The genetic structure of Rhizobium populations

Soil Bid.

Pergamon

THE GENETIC

D. M. GORDON,‘*

Biochem.

Vol. 21, No. 4/5, pp. 491499, 1995 Copyright 0 1995 Ei&ier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/95 $9.50 + 0.00

STRUCTURE OF RHIZOBIUM POPULATIONS

M. WEXLER,* T. B. REARDON

and P. J. MURPHY*

‘Division of Botany and Zoology, Australian National University, Canberra, ACT 0200, Australia, Department of Crop Protection, Waite Campus, University of Adelaide, Glen Osmond, SA 5064, Australia and 3Evolutionary Biology Unit, South Australia Museum, Adelaide, SA 5000, Australia Samnary-Allelic profiles for 14 loci were determined for 48 strains of Rhizobium dilofi and 43 strains of R. leguminosarum bv. oiciae using multi-locus enzyme electrophoresis. From these results, and using data from published studies, estimates of multi-locus linkage disequilibrium were obtained for a number of rhizobial species. The magnitude of linkage disequilibrium estimates suggest that the genetic structure of rhizobial populations spans the range from strictly clonal to essentially panmictic. R. meliloti appears to consist of two ecologically isolated subpopulations of strains, where recombination appears to be frequent within populations but not between populations. Symbiotically-effective populations of R. etli appear to

maintain a clonal population structure, while non-symbiotically-effectivestrains of this species,those lacking the symbiotic plasmid, exhibit a panmictic population structure. No linkage disequilibrium was detected among strains of R. leguminosarum bv. uiciae, suggesting frequent recombination, whereas recombination appears to be less frequent among strains of R. leguminosarum bv. frifolii.

WTRODUCMON

The economic

significance

of the Rhizobium-legume

association has resulted in extensive research to improve the efficiency of the symbiosis. Many attempts are under way to select naturally-occurring strains that are both symbiotically effective and able to persist in the environment. Efforts are now extending beyond the simple selection of strains to the genetic engineering of rhizobia with specific attributes, with the final goal being the release of such strains into the environment. Achieving this goal depends on having a sound understanding of the forces determining the genetic structure of rhizobial populations in order to assess the likelihood of gene transfer between introduced genetically-engineered organisms and the indigenous rhizobia and in predicting the potential genetic stability of the released organism. In bacteria, reproduction and the exchange of chromosomal genes are separate events. In the absence of chromosomal recombination, asexual reproduction will lead to all individuals of a species being related by clonal descent. However, chromosomal recombination may result from horizontal gene transfer by transduction, conjugation and transformation (Levy and Miller, 1989). Recombination disrupts the basic clonal population structure resulting from asexual reproduction and leads to a genetic structure that approaches panmixis. Selander and Levin (1980) examined genetic variation in Escherichiu coli using multi-locus enzyme electrophoresis (MLEE) and demonstrated that *Author for correspondence.

despite the presence of extensive allelic variation, there was only a relatively modest level of genotypic variation. This occurred because certain alleles at a locus were almost invariably associated with particular alleles at another locus. The scarcity of some allele combinations relative to expectations under complete recombination is an indication of linkage disequilibrium. Therefore, the presence of significant levels of linkage disequilibrium is good evidence for a clonal population structure and infrequent recombination. Subsequent studies have provided more support for the belief that recombination is rare in E. coli because linkage disequilibrium coefficients are near their theoretical maxima (Caugant et al., 1981), the magnitude of linkage disequilibrium coefficients is independent of the map distance between the genes (Whittam et al., 1983) and variation in geographic scale does not have a significant effect on linkage disequilibrium (Whittam et al., 1983). In addition, some genotypes have a global distribution and can be repeatedly isolated over many decades (O&man and Selander, 1984). Thus, the evidence that E. coli exhibits a clonal population structure is persuasive. This does not mean that chromosomal recombination has not influenced the evolution of the E. coli chromosome. Dykhuizen and Green (1991) showed that the apparent phylogenetic relationships among E. coli strains based on DNA sequence data depended upon which gene was analysed, and concluded that this observationcould only be a result ofchromosomal recombination. Although important in the genomic evolution of E. coli, recombination does not appear to occur frequently enough to disrupt a basically clonal population structure. 491

