- Email: [email protected]
Variation in the subtropical group of Dactylis glomerata L.—1. Evidence from allozyme polymorphism
Pergamon
Biochemicai~stematics and Ecology.Vol. 23, Na 4, pp. 407-418.1995 Copyright(D1995 ElsevierScience Ltd Printedin Great Britain.All rightsreserved 0305-1978/95 $9.50+0.00
0305-1978(95)00020-8
Variation in the Subtropical Group of Dactyhs glomerata L.1. Evidence from Allozyme Polymorphism ELVIRA SAHUQUILLO and ROSELYNE LUMARET DBpartement de Biologie des Populations, Centre d’Ecologie Fonctionnelle et Evolutive L. Emberger, CNRS, B.l? 5051,34033 Montpellier Cedex. France
Key Word
Index-Dacty/isglomerata;
Poaceae; allozymes; population variation; polyploidy; subtropical group.
Abstract-Allozyme variation at eight polymorphic loci was analysed in 15 populations collected over the Atlantic part of the subtropical distribution area of Dactylis glomerafa L. Four genetically distinct entities were identified. One included the diploid populations of subspecies smithji, endemic of Tenerife Island. A second included the tetraploid populations from Grand Canary island which also constituted a morphologically distinct group. A third group clusted together inland and coastal tetraploid populations from Madeira Island (likely two ecotypes of subspecies hylodes) which had been described previously as two distinct subspecies according to morphological traits. A fourth group gathered together the tetraploid populations from the coasts of Portugal (ssp. marina). The substantial allozyme variation observed in that subtropical group may be attributed partly to the occurrence of large geographic discontinuities in the distribution area and, at a particular locus (GOT7). the observed variation was assumed to be due mostly to adaptive selection related to large variation for humidity conditions in that Atlantic area.
Introduction The perennial outcrossing grass Dactylis ghmerata L., is one of the best-documented examples of a natural infraspecific polyploid complex. Evidence from its geographic distribution, comparative morphology, cytogenetic structure, its variation for allozymes, phenolic compounds and chloroplast DNA has been used to study phylogenetic relationships among the Da&y/is taxonomic groups (Stebbins and Zohary, 1959; Borrill, 1978; Jay et a/., 1984, 1989; Ardouin et a/., 1987; Fiasson et a/., 1987; Lumaret, 1988; Lumaret et a/., 1989; Lumaret and Barrientos, 1990). In Dactylis, the diploid (2n = 14) and tetraploid (213= 28) cytotypes were classified into either two groups of subspecies, namely Mediterranean and Eurasian groups, according to morphogeographical criteria, or into three groups, namely Subtropical, Mediterranean and Temperate groups, according to climate (Borrill, 1978; Lumaret, 1988). Plants of the subtropical group were mostly recorded from the Macaronesian islands (Madeira, Canary and Cape Verde), from the coasts of Portugal and, to a lesser extent, from several other coastal habitats in the Mediterranean Basin (Borrill, 1961; Parker, 1972; Lumaret, 1988). Whatever their geographic origin, plants of the subtropical area possess several particular morphological characters in common such as woody stems and papillose epidermal cells, suggesting that the several morphological forms identified in that group are closely related (Borrill, 1957,196l; Parker,1972; Borrill, 1978). In Dactylis, the papillose epidermal cells have been shown to play an important role in water use efficiency from the atmosphere (Wilson et al., 1977) and the papillose forms are restricted to cloudy or coastal habitats in regions of high summer temperature (Benson and Borrill, 1969). Dactylis plants of the subtropical group also show substantial cytological and morphological variation which may be related to their discontinuous pattern of spatial distribution (mostly between islands) as well as to the contrasted climatic conditions (more particularly for annual precipitations) occurring among the distribution (Received 11 July 1994)
408
E. SAHUQUILLOAND R. LUMARET
areas and even within a single island (De Lemps, 1959; Benson and Borrill, 1969). At least three subspecies have been already identified in the Atlantic part of the subtropical group. Those are the diploid smithii (Link) Stebbins and Zohary, endemic in Tenerife and Cape Verde islands (Stebbins and Zohary, 1959), the tetraploid hylodes Parker, endemic of the inland part of Madeira island (Parker, 1972) and the tetraploid marina Borrill distributed along the coasts of Portugal (Borrill, 1957, 1961; Benson and Borrill, 1969) and, according to Parker (1972) on the coasts of Madeira. Subspecies smithii is closely associated with the subtropical flora that is considered to date from the middle of the Tertiary period and this subspecies or its precursors should date therefore from the Miocene epoch whereas the tetraploid ssp. hylodes and marina should be of more recent origin and may have arisen from diploids closely related to ssp. smithii (Parker, 1972; Borrill, 1978). Additional morphologically distinct D. glomerata entities were discovered recently by Ramos and Marrero (pers. comm.) in several islands of the Canaries and more particularly in the central part of Grand Canary island. Further investigation of natural variation in Dactylis plant material from the subtropical group was therefore necessary to clarify the relationships among the several entities of that group. To be effective, those studies combined the use of several markers (e.g. morphological traits, enzyme and DNA polymorphism, variation for phenolic compounds, etc.) on the same plant material. Those markers are known indeed to have distinct evolutionary rates and various sensitivity vs environmental selection. Results obtained in those investigations are reported in a series of papers. The present paper is the first of the series and deals with the geographical distribution of isozyme polymorphism in populations of diploid and tetraploid subspecies or morphologically distinct types of Dactylis glomerata occurring in the Atlantic part of the subtropical group. Enzyme markers are considered most generally neutral (Sarich, 1977). However, in Dactylis, extensive studies have shown that the frequency of several alleles at the GOTI locus, which codes for glutamate oxaloacetate transaminases, were significantly correlated with soil humidity even if no evidence could be obtained to show that selection was acting directly on that locus (Lumaret, 1984; Roy and Lumaret, 1987). The main objectives of the present work are therefore (1) to estimate the genetic differentiation among the subtropical entities from the study of allele variation at eight polymorphic enzyme loci, and (2) to assess the part of adaptive differentiation related to climate variation by studying separately allozyme polymorphism at the GOTI locus. Materials and Methods Plant material Fifteen populations distributed over the Atlantic part of the subtropical Dactylis geographic distribution were studied (Table 2). Three populations of diploid plants (Te-1, Te-2 and Te-3) were collected in Tenerife island and were identified as ssp. smithii according to the formal description of Stebbins and Zohary (1959). Two populations of tetraploid plants (Ca-4 and Ca-5) were collected in the centre of Grand Canary island and are considered to belong to a morphologically distinct type (Ramos, pers. comm.), although it has not been yet described formally as a subspecies. Three populations of diploid plants (Md-6, Md-7 and Md-8) and four populations of tetraploid plants (Md-9, Md-10, Md-11 and Md-12) were collected from the inland part and the coastal part of Madeira island, respectively. The plants had been identified as ssp. hylodes (inland populations) and ssp. marina (coastal populations) by Parker (1972). Three populations of tetraploid plants (Po~13, Po-14 and Po-15) were collected along the coast of Portugal and were identified as ssp. marina according to Borrill (1961). The main morphological and environmental characteristics of the several subspecies involved in the present study are summarised in Table 1. The location of the collecting sites and the number of plants sampled are indicated in Table 2. The four plants collected from site Te-3 were not involved in the population genetics analysis. Plant material from sites Te-2, Po-13, Po-14 and Po-15 came from the collection of M. Borrill (Ref. BC 6425, BC 5221, BC 5217 and BC 5215 respectively, Welsh Plant Breeding Station, Aberystwyth). For plant and site description of the Portuguese material, see Benson and Borrill (1969). Sites Md-6, Md-8, Md-9, Md-10 and Md-11 in the present study correspond to sites 37, 27, 1, 50 and 19, respectively, which were described by Parker (1972).
Ssp. or type
srnithii
Canarian type
hylodes
marina
marina
Location
Tenerife
Grand Canary
Madeira inland
Madeira coast
Coast of Portugal
4x
4x
4x
4x
2x
Ploidy level
Bushy plants and dense tillering Bushy plants and dense tillering
Lax Shrubby
Shrubby and dense tillering
Lax Shrubby
Plant habit
20-50
20-70
20-50
90-10O
35-95
Culm. height (cm)
Prostrate
Prostrate or hanging
Erect or hanging
Erect
Erector hanging
Habit
Stems
+
+
+
+
+
Woody
+
+
-
-
+
Secondary branches
Glaucous
Glaucous
Light green
Glaucous
Light green
Co/our
+++
++
+
+
+
Epidermal papillae
Leaves
+++
++
+
++
-
3.5
3.2
2.7
5.0
6.0
Panicle Lobed Average lemma florets per apex spikelets
E
18002675
12001530
F G H
D
850980
415850
B C
430470
Dry mostly shaded crevices in volcanic rocks (150--600) Dry open rocky areas with bushy plants ( > 1200) inland cliffs or rocky areas (1 0-1300) Coast cliffs (0-50O)
Sand coasts rocks or maritime pasture by sea shore (0-500)
A
Annual rainfall (mm)
Habitat (altitude) (m)
Ref.
TABLE 1. GEOGRAPHICAL LOCATION, MORPHOLOGICAL TRAITS, HABITAT AND CLIMATE CHARACTERISTICS OF FIVE SUBSPECIES OR MORPHOLOGICAL TYPES IDENTIFIED IN THE SUBTROPICAL GROUP OF DACTYLIS GLOMERATA L. A, B, C, D, E, F, G and H refer to Stebbins and Zohary (1959), authors of the present paper, Ramos (pars. comm.), Parker (1972), Borrill (1957), Borrill (1961 ) and Benson and Borrill (1969), respectively. Annual rainfall data are from De Lamps (1969) and Walter eta/. (1979).
410
E. SAHUQUILLOAND R. LUMARET
TABLE2. Geographicorigin, locality,altitudeand numberof plantssampledin 15 collectingsitesdistributedoverthe subtropical areaof Dactylisglomerata Code
Origin
Locality
Altitude (m)
Plantno.
