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Recombination indices in species ofErythrinaL. (Leguminosae, Papilionoideae)
Botanical Journal of the Linnean Society (1996), 122: 163–170. With 1 figure
Recombination indices in species of Erythrina L. (Leguminosae, Papilionoideae) ELIANA REGINA FORNI-MARTINS Departamento de Botˆanica/IB/UNICAMP, caixa postal 6109, Campinas, 13081-970, SP, Brasil AND NEUSA DINIZ da CRUZ1 Seç˜ao de Citologia/Instituto Agronˆomico, caixa postal 28, Campinas, 13020-970, SP, Brasil Received March 1996, accepted for publication June 1996
Darlington’s recombination indices were calculated for eight species of Erythrina by adding the mean chiasma frequency and the haploid chromosome number of each species. The recombination indices distinguished subgenus Erythraster (two species with greater mean chiasma frequency) from subgenera Micropteryx and Erythrina. This discrimination corroborates literature data on pollen morphology. It was not possible to separate groups at sectional level using recombination index. The recombination indices were also compared with data on breeding system and pollinators, available in literature, to investigate Grant’s hypothesis on the occurrence of compensatory mechanisms in the regulation of recombination in plants. The data on breeding systems in Erythrina are very incomplete and it was not possible to correlate values of recombination indices with autogamy or allogamy. Species visited by hummingbirds showed lower recombination indices than species visited by other kind of pollinators. ©1996 The Linnean Society of London
ADDITIONAL KEY WORDS: — chiasma – reproductive biology. CONTENTS Introduction . . . . . . . . . . Material and methods . . . . . . . Results and discussion . . . . . . . Chiasma frequency . . . . . . Breeding systems and recombination Pollination and recombination . . References . . . . . . . . . . .
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163 165 165 165 167 168 169
INTRODUCTION
The genus Erythrina L. comprises 112 species distributed throughout the tropical regions and also occurs in some warm temperate areas, such as South Africa, the 1
In memoriam
0024–4074/96/010163 + 8 $25.00/0
163
©1996 The Linnean Society of London
164
E. R. FORNI-MARTINS AND N. D. DA CRUZ
Himalayas and southern China, Argentina (Rio de la Plata region) and the southern United States (Krukoff & Barneby 1974). The species are mainly trees or shrubs, but ten are perennial herbs. They occur in varied habitats, from lowland tropical rainforests to subtropical deserts and highland coniferous forests above 3000 m (Neill, 1988). Most of the species are ornamental and some are economically important, mainly in pharmacology, food, and in the production of stains and softwood (Krukoff, 1941; Burkart, 1952). Chromosome numbers of about 75% of the species of Erythrina are known. The majority have 2n = 42. There are reports of polyploidy (2n = 84) or different ploidy levels in 6% of the species. Polyploidy arose independently at least four times in the genus, and therefore the comparison of chromosome numbers is not useful for cytotaxonomic study (Neill, 1988). Studies on meiotic chromosome behaviour of some species were presented by Cruz, Ungaro & Medina (1976) and Neill (1988). Other papers showed chiasma frequencies in three species of Erythrina and in an interspecific hybrid ( Jalil, Srivastava & Khoshoo, 1982; Neill, 1988). However, there are no karyotypes available for the species of the genus. Chiasma frequency is genetically controlled ( Jones, 1987), although it may be altered by environmental variation, such as temperature, age, water content and exposure to mutagenic agents, as ionizing radiation and chemical products (Sybenga, 1972). Due to its genetic control, the chiasma frequency may be considered a hereditary expression of the species, and is sometimes more useful and accurate than karyotypic analysis for comparison of species (Roy & Singh, 1968). Chiasma frequency is a good estimator of genetic recombination in a population or in a species. It is used by some authors to measure genetic recombination in plants, as Darlington’s recombination index (Darlington, 1958), or the number of excess chiasmata (Burt & Bell, 1987), or Koellas’s recombination index (Koella, 1993). Recombination in plants is regulated by many factors, collectively known as regulatory factors. The regulatory factors can operate at different levels, from chromosome (e.g. chiasma frequency, chromosome number) to population (e.g. population size, competition) through individuals (e.g. breeding system, pollination, dispersal). Collectively, the regulatory factors are known as the recombination system, the main function of which is to achieve an optimum balance in the amount of genetic variability released for selection. The balance is between reproductive constancy favoured in the existing parental environment and reproductive variability thought to be necessary for long term flexibility (Lawrence, 1985). Grant (1958, 1975) had suggested the existence of compensatory mechanisms controlling the production of variability in a species. Some mechanisms that would lead to few recombinant types would be compensated by others operating at different stages of life cycle that would enhance the production of recombinants. Thus, for instance, the recombination index of a predominantly autogamous species should be greater than that of a predominantly allogamous one in order to counterbalance the number of recombinants in each case. In the same way, pollinators that carry pollen grains far and wide would be related to a more open recombination system than pollinators that visit restricted areas. Taking all this into account, a species showing a high recombination index is expected to be predominantly autogamous and/or pollinated by small-range pollinators, and/or dispersed for short distances. On the other hand, a species showing a low recombination index is expected to be predominantly allogamous and/or pollinated by large-range pollinators and/or dispersed for long distances.