D. M. Gordon et al

492

The clonal nature of E. coli populations has been raised to the status of a paradigm for all bacterial populations. The notion that all bacteria are clonal has been challenged by Souza et al. (1992), Istock et al. (1992) and Maynard Smith et al. (1993). Much of the data concerning the clonicity of bacterial populations depends on showing the occurrence of significant levels of linkage disequilibrium. However, Maynard Smith et at. (1993) observed that there are a number of ways linkage disequilibrium can occur in the face of frequent recombination. Linkage disequilibrium may arise by genetic drift or as a result of epistatic fitness interactions between loci. It may also be the consequence of analysing a sample of strains that is composed of a mixture of isolates from several populations that are geographically or ecologically isolated. What is the genetic structure of rhizobial populations? Our purpose in this paper is to present an analysis of the population genetic structure of several species of Rhizobium based on published data and our data obtained by MLEE. MATERIALS AND METHODS

Bacterial strains

The strains of R. meliloti and R. leguminosarum bv. in our study were obtained by

uiciae analysed

Table 1. Enzvme allelic orofiles for ETs of R.

subsampling existing strain collections from around the world in an ad hoc manner. The identities of the strains examined are presented in Table 1. Further details concerning the host plant, country of origin and the source of the strains are given in Wexler et al. (1995). Multi-locus

enzyme electrophoresis

(MLEE)

MLEE distinguishes between different alleles at a particular locus on the basis of the mobility of proteins in an electric field. The method has the limitation that only ca. 30% of amino acid changes are electrophoretically detectable, but this lack of sensitivity is balanced by low cost and rapid screening for many loci. Strains were grown in 150 ml of tryptone-yeast broth (Beringer, 1974) to late-log phase. The cultures were harvested by centrifugation, washed twice in 0.15 M phosphate buffer (pH 7.0), transferred to microcentrifuge tubes and pelleted. Lysis buffer, 50 ~1 (100 mg NADP and 500 ~1 of 2-mercaptoethanoll‘), were added to the cell pellet then sonicated using three 5-s bursts. The extracts were centrifuged and aliquots of the supernatant transferred to glass capillary tubes and stored at - 20°C. Enzyme extracts of each strain were separated by electrophoresis on cellulose-acetate gels (Cellogel, Chemetron) and stained for specific mdiloti

and R. lemmkmuwn

bv. vi&e

Alleles at indicated enzyme loci* Strain R. meliloti

CC169 Ml61 M254 M75 M58 WSM540 M7 Ml02 s33 102F51 9930 M275 74B3 128A7 15A6 N6B5 CC2003 1322 Ml19 SU258 102F34 CC2301 CC2068 KRC72 WSM922 CC2165 CC2160 cc2 I57 CC2153 RM220-3 102F77 WSM826 MSURSZa CC8076 cc201 3 CC2163a

I

2

3

4

5

6

7

8

9

10

11

12

13

14

at

b

a

b b b b b b b b a a a a a a a a a a

b d b d b b b b a a c c c c c c a a c _ c a a a c a _

c c c

a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a

a a a a a a a

d d d d d d d d a a a a a _ c

E

f b b

h h h h h h I f a

b

: b b b

d d b d d d d a c c c c c c c c c c c c c c c c c c c c c c c c c c c c

a a a a a a a a a a b b b b

a a a a a a a

; b -: b b b b b b b b b b b b b b b b b b b b b b b b

L c c c c c c c c c c ; c c : c c c c c c c c c c c c

: b e e e e e e e e e e e e e f e e e e e e e e e e e : d

: : a e a a a a b d a a a a a a

: a a a a a a

d

: b a a a a

d” b

: b

; _ d

I

; b b b

b”c c c

; a a a a a a a a a a a a a a a a a a a a a a a a a a a a

;

b b b b b b ; b b ; b b 6 b c c k b b b b c c

: d d d d d f d d

: a

s d” : d d b c d” d d d d d g g

a !4 a

a a a a c e f g

a

a a a a e e

b : b b k b b b b b b b b b c L b b : b : L b b 6 c c

: b b b b b b b b b b b b b b a a a ; b

continued opposite

Rhizobium population

493

structure

Table l-continued Alleles at indicated enxyme loci* Strain

2 bv.

