Abstract. Franceschinelli, E. V.; J. R. Stehmann, C. Melo-Silva & J. N. Mesquita Neto. 2013. Preliminary study on genetic diversity of endemic and threatened species of Petunia (Solanaceae). Darwiniana, nueva serie 1(2): 220-226.

Rare and narrowly endemic species are more vulnerable to extinction due to their restricted geographical distribution. the genus Petunia has 14 species and most of them are endemic to a restricted area. Four Petunia species that grow in Southern Brazil were studied: P. bonjardinensis, P. reitzii, P. integrifolia subsp. depauperata, and P. exserta. We presented a preliminary study on the genetic diversity of five populations of Petunia, using allozyme markers. For the genetic analyses, allozyme electrophoresis was carried out and eight enzyme systems were reliably scored. Standard measures of allozyme diversity were calculated. The highest genetic diversity and the highest fixation index was found in the two studied populations of P. bonjardinensis (P = 90, Â_p= 2.2 and 1.9, = 0.33 and 0.30, F = 0.31 and 0.27); this species occurs in small isolated patches, that may restrict gene flow among populations. Petunia reitzii showed the second highest genetic diversity values (P = 78, Â_p= 1.78, = 0.28), and Petunia integrifolia subsp. depauperata showed moderate values (P = 40, Â_p= 1.4 and = 0.11). Petunia exserta did not show any polymorphic locus. Our results show the urgency of preserving the studied population of P. exserta that did not show any allozyme variation and occurs in a very particular and threatened habitat.

Keywords. Allozyme; rare species; tropical plants.


Resumen. Franceschinelli, E. V.; J. R. Stehmann, C. Melo-Silva & J. N. Mesquita Neto. 2013. Estudio preliminar sobre la diversidad genética de las especies endémicas y amenazadas de Petunia (Solanaceae). Darwiniana, nueva serie 1(2): 220-226.

Las especies raras y estrictamente endémicas son más vulnerables a la extinción debido a su distribución geográfica restringida. El genero Petunia tiene 14 especies y la mayoría de ellas son endémicas de áreas restringidas. Se estudiaron cuatro taxones de Petunia que crecen en el sur de Brasil: P. bonjardinensis, P. reitzii, P. integrifolia subsp. depauperata y P. exserta. Se presenta un estudio preliminar de la diversidad genética de cinco poblaciones de Petunia utilizando marcadores de alozimas. Para los análisis de electroforesis de alozimas se llevaron a cabo ocho sistemas enzimáticos. Se calcularon las medidas estándar de diversidad. La mayor diversidad genética y también el índice de fijación más alto se encontró en las dos poblaciones estudiadas de P. bonjardinensis (P = 90, Â_p= 2.2 y 1.9, = 0.33 y 0.30, F = 0.31 y 0.27); esta especie se encuentra en pequeñas zonas aisladas entre sí, lo que puede dificultar el flujo génico interpoblacional. Petunia reitzii mostró los segundos valores más altos de diversidad genética (P = 78, Â_p= 1.78, = 0.28) y P. integrifolia mostró valores moderados (P = 40, Â_p= 1.4 y = 0.11). Petunia exserta no mostró loci polimórficos. Nuestros resultados muestran la urgencia de preservar la población estudiada de P. exserta, que no mostró ninguna variación alozímica y se encuentra en un hábitat muy particular y amenazado.

Palabras clave. Alozimas; especies raras; plantas tropicales.

