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Network analysis of genetic resources data: II. The use of isozyme data in elucidating geographical relationships
Agro-Ecosystems, 6 (1980) 111--118
111
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
NETWORK A N A L Y S I S OF GENETIC RESOURCES DATA: II. THE USE OF ISOZYME D A T A IN ELUCIDATING GEOGRAPHICAL RELATIONSHIPS
P.J. R O B I N S O N ,
R.L. B U R T
and W.T. W I L L I A M S
Division of Tropical Crops and Pastures,Davies Laboratory, CSIRO, PrivateMail Bag, Post
Office, Townsville, Qld. 4810 (Australia) (Accepted 23 January 1980)
ABSTRACT Robinson, P.J., Burt, R.L. and Williams, W.T., 1980. Network analysis of genetic resources data: II. The use of isozyme data in elucidating geographical relationships. Agro-Ecosystems, 6: 111--118. Seed proteins from 226 accessions of Stylosanthes (mostly S. hamata) were examined electrophoretically, providing 27 esterase bands from the single enzyme substrate anaphthylacetate. The accessions came from 23 geographical areas, and an inter-area distance matrix was obtained from the isozyme data. The resulting network delineated the geographical relationships almost as clearly as that from morphological/agronomic data. The relations between isozyme bands and morphological/agronomic attributes were explored by an "inverse" network procedure; it is shown that the bands are not only standing in lieu of morphological attributes, but may sometimes recover independent information of an u n k n o w n nature.
INTRODUC~ON
Traditional plant taxonomy is based largely on external morphology; but there is ample evidence (see, e.g. Boulter and Derbyshire, 1971; Gottlieb, 1977) that accepted taxonomic distinctions m a y be reflected in the structure of plant proteins. It follows that the configuration of the proteins m a y serve, at least in part, to stand in place of morphological attributes and recover the taxonomy of the group under study. Robinson and Megarrity (1975), in a study of 182 Stylosanthes accessions, were in fact able to recover m u c h of the classificationat species level from a study of seed proteins. In addition, there has been m u c h work on the identification of individual cultivars in a number of genera by electrophoretic techniques (see, e.g.,Bassiri and Rouhani, 1977, and references therein cited). The special interest of such techniques in studies of the interrelations of plant introduction materials lies in the fact that the data m a y be obtained from a single seed, thus obviating the necessity for the years normally spent in the quarantine glasshouse and/or spaced-plant experiments to record morphological information. 0304--3746/80/0000--0000/$02.25, © 1980 Elsevier Scientific Publishing Company
112 N o t all proteins are equally appropriate for such studies. Thurman (1971) has stressed the advantages of the esterase isozymes, since in electrophoretic studies these p r o d u c e a large n u m b e r of well-defined and easily-discriminated bands. Usberti and Jain (1979) used a combination of morphological and esterase characters in a s t u d y of populations of Panicum maximum; roots or leaves were used for electrophoretic assay. Robinson et al. (1976) used esterases together with acid phosphatases, in a s t u d y of seed proteins of Stylosanthes guianensis, which recovered a considerable a m o u n t of information concerning the patterns of nodulation with 22 strains of Rhizobium. In the present series of studies, it therefore seemed possible that a s t u d y of the esterase patterns from seeds of the 226 Stylosanthes accessions defined in the first paper of this series (Williams et al., 1980) might similarly stand in lieu of the morphological data, and similarly recover the floristic relationships of the 23 areas defined in that study. We shall show in this paper that this possibility has been largely realized. There then remains the problem of the relations between the t w o sets of attributes -- morphological and biochemical -- used in the investigation. There have been several studies concerned to relate protein patterns to particular physiological properties; see e.g., Powers et al. (1979) and the review b y J o h n s o n (1974); b u t we k n o w of no work relating protein patterns to specific morphological attributes. Moreover, plant adaptation is n o t a p r o p e r t y of single morphological attributes, b u t of a whole suite of them; and we have similarly been unable to trace any work concerned with relating a complete set of morphological attributes to a canonical set o f biochemical attributes. The recently-developed "inverse" technique of defining distances b e t w e e n attributes of mixed types (Lance and Williams, 1979) n o w makes this possible; if the 226 accessions are defined by both their morphological and their biochemical attributes, the technique enables us to explore the joint configuration of b o t h sets o f attributes simultaneously. These are the aims of the present paper. MATERIAL AND METHODS
Plant material The 226 Stylosanthes accessions, the morphological and agronomic data recorded, and the 23 defined geographical areas are as set o u t in the first paper of this series (Williams et al., 1980).
