Meiotic studies of some South African cultivars of Lantana camara ( Verbenaceae )

Lantana camara L. is a polyploid species with a basic chromosome number of 11 ( x = l l ) . Chromosome association in 39 cultivars indicated the occurrence of univalents to heptavalents with bivalents predominating. Multivalent association analysis revealed the presence in South Africa of at least four different groups of L. camara at the diploid level. The potential for sexual reproduction must exist, at least at the diploid level, to account for differences in chromosomal behaviour that can only be attributed to hybridization. The possibility exists that the basic chromosome number may be lower than 11, or else postspeciation genomic evolution must have occurred. No cytogenetical correlation exists between the South African and Indian cultivars. The number of chiasmata per genome increases with an increase in the polyploid level. Most multivalents are of the chain type. Univalents during diakinesis are the result of asynapsis. Triploid and pentaploid plants display a markedly abnormal meiosis. L. camara is a segmental allopolyploid species. INTRODUCTION Lantana camara L. is a widespread tropical weed noted for its toxicity to livestock and for its rapid invasion of natural pasturage, waste ground, forest margins and derelict or cultivated lands. Experience gained from a number of areas of the tropics over many decades has shown that L. camara is not a homogeneous species, but actually consists of a number of forms. The origin, nature and spread of these forms have been discussed in the first paper of this series (Stirton, 1977), L. camara is today recognized as a polyploid aggregate species with a basic chromosome number of x = l l and having diploid, triploid, tetraploid, pentaploid and hexaploid representatives (Schnack & Covas, 1947; Tjio, 1948; Singh, 1951; Sen & Sahni, 1955; Tandon & Bali, 1955; Tandon & Chandi, 1955; Natarajan & Ahuja, 1957; Hender­ son, 1969; Spies & Stirton, 1982). The need for an understanding of these different forms and their taxonomic relationships in South Africa prompted the biological control section of the Department of Agriculture and Fisheries to approach the Botanical Research Institute, of the same Department, to initiate a taxonomic study of L. camara in South Africa. This paper reports in depth on the value of meiotic analysis in circumscribing the more than 50 naturalized cultivars of the weed that are presently recognized in South Africa (Stirton & Spies, 1982). ) MATERIALS AND METHODS The plants used in this study are naturalized cultivars collected throughout South Africa and transplanted under quarantine in the Pretoria National Botanical Garden. Thirty-nine different cultivars, representative of all polyploid levels, were used in this study, 37 specimens being sampled from a hybrid swarm from the Transvaal lowveld. * Botanical Research Institute, Department of Agriculture and Fisheries, Private Bag X101, Pretoria, 0001. ** New address: c/o Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom. Young inflorescences were collected between 09h00 and 12h00 hours and fixed for 24 hours in Camoy’s fixative. Anthers were then squashed in aceto-carmine (Darlington & La Cour, 1976). The multivalent configurations during diakinesis were emphasized for meiotic analysis. RESULTS The sample studied comprised 13 diploids, 12 triploids, 9 tetraploids, 3 pentaploids and 2 hexaploids (Table 1) and confirms our earlier report (Spies & Stirton, 1982) that L. camara in South Africa is a polyploid complex. The early stages of meiosis were superficially similar in all the different cultivars although, during pachytene and diplotene, more chromosomes were visible in the higher polyploids (Fig. 1). Two chromosomes were always associated with the nucleolus at all polyploid levels. Chromosome configurations during diakinesis ranged from univalents to heptavalents, but were predominantly bivalents (Fig. 2). The number of chiasmata during diakinesis, as well as the different configurations formed, are shown in Table 1. From Table 2 it can be deduced that there are big genome differences between different cultivars at the same polyploid level. These genome differences result in different chromosome associations. A high frequency of unpaired chromosomes and laggards (Fig. 3) were encountered during anaphase I (Table 3). Secondary association of chromosomes during telophase I usually occurred (Fig. 4). Neither heteromorphic bivalents nor B-chromosomes were found. Instead of studying metaphase II and anaphase II, we concentrated our attention on the number of micronuclei found during telophase II (Table 4). The triploids and pentaploids usually deviated from the expected 4 nuclei. The size and number of the additional nuclei varied significantly (Fig. 6). In Stirton 7387, a triploid plant, chromatid segregation in two cells was noticed instead of chromosome segregation. No bridges were obser102 MEIOTIC STUDIES OF SOME SOUTH AFRICAN CULTIVARS OF LANTANA CAMARA (VERBENACEAE)


