differentiation within and between canine breeds ( Fredholm and Wintero 1995; Pihkanen et al. 1996; Zajc et al. 1997). Previous studies about the genetic structure of the Spanish dog breeds and the relationships among them involved calculating genetic distances and using the F statistics from morphological characters and biochemical polymorphisms (Jordana et al. 1992a–c). However, to our knowledge no reports about this subject have been published to date using microsatellites. Previously we carried out a preliminary study to detect genetic variation in the Spanish Alano using microsatellite markers (Morera et al. 1998). In this article we report the results obtained in a more complete study involving four other Spanish breeds.

Materials and Methods This study deals with five Spanish dog breeds: Alano (Spanish alano, AL) Galgo (Spanish greyhound, GE), Podenco Andaluz (Andalusian hound, PA), Perro de agua (Spanish water dog, PAE), and Maneto (MA). All of them have been previously described ( Barba and Moreno-Arroyo 1997a,b; Salamanca 1982; Sanz and Marıń 1982). The Real Sociedad Canina de Espanã (RSCE) officially recognizes GE, PA, and PAE and is in the process of recognizing AL and MA. GE is recognized by the Fe´de´ration Cynologique Internationale ( FCI), which also has tentatively recognized PAE. Blood samples for amplifications were collected from unrelated animals located in different places of the area of distribution. Four microsatellite loci, chosen on the basis of their reported polymorphism information content (PIC) values, were

used: AHT101 ( Holmes et al. 1993), VIASD10 (Primmer and Matthews 1993), and CXX.30 and CXX.140 (Ostrander et al. 1993). PCRs were carried out on 100 ng of DNA, prepared according to the salting out method of Miller et al. (1988), or 5 ml of a 1/100 diluted suspension of frozen then thawed lymphocytes. The reaction mixture included 200 mM dNTPs, 0.5 mM of each primer, and 0.5 U Taq polymerase in a final volume of 25 ml. The thermocycling conditions were 5 min at 948C, 30 cycles of 1 min at 948C, 1 min at the annealing temperature (AHT101 and VIAS-D10: 558C; CXX.30 and CXX.140: 598C), 1 min at 728C, and a final extension step of 10 min at 728C. Magnesium concentrations were 1 mM (AHT101) or 1.5 mM (VIAS-D10, CXX.30, and CXX.140). For electrophoresis, 5 ml of the amplification product was mixed with an equal volume of formamide dye solution, denatured 5 min at 948C, and loaded into a 6% polyacrylamide 7 M urea sequencing gel. The bands in the gels were visualized by silver staining ( Vega Pla 1996). Band sizes were determined by comparison to a molecular weight marker ladder (pBR322 DNA-MspI digest). Alleles were named according to relative migration speed of the corresponding bands. Unbiased estimates of heterozygosity (h), average heterozygosity (H), and their standard errors were obtained according to Nei (1987). The TFPGA program (Miller 1997) was used to calculate the deviations from Hardy–Weinberg equilibrium ( HWE) using a Markov chain method, and to calculate Wright’s F statistics and pairwise comparisons among populations obtain the significance of allele frequency differences. This program was also used to obtain the allele frequencies directly from the dataset. PIC was calculated as 1 2 Spi2 2 2Spi2pj2 ( Botstein et al. 1980).

Results The genetic variability detected, estimated by the number of alleles, heterozygos-

Table 1. Results obtained for each microsatellite locus in the five Spanish dog breeds a Breeds

b

Locus

AHT101

VIAS-D10

CXX.30

CXX.140

All loci

Number of alleles Range of sizes ( bp) Range of frequencies h 6 SEc PICc Number of alleles Range of sizes ( bp) Range of frequencies h 6 SE PIC Number of alleles Range of sizes ( bp) Range of frequencies h 6 SE PIC Number of alleles Range of sizes ( bp) Range of frequencies h 6 SE PIC Mean number of alleles H 6 SEc PIC

AL (35)

GE (30)

MA (32)

PAE (28)

PA (37)

4 130–136 0.03–0.62 0.56 6 0.05 0.50 6 109–133 0.01–0.3 0.77 6 0.02 0.72 7 151–173 0.1–0.24 0.83 6 0.02 0.80 3 139–143 0.18–0.44 0.64 6 0.02 0.55 5 0.70 6 0.06 0.64

10 124–142 0.05–0.25 0.87 6 0.02 0.84 6 109–129 0.07–0.37 0.77 6 0.03 0.72 5 151–159 0.03–0.45 0.69 6 0.04 0.62 5 137–145 0.05–0.55 0.64 6 0.05 0.59 6.5 0.74 6 0.05 0.69

9 124–142 0.03–0.28 0.82 6 0.02 0.78 6 109–129 0.03–0.36 0.74 6 0.03 0.68 6 151–159 0.02–0.41 0.72 6 0.03 0.66 7 133–145 0.02–0.39 0.77 6 0.03 0.72 7 0.76 6 0.02 0.71

8 124–142 0.05–0.23 0.85 6 0.02 0.82 5 109–125 0.02–0.39 0.72 6 0.03 0.66 5 151–159 0.04–0.39 0.72 6 0.03 0.66 5 133–145 0.09–0.25 0.79 6 0.02 0.74 5.75 0.77 6 0.03 0.72

7 124–142 0.01–0.29 0.81 6 0.02 0.76 7 109–133 0.06–0.19 0.85 6 0.01 0.82 4 151–157 0.05–0.69 0.49 6 0.06 0.44 7 133–145 0.01–0.32 0.71 6 0.03 0.66 6.25 0.71 6 0.08 0.67

See Materials and Methods for codes. Number of animals for each breed is in parentheses. c h 6 SE: heterozygosity 6 standard error. H 6 SE: average heterozygosity 6 standard error. PIC: polymorphism information content.

a

b

ity, and PIC value for each locus, and average heterozygosity and PIC value in each breed are summarized in Table 1. The four loci were polymorphic in the five breeds studied. Tests for HWE revealed significant deviations in six cases: AHT101 in AL (p , .001), CXX.30 in GE (p , .05) and MA (p , .001), and CXX.140 in AL (p , .01), MA (p , .05), and PA (p , .01). We used the Wright’s F statistics to analyze the differences between the observed and expected heterozygosities, assuming HWE, at the intra- and interbreed levels. The estimates of FIS, which measures the excess or deficit of average heterozygotes in each breed, and FST, which measures the degree of genetic differentiation among breeds, were 0.085 6 0.034 and 0.108 6 0.015, respectively. Both estimates were significant at the 99% level. The genetic profile shown by these microsatellites was clearly different among the five breeds. All the pairwise comparisons for differences in allele frequencies among breeds were significant (p , .001). Moreover, we found at locus CXX.30 two breed-specific alleles, 1 and 3 (decreasing electrophoretic mobility), which were detected only in the population of Spanish alano. As the overall loci estimate of FST as well as all the pairwise comparisons for differences in allele frequencies were significant, we obtained the FST values and their

standard errors for all the pairwise comparisons between breeds. FST takes values from 0 to 1 and can be used as a measure of genetic distance (Jordana et al. 1992c). The lowest FST values were obtained for the GE/PA (0.067 6 0.014), PA/PAE (0.071 6 0.018), and GE/PAE (0.077 6 0.024) pairs, and the highest for the AL/GE (0.154 6 0.029), AL/MA (0.146 6 0.059), and AL/ PA (0.145 6 0.054) pairs. All the FST values were significant at the 99% level except for MA/PA, which was significant at the 95% level.

Discussion We evaluated the degree of genetic variation that can be detected in Spanish dog breeds by using microsatellites. We analyzed five breeds for variability at four microsatellite loci, chosen from those described in the literature. If we consider the results for each locus, the degree of genetic variation, measured by the number of alleles, average heterozygosities, and PIC values, is similar to that previously reported for the loci AHT101 (7 alleles, PIC 5 0.72; Holmes et al. 1993) and VIAS-D10 (6 alleles, PIC 5 0.71; Primmer and Matthews 1993) and somewhat lower for CXX.30 and CXX.140 (PIC 5 0.77 and 0.76, respectively; Ostrander et al. 1993). On the other hand, our values of genetic variation within breeds can be considered to

be similar to those reported in the German shepherd (5 alleles/locus, H 5 0.60; Pihkanen et al. 1996), but clearly greater than those found in the flat-coated retriever (4.5 alleles/locus, H 5 0.52) and the dachshund (5.6 alleles/locus, H 5 0.55; Fredholm and Wintero 1995) or in the greyhound (2.5 alleles/locus, H 5 0.36), the Labrador retriever (3.3 alleles/locus, H 5 0.48), and the German shepherd (3.3 alleles/locus, H 5 0.43) ( Zajc et al. 1997). From previously reported data for HWE in dog breeds, with allozyme (Jordana et al. 1992c) or microsatellite ( Zajc et al. 1997) loci, a high proportion of significant deviations from equilibrium would be expected. Our results agree with this expectation, as we found 6 (30%) significant deviations out of a total of 20 (4 loci 3 5 breeds) possibilities. Only one locus, VIASD10, did not deviate significantly in any breed and only one breed, PAE, showed HWE for the four loci. Our data show a significant average deficit of heterozygotes (FIS 5 0.085) for each breed, which is in accordance with the findings of Jordana et al. (1992c) in a group of 10 Spanish dog breeds, although these authors obtained a lower estimate (FIS 5 0.040) for this deficit. On the other hand, our estimate of the average genetic differentiation among breeds (FST 5 0.108) is clearly lower than that reported by Pihkanen et al. (1996) for the German shepherd (FST ù 0.30) when this breed is compared with a composite mixture sample of 34 different breeds using microsatellite loci, but similar to that found by Jordana et al. (1992c) in Spanish breeds ( FST 5 0.099). This similarity in the estimates of genetic differentiation among Spanish breeds by using allozyme or microsatellite data argues in favor of the existence of a lower degree of genetic differentiation among Spanish breeds. We used the FST statistic to obtain the genetic distances among the five dog breeds. Our estimates are of similar magnitude to those found in Spanish breeds using allozyme data (Jordana et al. 1992c). We obtained the lowest genetic distance for the GE/PA pair and the lowest distance of this group was with respect to PAE. A similar group was also obtained by Jordana et al. (1992c), although we have additionally analyzed the Galgo, Podenco, and Gos d’Atura (Catalonian sheepdog) breeds. Gos d’Atura and PAE are two related breeds that are supposed to be descendants of the Bergamasco breed ( Barba and Moreno-Arroyo 1997a; Jordana et al. 1992c). The breed most distant from

