Electrophoretic studies of genetic variation in natural populations of allogamous Hedysarum capitatum and autogamous Hedysarum euspinosissimum

Plant S ~ e .

69 (1990) 4 9 - 6 4

49

Elsevier Scientific Publishers Ireland Ltd.

ELECTROPHORETIC STUDIES OF GENETIC VARIATION IN NATURAL POPULATIONS OF ALLOGAMOUS HEDYSARUM CAPITATUM AND AUTOGAMOUS HEDYSARUM EUSPINOSISSIMUM

H. BAATOUT'. M. MARRAKCHI~ and J. PERNES"

*Laboratoire d'Hi~tologte.Embr~yologte et Biologte CeUulaire. Facult~ de M~decine. 9 Rue Zouheir ESSAFI. 1006. Tunis. ~Laboratoire de G~n~tique. Facult~ des Sciences. 1060. Tunu fTuniJial and 'Universit~ Paris Sua~ Centre d'Orsay /France/

(Received October 2nd. 19891 (Revision received January 2nd, 19901 (Accepted January 23rd. 19901

Population genetic structure in Hedysarum spinosissimum Ls.L. was analyzed in fifteen natural populations from the western mediterranean basin. Starch gel electrophoresis was used to examine genetic variation within and among the diploid 12n = 2x = 16) widespread subspecies H. cap~tatum (alJogamous}and H. eu~pimosissimum iautogamous}. Allozyme variation at 13 loci detected for seven enzyme systems showed H. capitatum to be highly polymorphie in contrast to the monomorphism within 14. euJpinosissimum populations. The average number of alleles per locus in 11. capitatum and H. c~pinosissimum was 1.37 and 1.15 respectively, and on average, 11. capitatum and 11. eu~pmostssimum populations presented 23.10% and 11.55% loci with polymorphism, respectively. The autogamous species has less variation per population than the allogamous one with 0.055 as mean heterozygosity in tt. et~zpim)&izsimumv e r s u s 0.100 in H. cap~tatum. Several populations of I1. euJpinosizsimurn have a high rate of heterozygosity as found in H. capitatum populations. The mean genetic distance between pairs of populations were 0.023 in H. capitatum and 0.007 in H. eu~p~nosi~simum. In addition, principal components analysis revealed that few populations were clustered into regions representing the geographical origin. Key words: Hedysarum; isozymes: electrophoresis, populations; genetic diversity

Introduction The genus H e d y s a r u m (leguminosae) contains various species distinguishable by different morphology, m a t i n g s y s t e m s , biological cycles and geographical origins [1]. A m o n g the species of the m e d i t e r r a n e a n group, the diploids (2n = 2x = 16) 1t. c a r n o s u m L., H. c o r o n a r i u m L., 1t. f l e x u o s u m L., 1t. spinosiss i m u m L. subspecies c a p i t a t u m D e s f and e u s p i n o s i s s i m u m Briq are the m o s t widely s p r e a d in the w e s t e r n basin, w h e r e t h e y grow wild. T h e s e diploids are nutritious and highly palatable to s h e e p on the basis of morphological f e a t u r e s and can r e p r e s e n t an agricultural advantage. Up to now v e r y little is known a b o u t the e x t e n t of genetic variability p r e s e n t in each

species, a b o u t the organization of genetic variability and a b o u t the p h y l o g e n e t i c relationships b e t w e e n the species within the g e n u s H e d y s a rum. As m a t i n g s y s t e m s h a v e i m p o r t a n t factors d e t e r m i n i n g the genetic composition of population, it has been d e m o n s t r a t e d t h a t s e v e r a l ecological p r e s s u r e s a r e e x p e c t e d in o r d e r to c o n t r i b u t e to high intrapopulation variability and to r e s t r i c t the accumulation of genetic differences b e t w e e n populations [2]. S e p a r a t e d a s p e c t s of H e d y s a r u m biology have been considered s e p a r a t e l y in earlier studies. T h e y concern the m o r p h o l o g y and floral biology of the plants, their geographical and ecological distributions, and some of their biochemical characteristics. T h e s e plants all possess flowers of the papillionacea t y p e and articulated cloves. S e v e r a l ecological studies,