R. lewninosarum

P342L126 $1 sp75 P2S6 NA533 Ll41 Ml325

15

d : d : d d c

3

: c? d d d : d d d d d d d d d d d d d d

6

16

9

10

12

13

17

18

19

20

b

d d

c c c c c c c c

b b b b

b a a a

e e e

8 g g

c c c c

c c c c

: d d f

a a c c c

f

: b b b b b b b b

c c c c c c c c c c c c c c c c

: b b b b

? d b d d a c

5

:

c e e e c c c c c c c c c c

; j j f h j e

viciae

a a a a a : b a

B c B B

L e e e

f &5 Pl15 Ml201 CC321 L364 cc319 Nitrogerm sp22 WSMIOl5 P444 P233 Sp89 WSM1014 300 CC328 NA526 Ll6S Ml418 CC320 Ml934 Sp18 L241

5

6 : b b b a a a a a a a

: e e g e e ag d e ; d e

b” :

: b : b b :: b b : b b : : b b a : b

: : d d d 1 d b” b b b b : z : d d

C

e c c c c c :

b c c c e c f a

: : E : b

; e e

: b b b c

: b d d d

: c c c c

b” b b b b b b b b b

: d e f

8 d : : d d

f d e e e c e e e e f g : ; e e e c e e e e c g e g e

B 8 8 j

Fi i g g f j c

: a c C

: a a c c c c c c c c a c a c a c c c a

: g j

C C C e C e C C C C C C C C C C C C C C

d”

C

:

B

c

*The following enaymcs were assayed: 1, aconitase hydratase; 2, adenylate kinase; 3, timmrate hydratase; 4, glyceraldehyde-3-phosphate dehydrogenase; 5, guanine deaminase; 6, ghtwse&phosphate debydrogenase; 7, 3-hydroxybutyrate dehydrogenasc; 8, hexokinase; 9, isccitmte dehydrogenase; 10, male dehydrogenasc; 11, mannose+phosphate isomerase; 12, dipeptidase; 13, phophoghmonate dehydrogenase; 14, triosophosphate isomerasc; 15, enolape, 16, gJucose+phosphate isomerase; 17, phospboglucomutase; 18, shikimatc dehydrogenase; 19, supcroxide dismutase; 20, UTP-glucose-1-phosph uridylyltransfe.rase. tFor each enxyme locus, the letters denote a dit allele for that locus. $The absence of enxyme activity is scored as a null character state (-).

activity as described by Richardson et al. (1986) and Selander et al. (1986). All procedures were carried out at 5°C. Allelic profiles at 14 loci were determined for each species (Table 1). However, the same loci were not examined for each species, nor in cases of common loci can the results be compared between species. Estimates of the genetic relationships between strains were obtained using cluster analysis by the unweighted pair group method with averages (UPGMA) (Sneath and Sokal, 1973). enzyme

Index of linkage disequilibrium Brown et al. (1980) developed a multi-locus index of linkage disequilibrium that is particularly appropriate for data sets derived from enzyme electrophoresis. The index is calculated as follows. A number of loci, m, have been analysed in n individuals. The frequency of the ith allele at thejth locus is pij. The probability that two individuals differ at the jth locus is h,, hj = 1 - Zp$ Let K be the total difference between two individuals, i.e. the number of loci at which they differ. There are n(n - I)/2 such pairs in a sample of n

individuals. The individuals is

mean

difference

between

two

K=Zh,.

If the alleles present at different loci in an individual are independent, i.e. if there is no linkage disequilibrium, then the expected value of the variance of Kis V, = Z h,(l - hj). By comparing V,, the observed variance of K, with Va one obtains a measure of the degree of association between loci. A convenient index is 4 = V,/VE - 1. The index has an expected value of zero if there is no association between loci. To determine if the observed value of Z* is significantly different from zero, the observed (V,) and expected (V,) variances are compared. The error variance of Vr is given by var( VE) = l/n{Xhj - 7X hj + 12C hj-62:

hf+2[C h,(l-h,)]*}.

