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3Departamento de Botânica, Instituto de Biologia, Universidade Estadual de Campinas, Cx.P. 6109, Campinas-SP, 13083-970, Brazil; and 4Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas, Cx.P. 6109, Campinas-SP, 13083-970, Brazil
Received for publication March 2, 2000. Accepted for publication June 6, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: isozymes • genetic variability • Orchidaceae • Pleurothallis • population genetics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Borba and Semir, 1998
). There are two main campo rupestre areas: the Espinhaço Chain, extending from the center of Minas Gerais to the center-north of Bahia states, and the mountain ranges ("serras") of the south of Minas Gerais, along the basin of the Rio Grande. These two areas are of different geological origin and present distinct floras (Giulietti and Pirani, 1988
). The Espinhaço Chain is formed by several "serras" also with two main areas, the Chapada Diamantina in Bahia and the second mainly formed by the Serra do Cipó and the Diamantina plateau in the center of Minas Gerais.
Because of the discontinuity of these mountain ranges and outcroppings, many species, especially the rupicolous ones, are distributed in disjunct populations. It has been suggested that this characteristic is responsible for the great diversity and high endemism of the campo rupestre vegetation, one of the highest among the vegetation types of Brazil (Joly, 1970
; Giulietti and Pirani, 1988
).
In the last two decades a great effort has been made to describe the campo rupestre flora (e.g., Harley and Simmons, 1986
; Giulietti et al., 1987
; Stannard, 1995
), but, up to now, information on reproductive biology of these species is scanty (e.g., Sazima, 1977
; Sazima, Vogel, and Sazima, 1989
; Sazima and Sazima, 1990
; Borba and Semir, 1998, 1999
; Borba, Shepherd, and Semir, 1999
), and nothing has been published about their genetic variability.
A large number of myophilous orchid species occur on campo rupestre outcrops (Giulietti et al., 1987
; Brito, 1995
), and our group has been working on a biosystematic approach to understanding these species better. These studies include floral biology, breeding systems, phytochemistry, morphometry, and population genetics. This paper is one of a series discussing these topics for a group of five rupicolous orchid species of the genus Pleurothallis occurring in the Brazilian campo rupestre vegetation. These species are taxonomically difficult and were recently reviewed by Borba et al. (2000)
who described a new species, P. fabiobarrosii Borba and Semir, known only from three localities in northern-central Minas Gerais state, and suggested that it is closely related to P. johannensis Barb. Rodr. The latter, found only in the "serras" of southern Minas Gerais and formerly considered a synonym with P. teres Lindl., was treated as a distinct species. Pleurothallis rupestris Lindl. was synonymized of P. teres, a species almost restricted to the campos rupestres of the Espinhaço Chain in Minas Gerais, except for one disjunct population found in Rio de Janeiro state. Pleurothallis ochreata, a species previously known only from Bahia and Pernambuco states, was reported for the first time in Minas Gerais; this is the only known population of this species in which the leaves are linear-cylindrical. Individuals of P. ochreata from other populations always have large conduplicate leaves. Pleurothallis adamantinensis Brade is a rare species known only from two localities; it is the most distinct species of this group in both vegetative and floral traits.
These five species are pollinated by small flies (Diptera; E. L. Borba and J. Semir, unpublished data), a typical trait for the genus Pleurothallis (van der Pijl and Dodson, 1966
; Dressler, 1981, 1993
; Christensen, 1994
). As pollinators, flies generally make long visits and visit several flowers in the same inflorescence, characteristics that usually promote self-pollination or cross-pollination among flowers of the same individual (Chase, 1985
; Meve and Liede, 1994
; Borba and Semir, 1998
). As a consequence, and because these insects usually fly only short distances (Disney, 1994
), whereas the orchid populations themselves are isolated, our expectation was that these species should exhibit low genetic variability within the populations, in contrast to high genetic variability among conspecific populations.