Brief Communications 655

the GE/PA group was AL, which belongs to another morphologic group: the moloses (Sanz and Marıń 1982). In addition, this breed shows the greatest distance from any other breed. This can be due in part to the drastic reduction in population size undergone by the breed during the last century, to such an extent that it was considered extinct. This could have caused a great change in the allele frequencies due to a bottleneck effect, thereby increasing the genetic distances between populations ( Nei 1987). In conclusion, our results seem to indicate a lower interbreed genetic variation in Spanish dogs than that found in other breeds and point to the existence of a high degree of genetic variation within breeds, which can be detected with microsatellites. We also demonstrated the usefulness of these markers in genetic studies. From the Veterinary Faculty, Department of Genetics, University of Co´rdoba, Research Group ‘‘Molecular genetic markers in domestic animals,’’ Avda. Medina Azahara, 9 Co´rdoba, 14005, Spain. Address correspondence to L Morera at the address above or e-mail: [email protected]. We would like to thank the dog breeders who kindly collaborated in obtaining blood samples and Eva Sarrioń for her technical assistance. This work was supported by Spanish research program PB93-0160-C02-02. q 1999 The American Genetic Association

Morera L, de Andre´s DF, and Barba CJ, 1998. Deteccioń de variabilidad gene´tica por microsate´lites en el Alano espanõl. Arch Zootec 48:63–70. Nei M, 1987. Molecular evolutionary genetics. New York: Columbia University Press. Ostrander EA, Sprague FG, and Rine J, 1993. Identification and characterization of dinucleotide repeat (CA)n markers for genetic mapping in dog. Genomics 16:207–213. Petit E, Aulagnier S, Vaiman D, Bouissou C, and CrouauRoy B, 1997. Microsatellite variation in a introduced mouflon population. J Hered 88:517–520. Pihkanen S, Vaïno¨la¨ R, and Varvio S, 1996. Characterizing dog breed differentiation with microsatellite markers. Anim Genet 27:343–346. Primmer CR and Matthews ME, 1993. Canine tetranucleotide repeat polymorphism at the VIAS-D10 locus. Anim Genet 24:332. Salamanca D, 1982. Galgo espanõl. In: I Symposium Nacional de las Razas Caninas Espanõlas, Co´rdoba, 19–21 March 1982. Co´rdoba, Spain: Universidad de Co´rdoba; 283–291. Sanz JM and Marıń H, 1982. Los molosos de presa espanõles. In: I Symposium Nacional de las Razas Caninas Espanõlas, Co´rdoba, 19–21 March, 1982. Co´rdoba, Spain: Universidad de Co´rdoba, 187–195. Vega Pla JL, 1996. Polimorfismo de ADN equino. Obtencioń de marcadores moleculares y su aplicacioń al control de filiacioń (PhD dissertation). Co´rdoba, Spain: Universidad de Co´rdoba. Zajc I, Mellersh CS, and Sampson J, 1997. Variability of canine microsatellites within and between different dog breeds. Mamm Genome 8:182–185. Received November 20, 1998 Accepted May 31, 1999 Corresponding Editor: Robert Wayne

References Barba CJ and Moreno-Arroyo B, 1997a. El Perro de Agua Espanõl. Jaeń, Spain: Editorial Jabalcuz. Barba CJ and Moreno-Arroyo B, 1997b. El Podenco Andaluz y el Perro Maneto. Jaeń, Spain: Editorial Jabalcuz. Botstein D, White R, Skolnik M, and Dawis RW, 1980. Construction of a genetic linkage map using restriction fragment length polymorphisms. Am J Hum Genet 32: 314–331. Fredholm M and Wintero AK, 1995. Variation of short tandem repeats within and between species belonging to the Canidae family. Mamm Genome 6:11–18. Goldstein DB and Pollock DD, 1997. Launching microsatellites: a review of mutation processes and methods of phylogenetic inference. J Hered 88:335–342. Holmes NG, Mellersh CS, Humphreys SJ, Binns MM, Holliman A, Curtis R, and Sampson J, 1993. Isolation and characterization of microsatellites from the canine genome. Anim Genet 24:289–292. Jordana J, Piedrafita J, and Sańchez A, 1992a. Genetic relationships in Spanish dog breeds. I. The analysis of morphological characters. Geńe´t Se´l Evol 24:225–244. Jordana J, Piedrafita J, and Sańchez A, 1992b. Genetic relationships in Spanish dog breeds. II. The analysis of biochemical polymorphism. Geńe´t Se´l Evol 24:245–263. Jordana J, Piedrafita J, Sańchez A, and Puig P, 1992c. Comparative F statistics analysis of the genetic structure of ten Spanish dog breeds. J Hered 83:367–374. Miller SA, Dykes DD, and Polesky HF, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215. Miller MP, 1997. Tools for population genetic analyses ( TFPGA): a Windows program for the analysis of allozyme and molecular population genetic data. Northern Arizona University, Flagstaff.

656 The Journal of Heredity 1999:90(6)

Comparison of Multilocus DNA Fingerprints and Microsatellites in an Estimate of Genetic Distance in Chicken S. Ponsuksili, K. Wimmers, F. Schmoll, P. Horst, and K. Schellander We estimated genetic variation based on DNA fingerprinting and microsatellites in samples of chicken lines ranging from commercial high-yielding lines, to exotic local populations, to an experimental inbred line. Heterozygosity estimates based on DNA fingerprints and microsatellites were highly correlated (r 5 0.88). The potential of DNA fingerprints and microsatellites as precise tools to measure heterozygosity was demonstrated. Concerning estimation of genetic distance between populations, it was shown that the application of DNA fingerprints on DNA pools is suitable, fast, and economical and therefore competitive with the use of microsatellites, especially in species where

information to design primer pairs to detect microsatellites is limited. Multilocus DNA fingerprints ( DFPs) and microsatellite markers are based on length polymorphism of repetitive genomic regions. Multilocus DFP analysis (Jeffreys et al. 1985) has been widely used in population genetic studies in the chicken ( Dunnington et al. 1994; Kuhnlein et al. 1989; Mafeni et al. 1997; Ponsuksili et al. 1998; Siegel et al. 1992). Today the determination of heterozygosity and genetic distance based on microsatellite analyses is regarded as the most convenient method. However, reports about the use of microsatellites to study genetic relationships between chicken populations are scarce ( Liu et al. 1996; Ponsuksili et al. 1996; Takahashi et al. 1998). DFPs have the advantage that no specific knowledge about any genomic DNA sequences are necessary, whereas the application of microsatellites depends on the availability of specific primers. The main advantage of microsatellites is their character as a codominant monolocus system, allowing the identification of each allele per locus, which makes it useful for obtaining basic population genetic data, such as allele frequencies. The objective of the current study was to compare estimates of genetic variation based on DFP markers and microsatellite markers in the same samples from different chicken lines, including commercial high-yielding lines, exotic local populations, and an experimental inbred line.