0168-9452/90~T03.50 ~- 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

50 followed by genetic evaluation studies, were started to improve these species by breeding: Tunisia 1975; southern France 1976, 1977; Morocco 1979; Tunisia and Algeria 1981; Sardinia and Malta 1983. In a recent study of biochemical characteristics of Hedysarurn species, two types of acyanic mutants in H. coronarium and H. capitatum have been described 13]. Variability in carboxylic esterases has been shown to occur in natural populations of H. coronarium 14] and H. carnosum 151. A large genetic variability was found within each diploid species [1,6-9]. Previous results of comparison of the mitocbondrial DNA restriction patterns indicate that Pstl, BamHI, SrnaI, and EcoRI restriction patterns from the subspecies H. capitatum and H. euspinosissimum possess differences limited to one restriction site [10l. Other results from studies on Hedysarum species populations show that gene exchanges among the various compartments of known species complexes are restricted [7,9]. Among the species of the mediterranean group, H. spinosissimum L. subspecies capitaturn Desf and subspecies euspinosissimum Briq are widely distributed in the western basin, where they grow wild. The present electrophoretic study was undertaken to investigate the genetic structure of natural populations of the two morphologically related subspecies H. capitatum and H. euspinosissimum using allozymes as genetic markers. The subspecies capitatum is predominantly cross-fertilizing, noticeable by 6 - 1 2 big, violet corolla flowers per inflorescence, and pollinated almost exclusively by lepidopterans 13,7] and the subspecies euspinosissimum is predominantly self-fertilizing producing cleistogamy associated with 3 - 6 small, pale pink corolla flowers per inflorescence and little quantity of seed production in the absence of pollinating insects [7}. in this paper we provide an estimate of the genetic variation within and between natural populations of H. spinosissimum L. Our objectives are to describe the amount of variation within populations as measured by the average level of heterozygosity and the proportion of polymorphic loci. In addition genetic dif

ferentiation between populations and the genetic relationships among populations of the two morphologically related H. capitatum and H. euspinosissimum are examined. Materials and Methods

Plant material Allozymic variation was analysed in 11 populations of H. capitatum and 4 populations of H. euspinosissimum from Tunisia, Algeria, Morocco, Sardinia and Malta (Fig. 1~. The sites were chosen on the basis of avaiable seed, with the objective of sampling each subspecies as completely as possible with respect to its geographical distribution. Each population sample was an assemblage which contained several hundreds of seeds harvested in a widespread area during the different prospection programmes (Tunisia 1975, 1977, 1985; Morocco 1979; Algeria 1981; Sardinia and Malta 1983, data not yet publishedl.

Extraction of enzymes Extraction procedures, gel and buffer compositions have been described previously [11,12]. Electrophoresis was performed on seedlings approximately 3 - 4 days old following germination in Petri dishes at 25°C in the dark. For every single seedling, the tissue was handground in 250 ~i of extraction buffer [Sodium ascorbate buffer (pH 8.41 made up of 8.3% sodium ascorbate, 16.7% t~-saccharose and 0.03% 2-/~-mercaptoethanol] with a prechilled pestle and mortar at 4 °C. The homogenate was centrifuged at 4°C at 30 000 x 9 for 15 rain. The buffer was made up in sucrose to provide density.

Electrophore tic procedures The seven enzymes (Table l~ were separated on 13o/o (w/v~ horizontal starch gels electrophoresis using two buffer systems. System I was composed of buffer A (0.04 M lithium hydroxide. 0.19 M boric acid. pH 8.3) and buffer B (0.05 M Tris. 0.007 M citric acid monohydrate, pH 8.31. The gel buffer was composed of nine parts

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Fig. I. Approximate locations of the populations: Populations of If. capstatum [I, NAC (TunisiaL 2. KO (Tun(siaL 3. BI (Tun( sial 4. Kef (Tunisia}. 5. SPL (Tunisia}, 6, AL 31 (Algeria}. 7. AL 9 (Algeria}. 8. MM I 1 (Morocco}. 9. SA 13 (SardiniaL I0. SA 20 (Sardinia) and 1 I. MA 07 (Malta)] are represented by dark circles ( • L and the populations of H. eusp/nos/ssim um {12. J 1 (Tun( sial 13, GBI {TunisiaL 14, BK 1 (Tun,sia} and 15. AL 33 (Algeria}] are represented by dark triangles {A).

buffer B and one part buffer A, while the electrode buffer consisted only of buffer A. System I was used to resolve ADH and GOT enzymes. System II consisted of an electrode buffer (0.065 M L-histidine, 0.007 M citric acid monohydrate, pH 6.5) and a gel buffer corn posed of one part electrode buffer and three parts distilled water. This system was utilized for resolving PGM, PGI, ICD, MDH and 6PGD enzymes.