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This paper is aimed at verifying whether chiasma frequency and recombination index vary among species of Erythrina and how they can be related to taxonomy and to mechanisms that regulate recombination in plants, such as breeding systems and pollinators.
MATERIAL AND METHODS
Although some of the species here studied are native to Brasil, all of them were collected as cultivated plants at the Agronomic Institute (IAC) and on the campus of the Campinas State University (UNICAMP), municipality of Campinas, State of S˜ao Paulo, southeastern Brasil. Vouchers (Table 1) were deposited in the herbarium at UNICAMP (UEC). Floral buds of different sizes were fixed in Carnoy (3 volumes of ethanol : 1 volume of glacial acetic acid) for 24 hours at room temperature and then stored in a freezer. Cytological preparations were obtained by squashing anthers in a drop of 1.2% acetic carmine (Medina & Conagin, 1964). About 30 cells in metaphase I were analysed per species. Some cells were photographed using a photomicroscope. The recombination index was calculated according to Darlington (1958), by the addition of the haploid number to the mean chiasma number per cell. The standard deviation and the variation coefficient (CV%) were calculated to evaluate the variation of number of chiasmata among different cells of each species. The average number of chiasmata per species was compared by analysis of variance, Tukey test and Student t-test (Sokal & Rohlf, 1969).
RESULTS AND DISCUSSION
Chiasma frequency The chiasmata of Erythrina cells were mostly terminal (Fig. 1). Bivalents with up to TABLE 1. Average number of chiasmata per cell (×), standard deviation (SD), coefficient of variation (CV%) and recombination indices (RI) of samples of about 30 cells of different species of Erythrina, all with n = 21. Infrageneric categories after Krukoff & Barneby (1974). KP = kind of pollinator after Hemsley & Ferguson (1985): B = non hovering birds, H = hummingbirds. UEC = registration number of vouchers in the Herbarium of the Campinas State University. Different letters in Tukey mean significant differences at 5% level Tukey
×
SD
CV%
RI
KP
UEC
Cristae-galli
a,b
25.23
1.50
5.94
46.23
B
31.561
Micropteryx Erythraster Erythraster Stenotropis Erythrina Erythrina Corallodendra
a,b c c a,b a a,b b
26.23 28.30 29.18 26.03 24.91 26.16 26.30
1.45 2.19 1.43 1.97 1.68 1.23 1.23
5.54 7.77 4.90 7.58 6.77 4.70 4.70
47.23 49.30 50.18 47.03 45.91 47.16 47.30
B* ** B H H H H
31.566 31.980 31.567 31.562 31.564 31.563 31.979
Species
Subgenus
Section
E. crista-galli L E. poeppigiana (Walp) O. F. Cook E. sandwicensis O.Deg. E. velutina Willd. E. speciosa Andrews E. hondurensis Standl. E. guatemalensis Krukoff E. corallodendrum L.
Micropteryx Micropteryx Erythraster Erythraster Erythrina Erythrina Erythrina Erythrina
*Also visited by hummingbirds and insects (Raven 1977). **Chiropterophilic syndrome (personal observation).
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Figure 1. Metaphase I of meiosis in Erythrina sandwicensis O.Deg. (n = 21), with 17II, 2II commencing anaphase disjunction (d), and 4I already separated (m). Scale bar = 10 µm.