Materials and Methods Animals Twelve lines of chickens were used including an inbred line, commercial lines, and exotic breeds. These populations are the White Leghorn inbred line ETH77 ( IBL), three commercial high-yielding strains—a broiler male strain ( BRO), a Rhode Island Red layer line (RIR), and a White Leghorn layer line ( LEG)—and eight exotic breeds: Bankiva ( BAN), Dandrawi ( DAN), Fayoumi ( FAU), Kadakanath ( KAD), Nunukan ( Nun), Silky (SIL), Taiwan White-darkmeat broiler line ( TWW), and Taiwan Browndarkmeat broiler line ( TWB) (Ponsuksili et al. 1998). Detection of DFPs DFPs were prepared as described elsewhere (Ponsuksili et al. 1998). In brief, 10 mg of DNA were digested with AluI or HinfI. The DNA fragments were separated in

0.7% agarose gels (1 V/cm for 40 h) and transferred to nylon membranes. Hybridization was performed with the oligonucleotide probes [CAC]5, [CA]8, [GACA]4, and [GGAT]4. Two types of DFPs were made: those of individual DNA samples and those of DNA pools from each animal within each line. DFPs of individual DNA samples were used to determine the band sharing degree and the band frequencies within populations. Pooled DNA from different lines were used to produce DFP patterns, which were representative for the populations, allowing us to compare the patterns of many populations on a few DFPs. These DFPs were repeated several times with different arrays of the samples in order to compare each lane with another in close proximity. All banding patterns were compared with the next and the next but one to classify shared and unshared bands. Experiments were duplicated and were evaluated by two persons independently. Only bands representing fragments larger than 2 kb were scored. Bands were regarded as unshared if they differed in their position more than the half of the band width. For DFPs of individual DNA samples, band intensity was taken into account. Bands were scored as unshared if the ratio of the intensities was less than 1:2. Detection of Microsatellites Genotyping of DNA samples at 15 microsatellites ( Table 1) was carried out using 50 ng of genomic DNA in a reaction mixture containing 1.5 mM MgCl2, 200 mM dNTPs, 0.50 mM of each primer, 1 U Taq polymerase (30 cycles: 1 min at 948C, 1 min at 558C–608C, 1 min at 728C). Polymer-

ase chain reaction (PCR) products were then separated on a denaturing polyacrylamide gel, transferred to nylon membranes and detected by hybridization with (CA)8. Alternatively, fluorescent-labeled primers were used and analyses were performed on an automated DNA sequencer (A.L.F. Pharmacia-Biotech, Uppsala, Sweden). Measures of Genetic Variation For DFP analysis, heterozygosity was calculated according to Stephens et al. (1992). For microsatellite genotyping, heterozygosity was calculated per strain per marker according to Nei (1987). In DFP analyses, genetic distance between populations was estimated as the average band difference, the additive inverse of average band sharing between the representative banding patterns (Gilbert et al. 1990; Jeffreys et al. 1985). Based on microsatellite genotyping, CHORD genetic distance (dij) between populations was estimated (Cavalli-Sforza and Edwards 1967). These results were used to construct genetic trees by neighbor-joining cluster analyses. The NTSYS-pc (Rohlf 1993) and the PHYLIP (Phylogeny Interference Package) computer package version 3.5p ( Felsenstein J, University of Washington, Seattle) were used.

Results Genetic Variability Within Populations The DFP banding patterns of individual DNA samples were reproducible and contained about 40–45 bands. Band-sharing values obtained within the 12 populations ranged between 31.5% ( BRO) and 93.5%

Table 1. Populations used, number of animals per population, mean number of bands, mean band sharing, heterozygosity obtained by DNA fingerprints, and heterozygosity and PIC values obtained with 15 microsatellites DNA fingerprints Population

Number of animals

Mean number of bands (x 6 SD)

BRO KAD TWB TWW NUN RIR LEG DAN SIL BAN FAU IBL

25 20 9 8 21 22 20 26 18 12 10 26

44.3 44.9 44.4 43.4 44.7 41.1 44.3 45.9 44.1 43.0 42.2 41.3

6 6 6 6 6 6 6 6 6 6 6 6

4.11 6.49 6.80 6.83 7.13 6.26 4.79 8.37 5.23 8.89 8.76 8.43

Mean band sharing (x 6 SD) 31.5 38.5 42.2 46.7 48.6 49.9 53.1 60.9 62.1 62.6 66.8 93.5

6 6 6 6 6 6 6 6 6 6 6 6

7.78 8.06 6.78 7.09 8.56 7.40 7.09 8.18 9.63 7.40 9.49 3.88

( IBL). For different combinations of enzymes and probes between 18 and 31 loci were detected, as estimated based on the number of bands and band sharing as well as on band frequencies (Gilbert et al. 1990; Lynch et al. 1990) ( Table 1). For the 15 microsatellites, between 2 and 12 alleles were detected. Polymorphic information content (PIC) ranged from 0.25 for microsatellite HUJ6 to 0.51 for ADL20 when averaged over all populations ( Table 1). Mean PIC values, calculated over all 15 microsatellites, ranged from 0.56 for KAD to 0.16 for IBL ( Table 1). Based on band frequencies of DFPs, heterozygosity estimates ranged from 5.1% to 60.6%. Considering the group of commercial lines, BRO had the highest heterozygosity, whereas RIR and LEG had intermediate values. Within the group of exotic lines, KAD showed the highest value and FAU the lowest ( Table 1). The heterozygosity calculated based on all 15 microsatellites ranged from 56.8% in LEG to 12.1% in the IBL. Considering the group of commercial lines, BRO had the highest heterozygosity. TWB, FAU, and NUN showed the same level of heterozygosity, about 45%. SIL and DAN had significantly lower values (about 35%) and TWW had the lowest values within the group of exotic breeds (26%) ( Table 1). Microsatellites provided higher heterozygosity values than DFPs except for BRO and KAD. Both of these markers support that KAD and IBL have the highest and lowest heterozygosity, respectively. In total the results of both marker systems were highly correlated. In comparisons of DFPs for different combinations of enzymes and probes with the microsatellite data, coefficients of correlation between r 5 0.77 and 0.92 were found, all being highly significant. The highest correlation was found for the comparison of estimates from microsatellite data and DFPs produced with the probe [CA]8.

Microsatellites Heterozygosity

Hetero- Locus zygosity name

Mean PIC (x 6 SD)

59.7 38.6 36.2 21.9 16.0 22.8 60.6 37.7 22.4 30.1 42.1 5.1

50.1 45.6 37.6 25.6 42.6 36.4 56.8 46.1 32.6 41.6 43.6 12.1

0.51 0.41 0.36 0.10 0.34 0.47 0.49 0.34 0.25 0.37 0.22 0.35 0.35 0.38 0.31

ADL20 ADL23 ADL34 ADL37 ADL39 ADL44 HUJ1 HUJ2 HUJ6 HUJ7 HUJ8 HUJ12 MCW1 MCW4 MCW7

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

0.20 0.14 0.26 0.16 0.16 0.20 0.16 0.25 0.18 0.23 0.15 0.24 0.20 0.21 0.21

Genetic Distance Between Populations The representative banding patterns of all 12 populations were highly reproducible with about 50 bands. In the dendrogram, based on DFPs, two main clusters were classified. Both main clusters could be divided in two subgroups each ( Figure 1). Within the tree based on microsatellite analysis there was one main cluster containing all populations but SIL and KAD. Within the main cluster two subclusters could be identified. Bootstrapping values ranged from 9% to 89% ( Figure 1). In total the correspondence between the two den-


Figure 1. Dendrograms of 12 strains of chicken revealed by the neighbor-joining method based on DNA fingerprints and microsatellites. Numbers on the nodes are percentage bootstrap values from 1,000 replications of resampled loci.

drograms was low. The highest matrix correlation was observed when comparing microsatellite results with DFP data from the combination of restriction enzyme AluI and oligonucleotide probe [CA]8 (0.3).

Discussion This study was designed to evaluate the effect of different DNA markers on the estimates of genetic variation within and between different chicken populations. Populations were chosen which were diverse in their genetic makeup, representing commercial lines, a laboratory inbred line, and a broad range of exotic local populations and fancy breeds. These populations make a useful model to evaluate the suitability of DNA markers to detect genetic divergence. But it should be mentioned that the individuals of the exotic local breeds are members of small flocks. It should be noted that these animals represent only a subset of the original populations due to founder effect, genetic drift, and/or inbreeding, though efforts were made to reduce these effects by controlled mating and repeated supply of individuals. Therefore this study cannot provide a description of the genetic structure of the whole population. Genetic Variation Within the Populations Population data on DFPs do not provide a complete characterization of the genetic variation in terms of allele frequency distributions. Stephens et al. (1992) proposed a statistical parameter to estimate hetero-


zygosity based on band frequency. He stated that this parameter is insensitive for the lack of knowledge. In this study, and in agreement with some other publications (Gilbert et al. 1990; Kuhnlein et al. 1989; Siegel et al. 1992), the usefulness of DFPs for the purpose of estimating genetic variation has been successfully demonstrated. Compared to mammalian species, microsatellite variability in the chicken was moderate. This corresponds to data of Khatib et al. (1993). Heterozygosity estimates from microsatellite data reflected well the genetic variation within the populations, as is expected from the breeding history of the inbred and the commercial lines. The results provided by DFPs and microsatellite analyses were highly correlated, indicating that heterozygosity was reflected by both markers. The high correspondence might be supported by the fact that both markers detect the same kind of polymorphism in highly repetitive, noncoding regions of the genome. It is remarkable that the highest correlation between the heterozygosity estimates were found when comparing the microsatellite results with the results from DFPs produced with [CA]8. A reason for this might be that the microsatellites used here also contain CA repeats and that a higher degree of overlapping of the detected loci is given here than with other probes. Genetic Variation Between the Populations The additive inverse of average band sharing was shown to be an adequate measure