To test this hypothesis we used isozyme electrophoresis to estimate the genetic variability for 22 populations of the five species mentioned above, to investigate the partitioning of the genetic diversity among conspecific populations, and to compare the results with the taxonomic realignments proposed by Borba et al. (2000)
for these species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The extracts were absorbed in Whatman number 3 paper and then applied to a Sigma starch gel. We used four buffer systems: system 1—electrode: boric acid 0.3 mol/L, NaOH 0.06 mol/L, pH 8.0; gel: Tris 0.01 mol/L, pH 8.5; modified from Shaw and Prasad (1970)
; system 2—electrode: citric acid 0.04 mol/L adjusted to pH 6.1 with N-(3- aminopropyl)-morpholine; gel: electrode buffer diluted 1:20 (Clayton and Tretiak, 1972
); system 3—electrode: histidine 0.065 mol/L adjusted to pH 6.5 with citric acid; gel: electrode buffer diluted 1:4; modified from Stuber, Goodman, and Johnson (1977)
; system 4—electrode: Tris 0.17 mol/L, citric acid 0.04 mol/L, pH 8.6; gel: tris 0.02 mol/L, citric acid 0.0013 mol/L, pH 8.6. Standard horizontal electrophoresis was performed until the inner marker (bromophenol blue) reached 9 cm from the application site using the following running conditions: system 1—30 mA; system 2—180 V; system 3—50 mA; system 4—60 mA. Nine enzymatic systems gave enough resolution for reading and were used: buffer system 1: esterase (EST; EC 3.1.1.1), acid phosphatase (ACP; EC 3.1.3.2), leucine aminopeptidase (LAP; EC 3.4.11.1), glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1); buffer system 2: phosphoglucomutase (PGM; EC 2.7.5.1); buffer system 3: 6-phosphogluconate dehydrogenase (6PGD; EC 1.1.1.44), phosphoglucoisomerase (PGI; EC 5.3.1.9), malate dehydrogenase (MDH; EC 1.1.1.37); buffer system 4: isocitrate dehydrogenase (IDH; EC 1.1.1.42). The staining recipes were similar to but slightly adjusted from Brune, Alfenas, and Junghans (1998
; ACP, EST, LAP), Corrias et al. (1991
; IDH, 6PGD, GOT, PGI), and Soltis et al. (1983
; PGM, MHD). Enzymatic systems showing more than one locus were numbered in ascending order from the locus with lowest mobility. The alleles were numbered according their mobility relative to the allele of a standard individual of P. teres present in all gels and designated as 100.
Data analysis
The allelic frequencies were determinated by manually counting the banding patterns of the homozygotes and heterozygotes stained in the gels. Genetic variability for every population was estimated by the following parameters: proportion of polymorphic loci (P; 0.95 criterion), mean number of alleles per locus (A), observed (Ho) and expected (He) mean heterozygosity per locus. Departures from the expected mean heterozygosity under Hardy-Weinberg (HW) equilibrium were tested using 
2 with a correction for small samples according to Levene (1949)
. Partitioning of genetic diversity among conspecific populations was estimated by F statistics (Fis, the inbreeding coeficient measures the reduction in heterozygosity due to nonrandom mating within a population; Fst, measures the differentiation among populations; Wright, 1978
). Matrices of genetic distances (Nei's [1978
] unbiased genetic distance) and genetic identities (Nei's [1978
] unbiased genetic identity) were calculated for populations and species. Cluster analysis was performed with the genetic identities matrix of the populations using UPGMA (Sneath and Sokal, 1973
). Principal coordinate analysis (PCO) was performed with the matrix of genetic distances of the populations using the FITOPAC 1.0 statistical package (Shepherd, 1995
). The matrix of the three first axis scores was graphically represented using NTSYS 1.8 (Rohlf, 1993
). All analyses were done using the BIOSYS 1.0 software package (Swofford and Selander, 1989
) except for the PCO.
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RESULTS |
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Species genetic structure
Five to 20% of species variability may be accounted for by interpopulational differences (Table 4). Pleurothallis johannensis, P. fabiobarrosii, and P. adamantinensis showed moderately low values of Fst, interpreted as a low level of genetic structuring. Pleurothallis teres and P. ochreata showed high average Fst. However, the high value of Fst in P. teres (0.21) is mainly due to the sample from SM (Table 4), which is very far from the core area of this species (Fig. 1, Appendix). This population differed from the others mainly in two loci, GOT-2 and IDH-2 (Table 2). The removal of this population from the analysis results in a much lower Fst (0.06), similar to the values obtained in P. johannensis, P. fabiobarrosii, and P. adamantinensis (Table 4).