three chiasmata were observed, but one chiasma was the most frequent observation. Bivalents with three chiasmata were not observed in E. corallodendrum or E. hondurensis. The greatest number of bivalents with three chiasmata occurred in E. sandwicensis, in about 50% of the cells analysed. The intraspecific variation may be considered low (Table 1), with CV% always less than 10% and classified as low after Gomes (1978). The greatest value of CV was 7.77%, observed in E. sandwicensis. Intraspecific variation of chiasma frequency may be caused by many factors, such as genetic variation between cells, failures in observation and interpretation of chiasma number, and analysis of chromosomes with different levels of chiasma terminalization. Recombination is effectively increased if the increased chiasma frequency results from random distribution of the chiasmata along the chromosome length (Grant, 1958). The predominantly terminal chiasmata observed in Erythrina, scored at metaphase I, may have greater effect in genetic recombination if they result from terminalization of chiasmata in interstitial positions during prophase I. The recombination indices apparently vary little among the species because one of the terms used in their calculation is the haploid chromosome number (n), which is constant for all the species (n = 21). However, statistical comparison (Tukey test) of the mean frequency of chiasma by analysis of variance showed significant differences among the species (d.f. = 7, n = 232, F = 25,595, P = 0.05), in Table 1. The eight species studied (Table 1) are classified in three subgenera: Micropteryx (E. crista-galli and E. poeppigiana), Erythrina (E. speciosa, E. hondurensis, E. guatemalensis and E. corallodendrum) and Erythraster (E. sandwicensis and E. velutina). Species of the other subgenera (Tripterolobus and Chirocalyx) were not analysed in this study. The infrageneric classification of Erythrina is based mainly on the floral structure,
RECOMBINATION INDICES IN ERYTHRINA
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inflorescence orientation, fruit morphology, and vestiture and epidermal ornamentation of foliage and calices (Krukoff & Barneby, 1974). Comparison of the average frequency of chiasmata per cell showed a relationship with the taxonomic arrangement at subgenus level. The Tukey test (Table 1) suggested that two groups could be distinguished: one formed by the species E. sandwicensis and E. velutina, both of subgenus Erythraster; and another formed by the other six species, belonging to the subgenera Micropteryx and Erythrina. The species of subgenus Erythraster presented the highest recombination indices (Table 1). These results reinforce the differentiation of the subgenus Erythraster, already pointed out by Graham & Tomb (1977) based on pollen characters. Chiasma frequencies in the group formed by the six species of the subgenera Micropteryx and Erythrina did not show any relationship with the taxonomy at section level. E. guatemalensis (section Erythrina) showed mean chiasma frequency closer to that of E. corallodendrum (section Corallodendra) than to that of E. hondurensis, which is placed in the same section Erythrina (Table 1). Values reported in literature for chiasma frequencies of other species of Erythrina are greater than those presented in this study. Jalil et al. (1982) presented values of 31.35 ± 0.54 and of 31.25 ± 0.61 for E. variegata and E. resupinata (subgenus Erythrina), respectively, and 32.08 ± 0.7 for the interspecific hybrid. Neill (1988) presented 31.5 ± 84 for E. macrophylla (subgenus Erythraster). These results do not confirm the separation of subgenus Erythraster. The higher values of the recombination indices found by these authors are expected, because the analyses were made in diakinesis, a phase in which chiasmata terminalization is less advanced than in metaphase I. Failures in standardization of the stage in which observations were made during diakinesis and/or in the visualization of the chiasmata may have produced the lack of difference between subgenera Erythraster and Erythrina, as studied by those authors. Breeding systems and recombination There are very few studies investigating Grant’s (1958, 1975) hypothesis about the opposite mechanisms involved in the regulation of recombination in plants. Gibbs, Milne & Vargas-Carillo, (1975) observed that two predominantly autogamous species of Senecio had greater recombination indices than three other congeneric species with a low rate of allogamy. However, the predominantly autogamous species were polyploid and so the recombination index was influenced by this fact. Nevertheless, Senecio vulgaris, a predominately autogamous species, did show the greatest average frequency of chiasmata per bivalent. Lawrence (1985) studied the recombination systems of 32 species of Senecio and concluded that Darlington’s recombination index cannot be applied to this genus, since several ploidy levels are represented. However, the chiasma frequency of the species studied was closely correlated with breeding system: most of the self-incompatible species had a low chiasma frequency ( < 1.5 per bivalent) while most self-compatible species had a high chiasma frequency ( > 1.5 per bivalent). Koella (1993) compared published chiasma frequencies of 194 plant species of 15 angiosperm families. He did not use Darlington’s (1958) recombination index, but another recombination index (Koella 1993) and the number of excess chiasmata (Burt & Bell, 1987). He observed that genetic recombination was higher in animal-dispersed species than in others, and
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increased as dispersal distance decreased. This observation is consistent with Grant’s (1958, 1975) hypothesis about regulation of recombination in plants. It is difficult to establish a relationship between breeding systems and recombination indices for Erythrina. Very little is known about recombination indices for the genus as a whole. Only a small number of species has been studied methodically in relation to their breeding systems so far. Moreover, different results obtained by different authors in the same species have led to opposite conclusions. Preliminary information pointed out the heterogeneity of breeding systems in Erythrina (Raven, 1977). Among the species studied here (Table 1), E. crista-galli (Fryxell 1957) and E. poeppigiana (Arroyo 1981) are considered allogamous. Arroyo (1981) reviewed the breeding systems of leguminous plants, mentioning that in eleven experimentally tested species of Erythrina five were allogamous. Hernandez & Toledo (1979) pointed out that E. leptorhyza is allogamous. These authors observed dimorphism in the number and size