P1. Syst. Evol. 170, 97-106 (1990) --Plant

Systematics and

Evolution © by Springer-Verlag 1990

Ribosomal DNA variation within and among individuals of Lisianthius ( Gentianaceae) populations

KENNETH J. SYTSMA and BARBARA A. SCHAAL

Received March 1, 1989

Key words: Angiosperms, Gentianaceae, L&&nthius.-Population variation, ribosomal DNA, isozymes.

Abstract: Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of the Lisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site dif- ferences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18 S and 5.8 S rRNA genes, a region in Lisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.

Considerable interest has developed in examining DNA sequences for variation, not only among species and populations, but also within and among individuals comprising natural populations. Multigene families have been particularly useful for these types of studies. Assessment of levels of variation in histone genes and ribosomal DNA (rDNA) in Drosophila (COEN & al. 1982a, WILLIAMS & al. 1985) and globin genes in primates (Z~MMER & al. 1980) have demonstrated homoge- nization of repeats w i t h i n species, populations, and individuals but divergence of repeats a m o ng species. The phenomenon of homogenization within individuals, populations or species of such diverging repeats has been described as molecular drive (DovER 1982) or concerted evolution (ZIMMER & al. 1980). These homoge- nizations are the result of non meitoic processes such as unequal crossover of repeats (SMrrH 1975) and/or gene conversion (DOVER & al. 1982, ARNHEIM 1983).

Divergence of repeats within multigene families such as rDNA is expected and is due to both sequence and length changes. Ribosomal DNA is the stretch of DNA encoding the genes for the 18 S, 5.8 S, and 25 S ribosomal RNAs, and is composed of a tandem array of transcribed coding regions separated by intergenic

98 K.J. SYTSMA • B. A. SCHAAL:

spacer (IGS) regions (also called the non-transcribed spacer or NTS). These repeats, each comprising a coding and spacer region, can be present in up to 10 000 copies in plants (APPELS 84 HONEYCUTT 1986). Nucleotide sequence divergence and length heterogeneity, the latter due to unequal crossover among subrepeating elements within the IGS (APPELS & DVORAK 1982, YAKURA & al. 1984), are seen both among and within species (see APPELS & HONEYCUTT 1986 for recent review).

The levels of rDNA variation in plants, from the species down to the individual, suggest that the process of homogenization in rDNA may be gradual or rapid. A gradual process of homogenization of rDNA units was suggested for Phlox divari- cata because a mean number of between two and three rDNA repeat length variants per individual were found (SCHAAL 84 al. 1987). In contrast, rapid change has been seen in other species. Novel rDNA repeat types in Vicia faba can be generated within two generations by selfing within an inbred line (RO~ERS & al. 1986). Heritable change in copy number of rDNA repeat units due to genotype and environment interactions has been documented within one generation for Linum (reviewed in WALBOT & CULLIS 1985). Similarly, novel rDNA repeats may only appear on average every 10 3- 10 4 generations within Drosophila melanogaster (CoEy 84 al. 1982b), but appear in a single generation within D. mercatorurn (WILLIAMS 84 al. 198 5). No detectable evidence ofrDNA variation within individuals and/or populations, suggestive of fixation of one repeat type, is seen in other species of gicia (LAMPPA 84 al. 1984), Lupinus luteus (RAFALSKI 84 al. 1983), and some species of Glycine (DOYLE 84 BEACHY 1985).

The high levels of variation in rDNA in some plant populations and the lack of variation in other populations is of considerable evolutionary interest. Several studies have specifically attempted to quantify levels of rDNA variation within and among individuals and to delineate the evolutionary forces responsible for gen- erating the variability and affecting its distribution. Natural selection has been suggested as a force involved in the frequency and geographical distribution of rDNA length variants in native populations of cereal species and their wild ancestors (SAGHAI-MAROOF 84 al. 1984, CLUSTER 84 al. 1984, FLAVELL 84 al. 1986). In other studies random events such as genetic drift accompanied by limited gene flow, small population size, and geographical isolation has been implicated in producing the observed pattern of rDNA variation (ScHAAL 1985, LEARN 84 SCHAAL 1987, SCHAAL 84 al. 1987).