Te-1 Te-2 Te-2b Ca-3 Ca-4 Md-5 Md-6 Md-7 Md-8 Md-9 Md-10 Md-11 Po-12 Po-13 Po 14
Tenerife Tenerife Tenerife GrandCanary GrandCanary Madeira Madeira Madeira Madeira Madeira Madeira Madeira Portugal Portugal Portugal
Tigaiga Barrancode Ruiz Los Silos Degolladade Las Palomas AriSez Serrade Agua Lombode Cima Eirado Serrado CaboGirao SaoVieente Portoda Cruz Pontado Pargo Fozde Arelho Praiadas Macas CaboSao Vicente
580 620 350 1600 1400 300 350 850 580 5 5 250 15 10 20
44 37 4 17 27 32 19 20 16 27 13 15 28 48 45
Allozyme assays were performed on leaf tissue, from plants collected in situ in the case of the populations from Madeira and in all other cases from seedlings grown from seeds collected in nature. The seeds were germinated and seedlings grown in the greenhouse for approximately 1 2 weeks until they were assayed. One seedling individual was examined from each mother plant. Plant ploidy level was determined from DNA quantification using flow cytometry and a method adapted to Dactylis material (Maceira et al., ! 992). Chromosomes were numbered in root tip squashes of a few individuals which were used as reference for the DNA quantification analysis. Aneuploidy and the occurrence of B chromosomes were not observed in our plant material. Similar result was obtained by A. Ramos, who made chromosome counts in Dactylis plants from the same sites in the Canary islands (pers. comm.). AIIozyme analysis. The 392 plants (26 individuals per population on average) were scored for polymorphism at eight loci. Inheritance of allozymes as well as electrophoretic and staining techniques used to detect them, have been described previously for glutamate oxaloacetate transaminases (locus GOT1) (Lumaret and Valdeyron, 1978), for acid phosphatases (locus AcPH1) (Lumaret, 1981a), for peroxidases (locus PX1) (Lumaret, 1982), for phosphoglucose isomerases (duplicated loci PGI1-2) (Lumaret, 1986), for tetrazolium oxidases (locus TO1) and alcohol dehydrogenases (duplicated loci ADH1-2) (Lumaret, 1981 b). Data treatment. The mean number of alleles and mean heterozygosity were calculated for each population and averaged over the populations of the same subspecies. The mean allelic diversity per population, estimated by the Shannon-Weaver index (1963) and averaged over the eight loci, was then apportioned (following Levontin, 1972) within populations, among populations of each subspecies (or morphological group) and among subspecies. The proportion of diversity due to gene differentiation among populations was also estimated by the Gst value (Nei, 1978). In addition, identities and distances of Nei (1978) which are mostly sensitive to substantial allele frequency variation and the distance of X 2 weighted by mean allele frequency to maximize the effect of rare alleles (Lumaret, 1984) were determined between pairs of populations from their allelic distributions. The respective positions of the populations estimated by the distances between them were plotted in multidimensional space and then projected on to a plane by non-metric multidimensional scaling (or proximity analysis) (Escourier, 1975). The same distances were used as the basis for a cluster analysis (unweighted pair group means analysis in that case). Clusters at different levels of agglomeration (total ranging from 0 to 100%) were mapped on to the diagram obtained from multidimensional scaling so that the agreement between the results from the two methods could easily be compared. These analyses were performed using the BIOMECO computer package (Lebreton et al., 1987).
Results Organization o f enzyme polymorphism in the subtropical group of D a c t y l i s g l o m e r a t a A l l e l e d i s t r i b u t i o n at t h e e i g h t s t u d i e d e n z y m e loci in t h e t h r e e d i p l o i d p o p u l a t i o n s f r o m Tenerife a n d t h e 12 t e t r a p l o i d p o p u l a t i o n s f r o m G r a n d Canary, M a d e i r a (inland a n d c o a s t a l p a r t s c o n s i d e r e d s e p a r a t e l y ) a n d P o r t u g a l are s h o w n in Table 3. S i x t e e n alleles w e r e i d e n t i f i e d in t h e d i p l o i d s w h e r e a s in t h e t e t r a p l o i d s , f r o m 2 4 t o 2 5 alleles w e r e o b s e r v e d a c c o r d i n g t o t h e e n t i t y a n d 3 7 alleles w e r e f o u n d in t h e w h o l e . All t h e alleles p r e s e n t in t h e d i p l o i d s a l s o o c c u r r e d in t h e t e t r a p l o i d s w h e r e a s a m o n g t h e 21
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
411
TABLE 3. ALLELE FREQUENCIES DISTRIBUTION AT EIGHT (PGI1-2 and ADH1-2 are duplicated loci) LOCI CODING FOR ENZYMES IN DIPLOID POPULATIONS FROM TENERIFE (Te, ssp. srnithii) AND TETRAPLOID POPULATIONS FROM GRAND CANARY (Ca, Canarian type), INLAND MADEIRA (Md-I, ssp. hylodes), COASTAL MADEIRA (Md-C, ssp. marina according to Parker, 1972) AND FROM THE COAST OF PORTUGAL (Po, ssp. marina). The number of studied populations is indicated between brackets (~=allele frequency < 0.01 )
Loci
Alleles
GOT
1.26 1.00 0.90 0.72 0.62 0.38 0.26 1.07 1.04 1.00 0.93 0.92 0.90 null 1.02 1.00 0.95 0.88 0.86 0.82 1.12 1.03 1.02 1.00 0.88 1.50 1.25 1.00 0.75 0.50 0,25 1,07 1,03 1.00 0.95 0.92 0.81
PX1
AcPh I
TO1
PGII-2
ADH1-2
Total allele No.
37
Te (3)
1.00
Ca (2)
0.08 0.86 0.03
Md-I (3)
Md-C (4)
Po (3)
0.19 0.74
0.26 0.66
0.01
0.07
0.08
0.72 0.27
~ 0.79
0.03 0.17 0.98 0.01
0.01 0.02 0.98
0.07 0.53 0.40
0.03 0.06 0.06 0.73 0.09 0.05 ~ 0.21 0.56 0.09 0.04
0.66 0.29 0.01 0.03 ~ 0.03 0.83 0.13 ~
0.66 0.32
0.45 0.51
0.01 ~ 0.03 0.65 0.30 0.02
0.03
0.29 0.66
0.01 0.62
0.05
0,37 ~ 0.07 0.78 0.14
0.30 0.03 0.09 0.60 0.28
0.02 0.07 0.70
0.01 0.69 0.28 0.01 c
0.03
0.67 0.03
0.13 0.72 0.08 0.03 0.04 0.36 0.45 0,14 0,05 0,01 0.18 0.53 0.28
0.68
0.34
0.03 0.02 0.72
0.29
0.65 0.01
0.22 0.01
0.19 0.02
0.82 0.14 0.02 0.02
16
25
25
24
24
alleles specific to the tetraploids, six were peculiar to the Canarian type, three to both the inland and coastal populations of Madeira, one to the coastal populations of Madeira, four to the Portuguese populations and seven were common to several of the tetraploid entities. The alleles GOT1 0 6• 2 , P X I 107 , PX1 0 9• 2 and T O 1102 were never found previously in Dactylis. At theAcPH1, PGI1-2 and A D H 1 - 2 loci, the predominant alleles (frequency higher than 50%) were the same in all the populations. At the G O T I locus, allele 1.00 was predominant in the populations from Madeira (67% on average and a range from 49% to 94%) whereas the 0.72 allele is predominant in populations from the other origins (85% on average with a range from 64% to 100%). The three Portuguese populations have no predominant allele at the TO1 locus and, at the PX1 locus, allele 0.93 is predominant whereas 1.00 is predominant in populations from all the other accessions.
412
E. SAHUQUILLO AND R. LUMARET
TABLE 4. NUMBER OF ALLELES/POPULATION (At), OBSERVED HETEROZYGOSlTY [Ho), DIVERSITY (Hpop) AND EQUITABILITY (E) WITHIN POPULATIONS, DIVERSITY AND EQUITABILITY WITHIN GEOGRAPHIC GROUPS (HG and EG, respectively) AVERAGED OVER EIGHT ENZYME LOCI IN 14 DACTYLIS POPULATIONS FROM THE SUBTROPICAL GROUP Population
At
Ho
Hpop
E
Te-1 Te-2 Ca-3 Ca-4 Md I-5 Md I-6 Md I-7 Md C-8 Md C-9 Md C-10 Md C-11 Po-12 Po-13 Po-14 Average
13 10 16 21 19 17 21 15 19 18 18 21 18 20 17.6
0.25 0.19 0.39 0.44 0.39 0.41 0.60 0.31 0.44 0.50 0.44 0.45 0.44 0.51 0.40
0.59 0,44 0.89 1.10 0.96 0.99 1.24 0.73 1.07 1.18 1.07 t .21 1.09 1.24 0.98
0,38 0.36 0.65 0.61 0.57 0.69 0.71 0.87 0.65 0.79 0.69 0.67 0.69 0,75 0.65
HG
EG
0.53
0.34
1,11
0.57
1.11
0.58
1.10
0.58
1,30
0.65
1.02
0.54
In each population, genotype distribution at the studied loci (except PGI1-2 and ADH1-2 which are duplicated loci in both diploids and tetraploids) (Lumaret, 1988) was compared to expected values assuming panmixia and postulating tetrasomic inheritance with the index of separation being equal to 0 in the tetraploids (Demarly, 1963). Using the Z2 test, no significant differences were found between observed and expected values except, at the GOTllocus in population Po-15 (P < 0.05), at the PX1 locus in population Po-13 (P < 0.01), at the TO1 locus in population Md-7 (P < 0.05) and at the AcpHl locus in populations Md-6 (P < 0.05), Md-7 (P < 0.05) and Md-12 (P < 0.05). In all those situations heterozygote deficiency was found. Observed heterozygosity and allele diversity averaged over the eight loci studied were calculated in each population and in the geographic groups (Fable 4). Average heterozygosity was twice higher in tetraploids than in diploids and 26% of the tetraploids were observed to be trigenic at least at one non-duplicated locus. Allele diversity estimated by the Shannon-Weaver index and equitability (the ratio of the Shannon index to its value when the alleles are equifrequent) were found to be twice lower in diploids from Tenerife than in tetraploids for which mean diversity values were rather stable and ranged from 1.10 in the coastal populations from Madeira to 1.30 in the Portuguese populations. The Gst value ranged from 0.10 at locus Pxlto 0.47 at Got1, the mean value over the studied loci being equal to 0.23. Using the Shannon index, parts of allelic diversity attributable to the three levels of variation, namely within populations, among populations of each subspecies (or morphological group) and among subspecies were equal to 70%, 4% and 26%, respectively. Genetic distances among the subtropical entities Average Nei's identities a n d ~2 distances among the 14 populations clustered into several geographical groups are indicated in Table 5. Mean identity within groups ranged from 96% in the group from the coast of Madeira and from Portugal to 99% in populations from Tenerife and those of Grand Canary. Identity between groups ranged from 65%, between the populations from the coast of Madeira and those of Grand Canary, to 97%, between the coastal and the inland populations of Madeira which show as high values as those observed within groups. The same results were obtained using the Z2 distances except that a higher distance value was obtained between the two populations from Grand Canary which had several distinct local
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
413
low frequency alleles. Genetic relationships among the 14 populations studied from their allozyme polymorphism and using t h e ~,2 distance are indicated in Fig. 1.The two diploid populations from Tenerife on the one hand and the seven tetraploid populations from Madeira (from the inside as well as from the coast of the island) on the other hand clustered closely together (level 55% of the UPGMA tree). At the 90% agglomeration level, the two tetraploid populations from Grand Canary clustered together as well as the three populations from Portugal whereas the populations TABLE 5. MATRIX OF MEAN NEI'S IDENTITIES (%) (upper right) AND 7,2 DISTANCES ( x l 0 0 ) (lower left) BETWEEN DACTYLIS POPULATIONS GROUPED ACCORDING TO THEIR GEOGRAPHIC ORIGIN IN THE SUBTROPICAL AREA. Minimum and maximum values are indicated between brackets Origin
Tenerife
Grand Canary
Madeira I
Madeira C
Portugal
Tenerife
99 -03.7
91 (88-93)
78 (70-82)
69 (57-81)
83 (77-92)
99 -13.0
73 (66-75)
65 (55-73)
80 (75-89)
98 (97-99) 06.1 (05.9-06.3)
97 (88-100)
74 (71-78)
96 (93-99) 07.6 (06.6-09.4)
70 (61-77)
Grand Canary
12.8 (12.6-13.0) Madeira I
11.7 (10.5-13.0)
14.2 (13.4-15.1 )
Madeira C
13.1 (11.0-14.7)
15.3 (14.1-16.4)
06.9 (04.3-09.0)
Po~ugal
96
13.8 (12.3-15.7)
15.3 (14.2-16.6)
14.0 (13.1-15.1 )
14.7 (13.1-16.7)
(94-97) 10.0 (09.6-10.4)
=Axis 2
...90
/._.,
1-
, ~,,',-,,'Mr8/-,1/ _,acr1~t , / -4/.c.".:.. / / M d 1 0 I~d9 /
/(¢dll
(
/
/ I,
3'
~---AxisI
-1-
FIG. 1. POSITION OF DIPLOID AND TETRAPLOID POPULATIONS FROM THE DACTYLIS SUBTROPICAL GROUP ACCORDING TO POLYMORPHISM AT LOCI GOT1. PXl, AcPHT, TO1, PGI1-2 AND ADH1-2. Multidimensional scaling from 7.2 distances. Populations are clustered at levels 55 and 90 of the hieramhical clustering.