D. M. Gordon et al.

494

The upper 95% confidence limit (L) for V, is L = VE+ S{var( V,)}t’2. If VOis less than the upper 95% confidence limit (L) of FE, then I* is not significantly different from zero at the 0.05 level of significance. RESULTS AND DISCUSSION

From the 48 R. meliloti strains examined, there were 36 electrophoretically distinguishable types (ETs). All loci examined were polymorphic, with an average of 4.6 alleles observed per locus, and the average genetic distance between the ETs (D) was 0.515 (Tables 1 and 2). Cluster analysis (Fig. 1) revealed that the ETs were clumped into two divisions (A and B) separated by a genetic distance of 0.83. A further subgrouping occurred within division A (subgroups Al and A2), with the strains in these two subgroups being separated by a genetic distance of 0.52. A significant level of multi-locus linkage disequilibrium was detected when all 36 ETs were analysed (Table 2). When the division A and B strains were analysed independently, linkage disequilibrium was found among the division A strains, but not the division B strains (Table 2). No linkage disequilibrium was detected when the subgroups Al and A2 were analysed separately (Table 2). These results are similar to those of Eardly et al. (1990) in their MLEE and restriction fragment length polymorphism analysis (RFLP) of 232 isolates of R. meliloti. This was the first study to demonstrate the existence of two major divisions in R. meliloti, and Eardly et al. (1990) suggest that the differences observed between division A and B strains are sufficient to warrant assigning them specific status. Subsequent phylogenetic analysis of 16s rRNA Table 2. Measures of disequilibrium Species R. meliloti

R. Iegwninosarum bv. vi&e bv. trifdii

SOUWZ’

No. of loci

1

14

1 2

3

4

Mean No. of alleles/locus

Dt

A Al A2 B

36 28 22 6 8

4.6 3.4 2.1 1.8 1.9

0.515 0.345 0.232 0.262 0.236

3.99 1.46” -0.17NS -0.27NS -0.16NS

32 13 4 9 32 14 18 37 15 9 7 6 70

4.6 4.1 2.1 3.1 5.2 3.0 3.2 5.9 2.9 2.3 2.7 2.5 4.1

0.493 0.566 0.482 0.507 0.570 0.289 0.363 0.650 0.370 0.404 0.530 0.529 0.513

0.37NS 1.30” 0.93NS 0.94NS 1.47” -0.37NS O.IONS 1.33” 0.19NS 0.95” 1.39’ 1.84” - 0.05NS

14 28

8

15 A B C D

R. et/i (non-symbiotic)

5

8

between enzyme loci in Rhizobwn No. of ETs

A B R. et/i (symbiotic)

T. suavissima).

group

A B R. tropici

sequence data supports this conclusion (Segovia et al., 1993). Division B strains appear to be restricted to annual Medicago species growing in the eastern Mediterranean basin, while division Al strains can be isolated from a variety of annual and perennial medics, and are apparently cosmopolitan (Eardly et al., 1990; Wexler et al., 1995). The study by Eardlyet af. (1990) provided some evidence for the existence of two subgroups within division A. However, they had only a single representative for the subgroup A2, an unusual strain, in that it is able to form an effective symbiosis with Trigonella suavissima, an endemic Australian species that appears to have quite specific rhizobial requirements (Brockwell and Hely, 1966). For our study, we specifically selected a number of strains either isolated from T. suavissima or able to form an effective symbiosis with this species. All of these strains were subsequently identified as members of the A2 subdivision (Fig. 1). The two major divisions in R. meliloti sensu Iato apparently represent distinct species. If this is the case, then it is not surprising, when ETs of both “species” are analysed together, that significant levels of linkage disequilibrium are detected. It would be expected that recombination would occur much less frequently between species than within a species. The absence of linkage disequilibrium within the division B strains suggested that recombination may be frequent among strains of this division. Analysis revealed a significant level of linkage disequilibrium among all of the division A strains, but not within the subgroups Al and A2, which suggested that recombination is frequent among Al and A2 strains, but not between the two subgroups. This may be explained by ecological isolation of the subgroups due to their different host plant specificities (Medicago spp vs

IA

signif.