INTRODUCTION

Rare and narrowly endemic species has been the main target of conservation initiatives, because they are more vulnerable to extinction due to their restricted geographical distribution. Frequently, these species occur only in specific and vulnerable habitats threatened by human activity. The dispersion pattern of organism affects the genetic diversity and gene flow among and within populations. Species with restricted geographic distribution usually have low genetic diversity (Loveless & Hamrick, 1984; Hamrick & Godt, 1989; 1996), specially compared to related species with wider geographic distribution (Gitzendanner & Soltis, 2000).
Ecological and evolutionary processes may equally interfere in the genetic diversity and structure of natural populations. Ecological factors, such as pollination and seed dispersal are important processes responsible for the gene flow within and among plant populations. Gene flow in plants may be restricted when pollen and seeds are dispersed to short distances, decreasing neighbourhood size and area (Wright, 1943; Levin & Kerster, 1974; Crawford, 1984; Franceschinelli & Kesseli, 1999).
Levels of genetic diversity may also depend on population size and plant density in the area (Barret & Kohn, 1991; Ellstram & Elam, 1993). For example, rare species with large populations may show high levels of genetic diversity (Ellstram & Elam, 1993; Ægisdóttir et al., 2009). However, endemic species with a particular microhabitat requirement would be prone to have a disjunctive distribution and thus be affected by founder effect and selective pressure (Godt et al., 1997; Franceschinelli et al., 2006). Gitzendanner & Soltis (2000) suggested that genetic data for a rare species are more informative if compared with data for a widespread related species.
The genus Petunia Juss. has 14 species that are exclusively from South America, and 13 of them are found in southern and south-eastern Brazil (Stehmann et al., 2009). Intrinsic reproductive barriers among the species of Petunia are weak and genetic isolation is a consequence mainly of geographical separation and ecological diversification (Stehmann et al., 2009). Most Petunia species are endemic, and occur in very restricted areas. They are annual or biannual herbs or shrubs, mostly pollinated by bees, and seed are dispersed close to their mother plants (Lorenz-Lemke et al., 2006). Eight endemic taxa of Petunia occur in southern Brazil (States of Santa Catarina and Rio Grande do Sul); four of them were selected for this study: P. bonjardinensis T. Ando & Hashim, P. reitzii L. B. Sm. & Downs, P. exserta Stehmann, and P. integrifolia (Hook.) Schinz & Thell. subsp. depauperata (R. E. Fr.) Stehmann. The three first species have restricted distributions.
Petunia bonjardinensis and P. reitzii occur in the State of Santa Catarina and their geographical distributions are restricted to high elevation fields of the municipalities of Bom Jardim da Serra and Bom Retiro, respectively (Lorenz-Lemke et al., 2006); both are self-incompatible and are visited by small bees (J. R. Stehmann, unpublished data). Petunia exserta is endemic of Serra do Sudeste, in the State of Rio Grande do Sul, and grows in sandstone towers in shady cracks within the rock, which is a very restricted and specific habitat (Stehmann, 1987); this species is self-compatible (Watanabe et al., 1996), has red flowers, and it is pollinated by hummingbirds (Lorenz-Lemke et al., 2006). Petunia integrifolia subsp. depauperata grows in sandy soils of beaches along the coast from Rio Grande do Sul to the island of Florianópolis, Santa Catarina (Stehmann et al., 2009), its area of distribution is larger compared to the rest of the species; this subspecies is self-incompatible and it is pollinated by bees (J. R. Stehmann, unpublished data). The natural habitats in which these four species grow are not protected by law.
All four species are herbs, with a short life cycle. They flower generally from October to December, and die after fruit production (monocarpy), except for P. integrifolia subsp. depauperata that has a thick subterraneous system (Stehmann et al., 2009). Petunia exserta can flower twice a year, in May and from September to December (Stehmann, 1987), and generally, it occurs in small patches of few individuals. Plants of P. bonjardinensis and P. reitzii occur isolated or in small patches of two to eight individuals. These patches may be considered demes or subpopulations. On the other hand, Petunia integrifolia subsp. depauperata has larger population size (from 10 to 100 plants).
We undertake a preliminary study using allozyme markers to evaluate the population genetic diversity of four Petunia species in southern Brazil.

MATERIAL AND METHODS

Studied species
For the genetic analyses, we collected leaves of two populations (20 and 12 plants of each population) for P. bonjardinensis, 40 plants of one population of P. exserta, 30 plants of one population for P. integrifolia subsp. depauperata, 20 plants of one population for P. reitzii (

Table 1

).

The small sample size is due to species rareness. The plants were numbered and tagged in the field. The location of the studied populations are shown in

Fig. 1

.

