Discriminant alleles and discriminant analysis: efficient characters to separate closely related...

Botanical Journal of the Linnean Society (1991), 107: 139-161. With 3 figures

Discriminant alleles and discriminant analysis: efficient characters to separate closely related species: the example of Lathyrus Zutifolius L. and Lathyrus sylvestris L. (Leguminosae)

MYRIAM VALERO

Laboratoire de Gknnttique Ecologique et de Biologie des Populations Vbgbtales, URA CNRS 1185, U S . T.L.F.A., F-59655 Villeneuve d'Ascq cedex, France

AND

MARTINE HOSSAERT-MCKEY *

I.B.E.A.S, URA CNRS 1339, Universitt! de Pau et des Pays de l'ddour, Avenue de l'UniversitL, 64000 Pau, France

Received August 1989, accepted for publication January 1990

VALERO, M. & HOSSAERT-MCKEY, M., 1991. Discriminant alleles and discriminant analyda: efficient characters to separate closely related species: the e m p l e of hatliytwa laatifoliam L. and Latliytwa sylvcstris L. (Leguminosae). Two species of Lathyrus (L . latifolius and L. sylvestris) were compared using allozymes and quantitative vegetative and reproductive traits. The variation in these characters was analysed in several populations of the two species located in south-western, central and northern France. In a site where the two species are sympatric, intermediate plants are described. The two species can be identified by discriminant alleles. The genetic structure observed for intermediate plants in the zone of sympatry indicates, paradoxically, the occurrence of a strong reproductive barrier between the two species. The discriminant analysis performed on the quantitative traits readily distinguishes the two species and confirms the reproductive isolation between them. The quantitative character that best discriminates between the species is the number of ovules per ovary, a variable which has non- overlapping distributions in the two species. We discuss how this character, usually ignored, may be better for distinguishing species than some traditionally used reproductive traits such as the number of seeds per fruit. The vegetative traits studied (e.g. degree and intensity of branching) also differ significantly between the two species. Divergent evolution of these traits in the two species is discussed in relation to differences in the original habitats of the two species.

ADDITIONAL KEY WORDS:-Multivariate analysis - breeding systems - ovule and seed production - taxonomic characters.

*Present address: Department of Biology, University of Miami, PO Box 2491 18, Coral Gables, FL 33124,

139 USA.

0024-4074/91/100139 + 23 $03.00/0 0 1991 The Linnean Society of London

140 M. VALERO AND M. HOSSAERT-MCKEY

CONTENTS Introduction . . . . . . . . . . . . . . . Material and methods . . . . . . . . . . . . .

lnfrageneric systematics of Lalhyrus . . . . . . . . Study species and study sites . . . . . . . . . . Methods of allozyme analysis . . . . . . . . . . Study of morphological characters . . . . . . . . . Statistical analysis . . . . . . . . . . . .

Results . . . . . . . . . . . . . . . . Genetic studies . . . . . . . . . . . . . Differentiation of the two species by morphological characters . . Morphological characters in contact zones . . . . . . .

Discussion . . . . . . . . . . . . . . . . Are the two species reproductively isolated? . , , . , .

. . 140

. . 141

. . 141

. . 142

. . 142

. . 142

. . 144

. . 145

. . 145

. . 150

. . 152

. . 154

. . 154

. . 154 Which characters are useful in differentiation between species in the field? What is the contribution of plant population biology in the characterization of species? 158

Acknowledgements . . . . . . . . . . . . . . . . . 159 References . . . . . . . . . . . . . . . . . . . 160

INTRODUCTION

The taxonomic significance of reproductive characters for distinguishing among families and among species was pointed out by Ornduff (1969). He emphasized: “It is important that we systematists should be fully aware of the morphological patterns that are associated with different reproductive systems. Such an awareness can immeasurably aid us in making intelligent taxonomic assessments and also can lead to an understanding of much of the floral variation that provides the raw material for taxonomic classification”. The magnitude of variation for these characters at the species level has been addressed recently by Waser & Price (1984) in Ipomopsis aggregata (Polemoniaceae). These authors criticize the common practice of using reproductive structures including flowers, fruits and seeds as diagnostic taxonomic characters in higher plants. These characters “are taken ips0 facto as fixed within a taxon such as a species, subspecies, or ecotype”. Although within-population variation in these traits is glossed over in many systematic treatments, geographic variation evokes considerable debate about the taxonomic status of each variant. In Ipomopsis aggregata, morphological variability in reproductive structures occurred within populations and affected the relative fitness of individuals.

In regard to this consideration, the main focus of this paper is 1. to investigate, within several natural populations, new characters that can be used to distinguish Lathyrus LatifoLius and Lathyrus. sylvestris, two closely related Leguminosae sometimes distinct only at the level of subspecies (Fournier, 1946; Foury, 1954) and 2. to discuss these characters in light of differences in the reproductive system of the two species (Valero, 1987; Hossaert & Valero, 1988). Two kinds of characters are considered, allozymes and quantitative reproductive and vegetative traits. Allozymes are good markers of gene flow and reveal the occurrence of reproductive isolation (Gottlieb, 1984; Crawford, 1985; Valero & Olivieri, 1985), although allozymes underestimate genetic variation and sample only genes involved in basic metabolism (see Brown, 1979 and Gottlieb, 1981 for review). However, allozymes cannot be used to identify individuals to species in the field; morphological characters are needed for distinguishing species in the field because they allow a quicker identification. Finally, we discuss how some

DISCRIMINANT CHARACTERS IN LATHYRUS 141

reproductive characters usually ignored (e.g. number of ovules) may be better for distinguishing species than some traditionally utilized characters (e.g. number of seeds).