of distance (Gilbert et al. 1990; Ponsuksili et al. 1998). In microsatellite analysis, CHORD genetic distance was calculated because it is a frequently used distance measure, and according to Nei and Takezaki (1996), it is still is one of the most suitable measures. Here we found marked differences in the topologies of the population trees derived from the two marker systems. Similar to the results of the heterozygosity estimation, again best fit between microsatellite and DFP results is found for banding patterns obtained with [CA]8. In general, when constructing a dendrogram it is difficult to judge which is the best one with regard to the genetic relationship of the populations. With the use of different criteria (morphological, biochemical, etc.) to determine genetic similarity/distance, one has to expect to end up with different trees, each of which reflect only part of the whole tree. The first main cluster of the DFP dendrogram indicated a close relationship between TWB, TWW, and BRO. This is very conclusive and may reflect the intensive selection for growth traits resulting in many common DFP bands. The other alternative is that these strains are of the same origin and therefore the bands conserved are common in DFPs or they may have been crossbred. NUN and RIR are more similar to each other than KAD and SIL. One possible interpretation of these findings is that NUN and RIR might have common ancestors, so the morphology and body size of NUN and RIR are quite similar. Knowledge of the history of NUN gives no hints of this interpretation. For the relationship between KAD and SIL, only the geographic record showed that both strains originated in Asia. Their morphology is different, but the dark-meat characteristic and the light body weight are the same. Other references about the relationship of KAD and SIL have not been found. The second main cluster consisting of FAU, DAN, IBL, LEG, and BAN reflected the history of the stocks. The DFP analysis confirmed that IBL was derived from LEG by selection and subsequent inbreeding (Somes 1988). DAN and FAU are more distinct from BAN than LEG. DAN and FAU are populations developed in two regions of lower and upper Egypt and LEG was disseminated via the Mediterranean from its origin in Asia. The clustering of these five strains suggests that the light strains are more closely related to BAN, the common ancestor of all domestic chickens, than broiler types. This corresponds to the re-

sults of Siegel et al. (1992) that domestic jungle fowls resemble more closely the layer populations than broiler types. But this is not in agreement with the results of Hashiguchi et al. (1981), who found that Red Jungle Fowl is more closely related to White Plymouth Rock and White Cornish than to White Leghorn and Rhode Island Red. In the dendrogram based on microsatellites, BAN is located in a cluster consisting of TWW, TWB, and DAN. TWW and TWB originated in China, from where the samples of BAN were received, but TWW and TWB are based on the cross of White Cornish, a broiler type, and Silky. Here the tree based on microsatellite supports the results obtained with 18 protein markers by Hashiguchi (1981). The relationship of DAN to TWW, TWB, and BAN is hard to explain. Another cluster on the microsatellite-based tree consists of LEG, IBL (which is in good agreement with the DFP tree), FAU, and NUN. The common ground of the breeds FAU and LEG/IBL is found in their common path of distribution from their origin in Asia; NUN is an Asian breed. This is reflected in the microsatellite tree. RIR and BRO are commercial breeds that were established in the United States in the late 1800s. Finally, for KAD and SIL high genetic distance to the other populations is indicated by the microsatellite dendrogram. Both marker systems provided dendrograms that could be explained by the history and/or morphology and other specific traits of the populations. Even though the DFP data give no complete genetic information concerning the number of loci or allelic relationships, the method is useful to screen for variability within populations, especially in animal species where information for designing primer pairs to detect microsatellites is limited. The application of DFP analysis on DNA pools is suitable for estimating genetic relationships among chicken populations, making this kind of analysis fast and economical, and competitive with microsatellite analysis. From the Institut fu¨r Tierzuchtwissenschaft, Landwirtschaftliche Fakulta¨t der Rheinischen Friedrich-Wilhelms Universita¨t Bonn, Endenicher Allee 15, D-53115 Bonn, Germany (Ponsuksili, Wimmers, Schmoll, and Schellander), and Institut fu¨r Nutztierwissenschaften, Landwirtschaftlich-Ga¨rtnerische Fakulta¨t der Humboldt-Universita¨t zu Berlin, Berlin, Germany ( Horst). Address correspondence to S. Ponsuksili at the address above or e-mail: [email protected]. q 1999 The American Genetic Association

References Cavalli-Sforza LL and Edwards AWF, 1967. Phylogenetic analysis: models and estimation procedures. Evolution 32:550–570. Dunnington EA, Stallard LC, Hillel J, and Siegel PB, 1994. Genetic diversity among commercial chicken populations estimated from DNA fingerprints. Poult Sci 73:1218–1225. Gilbert DA, Reid YA, Gail MH, Pee D, White C, Hay RJ, and O’Brien SJ, 1990. Application of DNA fingerprints for cell-line individualization. Am J Hum Genet 47:499– 514. Hashiguchi T, Tsuneyoshi M, Nishida T, Higashiuwatoko H, and Hiraoka E, 1981. Phylogenetic relationships determined by the blood protein types of fowls. Jpn J Zootech Sci 52:713–729. Jeffreys AJ, Wilson V, and Thein SL, 1985. Individual specific ‘‘fingerprints’’ of human DNA. Nature 316:76– 79. Khatib H, Genislav E, Crittenden LB, Bumstead N, and Soller M, 1993. Sequence-tagged microsatellite sites as markers in chicken reference and resource populations. Anim Genet 23:355–362. Kuhnlein U, Dawe Y, Zadworny D, and Gavora JS, 1989. DNA fingerprinting: a tool for determining genetic distances between strains of poultry. Theor Appl Genet 77:669–672. Liu JS, Wu XH, Zhang XQ, Wei CF, Lian SD, Liu JS, Wu XH, Zhang XQ, Wei CF, and Lian SD, 1996. Detection and genetic difference of microsatellite polymorphisms in yellow broiler lines. Anim Biotechnol Bull 5(suppl):59– 62. Lynch M, 1990. The similarity index and DNA fingerprinting. Mol Biol Evol 7:478–484. Mafeni MJ, Wimmers K, and Horst P, 1997. Genetic diversity in indigenous Cameroon and German Dahlem Red Fowl populations estimated from DNA fingerprints. Archiv fu¨r Tierzucht, 40:581–589. Nei M. 1987. Molecular evolutionary genetics. New York: Columbia University Press. Nei M and Takezaki N, 1996. Reconstruction of phylogenetic trees from microsatellite (STR) loci. In: Proceedings of the XXVth International Conference on Animal Genetics. Toulouse, France: COREP; 2–5. Ponsuksili S, Wimmers K, and Horst P, 1996. Genetic variability in chickens using polymorphic microsatellite markers. Thai J Agric Sci 29:571–580. Ponsuksili S, Wimmers K, and Horst P, 1998. Evaluation of genetic variation within and between different chicken lines by DNA fingerprinting. J Hered 89:17–23. Rohlf FJ, 1993. NTSYS-pc numerical taxonomy and multivariate analysis system, version 1.80. Setauket, NY: Exeter. Siegel PB, Haberfeld A, Mukherjee TK, Stallard LC, Marks HL, Anthony NB, and Dunnington EA, 1992. Jungle fowl domestic fowl relationships—a use of DNA fingerprinting. World Poult Sci J 48:147–155. Somes RG, 1988. International registry of poultry genetic stocks. Bulletin 476. Storrs Agricultural Experiment Station. Stephens JC, Gilbert DA, Yuhki N, and O’Brien SJ, 1992. Estimation of heterozygosity for single probe multilocus DNA fingerprints. Mol Biol Evol 9:729–743. Takahashi H, Nirasawa K, Nagamine M, Tsudzuki M, and Yamamoto Y, 1998. Genetic relationships among Japanese native breeds of chicken based on microsatellite DNA polymorphism. J Hered 89:543–546. Received December 28, 1998 Accepted May 31, 1999 Corresponding Editor: Stephen E. Bloom

Constraints on Elevated Ploidy in Hybrid and Nonhybrid Parthenogenetic Snails S. G. Johnson, R. Hopkins, and K. Goddard We used flow cytometry to determine the genome size of sexual, hybrid parthenogenetic, and spontaneous (nonhybrid) parthenogenetic snails in the genus Campeloma. All hybrid parthenogens were allotriploids (5.07 pg/nucleus 6 0.09) and all spontaneous parthenogenetic snails were autodiploids (3.69 pg/nucleus 6 0.11). In conjunction with previous results, these findings confirm that allotriploidy arose through interspecific hybridization between a sexual C. limum female and a male C. geniculum followed by backcrossing with a male C. geniculum. There was no indication of variable ploidy levels within allotriploid or autodiploid parthenogens. We argue that numerous mechanisms may prevent elevated ploidy level. These include destabilizing effects of continued backcrossing to sexual ancestors, and increased nutrient requirements and lower growth rates of tetraploids in stressful environments. There is much speculation and controversy about the origin and maintenance of polyploid parthenogens ( Kondrashov 1997; Levin 1983; Maynard Smith 1978; Suomalainen 1950; Thompson and Lumaret 1992). Two mechanisms can account for the origin of parthenogenetic polyploids. In the classical model, parthenogenesis originates in a diploid lineage, and elevated ploidy levels occur by fertilization of parthenogenetic ova (Suomalainen 1950; White 1973). Although hybridization is not necessary, hybridization between two species and subsequent backcrossing lead to elevated ploidy and heterozygosity. An alternative hypothesis is that triploid parthenogens originate first, and parthenogens with higher or lower ploidy levels evolve from triploid ancestors (Saura et al. 1993). In most vertebrate and weevil polyploid hybrid parthenogens, triploidy is the most common ploidy level (Saura et al. 1993; Suomalainen et al. 1987; Vrijenhoek et al. 1989). In this alternative hypothesis, ploidy elevation to triploidy is concurrent with a switch to parthenogenesis. Triploids arise through fertilization of diploid ova by haploid sperm according to this view, and parthenogens with higher


Table 1. Individual DNA content for various sexual and parthenogenetic Campeloma populations from the southeastern United States

Figure 1. Collecting localities and reproductive mode of southeastern U.S. Campeloma.