Allozyme analyses
The leaf material was preserved in liquid nitrogen (-196°C) from the moment of field collection and transported to the Universidade Federal de Minas Gerais, where it was stored at -80°C until enzyme extraction. A small piece of each leaf was grounded in liquid nitrogen and the powdered tissue was mixed with extraction buffer number 1 of Alfenas et al. (1998) and absorbed onto filter paper wicks. Allozyme electrophoresis was carried out on horizontal 13% starch-gel. Twenty enzyme systems were screened with three electrophoretic buffers. Eight enzyme systems showed simple banding patterns and could be reliably scored: shikimate dehydrogenase (SKDH, EC 1.1.1.25), 6-phosphogluconic dehydrogenase (6PG, EC 1.1.1.44), phosphoglucomutase (PGM, EC 5.4.2.2), uridine diphosphoglucose pyrophosphorylase (UGPP, EC 2.7.7.9), isocitrate dehydrogenase (IDH, EC 1.1.1.41), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), phosphoglucose isomerase (PGI, EC 5.3.1.9), and aspartate aminotransferase (GOT, EC 2.6.1.1). UGPP, 6PG, and PGM were resolved on a morpholine citrate buffer system at pH 6.5 (Alfenas et al., 1998). IDH, SKDH, and G6PDH were resolved on a histidine buffer at pH 7.0 (Alfenas et al., 1998). GOT and PGI were assayed on a lithium borate buffer at pH 8.3 (Soltis et al., 1983). Locus banding patterns were consistent with typical subunit structures. Different loci and alleles for a given system were designated sequentially, with the lowest number corresponding to the most anodally migrating locus or allele.

Data analyses
Standard measures of allozyme diversity were calculated: the percentage of polymorphic loci following the 0.99 criterion (P_0.99), the mean number of alleles per locus (Â) and per polymorphic locus (Â_p), the observed heterozygosity (), the expected heterozygosity (= 1 - ∑pi²), and Wright’s fixation index (F = 1 - / ). Significant deviations from Hardy-Weinberg expectations were verified through chi-square tests within populations. These parameters were calculated using BIOSYS-2 (original version of Swofford & Selander, 1989; modified by Black, 1997).

RESULTS

For Petunia bonjardinensis, seven enzyme systems showed simple banding patterns and were reliably scored: UGPP, PGM, IDH, SKDH, GOT (loci 1 and 2), PGI (loci 1 and 2) and G6PDH. For P. reitzii, six enzyme systems were scored: UGPP (loci 1 and 2), G6PDH, IDH, SKDH, GOT (loci 1 and 2), PGI (loci 1 and 2). Thus, nine loci were used to analyze the genetic diversity of P. bonjardinensis and P. reitzii. For P. integrifolia subsp. depauperata, seven enzyme systems were reliably scored: UGPP (loci 1 and 2), PGM, IDH, 6PG, SKDH, GOT (loci 1 and 2), PGI (loci 1 and 2). For P. exserta, seven enzyme systems were scored: UGPP (loci 1 and 2), PGM, IDH, 6PG, SKDH, GOT (loci 1 and 2), PGI (loci 1 and 2). Thus, 10 loci were used to analyze the genetic diversity of P. exserta and P. integrifolia subsp. depauperata. Significant deviations from Hardy–Weinberg expectations were detected in UGPP and PGI-2 for the P. bonjardinensis population 1, and in GOT-1 and PGI-2 for the population 2. Only one locus (UGPP) showed significant deviation from the genotypic proportion expected by the Hardy–Weinberg equilibrium for P. reitzii population. Petunia integrifolia subsp. depauperata did not show any Hardy–Weinberg equilibrium deviation.
The highest genetic diversity was found in the two populations of P. bonjardinensis ( Table 2 ). They both have a high percentage of polymorphic loci (P = 90) and a high mean number of alleles per polymorphic locus (Â_p 2.2 and 1.9). Both populations showed high values of expected heterozygosity (0.33 and 0.30) and a low value of observed heterozygosity (0.23), resulting in high but not significant fixation indices (F = 0.31 and 0.27).
The population of P. reitzii also showed high percentage of polymorphic loci (P = 78) and Â_p=1.78. The expected heterozygosity (0.28) was slightly lower than the value found for P. bonjardinensis. Petunia reitzii population showed < , and a positive but not significant fixation index (F = 0.13). The population of P. integrifolia subsp. depauperata showed only 40% of polymorphic loci and Â_p = 1.4. The expected and observed heterozygosity were low (0.11 and 0.09, respectively). The fixation index was positive but not high (F = 0.12). Petunia exserta did not show any polymorphic locus. All analysed loci showed only one allele and did not show heterozygosity.