Separation of esterase enzymes Seed was harvested from all accessions, the testas removed and the seed meal e x t r a c t e d w i t h tris-borate buffer at pH 8.3 (50 mg seed per ml buffer}. 30 ~1 samples of extracts were applied to 2.5--27% concave gradient polyacrylamide slabs (Margolis and Kendrick, 1968} and electrophoretic separation carried o u t
113 in tris buffer at pH 8.3 for 1500 volt-h. Fresh extracts of a minimum of t w o accessions of k n o w n esterase band pattern were used on each gel plate to standardise migration distance of bands. No variation in either relative band migration or band intensity was detected within any of the ten accessions used as standards, regardless of seed source. The gels were stained for esterase activity using a-naphthylacetate as substrate b y the m e t h o d of WiUiamson et al. (1968). Identification o f isozyme bands in the lower (anodic) region of the slab were verified using 16--28% concave gradient gels, separation being carried o u t as before. On completion of isozyme separations for all accessions, a total o f 27 different bands were determined. For c o m p u t a t i o n these were scored as 27 4state ordinal attributes, from 1 (absent) to 4 (most intense).
Numerical methods For the area s t u d y the accessions were regarded as defined solely by the 27 ordinal attributes from electrophoresis; the 226 X 226 inter-accession matrix and the 23 X 23 inter-area matrix were calculated as explained in the first paper, and the n e t w o r k obtained as before. For the canonical attribute study, the accessions were defined b y the 55 morphological-agronomic attributes plus the 27 esterase attributes. A special difficulty may arise in the c o m p u t a t i o n of an "inverse" (inter-attribute) distance matrix. If the data contains discontinuities, such as b e t w e e n species, a number of attributes m a y change together at the species/species boundary. The most powerful of these m a y act as an "indicator" attribute to which the remaining members o f the group b e c o m e attached, thus obscuring any other relations t h e y possess. Such attributes can be recognised early in the computation, and are better removed. We have for this reason deleted three attributes; these were length of beak plus pod, bristle distribution on upper leaf surface and awn-length (Nos. S5, V17 and V22 in Table I of the first paper), leaving us with a 79 × 79 inter-attribute distance matrix. R ESULTS
(I) Data matrices The presentation of matrices of enzyme data (226 accessions × 27 bands) and morphological-agronomic data (226 accessions X 55 attributes) is impractical in this situation. These matrices are available from the authors on request. Schematic diagrams of selected isozyme patterns have been illustrated by Robinson (1978).
(II) Inter-area relations The n e t w o r k of the 23 areas based solely on the esterase data is presented as Fig. 1. Comparison with Fig. 2 of the previous paper, the n e t w o r k obtained
114
Z T
t
i
I.
i .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Fig. 1. N e t w o r k o f 23 areas b a s e d o n i s o z y m e data. Progressive increase in linkage s t r e n g t h is s h o w n as , - - , ."
from morphological-agronomic data, shows some striking similarities. Groups I and II, the western and eastern sections of the Caribbean chain of islands, are again clearly delimited. The unexpected link between Florida (area 1) and Antigua (area 6) is still strong, as is the disjunction between Antigua and Puerto Rico (area 5). There are, however, some interesting differences in the mainland configuration. Paraguana (area 13) is no longer closely linked to Maracaibo (14), but is instead linked with the two neighbouring inland areas 15 (Baranquilla) and 17 (Barinas), an entirely reasonable alternative. The naturalized Australian material (area 20), though no longer directly linked with Villavincencio (area 23), is still linked more closely to Central America (area 16) than to Brazil (area 22). The overall configuration, in fact, once more shows a strong resemblance borth to the map of the area and to the patterns of floristic distribution proposed by Howard (1973).