The crude protein extracts {10 ~1) were absorbed on 3 × 12 mm wicks of Whatman 3 MM chromatography paper and inserted into slits in the gels (24 per gel). Electrophoresis was performed horizontally with ice on the gels to prevent overheating. Electrophoresis was terminated after the tracking dye front had moved 4 cm toward the anode, and it was conducted at 40 mA (constant current) for 6 h at 4 °C for the buffer system I. For the buffer sys-

52 tern II, the electrophoresis was carried out at 4°C for 7 h at 16 W (constant power). After electrophoresis, the gel was cut horizontally and each slice was stained for a particular enzyme. The procedure for staining ADH activity was from Scandalios [13]. The stain procedure for 6 PGD was described by Stuber et al. [14]. Procedures described by Cardy et al. [15] were used for MDH, PGM, PGI and ICD. and the staining procedure for GOT was from Goodman et al. [16].

A llozym e nomenclature The bands of each zymogram were interpreted in terms of loci and alleles, and their frequencies were estimated. The naming of the bands was done according to the methods of Salanoubat and coworkers [11,12]: letters are used to designate the gene coding for the enzyme. The locus specifying the least anodal form is designated A, the next B, etc. At each locus, the allele with the least mobility has been arbitrarly named l, the next is 2, and so forth.

Data analysis Three parameters were estimated from the data on allelic frequencies determined for each individual in both H. capitatum and H. euspinosissirnum populations: mean number of alleles per locus and mean heterozygosity per locus have been measured by Nei's parameters [17], the percentage of polymorphic loci was estimated by Cavalli-Sforza's parameters [18]. Estimates of genetic relationships (between populations and among taxa) were calculated for each pair-wise comparison of populations by measuring genetic distance (D} using these methods [17,19-21]. Dendrograms were constructed by using WPGMA algorithm [22] (WPGMA, weighted pair group method using arithmetic averages). The distribution of genetic variation within and among the populations was analysed using F-statistics [19,23] as calculated by BIOSYS-1 [24]. The genetic population parameters used in this study were calculated by the BIOSYS-1 program with little modifications and adapted on I.B.M. 370.168 at the Centre Inter R6gionai de Calcul Electro-

nique (C.N.R.S., d'Orsay).

Universit6

PXI-Centre

Results

Loci The allozyme data presented in this study are based on seven enzyme systems (Table I). Thirteen loci were interpreted: one locus each for PGM (PGMA), PGI (PGIA), ICD (ICDA) and ADH (ADHA); three loci each for MDH (MDHA, MDHB, MDHC), 6 PGD (PGDA, PGDB, PGDC) and GOT (GOTA, GOTB, GOTC). The observed enzyme bands migrated anodally for all enzymes. ADHA, MDHB and MDHC loci were monomorphic in all seedlings examined in all populations of H. capitatum and H. euspinosissimum. The other twelve loci were polymorphic in at least one seedling per population. Allelic frequencies at the 13 variable loci in the fifteen populations of H. capitatum and H. euspinosissimum are given for each population in Table II. A total of 28 alleles were detected for all loci. Observed allelomorphs per polymorphic locus varied from two (PGMA, MDHA, 6PGDC, GOTA, GOTC) to three (PGIA, ICDA, 6PGDB, GOTB).

Table I. Abbreviations. structure and enzyme commission (E.C.) recommended name of enzyme systems analysed by horizontal starch gel electrophoresis.

Abbrevi. Subunit E.C. recommended ation structure name

E.C. No.