INTRODUCTION
Lantana camara L. is a widespread tropical weed noted for its toxicity to livestock and for its rapid invasion of natural pasturage, waste ground, forest margins and derelict or cultivated lands.
Experience gained from a number of areas of the tropics over many decades has shown that L. camara is not a homogeneous species, but actually consists of a number of forms.The origin, nature and spread of these forms have been discussed in the first paper of this series (Stirton, 1977), L. camara is today recognized as a polyploid aggregate species with a basic chromosome number of x = ll and having diploid, triploid, tetraploid, pentaploid and hexaploid representatives (Schnack & Covas, 1947;Tjio, 1948;Singh, 1951;Sen & Sahni, 1955;Tandon & Bali, 1955;Tandon & Chandi, 1955;Natarajan & Ahuja, 1957;Hender son, 1969;Spies & Stirton, 1982).The need for an understanding of these different forms and their taxonomic relationships in South Africa prompted the biological control section of the Department of Agriculture and Fisheries to approach the Botanical Research Institute, of the same Department, to initiate a taxonomic study of L. camara in South Africa.This paper reports in depth on the value of meiotic analysis in circumscribing the more than 50 naturalized cultivars of the weed that are presently recognized in South Africa (Stirton & Spies, 1982). )

MATERIALS AND METHODS
The plants used in this study are naturalized cultivars collected throughout South Africa and transplanted under quarantine in the Pretoria National Botanical Garden.Thirty-nine different cultivars, representative of all polyploid levels, were used in this study, 37 specimens being sampled from a hybrid swarm from the Transvaal lowveld.
Young inflorescences were collected between 09h00 and 12h00 hours and fixed for 24 hours in Camoy's fixative.Anthers were then squashed in aceto-carmine (Darlington & La Cour, 1976).The multivalent configurations during diakinesis were emphasized for meiotic analysis.
The early stages of meiosis were superficially similar in all the different cultivars although, during pachytene and diplotene, more chromosomes were visible in the higher polyploids (Fig. 1).Two chromosomes were always associated with the nucleolus at all polyploid levels.Chromosome configurations during diakinesis ranged from univalents to heptavalents, but were predominantly bivalents (Fig. 2).The number of chiasmata during diakinesis, as well as the different configurations formed, are shown in Table 1.From Table 2 it can be deduced that there are big genome differences between different cultivars at the same polyploid level.These genome differences result in different chromosome associations.
A high frequency of unpaired chromosomes and laggards (Fig. 3) were encountered during anaphase I (Table 3).Secondary association of chromosomes during telophase I usually occurred (Fig. 4).Neither heteromorphic bivalents nor B-chromosomes were found.
Instead of studying metaphase II and anaphase II, we concentrated our attention on the number of micronuclei found during telophase II (Table 4).The triploids and pentaploids usually deviated from the expected 4 nuclei.The size and number of the additional nuclei varied significantly (Fig. 6).
In Stirton 7387, a triploid plant, chromatid segregation in two cells was noticed instead of chromosome segregation.No bridges were obser-