(III) Inter-attribute relations The inverse network, showing the relations between the 27 enzyme bands and the 52 morphological-agronomic characters is presented as Fig. 2. We note that the network falls conveniently into ten groups (labelled I to X in the figure) separated by weak links. Three of these (IV, VIII and IX) consist entirely of morphological-agronomic characters; two (V and VII) entirely of
115
esterase bands; the remaining five contain cross-linkages b e t w e e n b o t h types. The cross-linkages all reflect t a x o n o m i c distinctions. In group I, b e a k / p o d ratio ($6), is strongly linked with beak length ($3), and these are strongly linked with the presence of band 25. This band is in turn linked with the presence of a shoulder on the seed (S1). Examination o f the data shows that the presence of a shoulder, or a beak length of less than 2 mm, or the presence of band 25, m a y be used to separate accessions of S. scabra, S. sp. aft. hamata
i
I-
X
Fig. 2. Inverse network of isozyme bands, morphological and agronomic characters. For list of vegetative (V), floral (F), seed (S) and agronomic (A) characters see Williams et al. (1980).
116
and S. sympodialis from the other accessions. The same three species are implicated in group X. In this, band 22 is linked with stem thickness (A8), dry matter yield (All), length o f main stem (A3) and length of longest lateral (A4). All the agronomic characters reflect overall plant size, and high values of these are characteristic of the three species in question. It is relevant to note t h a t the band pattern of these w o o d y species is also exhibited by accessions, conventionally ascribed to S. hamata from the extremes Of the geographical range o f the latter species (two from Florida and one from southern Brazil). These are w o o d y plants bearing a distinct morphological resemblance to S. scabra, and this resemblance extends to the esterase band pattern. In group II, if the angle of the main stem to the horizontal (character A6) is greater than 60 °, or if the seeds are round ($8), or if the accessions possess bands 24 or 27, t h e y are then either S. scabra or S. sp. aft. hamata, which can thus be separated from S. sympodialis. Region III contains seven enzyme bands linked with the single vegetative character V10, the degree of acuteness of the leaf apex. The presence of these bands, or the possession of leaves of the most acute type, indicates S. sp. aft. hamata from Brazil, S. hamata from Maracaibo, S. humilis or S. sympodialis. The bands of Region VI are linked with seed colour ( S l l ) , the length/breadth ratio of the last expanded leaf (V9), and the presence of a basal articulation (F1). Dark seeds and high values Of V9 are characteristic of S. hamata from Maracaibo and S. humilis. The absence of a basal articulation is characteristis of S. humilis. We now need to consider those groups which contain no esterase bands. The floral characters of group IV are interesting, since t h e y include the axis rudiment, the presence or absence of which is traditionally used to define the two subgenera within Stylosanthes. Accessions with a long axis r u d i m e n t and long inflorescences (F4) are S. sympodialis or diploid S. hamata f r o m Florida. Group IX is best considered as two sub-groups. The pair o f attributes (bristle characters) V2 and V6 serve simply to exclude the diploid and tetraploid true S. hamata accessions; the remaining attributes similarly serve to exclude the accessions of S. scabra. The attributes of group VIII have no interspecific significance; t h e y are entirely concerned with the patterns o f occurrence of hairs and bristles. Finally, there are the two groups V and VII which contain only esterase bands. The accessions which exhibit these bands show no consistent pattern, either of t a x o n o m y or geographic distribution. These accessions presumably have some properties in c o m m o n which are n o t reflected in the morphological and agronomic characters we have chosen to record, and their nature must for the present remain u n k n o w n . DISCUSSION The most striking feature of these results is the fidelity with which a geographic distributiOn h a s been retrieved from isozyme data which could have been obtained from a single seed of each accession, and which used only a
117
single enzyme substrate. Although the end result is sufficiently clear, the mechanism b y which it has been attained is not. The isozyme bands d o n o t appear simply to be standing in lieu o f morphological characters; f o r if they were we should have expected a more intimate relation between the t w o sets of characters than is evident in Fig. 2. The bands evidently suffice to recover some few aspects o f the t a x o n o m y of the set; b u t alone they do not recover enough to a c c o u n t for the complete distribution. It has been suggested that, at least in specific instances, esterases m a y serve as a measure of genetic diversity (Brown, 1978, and references therein cited). Brown, however, states "...the evaluation of genetic diversity in a sample is a problem different from the evaluation for its agronomic merit". Our results suggest that this statement is n o t entirely true. Our o w n earlier work (Burr et al., 1979) has shown that there can be strong connexion between geographic distribution and agronomic merit for a specific environment; and the results presented in this paper show that there is a connexion b e t w e e n esterase patterns and geographic distribution. If, therefore, it can be accepted that esterase patterns are in some sense a measure of genetic variation, the results we have n o w presented provide, even if insecurely, a bridge between the four properties o f isozymes, genetic variation, geographic distribution, and agronomic merit. ACKNOWLEDGEMENTS
We are much indebted to the Australian Meat Research Council for their continued encouragement and support; to Mr. L.L. Conlan for his skilled assistance in the electrophoretic measurements; to Mr. B.C. Pengelly for assistance in the organization of the data and interpretation of the results; and to Mr. H.J. Clay for extensive computational assistance.