MATERIAL AND METHODS

Criteria classically used in Floras to distinguish between the two species concern differences in flower colour and in sizes of stems, stipules, leaves, flowers and pods (Coste, 1901; Bonnier, 1934; Fournier, 1946; Foury, 1954; Clapham, Tutin & Warburg, 1962; Tutin et al., 1968; Delanghe et al., 1978). Those traits do not clearly discriminate between the two species because their distributions widely overlap (Tutin et al., 1968; Delanghe et al., 1978). Based on the criteria described in Floras, an individual of L. la t i f l ius observed in south-western France presents the same characteristics as a plant of L. sylvestris found in northern France (Valero, 1987; Hossaert-McKey, 1988).

None of the traits used by Kupicha (1983) in a systematic revision of the genus are useful in differentiating these two closely related species.

Whereas it is easy for someone regularly working on morphologically similar plant species to distinguish between them because he has integrated a number of covarying criteria, it is generally difficult for another worker who is unfamiliar with species to differentiate them. Our approach consists of a study of several populations that we have classified, a priori (because of our ‘experience’), as L. lat$olius or L. sylvestris. We then test a posteriori the capacity of measured characters to differentiate both groups of populations. Finally, we examine results from the only site we found where the two species are sympatric. Here we have found individuals (‘intermediate’ plants) presenting some of the characteristics of each species. The position of intermediate plants for the characters measured will be of special importance in testing the validity of our classification.

Infrageneric systematics of Lathyrus

In the genus Lathyrus, c. 150 species have been described (Allkin et al., 1983). Most of the species are diploids with the same basic chromosome number ( 2 n = 14). They are mostly distributed in temperate zones of the northern hemisphere and their centre of diversification lies in the Mediterranean region (Allkin et al., 1985). The most recent classification of this genus was published by Kupicha in 1983. All the principal characters she used to define 13 sections within the genus are morphological: anatomy of stipules and nodes; number of leaflets per leaf and number of flowers per inflorescence, leaflet venation and the structure of leaf epidermis. The kind of venation (parallel or pinnate) and the number of leaflets are both major basic criteria in current systematic treatment of the genus (Simola, 1968; Kupicha, 1983). Evolution in the genus is supposed to have proceeded from pinnate towards parallel venation and simultaneously towards a decreased number of leaflets and flowers per inflorescence. Perennial life histories are considered primitive within the genus, and the annual habit derived.

The secondary characters that Kupicha described are of three types: 1. qualitative (presence or absence of tendrils); 2. quantitative (size

I42 M. VALERO AND M. HOSSAERT-MCKEY

characteristics: chromosomes, pollen grains and seeds), and 3. biochemical (flower colours and non-protein amino acids; Bell, 1962). Kupicha (1983) placed the two species of the present study into the same section, Lathyrus subgenus Lathyrus group sylvestris.

Study species and study sites

The individuals of the two species are bushy vines 1 to 2 m high. They are found in disturbed habitats with calcareous and stony soil: mountain screes, roadsides and railways. Nevertheless, they differ in their geographical distribution: L. sylvestris is found in all of northern Europe, but only rarely in the Mediterranean basin; it is found in low-elevation sites as well as in montane sites to 1700 m. Lathyrus latifolius is very frequent in the Mediterranean region, and the northern limit of its distribution is in northern France. This species is rare at high-elevation sites. The populations are patchily distributed over the landscape. The two species are very rarely found together, despite the fact that they occur in the same type of habitat. The populations are often quite restricted in size (from 10 to 3000 individuals) and even within populations, individuals are generally patchily distributed (Hossaert-McKey, 1988).

Several natural populations distributed from northern to south-western France (Btarn) were studied for both species (Table 1): four populations of L. latifolius (L1 to L4: L as latifoolius), eight populations of L. sylvestris (S1 to S8: S as fylvestris) and one site where both species are sympatric. In this site, individuals of L. latzfolius (population CL: as ‘contact latifolius’) are escaped from an old garden which is surrounded by a natural population of L. sylvestris (CS: as ‘contact sylvestris’). The population CS is very large and spreads over 2 km in discontinuous patches. Two sites of sampling were chosen; one near L. latzfolius (site CSa) and the other 1 km away from the contact zone (site CSb). Some individuals appearing to be intermediate between both species (population CI: as ‘contact intermediate’) were observed. These individuals (CI) were similar to L. latifolius (CL) with regard to their vegetative system, but have flowers of the same size, number and colour as those of L. syluestris (CS). Most of the populations studied are found in the same type of habitat (Table 1): calcareous, stony soils and on sloping ground, except for sites L1 and S1, located in northern France, which were on colliery heaps (characterized by a strong magnesium/ calcium disequilibrium) and site S5 which was located in the Pyrtntes mountains at 1700 m elevation.

Methods o f allozyme analysis

Four enzymatic systems (GOT, LAP, EST and ACPH) were revealed on acrylamide gels (Valero, 1987). The genetic determination of banding patterns of five loci (GOT1, GOT2, LAP1, EST1 and ACPH1) was confirmed by controlled crosses (Valero, 1987).

S t u 4 of morphological characters

Quantitative reproductive and vegetative characters were measured in natural populations at most sites only once, but at some sites during two

TA

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este

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ranc

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Bel

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L. s

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Spec

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Loc

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50 m

50 m

160

m

160

m

20 m

100

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50 m

500

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1700

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60 m

700

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50 m

1984

(L

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) 19

85 (

Ll-

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1983

(L

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85 (

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1984

(L3-

84)

1983

(L4

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19

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1984

(S1-

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1985

(S1

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1984

(S2

-84)

1984

(S3

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1983

(S4-

83)

1984

(S4-

84)

1984

(S5

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1984

- (S

6-84

)

1984

(S7

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1985

(C

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5)

1985

(C

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1985

(C

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1985

(C

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25

10

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14

29

34

21

40

19

23

28

25

19

33

15

30 8 5 10

10

No.

Y

ears

in

divi

dual

s -


consecutive years; the sample size varied from 10 to 40 individuals per population (Table 1).