of ovules, size of pollen grains and length of pistil, but they did not investigate reproductive function in each case. They pointed out that the selfincompatibility would not be absolute in the species, for a low proportion of cleistogamous flowers produced fruits. Based on analysis of the number of fruits produced by self pollination, Feinsinger et al. (1979) concluded that E. pallida and E. fusca do not have complete self-incompatibility. Neill (1988), basing on experimental studies on interspecific relationships in Erythrina, stated that there is no species in the genus with a genetic incompatibility system, but he related endogamic depression with autogamy. Some of the species studied by Neill (1988) are E. crista-galli, E. sandwicensis, E. speciosa and E. guatemalensis whose recombination indices are presented in Table 1. Neill (1988) based his conclusions on the study of only 19 species, and for some of these he did not obtain fruit formation either after self-pollination or after cross-pollination. Apparently, Neill’s (1988) conclusion that all the species of Erythrina are autogamous should be treated with some caution. Hence, in face of the information gathered from the literature and considering the data presented here (Table 1), there is no secure basis for discussion of Grant’s (1958, 1975) hypothesis in relation to breeding systems in Erythrina, because not enough is known about the reproductive system of each species. More conclusive studies are needed. Pollination and recombination The behaviour of different kinds of pollinators can promote different levels of pollen flux among individuals of different species. Thus, this behaviour can regulate the flux of variability among species of Erythrina, together with breeding system, dispersion range and population size. According to Grant (1958, 1975), it is expected that species with greater pollen flux will have a lower recombination index than others with restricted distribution of pollen. Pollination in Erythrina is mainly by hummingbirds (Trochilidae) and other kinds of birds of the order Passeriformes (Neill, 1987), besides a confirmed mention of bats (Raven, 1979). Species pollinated by hummingbirds have erect inflorescences with tubular flowers orientated outward with the reproductive structures protected. Species pollinated by other birds have open flowers, with exposed reproductive parts, and the inflorescences are orientated in such a way as to allow the animal to perch while feeding on floral nectar (Neill, 1988). Chloroplast DNA relationships in 60
RECOMBINATION INDICES IN ERYTHRINA
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species of Erythrina indicated that hummingbird pollination, considered derived relative to passerine pollination, evolved convergently in at least three distinct lineages and that reversals to passerine pollination also may have occurred (Bruneau & Doyle, 1993). Table 1 presents the pollinators of the species studied here, according to Hemsley & Ferguson (1985). The structure and arrangement of inflorescences and flowers of E. sandwicensis, observed when the vouchers were collected for this study, suggest a chiropterophilic syndrome (Faegri & van der Pijl, 1971). In the evolution of the genus Erythrina, bats have been considered relatively unspecialized floral visitors (Raven, 1977). Neill (1988) pointed out that the hummingbirds with non-territorialist behaviour (trapliners) visit more individuals of Erythrina and promote greater flux of pollen than the generalist birds. Therefore, lower recombination indices would be expected in species pollinated by this type of hummingbird. However, the specific recombination indices in Table 1 did not show a strong relation with the pollinator type. The species pollinated by hummingbirds showed intermediate figures (45.9–47.3). The greatest indices were obtained for species whose flowers are pollinated by other birds and bats, but these included both the highest and the lowest figures. Yet, when all the hummingbird-pollinated species of Table 1 are considered they showed a lower average chiasma frequency (25.96 ± 1.61) than the set of the other species (27.32 ± 2.31). The Student t–test showed that the difference between them is significant at 5% level. Therefore, the average chiasma frequency suggests the existence of compensatory mechanisms in the recombination of species of Erythrina with different pollinators.
REFERENCES Arroyo MKT. 1981. Breeding systems and pollination biology in Leguminosae. In: Polhill RM, Raven RM, eds. Advances in legume systematics. Kew: Royal Botanic Gardens, 723–769. Bruneau A, Doyle JJ. 1993. Cladistic analysis of chloroplast DNA restriction site characters in Erythrina (Leguminosae: Phaseoleae). Systematic Botany 18: 229–247. Burkart A. 1952. Las leguminosas argentinas silvestres y cultivadas. Buenos Aires: ACME Agency. Burt A, Bell G. 1987. Chiasmata. Mammalian chiasma frequencies as a test of two theories of recombination. Nature 326: 803–805. Cruz ND, Ungaro MRG, Medina DM. 1976. Observa¸co˜ es citol´ogicas em trˆes esp´ecies de Erythrina. Bragantia 35: 133–139. Darlington CD. 1958. The evolution of genetic systems. New York: Inc. Publishers. Faegri K, van der Pijl L. 1971. The principles of pollination ecology. New York: Pergamon Press. Feinsinger P, Kinhart YB, Swarm LA, Wolfe JA. 1979. Aspects of pollination biology of three Erythrina species on Trinidad and Tobago. Annals of the Missouri Botanical Garden 66: 451–471. Fryxell PA. 1957. Mode of reproduction in higher plants. Botanical Review 25: 135–233. Gibbs PE, Milne C, Vargas-Carillo M. 1975. Correlation between the breeding system and recombination indexes in five species of Senecio. New Phytologist 75: 619–626. Gomes FP. 1978. Curso de estatistica experimental. S˜ao Paulo: Nobel. Graham A, Tomb AS. 1977. Palynology of Erythrina (Leguminosae:Papilionoideae): the subgenera, sections, and generic relationships. Lloydia 40: 413–435. Grant V. 1958. The regulation of recombination in plants. Cold Spring Harbor Symposia of Quantitative Biology. 23: 337–363. Grant V. 1975. Genetics of flowering plants. New York: Columbia University Press. Hemsley AJ, Ferguson IK. 1985. Pollen morphology of the genus Erythrina (Leguminosae: Papilionoideae) in relation to floral structure and pollinators. Annals of the Missouri Botanical Garden. 72: 570–590. Hernandez AJ, Toledo VM. 1979. The role of nectar robbers and pollinators in the reproduction of Erythrina leptorhiza. Annals of the Missouri Botanical Garden 66: 512–520. Jalil R, Pal M, Srivastava GS, Khoshoo TN. 1982. Cytogenetics of Erythrina x resuparcelli Srivastava. Allertonia 3: 19–24. Jones GH. 1987. Chiasmata. In: Moens PB. Meiosis. Orlando:Academic Press, 213–244.