DISCUSSION
According to Hamrick & Godt (1996), short-living plants show on average between 30 to 53.6% of polymorphic loci and 0.08–0.17 of expected heterozygosity. These authors found that endemic to regional distributed species have on average between 13.5 to 60% of polymorphic loci and 0.03 to 0.21 of expected heterozygosity. The analysed populations of Petunia bonjardinensis and P. reitzii showed higher values of genetic diversity parameters than the means found by those authors, suggesting that they may be well established species. However, both populations of P. bonjardinensis showed lower values of observed heterozygosity than of expected heterozygosity and high fixation indices. This species occur in fields located at the edges of Serra Geral in the State of Santa Catarina. The main economical activity in this area is cattle grazing, which may have contributed to the low population number and size and consequently to the high fixation indices of P. bonjardinensis. The isolated occurrence of these plants in small isolated patches may restrict gene flow among them. The main pollinators of Petunia species are bees that may fly short distances (Stehmann, 1999), making more difficult the exchange of pollen among far apart patches. Besides, Petunia species have restricted seed dispersal, which may cause genetic substructure of their populations. The studied populations of P. bonjardinensis may be going through a process of endogamy, which is most probably biparental since this species is self-incompatible. Crossings among genetically similar plants are common in small populations due to genetic drift. Apart from cattle grazing, the increments of agricultural activities, such as fruit-producing orchards, may threat these populations.
Although the distribution range of P. integrifolia subsp. depauperata is the largest among the species studied (35000 km²), the population analysed showed low values of genetic diversity parameters compared to P. bonjardinensis and P. reitzii (with a distribution area of 8000 and 6000 km², respectively). Petunia integrifolia subsp. depauperata occurs in the beaches and has larger population size. Probably, the population analysed may have undergone through a bottleneck process, followed by a recent expansion.
Petunia exserta is endemic of a very small area (about 500 km²) of the Serra do Sudeste region. It grows at a very specific habitat (Stehmann, 1987) on sandstone towers in shady cracks within the rock (shelters). Despite of being the only ornithophilous species of the genus (Lorenz-Lemke et al., 2006), it did not show any allozyme diversity. A very low allozyme diversity (or not at all) has been found in other endemic herbaceous species such as Harperocallis flava McDaniel (Godt et al., 1997) and even for long lived trees such as Pilgerodendron uviferum Florin (Premoli et al., 2001). In a review on plant allozyme literature, Hamrick & Godt (1989) found several widespread to narrowly distributed plant taxa that lacked allozyme diversity. Lorenz-Lemke et al. (2006) also found low nucleotide diversity in P. exserta and suggested that this species may have experienced a recent process of population bottleneck followed by expansion. Besides, they found low diversity within populations, which may have been caused by low gene flow between populations due to the autochoric seed dispersal system. These features and the specific habitat where this species occurs may explain the complete lack of allozyme diversity found in this species.
Our results suggest the urgency of the re-evaluation of the conservation status of Petunia exserta in order to preserve the only known population of this species. Petunia exserta was classified as threatened species by the Brazilian scientific community (http://www.biodiversitas.org.br/boletim/EAO). Moreover, the Brazilian government excluded it from the list of threatened species in the country, considering it only as a species with deficient data (Normative Instruction No. 6/2008 of the Brazilian Ministry of Environment). This species did not show any allozyme variation and probably it is not able to growth in any habitat other than the shady cracks within rocks. The transformation of the area of distribution of P. exserta in a conservation unit would also preserve other 30 endemic species (Guadagnin et al., 2000).
Petunia bonjardinensis, P. reitzii and P. integrifolia subsp. depauperata showed higher genetic diversity indices although a higher number of populations should be studied to get better conclusions on the genetic variability of these species.

ACKNOWLEDGEMENTS
We thank Igor Baldo de Castro for his help in the field. We are indebted to Giuliana M. P. Vasconcelos, Roselaini M. do Carmo, and Virginia Tenório for their assistance in the laboratory. This research was supported by grants from CNPq (N. 478756/2001-1 and N. 305589/2009-1).

BIBLIOGRAPHY

Ægisdóttir, H. H.; P. Kuss & J. Stöcklin. 2009. Isolated populations of a rare alpine plant show high genetic diversity and considerable population differentiation. Annals of Botany 104: 1313–1322.

Alfenas, A. C.; I. Peters, W. Brune & G. C. Passador. 1998. Eletroforese de proteínas e isoenzimas de fungos e essências florestais. Viçosa: Imprensa Universitária, Universidade Federal de Viçosa.

Barrett, S. C. H. & J. R. Kohn. 1991. Genetic and evolutionary consequences of small population size in plants: implications for conservation, in D. A. Falk & R. K. E. Holsinger (eds.), Genetic and conservation of rare plants. New York: Oxford University Press.

Black, W. C. 1997. BIOSYS-2: A computer program for the analysis of allelic variation in population genetics and biochemical systematics, release 1.7. Illinois: Illinois History Survey.