Individuals were collected in natural populations at the end of the flowering period when plants were bearing mature pods. For each individual we counted on one or two principal stems per individual: 1. the number of secondary stems; 2. the number of tertiary stems; 3. the number of inflorescences; 4. the number of flower buds per inflorescence (at the period of the harvest, the buds were no longer present, but it was possible to count them by the scars still present on the inflorescence axis); 5 . the number of pods per inflorescence; 6. the number of ovules per flower (it is very easy to count them in the pod by adding up the number of unfertilized ovules, aborted and mature seeds; Hossaert & Valero, 1988); 7. the number of seeds per pod.

Statistical analysis

Firstly, to test the differences among years, populations and species, each of the seven variables was studied in a three-level nested analysis of variance (Sokal & Rohlf, 1981); the species level was a fixed effect and all the other levels were random effects. Thus, we could not estimate the proportional contribution of the species effect to the overall variance. We will only discuss the results using the F-values. Because of the unbalanced design, the Satterthwaite approximation (Sokal & Rohlf, 1981: 301) was used. T o test the homogeneity of variances, the Scheffe-Box test was applied (Sokal & Rohlf, 1981: 405) and transformations of variables were performed when data were not homoscedastic. For those variables in which transformations were unable to make the data meet the assumptions of analysis of variance, we used a non-parametric method: the Kruskal-Wallis test, analogous to a single classification analysis of variance (Sokal & Rohlf, 1981: 429). Because the nested design could not be applied, it was necessary to test differences separately:

1. The differences between species using means calculated over populations (populations studied for two consecutive years were pooled together);

2. The differences between populations within each species (populations studied for two consecutive years were pooled together);

3. The differences between years within the populations studied for two consecutive years.

Secondly, to determine the most discriminant characters between species, all the characters were simultaneously studied by a discriminant analysis (Fisher, 1936; Dagnelie, 1975), using species as the classification criteria.

Univariate and multivariate analyses did not concern exactly the same variables. In the univariate study, we focused on the seven variables formerly mentioned because they are easily observable in the field. For the multivariate study, the variables utilized should not be directly causally related to one another and some of the variables had to be transformed. The seven following variables were studied: 1. number of secondary stems per principal stem; 2. number of tertiary stems per secondary stem; 3. number of inflorescences per ramification; 4. number of flower buds per inflorescence; 5. number of pod per flower bud; 6. number of ovules per flower; 7. number of seeds per ovule. Variables 1, 4 and 6 are the same as for the univariate analysis, directly collected

DISCRIMINANT CHARACTERS IN LA THYRUS 145

-7 J -6 -5 - 4 -3 - 2 -I 0

L I 1 2

Discrirninont values

0 L. foflfo/lus

L . sylvestris

L 6 7 8

L. latijolius L. sylvestris

Northern L184: N = 18 S184: N = 17 populations L185: N = 10 S185: N = 10

S284: N = 8 S384: N = 1 1

Central L285: X = 1 I populations

South-western L383: N = 13 S483: N = 6 populations L384 N = 8 S484: N = 13

S585: N = 10

N, Sample size of the population.

Figure 1. Location of individuals on the discriminant function.

in the field, and the others are transformed variables. This analysis was performed on the populations for which complete data were available: five populations of L. l a t i f l i u s and seven of L. sylvestris. For this multivariate analysis, the number of individuals and the different populations studied are given in Fig. 1.

In the contact zone, significant differences of means were tested using the multiple comparison method of Games and Howell (Sokal & Rohlf, 1981: 409).

RESULTS

Genetic studies

The strongest evidence for species level differences that can be obtained by allozyme studies is when one or more diagnostic loci are present. A diagnostic locus is a locus presenting two alleles, one fixed in one species and the other fixed

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TABLE 3. Genotype frequencies (homozygotes and heterozygotes) at different loci studied in the sympatric site

LAPl EST 1 GOTl GOT2 ACPH 1 Loci Genotypes Ha He N Ho He JV Ho He N Ha He N Ha He N

L. latijolius 0.82 0.18 28 0.67 0.33 33 1.00 - 28 0.41 0.59 17 1.00 - 10

Intermediates 0.86 0.14 21 1.00 ~ 15 1.00 - 21 0.76 0.24 21 ~ - -

0.40 0.60 10 0.92 0.08 11 0.75 0.25 12 0.83 0.17 12 1.00 - 12

(CL)

(CI) L. sylvestris

close to L. latijolius

L. sylvestris 0.70 0.30 44 1.00 - 45 1.00 - 45 1.00 ~ 45 0.44 0.56 18 far from

L. latijolius

( C W

(CSb)

JV, Sample size; Ha, homozygotes; He, heterozygotes.

in the other species, so that each individual can be classified unambiguously. The results of our study (Table 2) show that none of the loci studied were diagnostic. Nevertheless, discriminant alleles (i.e. one allele observed but not fixed in one species and absent in the other species) were observed at high frequency for all the loci studied except for locus ACPHl. Examples of discriminant alleles were allele 4 at locus LAPl and allele 2 at locus G O T l which are observed with a frequency of 0.50 and greater in L. sylvestris but which are not present in L. latifolius; and alleles 2 at loci EST1 and GOT2, which were only observed in L. latifolius. For the ACPHl locus, the two species differed only by allelic frequencies.