REFERENCES
Bassiri, A. and Rouhani, I., 1977. Identification of broad bean cultivarsbased on enzyme patterns. Euphytica, 26: 279--286. Boulter, D. and Derbyshire, E., 1971. Taxonomic aspects of the structure of legume proteins. In: J.B. Harborne, D. Boulter and B.L. Turner (Editors), C h e m o t a x o n o m y of the Leguminosae. Academic Press, London, pp. 285--308. Brown, A.H.D., 1978. Isozymes, plant population genetic structure and genetic conservation. Theor. Appl. Genet., 52: 145--157. Burt, R.L., Isbell,R.F. and Willian~, W.T., 1979. Strategy of evaluation of a collection of tropical herbaceous legumes from Brazil and Venezuela. I. Ecological evaluation at the point of collection. Agro-Ecosystems, 5: 99--117. Gottlieb, L.D., 1977. Electrophoretic evidence and plant systematics. Ann. Miss. Bot. Garden, 64: 161--180. Howard_, R.A., 1973. The vegetation of the Antilles. In: A. Graham (Editor), Vegetation and Vegetational History of Northern Latin America. Elsevier, Amsterdam, pp. 1--38. Johnson, G.B., 1974. E n z y m e polymorphism and metabolism. Science, 184: 28--37. Lance, G.N. and Williams, W.T., 1979. I N V E R : a program for the computation of distancemeasures between attributes of mixed types. Aust. Comput. J., 11: 27--28.
118 Margolis, J. and Kendrick, J.G., 1968. Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient. Anal. Biochem., 25: 347--362. Powers, D.A., Greany, G.S. and Place, A.R., 1979. Physiological correlation between lactate dehydrogenase genotype and haemoglobin function in killifish. Nature (London), 227: 240--241. Robinson, P.J., 1978. Chemot~xonomy In: Ann, Rep. Div. Trop. Crops & Pastures, C.S.I.R.O., Brisbane, pp. 48. Robinson, P..J. and Megarrity, R.G., 1975. Characterization of Styiosanthes introductions by using seed protein patterns. Aust. J. Agric. Res., 26: 467--479. Robinson, P.J., Date, R.A. and Megarrity, R.G., 1976. Nodulation of Stylosanthes guyanens/s: prediction of Rhizobium effectiveness response with seed enzyme patterns. Aust. J. Agric. Res., 27: 381--389. Thurman, D.A., 1971. Comparative study of legume enzymes. In: J.B. Harborne, D. Boulter and B.L. Turner (Editors), Chemotaxonomy of the Leguminosae. Academic Press, London, pp. 463--484. Usberti, J.A. and Jain, S,K., 1979. Ecotypic differentiation in guinea grass (Panicum maximum Jacq.). Agro-Ecosystems, 5: 147--158. Williams, W.T., Burt, R.L., Pengelly, B.C. and Robinson, P.J., 1980. Network analysis of genetic resources data. I. Geographical relationships. Agro-Ecosystems, 6: 99--109. Williamson, J.A., Kleese, R.A. and Snyder, J.R., 1968. Electrophoretic variation of esterase of three varieties of oats (Arena sativa). Nature (London), 220: 1134--1136.