‘Source of data: 1, this study; 2, Demezas et al. (1991); 3, Martinez-Romero etal. (1991); 4,Pihero et al. (1988); 5, Segovia et al. (1991). tGenetic distance: calculated as the arithmetic average of h, for all loci, tSignificance level (0.05): ‘I* different from zero; Ns I* not different from zero.

Rhizobiumpopulation structure

495

STRAIN WSM922 Ml 19

N6B5 M275 128A7 CC8076 ATCC 9930 RM220-3 102F34 102F77 MSUR52a 74B3 CC2165 CC2003 322 102F51 CC2068 s33 WSM826 CC2301 15A6 KRC72 CC2157 SU258 CC2153 CC2163a CC2013 cc2160 Ml02 M254 M58 WSM540 CC169 M75 M7 Ml61

r

Al

B

10

20

30 Genetic

40

50

60

70

80

90

Distance

Fig. 1. Genetic relationships among the 36 ETs of R. meliloti,based on allelic profiles at 14 .loci. In the 43 R. leguminosarum bv. viciae isolates examined, 32 ETs were identified. There was a mean of 4.6 alleles per locus and the average genetic distance between strains was 0.493 (Tables 1 and 2). Cluster analysis did not reveal the existence of any distinct clusters of strains (Fig. 2). The apparent absence of multi-locus linkage disequilibrium, even when all ETs were considered, suggested that recombination may be frequent among strains of this R. legurninosarum bv. Demezas et al. (1991) presented the results of MLEE and RFLP (chromosomal gene probes) analyses of 13 ETs of R . leguminosarum bv. trifolii. A significant level of multi-locus linkage disequilibrium was detected when all ETs were used to estimate ZA(Table 2). Cluster analysis of the MLEE results showed that there were two groups of strains separated by a genetic distance of0.62(Demezasetal., 1991). WhentheETsin the two groups were analysed independently linkage disequilibrium could not be detected in either subgroup. However, the division of the strains into two groups based on the MLEE data likely has no biological

meaning, as the strain clustering patterns obtained were different when the RFLP data were analysed (Demezas et al., 1991). As discussed later, the lack of significant levels of linkage disequilibrium among strains in these two subgroups is likely due to loss of statistical power resulting from small sampk sizes. Three biovars of R. leguminosarum are recognized: bv. viciae, capable of nodulating plants of the genera Lens, Prkum and Vicia; bv. trifolii, nodulating Trifolium spp; and bv. phaseoli, symbionts of Phaseolus vulgaris (Jordan, 1984). In R. leguminosarum host specificity is largely a symbiotic plasmid determined trait (Laguerre et al., 1993). The results of population genetic studies suggest that R. leguminosarum constitutes a fairly homogenous group. In general, strains from the three biovars cannot be distinguished using techniques that assess chromosoma1 variation. This has been the case for studies exploiting MLEE analysis (Young, 1985; Segovia et al., 1991), RFLP analysis using chromosomal gene probes (Laguerre et al., 1993), two-dimensional gel electrophoresis of total cellular protein (Roberts ef al.,

D. M. Gordon et al.

496 STRAIN

I

Sp89 NAj26 P256 Ml934 P115 Ml418 8401 la P324 L126 Ml325 L364 CC328 L141 NA533 L165 L241 Sp45 Sp18 sp22

I

sp75 30

40

50

60

70

80

Genetic Distance Fig. 2. Genetic relationships among the 32 ETs of R.leguminosuruimbv. vi&e, based on allelic profiles at 14 loci. 1980) and randomly amplified polymorphic DNA profiles (Dooley and Harrison, 1993). Furthermore, studies examining isolates within bv. trifolii or bv. viciue have not revealed the presence of major subgroups of strains that are sufficiently distinct genetically to suggest the possibility of multiple rhizobial species exploiting the hosts of these biovars (Young et al., 1987; Young and Wexler, 1988; Harrison et al., 1989; Demezas et al., 1991). In light of the difficulty in distinguishing the biovars of R. leguminosarum on the basis of chromosomally determined characteristics, the absence of any indication of multi-locus linkage disequilibrium among bv. viciue strains and the presence of linkage disequilibrium in the bv. trifofii strains is perplexing. Unfortunately, there are no adequate MLEE data for bonafide bv. phaseoli strains, so we have no indication of the population structure of this biovar. Nor are there results available of an MLEE study that has examined sufficient numbers of isolates of all three biovars simultaneously. However, if such data were available it is probable that analysis would reveal significant levels of linkage disequilibrium for the species as a whole.