Even the species with low Fst presented exclusive alleles in some populations (Table 2), chiefly in PGI-1. In this system, for example, allele 77 was frequent in P. johannensis sample C3, but was not found in the other two (C1 and C2) populations from nearly localities (Table 2, Appendix).
Phenetic relationships
All species showed high genetic identity within their populations (above 0.86). The only exception was P. teres, in which the population from SM had lower identities than all the others (Table 5, Fig. 2). Except for this population, cluster analysis and ordination grouped conspecific populations together, indicating that this procedure may be useful in identifying and delimiting species in this genus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Hamrick, Linhart, and Mitton, 1979
; Loveless and Hamrick, 1984
; Hamrick, 1989
; Hamrick and Godt, 1990
) and higher than values previously reported in the Orchidaceae (Scacchi and De Angelis, 1989
; Schlegel et al., 1989
; Scacchi, De Angelis, and Lanzara, 1990
; Corrias et al., 1991
; Klier, Leoschke, and Wendel, 1991
). These values are also higher than the average reported for monocots, herbs, long-lived perennials, plants with restricted geographical distribution, wind-dispersed seeds, and sexual reproduction (Hamrick and Godt, 1990
).
We did not expect to find this high genetic variability, which, in plants, is typically dependent on pollinator behavior (Levin and Kerster, 1968
; Schaal, 1980
; Schmitt, 1980
; Waser, 1982
; Loveless and Hamrick, 1984
; Ellstrand and Marshall, 1985
; Hamrick, 1989
; Fenster, 1991
; Godt and Hamrick, 1993
; Oshsawa, Furuya, and Ukai, 1993
). The Pleurothallis species we studied are pollinated by small Diptera of the families Phoridae and Chloropidae (E. L. Borba and J. Semir, unpublished data). These insects usually remain for a long time on the flowers and inflorescence, thus being able to promote pollination between flowers of the same plant (Chase, 1985
; Meve and Liede, 1994
; Borba and Semir, 1998
). E. L. Borba and J. Semir (unpublished data) observed that this behavior occurs at least in P. johannensis, P. fabiobarrosii, and P. adamantinensis.
Although orchids are usually self-compatible (van der Pijl and Dodson, 1966
; Dressler, 1981, 1993
; Borba, Shepherd, and Semir, 1999
), these five species have weak self-incompatibility, together with inbreeding depression and mechanical barriers preventing self-pollination (E. L. Borba and J. Semir, unpublished data). These mechanisms in conjunction seem to be effective in promoting seed set by cross-pollination as suggested by the high degree of genetic variability actually found. The genetic variability found in Pleurothallis is similar to or higher than that usually found in cross-pollinated species, while autogamous ones tend to have much lower values (Hamrick and Godt, 1990
). The values of Fis are low, supporting our hypothesis of low selfing in these species. Borba and Semir (1999)
noted that these combined mechanisms frequently occur in fly-pollinated orchids or for species in which the pollinators stay for a long time in flowers of the same individual. This, in turn, may help to maintain high genetic variability in these species.
Intraspecific phenetic relationship and genetic structure
The genetic identities found among conspecific populations were similar to or higher than those reported in other plant species (Thorpe, 1982
; Crawford, 1989
). Only the identities between the population of SM and the other P. teres populations (0.61–0.70) were lower than the values usually found for allopatric conspecific populations (Thorpe, 1982
; Crawford, 1989
; Harrison, 1991
). The affinities of this population, however, are clearly closest to P. teres as can be seen in the ordination (Fig. 3). Although we could not detect any morphological differences in this population, these data indicate a high degree of genetic differentiation, and this is also supported by the chemical differences found between this population and the pattern of the others (Borba, Trigo, and Semir, 2001)
. This population is located very far from the core area of the species (Fig. 1, Appendix), with forested areas between them. Therefore, it is reasonable to suppose that SM has been isolated for a long time and may be in the process of allopatric speciation. For the moment, we prefer a more conservative position of not proposing a new species, mainly due to the problems of identifying it in collections.