Presented here is a survey of rDNA variation within populations of the Lis- ianthius skinneri (Gentianaceae) species complex. This species complex has been the subject of a multi-disciplinary biosystematic approach. Phylogenetic analysis among populations has encompassed morphology and reproductive biology (SYTSMA 1988), isozymes (SYTSMA 84 SCHAAL 1985 a), chloroplast DNA and rDNA (SYTSMA 84 SCHAAL 1985b). Lisianthius skinneri is widespread in C. America and occupies mid-elevational, moist or wet tropical forests. Two lineages have evolved indepen- dently from the ancestral L. skinneri on geographically isolated, cloud forest peaks in C. Panama. Four of these five derived species are confined to a single or a series of isolated cloud forest "islands". All taxa in the complex are composed of small, widely separated populations. The entire genus is apparently tetraploid with a gametic chromosome number n = 18 (WEAVER 1972).

The different populations of the Lisianthius skinneri species complex vary con- siderably in floral characters affecting pollinator behavior, degree of selfing or

rDNA population variation in Lisianthius 99

outcrossing, and levels of isozymic variability. This study examines within and among individual r D N A variation within the seven populat ions of the Lisianthius skinneri species complex previously sampled and analyzed for isozyme variation (SYTSMA & SCHAAL 1985a). The current study addresses three specific questions: (1) What is the level of variation within and among individuals? (2) Where is this r D N A variation located? (3) Does the level of r D N A variation correlate with levels of isozymic variation within each populat ion?

Material and methods

Plant material and D N A isolation. Seven populations of 30 to 70 individuals in the Lisianthius skinneri species complex were examined for rDNA variation (Table 1, vouchers deposited in MO, WIS). These included one population each of the cloud forest species L. jefensis, L. habuensis, L. aurantiacus, L. peduncularis, and three populations of the lower elevation L. skinneri (El Llano-Carti, Cerro Jefe, Cascajal). 25 reproductively mature plants were sampled within each population. Direct comparisons of the distribution and level of rDNA and isozyme variation are possible as the individuals and populations surveyed are the same as those used in the study of isozymic variation (SYn'SMA & SCHAAL 1985a).

Leaves from each individual were collected, placed on wet ice, flown back to the laboratory, and stored at --70 °C. The miniprep DNA isolation technique of COEN & al. (1982b) was used to obtain total cellular DNA. This miniprep procedure is fast (4h) and uses small amounts of leaf tissue (0.2- 0.5 g).

Restriction enzyme analysis. Seven restriction endonucleases were used to digest total DNA: BamHI, BglI, EcoRI, HincII, SstI, HindIII, and XbaI. The first five restriction enzymes were selected because they recognize several sites in Lisianthius rDNA and all, except for BamHI, recognize sites within the IGS. HindIII and XbaI each have a single cleavage site within Lisianthius rDNA (except for population LH 1 with two Xba I sites). Approximately 0.5 - 1.0 pg of DNA were digested with 10 units of restriction endonuclease for 8 - 16 h following manufacturer's specifications. Greater amounts of enzyme with longer incubation periods were used with BamHI, BglI, and SstI in several populations to insure that fragment patterns seen were not simply due to incomplete digestion. Restriction frag- ments were separated in Tris-EDTA-acetate agarose gels, denatured, neutralized, and trans- ferred to nitrocellulose membrane, as described elsewhere (SYTSMA & SCHAAL 1985b; SYTSMA, unpubl, data).

Hybridization. A complete rDNA repeat from Glycine max (soybean) cloned in pBR 325 (pGmr- 1, kindly provided by E. A. ZIMMER) and several subclones were used as heterologous probes for rDNA. Lisianthius rDNA fragments bound to membranes were visualized by

Table 1. Lisianthius populations in Panama examined for within and among individual nuclear rDNA variation (and sampled for isozyme variation, SYTSMA & SCHAAL 1985a)

Population Location Collection nO.