111
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
NETWORK A N A L Y S I S OF GENETIC RESOURCES DATA: II. THE USE OF ISOZYME D A T A IN ELUCIDATING GEOGRAPHICAL RELATIONSHIPS
P.J. R O B I N S O N ,
R.L. B U R T
and W.T. W I L L I A M S
Division of Tropical Crops and Pastures,Davies Laboratory, CSIRO, PrivateMail Bag, Post
Office, Townsville, Qld. 4810 (Australia) (Accepted 23 January 1980)
ABSTRACT Robinson, P.J., Burt, R.L. and Williams, W.T., 1980. Network analysis of genetic resources data: II. The use of isozyme data in elucidating geographical relationships. Agro-Ecosystems, 6: 111--118. Seed proteins from 226 accessions of Stylosanthes (mostly S. hamata) were examined electrophoretically, providing 27 esterase bands from the single enzyme substrate anaphthylacetate. The accessions came from 23 geographical areas, and an inter-area distance matrix was obtained from the isozyme data. The resulting network delineated the geographical relationships almost as clearly as that from morphological/agronomic data. The relations between isozyme bands and morphological/agronomic attributes were explored by an "inverse" network procedure; it is shown that the bands are not only standing in lieu of morphological attributes, but may sometimes recover independent information of an u n k n o w n nature.
INTRODUC~ON
Traditional plant taxonomy is based largely on external morphology; but there is ample evidence (see, e.g. Boulter and Derbyshire, 1971; Gottlieb, 1977) that accepted taxonomic distinctions m a y be reflected in the structure of plant proteins. It follows that the configuration of the proteins m a y serve, at least in part, to stand in place of morphological attributes and recover the taxonomy of the group under study. Robinson and Megarrity (1975), in a study of 182 Stylosanthes accessions, were in fact able to recover m u c h of the classificationat species level from a study of seed proteins. In addition, there has been m u c h work on the identification of individual cultivars in a number of genera by electrophoretic techniques (see, e.g.,Bassiri and Rouhani, 1977, and references therein cited). The special interest of such techniques in studies of the interrelations of plant introduction materials lies in the fact that the data m a y be obtained from a single seed, thus obviating the necessity for the years normally spent in the quarantine glasshouse and/or spaced-plant experiments to record morphological information. 0304--3746/80/0000--0000/$02.25, © 1980 Elsevier Scientific Publishing Company
112 N o t all proteins are equally appropriate for such studies. Thurman (1971) has stressed the advantages of the esterase isozymes, since in electrophoretic studies these p r o d u c e a large n u m b e r of well-defined and easily-discriminated bands. Usberti and Jain (1979) used a combination of morphological and esterase characters in a s t u d y of populations of Panicum maximum; roots or leaves were used for electrophoretic assay. Robinson et al. (1976) used esterases together with acid phosphatases, in a s t u d y of seed proteins of Stylosanthes guianensis, which recovered a considerable a m o u n t of information concerning the patterns of nodulation with 22 strains of Rhizobium. In the present series of studies, it therefore seemed possible that a s t u d y of the esterase patterns from seeds of the 226 Stylosanthes accessions defined in the first paper of this series (Williams et al., 1980) might similarly stand in lieu of the morphological data, and similarly recover the floristic relationships of the 23 areas defined in that study. We shall show in this paper that this possibility has been largely realized. There then remains the problem of the relations between the t w o sets of attributes -- morphological and biochemical -- used in the investigation. There have been several studies concerned to relate protein patterns to particular physiological properties; see e.g., Powers et al. (1979) and the review b y J o h n s o n (1974); b u t we k n o w of no work relating protein patterns to specific morphological attributes. Moreover, plant adaptation is n o t a p r o p e r t y of single morphological attributes, b u t of a whole suite of them; and we have similarly been unable to trace any work concerned with relating a complete set of morphological attributes to a canonical set o f biochemical attributes. The recently-developed "inverse" technique of defining distances b e t w e e n attributes of mixed types (Lance and Williams, 1979) n o w makes this possible; if the 226 accessions are defined by both their morphological and their biochemical attributes, the technique enables us to explore the joint configuration of b o t h sets o f attributes simultaneously. These are the aims of the present paper. MATERIAL AND METHODS
Plant material The 226 Stylosanthes accessions, the morphological and agronomic data recorded, and the 23 defined geographical areas are as set o u t in the first paper of this series (Williams et al., 1980).