DISCUSSION
L. camara is a polyploid species with a basic chromosome number of 11 (Tandon & Chandi, 1955).The South African cultivars of this weedy species represent all the known polyploid levels (Spies & Stirton, 1982).Sharma & Mukhopadhyay (1963) quoted reports indicating aneuploidy in L. camara.The only aneuploid form deviating from a basic number of 11 is the 2n=32 plant found by Schnack & Covas (1947).The camera lucida drawing presented by them, indicates that their plant could be a triploid (2n=33) with a 17-16 chromosome distribution in the late anaphase I illustrated.
The number of different chromosome configur ations found increased with an increase in polyploid level.Significant variation within each polyploid level was found.Each polyploid level will now be discussed separately.a) The diploid group In the diploid group of 13 plants, one plant (Stirton 7066) showed bivalents only, whereas the other extremity (Stirton 7315) had 6,14% trivalents and 18,41% univalents, leaving 75,45% bivalents (Table 1).These differences clearly indicate a heterogeneous group with Stirton 6882,7066, and 7374 being the only homogeneous diploid plants.Analysis of the bivalent configuration types indicated that Stirton 7066 and 7374 are more closely related to each other (r=0,9791) than they are related to Stirton 6882 (r=0,8836 and 0,9565 respectively), because a higher correlation indicates a higher degree of similarity.Stirton 7315 might be distantly related to Stirton 7306 (r=0,5466), whereas Stirton 7314 showed no correlation with and is, therefore, not related to any other diploid plant.

Multivalent association analysis of the asso ciations presented in
The diploid plants displayed little variation (s=0,53) with respect to the total number of chiasmata formed per genome.The chiasmata always occurred in the near terminal or terminal parts of the chromosome.Table 1 shows that in 89,09% of the bivalents formed, only one chiasma was found.Of this number, 63,92% of the chiasmata were terminal.Notwithstanding this high figure of terminal associations, cases with two terminal chiasmata (4,01%) were much lower than cases with one terminal and one near terminal chiasma (6 ,88%).
All the trivalents encountered were of the chain type with two chiasmata.No correlation exists between the number of chiasmata found and the occurrence of trivalents.
During anaphase I regular chromosome segrega tion occurred in 93,85% of the cells studied (Table 3).In a single plant (Stirton 7294) less than 90% normal anaphases were encountered (60%).One cell had a 11-10-1 distribution, whereas three cells had two laggards in a 11-9-2 distribution.No explanation can be advanced as to why this plant with its relatively regular metaphase I exhibits such an irregular anaphase I.
The second meiotic division seems quite normal in this diploid group.Only two cells (1,54%) had an additional micro-nucleus during telophase II.
Published information describes bivalents only during metaphase I and a regular anaphase I with a high pollen fertility (Tandon & Bali, 1955;Tandon & Chandi, 1955;Natarajan & Ahuja, 1957).The only indication of abnormality was found by Khaleel & Nalini (1972) where a large number of pollen grains remained uninucleate.It is, therefore, clear that the South African diploid cultivars of L. camara differ greatly from their Indian counterparts as regards chromosomal behaviour.
The high average number of univalents (7,83%) found in South African diploid cultivars, indicates a high degree of incomplete homology between chromosomes, resulting in asynapsis.This might imply that most of the diploid plants encountered in this study, originated as hybrids between distantly related cultivars or possibly different subspecies of L. camara.
Trivalents were found in 2,02% of the chromo some associations of diploid plants.This figure is also too high to account for the occasional trivalent that may sometimes be found.Three Causes for this high percentage of trivalents might exist.Either 11 is not the basic chromosome number or these diploid plants are actually allopolyhaploids derived from allotetraploids or minor genomic differences exist in this species resulting in abnormal hybrids.To determine which cause is responsible, the probabil ity of occurrence of each one must be discussed.
If L. camara had originated as a hybrid between two related plants with two different basic chromo some numbers (e.g. 5 and 6), partial homology between the genomes must exist in some cases resulting in occasional trivalent formation.The expected quadrivalent formation was not encoun tered because of the low frequency of chiasmata and the relative low number of cells studied.No homology between the parental genomes would have resulted in normal 'diploids' (actually allotetra ploids) which seems to be the case with Stirton 7066.
The results of this study indicate that at least three genomes, of which two are at least partially homologous and the third is nonhomologous to either of them, could have contributed to the development of L. camara.
The frequency of trivalents is too high and the rate of haplodization too low for secondary haplodization to play any major part in this case of speciation.
In the third case, minor genomic differences existin different cultivars of L. camara.These differences could be the results of prespeciation differences (described above) or post-speciation genomic evolu tion in the form of translocations, inversions, duplications and deletions.In order to determine which process actually occurred, a study of chromosome morphology is absolutely essential.In either case, however, hybrids between cultivars with minor differences in genomes, will result in a group of diploid plants with different chromosomal behaviour to that encountered in this South African hybrid swarm.
The occurrence of lower basic chromosome numbers in the Verbenaceae (x=5,6 and 7) (Lewis & Oliver, 1961), supports the theory of a possible lower basic chromosome number for L. camara.
Further support for this theory might be the occurrence of 12 as a basic chromosome number in other species of Lantana, where different composi tions from the low basic numbers are responsible for this difference rather than aneuploidy at the higher chromosome level.
Contrary to the findings of this study Natarajan & Ahuja (1957) found no evidence at any polyploid level for a basic chromosome number lower than 11.This clearly indicates that either post introduction genomic evolution occurred in either group or that totally different cultivars of L. camara were introduced into South Africa and India respectively.
A high frequency of univalents was encountered.
In addition to the univalents resulting from asynapsis, univalents are expected in triploid plants when bivalents are formed.The higher chromosome number produced more homologue chromosomes in the triploid compared with the diploid plants, so the role of asynapsis was diminished.The formation of quadrivalents is insignificant.
Very little variation (s=0,26) in the number of chiasmata formed was encountered.The number of terminal one chiasma bivalent configurations is lower in the triploid than in the diploid group.The number of terminal two chiasmata bivalents and terminal-subterminal two chiasmata bivalents is almost doubled.This led to a considerable increase in the number of chiasmata per genome compared to the diploids.
In addition to the chain type trivalents, a star type trivalent was encountered in 8,48% of the trivalents formed.This indicates that two chiasmata can occur close to each other, since this type of configuration requires a terminal chiasma between two chromoso mes and a subterminal chiasma between one of them and another chromosome.All quadrivalents were of the chain type.
Anaphase I revealed vast differences between the triploid plants.In most of the cells studied a 18-15 (25%) or 17-16 (20,5%) distribution was found.One cell of Stirton 7384 acted as an allotriploid plant should, resulting in a 11-11-11 distribution.The other cells studied contradicted this observation.
In two cells of another plant (Stirton 7387) chromatid segregation occurred during anaphase I.If these cells had reached maturity, they would have had triploid pollen with the potential to increase the polyploid level of L. camara once again.
The abnormal anaphase I is reflected in an irregular second division.Only 30,08% telophase II cells had the expected 4 nuclei.As this does not guarantee that all the normal telophase II cells will develop into fertile pollen, the South African triploid L. camara will have less fertile pollen than their Indian counterparts (Tandon & Bali, 1955).
In both South African and Indian material an irregular meiosis was observed (Khoshoo & Mahal, 1967).Whereas the South African material mostly had a high number (more than 11) of bivalents, the Indian material had mostly 31U 6n 12t (Tandon Bali, 1955) associations.The occurrence of more than 11 bivalents and trivalents in the South African material indicates homology between more than two genomes, therefore a basic chromosome number of less than 11 seems possible.

