Population genetic structure in Phyla scaberrima from Mexico and Colombia assessed by AFLP markers and implications for conservation.

Phyla scaberrima (Verbenaceae) is a herbaceous perennial species that is distributed from Mexico (center of origin) to Colombia, growing in forest and swamp edges or grasslands from sea level up to an altitude of 1800 m. The chemical properties and uses in popular medicine have drastically affected the population size of this species. In this study, we investigated genetic variability in populations of P. scaberrima using AFLP markers. Three AFLP primer combinations rendered a total of 997 markers in a sample of 131 individuals from five populations, including two populations from Mexico and three from Colombia. The average percentage of polymorphic loci, gene diversity and Shannon-Wiener index were 46.62, 0.0695, and 0.119, respectively. Analysis of molecular variance showed that the distribution of the genetic variability within populations (85.41%) was higher than between groups (8.11%) and between populations (6.48%). Principal coordinate analysis and Bayesian analysis for the K number of clusters showed that the individuals were dispersed in five (K= 5) clusters. The low levels of genetic diversity observed in these populations demonstrated that the populations from Mexico and Colombia need urgent management to recover their genetic variability.

Isolation and characterization of simple sequence repeat markers for the herbaceous species Phyla scaberrima (Verbenaceae).

Phyla scaberrima (Verbenaceae) is a herbaceous species distributed from Mexico to Panama. Because of its well-known sweet properties and other medicinal uses, this species is cultivated in South America and the Caribbean. Phyla scaberrima has been arbitrarily extracted from nature, resulting in a severe reduction in its gene pool. In this study, we developed and characterized 11 simple sequence repeat markers for P. scaberrima to determine the genetic variability and patterns of population structure of the species. Fifty-six alleles were detected in a sample of 48 individuals belonging to 3 different populations. The average number of alleles per locus was 5.09, while the polymorphic information content ranged from 0.000-0.587. The observed and expected heterozygosities varied from 0.000-0.543 and from 0.000-0.651, respectively. Two loci exhibited significant deviation of the expected Hardy-Weinberg proportion. The 11 primer pairs were also tested for cross-amplification to 6 species of the related genus Lippia. The transferability rate ranged from 4 loci in Lippia florida and L. rotundifolia to 6 loci in L. corymbosa and L. microcephala. The 11 primer sets were shown to be valuable tools for population genetic studies in P. scaberrima and in species of the genus Lippia in which primer transferability was detected.

Development and characterization of 14 microsatellite loci in the Neotropical fish Geophagus brasiliensis (Perciformes, Cichlidae).

Fourteen polymorphic microsatellite loci were isolated and characterized for the Neotropical cichlid Geophagus brasiliensis and tested on 30 individuals belonging to a single population. Among the 14 loci described, four showed potential presence of null alleles, inferred from the excess of homozygous genotypes, and three of these loci showed significant deviations from Hardy-Weinberg equilibrium. Fifty-nine different alleles were detected (ranging from two to eight alleles per locus), with estimates of observed and expected heterozygosity ranging from 0·167 to 0·700 and from 0·269 to 0·825. Cross-amplification of primers was successful in five other cichlid species.

Isolation and characterization of 8 microsatellite loci from Chrysophyllum gonocarpum (Sapotaceae).

Chrysophyllum gonocarpum is a tropical tree species that is very important in the recovery of heterogeneous forests and of degraded areas of permanent preservation. We identified microsatellite loci for C. gonocarpum to assess the genetic variability and the patterns of the population structure of the species. We isolated 8 microsatellite primers by using CT- and GT-enriched genomic libraries. We detected 2-4 alleles with 2.9 alleles per locus on average, by polymerase chain reaction. Test for cross-amplification showed that some loci were successfully amplified in 2 other Chrysophyllum species. The microsatellites can be used to assess the genetic diversity and population structure of C. gonocarpum. Some primer pairs can be amplified in C. marginatum and C. splendens.

Karyotype studies on populations of two Hypochaeris species (H. catharinensis and H. lutea), Asteraceae, endemics to southern Brazil.

Hypochaeris is an excellent system for studying different modes of chromosome evolution in plants. We carried out a cytogenetic analysis on populations of 2 Hypochaeris species, comprising 10 populations of H. catharinensis and 5 of H. lutea, to assess possible changes on chromosome organization in this interesting genus. Conventional Feulgen staining and fluorescent banding revealed that the general aspects of chromosome morphology for all populations of both species were similar, evidence of the typical bimodal karyotypes with 2n = 8 chromosomes that characterize the South American Hypochaeris. Comparative analysis of the karyotypes identified minor variations in the absolute size and arm ratio of corresponding chromosome pairs. One population of H. lutea was entirely polyploid adding a novel cytotype to this species. Fluorescent banding revealed strong chromomycin A3 (CMA3)-positive signals on both arms of chromosomes 3 and 4 of H. catharinensis, revealing a new pattern for the distribution of GC-rich heterochromatin in Hypochaeris. A strong CMA-positive signal was observed on the short arm of chromosome 3 in one population of H. lutea, while the other populations validated the CMA3 pattern already described for this species. While the overall karyotype similarities of the 2 species are in compass with all South American Hypochaeris, the presence of unusual large blocks of GC-rich heterochromatin suggests that chromosome rearrangements, related to dispersion of heterochromatin, are taking place in the karyotype of H. catharinensis. The novel polyploid cytotype identified in H. lutea provides support that polyploidization is an active process in the mode of chromosome evolution in Hypochaeris.