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Koella JC. 1993. Ecological correlates of chiasma frequency and recombination index of plants. Biological Journal of the Linnean Society 48: 227–238. Krukoff BA. 1941. Supplementary notes on the American species of Erythrina. American Journal of Botany. 28: 683–691. Krukoff BA, Barneby RC. 1974. A conspectus of the genus Erythrina. Lloydia 37: 332–459. Lawrence ME. 1985. Senecio L. (Asteraceae) in Australia: recombination systems of a polyploid series. Australian Journal of Botany 33: 209–219. Medina DM, Conagin, CHTM. 1964. T´ecnica citol´ogica. Campinas: Instituto Agronˆomico, publica¸ca˜ o 2610. Neill DA. 1987. Trapliners in trees: hummingbird pollination of Erythrina Sect. Erythrina (Leguminosae Papilionoideae). Annals of the Missouri Botanical Garden 74: 27–41. Neill DA. 1988. Experimental studies on species relationships in Erythrina (Leguminosae Papilionoideae). Annals of the Missouri Botanical Garden 75: 886–969. Raven PH. 1977. Erythrina Symposium II. Erythrina (Fabaceae: Faboideae): Introduction to Symposium II. Lloydia 40: 401–406. Raven PH. 1979. Erythrina symposium III. Erythrina (Fabaceae: Faboideae): Introduction to Symposium III. Annals of the Missouri Botanical Garden 66: 417–421. Roy RP, Singh MK. 1968. Meiotic studies in the genus Lathyrus L. The Nucleus 11: 7–12. Sokal RR, Rohlf FJ. 1969. Biometry. San Francisco: WH Freeman Company. Sybenga J. 1972. General cytogenetics. Amsterdam: North-Holland Publishing Company.
Recombination indices in species of Erythrina L. (Leguminosae, Papilionoideae) ELIANA REGINA FORNI-MARTINS Departamento de Botˆanica/IB/UNICAMP, caixa postal 6109, Campinas, 13081-970, SP, Brasil AND NEUSA DINIZ da CRUZ1 Seç˜ao de Citologia/Instituto Agronˆomico, caixa postal 28, Campinas, 13020-970, SP, Brasil Received March 1996, accepted for publication June 1996
Darlington’s recombination indices were calculated for eight species of Erythrina by adding the mean chiasma frequency and the haploid chromosome number of each species. The recombination indices distinguished subgenus Erythraster (two species with greater mean chiasma frequency) from subgenera Micropteryx and Erythrina. This discrimination corroborates literature data on pollen morphology. It was not possible to separate groups at sectional level using recombination index. The recombination indices were also compared with data on breeding system and pollinators, available in literature, to investigate Grant’s hypothesis on the occurrence of compensatory mechanisms in the regulation of recombination in plants. The data on breeding systems in Erythrina are very incomplete and it was not possible to correlate values of recombination indices with autogamy or allogamy. Species visited by hummingbirds showed lower recombination indices than species visited by other kind of pollinators. ©1996 The Linnean Society of London
ADDITIONAL KEY WORDS: — chiasma – reproductive biology. CONTENTS Introduction . . . . . . . . . . Material and methods . . . . . . . Results and discussion . . . . . . . Chiasma frequency . . . . . . Breeding systems and recombination Pollination and recombination . . References . . . . . . . . . . .