Pifiero et al. (1988) analysed a number of rhizobia isolates from Phaseolus using MLEE. They found extremely high levels of genetic diversity and the existence of several subgroups among these isolates, with large genetic distances separating some of the subgroups. Their conclusion, that rhizobia which nodulate beans are a polyphyletic assemblage of species, has been amply supported by systematic studies that described several new species and reclassified others (Martinez et al., 1988; Martinez-Romero et al., 1991; Eardly et al., 1992; Segovia et al., 1993; Laguerre et al., 1993). In their description of the new species, R. tropici, Martinez-Romero et al. (1991) presented the results of a number of analyses including MLEE. Cluster analysis of the MLEE data revealed the presence of two major divisions of strains (A and B) separated by a genetic distance of 0.79; a result supported by their analysis of phenotypic characters and 16s rRNA sequence data. A significant level of multi-locus linkage disequilibrium was found when all ETs of R. tropici were considered, but none was detected when the two divisions were analysed independently (Table 2).

Rhizobium population structure The presence of linkage disequilibrium between groups A and B strains but not within groups may be a consequence of A and B strains actually representing phylogenetically distinct species. There is no evidence to suggest that the groups are geographically isolated, nor does there appear to be ecological isolation due to differences between the groups in their host plant specificities (Martinez-Romero et al., 1991). A number of the strains classified as R. leguminosarum bv. phaseoli (ETs l-37) in the MLEE study ofPiiiero et al. (1988) have since been reclassified as R. etli (Segovia et al., 1993). Significant multi-locus linkage disequilibrium was detected when all R. etli ETs were considered, as well as in three of the four subgroups identified by Piiiero et al. (1988) by means of cluster analysis (Table 2). Although there is no evidence to suggest that these are biologically meaningful subgroups of strains, the presence of significant levels of linkage disequilibrium among strains overall and in the majority of subgroups suggests that R. etli maintains a clonal population structure with recombination being an infrequent occurrence. We conclude that R. etli maintains a clonal population structure as our anaylsis showed, that even when broken down into subgroups, significant levels of linkage disequilibrium are observed. Souza et al. (1992) concluded from the results of their MLEE analysis of a very large collection of R. etli isolates that recombination may occur frequently within local populations of this species. Our conclusions do not disagree. We suggest that compared to other rhizobial species, R. etli populations appear to maintain a more clonal genetic structure. Segovia et al. (1991) investigated the genetic structure of a soil population of non-symbiotic R. etli (then considered as R. leguminosurum). They isolated strains from a single field previously cultivated with beans using a range of selective procedures, and screened those strains showing a phenotype consistent with symbiotic strains of R. etli for the presence of the symbiotic plasmid by means of southern hybridization of a nifH gene probe (a sym-plasmid-borne gene). Using a variety of approaches, including MLEE, RFLP (chromosomal gene probe) and 16s rRNA sequence analysis, they demonstrated that the symbiotic and non-symbiotic (those lacking the sym-plasmid) R. etli strains were indistinguishable, athough the same approaches were able to distinguish between all R. etli strains and other Rhizobium spp. When we analysed their MLEE data for 70 non-symbiotic ETs, we were unable to detected any indication of multi-locus linkage disequilibrium among these ETs (Table 2). Why symbiotic and non-symbiotic populations of R. etli should have an apparently different population structure is unknown. Although the genetic diversity of the non-symbiotic isolates (0.513) is lower than the symbiotic isolates (0.650), they are as genetically diverse as those subgroups of symbiotic isolates where