ploidy levels evolve from triploid ancestors. Diploid parthenogens can evolve from triploid ancestors by meiotic plate disjunction (Suomalainen 1950). Ploidy level may affect the persistence of parthenogens. Triploid parthenogens may be more common because they have a fitness advantage over diploid or tetraploid parthenogens. Triploids may more effectively mask the effects of deleterious mutations compared to diploids ( Kondrashov 1997; Stebbins 1971), but continued backcrossing leading to tetraploidy and higher ploidy may have destabilizing effects ( Lynch 1984). There are both sexual and parthenogenetic forms of the freshwater snail Campeloma. In a recent study of allozyme and mtDNA sequence variation in the freshwater snail Campeloma, we found spontaneous parthenogens within one species and interspecific hybrid parthenogens through out the southeastern United States (Johnson and Bragg, in press; Johnson and Leefe, in press). We discovered numerous allfemale populations of C. limum from the headwaters of the Suwannee River in northern peninsular Florida to the Savannah River ( Figure 1). Although parthenogens and sexuals do coexist in some river drainages, parthenogens occur in disturbed habitats in the upper headwaters and main river channels. We ascribed spontaneous (nonhybrid) origins of parthenogenesis in C. limum because parthenogens had significantly lower heterozygosity and genotypic diversity compared to sexual populations, and most alleles in spontaneous parthenogens are present in sexual C. limum populations. Based on mtDNA sequence divergence, most spontaneous parthenogens arose during the Pleistocene from sexual C. limum. We suggested that these spontaneous partheno-


Species/population

Average DNA content per individual (pg/nucleus)

C. geniculum Holmes Creek ( HL) Ten-Mile Creek ( TM)

3.49, 3.58, 3.36, 3.25, 3.25 3.53, 3.51, 3.33, 3.61, 3.36, 3.55

C. parthenum Holmes Creek ( HC) Wright’s Creek (WC)

5.67, 5.58, 4.73, 4.83, 5.33, 4.77 5.3, 4.84, 4.74, 4.81, 5.09, 5.31, 4.95

C. limum (sexual) Horse Creek ( HR) Lower Three Runs Creek ( TR)

3.24, 3.84, 3.51 4.03, 3.63, 3.87, 3.67, 4.05

C. limum (parthenogenetic) Alapaha River (AL) Brier Creek ( BC)

3.49, 3.11, 4.03, 4.13, 3.32 3.47, 4.08, 3.91, 3.63, 3.75

Refer to Figure 1 for locations.

gens were diploid because there was no unbalanced staining intensity in heterozygous genotypes at mono- or dimeric enzymes (Johnson and Leefe, in press). However, determination of ploidy by allozyme staining intensity is unreliable (e.g., Goddard and Schultz 1993; Vrijenhoek 1974; Whitt et al. 1972). Hybrid parthenogens, collectively called C. parthenum, occur in disturbed habitats in the Florida panhandle, and they are fixed heterozygotes at four of six enzyme loci (Johnson and Leefe, in press). Earlier cytological work on Little River hybrid parthenogens in the Ochlockonee River drainage indicate that they are triploids ( Figure 1) ( Dougherty 1982). Possession of C. limum mtDNA indicates that C. limum from the Ogeechee River in Georgia was the maternal ancestor of these clones (Johnson and Bragg, in press). Allozyme staining intensity suggests that these triploids have one copy of the C. geniculum genome and two copies of the C. limum genome. We argued that the most likely explanation for allotriploidy in these hybrids was insemination of a diploid parthenogenetic C. limum by a male C. geniculum because diploid parthenogenetic C. limum are common. Thus parthenogenesis arose before hybridization and genome addition results in allotriploidy. Mitochondrial DNA and allozyme evidence suggest a different origin for triploid hybrids in the upper headwaters of Holmes Creek and Wright’s Creek ( Figure 1). The maternal ancestor of these hybrid parthenogens was C. limum from the Santa Fe River in northern peninsular Florida ( Figure 1). Individuals with three alleles occur at one enzyme locus, and they appear to have two copies of the C. geniculum genome and one copy of the C. limum

genome. Because they carry C. limum mtDNA, they could not have arisen from diploid C. geniculum parthenogens. This supports the classical model for the origin of allotriploidy: a diploid parthenogen arose by hybridization between a sexual C. limum female and a male C. geniculum, and allotriploidy was achieved by backcrossing by a male C. geniculum. At present we have not found diploid hybrid parthenogens. In the present study we infer ploidy level of spontaneous and hybrid parthenogens through flow cytometry. While the possession of three allozymes seems an indisputable indicator of triploidy, a direct method by which to assess ploidy levels is needed to specifically address the following questions: First, does flow cytometry support the indirect evidence from allozymes of diploidy and triploidy in spontaneous and hybrid parthenogens, respectively? Second, is there variation in ploidy level within parthenogenetic populations? Concerning these questions, we hypothesize that if mating behavior does not decay rapidly in parthenogens, there is ample opportunity for elevation of ploidy level in hybrid and spontaneous parthenogens because sexuals occur within the same river drainages. If single ploidy levels occur in both groups of parthenogens, selection against elevated ploidy may be operating.

Materials and Methods We sampled eight populations of the various Campeloma species from southeastern U.S. rivers ( Figure 1, Table 1). From the Choctawhatchee River drainage we collected individuals from two populations ( HL and TM) of the Florida sexual

species, C. geniculum, and two populations ( HC and WC) of the hybrid parthenogen, C. parthenum. We collected parthenogenetic C. limum from the Alapaha River (AL) and Brier Creek ( BC) in the Savannah River drainage. We sampled sexual C. limum from the Ogeechee River ( HR) and Lower Three Runs Creek ( TR) in the Savannah River drainage. We isolated snail cells from digestive gland by grinding in a freezing solution (250 mM sucrose, 40 mM trisodium citrate dihydrate, and 5% dimethylsulfoxide; Vindelov et al. 1982a). Other tissues contained mucus and other materials that interfered with the analysis. We used two internal standards to determine the genome size of Campeloma individuals: chick (2.5 pg/nucleus; Rasch et al. 1982) or trout red blood cells (5.45 pg/nucleus; Dawley 1992). Standards were also stored in the solution described above. We used a method developed by Vindelov et al. (1982b) with modification in which snail cells plus standard cells were lysed and their nuclei stained in 75 mM propidium iodide with 3.4 mM trisodium citrate, containing 0.1% Nonidet P-40 and 2500–5000 U RNase A (1 g/L). The snail plus standard cells were then frozen at 2808C overnight to improve intercalation of propidium iodide into the DNA. Ten thousand or more cells (snail plus standard) were measured in each sample using a FACScan flow cytometer ( Becton-Dickinson). All coefficients of variation were less than 5%, usually close to 2%. The average DNA content (picograms) per cell was calculated for each snail sample using the following formula: mean fluorescent intensity of snail DNA/mean fluorescent intensity of reference DNA 3 standard pg/nucleus 5 snail pg/nucleus. The haploid genome size was calculated by dividing the digestive gland genome size by two. Because our concern is the average DNA content of the two sexuals and two groups of parthenogens, we used a one-way analysis of variance (ANOVA) to test the null hypothesis that DNA content did not differ among these four groups. Data were log-transformed to equalize variances among groups. We used the GT2 pairwise comparison of means because there were unequal sample sizes among groups ( Day and Quinn 1989).

Results Table 1 presents the average DNA content for each individual in the various Campeloma. The average (61 SE) DNA content

Figure 2. Mean (695% confidence interval) DNA content in sexual C. geniculum, hybrid parthenogenetic C. parthenum, sexual C. limum, and parthenogenetic C. limum. Shared letters represent groups that are not significantly different from each other based on GT2 post hoc comparison of means.

of the diploid parthenogens was 3.69 6 0.11 pg/nucleus, and the average (61 SE) DNA content of the triploid parthenogens was 5.08 6 0.09 pg/nucleus. We rejected the null hypothesis that average DNA content did not differ among the four groups (F 5 74.86, df 5 3, 38, p , .0001). The DNA content of hybrid parthenogens was significantly higher (approximately 1.5 times higher) than sexuals and spontaneous parthenogens ( Figure 2). Although there is some variation in DNA content among C. parthenum individuals, the average DNA content per nucleus was 5.08 pg, close to the calculated 5.29 pg/nucleus expected for an individual with two haploid C. geniculum genomes and one haploid C. limum genome. No DNA values of individuals of C. parthenum are near the expected diploid (3.57 pg/nucleus) or tetraploid values (7.14 pg/nucleus) ( Table 1). Parthenogenetic and sexual C. limum have nearly identical mean DNA content, which is consistent with diploidy in spontaneous parthenogens ( Table 1).