414
E. SAHUQUILLO AND R. LUMARET
I
100-
a
605040302010O0 Q~
0 Po13 Po14 Po15 C04 CQ5 Tel
Te2 Md8 Md9 Md12 Mdll
Mdl0Md7 Md6
Populations
;0080-
b
70U3
605040-
2O 10 © Co4 Co5 Po13 Po14 Po15 Tel
Te2 Md8 Md9 Md12 Mdlo Md7 Md11 Md6
Populations FIG. 2. PHENOGRAMS SHOWING GENETIC RELATIONSHIPS AMONG DACTYLIS SUBTROPICAL GROUP OF POPULATIONS. (a) Hierarchical clustering based on Nei's Distances over eight studied enzyme loci (GOT1, PX1, AcPH1, TO1, ADH1-2 and PGI1-2). (b) Hierarchical clustering over seven loci (without GOT1).
from lenerife and Madeira constituted a single group. Similar results were obtained using Nei's distances (Fig. 2a) but two main differences were also observed in the agglomeration patterns: (1) populations fromTenerife and Grand Canary clustered at a lower agglomeration level, suggesting that the genetic differentiation between the populations from these two islands is due mostly to the occurrence of distinct low frequency alleles; and (2) populations from Madeira are genetically isolated from the other populations. The same data treatment was carried out without taking into account enzyme polymorphism at the GOT1 locus. Using Nei's distance, the results (Fig. 2b) were similar to those obtained when the whole loci were considered except that the populations from Madeira clustered more closely on the diagram with those from the other geographic accessions and more particularly with those fromTenerife. Such a result suggests that most of the differentiation observed between the populations from Madeira and the other subtropical populations is due to the occurrence of very distinct allele frequencies at the GOTI locus.
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
415
Discussion
Identification of distinct genetic entities in the Dactylis subtropical group and relationship among them In the present work, four genetic entities could be identified from the study of enzyme polymorphism in Dactylis populations from the Atlantic part of the subtropical group. A first entity is constituted by the diploid populations from Tenerife island. These diploids which have been described as ssp. smithfi (endemic in Tenerife and Cape Verde islands) according to specific morphological traits (Stebbins and Zohary, 1959) possess a lower genetic diversity for allozymes than the tetraploid entities. Lower allozyme diversity in diploids as compared to polyploids has been observed to be the rule in Dactylis glomerata (Lumaret, 1988) and has been found in many other infraspecific plant polyploid complexes (see Soltis and Soltis, 1993). A second entity consists of the tetraploid populations from Madeira island what ever be their local origin. This entity is the closest to ssp. smithi~ both morphologically and for isozyme variation (except for polymorphism at the GOT1 locus), suggesting that autopolyploidy likely occurred in Dactylis within the Macaronesian archipelago. Autopolyploidisation in endemics of oceanic islands has been observed very rarely (Borgen, 1979). According to Bramwell (1976), the flora of the Canary islands, mostly of very ancient origin, includes only 24.5% polyploids, most of them not being endemic in the Macaronesian islands. Autopolyploidisation of Dactylis in the Macaronesian islands likely constitutes therefore an exceptional event. Apart from two rare and/or low frequency alleles (PX11°4, TO1102 and T01112), Maderian populations from the inland part and those from the coastal part of the island show exactly the same aUelic pool with very similar frequency distribution so that they cannot be distinguished on the base of enzyme polymorphism In addition, according to the results of the present study, the Madeiran populations are clearly distinct genetically from the Portuguese populations which constitute a third subtropical entity identified morphologically as ssp. marina (Borrill, 1961). Such results do not support previous Parker's description (1972) based on morphological characters and according which the inland populations of Madeira constituted ssp. hylodes whereas those from the coast of the island belonged to ssp. marina. According to in situ observation made by the second author of the present paper, individual plants showing intermediate morphological traits between those of the inland and coastal populations were observed in several sites, suggesting that gene flow may occur among these two sets of populations. Up to now, all the subspecies which have been distinguished in Dactylis showed distinct allozyme patterns (Lumaret, 1988). The two groups of Madeiran populations may constitute therefore two ecotypes of the same subspecies (hylodes) which would be maintained distinct by environmental adaptive selection on several morphological or physiological traits (e.g. frequency of papillose cells). A fourth entity is constituted by the tetraploid populations from the centre of Grand Canary island. These populations also constitute a very morphologically distinct group growing at high elevation in dry habitats and under sub-humid Mediterranean climate which is not much influenced by the sea (Tables 1 and 2 and unpublished data). Within the subtropical group, the Canarian type possesses the highest number of alleles which are typical of the Mediterranean group (e.g. PGI1-2°"5 0 , PGI1-2° 25 , etc.) (Lumaret, 1988). Moreover, another population of Dactylis plants, all diploid and which were indistinguishable morphologically from the tetraploids was discovered very recently in the centre of Grand Canary island (A. Ramos, pers. comm. and unpublished data). Such a situation suggests that (1) within the Macaronesia area, early differentiation may have occurred in diploids to create two distinct entities, adapted to distinct climatic conditions, inTenerife and Grand Canary (two large islands), respectively, and (2) autopolyploidy likely occurred from each of these two diploids which are both endemic in the Macaronesian area. However, as both diploid and tetraploid plants
416
E. SAHUQUILLO AND R. LUMARET
from the centre of Grand Canary island combine morphological traits observed usually in Dactylis entities from either the subtropical or the Mediterranean groups, the possibility that the diploids may descend from hybridization between two diploid entities, one located in the subtropical group (Macaronesia) and the other originally from a mainland Mediterranean area (for instance, North Africa) cannot be ruled out. The present allozyme study in the Dactylis subtropical group shows that, on the whole, allele pool and frequency distribution are close but still distinct from those observed previously in the Mediterranean group (Lumaret, 1988). The subtropical taxa form therefore a distinct genetic group which may reflect a monophyletic origin for that group with reserves regarding origin of the Grand Canary type (see above). Low levels of allozyme divergence have been reported in several plant species endemic to oceanic island, e.g. in Tetrarnolopium species from Hawaiian islands (Lowrey and Crawford, 1985), in Dendroseris species from the Juan Fernandes islands (Crawford et al., 1987) and in the Chamaecytisus proliferus complex in the Canary islands (Francisco-Ortega et al., 1992). According to these authors, the occurrence of low levels of allozyme divergence is attributable to the combined effects of genetic bottlenecks associated with colonisation, small population size, and to either speciation on young island or, alternatively, recent colonization of old islands. By contrast, higher genetic divergence was observed in several other taxa endemic to oceanic archipelagoes, e.g. in Gossypium darwinii from the Galapagos islands (Wendel and Percy, 1990), in Avena canariensis from the Canary islands (Morikawa and Leggett, 1990) and in several species of Robinsonia endemic to the Juan Fernandez islands (Crawford et aL, 1992), and was considered to be mainly the result of progressive accumulation of mutations following early colonisation of the several islands. In the Dactylis subtropical group, substantial genetic divergence is observed among several (mainly tetraploid) taxonomic entities which grow in distinct islands of the Macaronesian archipelago or along Portugal coasts. In Dactylis, genetic divergence among the subtropical entities, estimated in the present study by either the Gst value or mean Nei's identity value, is much higher than divergence observed previously among mainland tetraploid subspecies (Lumaret, 1985). That result provides evidence for very early colonisation and adaptive radiation of the parental diploid entities in the Macaronesian area. In addition, substantial accumulation of new allozymes in the polyploids, endemic to the Macaronesian area and clearly related to the diploids, may reflect the occurrence of early autopolyploidisation in that region. However, accumulation of mutations was likely slower in the diploids than in the derived autopolyploids which possessed higher genetic inertia with much slower segregation rates (Stebbins, 1980). Moreover, most of the differentiation between ssp. hylodes and the other entities of the subtropical group is expressed at the GOT1 locus, suggesting that genetic variation at that locus may have a different evolutionary significance from that observed at the other studied allozyme markers.