In the site where the two species occurred sympatrically, the results (Table 2) show that the local population of L. latifolius (CL) had allelic frequencies that fell within the range typical for the species; similarly, the local L. sylvestris populations ( C S ) were typical for that species. Surprisingly, ‘intermediate’ plants (CI) did not show any electrophoretic traits expected of hybrids. Indeed, no increased heterozygosity was observed at any locus (Table 3) . On the contrary, the intermediate plants are often strongly homozygous. At locus LAPl, the intermediate plants possess, at a high frequency (0.79: Table 2), an allele restricted to L. sylvestris and found in the local L. sylvestris population (allele 4). These intermediate plants are not hybrids a t this locus because most of them are homozygotes for this allele (Table 3) . At locus GOT1, intermediate plants are homozygous for the allele frequent in the local L. latifolius population. Moreover, the intermediate plants sometimes exhibit a high frequency of rare alleles compared with adjacent populations. At locus GOT2, allele 2, specific to L. latifolius, is observed a t a high frequency in intermediate plants whereas it is rare in the local L. latifolius population. The only cases that suggest hybridization of the two species are found in individuals of site CSa (Table 2). These plants, presenting a L. sylvestris phenotype but closer to the contact zone than plants of site CSb, had some alleles of the adjacent population of L. latifolius at low frequencies (allele 1 of locus GOT1). At locus GOT2, they had allele 2, which is only observed in L. latifolius plants. At locus LAPI, the level of

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(39)

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0.17

+0.0

7

0.35

+0.1

6

0.14

+0.0

8

(29)

(34)

(21)

(6)

4.62

f0.6

8 (2

6)

3.84

f0.8

0 (1

9)

7.55

f 1.

09

(11)

1.26

f0.6

4

17.9

5f5.

73

34.1

0 f

14.7

7

9.51

k0.

81

(18)

(10)

(39)

~

6.79

f0.8

0

7.65

i 1.0

8

8.24

f0.9

7

(29)

(34)

(21)

(6)

(26)

(19)

(11)

14.0

4 f 4.

34

19.3

5k2.

27

20.6

3 5.

47

36.3

6k4.

82

0.39

f0

. 19

0.90

f0.6

1

0

(18)

( 10)

(39)

~

0

0.03

fO.0

3

0

(29)

(34)

(21)

(6)

(26)

(19)

(11)

0.22

f0.1

5

0.15

k 0.

10

0.79

f0.4

1

1.00

f0.5

6

6.47

f0.3

1

5.77

f0.5

9

3.22

f0.7

0

7.94

i0.5

0

(25)

(10)

(11)

(13)

-

0.82

f 0.

37

4.22

f0.9

5 13

)

(8)

(6)

(4)

(10)

4.74

f 1.

04

2.92

+O.

14

2.87

k0.2

6

4.33

f0.2

4 (2

3)

1.58

f0.3

0 3 z

1.45

f0.1

6

0.85

+0.1

1

(18)

U

(10)

(39)

R E

-

P 0.

31 k

0.07

j

0.23

+0.0

8 Q

O.lf

2!).

04

5 (2

9)

z

(21)

(6)

(26)

(19)

(11)

0.77

k0.2

5

0.26

k0.

03

0.79

f 0.

14

0.31

k0.

04

Lath

yrus

Syluestris

Nor

ther

n

Sout

h-w

este

rn

Nor

ther

n

Nor

ther

n

Nor

ther

n

Nor

ther

n

Sylu

estri

s m

ean

Nor

ther

n

Con

tact

N

orth

ern

site

N

orth

ern

Nor

ther

n

s3

s4

s5

S6

s7 CL

CI

CSa

CSb

1984

1983

1984

1984

1984

I984

1985

1985

1985

1985

12.2

7 +O

. 18

11.7

1 k0.

21

11.9

6+0.

23

13.7

6 f0.

27

12.8

3k0.

30

12.9

7k0.

24

(23)

(14)

(17)

(33)

(15)

(30)

(9)

(8)

(5)

(10)

(10)

12.3

9 f0.

08

20.5

0 k0

.69

15.0

0f0.

70

15.7

0 50.

53

12.5

0f0.

45

6.77

f 0

.18

3.29

f0.0

8

3.55

k0.1

3

3.17

f0.1

7

(27)

(25)

(19)

(10)

-

~

4.80

k0.5

7 (7

)

10.9

3 k0.

45

(7)

10.8

8f 1

.28

(4)

7.57

f0.3

5 (1

0)

6.19

k0.2

2

0.96

f 0.

82

9.28

+ 1.

49

5.37

f 1.

27

6.00

k 1.

60

(28)

(25)

(19)

(10)

~

24.7

3 f4

.23

63.1

3f13

.72

40.6

6k 1

0.52

24.6

0 f 5.

35

(28)

(25)

(19)

(10)

-

3.50

k2.0

0

9.80

f2.7

3

5.26

+ 1.

91

0.60

k0.3

4

(28)

(25)

(19)

(10)

-

6.80

k0.9

8 32

.78k

5.89

3.

02f

1.33

(7

) (7

) (7

)

4.31

k0.

31

2.42

k 0.

24

3.00

0.

29

3.28

k 0.2

7

5.86

f0.3

4

0.27

f0.2

4

(23)

(14)

(17)

(29)

(15)

(30)

(9)

3.69

k0.3

6

-

-

-

-.

0.42

f0.0

6 (2

7)

0.04

f0.0

1 (2

5)

0.02

kO.0

1 (1

9)

0.15

f0.0

3 (1

0)

-

-

0.28

k0. 10

(7

)

-

-

-

-

~ ~

~~

~~

~ ~

~ ~

N, Sa

mpl

e si

zes;

OV

/FL

, ovu

les

per

flow

er; B

UD

/IN

F, f

lora

l bud

s pe

r in

flore

scen

ce; S

2/PS

, sec

onda

ry st

ems p

er p

rinc

ipal

ste

m; I

NF

/PS,

inflo

resc

ence

s per

pri

ncip

al st

em;

S3/P

S, t

ertia

ry s

tem

s pe

r pr

inci

pal s

tem

; SE

ED

/PO

D, s

eeds

per

pod

; PO

D/I

NF

, pod

per

inflo

resc

ence

.


TABLE 5. Univariate analysis.

TABLE 5a. Results of the three-level nested analyses of variance (species, population, years) for the five variables studied

F F Variables species populations

~

F years

~~

OV/FL F(1,8) = 170.20"

BUD/INFb F(1,7) = 17.79"

SP/PSb F(1,7) = 19.21"

INF/PS' F(1,6) = 10.15"

SEEDjPOD F(1,5) = 2.48"

***

**

** *

n.s.