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163 165 165 165 167 168 169
INTRODUCTION
The genus Erythrina L. comprises 112 species distributed throughout the tropical regions and also occurs in some warm temperate areas, such as South Africa, the 1
In memoriam
0024–4074/96/010163 + 8 $25.00/0
163
©1996 The Linnean Society of London
164
E. R. FORNI-MARTINS AND N. D. DA CRUZ
Himalayas and southern China, Argentina (Rio de la Plata region) and the southern United States (Krukoff & Barneby 1974). The species are mainly trees or shrubs, but ten are perennial herbs. They occur in varied habitats, from lowland tropical rainforests to subtropical deserts and highland coniferous forests above 3000 m (Neill, 1988). Most of the species are ornamental and some are economically important, mainly in pharmacology, food, and in the production of stains and softwood (Krukoff, 1941; Burkart, 1952). Chromosome numbers of about 75% of the species of Erythrina are known. The majority have 2n = 42. There are reports of polyploidy (2n = 84) or different ploidy levels in 6% of the species. Polyploidy arose independently at least four times in the genus, and therefore the comparison of chromosome numbers is not useful for cytotaxonomic study (Neill, 1988). Studies on meiotic chromosome behaviour of some species were presented by Cruz, Ungaro & Medina (1976) and Neill (1988). Other papers showed chiasma frequencies in three species of Erythrina and in an interspecific hybrid ( Jalil, Srivastava & Khoshoo, 1982; Neill, 1988). However, there are no karyotypes available for the species of the genus. Chiasma frequency is genetically controlled ( Jones, 1987), although it may be altered by environmental variation, such as temperature, age, water content and exposure to mutagenic agents, as ionizing radiation and chemical products (Sybenga, 1972). Due to its genetic control, the chiasma frequency may be considered a hereditary expression of the species, and is sometimes more useful and accurate than karyotypic analysis for comparison of species (Roy & Singh, 1968). Chiasma frequency is a good estimator of genetic recombination in a population or in a species. It is used by some authors to measure genetic recombination in plants, as Darlington’s recombination index (Darlington, 1958), or the number of excess chiasmata (Burt & Bell, 1987), or Koellas’s recombination index (Koella, 1993). Recombination in plants is regulated by many factors, collectively known as regulatory factors. The regulatory factors can operate at different levels, from chromosome (e.g. chiasma frequency, chromosome number) to population (e.g. population size, competition) through individuals (e.g. breeding system, pollination, dispersal). Collectively, the regulatory factors are known as the recombination system, the main function of which is to achieve an optimum balance in the amount of genetic variability released for selection. The balance is between reproductive constancy favoured in the existing parental environment and reproductive variability thought to be necessary for long term flexibility (Lawrence, 1985). Grant (1958, 1975) had suggested the existence of compensatory mechanisms controlling the production of variability in a species. Some mechanisms that would lead to few recombinant types would be compensated by others operating at different stages of life cycle that would enhance the production of recombinants. Thus, for instance, the recombination index of a predominantly autogamous species should be greater than that of a predominantly allogamous one in order to counterbalance the number of recombinants in each case. In the same way, pollinators that carry pollen grains far and wide would be related to a more open recombination system than pollinators that visit restricted areas. Taking all this into account, a species showing a high recombination index is expected to be predominantly autogamous and/or pollinated by small-range pollinators, and/or dispersed for short distances. On the other hand, a species showing a low recombination index is expected to be predominantly allogamous and/or pollinated by large-range pollinators and/or dispersed for long distances.
RECOMBINATION INDICES IN ERYTHRINA
165
This paper is aimed at verifying whether chiasma frequency and recombination index vary among species of Erythrina and how they can be related to taxonomy and to mechanisms that regulate recombination in plants, such as breeding systems and pollinators.
MATERIAL AND METHODS
Although some of the species here studied are native to Brasil, all of them were collected as cultivated plants at the Agronomic Institute (IAC) and on the campus of the Campinas State University (UNICAMP), municipality of Campinas, State of S˜ao Paulo, southeastern Brasil. Vouchers (Table 1) were deposited in the herbarium at UNICAMP (UEC). Floral buds of different sizes were fixed in Carnoy (3 volumes of ethanol : 1 volume of glacial acetic acid) for 24 hours at room temperature and then stored in a freezer. Cytological preparations were obtained by squashing anthers in a drop of 1.2% acetic carmine (Medina & Conagin, 1964). About 30 cells in metaphase I were analysed per species. Some cells were photographed using a photomicroscope. The recombination index was calculated according to Darlington (1958), by the addition of the haploid number to the mean chiasma number per cell. The standard deviation and the variation coefficient (CV%) were calculated to evaluate the variation of number of chiasmata among different cells of each species. The average number of chiasmata per species was compared by analysis of variance, Tukey test and Student t-test (Sokal & Rohlf, 1969).
RESULTS AND DISCUSSION
Chiasma frequency The chiasmata of Erythrina cells were mostly terminal (Fig. 1). Bivalents with up to TABLE 1. Average number of chiasmata per cell (×), standard deviation (SD), coefficient of variation (CV%) and recombination indices (RI) of samples of about 30 cells of different species of Erythrina, all with n = 21. Infrageneric categories after Krukoff & Barneby (1974). KP = kind of pollinator after Hemsley & Ferguson (1985): B = non hovering birds, H = hummingbirds. UEC = registration number of vouchers in the Herbarium of the Campinas State University. Different letters in Tukey mean significant differences at 5% level Tukey
×
SD
CV%
RI
KP
UEC
Cristae-galli
a,b
25.23
1.50
5.94
46.23
B
31.561
Micropteryx Erythraster Erythraster Stenotropis Erythrina Erythrina Corallodendra
a,b c c a,b a a,b b
26.23 28.30 29.18 26.03 24.91 26.16 26.30
1.45 2.19 1.43 1.97 1.68 1.23 1.23
5.54 7.77 4.90 7.58 6.77 4.70 4.70
47.23 49.30 50.18 47.03 45.91 47.16 47.30
B* ** B H H H H
31.566 31.980 31.567 31.562 31.564 31.563 31.979
Species
Subgenus
Section
E. crista-galli L E. poeppigiana (Walp) O. F. Cook E. sandwicensis O.Deg. E. velutina Willd. E. speciosa Andrews E. hondurensis Standl. E. guatemalensis Krukoff E. corallodendrum L.