Adaptive differentiation in the Dactylis subtropical group At the GOT1 locus, the allele 0.72 which is present alone in the diploids (ssp. smithii) and is predominant in the tetraploid populations from Grand Canary and Portugal, occurs at a very low frequency in the populations from Madeira in which the allele 1.00 becomes predominant. In addition, all the Madeiran populations are characterised by the occurrence of allele GOT1126 (24% on average). Results from previous extensive studies in Dactylis glomerata have shown that several alleles investigated at the GOT1 locus in numerous diploid and tetraploid populations were non-randomly distributed. More particularly, the frequency of alleles 1.00 and 1.26 were shown to be statistically positively correlated with humidity whereas high frequency of allele 0.72 was related to the occurrence of a dry environment (Lumaret, 1984; Roy and Lumaret, 1987). Average annual rainfall and local humidity are much
ALLOZYMEPOLYMORPHISMIN DACTYLISGLOMERATA
417
higher and the summer drought period is much shorter (or most of the time is absent) in the Dactylis collecting sites of Madeira than in those of the other collecting areas in Tenerife, Grand Canary and the southern coast of Portugal (Walter et al., 1979). In Dactyiis subtropical group, allozyme differentiation observed at the GOT1 may have therefore an adaptive significance and may be the result of natural selection on that locus or on loci tightly linked to it. As shown in the present study, substantial allozyme variation occurs in the Dactylis subtropical group. This variation appears to be due to the great geographic discontinuity of the distribution area which favours the occurrence of distinct local alleles in the several islands, and, as shown at the particular locus GOT1, allozyme variation may be the result of adaptive selection related to significant climate variation, more particularly for humidity occurring in that Atlantic region. Acknowledgements--We thank A. Ramos-Martinez and A. Marrero-Rodriguez (Jardin Botanico "Viera y
Clavijo", Grand Canary) for their help in plant material collection in Grand Canary and Tenerife islands.
References Ardouin, P., Jay, M. and Lumaret, R. (1987) Etude d'une situation de sympatrie entre diplo(des et t~traplofdes de Dactylis glomerata L. sur la base du polymorphisme enzymatique et ph~nolique. Can. J. Bot. 65, 526-531~ Benson, M. and Borrill, M. (1969) The significance of clinal variation in Dactylis marina Borrill. New Phytol. 68, 1159-1173. Borgen, L. (1979) Karyology of the Canarian flora. In Plants and/s/ands (Bramwell, D., ed.), pp. 329-346. Academic Press, London. Borrill, M. (1957) A morphologically distinct ecotype of Dactylis glomerata L. Nature 179, 544-546. Borrill, M. (1961 ) Dactylis marina Borrill, sp. nov., a natural group of related forms. J. Linn. Soc. 56, 431 439. Borrill, M. (1978) Evolution and Genetic Resources in Cocksfoot. A. Rep. Welsh Plant Breeding Station, 1977, Aberystwyth, U.K., pp. 190-209. Bramwell, D. (1976) The endemic flora of the Canary islands; distribution, relationships and phytogeography. In Biogeography andEcology in the Canary/s/ands (Kunkel, G., ed.), pp. 207-248. Junk, The Hague. Crawford, D. J., Stuessy, T. F. and Silva, M. (1987) AIIozyme divergence and the evolution of Dendroseris (Compositae: Lactuceae) on the Juan Fernandez islands. Syst. Bot. 12, 435-443. Crawford, D. J., Stuessy, T. F., Haines, D. W., Cosner, M. B., Silva, O. M. and Lopez, P. (1992) Allozyme diversity within and divergence among four species of Robinsonia (Asteraceae: Senecioneae), a genus endemic to the Juan Fernandez islands, Chile. Am. J. Bot. 79, 962-966. De Lemps, H. (1969) Le Climat des iles Canaries. Pub. Facult~ lettres et sciences humaines, s(~rie recherches, Paris-Sorbonne (54). Demarly, Y. (1963) G~n~tique des t~traplo'Ldeset amelioration des plantes. Ann. Amdlior. P/antes 13, 307400. Escoufier, Y. (1975) Le positionnement multidimentioneL Rev. Star. Appl. 23, 5-14. Fiasson, J. L., Ardouin, P. and Jay, M. (1987) Phylogenetic groundplan of the specific complex Dactylis glomerata L. Biochem. Syst. Ecol. 15, 225-228. Francisco-Ortega, J., Jackson, M. T,, Catty, J. P. and Ford-Lloyd, B. V. (1992) Genetic diversity in the Chamaecytisus proliferus complex (Fabaceae: Genisteae) in the Canary islands in relation to in situ conservation. Genet. Res. Crop Evol. 39, 149--158. Jay, M., Plenet, D., Ardouin, P., Lumaret, R. and Jacquard, P. (1984) Flavonoid variation in seven tetraploid populations of Dactylis glomerata L. (Gramineae). Biochem. Syst. Ecol. 12, 193-198. Jay, M., Fiasson, J. L., Lumaret, R. and Ardouin, P. (1989) Tetraploidy and habitat differentiation reflected by flavonoid patterns in Dactylis glomerata L from Galicia (Spain). Biochem. Syst. Ecol. 17, 283-285. Lebreton, J. D., Roux, M., Banco, G. and Bacou, A. M. (1987). The BIOMECO package, Centre Emberger, Centre National de la Recherche Scientifique, Montpellier, France. Levontin, R. C. (1972) The apportionment of human diversity. In Evolutionary Biology 6 (Dobzhansky, T., Hecht, M. K. and Steere, W. C., eds), pp. 381-383. Appleton-Century-Crofts, New York. Lowrey, T. K. and Crawford, D. J. (1985) AIIozyme divergence and evolution in Tetramolopium (Compositae: Astereae) on the Hawaiian islands. Syst. Bot. 101, 64-72.
418
E. SAHUQUILLOAND R. LUMARET
Lumaret, R. (1981 a) Etude de I'h~r~dit~ de phosphatases acides chez le Dactyle (Dactylis glomerata L.) diplo'fde et t~traplofde. Can. J. Genet. Cytol. 23, 513-523. Lumaret, R. (1981 b) Structure g~n~tique d'un coplexe polyplotde: Dactylis glomerata L. Relations entre le polymorphisme enzymatique et certains aspects de la biologie, de I'~cologie et de I'~volution de I'esp~ce. State D; Thesis, Universit~ des Sciences et Techniques du Languedoc, Montpellier, France. Lumaret, R. (1982) Protein variation in diploid and tetraploid orchard grass (Dactylisglomerata L.): formal genetics and population polymorphism of peroxidases and malate dehydrogenases. Genetica 57, 207-215. Lumaret, R. (1984) The role of polyploidy in the adaptive significance of polymorphism at the GOT1 locus in the Dactylis glomerata complex. Heredity 52, 153-169. Lumaret, R, (1985) Phenotypic and genotypic variation within and between populations of the polyploid complex Dactylis glomerata L. In Structure and Functioning of Plant Populations, Phenotypic and Genotypic Variation in Plant Populations (Haeck, J. and Woldendorp, J. W., eds), pp. 343-354. North-Holland Publishing Co., Amsterdam. Lumaret, R. (1986) Doubled duplication of the structural gene for cytosolic phosphoglucose isomerase in the Dactylis glomerata L. polyploid complex. Molec. Biol. Evol. 3, 449-452. Lumaret, R. (1988) Cytology, genetics and evolution in the genus Dactylis, C.R.C. Crit. Rev. Plant Sci. 7, 55--91. Lumaret, R. and Valdeyron, G. (1978) Les glutamate oxaloac6tate transaminases du Dactyle (Dactyfis glomerata L.): G6n~tique formelle d'un locus. C. R. Acad. ScZ Paris 287 D, 705-708. Lumaret, R., Bowman, C. M. and Dyer, T. A. (1989) Autopolyploidy in Dactylis glomerata L.: further evidence from studies of chloroplast DNA variation. Theoret. Appl. Genet. 78, 393-399. Lumaret, R. and Barrientos, E. (1990) Phylogenetic relationships and gene flow between sympatric diploid and tetraploid plants of Dactylis glomerata (Gramineae). Plant Syst, Evol. 169, 81-96. Maceira, N. O., De Haan, A. A., Lumaret, R., Billon, M. and Delay, J. (1992) Production of 2n gametes in diploid subspecies of Dactylisglomerata L. 1. Occurrence and frequency of 2n pollen. Ann. Bot. 69, 335-343. Morikawa, T. and Leggett, J. M (1990) Isozyme polymorphism in natural populations of Avena canariensis from the Canary islands. Heredity 64, 403-411. Nei, M. (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583-590. Parker, P. F. (1972) Studies in Dactylis 2. Natural variation, distribution and systematics of the Dacty/is smithii Link. complex in Madeira and other Atlantic islands. New Phytol. 71,371-378. Roy, J. and Lumaret, R. (1987) Associated clinal variation in leaf tissue water relations and allozyme polymorphism in Oactylis glomerata L. populations. EvoL Trends Plants 1, 9-19. Sarich, V. M. (1977) Rates, sample sizes and the neutrality hypothesis for electrophoresis in evolutionary studies. Nature 265, 24-28. Shannon, C. E. and Weaver, W. (1963) The Mathematical Theory of Communication. Urbana Univ. Illinois Press. Soltis, D. E. and Soltis, P. S. (1993) Molecular data and the dynamic nature of polyploidy. C.R,C. Crit. Rev. Plant Sci. 12, 243-273. Stebbins, G. L. (1980) Polyploidy in plants: unsolved problems and prospects. In Polyploidy, Biological Relevance (Lewis, H., ed.), pp. 495-520. Plenum, New York. Stebbins, G. L and Zohary, D. (1959) Cytogenetics and evolutionary studies in the genus Dactylis 1. Morphology distribution and interrelationships of the diploid subspecies. Univ. California Pub/. Bot. 31, 1-40. Walter, H., Harnickell, E. and Mueller-Dombois, D. (1979) Climate Diagram Maps. Springer Verlag, Berlin. Wendel, J. F. and Percy, R. G. (1990) AIIozyme diversity and introgression in the Galapagos islands endemic Gossypium darwinii and its relationship to continental G. barbadense. Biochem. Syst. Ecol. 18, 517-528. Wilson, D., Abdullah, I. B. and Trickey, S. A. (1977) Variation in transpiration rate in Dactylis. Proc. X/I/ Int. Grassland Congress, Leipzig, pp. 71-79.