Characterization of nine microsatellite loci for the tree species Parapiptadenia rigida (Fabaceae-Mimosoideae) and their transferability.

Parapiptadenia rigida, locally known as angico, is a tropical tree common in the semideciduous Brazilian forest. Its wood is naturally resistant to insect attack and is useful for construction. Extracts from the tree have medicinal properties. We characterized nine microsatellite loci for P. rigida. Thirty-five alleles were detected in a sample of 45 individuals from 3 different populations, with an average of 3.9 alleles per locus. The average polymorphic information content ranged from 0.099 to 0.640. Observed and expected heterozygosities varied from 0.111 to 0.489 and from 0.106 to 0.707, respectively. One locus exhibited significant deviation from Hardy-Weinberg equilibrium and four pairs of loci showed significant linkage disequilibrium. All nine primers were tested for cross-amplification in species from the Fabaceae-Mimosoidea family, yielding a transferability success rate of 7 loci in Stryphnodendron adstringens to 0 transferred loci in Pithecellobium incuriale and Inga marginata. These microsatellites will be valuable to study population genetics of this and other species where primer transferability was detected.

Population genetic structure of the tropical tree species Aegiphila sellowiana (Lamiaceae).

The Tibagi River, located in southern Brazil, is associated with a significant degree of environmental heterogeneity, along its 550 km extension. There is concern about the integrity of this river's ecosystem, as human interference has been increasing. Aegiphila sellowiana (Lamiaceae) is an important pioneer tree species, commonly found near rivers; the fruit is consumed by avifauna. We studied this species along three ecological gradients, comprising the upper, middle, and lower regions of the Tibagi River basin. The genetic structure of nine subpopulations of A. sellowiana distributed along these gradients was investigated using RAPDs. Moderate levels of gene diversity (ranging from 0.091 to 0.132) were identified, inferred by a traditional approach and a Bayesian model-based method. The F-statistic, G(ST) parameters and molecular variance analysis showed high genetic differentiation among the three regions (39.5 to 50.26%). Analysis of molecular variance revealed high levels of genetic variation between populations (50.26%), while lower values of genetic variation (ranging from 9.56 to 16.35%) were seen between subpopulations within the upper, middle, and lower regions of the Tibagi River basin. The validity of these results was confirmed by principal coordinate analysis. Linear regression analysis showed significant correlations (r = 0.621, P = 0.0001) between the genetic and geographical distances. The differences observed in genetic variation between regions are probably due to habitat fragmentation; for conservation purposes, we recommend that at least one subpopulation from each region of the Tibagi River should be maintained.

Genetic differentiation in Aspidosperma polyneuron (Apocynaceae) over a short geographic distance as assessed by AFLP markers.

Studies on intraspecific variation can contribute to the development of conservation strategies by identifying units of conservation for threatened species. Aspidosperma polyneuron is a tropical tree of seasonal semideciduous forests that is currently endangered and protected because it has been heavily logged for lumber, although it was once common in Brazil and neighboring countries. We investigated genetic structure in two samples of A. polyneuron collected from steep hillsides and from flat areas of a natural forest fragment in northern Paraná State, Brazil. Seven AFLP primer combinations yielded 200 markers, with a polymorphic rate of 88.5% for samples from the flat area and 99% for samples from the high declivity area. Total genetic diversity (H(T)) was 0.387, while the genetic diversity within the populations (H(S)) was 0.307 and 0.372, for samples from the flat and the high declivity areas, respectively. Genetic differentiation between samples was high, with a mean F(ST) of 0.265 and a genetic distance of 0.148, indicative of a high degree of genetic structure over a short distance. Principal coordinate analysis separated the samples into three groups of individuals; the first group included individuals from the high declivity area, the second group consisted of individuals only from the flat area, and the third group had individuals from both areas. Bayesian analysis also showed K = 3 clusters. The unexpected high level of intraspecific variation of A. polyneuron in this small forest fragment should be taken into account when evaluating the genetic impact of forest degradation on this species in other semideciduous forest fragments.

Genetic diversity and flooding survival in Aegiphila sellowiana (Lamiaceae), a typical tree species from upland riparian forests.