Micropteryx Micropteryx Erythraster Erythraster Erythrina Erythrina Erythrina Erythrina
*Also visited by hummingbirds and insects (Raven 1977). **Chiropterophilic syndrome (personal observation).
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Figure 1. Metaphase I of meiosis in Erythrina sandwicensis O.Deg. (n = 21), with 17II, 2II commencing anaphase disjunction (d), and 4I already separated (m). Scale bar = 10 µm.
three chiasmata were observed, but one chiasma was the most frequent observation. Bivalents with three chiasmata were not observed in E. corallodendrum or E. hondurensis. The greatest number of bivalents with three chiasmata occurred in E. sandwicensis, in about 50% of the cells analysed. The intraspecific variation may be considered low (Table 1), with CV% always less than 10% and classified as low after Gomes (1978). The greatest value of CV was 7.77%, observed in E. sandwicensis. Intraspecific variation of chiasma frequency may be caused by many factors, such as genetic variation between cells, failures in observation and interpretation of chiasma number, and analysis of chromosomes with different levels of chiasma terminalization. Recombination is effectively increased if the increased chiasma frequency results from random distribution of the chiasmata along the chromosome length (Grant, 1958). The predominantly terminal chiasmata observed in Erythrina, scored at metaphase I, may have greater effect in genetic recombination if they result from terminalization of chiasmata in interstitial positions during prophase I. The recombination indices apparently vary little among the species because one of the terms used in their calculation is the haploid chromosome number (n), which is constant for all the species (n = 21). However, statistical comparison (Tukey test) of the mean frequency of chiasma by analysis of variance showed significant differences among the species (d.f. = 7, n = 232, F = 25,595, P = 0.05), in Table 1. The eight species studied (Table 1) are classified in three subgenera: Micropteryx (E. crista-galli and E. poeppigiana), Erythrina (E. speciosa, E. hondurensis, E. guatemalensis and E. corallodendrum) and Erythraster (E. sandwicensis and E. velutina). Species of the other subgenera (Tripterolobus and Chirocalyx) were not analysed in this study. The infrageneric classification of Erythrina is based mainly on the floral structure,
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inflorescence orientation, fruit morphology, and vestiture and epidermal ornamentation of foliage and calices (Krukoff & Barneby, 1974). Comparison of the average frequency of chiasmata per cell showed a relationship with the taxonomic arrangement at subgenus level. The Tukey test (Table 1) suggested that two groups could be distinguished: one formed by the species E. sandwicensis and E. velutina, both of subgenus Erythraster; and another formed by the other six species, belonging to the subgenera Micropteryx and Erythrina. The species of subgenus Erythraster presented the highest recombination indices (Table 1). These results reinforce the differentiation of the subgenus Erythraster, already pointed out by Graham & Tomb (1977) based on pollen characters. Chiasma frequencies in the group formed by the six species of the subgenera Micropteryx and Erythrina did not show any relationship with the taxonomy at section level. E. guatemalensis (section Erythrina) showed mean chiasma frequency closer to that of E. corallodendrum (section Corallodendra) than to that of E. hondurensis, which is placed in the same section Erythrina (Table 1). Values reported in literature for chiasma frequencies of other species of Erythrina are greater than those presented in this study. Jalil et al. (1982) presented values of 31.35 ± 0.54 and of 31.25 ± 0.61 for E. variegata and E. resupinata (subgenus Erythrina), respectively, and 32.08 ± 0.7 for the interspecific hybrid. Neill (1988) presented 31.5 ± 84 for E. macrophylla (subgenus Erythraster). These results do not confirm the separation of subgenus Erythraster. The higher values of the recombination indices found by these authors are expected, because the analyses were made in diakinesis, a phase in which chiasmata terminalization is less advanced than in metaphase I. Failures in standardization of the stage in which observations were made during diakinesis and/or in the visualization of the chiasmata may have produced the lack of difference between subgenera Erythraster and Erythrina, as studied by those authors. Breeding systems and recombination There are very few studies investigating Grant’s (1958, 1975) hypothesis about the opposite mechanisms involved in the regulation of recombination in plants. Gibbs, Milne & Vargas-Carillo, (1975) observed that two predominantly autogamous species of Senecio had greater recombination indices than three other congeneric species with a low rate of allogamy. However, the predominantly autogamous species were polyploid and so the recombination index was influenced by this fact. Nevertheless, Senecio vulgaris, a predominately autogamous species, did show the greatest average frequency of chiasmata per bivalent. Lawrence (1985) studied the recombination systems of 32 species of Senecio and concluded that Darlington’s recombination index cannot be applied to this genus, since several ploidy levels are represented. However, the chiasma frequency of the species studied was closely correlated with breeding system: most of the self-incompatible species had a low chiasma frequency ( < 1.5 per bivalent) while most self-compatible species had a high chiasma frequency ( > 1.5 per bivalent). Koella (1993) compared published chiasma frequencies of 194 plant species of 15 angiosperm families. He did not use Darlington’s (1958) recombination index, but another recombination index (Koella 1993) and the number of excess chiasmata (Burt & Bell, 1987). He observed that genetic recombination was higher in animal-dispersed species than in others, and