Separation of esterase enzymes Seed was harvested from all accessions, the testas removed and the seed meal e x t r a c t e d w i t h tris-borate buffer at pH 8.3 (50 mg seed per ml buffer}. 30 ~1 samples of extracts were applied to 2.5--27% concave gradient polyacrylamide slabs (Margolis and Kendrick, 1968} and electrophoretic separation carried o u t
113 in tris buffer at pH 8.3 for 1500 volt-h. Fresh extracts of a minimum of t w o accessions of k n o w n esterase band pattern were used on each gel plate to standardise migration distance of bands. No variation in either relative band migration or band intensity was detected within any of the ten accessions used as standards, regardless of seed source. The gels were stained for esterase activity using a-naphthylacetate as substrate b y the m e t h o d of WiUiamson et al. (1968). Identification o f isozyme bands in the lower (anodic) region of the slab were verified using 16--28% concave gradient gels, separation being carried o u t as before. On completion of isozyme separations for all accessions, a total o f 27 different bands were determined. For c o m p u t a t i o n these were scored as 27 4state ordinal attributes, from 1 (absent) to 4 (most intense).
Numerical methods For the area s t u d y the accessions were regarded as defined solely by the 27 ordinal attributes from electrophoresis; the 226 X 226 inter-accession matrix and the 23 X 23 inter-area matrix were calculated as explained in the first paper, and the n e t w o r k obtained as before. For the canonical attribute study, the accessions were defined b y the 55 morphological-agronomic attributes plus the 27 esterase attributes. A special difficulty may arise in the c o m p u t a t i o n of an "inverse" (inter-attribute) distance matrix. If the data contains discontinuities, such as b e t w e e n species, a number of attributes m a y change together at the species/species boundary. The most powerful of these m a y act as an "indicator" attribute to which the remaining members o f the group b e c o m e attached, thus obscuring any other relations t h e y possess. Such attributes can be recognised early in the computation, and are better removed. We have for this reason deleted three attributes; these were length of beak plus pod, bristle distribution on upper leaf surface and awn-length (Nos. S5, V17 and V22 in Table I of the first paper), leaving us with a 79 × 79 inter-attribute distance matrix. R ESULTS
(I) Data matrices The presentation of matrices of enzyme data (226 accessions × 27 bands) and morphological-agronomic data (226 accessions X 55 attributes) is impractical in this situation. These matrices are available from the authors on request. Schematic diagrams of selected isozyme patterns have been illustrated by Robinson (1978).
(II) Inter-area relations The n e t w o r k of the 23 areas based solely on the esterase data is presented as Fig. 1. Comparison with Fig. 2 of the previous paper, the n e t w o r k obtained
114
Z T
t
i
I.
i .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Fig. 1. N e t w o r k o f 23 areas b a s e d o n i s o z y m e data. Progressive increase in linkage s t r e n g t h is s h o w n as , - - , ."
from morphological-agronomic data, shows some striking similarities. Groups I and II, the western and eastern sections of the Caribbean chain of islands, are again clearly delimited. The unexpected link between Florida (area 1) and Antigua (area 6) is still strong, as is the disjunction between Antigua and Puerto Rico (area 5). There are, however, some interesting differences in the mainland configuration. Paraguana (area 13) is no longer closely linked to Maracaibo (14), but is instead linked with the two neighbouring inland areas 15 (Baranquilla) and 17 (Barinas), an entirely reasonable alternative. The naturalized Australian material (area 20), though no longer directly linked with Villavincencio (area 23), is still linked more closely to Central America (area 16) than to Brazil (area 22). The overall configuration, in fact, once more shows a strong resemblance borth to the map of the area and to the patterns of floristic distribution proposed by Howard (1973).
(III) Inter-attribute relations The inverse network, showing the relations between the 27 enzyme bands and the 52 morphological-agronomic characters is presented as Fig. 2. We note that the network falls conveniently into ten groups (labelled I to X in the figure) separated by weak links. Three of these (IV, VIII and IX) consist entirely of morphological-agronomic characters; two (V and VII) entirely of
115
esterase bands; the remaining five contain cross-linkages b e t w e e n b o t h types. The cross-linkages all reflect t a x o n o m i c distinctions. In group I, b e a k / p o d ratio ($6), is strongly linked with beak length ($3), and these are strongly linked with the presence of band 25. This band is in turn linked with the presence of a shoulder on the seed (S1). Examination o f the data shows that the presence of a shoulder, or a beak length of less than 2 mm, or the presence of band 25, m a y be used to separate accessions of S. scabra, S. sp. aft. hamata
i
I-
X
Fig. 2. Inverse network of isozyme bands, morphological and agronomic characters. For list of vegetative (V), floral (F), seed (S) and agronomic (A) characters see Williams et al. (1980).