L. skinneri (HEMs.) O. KUNT. (LS4) Panama, El-Llano Carti L. skinneri (LS 6) L. skinneri (LS 7) L. jefensis ROBYNS ~% ELIAS (LJ 1) L. habuensis SYTSMA (LH 1) L. peduncularis WILLIAMS (LP 1) L. aurantiacus SYTSMA (LA1)

Panama, Cerro Jefe Coc16, Cascajal Panama, Cerro Jefe Panama, El-Llano Carti Coc16, Cerro Carocoral Coc16, Cascajal

SYTSMA 4432 SYTSMA•KNAPP 4795 SYTSMA 3939 SYTSMA 1399 SYTSMA 4002 SYTSMA 3815 SYTSMA ~ al. 4379

100 K.J. SYTSMA & B. A. SCHAAL:

autoradiography. Mapping studies of Lisianthius rDNA indicated that enough homology exists between the coding and noncoding portions of Glycine and Lisianthius rDNAs that almost all Lisianthius fragments using these seven restriction enzymes are visible. Different fragments had different intensities reflecting differential homology to the Glycine rDNA probe. Nick translations, prehybridization, hybridization, and filter washes follow SYTSMA ~; SCHAAL (1985b).

Results and discussion

No restriction site polymorphisms were detected in populations. Likewise, no length heterogeneity was seen in the IGS within or among individuals of a given population, although this spacer region is the primary location for restriction site and length heterogeneity found among Lisianthius populations and species (SYTSMA & SCHAAL 1985b). Figures 1 and 2 depict the typical nonvarying fragment pattern obtained when populations are surveyed for restriction site or length variation in the IGS region, in this case with HindIII and BamHI. HindIII cleaves rDNA of all pop- ulations only once. A single fragment representing the one rDNA repeat size per individual is seen in the 10 individuals ofL. habuensis (LH 1) (repeat size of 12.4 kb) and the one individual of L. skinneri (LS4) (repeat size of 11.5 kb) illustrated in Fig. 1. Unlike the situation found within or among individuals of a given population,

C~ ° ~

• ~. ¢-

~ r - - t "

__1 __i I

I I

12 .4 Kb _.,,,. 11.5 =

Fig. 1. Autoradiographic fragment patterns of rDNA repeats digested with HindllI from one individual of Lisianthius skinneri (population LS4) and ten individuals of L. habuensis (population LH 1) illustrating rDNA repeat length variation among populations

rDNA population variation in Lisianthius 101

kb

--12.0-- - - 8 . 2 - -

- - 6 . 9 - -

- - 3 . 8 - -

- - 2 . 5 - -

- - I . 3 - -

Fig. 2. Autoradiographic fragment patterns of rDNA repeats digested with BamH! from 24 individuals of Lisianthius aurantiacus (population LA 1) indicating lack of IGS length variation within populations

different populations are often fixed for different IGS length variants as seen between LH1 and LS4 (Fig. 1) (see also SYTSMA (~ SCHAAL 1985b: 599, Fig. 3). TWO BamHI fragments, 6.9kb and 1.3 kb, span the IGS region in Lisianthius aurantiacus (Fig. 2). The 24 individuals shown here exhibit a single band for both of these fragments and show no detectable length variants. The larger fragments, 8.2 and 12.0 kb, are due to partial or differential cleavage of sites between adjacent fragments, a common occurrence with BamHI digests of methylated, repetitive nuclear DNA.

The small, transcribed spacer region (ITS) between the 18 S and 25 S genes is the only region of rDNA that exhibits variation within and among individuals of populations. This ITS region is also the site of a 50 bp deletion in all individuals of the LS7 population relative to other populations (SYTSMA • SCHAAL 1985b: 598, Fig. 2b). Figure 3 illustrates the SstI rDNA restriction fragment patterns in 10 individuals from Lisianthius habuensis (LH 1) and 11 individuals from L. skinneri (LS7). The rDNA repeat in LH 1 is cut by SstI at three sites generating 8.4, 2.2, and 1.6 kb fragments (Fig. 3 a, c). Individual 7 of LH 1 contains an additional 2.3 kb fragment (Fig. 3 a, c). Likewise, six of the 11 individuals of LS 7 depicted contain both a 2.15 and 2.25 kb fragment (Fig. 3 b, c).

Mapping studies with EcoRV have placed the position of both the LH 1 2.2/ 2.3 kb and the LS7 2.15/2.25 kb SstI fragments to the 3' end of the 18 S gene, the ITS, and the 5' end of the 25 S gene (see SYTSMA & SCHAAL 1985 b). EcoRV cleaves Lisianthius rDNA only once and this site has been mapped to the intervening sequence between the 18 S and 25 S genes (SYTsMA & SCHAAL 1985b). Sequence studies on the 5.8 S gene that occurs in this ITS region indicate that all angiosperms so far examined possess an EcoRV site within the 5.8 S gene (ERI)MAYN 1982).