Discussion The flow cytometric data clearly confirm triploidy in the hybrid parthenogen C. parthenum and diploidy in spontaneous parthenogenetic C. limum. In conjunction with the allozyme and mtDNA data, flow cytometry confirms that C. parthenum are allotriploids that originated by the classical mechanism of genome addition: triploidy evolved from a C. limum 3 C. geniculum diploid hybrid through genome addition by a male C. geniculum. Triploid

Campeloma parthenogens do not arise through a single event in which a diploid gamete that arises through defective meiosis fuses with a haploid gamete, as hypothesized by Saura et al. (1993). The parthenogenetic C. limum that we examined arose spontaneously through diploidy. There is no indication that diploid parthenogenetic C. limum arise from triploids by metaphase plate disjunction (Saura et al. 1993). What is perhaps more interesting is why higher ploidy levels do not occur within parthenogenetic C. limum and C. parthenum. Although parthenogenetic C. limum and C. parthenum occur in disturbed habitats, mating opportunities exist or existed in the past. There is mtDNA evidence that both parthenogens arose recently and dispersed long distances, probably during Pleistocene low sea stands (Johnson, in press; Johnson and Bragg, in press). During these dispersal events, opportunities for mating and thus ploidy elevation would be likely. Because sexuals exist in the same river drainages, we might expect higher ploidy levels in both parthenogens. In the upper headwaters of Holmes Creek and Wright’s Creek, male C. geniculum cooccur at low frequency (1–10%) with allotriploids (Johnson and Leefe, submitted). Two alternative hypotheses seem likely explanations for limited ploidy: decay of mating behavior in parthenogens or selection against higher ploidy. Although we have no direct evidence on mating propensity in Campeloma parthenogens, studies of mating behavior indicate that parthenogens mate with sexuals ( Darevsky 1966; Lynch 1984; Maslin 1971; White and Contreras 1979). Even in Drosophila where some parthenogenetic strains show reduced mating speed, other parthenogenetic strains show mating speeds comparable to sexual females (Carson et al. 1982). Clearly at least one diploid hybrid Campeloma mated with a C. geniculum male, although the time of this event is unknown. We believe that strong selection against higher ploidy levels is a better explanation for ploidy levels in parthenogenetic Campeloma. Given the absence of triploidy in C. limum parthenogens and tetraploidy in C. parthenum, we argue that there has been ongoing selection against individuals with these ploidy levels. We recognize that the selective pressures against ploidy elevation may be different on diploids than triploids. Lynch (1984) outlined numerous mechanisms that can destabilize parthenogens and lead to reduced fitness upon


backcrossing. Lynch pointed out that the cause of disjunct geographic distributions of parthenogens and sexuals could be the deleterious effects of hybridization between parthenogens and sexual relatives. One destabilizing mechanism is the simultaneous increase in ploidy level and potential return to sexuality. Lynch (1984) cites many examples in which matings between diploid parthenogens and a sexual relative produces sterile male offspring and female offspring with a high incidence of sterile eggs and limited parthenogenetic capacity. Sterility probably derives from the presence of fatal multivalents in autopolyploids. This mechanism may underlie the absence of polyploid parthenogenetic C. limum and the restriction of diploid parthenogens to marginal habitats. Parthenogenetic C. limum may only persist in habitats where backcrossing to sexual C. limum is absent. We plan to compare the fecundity and survivorship of diploid C. limum parthenogens and experimentally induced autotriploids. Tetraploidy is apparently absent in C. parthenum and is rare or unknown in many polyploid parthenogens (Suomalainen et al. 1987; Vrijenhoek et al. 1989). We hypothesize that triploidy may be the optimal ploidy level in subtropical Campeloma because increasing cell volume in tetraploids could result in higher nutrient requirements and longer mitotic cycles. If there is a positive correlation between genome size and cell size (Cavalier-Smith 1985), polyploid gigantism can occur. There are numerous cases of larger body size in polyploids compared to related diploids (Guo and Allen 1996; Levin 1983; Walsh and Zhang 1992). If allotetraploids have slower growth rates, allotriploids may outcompete them during colonization of marginal, resource-poor habitats. We can address this hypothesis by comparing the fitness of allotriploids and experimentally induced allotetraploids in resourcepoor environments. To understand the constraints on ploidy in Campeloma and other parthenogenetic organisms, we need experimental investigations into the ecological and evolutionary consequences of varying ploidy. From the Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148 (Johnson), and Department of Biology, Ursinus College, Collegeville, Pennsylvania ( Hopkins and Goddard). Address correspondence to Steven G. Johnson at the address above or e-mail: sgjohnso@ uno.edu. We thank R. Dawley and R.J. Schultz for discussion about these ideas, and the National Science Foundation ( DEB-9629287) for supporting this project. q 1999 The American Genetic Association


References Carson HL, Chang LS, and Lyttle TW, 1982. Decay of female sexual behavior under parthenogenesis. Science 218:68–70. Cavalier-Smith T, 1985. Cell volume and the evolution of eukaryotic genome size. In: The evolution of genome size (Cavalier-Smith T, ed). New York: John Wiley & Sons; 105–184.

Vindelov LV, Christensen IJ, and Nissen NI, 1982b. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3: 323–327. Vrijenhoek RC, 1974. Gene dosage in diploid and triploid unisexual fishes (Poeciliopsis, Poeciliidae). In: Isozymes. Vol 4, Genetics and evolution (Markert CL, ed). New York: Academic Press; 463–476.

Darevsky IS, 1966. Natural parthenogenesis in a polymorphic group of Caucasian rock lizards related to Lacerta saxicola Eversmann. J Ohio Herpetol Soc 5:115– 152.

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Dawley RM, 1992. Clonal hybrids of the common laboratory fish Fundulus heteroclitus. Proc Natl Acad Sci USA 89:2485–2488.

Walsh EJ and Zhang L, 1992. Polyploidy and body size variation in a natural population of the rotifer Euchlanis dilatata. J Evol Biol 5:345–353.

Day RW and Quinn GP, 1989. Comparisons of treatments after an analysis of variance in ecology. Ecol Monogr 59:433–463.

White MJD, 1973. Animal cytology and evolution, 3rd ed. Cambridge: Cambridge University Press.

Dougherty BJ, 1982. Comparative karyology of some sexual and parthenogenetic Campeloma (Mesogastropoda: Viviparidae) in the southeastern United States (Masters thesis). Tallahassee, FL: Florida State University. Goddard KA and Schultz RJ, 1993. Aclonal reproduction by polyploid members of the clonal hybrid species Phoxinus eos-neogaeus (Cyprinidae). Copeia 1993:650– 660. Guo X and Allen SK, 1996. Sex determination and polyploid gigantism in the dwarf surfclam (Mulinia lateralis Say). Genetics 138:1199–1206.

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RAPD Variation in a Norway Spruce Seedlot: Consequences of Somatic Embryogenesis E. Passerieux, S. Baud, H. Dulieu, and M. Paˆques Norway spruce (Picea abies L. Karst) somatic embryos can be obtained from about 25% of the seedlings from a provenance. Random amplified polymorphic DNAs (RAPDs) were used to investigate if plant production by somatic embryogenesis significantly eroded the genetic diversity of a provenance as a consequence of selection. A sample of 124 seedlings from a natural Norway spruce population, split into two subpopulations, that is, embryogenic (E; n 5 31) and nonembryogenic (NE; n 5 93) individuals, generated 210 RAPD fragments, each assumed to correspond to a locus. Assuming random mating under Hardy–Weinberg equilibrium, allele frequencies and differentiation indexes were estimated. The mean differentiation index over all loci was included within the distribution of a hundred differentiation indexes calculated after 100 simulations by random sampling 31 and 93 individuals. Moreover, the distributions of allele frequencies of the amplified alleles over all loci were similar between the E and NE subpopulations, and the distribu-

tion of the E individuals in clusters based upon genetic similarity was at random. Thus it was concluded that the selective effects of the embryogenesis process in the embryogenic subpopulation were not significant and that the erosion of the genetic diversity is mainly determined by bottleneck effects. Since somatic embryogenesis was first demonstrated in Norway spruce (Picea abies L. Karst) by Hakman et al. (1985), this technique has proved to be the most powerful method for the vegetative multiplication of conifers (Smith et al. 1994). Indeed, thousands of somatic embryos can be produced per liter of culture medium. Somatic embryogenesis is considered to be an effective system that can contribute to reducing the duration of breeding programs and accelerate reforestation with progenies selected for high wood quality and production (Cheliak and Rogers 1990). Currently only 5–25% of the seeds of a Norway spruce seedlot have been cloned by somatic embryogenesis (Ruaud et al. 1992). If the somatic embryogenesis response depends on genotypes, as shown in some annual plants (such as cotton; Trolinder and Xhixian 1989) and in forest trees [such as white spruce (P. glauca Moench Voss)], especially during the initiation of embryogenic tissue (Park et al. 1993, 1994), and if there is a genetic association between somatic embryogenesis (SE) and other traits, SE could result in selection effects. Therefore it seems important to investigate if the increased use of somatic embryogenesis could either significantly erode the genetic diversity of conifers by selecting genes involved in embryogenesis or linked to them or could simply result from subsampling (random genetic drift). The integration of somatic embryogenesis into conifer breeding and reforestation programs requires evaluation of the risk of reduced genetic diversity (Adams et al. 1994; Attree and Fowke 1993). Genetic diversity is assumed to be necessary to maintain the ability of the population to evolve in response to environmental changes or pest and pathogen attacks. Fortunately most woody species have high levels of genetic diversity ( Hamrick et al. 1992; Mitton 1983; Tigerstedt 1973). Nevertheless, the time required for recovery is very long following a loss of genetic diversity in long-lived, long-generation organisms like trees (Mosseler et al. 1992). Therefore it is important to maintain a sufficiently large genetic base ( Baradat et al. 1995).

