phlox divaricatal - molecular biology and evolution

Ribosomal DNA Variation in the Native Plant Phlox divaricatal

Barbara A. Schaal, * Wesley J. Leverich, =f and Jorge Nieto-Sotelo* *Department o f Biology, Washington University; and TDepartment of Biology, Saint Louis University

Variation of ribosomal DNA (rDNA) was analyzed within and among individuals, populations, and subspecies of Phlox divaricata. Phlox divaricata is a widespread woodland perennial of the midwestern United States. It consists of a large eastern subspecies, ssp. divaricata, and a more restricted western subspecies, ssp. Zaphami. As in other plant species, individual plants may contain as many as five types of rDNA length variants. rDNA repeat-type frequencies were determined within eight populations, four of each subspecies. There is clear differentiation among populations of a subspecies, and there also are differences between the two subspecies in both the number and types of rDNA repeats. Subspecies divaricata has more variation than does ssp. Zaphami, both in the number of rDNA repeat types (nine vs. eight, respectively) and in the average number of repeat types within an individual (2.47 vs. 1.52, respectively). rDNA diversity was analyzed using the Shannon diversity index. Of the total diversity in the species, 6 1% is found among individuals of a population, 2 1% is found within subspecies among populations, and 18% is found between the two subspecies. On the basis of morphological criteria, ssp. Zamphami is considered derived from ssp. divaricata. The rDNA data are not completely consistent with this interpretation. The diversity of rDNA found within ssp. Zaphami is less than that in ssp. divaricata, as would be expected for a derived taxon. On the other hand, laphami has three repeat types not present in divaricata.

Introduction

The recent application of molecular techniques to the study of evolution and population biology has revealed a host of processes that occur at the molecular level and strongly influence the genetics of populations; for example, the occurrence of transposition and its effect on hybrid dysgenesis has potentially far-reaching effects on gene dispersal and speciation. Consideration of molecular processes has led to potentially different modes of evolution, such as selfish DNA (Doolittle 1982) or concerted evolution. Studies of molecular evolution in multigene families have frequently observed sequence homogenization within species; for example, nucleotide sequences in rRNA-coding DNA (rDNA) and histone-gene families of Drosophila may be highly conserved within a species but show major discontinuities between species (Coen et al. 1982). Similar sequence homogenization is observed in the globin genes of primates (Zimmer et al. 1980). The process of homogenization has been termed molecular drive and results from the process of unequal crossover and/or gene conversion (Dover 1982). rDNA of many species appears to have undergone similar homogenization of

1. Key words: ribosomal DNA, restriction-fragment-length polymorphism (RFLP), population differentiation, native plant species.

Address for correspondence and reprints: Dr. Barbara Schaal, Department of Biology, Washington University, St. Louis, Missouri 63 130.

Mol. Biol. Evol. 4(6):611-621. 1987. 0 1987 by The University of Chicago. All rights reserved. 0737-4038/87/0406-0005$02.00

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variation. Length heterogeneity is common within individual plants and suggests that homogenization is a gradual process.

The transcribed sequences of rDNA that code for the 18s and 28s ribosomal subunit, plus an intervening spacer, appear to be evolutionarily conservative, although even in this situation intraspecific variation is observed (Sytsma and Schaal 1985). The large nontranscribed spacer region that separates the tandem repeats of rDNA is, on the other hand, highly variable within species. Both restriction-site variation and, more commonly, length variation occur (Appels and Dvorak 1982). In a study of Hordeum vulgare (barley) populations Saghai-Maroof et al. ( 1984) have observed high levels of rDNA variation within single plants, and in Vicia faba (broad bean) as many as 20 length variants per individual have been detected (Rogers et al. 1986). CZematis fremontii (leather flower) also contains spacer-length heterogeneity, and in this case spatial differentiation for rDNA length variants occurs on a microgeographical scale within populations (Learn and Schaal 1987).

The purpose of the present study is to apportion the levels of rDNA variation within a native plant species, Phlox divaricata (blue phlox). The study addresses the following specific questions: ( 1) What are the levels of rDNA variation within populations? (2) Is there significant differentiation of rDNA among populations? (3) Are there discontinuities in rDNA variation between subspecies of P. divaricata?