The high values of genetic identities between conspecific populations were surprising. The plants grow on rocky formations, as isolated islands of campo rupestre vegetation, a type of distribution that may explain the great number of endemic species in this environment (Giulietti and Pirani, 1988
). The Pleurothallis species we studied have pollinators that fly over short ranges (Disney, 1994
), and so we expected to find higher genetic distances between populations. On the other hand, Orchidaceae seeds are very light and may be wind dispersed for long distances (Dressler, 1981, 1993
); this may be important in promoting gene flow and, as a consequence, in maintaining species cohesion. Contrary to this hypothesis is the presence of alleles in significant frequencies that are found in a given population and not in nearby ones (e.g., the alleles of PGI in populations C1, C2, and C3 of P. johannensis, just a few kilometers apart). This may indicate that seed dispersal is not very efficient and so the low genetic distance between populations could also be due to recent isolation. We also cannot discard the importance of natural selection in keeping morphological cohesion in spite of the absence of gene flow.
Except for P. ochreata, which has the highest Fst and also the widest geographical distribution, the values of Fst were similar to wind-pollinated plant species (Hamrick and Godt, 1990
). In P. ochreata, there is just one population known in Minas Gerais. We found no remarkable genetic difference between this population and the others. It does, however, show a peculiar vegetative feature: it is the only one with linear, almost cylindrical leaves, while in all other populations the leaves are large and conduplicate. In addition, this population shows the same degree of chemical differentiation in relation to the other P. ochreata populations as the population of SM in relation to the other P. teres populations (Borba, Trigo, and Semir, 2001)
. Although the genetic markers do not indicate genetic differentiation corresponding to a distinct taxon, morphological and chemical data indicate that the population of Grão Mogol could be treated as a distinct subspecies (Borba, Trigo, and Semir, 2001)
.
Among the species with narrow distributions, P. fabiobarrosii had the greatest interpopulational differentiation. This species consists of distant populations that occur in mountains that do not belong to the core area of the Espinhaço Chain (Fig. 1, Appendix), and showed a number of rare alleles restricted to a given population. Our group has also found high genetic differentiation in populations of Proteopsis (Asteraceae) collected at these same localities (F. F. Jesus, V. N. Solferini, J. Semir, and P. I. K. L. Prado, unpublished data). These data agree with suggestions that species distribution in disjunct populations is responsible for the great degree of differentiation and endemism in campos rupestres (Giulietti and Pirani, 1988
).
Duplication of PGM-1 in P. fabiobarrosii
In the Joaquim Felicio population, we found evidence of duplication at the PGM-1 locus in six plants. This duplication was found in only one locus, and so we are convinced that it is not a polyploidization event (Gottlieb, 1982
). Its presence in only 19% of the individuals suggests that it may be a recent event. However, we may be underestimating the frequency of the duplication, because these alleles may be shared by the original and the duplicated loci. The distance between the populations and the discontinuity of the outcrops make gene flow very difficult. If this duplication has any adaptive value, it may be important to local adaptation and the differentiation of this population may ultimately lead to speciation (Kreitman and Akashi, 1985
).
Interspecific phenetic relationships
Excluding the population of P. teres from Santa Maria Madalena, each species can be recognized by its electrophoretic profile, in agreement with the species limits proposed by Borba et al. (2000)
. Similar agreement between mophological species delimitations and electrophoretic profiles have been observed in other orchids (Steinbrück et al., 1986
; Schlegel et al., 1989
; Corrias et al., 1991
; Klier, Leoschke, and Wendel, 1991
). As shown in Figs. 2 and 3, P. ochreata is well separated from the other species, as expected due to its peculiar morphological traits. On the other hand, the grouping of P. fabiobarrosii and P. adamantinensis was surprising. These two species have very different flowers, but they both have conduplicate leaves (except P. adamantinensis from Diamantina) and secondary stems that are as long as the leaves (found in P. ochreata but not in P. teres and P. johannensis, which have cylindrical leaves and short secondary stems).
In spite of floral differences, there were some difficulties in delimiting P. teres and P. johannensis, mainly due to vegetative similarity. Based on the floral morphology, Borba et al. (2000)
suggested that P. fabiobarrosii is related to P. johannensis, in spite of many vegetative differences. Our current results suggest that P. johannensis and P. teres are closely related but distinct species, and that P. johannensis and P. fabiobarrosii are a good example of taxa with convergence of floral form, probably due to similar pollination mechanisms (E. L. Borba and J. Semir, unpublished data). In this group, as in other orchids (Chase and Palmer, 1992
), it appears that vegetative characters may be more informative in assessing phylogenetic relationships than floral ones.
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FOOTNOTES |
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2 Author for correspondence (e-mail: borba{at}unicamp.br
)
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