Contrasting genetic diversity and differentiation of populations of two successional stages in a neotropical pioneer tree (Eremanthus erythropappus, Asteraceae)

Eremanthus erythropappus, commonly known as “candeia”, is an abundant pioneer tree species, forming dense populations known as “candeial”, but it is also found in forests at middle stages of succession. Trees from forests are bigger and occur in lower density than in the “candeial”. The objectives of the present study were to investigate if the decrease in population density during successional process is accompanied by 1) changes in within-population genetic diversity, and 2) differentiation of populations. Eight populations, four of early successional stage (“candeial”) and four of middle successional stages (forest), were analyzed with RAPD markers. The genetic diversity found was high compared to other tree species analyzed with RAPD markers. AMOVA revealed that most of the genetic variations of E. erythropappus were found within populations (85.7%), suggesting that this species is predominantly outcrossing. The relatively low differentiation among the populations can be attributed to small distances among the populations analyzed (0.2 to 10.8 km). No indication that populations from middle successional habitats show lower genetic variation than populations from early successional stages was found. The percentage of polymorphic fragments (82.8 and 84.8%) and the Shannon indexes (0.442 and 0.455) were similar in “candeial” and forest, respectively. These results suggest that if an increase in selection intensity occurred during succession, it did not result in a decrease in genetic diversity or that the selection effect was balanced by other factors, such as gene flow. Higher significant differentiation among E. erythropappus populations from “candeial” in relation to that among populations from forest was also not detected.

Comparative analysis of different DNA extraction protocols in fresh and herbarium specimens of the genus Dalbergia

Five published DNA extraction protocols were compared for their ability to produce good quality DNA from fresh and herbarium leaves of several species of the genus Dalbergia. The leaves of these species contain high amounts of secondary metabolites, which make it difficult to perform a clean DNA extraction and thereby interfering with subsequent PCR amplification. The protocol that produced the best DNA quality in most of the Dalbergia species analyzed, utilizes polyvinylpyrrolidone to bind the phenolic compounds, a high molar concentration of NaCl to inhibit co-precipitation of polysaccharides and DNA, and LiCl for removing RNA by selective precipitation. The DNA quality of herbarium specimens was worse than that for fresh leaves, due to collecting conditions and preservation of samples. We analyzed 54 herbarium specimens, but the recovered DNA allowed successful PCR amplification in only eight. For the genus Dalbergia, the herbarium is an important source of material for phylogenetic and evolutionary studies; due to the occurrence of the different species in various geographical regions in Brazil, it is difficult to obtain fresh material in nature. Our results demonstrated that for Dalbergia species the methods used for the collection and preservation of herbarium specimens have a mayor influence on DNA quality and in the success of phylogenetic studies of the species

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.