Biochemicai~stematics and Ecology.Vol. 23, Na 4, pp. 407-418.1995 Copyright(D1995 ElsevierScience Ltd Printedin Great Britain.All rightsreserved 0305-1978/95 $9.50+0.00
0305-1978(95)00020-8
Variation in the Subtropical Group of Dactyhs glomerata L.1. Evidence from Allozyme Polymorphism ELVIRA SAHUQUILLO and ROSELYNE LUMARET DBpartement de Biologie des Populations, Centre d’Ecologie Fonctionnelle et Evolutive L. Emberger, CNRS, B.l? 5051,34033 Montpellier Cedex. France
Key Word
Index-Dacty/isglomerata;
Poaceae; allozymes; population variation; polyploidy; subtropical group.
Abstract-Allozyme variation at eight polymorphic loci was analysed in 15 populations collected over the Atlantic part of the subtropical distribution area of Dactylis glomerafa L. Four genetically distinct entities were identified. One included the diploid populations of subspecies smithji, endemic of Tenerife Island. A second included the tetraploid populations from Grand Canary island which also constituted a morphologically distinct group. A third group clusted together inland and coastal tetraploid populations from Madeira Island (likely two ecotypes of subspecies hylodes) which had been described previously as two distinct subspecies according to morphological traits. A fourth group gathered together the tetraploid populations from the coasts of Portugal (ssp. marina). The substantial allozyme variation observed in that subtropical group may be attributed partly to the occurrence of large geographic discontinuities in the distribution area and, at a particular locus (GOT7). the observed variation was assumed to be due mostly to adaptive selection related to large variation for humidity conditions in that Atlantic area.
Introduction The perennial outcrossing grass Dactylis ghmerata L., is one of the best-documented examples of a natural infraspecific polyploid complex. Evidence from its geographic distribution, comparative morphology, cytogenetic structure, its variation for allozymes, phenolic compounds and chloroplast DNA has been used to study phylogenetic relationships among the Da&y/is taxonomic groups (Stebbins and Zohary, 1959; Borrill, 1978; Jay et a/., 1984, 1989; Ardouin et a/., 1987; Fiasson et a/., 1987; Lumaret, 1988; Lumaret et a/., 1989; Lumaret and Barrientos, 1990). In Dactylis, the diploid (2n = 14) and tetraploid (213= 28) cytotypes were classified into either two groups of subspecies, namely Mediterranean and Eurasian groups, according to morphogeographical criteria, or into three groups, namely Subtropical, Mediterranean and Temperate groups, according to climate (Borrill, 1978; Lumaret, 1988). Plants of the subtropical group were mostly recorded from the Macaronesian islands (Madeira, Canary and Cape Verde), from the coasts of Portugal and, to a lesser extent, from several other coastal habitats in the Mediterranean Basin (Borrill, 1961; Parker, 1972; Lumaret, 1988). Whatever their geographic origin, plants of the subtropical area possess several particular morphological characters in common such as woody stems and papillose epidermal cells, suggesting that the several morphological forms identified in that group are closely related (Borrill, 1957,196l; Parker,1972; Borrill, 1978). In Dactylis, the papillose epidermal cells have been shown to play an important role in water use efficiency from the atmosphere (Wilson et al., 1977) and the papillose forms are restricted to cloudy or coastal habitats in regions of high summer temperature (Benson and Borrill, 1969). Dactylis plants of the subtropical group also show substantial cytological and morphological variation which may be related to their discontinuous pattern of spatial distribution (mostly between islands) as well as to the contrasted climatic conditions (more particularly for annual precipitations) occurring among the distribution (Received 11 July 1994)
408
E. SAHUQUILLOAND R. LUMARET
areas and even within a single island (De Lemps, 1959; Benson and Borrill, 1969). At least three subspecies have been already identified in the Atlantic part of the subtropical group. Those are the diploid smithii (Link) Stebbins and Zohary, endemic in Tenerife and Cape Verde islands (Stebbins and Zohary, 1959), the tetraploid hylodes Parker, endemic of the inland part of Madeira island (Parker, 1972) and the tetraploid marina Borrill distributed along the coasts of Portugal (Borrill, 1957, 1961; Benson and Borrill, 1969) and, according to Parker (1972) on the coasts of Madeira. Subspecies smithii is closely associated with the subtropical flora that is considered to date from the middle of the Tertiary period and this subspecies or its precursors should date therefore from the Miocene epoch whereas the tetraploid ssp. hylodes and marina should be of more recent origin and may have arisen from diploids closely related to ssp. smithii (Parker, 1972; Borrill, 1978). Additional morphologically distinct D. glomerata entities were discovered recently by Ramos and Marrero (pers. comm.) in several islands of the Canaries and more particularly in the central part of Grand Canary island. Further investigation of natural variation in Dactylis plant material from the subtropical group was therefore necessary to clarify the relationships among the several entities of that group. To be effective, those studies combined the use of several markers (e.g. morphological traits, enzyme and DNA polymorphism, variation for phenolic compounds, etc.) on the same plant material. Those markers are known indeed to have distinct evolutionary rates and various sensitivity vs environmental selection. Results obtained in those investigations are reported in a series of papers. The present paper is the first of the series and deals with the geographical distribution of isozyme polymorphism in populations of diploid and tetraploid subspecies or morphologically distinct types of Dactylis glomerata occurring in the Atlantic part of the subtropical group. Enzyme markers are considered most generally neutral (Sarich, 1977). However, in Dactylis, extensive studies have shown that the frequency of several alleles at the GOTI locus, which codes for glutamate oxaloacetate transaminases, were significantly correlated with soil humidity even if no evidence could be obtained to show that selection was acting directly on that locus (Lumaret, 1984; Roy and Lumaret, 1987). The main objectives of the present work are therefore (1) to estimate the genetic differentiation among the subtropical entities from the study of allele variation at eight polymorphic enzyme loci, and (2) to assess the part of adaptive differentiation related to climate variation by studying separately allozyme polymorphism at the GOTI locus. Materials and Methods Plant material Fifteen populations distributed over the Atlantic part of the subtropical Dactylis geographic distribution were studied (Table 2). Three populations of diploid plants (Te-1, Te-2 and Te-3) were collected in Tenerife island and were identified as ssp. smithii according to the formal description of Stebbins and Zohary (1959). Two populations of tetraploid plants (Ca-4 and Ca-5) were collected in the centre of Grand Canary island and are considered to belong to a morphologically distinct type (Ramos, pers. comm.), although it has not been yet described formally as a subspecies. Three populations of diploid plants (Md-6, Md-7 and Md-8) and four populations of tetraploid plants (Md-9, Md-10, Md-11 and Md-12) were collected from the inland part and the coastal part of Madeira island, respectively. The plants had been identified as ssp. hylodes (inland populations) and ssp. marina (coastal populations) by Parker (1972). Three populations of tetraploid plants (Po~13, Po-14 and Po-15) were collected along the coast of Portugal and were identified as ssp. marina according to Borrill (1961). The main morphological and environmental characteristics of the several subspecies involved in the present study are summarised in Table 1. The location of the collecting sites and the number of plants sampled are indicated in Table 2. The four plants collected from site Te-3 were not involved in the population genetics analysis. Plant material from sites Te-2, Po-13, Po-14 and Po-15 came from the collection of M. Borrill (Ref. BC 6425, BC 5221, BC 5217 and BC 5215 respectively, Welsh Plant Breeding Station, Aberystwyth). For plant and site description of the Portuguese material, see Benson and Borrill (1969). Sites Md-6, Md-8, Md-9, Md-10 and Md-11 in the present study correspond to sites 37, 27, 1, 50 and 19, respectively, which were described by Parker (1972).
Ssp. or type
srnithii
Canarian type
hylodes
marina
marina
Location
Tenerife
Grand Canary
Madeira inland
Madeira coast
Coast of Portugal
4x
4x
4x
4x
2x
Ploidy level
Bushy plants and dense tillering Bushy plants and dense tillering
Lax Shrubby
Shrubby and dense tillering
Lax Shrubby
Plant habit
20-50
20-70
20-50
90-10O
35-95
Culm. height (cm)
Prostrate
Prostrate or hanging
Erect or hanging
Erect
Erector hanging
Habit
Stems
+
+
+
+
+
Woody
+
+
-
-
+
Secondary branches
Glaucous
Glaucous
Light green
Glaucous
Light green
Co/our
+++
++
+
+
+
Epidermal papillae
Leaves
+++
++
+
++
-
3.5
3.2
2.7
5.0
6.0
Panicle Lobed Average lemma florets per apex spikelets
E
18002675
12001530
F G H
D
850980
415850
B C
430470
Dry mostly shaded crevices in volcanic rocks (150--600) Dry open rocky areas with bushy plants ( > 1200) inland cliffs or rocky areas (1 0-1300) Coast cliffs (0-50O)
Sand coasts rocks or maritime pasture by sea shore (0-500)
A
Annual rainfall (mm)
Habitat (altitude) (m)
Ref.
TABLE 1. GEOGRAPHICAL LOCATION, MORPHOLOGICAL TRAITS, HABITAT AND CLIMATE CHARACTERISTICS OF FIVE SUBSPECIES OR MORPHOLOGICAL TYPES IDENTIFIED IN THE SUBTROPICAL GROUP OF DACTYLIS GLOMERATA L. A, B, C, D, E, F, G and H refer to Stebbins and Zohary (1959), authors of the present paper, Ramos (pars. comm.), Parker (1972), Borrill (1957), Borrill (1961 ) and Benson and Borrill (1969), respectively. Annual rainfall data are from De Lamps (1969) and Walter eta/. (1979).
410
E. SAHUQUILLOAND R. LUMARET
TABLE2. Geographicorigin, locality,altitudeand numberof plantssampledin 15 collectingsitesdistributedoverthe subtropical areaof Dactylisglomerata Code
Origin
Locality
Altitude (m)
Plantno.