PGM PGI

2.7.5.1 5.3.1.9

ICD ADH MDH 6-PGD GOT

Monomer Phosphoglucomut&se Dimer Phosphoglucose isomerase Dimer Isocitrase dehydrogenase Dimer Alcoholdehydrogenase Dimer MalatedehydrogenL,e D i m e r 6-Phosphogluconate. dehydrogenase Dimer Glutamicoxalictransaminase

1.1.1.42 1.1.1.1 I.I.I.37 1.1.1.40 2.6.1.1

53

Genetic interpretations of enzyme phenotypes Interpretations of enzyme phenotypes are based on banding patterns of variability in all populations studied {Fig. 2}. Alcohol dehydrogenase fADHL One zone of enzyme activity was detected on gels stained for ADH in H. capitatum and H. euspinosissimum. One zone with 3 bands was rarely observed in H. coronariurn [4]. ADH was dimerie and encoded by one locus, and presented an intragenic heterodimer formed between the products of the 2 alleles detected. This dimeric structure is in agreement with that observed in a number of plant species [4,5,13,25- 30].

Malate dehydrogenase fMDHL H. euspinosissimum MDH banding patterns are complex. Three zones of activity (MDHA, MDHB and MDHC) were found on gels stained. MDHB and MDHC were monomorphie. Locus A appeared polymorphic with two alleles. Both intragenic and intergenic heterodimers were formed between the products of these three loci. MDH is usually encoded by many enzyme loci in other plant species [4.5,15,30- 36). Isocitrate dehydrogenase flCD). ICD showed only one zone of activity. The zymotypes, either one or three banded, found in diploids were consistent with the existence of at least three alleles and a dimeric structure for this enzyme. One staining region of activity is observed for ICD with a single band which is interpreted tentatively as a single monomorphic locus in H. euspinosissimum and slightly polymorphic with three alleles in H. capitatum. Heterozygous ICDA individuals showed three banded phenotypes, indicating that this locus codes for a functionally dimeric protein, as found in other species studied [5,12,34,36- 41].

6-Phosphogluconate dehydrogenasc ¢6-PGDI. H. capitatum and H. euspinosissimum banding patterns are complex. Three staining regions were apparent. It appeared that three loci govern the expression of 6-PGD in H. capitatum and H. euspinosissimum. There were three alleles, which in heterozygotes interact to form three banded phenotypes at the 6-PGDA and 6PGDB locus, respectively. At locus C, two

alleles were detected. The positions of the bands were consistent with two or three alleles at the loci and a dimeric structure of the enzyme with intragenic and intergenic interaction. Other high plant species have been shown 6-PGD interlocus dimerization [4,16,30,36,3845]. Glutamic oxalic transaminase IGOT). Three regions of staining were observed in gels stained for GOT and probably correspond to the GOTA, GOTB, and GOTC loci. Two, three and two alleles were detected for GOTA, GOTB and GOTC, respectively. The dimeric structure of GOT could be seen in the three banded pattern of heterozygous phenotypes. Other plant species have been shown to possess a dimeric structure for GOT with intra and inter loci interaction [4,5,39,46,47]. Phosphoglucomutase (PGMI. One zone of activity was observed for PGM in all material studied, which correspond at locus PGMA with two alleles. The rare heterozygous individuals of H. capitatum displayed two banded monomerie patterns. In other diploid plant species similar results have been found for the monomerie structure of PGM [30,32,34 - 36,38,48 - 511. Phosphoglucose isomerase (PGI). PGI exhibited one zone of enzymatic activity: PGIA locus. Several individuals have provided three banded heterozygous phenotypes, indicating that the PGIA locus codes for a functionally dimerie protein, as found in several other plant species studied [5,31,34 - 38,41.52 - 55]. The genetic analysis of enzyme banding patterns of GOT, 6-PGD and PGI were verified by examining the segregation ratios in the selfed progenies and have been confirmed by TrifiFarah 141.

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57 number of alleles per locus (A) per population, was 1.37 in H. eapitatum (range: 1.10 - 1.60} and 1.15 in H. euspinosissimum (range: 1.10-1.20). An average of 23.10 (range: 7.70-38.501 and 11.55 (range: 7.70-15.40} of loci were polymorphic per population in H. capitatum and in H. euspinosissimum, respectively; and an average of 0.100 (range: 0.039-0.158) and 0.055 (range: 0.039-0.078) of mean heterozygosity were detected in H. capitatum and H. euspinosissimurn, respectively. The taxon H. capitatum appear to have a greater diversity of alleles, a higher proportion of polymorphic loci and a higher mean of heterozygosity than the noncompletely monomorphic taxon H. euspinosissimum which shows no strong differences between the population variabilities. Across all populations of the allogamous H. capitatum, Fig. 3 shows high heterozygozity per locus per population observed at loci PGDA, GOTB and GOTC in 'NAC' population; at locus GOTA in 'KO'; at GOTA, GOTB and GOTC in 'BI'; at PGDA, GOTA and GOTC in 'Kef; at GOTA and GOTC in 'SPL'; at ICDA, PGDA, PGDB and GOTA in 'Al 31'; at PGIA, PGDA, and GOTA in 'AL 9'; at GOTA in 'MM 11'; at GOTA and GOTB in 'SA 13'; at PGDA, GOTA, and GOTB in 'SA 20'; and at PGDA and GOTA in 'MA 07' population. However populations of the autogamous H. euspinosissimum have shown high heterozygosity only at locus GOTA in four populations, and at locus PGDA in two populations.