Kb

8.4

2.5 2.2

1.6

102 K.J. SYTSMA • B. A. SCHAAL:

a. LHI b. LS7

I 2 3 4 5 6 7 8 9 I0 I 2 5 4 5 6 7 8 9 I0

Kb

8.2

.3.8

2.25 2.15

1.6

C.

LHI

LS7

i 'Ts I I I

, -( ,

(8.4)

IGS 18S

(8.2)

Sst I

F

Sst

Fig. 3. Autoradiographic patterns of rDNA repeats digested with SstI from individuals of a Lisianthius habuensis (population LH 1) and b L. skinneri (population LS7) depicting ITS length variation within and among individuals in populations, c Restriction site map of an rDNA repeat in L. habuensis (LH 1) and L. skinneri (LS7) indicating SstI and EcoRV sites and variable SstI fragment lengths

Furthermore, EcoRV double digests with EcoRI, SstI, ~/nd BamHI indicate that the 100bp difference between the 2.2 and 2.3kb fragments maps to the region between the EcoRV site and the EcoRI, SstI, and BamH! sites in the 18 S gene.

These lines of evidence indicate that a 100 bp insertion has occurred in the region surrounding the 3' end of the 18 S gene and the 5' end of the 5.8 S gene, most likely in the spacer between the 18 S and 5.8 S genes. This insertion is found in a portion of the rDNA repeats of some individuals in many of the populations examined. Five of the seven Lisianthius populations sampled contained this longer repeat type but in varying frequencies (Table 2). Lisianthius skinneri populations LS4 & LS6 lack the ITS length variation completely. Lisianthius aurantiacus (LA 1), however, exhibits the 100 bp ITS length variant in all 25 individuals examined. The rDNA repeats containing the 100bp insertion are always present with the shorter and typical ITS repeats. The 2.3 kb (or 2.25 kb in LS7) repeat type never comprises the only repeat type in an individual of any population so far examined.

The autoradiographic intensities of the two variant ITS spanning fragments in

rDNA population variation in Lisianthius 103

Table 2. Frequency of individuals heterozygous for the ITS length variant (F) and com- parison to isozyme variation (Sva'SMA & SCHAAL 1985a): P percentage of polymorphic loci, k mean number alleles per locus, Ho mean observed loci heterozygous, HET hetero- zygosity

Population F P k Ho HET

LS4 0 0 1.00 0 0 LS6 0 0 1.00 0 0 LS 7 0.48 0 1.00 0 0 LJ 1 0.60 16.7 1.17 0.030 0.061 LH 1 0.64 8.3 1.08 0.017 0.015 LP1 1.00 33.3 1.33 0.073 0.082 LA 1 0.48 33.3 1.33 0.106 0.079

Mean 0.46 13.1 1.13 0.032 0.035

some individuals (see Fig. 3 a, b) indicate that the two repeat types are found in approximately equal copy number. Many individuals, however, possess different amounts of the two fragments (see Fig. 3b), although these differences have not been quantified. The novel ITS variability observed within individuals may arise and persist due to multi-chromosomal localization of the rDNA arrays. Multiple rDNA arrays would be consistent with the apparent (ancient) tetraploid nature of Lisianthius, but, without the necessary genetic crosses and progeny screening of these woody perennials, the number and localization of rDNA arrays in Lisianthius is only speculative.

Associated with the 100 bp insertion in most polymorphic populations is a 3.8 kb SstI fragment, representing partial digestion, methylation, or loss of the SstI re- striction site between the adjacent 2.2 and 1.6 kb fragments. The presence of the 3.8 kb fragment does not permit accurate quantification of the relative copy number of the two repeat types as the 3.8 kb fragment also contains some of either the 2.2 or 2.3 kb SstI variant fragments. It is unclear why the presence of the 3.8 kb fragment in individuals is associated with the presence of the 100bp ITS insertion. One possible explanation for this association of the length and site variation might be that both the SstI site modification and the length variation are present on the same rDNA repeats. Cloning, characterization, and sequencing of both length variants is needed to fully explain this phenomenon.