Te-1 Te-2 Te-2b Ca-3 Ca-4 Md-5 Md-6 Md-7 Md-8 Md-9 Md-10 Md-11 Po-12 Po-13 Po 14
Tenerife Tenerife Tenerife GrandCanary GrandCanary Madeira Madeira Madeira Madeira Madeira Madeira Madeira Portugal Portugal Portugal
Tigaiga Barrancode Ruiz Los Silos Degolladade Las Palomas AriSez Serrade Agua Lombode Cima Eirado Serrado CaboGirao SaoVieente Portoda Cruz Pontado Pargo Fozde Arelho Praiadas Macas CaboSao Vicente
580 620 350 1600 1400 300 350 850 580 5 5 250 15 10 20
44 37 4 17 27 32 19 20 16 27 13 15 28 48 45
Allozyme assays were performed on leaf tissue, from plants collected in situ in the case of the populations from Madeira and in all other cases from seedlings grown from seeds collected in nature. The seeds were germinated and seedlings grown in the greenhouse for approximately 1 2 weeks until they were assayed. One seedling individual was examined from each mother plant. Plant ploidy level was determined from DNA quantification using flow cytometry and a method adapted to Dactylis material (Maceira et al., ! 992). Chromosomes were numbered in root tip squashes of a few individuals which were used as reference for the DNA quantification analysis. Aneuploidy and the occurrence of B chromosomes were not observed in our plant material. Similar result was obtained by A. Ramos, who made chromosome counts in Dactylis plants from the same sites in the Canary islands (pers. comm.). AIIozyme analysis. The 392 plants (26 individuals per population on average) were scored for polymorphism at eight loci. Inheritance of allozymes as well as electrophoretic and staining techniques used to detect them, have been described previously for glutamate oxaloacetate transaminases (locus GOT1) (Lumaret and Valdeyron, 1978), for acid phosphatases (locus AcPH1) (Lumaret, 1981a), for peroxidases (locus PX1) (Lumaret, 1982), for phosphoglucose isomerases (duplicated loci PGI1-2) (Lumaret, 1986), for tetrazolium oxidases (locus TO1) and alcohol dehydrogenases (duplicated loci ADH1-2) (Lumaret, 1981 b). Data treatment. The mean number of alleles and mean heterozygosity were calculated for each population and averaged over the populations of the same subspecies. The mean allelic diversity per population, estimated by the Shannon-Weaver index (1963) and averaged over the eight loci, was then apportioned (following Levontin, 1972) within populations, among populations of each subspecies (or morphological group) and among subspecies. The proportion of diversity due to gene differentiation among populations was also estimated by the Gst value (Nei, 1978). In addition, identities and distances of Nei (1978) which are mostly sensitive to substantial allele frequency variation and the distance of X 2 weighted by mean allele frequency to maximize the effect of rare alleles (Lumaret, 1984) were determined between pairs of populations from their allelic distributions. The respective positions of the populations estimated by the distances between them were plotted in multidimensional space and then projected on to a plane by non-metric multidimensional scaling (or proximity analysis) (Escourier, 1975). The same distances were used as the basis for a cluster analysis (unweighted pair group means analysis in that case). Clusters at different levels of agglomeration (total ranging from 0 to 100%) were mapped on to the diagram obtained from multidimensional scaling so that the agreement between the results from the two methods could easily be compared. These analyses were performed using the BIOMECO computer package (Lebreton et al., 1987).
Results Organization o f enzyme polymorphism in the subtropical group of D a c t y l i s g l o m e r a t a A l l e l e d i s t r i b u t i o n at t h e e i g h t s t u d i e d e n z y m e loci in t h e t h r e e d i p l o i d p o p u l a t i o n s f r o m Tenerife a n d t h e 12 t e t r a p l o i d p o p u l a t i o n s f r o m G r a n d Canary, M a d e i r a (inland a n d c o a s t a l p a r t s c o n s i d e r e d s e p a r a t e l y ) a n d P o r t u g a l are s h o w n in Table 3. S i x t e e n alleles w e r e i d e n t i f i e d in t h e d i p l o i d s w h e r e a s in t h e t e t r a p l o i d s , f r o m 2 4 t o 2 5 alleles w e r e o b s e r v e d a c c o r d i n g t o t h e e n t i t y a n d 3 7 alleles w e r e f o u n d in t h e w h o l e . All t h e alleles p r e s e n t in t h e d i p l o i d s a l s o o c c u r r e d in t h e t e t r a p l o i d s w h e r e a s a m o n g t h e 21
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
411
TABLE 3. ALLELE FREQUENCIES DISTRIBUTION AT EIGHT (PGI1-2 and ADH1-2 are duplicated loci) LOCI CODING FOR ENZYMES IN DIPLOID POPULATIONS FROM TENERIFE (Te, ssp. srnithii) AND TETRAPLOID POPULATIONS FROM GRAND CANARY (Ca, Canarian type), INLAND MADEIRA (Md-I, ssp. hylodes), COASTAL MADEIRA (Md-C, ssp. marina according to Parker, 1972) AND FROM THE COAST OF PORTUGAL (Po, ssp. marina). The number of studied populations is indicated between brackets (~=allele frequency < 0.01 )
Loci
Alleles
GOT
1.26 1.00 0.90 0.72 0.62 0.38 0.26 1.07 1.04 1.00 0.93 0.92 0.90 null 1.02 1.00 0.95 0.88 0.86 0.82 1.12 1.03 1.02 1.00 0.88 1.50 1.25 1.00 0.75 0.50 0,25 1,07 1,03 1.00 0.95 0.92 0.81
PX1
AcPh I
TO1
PGII-2
ADH1-2
Total allele No.
37
Te (3)
1.00
Ca (2)
0.08 0.86 0.03
Md-I (3)
Md-C (4)
Po (3)
0.19 0.74
0.26 0.66
0.01
0.07
0.08
0.72 0.27
~ 0.79
0.03 0.17 0.98 0.01
0.01 0.02 0.98
0.07 0.53 0.40
0.03 0.06 0.06 0.73 0.09 0.05 ~ 0.21 0.56 0.09 0.04
0.66 0.29 0.01 0.03 ~ 0.03 0.83 0.13 ~
0.66 0.32
0.45 0.51
0.01 ~ 0.03 0.65 0.30 0.02
0.03
0.29 0.66
0.01 0.62
0.05
0,37 ~ 0.07 0.78 0.14
0.30 0.03 0.09 0.60 0.28
0.02 0.07 0.70
0.01 0.69 0.28 0.01 c
0.03
0.67 0.03
0.13 0.72 0.08 0.03 0.04 0.36 0.45 0,14 0,05 0,01 0.18 0.53 0.28
0.68
0.34
0.03 0.02 0.72
0.29
0.65 0.01
0.22 0.01
0.19 0.02
0.82 0.14 0.02 0.02
16
25
25
24
24
alleles specific to the tetraploids, six were peculiar to the Canarian type, three to both the inland and coastal populations of Madeira, one to the coastal populations of Madeira, four to the Portuguese populations and seven were common to several of the tetraploid entities. The alleles GOT1 0 6• 2 , P X I 107 , PX1 0 9• 2 and T O 1102 were never found previously in Dactylis. At theAcPH1, PGI1-2 and A D H 1 - 2 loci, the predominant alleles (frequency higher than 50%) were the same in all the populations. At the G O T I locus, allele 1.00 was predominant in the populations from Madeira (67% on average and a range from 49% to 94%) whereas the 0.72 allele is predominant in populations from the other origins (85% on average with a range from 64% to 100%). The three Portuguese populations have no predominant allele at the TO1 locus and, at the PX1 locus, allele 0.93 is predominant whereas 1.00 is predominant in populations from all the other accessions.
412
E. SAHUQUILLO AND R. LUMARET
TABLE 4. NUMBER OF ALLELES/POPULATION (At), OBSERVED HETEROZYGOSlTY [Ho), DIVERSITY (Hpop) AND EQUITABILITY (E) WITHIN POPULATIONS, DIVERSITY AND EQUITABILITY WITHIN GEOGRAPHIC GROUPS (HG and EG, respectively) AVERAGED OVER EIGHT ENZYME LOCI IN 14 DACTYLIS POPULATIONS FROM THE SUBTROPICAL GROUP Population
At
Ho
Hpop
E
Te-1 Te-2 Ca-3 Ca-4 Md I-5 Md I-6 Md I-7 Md C-8 Md C-9 Md C-10 Md C-11 Po-12 Po-13 Po-14 Average
13 10 16 21 19 17 21 15 19 18 18 21 18 20 17.6
0.25 0.19 0.39 0.44 0.39 0.41 0.60 0.31 0.44 0.50 0.44 0.45 0.44 0.51 0.40
0.59 0,44 0.89 1.10 0.96 0.99 1.24 0.73 1.07 1.18 1.07 t .21 1.09 1.24 0.98
0,38 0.36 0.65 0.61 0.57 0.69 0.71 0.87 0.65 0.79 0.69 0.67 0.69 0,75 0.65
HG
EG
0.53
0.34
1,11
0.57
1.11
0.58
1.10
0.58
1,30
0.65
1.02
0.54
In each population, genotype distribution at the studied loci (except PGI1-2 and ADH1-2 which are duplicated loci in both diploids and tetraploids) (Lumaret, 1988) was compared to expected values assuming panmixia and postulating tetrasomic inheritance with the index of separation being equal to 0 in the tetraploids (Demarly, 1963). Using the Z2 test, no significant differences were found between observed and expected values except, at the GOTllocus in population Po-15 (P < 0.05), at the PX1 locus in population Po-13 (P < 0.01), at the TO1 locus in population Md-7 (P < 0.05) and at the AcpHl locus in populations Md-6 (P < 0.05), Md-7 (P < 0.05) and Md-12 (P < 0.05). In all those situations heterozygote deficiency was found. Observed heterozygosity and allele diversity averaged over the eight loci studied were calculated in each population and in the geographic groups (Fable 4). Average heterozygosity was twice higher in tetraploids than in diploids and 26% of the tetraploids were observed to be trigenic at least at one non-duplicated locus. Allele diversity estimated by the Shannon-Weaver index and equitability (the ratio of the Shannon index to its value when the alleles are equifrequent) were found to be twice lower in diploids from Tenerife than in tetraploids for which mean diversity values were rather stable and ranged from 1.10 in the coastal populations from Madeira to 1.30 in the Portuguese populations. The Gst value ranged from 0.10 at locus Pxlto 0.47 at Got1, the mean value over the studied loci being equal to 0.23. Using the Shannon index, parts of allelic diversity attributable to the three levels of variation, namely within populations, among populations of each subspecies (or morphological group) and among subspecies were equal to 70%, 4% and 26%, respectively. Genetic distances among the subtropical entities Average Nei's identities a n d ~2 distances among the 14 populations clustered into several geographical groups are indicated in Table 5. Mean identity within groups ranged from 96% in the group from the coast of Madeira and from Portugal to 99% in populations from Tenerife and those of Grand Canary. Identity between groups ranged from 65%, between the populations from the coast of Madeira and those of Grand Canary, to 97%, between the coastal and the inland populations of Madeira which show as high values as those observed within groups. The same results were obtained using the Z2 distances except that a higher distance value was obtained between the two populations from Grand Canary which had several distinct local
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
413
low frequency alleles. Genetic relationships among the 14 populations studied from their allozyme polymorphism and using t h e ~,2 distance are indicated in Fig. 1.The two diploid populations from Tenerife on the one hand and the seven tetraploid populations from Madeira (from the inside as well as from the coast of the island) on the other hand clustered closely together (level 55% of the UPGMA tree). At the 90% agglomeration level, the two tetraploid populations from Grand Canary clustered together as well as the three populations from Portugal whereas the populations TABLE 5. MATRIX OF MEAN NEI'S IDENTITIES (%) (upper right) AND 7,2 DISTANCES ( x l 0 0 ) (lower left) BETWEEN DACTYLIS POPULATIONS GROUPED ACCORDING TO THEIR GEOGRAPHIC ORIGIN IN THE SUBTROPICAL AREA. Minimum and maximum values are indicated between brackets Origin
Tenerife
Grand Canary
Madeira I
Madeira C
Portugal
Tenerife
99 -03.7
91 (88-93)
78 (70-82)
69 (57-81)
83 (77-92)
99 -13.0
73 (66-75)
65 (55-73)
80 (75-89)
98 (97-99) 06.1 (05.9-06.3)
97 (88-100)
74 (71-78)
96 (93-99) 07.6 (06.6-09.4)
70 (61-77)
Grand Canary
12.8 (12.6-13.0) Madeira I
11.7 (10.5-13.0)
14.2 (13.4-15.1 )
Madeira C
13.1 (11.0-14.7)
15.3 (14.1-16.4)
06.9 (04.3-09.0)
Po~ugal
96
13.8 (12.3-15.7)
15.3 (14.2-16.6)
14.0 (13.1-15.1 )
14.7 (13.1-16.7)
(94-97) 10.0 (09.6-10.4)
=Axis 2
...90
/._.,
1-
, ~,,',-,,'Mr8/-,1/ _,acr1~t , / -4/.c.".:.. / / M d 1 0 I~d9 /
/(¢dll
(
/
/ I,
3'
~---AxisI
-1-
FIG. 1. POSITION OF DIPLOID AND TETRAPLOID POPULATIONS FROM THE DACTYLIS SUBTROPICAL GROUP ACCORDING TO POLYMORPHISM AT LOCI GOT1. PXl, AcPHT, TO1, PGI1-2 AND ADH1-2. Multidimensional scaling from 7.2 distances. Populations are clustered at levels 55 and 90 of the hieramhical clustering.