Population differentiation Some additional analyses were performed to further examine the nature of population differentiation: Genetic distance. Nei's [17,20] genetic distance (D) was calculated for every pair-wise comparison of populations at 13 loci and 28 alleles. Table IV summarizes genetic distance (D) [(D) = - Log I] values among all populations examined within and between the two subspecies H. capitatum and H. euspinosissimum. The values of (D) represent the extent of difference in allele frequencies summed over loci between populations. For H. capitatum populations, (D) measures vary from 0.000 (between 'KO' -

'MM 113 to 0.081 (between 'NAC' - 'AL 31'). For H. euspinosi~simum populations, (D) values range from 0.000 (between 'AL 33' - 'GBI') to 0.066 (between 'NAC' - 'AL 33'). Mean (D) values for H. capitatum populations and for H. euspinosissimum populations are 0.023 and 0.007, respectively. Mean distance measure detected between subspecies H. capitatum and H. euspinosissimum is 0.015. For example results of Nei's genetic distance were represented diagramatically in a dendrogram in Fig. 4.

Genetic variation within and amon 9 populations. F-statistics. [19-231 were calculated to analyse genetic structure within and among populations: a high genetic differentiation has been found among populations at PGIA, PGDB, GOTB and GOTC loci. The mean Fsr equal to 0.022 [representing differentiation between populations) indicated slight genetic heterogemeity among the fifteen populations studied. Principal components analysis. Results of principal components analysis (A.C.P.) was presented [56]. The input data matrix consisted of allele frequencies for 28 alleles in 15 populations. Population relationships were visualized by projection of population positions in 28 dimensional space using a plane defined by the first two principal components, as illustrated in Fig. 5: the first two principal components accounted for 22.36% and 19.32°/0 of the total variance, respectively. The notable feature of Fig. 5 is the apparent clustering of H. euspinosissimum populations into one group ('J 1' - 'BKI' and 'GBI' - 'AL 33'1 relatively representing the geographic areas South Tunisia and Center-East Algeria. This group is noticeable by a low genetic differentiation between populations which have almost overlap in their variability ranges. However, in the taxon H. capitatum the degree of genetic differentiation between populations is distinctly higher and it is not correlated with geographical distance. Thus, the populations tended to cluster into groups representing the geographic areas [North Tunisia ('NAC')]; [North-West Tunisia ('Kef) - East-Center Tunisia ('SPL')]; [North Tunisia ('BI') - North-East Tunisia ('KO') North-East Morocco ('MMll'); [West Algeria

58

Table 111.

Genetic variability at 13 loci in all populations (S.E. in parentheses}.

Population

Mean sample size per locus

Mean no. of alleles per locus

% of loci polymorphic"

Mean hetero zygosity HDYWBG expected'

26.0 (0.0) 24.0 (0.0)

1.6 (0.2) 1.4 (0. I )

30.8

0.135 {0.061 ) 0.073 (0.040)

15.0 (0.0) 48.0 (0.0)

1.3 {0.2) 1.6 (0.2)

23.1

48.0 (0.0)

1.5 (0.2)

23.1

0.080 (0.042}

26.0 (0.0) 26.0 (0.0) 24.0 (0.0)

1.5 (0.2) 1.5 (0.2) 1.1 (0.1)

38.5

0.158 (0.064) 0.117 (0.051) 0.039 (0.039)

24.0 (0.0)

1.2 (0.1)

15.4

0.076 (0.0521

24.0 {0.0)

1.2 {0.1 )