~~

F(8,3) = 9.11"

F(7,4) = 6.86"

F(7,4) = 5.92" ns.

F(7,4) = 2.98" ns.

F(8,5) = 1.30 n.s.

n.s.

*

F(5,270) = 1.25

F(4,272) = 10.37

F(4,276) = 2.19 ns.

F(4,276) = 1.56 ns.

F(5,266) = 19.37

n.s.

***

***

"Satterthwaite approximation (Sokal & Rohlf, 1981: 301) was applied; 'A square root transformation of this variable was applied in order to make the data homoscedastic; 'A sixth root transformation of this variable was applied in order to make the data homoscedastic.

TABLE 5b. H values of the Kruskal-Wallis test for the number of tertiary stems per principal stem and the number of pods per inflorescence

Levels

Species Populations within species

lafijolius syiuestris Number of

groups 2 4 5

Years within populations ~-

L1 L4 s1 s4

2 2 2 2

S3/PS 4.90 22.29 21.25

POD/INF 0.24 67.79 63.21 ns. *** ***

* *** *** 0.40 0.62 2.44 1.34 n.s. ns. n.s. n.s.

0.12 0.80 13.20 1.99 n.s. ns. n.s. ***

n.s. Non-significant; *P < 0.05; **P < 0.01; ***P < 0.001.

heterozygosity in the CSa intermediates is higher than in either L. Latifolius or L. sylvestris (CSb) (Table 3): allele 2, found at a moderate frequency in the adjacent population (CI) and absent from L. sylvestris CSb, is observed at a non- negligible frequency (0.15) in population CSa, along with allele 4, which is restricted to L. sylvestris (Table 2 ) .

Dzferentiation of the two species by mor-hoLogical characters

Univariate study Means and standard errors of the seven variables, measured in each

population studied and calculated by species, are given in Table 4. The results of the nested analysis of variance are given in Table 5a for each variable studied except for the number of tertiary stems per principal stem and the number of


pods per inflorescence. For these two last variables, data did not meet the assumptions of the nested analysis of variance and Kruskal-Wallis tests were applied (Table 5b).

The highest value for F between species is observed for the number of ovules per flower (P(F(1,8) = 170.2) << Table 5a). The flowers of individuals of L. latifolius contained c. 19 ovules whereas those of L. sylvestris contained C. 12 ovules. Within each species the differences among populations were not significant. The variation for this character between two consecutive years was not significant.

The number of secondary stems per principal stem and the number of floral buds per inflorescence were the characters with the next highest F values between species (level of significance P < 0.01: Table 5a). The number of secondary stems was higher for individuals of L. sylvestris (seven on average) than L. latifolius (one on average). This character did not vary significantly between populations or between years (Table 5a).

Plants of L. latifolius had on average ten floral buds per inflorescence, twice as many as in L. sylvestris. In spite of the strong difference between species the variation among populations and especially between two years remains very important and reveals the sensitivity of this variable to environmental fluctuations. The differences among populations appear to be related to latitude (Table 4) in both species, but particularly in L. sylvestris, where the number of floral buds per inflorescence was significantly greater for the individuals of northern populations compared with the south-western ones of the same species (two-level nested anova; level 1 : among geographical locations, level 2: among populations within locations, P(F, , , , , = 16.97) among locations < 0.05).

The two other characters that may allow differentiation between these two species were the number of inflorescences per principal stem (P(F(1,7) = 10.15) < 0.05; Table 5a) and the number of tertiary stems per principal stem (P(H(, d,f,) = 4.91) < 0.05; Table 5b). The number of inflorescences was significantly higher in L. sylvestris (33 on average) than in L. latifolius (14 on average). This character did not vary significantly among populations or between years (Table 5a). The number of tertiary stems per principal stem was significantly higher in L. sylvestris (3.02 on average) than in L. latifolius (0.22 on average). This character varied strongly between populations but is stable between years (Table 5b).

The two other characters studied did not vary significantly between the two species: the number of pods per inflorescence (Table 5a) and the number of seeds per pod (Table 5b). These two variables differed significantly between years (at least in one population). The number of seeds per pod did not vary significantly among populations. The number of pods per inflorescence did vary significantly.

Multivariate study The results obtained in the discriminant analysis are given in Table 6. The

canonical discriminant function was significant for P < (Table 6) and separated individuals from the two species very well (the classification variable: species, explains 93% of the overall variance, Table 6). The number of ovules per flower, and to a lesser extent, the number of floral buds per inflorescence are the two characters best correlated with the discriminant function (Table 6). The individuals of L. latifolius, with more ovules per flower and more floral buds per

152

Variables

M. VALERO AND M. HOSSAERT-MCKEY

TABLE 6. Results of the discriminant analysis

Correlations with the discriminant function

Ovules/Flower Floral buds/Inflorescence Tertiary stemsiprincipal stem Seeds/Ovule Secondary Stems/Principal stern Inflorescences/Ramification Pod/Floral bud

Eigenvalues R2 = Canonical correlation R2 = Contribution of the

classification factor (species) to the overall variance

Significance Percentage of wrongly classified

individuals

0.8821 0.4493 0.2356

-0.2232 -0.1820

0.1439 -0.1 140

13.52 0.97

0.93

0 % <

inflorescence, are all located towards positive values while all L. ylvestris plants present negative values (Fig. 1: percentage of wrongly classified individuals =

0%).