Phlox divaricata is an herbaceous, diploid, perennial plant of widespread occurrence in eastern North America, from Minnesota to Texas on the west and from southern Quebec to Florida on the east. It is primarily a woodland species and is a conspicuous component of the spring flora of the eastern deciduous forest, forming populations ranging from a few to > 100 plants. The plants have both prostrate and erect stems; rooting at nodes results in clonal spread of individuals. Individuals are self-incompatible, and flowers are pollinated by bees and lepidopterans. The seed set per plant is 30-50 seeds. Seed dispersal is by explosive dehiscence of capsules. Phlox divaricata has two morphologically distinct and largely allopatric subspecies, ssp. divaricata in the eastern part of the species range and ssp. Zaphami in the western part of the species range. The subspecies are distinguished by flower petal shape, leaf shape, stature, and flowering time (Levin 1967).

Material and Methods

Plants were collected from eight populations of Phlox divaricata, four from each subspecies. Sample locations are listed in table 1. Individuals were collected several

Table 1 Collection Locations

Population (Location) Subspecies

(No. of Plants Analyzed)

CC (Creve Coeur, St. Louis) ................. . . . laphami (8) Ty (Tyson Research Center, St. Louis Co., MO.) . . . . laphami (9) WH (Wild Horse Creek, St. Louis Co., MO.) .... . . . . laphami (8) F (Freeburg, St. Clair Co., Ill.) ............... . . . . laphami (8) TC (Tell City, Perry Co., Ind.) ............... . . . . divaricata (6) PH (Pounds Hollow, Gallatin Co., Ind.) ....... . . . . divaricata ( 10) B (Bacon, Orange Co., Ind.) ................. . . . . divaricata (8) M (Mansfield, Parke Co., Ind.) ............... . . . . divaricata (8)

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Ribosomal DNA Variation in Phlox 613

meters apart to minimize the probability of repeated sampling of the same clonal genotype. Six or more plants from each population were analyzed from among plants that had been transplanted into the greenhouse.

Fresh leaf tissue weighing lo-20 g was harvested from greenhouse-grown plants. Leaves were washed, sterilized in a 1% Chlorox solution, blotted dry, and frozen im- mediately in liquid nitrogen. The frozen leaf material was ground with a mortar and pestle and then stored at -70 C. DNA was isolated in one of two ways. DNA from plants used for the mapping studies was isolated according to the methods of Rivin et al. (1982), which use a cesium chloride-ethidium bromide density-gradient cen- trifugation. All of the powdered plant material was used in these isolations and yielded 245-500 pg DNA/plant. DNA obtained by means of this procedure is predominantly nuclear DNA, but - lo-20% is chloroplast DNA. For the survey of population variation, DNA was isolated by means of a “mini-prep” procedure using ~0.5 g of powdered plant material. The procedure is a modification of that of Coen et al. (1982), which isolates DNA by means of phenol extraction. Yields of DNA were 3-12 pg, and DNA was both plastid and nuclear.

Nuclear rDNA was analyzed by probing genomic Southern blots. Genomic DNA was digested with one of a battery of restriction endonucleases. Restriction digestion were performed according to manufacturer’s specifications or according to the method of Maniatis et al. ( 1982). Digestions were done routinely for 16 h with 3 units enzyme/ yg DNA. After digestion, DNA fragments were separated on horizontal 0.8% agarose TEA (0.04 M Tris, 0.005 M disodium ethylenediaminetetraacetate, 0.001 M sodium acetate) gels for 14- 18 h at constant 50 mA. Size standards were bacteriophage lambda- digested with either Hind111 or EcoRI and HindIII. The separated DNA fragments were denatured in 1.5 M NaCl, 0.5 M NaOH for 1 h and then neutralized in 3 M NaCl, 0.5 M Tris, pH 7.2, for 1 h. The resulting single-stranded DNA fragments were transferred to a nitrocellulose filter (Southern 1975). The nitrocellulose filter was wetted with Hz0 and then 20 X SSC (3 M NaCl, 0.3 M sodium citrate). The filter was placed on the gel and covered with three layers of Whatman 3-mm paper that was wetted with Hz0 and 20 X SSC. The filters were covered with 2 inches of stacked paper towels and weighted down. Blotting was done overnight. After blotting, the fragments were baked on to the filter by placing the filter in a 65 C oven for 2 h.