23.1

0.098 {0.052}

26.0 (0.0~

1,2 (0.2)

15.4

0.079 (0.055)

23.10

0.100

0.065 (0.045) 0.040 {0.040)

HedyJa~m capitatum 1. NAC (North Tunisia) 2. KO (North-east Tunisia) 3. BI {North Tunisia) 4. Kef (North-west Tunisia) 5. SPL (F.,ast ~ e n t e r Tunisia) 6. AL31 (West-Algeria) 7. ALC9 (North-Algeria) 8. MMI 1 (North-east Morocco) 9. SA 13 (South-Sardinia Island) 10. SA20 (South-centerSardinia} I I. MA07 (Island Malta) Mean"

1.37

23.1

23.1

30.8 7.7

0.102 (0.055) 0.I 12 (0.055)

Hedysarum eu~p~nosU#imum 12. J 1 (Jerba peninsula) 13. GBI (south-east. Tunisia) 14. BK 1 {South Tunisia) 15. AL33 (E~st-eenter Algeria) Mean"

72.0 (0.0) 15.0 (0.0)

1.2 (0.1) 1.1 (0.1)

15.4

24.0 (0.0) 24.0 (0.0)

1.2 (0. I 1.1 (0.1)

15.4

1.15

7.7

7.7

11.55

' A locus is considered polymorphic if the frequency of the mo~t common allele does not exceed 0.95. • Unbiased estimate {see NEI. 1978). • Mean value over all populations.

0.078 (0.053) 0.039 (0.039}

0.055

59

Heclyr,a r u r n

eu-sp,nostssirnurn

populal,on AL33

I

,K,

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ro

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Fig. 3. Schematic representation of heterozygosity per locus per population at 13 loci investigated (H = rate of heterozygosity).

['AL 31'] - South-Center Sardinia ('SA20')I; [North Algeria ('ALC9')]; [South Sardinia Island 1'SA13') - Malta Island ('MA07')].

Discussion u d Conclusions The information obtained in this study on

population structure in H. spinosissimum subspecies H. capitatum and subspecies H. eusp/nosissirnum is consistent with data for most angiosperms. Outcrossing populations exhibit a high degree of intrapopulation genetic structure, and the most highly inbreeding population maintains negligible genetic variation. The mean number of alleles per locus was 1.37 for the allogamous subspecies versus 1.15 for the autogamous subspecies. Not surprising, the selfing subspecies H. euspinosissimum has less variation per population than the outcrossing subspecies H. capitatum: mean heterozygozity in H. euspinosissimum was 0.055 versus 0.100 in H. capitatum. Percentage of loci polymorphic is 23.1 for H. capitatum versus 11.55 for H. euspinosissimum. The allogamous subspecies tend to have a greater diversity of alleles and higher proportion of polymorphic loci than the autogamous subspecies. Thus, the high level of variation within the populations of H. capitatum is not surprising in light of the high outcrossing rates revealed by mating system analysis [3,7]. The mean heterozygosity per locus per population (estimated on the basis of all loci} is of interest. Although the mean is lower for H. euspinosissimum (0.055) compared to 0.100 in H. capitatum, there are populations of H. euspinosissimum which have a high rate of heterozygozity, as found in H. capitatum populations. Consequently, the selling H. euspinosissimum is not completely monomorphic, and the outcrossing H. capitatum could not be easily distinguished from H. eusp/nosissimum with the loci examined. Of the 13 loci considered, 6-PGD, GOT and slightly MDH were polymorphic throughout the taxa of H. euspinosissimum studied. Previous electrophoretic studies, high values of (i} mean number of alleles per locus, (ii} percentage of loci polymorphic and (iii) heterozygosity have been found in H. coronarium and H. carnosum [51. However the two subspecies H. capitatum and H. euspinosissimum exhibit less higher genetic variation. High levels of autogamy in H. eusp/nosissimum [7] appear to be associated with a depression in the amount of heterozygosity in population of H. euspinosissimum by compari-

I I MA07 12Jl 13 GB 1 14BK 1 15AL33

I NAC 2 KO 3 BI 4 Kef 5 SPL 6 AI.,31 7 ALC9 8MMII 9SA13 I0 SA20

Population

1l E

HC

1

0.060 ....

2 0.053 0.006 ....

3 0.042 0.010 0.013 ....

4 0.049 0.002 0.005 0.006 ....