Morphological characters in contact zones

Concerning the sympatric site, we have chosen to focus only on the two best

1. The number of ovules per flower, 2. The number of floral buds per inflorescence. Table 4 gives the means obtained in each site of the contact zone. Tables 7

and 8 allow a multiple comparison of the results obtained in three sites with those, discussed previously, observed on an average of several populations in both species. The same discriminant variables distinguished the site CSb (the L. ylvestris population furthest from L. la t i f l ius ) from the site CL (L. latifolius population). The individuals in the two sites CI (intermediates) and CSa (‘sylvestris’) were very similar and were intermediate between the two species. Plants of sites CL (latiJolius) and CSb (sylvestris) had the same number of ovules per flower as those from typical populations of the two species (Table 7) . For the variable floral buds per inflorescence, site CL did not differ from the L. latifolius average, whereas individuals of site CSb had a greater number of floral buds per inflorescence ( > 6) than that observed on average in L. syluestris (5 ; Tables 4 and 8). Nevertheless, we have noticed before that this variable varies strongly among populations, especially in L. sylvestris where individuals from some populations in northern France had an average of about six floral buds per inflorescence (Table 4), i.e. twice as many as in individuals from populations in south-western France. This contact zone must be compared with populations of northern France because it is located in nearby Belgium. This site CSb did not differ from populations of the region for the variable number of buds per

discriminant variables to distinguish between species:


TABLE 7. Multiple comparison of the mean number of ovules per ovary in the sites of the contact zone compared to both species. The equality of means was tested by

the Games and Howell method (Sokal & Rohlf, 1981: 409)

1 2 3 4 5 6

I 0 1.02 4.23 3.53 6.73 6.83 2 1.97 0 5.25 4.55 7.75 7.86 3 6.45' 6.56* 0 0.70 2.50 2.61 4 6.73** 6.52.. 0.87 0 3.20 3.31 5 14.60*** 11.90*** 3.27 4.89* 0 0.11 6 22.49*** 14.24*** 3.83 5.95' 0.21 0

~ ~ ~ ~~~~

Group 1: mean over the 6 populations of L. fut?folrus studied. Group 2: mean over individuals studied in site CL (contact /attfoltus) Group 3: mean over individuals studied in site CI (contact intermediate). Group 4: mean over individuals studied in site CSa (contact sylvestris close to CI). Group 5: mean over individuals studied in site CSb (contact sylvestrzs far from CI) . Group 6: mean over the 8 populations of L. sylvestns studied. The differences between means are given below the diagonal and the corresponding

*Differences > MSD for 0.05 level of significance; **differences > MSD for 0.01 level of Minimum Significant Differences (MSD) are given above the diagonal.

significance *** differences > MSD for 0.001 level of significance

inflorescence; however, it differed significantly from south-western populations (Table 8).

Individuals from both intermediate sites CSa and CI appear very similar (Tables 7 and 8) and were intermediate between the two species (Table 4). The number of ovules per ovary differed significantly from both species in site CSa and was closer to L. syluestris in site CI (Table 7). The number of floral buds per inflorescence was not significantly different from both species in site CI and was closer to L. sylvestris in site CSa (Table 8).

TABLE 8. Multiple comparison of the mean number of floral buds per inflorescence in the sites of the contact zone compared to both species. The equality of means was tested by the Games and

Howell method (Sokal & Rohlf, 1981: 409) ~ ~ ~

1 2 3 4 5 6 7 8

0 1.71 0.98 3.50 6.62 5.25

11.43'. 6.27'

1.15 0 0.05 6.81**

11.97*** 8.00.

18.95*** 8.88**

1.10 0.05 0 3.26 4.99 4.61 7.76 5.43

2.22 3.79 3.93 6.44 5.01 3.36 4.93 5.08 7.59 6.16 3.31 4.88 5.03 7.54 6.10 0 1.57 1.72 4.23 2.79 4.57 0 0.14 2.66 1.22 2.89 0.28 0 2.51 1.08

12.83*** 14.28*** 4.86 0 1.44 4.27 2.05 1.41 2.45 0

Group 1-5 as in Table 7. Group 6: mean over the four northern populations of L. syluestris studied. Group 7 : mean over the three south-western populations of L . syluestris studied. Group 8: mean over the seven populations of L. syluestris studied. The differences between means are given below the diagonal and the corresponding Minimum Significant

*Differences > MSD for 0.05 level of significance; **differences > MSD for 0.01 level of significance; Differences (MSD) are given above the diagonal.

***differences > MSD for 0.001 level of significance.


DISCUSSION

Are the two species reproductive& isolated?

Discriminant alleles readily distinguished the study species (Table 2) and indicated reproductive isolation between them. Barriers to gene flow between the two species have been mentioned in the literature (Senn, 1937; Davies, 1957; Chai’b, Delbos & Combes, 1987). Nevertheless, the fact that for all the loci studied, some alleles are found in both species means that fixation for one allele in one species and for another allele in the other species has not yet occurred, suggesting that divergence between the two species could be considered as a relatively recent event.

The study of the sympatric zone shows that reproductive barriers can be partially broken, since intermediate individuals were observed. Nevertheless, the occurrence of such individuals seems to be quite rare. The literature does not mention it; furthermore, among the 30 sites we have studied, we have found intermediates at only one. The number of intermediate individuals is very low in that site (we have observed 20-30 plants in site CI, 20-30 in site CSa, c. 100 in site CL and over 1000 individuals in site CSb). Based on our studies of a relatively few individuals, these intermediates did not appear, electrophoretically, to be F, hybrids. They were not heterozygotes for L. latijolius and L. sylvestris alleles. In contrast, depending on the locus, an allele of one or the other species was nearly fixed, or, in one case, the intermediate plants were fixed for a ‘rare’ allele which is present in one of the parents (Table 2) . This weak heterozygosity level and the occurrence of a ‘local’ allele suggest that the intermediates arose from a very small number of individuals and do not interbreed with other plants from the contact zone. These intermediate individuals confirm then, paradoxically, the occurrence of a strong reproductive barrier between the two study species.

The discriminant analysis confirms the reproductive isolation between these species. This analysis shows that the criterion chosen readily distinguishes the species (Fig. 1 ) .

Many examples in the literature (Davis & Gilmartin, 1985; Gottlieb, Warwick & Ford, 1985) have noted a lack of concordance between enzymatic and morphological data on the level of differentiation. The results obtained in this study reveal quite a good concordance between the two kinds of characters.