497

significant levels of linkage disequilibrium were detected (Table 2). The non-symbiotic ETs were isolated from a single field, while the symbiotic strains were isolated from a number of localities in Central and South America. However, these differences in geographic scale may not account for the differences in population structure. Souza ef al. (1992) reported finding significant levels of linkage disequilibrium even among isolates of R. etli taken from individual host plants, as well as host plants grown in the same field. A cautionary note is appropriate at this stage. The index of multi-locus linkage disequilibrium (IA) is not entirely independent of the number of loci examined or the degree of polymorphism at a locus (Brown et al., 1980). Thus, care must be taken when comparing studies that have examined different or differing numbers of loci. The degree to which this lack of independence influences the index of linkage disequilibrium cannot be determined analytically. However, for the data considered in this paper the problem does not appear to be significant. In the case of the MLEE studies examining symbiotic and non-symbiotic isolates of R. etli (15 loci vs 8 loci, Table 2) random sets of 8 loci were selected from the data for the symbiotic isolates and IA was calculated for each set. In no case did a non-significant value of Z* result. Care must also be taken when comparing indices between all strains in a sample and subgroups within that sample. This is a particular problem when the subgroups are defined solely on the basis of the multi-locus enzyme data, as subgroups are often identified as a result of showing monomorphism at 1 or more loci. For example, in the R. meliloti data when all strains are considered together there are 13 polymorphic loci, however, the division B strains are polymorphic at only 8 loci (Table 1). As only polymorphic loci contribute information when calculating the degree of multi-locus linkage disequilibrium, the resulting indices are based on a different number of loci. The data were also reanalysed so that in calculating IA for the total sample only those loci that were polymorphic for the subgroup were used in the calculation; i.e. the 8 polymorphic loci of the division B R. meliloti were used to calculate IA for the total sample of R. meliloti ETs. In no case did this result in a change in the significance of ZA. Maynard Smith et al. (1993) demonstrated the usefulness of partitioning data sets into subgroups as an aid in assessing the genetic structure of bacterial populations. We have presented one reason why the results of partitioning must be interpreted cautiously and Lenski (1993) discusses another problem. Lack of significant levels of linkage disequilibrium among a group of strains may not suggest frequent recombination but may simply reflect a loss of statistical power. For example, the subgroups of the R. leguminosarum bv. trijolii ETs do not show significant levels of linkage disequilibrium, but the values of ZAdeclined only a modest amount compared to the Z, for all strains (0.94 vs 1.30). By contrast, the value of IA for the

498

D. M. Gc rrdon et al.

division B R. meliloti strains declined substantially from the value calculated for all strains (- 0.16 vs 3.95). The analysis of subgroups that results in significant levels of linkage disequilibrium being maintained can be interpreted with much more confidence, as was the case for the R. etli subgroups. Furthermore, conclusions derived from the analysis of subgroups, especially in those cases where the results suggest that recombination is occurring within but not between subgroups, will be much stronger when there is independent evidence that the subgroups are biologically meaningful entities. The results of our study graphically demonstrate the difficulty in predicting the population structure of a particular species, or in understanding the factors responsible in determining genetic structure. Symbiotically-effective populations of R. etli appear to maintain a more clonal population structure, while non-symbiotic R. etli populations are apparently panmictic. Why the presence of the symbiotic plasmid and the resulting ability to initiate an association with legumes should have such a significant effect on population structure is unknown. R. leguminosarum populations would probably show significant levels of linkage disequilibrium, yet within the species, populations of the bv. uiciae appear to be panmictic. while the bv. trifolii seems to maintain a clonal genetic structure. There is no evidence to suggest that the biovars represent true species. Perhaps factors related to host plant specificity and sym-plasmid type are influencing the rates of recombination in these biovars. In their analysis of a number of bacterial species Maynard Smith et al. (1993) concluded that different species may exhibit population genetic structures that range from clonal to panmictic. Our analysis of linkage disequilibrium in a variety of Rhizobium spp leads to a similar conclusion. It appears that within the genus Rhizobium different species experience different levels of recombination, so that while some species or biovars may have a panmictic population structure others are more clonal. Although it is now quite obvious that all bacterial species do not maintain an essentially clonal population structure, the reasons for the large variations in the frequency of recombination among different species of bacteria are unknown. Identifying the factors responsible for this variation should be the subject of further research. Acknowledgements-We wish to thank Natalie Cavanagh for technical assistance and Anthony Brown for helpful discussions. This work was supported by a grant from the Australian Research Council.

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