5 0.081 0.032 0.041 0.028 0.031 ....

6 0.057 0.010 0.018 0.004 0.008 0.025 ....

7 0.066 0.000 0.006 0.013 0.003 0.033 0.012 ....

8 0.029 0.028 0.019 0.030 0.021 0.071 0.043 0.031 ....

9 0.049 0.006 0.008 0.000 0.004 0.028 0.004 0.009 0.031 ....

10 0.047 0.005 0.015 0.005 0.005 0.026 0.003 0.006 0.039 0.003 ....

11

M a t r i x of g e n e t i c d i s t a n c e coefficients. A b o v c diagonal: N E I {1978) u n b i a s e d g e n e t i c d i s t a n c e . HC.

e~.spinosissimum.

Table IV.

0.059 0.002 0.011 0.005 0.003 0.025 0.003 0.003 0.036 0.003 0.001 ....

12

0.065 0.000 0.006 0.013 0.002 0.033 0.012 0.000 0.031 0.008 0.006 0.003 ....

13

0.060 0.014 0.026 0.004 0.012 0.025 0.001 0.017 0.051 0.005 0.004 0.004 0.017 ....

14

0.000 0.017

0.003

0.066 0.080 0.006 0.013 0.003 0.033 0.012 0.000 0.031 0.009 0.006

15

lledysarum capitatum: HE. Hedysarum

.

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0.031

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0.03

0 .+04 - - • . . . . . .O. . . . .0. .3. .

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0.04

Cluster analysis using weighted pair group m~.thod. Coefficient used: Nei 11978~ grnetic distance.

.

Fig. 4.

.

• . . . . 0.• 0. .9. .

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DISTANCE .

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0 .+0. .2. . . . . . ~"

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0.02 .

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IliqE; pl 0.006

4. .

62

AXIS 2 19.32~

'0 AXISI 22.36~

'e

Fig. 5.

Graphical representation of principal components analysis of allozyme frequencies from I I populations of H capita.

11. NAC (Tunisial. 2. KO (Tunisia). 3. BI (Tunisia). 4. Kef (Tunisial. 5. SPL (Tunisia}. 6. AI. 31 (Algeria~. 7. AI. 9 (Algeria1.8. MM 11 (Morocco).9. SA 13 tSardinia). 10. SA 20 (Sardinial and 11. MA 07 (Malta)]and 4 populations of !!. euspinosissimum [12. Jl (Tunisia). 13. GBI (Tunisia). 14. BK1 (Tunisia) and 15. AL 33 (Algerial]. Each symbol represents the position of a population in 28 dimensional space projected on to a plane defined by the first two principal components. turn

son with o u t c r o s s e r 1t. capitatum. Heterozygosity deficiency m a y be due to i m b r e e d i n g and n o n r a n d o m m a t i n g which m a y be responsible for the low levels of genetic variation. To sum up the r e s u l t s of this study, the outcrossing subspecies 1t. capitatum a p p e a r s to have g r e a t e r allozymic differentiation within and b e t w e e n populations than the selfing subspecies H. euspinosissimum. T h e e s t i m a t e s of genetic distance (D} a r e not c o m p l e t e l y geographically d e p e n d a n t in H. capitatum and in 1t. euspinosissimum: T h e y show low (D)'s b e t w e e n geographically d i s t a n t populations and large (D)'s b e t w e e n short g e o g r a p h i c distances a m o n g populations. In conclusion, with

the added f e a t u r e s of isozymes a n a l y s e s of more loci and population genetic information. we can e x a m i n e additional variability and allele frequency p a t t e r n s for the role of the b r e e d i n g s y s t e m and can elucidate phylogenetic relationships in 1t. spinosissimum s u b s p e c i e s capitaturn and subspecies euspinossimum.

Acknowledgements We t h a n k M. S a n d m e i e r for e l e c t r o p h o r e t i c technical assistance, E. N g u y e n Van for c o m p u t e r analyses and Prof. J.C. Mounolou for d i s cussion, advice and helpful c o m m e n t s in i n t e r p r e t a t i o n s of results. This work was

supported by grants from the Agence de Coop6ration Culturelle et Technique (A.C.C.T.), the Centre National de Recherche Scientifique (C.N.R.S.) and the International Foundation for Science (F.I.S.).

13

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