Which characters are useful in d~ferent iat~on between species in the Jield?

Daferences between species To answer this question, we have examined the frequency distributions for the

five variables that were significantly different between species (Fig. 2A-E). This kind of representation takes into consideration the level of individuals and shows if the character studied discriminates, without any ambiguity, between individuals of both species. It appears that the only variable that shows a strong bimodal distribution is the number of ovules per flower (Fig. 2A). The two modes correspond to individuals of each species. The two species are very well separated. Only one individual of L. sylvestris of the 285 studied is located in the histogram of L. la t i f l ius . Thus this character appears to be a good diagnostic

DISCRIMINANT CHARACTERS IN LA THI'RVS

E -

155

80

60

0 5

U

LL

2 40 E

20

Ovules per flower 50 I

-10 -5 0 5 10 15 20 25 30

Secondary stems per principal stem

40 :;

0 -20 0 20 40 60 80

Tertiary stems per principal stem

I

Figure 2. Frequency distributions of the five characters which are significantly different between species. A. Number of ovules per flower. B. Number of secondary stems per principal stem. C. Number of floral buds per inflorescence. D. Number of inflorescences per principal stem. E. Number of tertiary stems per principal stem. -U-, L. l ~ t i j ~ l i ~ ~ ; -+-, L. sylvestris.

I56 M. VALERO AND M. HOSSAERT-MCKEY

one: it allows a good discrimination between species with a probability of error of only 1/285. It is also a character that can be easily observed in the field.

For the four other variables (Fig. 2B-E), distributions of the two species overlap considerably more. For this reason, they are not as good for use as a taxonomic character, although they differ significantly between species.

The number of floral buds per inflorescence is even mentioned in Floras as a discriminant criterion. The strong geographical differentiation for this character, especially in L. sylvestris with northern populations presenting twice as many floral buds as south-western ones (Table 4)) could explain identification errors made by some authors (Fournier, 1946; Foury, 1954): individuals from south- western populations of L. latifolius with 6-10 floral buds per inflorescence may be confounded with those from northern populations of L. sylvestris with 4-8 floral buds (Fig. 2C).

The number of secondary and tertiary stems and the number of inflorescences per stem are characteristic of the branching system. Plants of L. sylvestris are more branched (more secondary stems, more tertiary stems and more inflorescences per stem) than those of L. latzfolius (Table 4) even though distributions of the two species overlap (Fig. 2B,D,E). An increase in the number of ramifications leads to an increase in the number of meristems and might allow faster development of the plant. We have shown (Hossaert & Valero, 1986) that the root reserves are extensive in L. syluestris individuals and permit a faster emergence of aerial stems than in L. latifolius plants. A hypothesis put forward by Hossaert-McKey (1988) suggests that the maintenance of the biological characteristics of the branching system specific to L. sylvestris may result from a selection for faster growth. We have seen that geographical ranges of these species differ. The original habitat of L. syluestris is supposed to be located in mountain regions. The colonization of valleys and northern regions of Europe presumably took place afterwards. Lathyrus latifolius originated in Mediterranean regions with the northern limit located in northern France. The differences in branching system of both species could be interpreted as the result of an adaptation for rapid growth in L. syluestris in habitats where the favourable period for plant growth is more restricted in time.

How well do the two chosen discriminant characters separate the dzjerent individuals of the contact zone?

Like allozyme markers, the number of ovules per flower classifies individuals from site CSb (sylvestris type) as typical L. sylvestris and those from site CL (latifolius type) as typical L. latifolius (Table 7 ) . The two kinds of intermediate plant described by allozymes (CI and CSa) present the same number of ovules per flower, around 15 on average (Table 4), and do not differ significantly from each other (Table 7 ) . Whereas this value corresponds to a value intermediate between the two species, the frequency distribution (Fig. 3A) shows that the number of ovules per flower varies from 13 to 16 in site CI and from 13 to 18 in site CSa. Individuals from CI site are closer to typical L. sylvestris than those from CSa site (Table 7). This result is not strictly similar to the one obtained with allozymes for intermediate plants. In this case, allozymes are more useful than multilocus quantitative characters in studying the affinities of individuals in intermediate or hybrid zones.


A

0 3 -

5 10 15 20 25

t !I

Ovules per flower

0 2 4 6 8 10 12 14 16 18

A ; \ 4 6 1

0 2 4 6 8 10 12 14 16 18

Floral buds per inflorescence

Figure 3. Frequency distribution of the two discriminant characters in the different sites of the contact zone. A. Number of ovules per flower. B. Number of floral buds per inflorescence. PO-, CL. --.-- CSb; .... O...., CI; -.-.A .-.-, CSa,

A similar result was obtained for the number of floral buds per inflorescence. The two sites CL and CSb do not differ significantly from their respective species (Table 8). The intermediates (CI and CSa) also do not differ from each other (Table 8). The frequency distribution (Fig. 3B) confirms this result and underlines the wider distribution observed in intermediate plants compared with plants typical of each species. This wide range of values for intermediate plants explains why individuals from site CI did not differ significantly from either species (Table 8).

In summary, both variables are quite good at identifying individuals typical of either species. The number of ovules per flower is a more consistent character for separation between the two species than the number of floral buds per inflorescence (Fig. 3A,B for sites CL and CSb). Nevertheless, these two quantitative characters appear to be not as consistent as allozymes in revealing gene flow, and consequently are more difficult to use to characterize intermediate plants.


W h a t is the contribution of plant population biology in the characterization of species?

Most of the characters studied in this paper are probably strongly related to individual fitness in natural populations. These characters are very often studied by population biologists, who are interested in their levels of variation within and among populations, and in measuring individual fitness. They have not often been used by systematists. In this last section we will outline how advances in plant reproductive biology have led to elucidation of new characters useful in biosystematics.