rDNA was detected by hybridizing the nitrocellulose filter with a pBR325 plasmid that contained the cloned sequence for the complete nuclear rDNA repeat of the soybean Glycine max (provided by E. Zimmer). The probe sequences were nick-trans- lated with 35 p.Ci 32P dCTP in 20 ~1 nick-translation mixture (20 mM Tris-HCl, pH 7.5, 10 mM MgCl*, 50 pg bovine serum albumin [BSA]/ml, 10 pM each of dTTP/ dGTP/dATP, 5 ng DNase/ml, and 5 units DNA polymerase) for 2-3 h at 15 C. The nick-translation mixture was separated on a Sephadex G-100 column, and the blue- dextran fraction was collected and then denatured at 100 C for 10 min. Nitrocellulose filters were prehybridized for 30 min at 65 C in the hybridization buffer (5 X SSC, 1% Na sarkosyl, 1 X Denhardt’s solution [0.02% each BSA, Ficoll, and PVP], 25 mM potassium phosphate) and hybridized for 12-24 h at 65 C with the labeled probe ( 1 06- 5 X lo6 cpm). The filters were washed twice for 15 min in 2 X SSC, 0.2% sodium dodecyl sulfate (SDS), 1 X Denhardt’s and then rinsed twice in 2 X SSC, 0.1% SDS. The blots were then washed twice in the latter solution for 1 h at 37 C with agitation. Filters were rinsed to 0.2 X SSC and air dried. The filters were loaded into an X-ray cassette with intensifying screens. The X-ray film was exposed at -70 C.

The Phlox rDNA repeat was mapped by means of a series of single and double

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digests using EcoRI, EcoRV, BamHI, BgZII, KpnI, and XbaI. The map was oriented to the conserved XbaI site located 166 bp 3’ of the 5’ end of the 18s coding sequence (Eckenrode et al. 1985). After the rDNA map was established, plants were surveyed for variation in rDNA by means of separate digestions with each of the six restriction endonucleases.

Results

The six restriction endonucleases cut the rDNA repeat of Phlox divaricata at 15 separate sites (fig. 1). Many of these sites are known conserved sites, such as the XbaI site located at the beginning of the 18s subunit or the EcoRV site in the 5.8s rDNA between the 18s and 26s sequence (Eckenrode et al. 1985). Phlox divaricata has little, if any, restriction-site polymorphism. No restriction-site variation was observed among the 65 plants analyzed in the present survey. However, the species is highly variable for rDNA length. Large amounts of length variation in the rDNA repeat are observed both within and among individual plants. The length of the P. divaricata rDNA repeat unit is 8.0-12.5 kb. Figure 2 shows an autoradiograph of rDNA from nine plants of a single population that contains different length variants. Single plants can contain as many as five length variants. The length variation is due to a series of insertions and/or deletions that map to the intervening nontranscribed spacer region (fig. 1). A total of six insertions/deletions were detected in the present survey. Table 2 shows the various combinations of insertions/deletions that lead to the specific length variants; for example, variant V-2 has the basic repeat length of 9.4 kb, whereas the 9.7-kb variant V-3 has that length plus the 0.3-kb insertion labeled E. Some of the different variants are indistinguishable on the basis of total repeat length; for example, length variants V-4 and V-5 are both 10.2 kb. These length variants result from the 9.4-kb

I

10 Kb

FIG. 1 .-Map of Phlox divaricuta rDNA. A-E denote locations of different insertions/deletions.

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Table 3 Frequency of Multiple Repeat Types in Individual Plants

FREQUENCY OF MULTIPLE REPEAT TYPES (No. of Plants)

No. OF VARIANTS All Populations Ssp. laphami Ssp. divaricata

1 . . . . . . . . . . . . . 0.27 (18) 0.55 (18) 0.00 2 3 :::::::::::::

0.52 (34) 0.39 (13) 0.66 (21) 0.14 (9) 0.06 (2) 0.22 (7)

4 . . . . . . . . . . . . . 0.05 (3) 0.00 0.09 (3) 5 . . . . . . . . . . . . . 0.02 (1) 0.00 0.03 (1)

Total . . . . . 65 33 32

variant V-5. In contrast, the M population of ssp. divaricata was polymorphic for seven repeat types, and each plant on average contained three different rDNA variants. The mean number of repeats per individual within populations of ssp. Zaphami was 1.2-2.0. The frequency of specific variants also differs significantly among the populations of a subspecies. In ssp. Zaphami, for example, the frequency of variant V-6 is 0.0-0.55. The frequencies of specific variants also differ among populations of ssp. divaricata. The frequency of the V-2 variant within populations is 0%-50%.

In addition to differentiation among the populations of a subspecies, significant differentiation for rDNA repeat types occurs between the two subspecies. First, the mean number of rDNA variants per individual in ssp. Zaphami (1.52) is significantly different from that in ssp. divaricata (2.47) (t = 3.42, P < 0.001) (table 4). The distribution of repeat types between the subspecies is also different. Variants V-l, V-2, V- 10, and V- 11 are confined exclusively to ssp. divaricata, whereas variants V-3, V-8, and V-9 are found only in ssp. Zaphami. Subspecies divaricata has one more variant than does Zaphami (nine vs. eight variants). The frequency of specific rDNA repeat types varies significantly among subspecies (x2 = 19.8, P < 0.05). Subspecies Zaphami has two variants in relatively high frequency, V-4 (38%) and V-6 (24%). Subspecies divaricata has a more even distribution of variants, with six variants having frequencies of lo%-20%.