414
E. SAHUQUILLO AND R. LUMARET
I
100-
a
605040302010O0 Q~
0 Po13 Po14 Po15 C04 CQ5 Tel
Te2 Md8 Md9 Md12 Mdll
Mdl0Md7 Md6
Populations
;0080-
b
70U3
605040-
2O 10 © Co4 Co5 Po13 Po14 Po15 Tel
Te2 Md8 Md9 Md12 Mdlo Md7 Md11 Md6
Populations FIG. 2. PHENOGRAMS SHOWING GENETIC RELATIONSHIPS AMONG DACTYLIS SUBTROPICAL GROUP OF POPULATIONS. (a) Hierarchical clustering based on Nei's Distances over eight studied enzyme loci (GOT1, PX1, AcPH1, TO1, ADH1-2 and PGI1-2). (b) Hierarchical clustering over seven loci (without GOT1).
from lenerife and Madeira constituted a single group. Similar results were obtained using Nei's distances (Fig. 2a) but two main differences were also observed in the agglomeration patterns: (1) populations fromTenerife and Grand Canary clustered at a lower agglomeration level, suggesting that the genetic differentiation between the populations from these two islands is due mostly to the occurrence of distinct low frequency alleles; and (2) populations from Madeira are genetically isolated from the other populations. The same data treatment was carried out without taking into account enzyme polymorphism at the GOT1 locus. Using Nei's distance, the results (Fig. 2b) were similar to those obtained when the whole loci were considered except that the populations from Madeira clustered more closely on the diagram with those from the other geographic accessions and more particularly with those fromTenerife. Such a result suggests that most of the differentiation observed between the populations from Madeira and the other subtropical populations is due to the occurrence of very distinct allele frequencies at the GOTI locus.
ALLOZYME POLYMORPHISM IN DACTYLIS GLOMERATA
415
Discussion
Identification of distinct genetic entities in the Dactylis subtropical group and relationship among them In the present work, four genetic entities could be identified from the study of enzyme polymorphism in Dactylis populations from the Atlantic part of the subtropical group. A first entity is constituted by the diploid populations from Tenerife island. These diploids which have been described as ssp. smithfi (endemic in Tenerife and Cape Verde islands) according to specific morphological traits (Stebbins and Zohary, 1959) possess a lower genetic diversity for allozymes than the tetraploid entities. Lower allozyme diversity in diploids as compared to polyploids has been observed to be the rule in Dactylis glomerata (Lumaret, 1988) and has been found in many other infraspecific plant polyploid complexes (see Soltis and Soltis, 1993). A second entity consists of the tetraploid populations from Madeira island what ever be their local origin. This entity is the closest to ssp. smithi~ both morphologically and for isozyme variation (except for polymorphism at the GOT1 locus), suggesting that autopolyploidy likely occurred in Dactylis within the Macaronesian archipelago. Autopolyploidisation in endemics of oceanic islands has been observed very rarely (Borgen, 1979). According to Bramwell (1976), the flora of the Canary islands, mostly of very ancient origin, includes only 24.5% polyploids, most of them not being endemic in the Macaronesian islands. Autopolyploidisation of Dactylis in the Macaronesian islands likely constitutes therefore an exceptional event. Apart from two rare and/or low frequency alleles (PX11°4, TO1102 and T01112), Maderian populations from the inland part and those from the coastal part of the island show exactly the same aUelic pool with very similar frequency distribution so that they cannot be distinguished on the base of enzyme polymorphism In addition, according to the results of the present study, the Madeiran populations are clearly distinct genetically from the Portuguese populations which constitute a third subtropical entity identified morphologically as ssp. marina (Borrill, 1961). Such results do not support previous Parker's description (1972) based on morphological characters and according which the inland populations of Madeira constituted ssp. hylodes whereas those from the coast of the island belonged to ssp. marina. According to in situ observation made by the second author of the present paper, individual plants showing intermediate morphological traits between those of the inland and coastal populations were observed in several sites, suggesting that gene flow may occur among these two sets of populations. Up to now, all the subspecies which have been distinguished in Dactylis showed distinct allozyme patterns (Lumaret, 1988). The two groups of Madeiran populations may constitute therefore two ecotypes of the same subspecies (hylodes) which would be maintained distinct by environmental adaptive selection on several morphological or physiological traits (e.g. frequency of papillose cells). A fourth entity is constituted by the tetraploid populations from the centre of Grand Canary island. These populations also constitute a very morphologically distinct group growing at high elevation in dry habitats and under sub-humid Mediterranean climate which is not much influenced by the sea (Tables 1 and 2 and unpublished data). Within the subtropical group, the Canarian type possesses the highest number of alleles which are typical of the Mediterranean group (e.g. PGI1-2°"5 0 , PGI1-2° 25 , etc.) (Lumaret, 1988). Moreover, another population of Dactylis plants, all diploid and which were indistinguishable morphologically from the tetraploids was discovered very recently in the centre of Grand Canary island (A. Ramos, pers. comm. and unpublished data). Such a situation suggests that (1) within the Macaronesia area, early differentiation may have occurred in diploids to create two distinct entities, adapted to distinct climatic conditions, inTenerife and Grand Canary (two large islands), respectively, and (2) autopolyploidy likely occurred from each of these two diploids which are both endemic in the Macaronesian area. However, as both diploid and tetraploid plants
416
E. SAHUQUILLO AND R. LUMARET
from the centre of Grand Canary island combine morphological traits observed usually in Dactylis entities from either the subtropical or the Mediterranean groups, the possibility that the diploids may descend from hybridization between two diploid entities, one located in the subtropical group (Macaronesia) and the other originally from a mainland Mediterranean area (for instance, North Africa) cannot be ruled out. The present allozyme study in the Dactylis subtropical group shows that, on the whole, allele pool and frequency distribution are close but still distinct from those observed previously in the Mediterranean group (Lumaret, 1988). The subtropical taxa form therefore a distinct genetic group which may reflect a monophyletic origin for that group with reserves regarding origin of the Grand Canary type (see above). Low levels of allozyme divergence have been reported in several plant species endemic to oceanic island, e.g. in Tetrarnolopium species from Hawaiian islands (Lowrey and Crawford, 1985), in Dendroseris species from the Juan Fernandes islands (Crawford et al., 1987) and in the Chamaecytisus proliferus complex in the Canary islands (Francisco-Ortega et al., 1992). According to these authors, the occurrence of low levels of allozyme divergence is attributable to the combined effects of genetic bottlenecks associated with colonisation, small population size, and to either speciation on young island or, alternatively, recent colonization of old islands. By contrast, higher genetic divergence was observed in several other taxa endemic to oceanic archipelagoes, e.g. in Gossypium darwinii from the Galapagos islands (Wendel and Percy, 1990), in Avena canariensis from the Canary islands (Morikawa and Leggett, 1990) and in several species of Robinsonia endemic to the Juan Fernandez islands (Crawford et aL, 1992), and was considered to be mainly the result of progressive accumulation of mutations following early colonisation of the several islands. In the Dactylis subtropical group, substantial genetic divergence is observed among several (mainly tetraploid) taxonomic entities which grow in distinct islands of the Macaronesian archipelago or along Portugal coasts. In Dactylis, genetic divergence among the subtropical entities, estimated in the present study by either the Gst value or mean Nei's identity value, is much higher than divergence observed previously among mainland tetraploid subspecies (Lumaret, 1985). That result provides evidence for very early colonisation and adaptive radiation of the parental diploid entities in the Macaronesian area. In addition, substantial accumulation of new allozymes in the polyploids, endemic to the Macaronesian area and clearly related to the diploids, may reflect the occurrence of early autopolyploidisation in that region. However, accumulation of mutations was likely slower in the diploids than in the derived autopolyploids which possessed higher genetic inertia with much slower segregation rates (Stebbins, 1980). Moreover, most of the differentiation between ssp. hylodes and the other entities of the subtropical group is expressed at the GOT1 locus, suggesting that genetic variation at that locus may have a different evolutionary significance from that observed at the other studied allozyme markers.