c) The tetraploid group
The tetraploid plants were a heterogeneous group (Table 1).The group had mostly bivalents, followed by trivalents, quadrivalents, univalents, a few pentavalents and even a hexavalent.Within the group large differences were encountered.The number of bivalents formed varied from 55,27% in Stirton 7230 to 82,8% in Stirton 7351.No evidence of a allotetraploid-like L. camara var.mista Bailey with only bivalents (reported by Natarajan & Ahuja, 1957) was found.
Whereas Stirton 6784, 7312 & 7351 had signifi cantly more quadrivalents than trivalents, Stirton 7060, 7230, 7253 & 7270 had significantly less quadrivalents than trivalents.Stirton 5288 and 7087 were intermediate between these groups.Although this finding may appear to indicate three groups, the cytogenetical differences between the plants in each 'group' are too large to group them together.
During this study a much lower incidence of univalents and a higher number of bivalents was encountered than Natarajan & Ahuja (1957) reported.
The high frequency of multivalents indicates a segmental alloploid origin with an inclination towards autopolyploidy.Slightly more chiasmata per genome were encountered in the tetraploid group than in the triploids (Table 1).A very high frequency (72,64%) of terminal one chiasma bivalents were formed.The trivalents were mostly of the chain type with a significant increase in the number of the star types (23,12%).Five different quadrivalent types were encountered.Usually the chain type (70,54%) and a ring of four (16,57%) were seen.The other types included a star (5,9%), a T (5,34%), and a terminal bivalent ring associated with a terminal bivalent chain (1,65%), to form a quadrivalent.
Pentavalents were encountered in one plant (Stirton 7230).The pentavalent consisted of a chain (33,333%) or of a ring of four with one chromosome associated in a near terminal chiasma with the ring.The hexavalent was in the form of a ring.
The few anaphase I cells suitable for analysis seemed to be normal.Telophase II was normal with four nuclei: d) The pentaploid group Unfortunately only three pentaploid plants were encountered during this study.Multivalent associa tion analysis indicated that these plants are totally different.The differences are evident in every cytogenetic aspect examined.
One plant (Stirton 5287) had no pentavalents, and the other two plants had 7,73% and 6,82% pentavalents respectively.The number of univalents (6,21%) is much lower than expected after studying the triploid group (10,37%).The high chromosome number obtained by segmental allopolyploidy could have increased the number of homologue chromo somes to rule out asynapsis at the higher polyploid levels.
The total number of chiasmata per genome differs from 5,89 to 6,99.Bivalents were usually of the chain type with terminal chiasmata.Only Stirton 7430 had bivalents with two chiasmata.All trivalents were of the chain type.With the exception of Stirton 7432, quadrivalents were usually of the chain type with approximately a quarter of all quadrivalents of the star type.In Stirton 7432 half the quadrivalents were of the star type with an equal amount of chains and rings.Pentavalents were usually of the chain type, followed by a T-type and an elongated star type.
Anaphase I was observed in Stirton 7432 only and it seemed to be quite irregular with between 2 and 10 laggards.Irregular meioses in pentaploid L. camara plants were also described by Khoshoo & Mahal (1967).Telophase II revealed an irregular meiosis with 4 nuclei in only 16,7% of the cells studied.