The apportionment of rDNA diversity was analyzed by means of a Shannon information measure, H (table 5). The average diversity of populations was 1.16, which represents 6 1% of the total species diversity (H&Hsmi,; table 5). Differences among the populations of a subspecies ([H, - Hwp]/Hspies) account for 2 1% of the total diversity, whereas differences between subspecies ([Hw, - H,J/Hspeci& account for 18% of the total diversity. Subspecies divaricata has a greater diversity (2.05) than does ssp. Zaphami (1.69). Thus, most of the diversity within the species for rDNA variation results from differences among (and within) individuals of a population. Substantial differences occur among the populations of a subspecies, whereas differences among subspecies add the least amount to the total rDNA diversity of the species.

Discussion

Levels of variation in rDNA of Phlox divaricata are high. Compared with animal species, plant species have relatively many copies of rDNA; rDNA copy number varies from -3,000 to > 10,000 copies per genome in plants, whereas mammals and insects usually have ~500 copies (Long and Dawid 1980). The occurrence of multiple copies per genome affords the possibility of polymorphism within individuals, and, indeed,

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Table 5 H Values by Group

Grouping Ha

Ssp. laphami: H :

?c ................. T .................. WH ................ F ..................

H, laphami ........... Ssp. divaricata: ...........

H POP:

TC ................. PH ................. B .................. M.. ................

H, divaricata .......... Overall R,i ....... Overall R, ........ Overall Htim ......

1.23 1.12 1.16 0.63 1.69

1.51 1.09 1.68 1.82 2.05 1.29 1.73 2.12

‘H = pilOg,pi, where pi is the frequency of the ith variant (Lewontin 1972).

this is observed for many plant species as well as for Drosophila (Williams et al. 1985). Individual P. divaricata plants contain as many as five different types of rDNA repeats. Similar variation within individuals is observed for the plants barley (Saghai-Maroof et al. 1984), wheat (Appels and Dvorak 1982), broad bean (Yakura et al. 1984), and CZematis fremontii (Learn and Schaal 1987). In all of these species the length of the rDNA repeat is variable and maps to the nontranscribed spacer region. This length variation is due to a series of insertions/deletions of 130 bp in wheat and of 325 bp in broad bean, occurring in the nontranscribed spacer region (Appels and Dvorak 1982; Yakura et al. 1984). The inheritance of such rDNA length variation has been analyzed in barley and is Mendelian in nature (Saghai-Maroof et al. 1984). Such length variation appears to be common in plants, although other studies indicate that such length variation is not ubiquitous; for example, studies of rDNA variation in the genus Lisianthius revealed very little repeat-length polymorphism within a species (Sytsma and Schaal 1985).

No restriction-site polymorphism was detected within P. divaricata. Restriction- site variation within rDNA has, however, been observed in other plant species; for example, in the goldenrod Solidago altissima polymorphic restriction sites are contained both in the coding sequences of the 28s gene and in the nontranscribed spacer region (Schaal 1985). Plants can show restriction-site variation due to methylation (Delseny et al. 1984). The pattern of rDNA variation in P. divaricata, which has high levels of length variation and lack of restriction-site polymorphisms, is similar to the variation found in natural populations of C. fremontii. In most of the plant species that exhibit such length variation, the mean number of variants per individual is between two and three and ranges within populations from one variant to as many as five in C. fremontii (Learn and Schaal 1987). Phlox divaricata falls within this range, with a mean number of repeat types per plant for the species of 1.98.

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Ribosomal DNA Variation in PhIox 6 19

With regard to rDNA type P. divaricata shows clear differentiation among populations. First, the mean number of repeat types per plant varies significantly among populations, with populations being highly variable in the number of variants contained per individual. Populations of P. divaricata can be fixed for the number of variants per individual; in the PH population all plants contain two variants. Other populations show variation in the number of rDNA variants that a plant contains, the range being from one to five variants per plant. Variation in the mean number of rDNA variants per plant is, in part, a function of the number of variants contained within populations. Phlox divaricata shows clear differences among populations in both the number and types of rDNA length variants contained within populations. The maximum number of variants found in a single population was seven, the minimum three.