Crawford, T. J. 1984. What is a population?, in B. Shorrocks (ed.) Evolutionary Ecology. Oxford: Blackwell.

Ellstram, N. C. & D. R. Elam. 1993. Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and Systematics 24: 217–242.

Franceschinelli, E. V. & R. Kesseli. 1999. Population structure and gene flow of the Brazilian shrub Helicteres brevispira. Heredity 82: 355-364.

Franceschinelli, E. V.; C. M. Jacobi, M. G. Drummond & M. F. S. Resende. 2006. The genetic diversity of two Brazilian Vellozia (Velloziaceae) with different patterns of spatial distribution and pollination biology. Annals of Botany 97: 585–592. 

Gitzendanner, M. A. & P. S. Soltis. 2000. Patterns of genetic variation in rare and widespread plant congeners. American Journal of Botany 87: 783–792.

Godt, M. J. W.; J. Walker & J. L. Hamrick. 1997. Genetic diversity in the endangered lily Harperocallis flava and a close relative, Tofieldia racemosa. Conservation Biology 11: 361–366.

Guadagnin, D. L.; J. Larocca & M. Sobral. 2000. Flora vascular de interesse para a conservação na bacia do arroio João Dias: avaliação ecológica rápida, in L. H. Ronchi & A. O. C. Lobato (eds.), Minas Do Camaquã. Um Estudo Multidisciplinar. Pag. 71–84. São Leopoldo: Editora Unisinos.

Hamrick, J. L. & M. J. W. Godt. 1989. Allozyme diversity in plant species, in A. H. D. Brown, M. T. Clegg, A. L. Kahler & B. S. Weir (eds.) Plant Population Genetics, Breeding and Genetic Resources. Sunderland: Sinauer Press.

Hamrick, J. L. & M. J. W. Godt. 1996. Effects of life history traits on genetic diversity in plant species. Philosophical Transactions of the Royal Society of London Biological Sciences 351: 1291–1298.

Levin, D. & H. W. Kerster. 1974. Gene flow in seed plants. Evolutionary Biology 7: 139–220.

Lorenz-Lemke, A. P.; G. Mäder, V. C. Muschner, J. R. Stehmann, S. L. Bonatto, F. M. Salzano & L. B. Freitas. 2006. Diversity and natural hybridization in a highly endemic species of Petunia (Solanaceae): a molecular and ecological analysis. Molecular Ecology 15: 4487–4497.

Loveless, M. D. & J. L. Hamrick. 1984. Ecological determinants of genetic structure in plant populations. Annual Review of Ecology and Systematics 15: 65–95.

Premoli, A. C.; C. Souto, T. A. Unutt & A. C. Newton. 2001. Effects of population disjunction on isozyme variation in the widespread Pilgerodendron uviferum. Heredity 87: 337–343.

Soltis, D. E.; C. H. Haufler, D. C. Darrow & G. J. Gastony. 1983. Starch gel electrophoresis of ferns: a compilation of grinding buffers, gel and electrode buffer, and staining schedules. American Fern Journal 73: 9–27.

Stehmann, J. R. 1987. Petunia exserta (Solanaceae): uma nova espécie do Rio Grande do Sul, Brasil. Napaea 2: 19–21.

Stehmann, J. R. 1999. Estudos taxonômicos na tribo Nicotianeae (Solanaceae): revisão de Petunia Jussieu, das espécies brasileiras de Calibrachoa La Llave & Lexarza e o estabelecimento do novo gênero Petuniopsis Stehmann & Semir. Tese de doutorado, Universidade Estadual de Campinas, Campinas.

Stehmann, J. R.; J. Semir, A. P. Lorenz-Lemke & L. B. Freitas. 2009. The genus Petunia, in T. Gerats & J. Strommer (eds.), Petunia: Evolutionary, Developmental and Physiological Genetics. New York: Springer Science, Business Media.

Swofford, D. L. & R. B. Selander. 1989. Biosys-1: A computer program for the analysis of genetic variation in population genetic and biochemical systematics, Version 1.7. Champaign: Illinois Natural History survey.

Watanabe, H.; T. Ando, S. I. Iida, A. Suzuki, K. I. Buto, T. Tsukamoto, G. Hashimoto & E. Marchesi. 1996. Cross compatibility of Petunia cultivars and P. axillaris with native taxa of Petunia in relation to their chromossome number. Journal of the Japanese Society for Horticultural Science 65: 625–634.

Wright, S. 1943. Isolation by distance. Genetics 28: 114–138.