116
and S. sympodialis from the other accessions. The same three species are implicated in group X. In this, band 22 is linked with stem thickness (A8), dry matter yield (All), length o f main stem (A3) and length of longest lateral (A4). All the agronomic characters reflect overall plant size, and high values of these are characteristic of the three species in question. It is relevant to note t h a t the band pattern of these w o o d y species is also exhibited by accessions, conventionally ascribed to S. hamata from the extremes Of the geographical range o f the latter species (two from Florida and one from southern Brazil). These are w o o d y plants bearing a distinct morphological resemblance to S. scabra, and this resemblance extends to the esterase band pattern. In group II, if the angle of the main stem to the horizontal (character A6) is greater than 60 °, or if the seeds are round ($8), or if the accessions possess bands 24 or 27, t h e y are then either S. scabra or S. sp. aft. hamata, which can thus be separated from S. sympodialis. Region III contains seven enzyme bands linked with the single vegetative character V10, the degree of acuteness of the leaf apex. The presence of these bands, or the possession of leaves of the most acute type, indicates S. sp. aft. hamata from Brazil, S. hamata from Maracaibo, S. humilis or S. sympodialis. The bands of Region VI are linked with seed colour ( S l l ) , the length/breadth ratio of the last expanded leaf (V9), and the presence of a basal articulation (F1). Dark seeds and high values Of V9 are characteristic of S. hamata from Maracaibo and S. humilis. The absence of a basal articulation is characteristis of S. humilis. We now need to consider those groups which contain no esterase bands. The floral characters of group IV are interesting, since t h e y include the axis rudiment, the presence or absence of which is traditionally used to define the two subgenera within Stylosanthes. Accessions with a long axis r u d i m e n t and long inflorescences (F4) are S. sympodialis or diploid S. hamata f r o m Florida. Group IX is best considered as two sub-groups. The pair o f attributes (bristle characters) V2 and V6 serve simply to exclude the diploid and tetraploid true S. hamata accessions; the remaining attributes similarly serve to exclude the accessions of S. scabra. The attributes of group VIII have no interspecific significance; t h e y are entirely concerned with the patterns o f occurrence of hairs and bristles. Finally, there are the two groups V and VII which contain only esterase bands. The accessions which exhibit these bands show no consistent pattern, either of t a x o n o m y or geographic distribution. These accessions presumably have some properties in c o m m o n which are n o t reflected in the morphological and agronomic characters we have chosen to record, and their nature must for the present remain u n k n o w n . DISCUSSION The most striking feature of these results is the fidelity with which a geographic distributiOn h a s been retrieved from isozyme data which could have been obtained from a single seed of each accession, and which used only a
117
single enzyme substrate. Although the end result is sufficiently clear, the mechanism b y which it has been attained is not. The isozyme bands d o n o t appear simply to be standing in lieu o f morphological characters; f o r if they were we should have expected a more intimate relation between the t w o sets of characters than is evident in Fig. 2. The bands evidently suffice to recover some few aspects o f the t a x o n o m y of the set; b u t alone they do not recover enough to a c c o u n t for the complete distribution. It has been suggested that, at least in specific instances, esterases m a y serve as a measure of genetic diversity (Brown, 1978, and references therein cited). Brown, however, states "...the evaluation of genetic diversity in a sample is a problem different from the evaluation for its agronomic merit". Our results suggest that this statement is n o t entirely true. Our o w n earlier work (Burr et al., 1979) has shown that there can be strong connexion between geographic distribution and agronomic merit for a specific environment; and the results presented in this paper show that there is a connexion b e t w e e n esterase patterns and geographic distribution. If, therefore, it can be accepted that esterase patterns are in some sense a measure of genetic variation, the results we have n o w presented provide, even if insecurely, a bridge between the four properties o f isozymes, genetic variation, geographic distribution, and agronomic merit. ACKNOWLEDGEMENTS
We are much indebted to the Australian Meat Research Council for their continued encouragement and support; to Mr. L.L. Conlan for his skilled assistance in the electrophoretic measurements; to Mr. B.C. Pengelly for assistance in the organization of the data and interpretation of the results; and to Mr. H.J. Clay for extensive computational assistance.
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