Adaptive differentiation in the Dactylis subtropical group At the GOT1 locus, the allele 0.72 which is present alone in the diploids (ssp. smithii) and is predominant in the tetraploid populations from Grand Canary and Portugal, occurs at a very low frequency in the populations from Madeira in which the allele 1.00 becomes predominant. In addition, all the Madeiran populations are characterised by the occurrence of allele GOT1126 (24% on average). Results from previous extensive studies in Dactylis glomerata have shown that several alleles investigated at the GOT1 locus in numerous diploid and tetraploid populations were non-randomly distributed. More particularly, the frequency of alleles 1.00 and 1.26 were shown to be statistically positively correlated with humidity whereas high frequency of allele 0.72 was related to the occurrence of a dry environment (Lumaret, 1984; Roy and Lumaret, 1987). Average annual rainfall and local humidity are much
ALLOZYMEPOLYMORPHISMIN DACTYLISGLOMERATA
417
higher and the summer drought period is much shorter (or most of the time is absent) in the Dactylis collecting sites of Madeira than in those of the other collecting areas in Tenerife, Grand Canary and the southern coast of Portugal (Walter et al., 1979). In Dactyiis subtropical group, allozyme differentiation observed at the GOT1 may have therefore an adaptive significance and may be the result of natural selection on that locus or on loci tightly linked to it. As shown in the present study, substantial allozyme variation occurs in the Dactylis subtropical group. This variation appears to be due to the great geographic discontinuity of the distribution area which favours the occurrence of distinct local alleles in the several islands, and, as shown at the particular locus GOT1, allozyme variation may be the result of adaptive selection related to significant climate variation, more particularly for humidity occurring in that Atlantic region. Acknowledgements--We thank A. Ramos-Martinez and A. Marrero-Rodriguez (Jardin Botanico "Viera y
Clavijo", Grand Canary) for their help in plant material collection in Grand Canary and Tenerife islands.
References Ardouin, P., Jay, M. and Lumaret, R. (1987) Etude d'une situation de sympatrie entre diplo(des et t~traplofdes de Dactylis glomerata L. sur la base du polymorphisme enzymatique et ph~nolique. Can. J. Bot. 65, 526-531~ Benson, M. and Borrill, M. (1969) The significance of clinal variation in Dactylis marina Borrill. New Phytol. 68, 1159-1173. Borgen, L. (1979) Karyology of the Canarian flora. In Plants and/s/ands (Bramwell, D., ed.), pp. 329-346. Academic Press, London. Borrill, M. (1957) A morphologically distinct ecotype of Dactylis glomerata L. Nature 179, 544-546. Borrill, M. (1961 ) Dactylis marina Borrill, sp. nov., a natural group of related forms. J. Linn. Soc. 56, 431 439. Borrill, M. (1978) Evolution and Genetic Resources in Cocksfoot. A. Rep. Welsh Plant Breeding Station, 1977, Aberystwyth, U.K., pp. 190-209. Bramwell, D. (1976) The endemic flora of the Canary islands; distribution, relationships and phytogeography. In Biogeography andEcology in the Canary/s/ands (Kunkel, G., ed.), pp. 207-248. Junk, The Hague. Crawford, D. J., Stuessy, T. F. and Silva, M. (1987) AIIozyme divergence and the evolution of Dendroseris (Compositae: Lactuceae) on the Juan Fernandez islands. Syst. Bot. 12, 435-443. Crawford, D. J., Stuessy, T. F., Haines, D. W., Cosner, M. B., Silva, O. M. and Lopez, P. (1992) Allozyme diversity within and divergence among four species of Robinsonia (Asteraceae: Senecioneae), a genus endemic to the Juan Fernandez islands, Chile. Am. J. Bot. 79, 962-966. De Lemps, H. (1969) Le Climat des iles Canaries. Pub. Facult~ lettres et sciences humaines, s(~rie recherches, Paris-Sorbonne (54). Demarly, Y. (1963) G~n~tique des t~traplo'Ldeset amelioration des plantes. Ann. Amdlior. P/antes 13, 307400. Escoufier, Y. (1975) Le positionnement multidimentioneL Rev. Star. Appl. 23, 5-14. Fiasson, J. L., Ardouin, P. and Jay, M. (1987) Phylogenetic groundplan of the specific complex Dactylis glomerata L. Biochem. Syst. Ecol. 15, 225-228. Francisco-Ortega, J., Jackson, M. T,, Catty, J. P. and Ford-Lloyd, B. V. (1992) Genetic diversity in the Chamaecytisus proliferus complex (Fabaceae: Genisteae) in the Canary islands in relation to in situ conservation. Genet. Res. Crop Evol. 39, 149--158. Jay, M., Plenet, D., Ardouin, P., Lumaret, R. and Jacquard, P. (1984) Flavonoid variation in seven tetraploid populations of Dactylis glomerata L. (Gramineae). Biochem. Syst. Ecol. 12, 193-198. Jay, M., Fiasson, J. L., Lumaret, R. and Ardouin, P. (1989) Tetraploidy and habitat differentiation reflected by flavonoid patterns in Dactylis glomerata L from Galicia (Spain). Biochem. Syst. Ecol. 17, 283-285. Lebreton, J. D., Roux, M., Banco, G. and Bacou, A. M. (1987). The BIOMECO package, Centre Emberger, Centre National de la Recherche Scientifique, Montpellier, France. Levontin, R. C. (1972) The apportionment of human diversity. In Evolutionary Biology 6 (Dobzhansky, T., Hecht, M. K. and Steere, W. C., eds), pp. 381-383. Appleton-Century-Crofts, New York. Lowrey, T. K. and Crawford, D. J. (1985) AIIozyme divergence and evolution in Tetramolopium (Compositae: Astereae) on the Hawaiian islands. Syst. Bot. 101, 64-72.
418
E. SAHUQUILLOAND R. LUMARET
Lumaret, R. (1981 a) Etude de I'h~r~dit~ de phosphatases acides chez le Dactyle (Dactylis glomerata L.) diplo'fde et t~traplofde. Can. J. Genet. Cytol. 23, 513-523. Lumaret, R. (1981 b) Structure g~n~tique d'un coplexe polyplotde: Dactylis glomerata L. Relations entre le polymorphisme enzymatique et certains aspects de la biologie, de I'~cologie et de I'~volution de I'esp~ce. State D; Thesis, Universit~ des Sciences et Techniques du Languedoc, Montpellier, France. Lumaret, R. (1982) Protein variation in diploid and tetraploid orchard grass (Dactylisglomerata L.): formal genetics and population polymorphism of peroxidases and malate dehydrogenases. Genetica 57, 207-215. Lumaret, R. (1984) The role of polyploidy in the adaptive significance of polymorphism at the GOT1 locus in the Dactylis glomerata complex. Heredity 52, 153-169. Lumaret, R, (1985) Phenotypic and genotypic variation within and between populations of the polyploid complex Dactylis glomerata L. In Structure and Functioning of Plant Populations, Phenotypic and Genotypic Variation in Plant Populations (Haeck, J. and Woldendorp, J. W., eds), pp. 343-354. North-Holland Publishing Co., Amsterdam. Lumaret, R. (1986) Doubled duplication of the structural gene for cytosolic phosphoglucose isomerase in the Dactylis glomerata L. polyploid complex. Molec. Biol. Evol. 3, 449-452. Lumaret, R. (1988) Cytology, genetics and evolution in the genus Dactylis, C.R.C. Crit. Rev. Plant Sci. 7, 55--91. Lumaret, R. and Valdeyron, G. (1978) Les glutamate oxaloac6tate transaminases du Dactyle (Dactyfis glomerata L.): G6n~tique formelle d'un locus. C. R. Acad. ScZ Paris 287 D, 705-708. Lumaret, R., Bowman, C. M. and Dyer, T. A. (1989) Autopolyploidy in Dactylis glomerata L.: further evidence from studies of chloroplast DNA variation. Theoret. Appl. Genet. 78, 393-399. Lumaret, R. and Barrientos, E. (1990) Phylogenetic relationships and gene flow between sympatric diploid and tetraploid plants of Dactylis glomerata (Gramineae). Plant Syst, Evol. 169, 81-96. Maceira, N. O., De Haan, A. A., Lumaret, R., Billon, M. and Delay, J. (1992) Production of 2n gametes in diploid subspecies of Dactylisglomerata L. 1. Occurrence and frequency of 2n pollen. Ann. Bot. 69, 335-343. Morikawa, T. and Leggett, J. M (1990) Isozyme polymorphism in natural populations of Avena canariensis from the Canary islands. Heredity 64, 403-411. Nei, M. (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583-590. Parker, P. F. (1972) Studies in Dactylis 2. Natural variation, distribution and systematics of the Dacty/is smithii Link. complex in Madeira and other Atlantic islands. New Phytol. 71,371-378. Roy, J. and Lumaret, R. (1987) Associated clinal variation in leaf tissue water relations and allozyme polymorphism in Oactylis glomerata L. populations. EvoL Trends Plants 1, 9-19. Sarich, V. M. (1977) Rates, sample sizes and the neutrality hypothesis for electrophoresis in evolutionary studies. Nature 265, 24-28. Shannon, C. E. and Weaver, W. (1963) The Mathematical Theory of Communication. Urbana Univ. Illinois Press. Soltis, D. E. and Soltis, P. S. (1993) Molecular data and the dynamic nature of polyploidy. C.R,C. Crit. Rev. Plant Sci. 12, 243-273. Stebbins, G. L. (1980) Polyploidy in plants: unsolved problems and prospects. In Polyploidy, Biological Relevance (Lewis, H., ed.), pp. 495-520. Plenum, New York. Stebbins, G. L and Zohary, D. (1959) Cytogenetics and evolutionary studies in the genus Dactylis 1. Morphology distribution and interrelationships of the diploid subspecies. Univ. California Pub/. Bot. 31, 1-40. Walter, H., Harnickell, E. and Mueller-Dombois, D. (1979) Climate Diagram Maps. Springer Verlag, Berlin. Wendel, J. F. and Percy, R. G. (1990) AIIozyme diversity and introgression in the Galapagos islands endemic Gossypium darwinii and its relationship to continental G. barbadense. Biochem. Syst. Ecol. 18, 517-528. Wilson, D., Abdullah, I. B. and Trickey, S. A. (1977) Variation in transpiration rate in Dactylis. Proc. X/I/ Int. Grassland Congress, Leipzig, pp. 71-79.