e) The hexaploid group
Only two plants were found and studied.In almost all cytogenetical aspects these two plants differ.No correlation could be found by multivalent association analysis.The same number of multiva lents are formed in each plant but the frequencies of the different multivalent configurations varies significantly.The figures obtained during this study correspond well with Natarajan & Ahuja's (1957) figures, except for the number of quadrivalents where twice their number was observed.
Anaphase I seemed normal and an extra micronucleus was encountered in only one telophase H cell (Table 4).

CONCLUSIONS
L. camara is a polyploid species.Since all polyploid forms encountered are multiples of 11 this number has been considered to be the basic chromosome number.The results of this study indicate either a lower basic chromosome number than 11, or postspeciation evolution of the genome must have occurred.
Cytogenetical differences at all polyploid levels indicate the occurrence of different genomic compositions in L. camara.This indicates either the introduction of different cultivars followed by hybridization in the past or a high rate of post introduction evolution has occurred within this species.At the diploid level at least four different groups exist with signs of hybridization.
The South African and Indian cultivars of L. camara reveal quite different chromosomal behavi our, implying that either different cultivars were introduced into these countries, or post introduction evolution in both countries resulted in different cultivar assemblages.
The polyploid forms of South African cultivars of L. camara are segmental allopolyploid plants.
one fragment in one cell of a pentaploid cultivar (Stirton 7430) was recorded.
The frequency of trivalents indicates allopoly ploidy with Stirton 7348 & 7394 tending towards autopolyploidy and Stirton 7381 & 7383 tending towards allopolyploidy.The remainder of the plants were distributed between these extremes.

TABLE 2 . -Multivalent associations in Lantana camara
Table 2 indicates a high correlation betweenStirton 7374, 6882, 7066, 7294,  7304, 7389, 7311 & 7361.In this group the homogeneous diploid plants(Stirton 6882, 7066 &  7314), or genetically similar plants, must have acted as some of the parental forms involved in hybridization during the formation of this group.Abnormal meiosis indicates that the rest of the group are hybrids.Morphological studies are required to indicate whether these three plants represent three different subspecies and also which related hybrids were formed by them.