The number of pods per inflorescence and the number of seeds per pod were not significantly different between species (Table 5A,B). The values of these two characters in natural populations result from flower abscission and ovule abortion. Two major factors have been proposed to account for abscission and abortion, namely 1. environmental unpredictability Uanzen, 1977; Schemske, 1980; Stephenson, 1981; Bertin, 1982; Heithaus, Stashko & Anderson, 1982; Travis, 1984; Hossaert & Valero, 1985) and 2. sexual selection (Charnov, 1979, 1982; Charlesworth & Charlesworth, 1981). Arguments based on sexual selection suggest that investment in male and female functions will vary with the breeding system. Many results have confirmed this assumption. Ornduff (1969), Cruden (1977), Lovett-Doust & Cavers (1982), Queller (1984) and Vesek & Weng (1988) have observed that pollen production is much lower in selfers; Waller (1979), Schoen (1982) and Sutherland (1987) have shown that flower and fruit abscission is more extensive in outcrossers; and Bubar (1958), Wiens (1984) and Sutherland (1987) have pointed out that seed abortion is higher in outcrossing plants.

These two species are essentially outcrossed (Valero, 1987; Hossaert & Valero, 1988), which could explain why fruit abscission and seed abortion appear to be very important (Table 4) and dependent upon environmental fluctuations. The number of seeds per pod and in some cases the number of pods per inflorescence differed significantly between two consecutive years (Tables 5A,B). These two characters appear then not very useful in discriminating between these two outcrossing species.

The number of ovules per ovary is a reproductive character not often used in plant systematics, where the number of seeds per fruit is more frequently chosen as a classification criterion. There is an important reason, however, why ovule number is to be preferred as a taxonomic trait in comparative studies. While ovule number is a phenotypic trait of the plant, number of seeds per fruit is due to the interaction of this trait with a host of environmental influences, such as pollinator success, resource availability and seed predation. Furthermore, ovule number, rather than seed number, may be more closely correlated with variation in reproductive systems among related species.

Many authors (Stephenson, 1981; Willson & Burley, 1983; Bawa & Webb, 1984; Lee & Bazzaz, 1986; Wiens et al., 1987) have suggested that the differential abortion of seeds within fruit in outcrossing plants could be the result of a selection which eliminates the more inbred genotypes (because the plants are outcrossers) and favours the more vigorous ones. An excess number of ovules may allow plants to selectively abort seeds sired by unsuitable (e.g. incompatible or too closely-related) pollen donors (Marshall & Ellstrand, 1988). In papers comparing numerous outcrossing and selfing species, Ornduff ( 1969) has noticed


the greater number of ovules per flower in outcrossers. We have observed such a difference in the two species of Lathyrus we studied. The number of seeds per fruit overlaps broadly in these species but they differ in the number of ovules per ovary. We have shown (Valero, 1987) that inbreeding is more frequent in L. sylvestris than in L. latifolius. The difference in the number of ovules per ovary may be the result of the difference in their reproductive system, with the number of ovules higher in the more outcrossing species ( 19 in latifolius and 12 in sylvestris, Table 4).

Number of ovules per ovary is an easily measured character, especially in legumes, and also shows great variation at several taxonomic levels within this family. Even within genera this variation can be striking. In Sesbania, some species have one or two ovules per ovary (5. vesicaria) (Marshall, Fowler & Levin, 1985), while others have up to 55 ovules (S. dummeri) (Gillett, Polhill & Verdcourt, 1971). In Lotus also, the ovule number per ovary varies from one to many (Zanstra & Grant, 1968), for example, up to 70 ovules per fruit in Lotus

jacobaeus (Bubar, 1958). Among the species found in Canada, the number of seeds per ovary varies from two ( L . denticulatus) to 35 (L . pedunculatus) (Zanstra & Grant, 1968).

In legumes, this trait appears to show substantial variation between species and relative constancy within species. Will this be the case in other families? As Webb ( 1984) has discussed, the constraints acting on reproductive characters may be different in different plant families. Traits that are phylogenetically constrained in one family may show great variation among species in another family. Webb’s data on ovule number in Gentiana (Gentianaceae) and Ranunculus (Ranunculaceae) (Webb, 1984), along with those of Wyatt (1984) on Arenaria (Caryophyllaceae), those of Preston (1986) on Cruciferae and those of Vasek & Weng (1988) on Ctarkia (Onagraceae) demonstrate that this trait shows very different patterns of variation, depending on the taxon involved. Because so little attention has been paid to this character, attempting to identify and explain patterns is premature. The tentative comparisons made so far lead to one insight, namely, that such comparisons must be made carefully. Webb (1984) found that ovule number per flower in Ranunculus is very variable within species, even within individuals, whereas in Gentiana this trait can be used to distinguish between species. However, in Ranunculus, with its apocarpous gynoecium composed of numerous simple ovaries, ‘number of ovules per flower’ is not the same reproductive trait as in Gentiana, with its syncarpous gynoecium. The variation in number of ovules per flower in Ranunculus is largely due to variation in number of carpels per flower, with the number of ovules per carpel being highly constrained within the genus, always one (Heywood, 1978). Legumes present a third case. Perhaps in this family, in which the flower always has a single, simple ovary, the character ‘number of ovules per flower’ is more likely to vary among species, and be constant within species, than in families where ovary construction is not faced with the same constraints.

ACKNOWLEDGEMENTS

We wish to thank Genevieve Tortay, Patrice Blanchard, Anissa Chaib, Daniele Magda, Florence Petit and Alida Yousef for valuable field assistance. The manuscript benefited from the helpful comments of Joel Cuguen, Jose


Godin, Marc Jarry, Carol Horvitz, Vincent Labeyrie, Josiane Lecorff, Doyle McKey, Jean Raymond and Philippe Vernet. Financial support was provided by a grant “Aide a la Biologie” from the Ministry of the National Education (MEN) of the French Government.

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