Phlox divaricata populations show clear genetic differentiation in rDNA, as might be expected considering the geographical distribution of P. divaricata populations. All the populations of both subspecies are geographically isolated from each other and are most likely genetically isolated except for very rare instances of gene flow. Both pollen and seed dispersal are highly restricted in Phlox (Levin 198 1). The two most similar populations of ssp. Zaphami, WH and T, are also the closest geographically, being separated by - 8 km. In contrast, the two most similar populations of subspecies divaricata, M and B, are separated by > 160 km. The relationship between geographical association and rDNA differentiation is not straightforward and P. divaricata does not show clear genetic isolation as a function of geographical distance. The pattern of genetic differentiation of rDNA in Phlox is similar to the allozyme differentiation observed for plant species with small, isolated populations. In such species (e.g., Des- modium nudiflorum [Schaal and Smith 19801 or the pitcher plant Sarracenia purpurea [Schwaegerle and Schaal 1979]), relatively large genetic differences occur between populations. The interpopulation component of diversity for S. purpurea is 0.37, that for D. nudiflorum is 0.20, and that for P. drummondii is 0.16 (Levin 1978). In contrast, plant species that have large population sizes are highly variable and have high levels of gene flow. These species show very little genetic differentiation among populations, and their H values for interpopulation differentiation are typically quite low, e.g., <0.05 in Pinus (Guries and Ledig 1982). There is little information concerning rDNA diversity among populations of native plant species, and it is difficult to evaluate the Phlox rDNA values in terms of diversity studies on allozymes. Nonetheless, the in- ter-population component of rDNA differentiation in P. divaricata, H = 0.2 1, falls within the range for plant species; it is relatively high and corresponds to the diversity values of other species with geographically isolated populations. Such high levels of interpopulation differentiation are consistent with the biology of Phlox populations. Limited gene flow, small population size, and geographic isolation among populations all contribute to genetic differentiation among populations.

Finally, we consider variation between the two subspecies. Wherry ( 1955), in a study of morphological differentiation between the subspecies, concluded that ssp. Zaphami was derived from the larger, widespread eastern ssp. divaricata. Levin ( 1967), in subsequent studies of P. divaricata, noted morphological similarities between ssp. Zaphami from Missouri and Arkansas and P. pilosa, a congener. On the basis of these morphological similarities Levin suggested the occurrence of hybridization and introgression of ssp. Zaphami with P. pilosa. Regardless of the origin of these differences between the subspecies, the delineation of the subspecies corresponds to a real dis- continuity in morphological variation between the taxa. Our analysis of rDNA variation also shows clear differences between the subspecies in both the types and frequency

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of variants. Subspecies divaricata, which Wherry considers ancestral, has greater variation, containing nine rDNA variants, four of which are not found in the other subspecies. Laphami, on the other hand, has only eight variants, three of which are unique and occur at low frequency. The average number of rDNA repeats per individual also reflects the difference in variability of the subspecies. Again, ssp. divaricata is more variable; these results are consistent with a hypothesized derived origin for Zaphami. If ssp. divaricata were ancestral and Zaphami were derived, one would expect greater diversity in divaricata than in the derived Zaphami, owing to limited sampling of rDNA variants during founder events. The occurrence of unique variants in ssp. Zaphami does not rule out such an origin, since a much larger population sample would be necessary to eliminate the possible occurrence of these variants in divaricata. If ssp. Zaphami is the result of a few founder populations, then rare variants of divaricata may occur even in high frequency, owing to founder events. The hybridization hypothesis is more difficult to support with these data. Possibly the two unique variants of ssp. Zaphami may be due to hybridization or introgression with P. pilosa. However, one might expect greater diversity in ssp. Zaphami than in ssp. divaricata if hybridization were adding variants. Such an increase in diversity has not been observed. The possibility of infrequent introgression introducing rare variants cannot, however, be ruled out. Since ssp. Zaphami has less rDNA diversity, the rDNA data suggest that geographical isolation and divergence are a reasonable hypothesis for origin.

Clearly, more work on introgression, hybridization, and their effects on rDNA variation is necessary to evaluate these hypotheses. Nonetheless, in P. divaricata the occurrence and distribution of rDNA variation shows clear differentiation between subspecific levels and thus suggests that the distribution of rDNA variants is influenced by both the population biology and the evolutionary history of the species.

Acknowledgments

We wish to thank D. Bedigian, K. Helenurm, L. King, G. Learn, and J. Matos for their comments on this study. This work was supported by National Science Foun- dation grants DEB 8 1 113 12 and BSR 82 07020.

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WALTER M. FITCH, reviewing editor

Received September 11, 1986; revision received June 10, 1987

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phlox divaricatal - molecular biology and evolution

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