The development of random amplified polymorphic DNA (RAPD) markers by Williams et al. (1990) and Welsh and McClelland (1990), which tend to be neutral and anonymous, has provided a powerful tool for the investigation of genetic variation ( Boscherini et al. 1994; Huff et al. 1993; Liu and Furnier 1993; Lynch and Milligan 1994; Mosseler et al. 1992). Other authors used RAPD markers to test the eventual occurrence of somaclonal variations in plantlets regenerated from cell culture via somatic embryogenesis; they were observed only between clone variations ( Fourre´ et al. 1997, on P. abies; Isabel et al. 1993, on P. mariana). Especially in conifers, the large amount of DNA per haploid genome (30–40 3 109 bp; Miksche and Hotta 1973; Ohri and Khoshoo 1986) and extended regions of repeated DNA ( Neale and Williams 1991) are the main factors that have enhanced the development of the RAPD technique. The primary objective of this study was to evaluate possible genetic differentiation among the embryogenic ( E) and nonembryogenic ( NE) individuals from a sample representing a seedlot from a natural population of P. abies L. Karst. using allele frequencies derived from RAPD phenotypes. A second objective is to investigate the usefulness of RAPD markers to develop sampling strategies for the preservation of genetic diversity.

Materials and Methods Seeds and Embryogenic Ability Open-pollinated seeds were collected by Vilmorin, Inc., in 1995 from 20 randomly selected trees in P. abies stands in the ‘‘Chapois area’’ (Jura, France; altitude 640 m). A sample of mixed seeds were surface sterilized during 20 min in a solution of hydrogen peroxide and rinsed three times in sterile water; they were germinated in sterile Sorbarodst and cultured at 258C with a photoperiod of 16 h. After 14 days from sowing, cotyledons were excised from 369 seedlings and their embryogenic ability evaluated in vitro according to the protocol described by Ruaud et al. (1992): the cotyledons were cultured in a medium similar to that of Gupta and Durzan (1986) but containing 21.5 mM naphthylacetic acid ( NAA) and 0.45 mM benzylaminopurine ( BAP). After 1 week the explants were transferred on a similar medium but containing 5 mM 2,4-dichlorophenoxyacetic acid, replacing NAA, and 2 mM BAP and 2 mM kinetin. The embryogenic calluses can be detected within 4 weeks. Seventy-two

individuals (19.5%) produced embryogenic structures on the cotyledon’s surface. The original 369 seedlings, each with two remaining cotyledons, were acclimatized and grown in a nursery. For molecular studies, 124 individuals were randomly sampled out of the 369, among which 31 were determined to be embryogenic ( E) and 93 nonembryogenic ( NE). DNA Extraction and Amplification Needles were sampled from each of the 124 individuals at the same stage of development after being grown 5 months in a nursery. Genomic DNA was isolated from needles (about 50 mg) according to the CTAB method described by Rogers and Bendich (1985). DNA amplifications were performed by using 18 preselected arbitrary 10-mer primers ( Bioprobe, Illkirch, Fr). Each RAPD reaction was performed in a volume of 25 ml containing 13 reaction buffer, 1.8 mM MgCl2, 200 mM of each dNTP (Gibco BRL-Life Technologies, Inc., Rockville, MD), 0.4 mM of primer, 20 ng of genomic DNA, and 0.75 U Taq polymerase (Gibco BRL). A control reaction containing all PCR components except the DNA template was included with each set of reactions to verify the lack of contaminating DNA. Amplifications were performed in a PTC 100 thermal cycler (MJ Research, Inc., Watertown, MA) programmed for 45 cycles of 1 min at 948C, 1 min at 378C, and 2 min at 728C. The last cycle was followed by a final extension of 7 min at 728C. Amplified products were electrophoresed in a 1.8% agarose gel, stained with ethidium bromide, and visualized under ultraviolet ( UV) light (254 nm). Data Analysis RAPD products were scored as presence (1) or absence (0) at each relative migration (Rm) on the gels using ImageMastery Vds software (Pharmacia, Inc., Uppsala, Sweden). This software was used in automatic mode (sensitivity 8) and amplified products with a relative intensity inferior to 0.05 were not scored. In these conditions only the main fragments, or clear fragments of each pattern, were considered. In order to reduce the possibility of classifying an amplification artifact as a polymorphism, less intense fragments were not included. Indeed, minor fragments and amplification products less than 500 bp seemed to possess the lowest levels of reproducibility, as discussed by Heun and Helentjaris (1993).


Table 1. Selected decamer oligonucleotides (primers) and numbers of loci detected

Primer

Sequence (59–39)

Number of RAPD loci scored

OPA 01 OPA 05 OPA 06 OPA 11 OPA 12 OPB 18 OPE 18 OPE 19 OPF 16 OPF 19 OPF 20 OPG 11 OPG 12 OPG 13 OPG 15 OPJ 01 OPJ 12 OPJ 17 Total

CAGGCCCTTC AGGGGTCTTG GGTCCCTGAC CAATCGCCGT TCGGCGATAG CCACAGCAGT GGACTGCAGA ACGGCGTATG GGAGTACTGG CCTCTAGACC GGTCTAGAGG TGCCCGTCGT CAGCTCACGA CTCTCCGCCA ACTGGGACTC CCCGGCATAA GTCCCGTGGT ACGCCAGTTC 18

14 11 11 7 9 11 13 12 15 12 13 12 11 12 11 11 11 14 210

Number of polymorphic RAPD loci 14 11 11 6 8 10 13 12 15 12 13 12 11 12 11 10 11 14 206

Table 2. Loci significantly more or less frequent in the embryogenic than in the nonembryogenic subsample

Primer OPA 01 OPA 05 OPA 06 OPA 11 OPF 16 OPF 19 OPF 20 OPG 11 OPG 15 OPJ01 OPJ12

Embryogenic subsample (n 5 31)

Nonembryogenic subsample (n 5 93)

Allelic frequencies pc

Fragment length ( bp) x2 a

FST

(1)

(2)

(1)

(2)

E

NE

1900 2000 2500 800 1635 1100 2400 1300 800 750 505 750 1100 1017 2035 1400 1017

0.016 0.012 0.024 0.017 0.013 0.012 0.010 0.015 0.065 0.028 0.104 0.014 0.011 0.015 0.037 0.0229 0.0140

13 3 16 8 4 11 1 14 30 26 23 8 12 5 28 9 13

18 28 15 23 27 20 30 17 1 5 8 23 19 26 3 22 18

59 28 23 7 2 52 18 61 76 59 88 8 19 37 67 7 58

34 65 70 86 91 41 75 32 17 34 5 85 74 56 26 86 35

0.238 0.050 0.304 0.139 0.067 0.197 0.016 0.260 0.820 0.598 0.492 0.139 0.217 0.084 0.689 0.158 0.238

0.395 0.164 0.132 0.038 0.011 0.336 0.102 0.413 0.572 0.395 0.768 0.044 0.108 0.224 0.471 0.038 0.387

4.42 5.18 7.79 7.31 5.84 3.88 4.66 4.06 4.25 4.50 10.3 6.12 4.14 5.81 4.34 9.57 3.97

b

b

b

b

Significance of chi-square tests at p # .05 and 1 degree of freedom. Presence (1) or absence (2) of the amplified product in the embryogenic and nonembryogenic subpopulation. c Allele frequency of the amplifiable allele in the embryogenic ( E) and nonembryogenic ( NE) subpopulations.

a

b

Genetic Analysis Each RAPD product at a given Rm, for a given primer, was assumed to signal a locus with one amplified allele (1) and one nonamplified allele (2). To assess random mating in the population and assume Hardy–Weinberg equilibrium, a chi-square analysis was used to test the random association between amplified products in all pair combinations (p # .05). Chi-square 2 3 2 tests were performed between each marker (1 or 2) and E or NE phenotype. Using the presence/absence matrix (individuals 3 loci), similarity coefficients were calculated between all the 124 individuals according to Jaccard (1908). A cluster analysis was performed based on these coefficients using the WARD method of the SAS statistical package (SAS 1987). Observed heterozygosity could not be determined for RAPD data because heterozygotes could not be distinguished from dominant homozygotes. Expected heterozygosity, however, could be estimated ( Liu and Furnier 1993; Lynch and Milligan 1994) if Hardy–Weinberg equilibrium is assumed. The frequency of a recessive allele has been estimated as the square root of the frequency of individuals lacking the band (the frequency of recessive homozygous genotypes) and the heterozygote frequencies were calculated for the whole sample (HT) and for the subsamples (HS). These parameters allowed estimation of the fixation index (F statistics, FST), according to Wright (1965, 1969), using Lynch and Milligan’s method (1994), which excludes monomorphic fragments


and where less biased estimates are obtained ( Isabel et al. 1995). Computer Simulation The significance of the global FST value, calculated over all the loci between the two subpopulations—E (n 5 31) and NE (n 5 93)—was tested by performing 100 simulations by random sampling 31 individuals of the 124. One hundred simulated global FST values were calculated and the FST obtained with E/NE was compared with the distribution of the 100 simulated FST.