Saplings of Aegiphila sellowiana were submitted to flooding and analysis of genetic diversity in order to investigate flooding tolerance as well as its genetic determination. This response is important because it means that some lines could be planted in degraded riparian areas. Leaves were sampled from each plant, and they were submitted to different flooding periods. Mortality of saplings was 40, 80, 50, 53.3, 33.3, and 33.3% in flooding for 15, 18, 25, 50, 80 days, and flooding for 50 days followed by re-aeration for 30 days, respectively. From the total number of flooded plants, 46.7% died in the first seven days of treatment, while 53.3% survived the flooding. The percentage of polymorphic loci (P(p)), Nei's genetic diversity (H) and the Shannon index (I) were slightly higher for the group that survived the stress of flooding (surviving: P(p) (%) = 67.48, H = 0.184, I = 0.287; not surviving: P(p) (%) = 66.67, H = 0.165, I = 0.261). Analysis of molecular variance showed that 5.88% of the genetic variability was due to the differences between groups of plants surviving and not surviving flooding, while 94.12% was due to genetic differences between individuals within these groups. Similar results were obtained by principal coordinate analysis. Based on these results, we can assume the existence of environment-specific genotypes and the genetic determination of flooding tolerance in A. sellowiana. Thus, some lines of A. sellowiana could be used in the reforestation of riparian habitats, especially in uplands along riverbanks.

Anatomy and genetic diversity of two populations of Schinus terebinthifolius (Anacardiaceae) from the Tibagi River basin in Paraná, Brazil.

Knowledge of the effects of flooding on plant survival is relevant for the efficiency of management and conservation programs. Schinus terebinthifolius is a tree of economic and ecological importance that is common in northeast Brazil. Flooding tolerance and genetic variation were investigated in two riparian populations of S. terebinthifolius distributed along two different ecological regions of the Tibagi River basin. Flooding tolerance was evaluated through the investigation of young plants, submitted to different flooding intensities to examine the morphological and anatomical responses to this stress. The growth rate of S. terebinthifolius was not affected by flooding, but total submersion proved to be lethal for 100% of the plants. Morphological alterations such as hypertrophied lenticels were observed in both populations and lenticel openings were significantly higher in plants from one population. Genetic analysis using DNA samples obtained from both populations showed a moderate degree of genetic variation between populations (13.7%); most of the variation was found within populations (86.3%). These results show that for conservation purposes and management of degraded areas, both populations should be preserved and could be used in programs that intend to recompose riparian forests.

Evidence of ecotypic differentiation between populations of the tree species Parapiptadenia rigida due to flooding.

The tree species Parapiptadenia rigida, native to southern South America, is frequently used in reforestation of riverbanks in Brazil. This tree is also a source of gums, tannins and essential oils, and it has some medicinal uses. We investigated flooding tolerance and genetic diversity in two populations of P. rigida; one of them was naturally exposed to flooding. Plants derived from seeds collected from each population were submitted to variable periods of experimental waterlogging and submergence. Waterlogging promoted a decrease in biomass and structural adjustments, such as superficial roots with aerenchyma and hypertrophied lenticels, that contribute to increase atmospheric oxygen intake. Plants that were submerged had an even greater reduction in biomass and a high mortality rate (40%). The two populations varied significantly in their RAPD marker profiles, in their ability to produce aerenchyma when waterlogged and to survive when submerged, suggesting ecotypic differentiation between them. Hence, the seasonal flooding that has been challenging the tropical riparian forest appears to be genetically modifying the P. rigida populations exposed to it by selecting individuals with increased ability to live under this condition.

Phylogeography of the invasive weed Hypochaeris radicata (Asteraceae): from Moroccan origin to worldwide introduced populations.

In an attempt to delineate the area of origin and migratory expansion of the highly successful invasive weedy species Hypochaeris radicata, we analysed amplified fragment length polymorphisms from samples taken from 44 populations. Population sampling focused on the central and western Mediterranean area, but also included sites from Northern Spain, Western and Central Europe, Southeast Asia and South America. The six primer combinations applied to 213 individuals generated a total of 517 fragments of which 513 (99.2%) were polymorphic. The neighbour-joining tree presented five clusters and these divisions were supported by the results of Bayesian analyses: plants in the Moroccan, Betic Sierras (Southern Spain), and central Mediterranean clusters are all heterocarpic. The north and central Spanish, southwestern Sierra Morena, and Central European, Asian and South American cluster contain both heterocarpic (southwestern Sierra Morena) and homocarpic populations (all other populations). The Doñana cluster includes two homocarpic populations. Analyses of fragment parameters indicate that the oldest populations of H. radicata are located in Morocco and that the species expanded from this area in the Late Quaternary via at least three migratory routes, the earliest of which seems to have been to the southwestern Iberian Peninsula, with subsequent colonizations to the central Mediterranean area and the Betic Sierras. Homocarpic populations originated in the southwestern Iberian Peninsula and subsequently spread across north and central Spain, Central Europe and worldwide, where they became a highly successful weed.