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increased as dispersal distance decreased. This observation is consistent with Grant’s (1958, 1975) hypothesis about regulation of recombination in plants. It is difficult to establish a relationship between breeding systems and recombination indices for Erythrina. Very little is known about recombination indices for the genus as a whole. Only a small number of species has been studied methodically in relation to their breeding systems so far. Moreover, different results obtained by different authors in the same species have led to opposite conclusions. Preliminary information pointed out the heterogeneity of breeding systems in Erythrina (Raven, 1977). Among the species studied here (Table 1), E. crista-galli (Fryxell 1957) and E. poeppigiana (Arroyo 1981) are considered allogamous. Arroyo (1981) reviewed the breeding systems of leguminous plants, mentioning that in eleven experimentally tested species of Erythrina five were allogamous. Hernandez & Toledo (1979) pointed out that E. leptorhyza is allogamous. These authors observed dimorphism in the number and size of ovules, size of pollen grains and length of pistil, but they did not investigate reproductive function in each case. They pointed out that the selfincompatibility would not be absolute in the species, for a low proportion of cleistogamous flowers produced fruits. Based on analysis of the number of fruits produced by self pollination, Feinsinger et al. (1979) concluded that E. pallida and E. fusca do not have complete self-incompatibility. Neill (1988), basing on experimental studies on interspecific relationships in Erythrina, stated that there is no species in the genus with a genetic incompatibility system, but he related endogamic depression with autogamy. Some of the species studied by Neill (1988) are E. crista-galli, E. sandwicensis, E. speciosa and E. guatemalensis whose recombination indices are presented in Table 1. Neill (1988) based his conclusions on the study of only 19 species, and for some of these he did not obtain fruit formation either after self-pollination or after cross-pollination. Apparently, Neill’s (1988) conclusion that all the species of Erythrina are autogamous should be treated with some caution. Hence, in face of the information gathered from the literature and considering the data presented here (Table 1), there is no secure basis for discussion of Grant’s (1958, 1975) hypothesis in relation to breeding systems in Erythrina, because not enough is known about the reproductive system of each species. More conclusive studies are needed. Pollination and recombination The behaviour of different kinds of pollinators can promote different levels of pollen flux among individuals of different species. Thus, this behaviour can regulate the flux of variability among species of Erythrina, together with breeding system, dispersion range and population size. According to Grant (1958, 1975), it is expected that species with greater pollen flux will have a lower recombination index than others with restricted distribution of pollen. Pollination in Erythrina is mainly by hummingbirds (Trochilidae) and other kinds of birds of the order Passeriformes (Neill, 1987), besides a confirmed mention of bats (Raven, 1979). Species pollinated by hummingbirds have erect inflorescences with tubular flowers orientated outward with the reproductive structures protected. Species pollinated by other birds have open flowers, with exposed reproductive parts, and the inflorescences are orientated in such a way as to allow the animal to perch while feeding on floral nectar (Neill, 1988). Chloroplast DNA relationships in 60
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species of Erythrina indicated that hummingbird pollination, considered derived relative to passerine pollination, evolved convergently in at least three distinct lineages and that reversals to passerine pollination also may have occurred (Bruneau & Doyle, 1993). Table 1 presents the pollinators of the species studied here, according to Hemsley & Ferguson (1985). The structure and arrangement of inflorescences and flowers of E. sandwicensis, observed when the vouchers were collected for this study, suggest a chiropterophilic syndrome (Faegri & van der Pijl, 1971). In the evolution of the genus Erythrina, bats have been considered relatively unspecialized floral visitors (Raven, 1977). Neill (1988) pointed out that the hummingbirds with non-territorialist behaviour (trapliners) visit more individuals of Erythrina and promote greater flux of pollen than the generalist birds. Therefore, lower recombination indices would be expected in species pollinated by this type of hummingbird. However, the specific recombination indices in Table 1 did not show a strong relation with the pollinator type. The species pollinated by hummingbirds showed intermediate figures (45.9–47.3). The greatest indices were obtained for species whose flowers are pollinated by other birds and bats, but these included both the highest and the lowest figures. Yet, when all the hummingbird-pollinated species of Table 1 are considered they showed a lower average chiasma frequency (25.96 ± 1.61) than the set of the other species (27.32 ± 2.31). The Student t–test showed that the difference between them is significant at 5% level. Therefore, the average chiasma frequency suggests the existence of compensatory mechanisms in the recombination of species of Erythrina with different pollinators.
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