Correlation between the frequency of individuals with the ITS length variant and different measures of isozymic diversity for each population was tested with the Spearman rank and Kendall tau-b tests. No significant correlations occur between the presence of rDNA length variation and percentage polymorphic loci, mean number of alleles per locus, mean heterozygous loci, or heterozygosity (Table 3). Most measures of isozymic diversity are significantly inter-correlated in the populations. Three populations of Lisianthius skinneri are completely mono- morphic at the 12 isozyme loci examined. However, only two of these populations are also lacking in rDNA length variation; the third has nearly V2 of its individuals exhibiting the ITS length variant. The only other plant study in which isozymic and rDNA variation have been tested for correlation has been in populations of

104 K.J. SYTSMA • B. A. SCHAAL;

Table 3. Spearman rank and Kendall tau-b correlations (upper right and lower left diagonals, respectively) of frequency of rDNA variants (F) and measures of isozymic diversity (SYTSMA & SCI-IAAL 1985a). Abbreviations see Table2

F P k Ho HET

F 1.000 0.640 0.640 0.519 0.679 P 0.520 1.000 1.000 0.969 0.994 k 0.520 1.000 1.000 0.969 0.994 H o 0.392 0.898 0.898 1.000 0.950 HET 0.549 0.980 0.980 0.840 1.000

Triticum dicoccoides in Israel (FLAVELL & al. 1986). This study presented evidence for positive correlation of rDNA IGS length variation with isozymic variation. Interestingly, in subterranean mole rats rDNA IGS variation is negatively correlated with isozyme variation but positively correlated with humidity and temperature variables (NEvo & BHLES 1988).

The lack of substantial variation in rDNA within populations of the Lisianthius skinneri species complex reflects the almost total lack of morphological variation seen within these populations (SYTSMA 1988). The lack of IGS rDNA variation is, however, surprising considering the amount of IGS length variation (and site variation) among these populations (SYTSMA & SCHAAL 1985 b) and the high amount of isozymic variation in some Lisianthius populations that are clearly outcrossing (SYTSMA & SCHAAL 1985a).

However, although the L. skinneri species complex has populations that are variable isozymically, the number of alleles per variable locus is extremely low in all populations. This low allelic diversity, despite the high frequencies of poly- morphic loci and high heterozygosity in some populations, has suggested that founder events were important evolutionary factors in the origin of the species complex (SYTSMA & SCHAAL 1985a). The pattern of rDNA variation within and among populations, being almost totally homogeneous within populations but fixed for alternative repeat lengths among populations, is consistent with the notion of spatial and genetic isolation and subsequent genetic drift among the populations of the L. skinneri species complex (SYTsMA & SCHAAL 1985a).

Similarly, genetic drift following spatial or genetic isolation has been implicated in the fixations of alternative rDNA repeats in different populations of both Phlox divaricata (SCHAAL & al. 1987) and Clematisfremontii (SCHAAL 1985, LEARN & SCI-IAAL 1987). Analysis of rDNA provided evidence for genetic drift among "is- land" populations in Triticum dicoccoides but ruled out founder events as important factors in the diversification of the species (FLAVELL & al. 1986). The interaction of molecular mechanisms giving rise to rDNA variation and subsequent homog- enization with features of plant populations such as size, structuring, reproductive biology, and amount of outcrossing or inbreeding is still vague. Additional species need to be examined in more detail before comparative generalizations can be made between rDNA and these population parameters, as has been done with isozymes (HAMRICK & al. 1979, LOVELESS & HAMRICK 1984).

rDNA population variation in Lisianthius 105

The authors are grateful to the Missouri Botanical Garden for travel funds to collect leaf tissue, to G. H. LEARN and J. S. SHOEMAKER for comments on the manuscript, to E. A. Z~MMER for providing the pGmr-1 clone, and to an anonymous reviewer for comments. Research was supported in part by NSF grants to B. A. SCHAAL (BSR 82 07020) and to K. J. SYTSMA (BSR 85 16573).

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Addresses of the authors: KENNETH J. SYTSMA, Department of Botany, University of Wisconsin, Madison, W153706, U.S.A. - BARBARA A. SCHAAL, Department of Biology, Washington University, St. Louis, Missouri, U.S.A.