Results RAPD Results and Genetic Differentiation A total of 210 RAPD markers from 18 selected primers were scored and analyzed. The average number of amplification products scored per primer was 11.6 6 0.5 and their sizes ranged from 500 to 2500 bp. The mean number of amplified fragments per primer per individual was 4.6. Of these 210 markers, only 4 were monomorphic across the total sample and therefore 206 (98%) of these bands were polymorphic ( Table 1). The 2 3 2 chi-square test, which was used to test random association for each of the loci pairs in all combinations, indicated that only 6% of the paired loci combinations showed evidence of significant association (p # .05). Hence the majority (94%) of the loci were randomly associated, suggesting that under random mating the as-

sumption of Hardy–Weinberg equilibrium made for estimating expected heterozygosities from RAPD data is reasonable. Large outcrossing (91%) was demonstrated by Xie and Knowles (1994) in P. abies, and Hardy–Weinberg equilibrium was observed, in spite of some slight excess of heterozygotes, in P. mariana by Isabel et al. (1995). The 2 3 2 chi-square tests performed for each marker (1 or 2) and the E versus NE phenotypic classification showed that, over 206 markers, 189 were not distributed differently (p # .05) in the two subpopulations E and NE. Seventeen markers were detected as significantly more or less frequent in the E versus NE subpopulation ( Table 2). However, these 17 markers were not associated with each other. Moreover, the E individuals were distributed homogeneously within the 11 groups of individuals determined by the cluster analysis. These three results favor the hypothesis that, if genotypes favoring embryogenesis exist, they seem to be produced by random combinations of unlinked genes. The allele frequency distribution of the (1) and (2) alleles for all 206 loci ( Figure 2) in the two subpopulations E and NE were not significantly different (df 5 9, x2 5 10.23) from the whole population. The 208 estimated FST values for each polymorphic locus ranged from 0.00 to 0.180, with an average value over all loci of 0.010 6 0.0019 ( Table 3). The distribution of the FST values ( Figure 1) over all the loci appeared nonsymmetrical. This was partially due to the 0.00–0.05 class corresponding

Figure 1. Distribution of allele frequencies of the amplified allele in the embryogenic sub-sample ( E), the nonembryogenic subsample ( NE), and the entire population (total).

to a low genetic differentiation, which is greatly represented (96.2%) and also to 1.9% of the markers with high FST values. Computer Simulations Average FST values obtained in the 100 simulations ranged from 0.005 to 0.013, with an overall average value of 0.008 ( Figure 3). In 97 of 100 cases, genetic differentiation (FST) did not differ significantly from values observed when subdivision was applied on embryogenic criteria (p # .05). These results indicate that the subdivision of the Chapois population into two subpopulations—E (n 5 31) and NE (n 5 93)—had no significant effect on the FST value. Moreover, the proportions of loci affected by the distribution into two sub-

Table 3. Estimates of FST values among embryogenic and nonembryogenic subpopulations a

Primer OPA 01 OPA 05 OPA 06 OPA 11 OPA 12 OPB 18 OPE 18 OPE 19 OPF 16 OPF 19 OPF 20 OPG 11 OPG 12 OPG 13 OPG 15 OPJ 01 OPJ 12 OPJ 17 Mean valuesb SEMc

Number of loci HT 14 11 11 6 8 10 13 12 15 12 13 12 11 12 11 10 11 14 206

0.293 0.210 0.198 0.232 0.189 0.212 0.265 0.288 0.263 0.248 0.275 0.270 0.313 0.312 0.327 0.253 0.310 0.228 0.267 (0.011)

HS

FST total loci

0.290 0.203 0.188 0.230 0.188 0.211 0.262 0.287 0.261 0.245 0.269 0.268 0.312 0.310 0.325 0.251 0.308 0.221 0.264 (0.011)

0.007 0.020 0.034 0.006 0.002 0.003 0.010 0.003 0.005 0.009 0.014 0.005 0.003 0.005 0.006 0.005 0.005 0.025 0.010 (0.002)

Means per primer and mean over all loci. The mean values are estimated by averaging the values obtained for each polymorphic locus, not by averaging the mean values obtained by primer. c Standard error of the mean.

Figure 2. Distribution of the FST values over the loci in the Chapois Norway spruce population. The degree of genetic differentiation is characterized by the FST values (Wright 1965): 0–0.05 5 low differentiation (96.2% of the loci); .0.05–0.15 5 moderate differentiation (1.9% of the loci); .0.15–0.25 5 strong differentiation (1.9% of the loci).

populations were about 8%, whether subdivision was based on embryogenic potential or on simulated random subdivisions. These results confirm that total genetic differentiation between the two subpopulations (31 and 93 individuals) subdivided on the embryogenic criteria can be considered as nonsignificant. Estimation of Effective Individual Numbers for Production of Somaclones Determining a minimum number of individuals that can maintain sufficient genetic diversity and might therefore be suitable for inclusion in a production of somaclones is useful for the management of genetic variability. Using the lower allele frequencies of amplified fragments, p 5 .05 or 0.1, the minimum sample N to be

multiplied to maintain the observed genetic diversity at a 99% level was estimated by N $ ln0.01/2 ln(1 2 p). At the P.01 level, N should be greater than 45 and 22, respectively.

Discussion In this study we investigated the potential erosion of genetic polymorphism when a multiclonal population was constituted from the embryogenic individuals, given that only 20% of seedlings from a seedlot collected in a natural population (or provenance) can be propagated by somatic embryogenesis. First, frequencies of the (1) and (2) alleles estimated at each locus showed that the frequency of the (1) allele was almost

a

b

Figure 3. Distribution of the 100 FST values obtained after randomly splitting the population by drawing lots of 31 individuals out of 124 and the FST value obtained by splitting the population into E and NE phenotypes.


always the lowest, as shown by the dissymmetrical distribution of the allele frequencies of the (1) alleles into classes where the 0.0–0.1 class was overrepresented. It can then be supposed that the actual polymorphism is largely higher than that estimated by the heterozygosity under a biallelic model, according to which the nonamplified genotype was assumed to be homozygous for a single null allele. The second aspect of this work was to elucidate the genetic consequences of splitting the population into two subpopulations based on in vitro embryogenic potential. The two subpopulations (31 embryogenic and 93 nonembryogenic individuals) appeared not to be genetically differentiated, as shown both by distributions of frequencies of the (1) allele and by the differentiation index (FST) over all the loci. Moreover, it seems obvious that splitting the sample into E and NE classes was similar to the 100 random samplings performed, for the same 208 loci. Therefore the question of the risk of losing genes after the embryogenesis process can be reduced to a genetic drift or ‘‘bottleneck’’ effect, if the sample size constituting the new multiclonal population is too small. However, this implies that, with an embryogenesis rate of about 20% for Norway spruce, at least 110 individuals must be tested in vitro in order to obtain at least 22 embryogenic clones. The results obtained and the interpretations presented are in accordance with those of several authors who studied genetic polymorphism and/or genetic drift risk within forest species by isozyme or molecular markers (Guries and Ledig 1982; Ledig and Conkle 1983; Yeh and Layton 1979). Moreover, the estimates of heterozygosity are not significantly different (H 5 0.268 6 0.011, this article) from that calculated by Hamrick et al. (1981; H 5 0.255), Lundkvist (1979; H 5 0.307), Gianini et al. (1991; H 5 0.318), Gomory (1992; H 5 0.186), and Goncharenko et al. (1995; H 5 0.185). It can then be concluded that the construction of a multiclonal population after vegetative multiplication of individuals by somatic embryogenesis does not present a significant risk of reducing genetic diversity, provided that the number of clones to be multiplied should be established as a function of the genetic richness of the population to be maintained. If the same procedure is maintained over several sexual generations, without identification of the clones of the first generation, with the same number of


presumed clones in the second generation, it would unavoidably lead to a ‘‘bottleneck’’ effect, because the effective number of individuals would be reduced to four to five for the next generation. It is thus advisable that the populations used as provenances should be maintained in the future, both for natural seed production and embryo production. In this work it was also shown that RAPD markers, in spite of their dominant nature, allowed for a rapid evaluation of polymorphism as well as defining principles for a practice of biotechnologies compatible with the preservation of genetic richness. From AFOCEL (Association Fore ˆt-Cellulose), Ressources du futur (Passerieux, Pa ˆques) and Laboratoire de Biome´trie ( Baud), Domaine de l’Etançon, F-77370 Nangis, France, and Universite´ de Bourgogne, Laboratoire de Geńe´tique Ve´ge´tale, Dijon, France ( Dulieu). Address correspondence to M. Pa ˆques at the address above or e-mail: [email protected]. This research was supported by A.N.R.T. grant C.I.F.R.E. n8 627/95 (to E.P.).

genetic variation in trees: influence of life history characteristics. In: Isozymes of North American trees and forest insects (Conkle MT, ed). Forest Service General Technical Report PSW-48. Washington, DC: U.S. Department of Agriculture; 35–41. Hamrick JL, Godt MJW, and Sherman-Broyles SL, 1992. Factors influencing levels of genetic diversity in woody plant species. New For 6:95–124. Heun M and Helentjaris T, 1993. Inheritance of RAPDs in F1 hybrids of corns. Theor Appl Genet 85:961–968. Huff DR, Peakall R, and Smouse PE, 1993. RAPD variation within and among natural populations of outcrossing buffalograss [Buchoe¨ dactyloides ( Nutt.) Engelm]. Theor Appl Genet 86:927–934. Isabel N, Tremblay L, Michaud M, Tremblay FM, and Bousquet J, 1993. RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.B. Theor Appl Genet 86:81–87. Isabel N, Beaulieu J, and Bousquet J, 1995. Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce. Proc Natl Acad Sci USA 92:6369–6373. Jaccard P, 1908. Nouvelles recherches sur la distribution florale. Bull Soc Vaud Sci Nat 44:223–270.

q 1999 The American Genetic Association

Ledig FT, and Conkle MT, 1983. Genetic diversity and genetic structure in a narrow endemic, Torrey pine (Pinus torreyana Parry ex Carr.). Evolution 37:79–85.

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