Fabiano Salgueiro

Optimal sampling strategy for estimation of spatial genetic structure in tree populations

Fine-scale spatial genetic structure (SGS) in natural tree populations is largely a result of restricted pollen and seed dispersal. Understanding the link between limitations to dispersal in gene vectors and SGS is of key interest to biologists and the availability of highly variable molecular markers has facilitated fine-scale analysis of populations. However, estimation of SGS may depend strongly on the type of genetic marker and sampling strategy (of both loci and individuals). To explore sampling limits, we created a model population with simulated distributions of dominant and codominant alleles, resulting from natural regeneration with restricted gene flow. SGS estimates from subsamples (simulating collection and analysis with amplified fragment length polymorphism (AFLP) and microsatellite markers) were correlated with the ‘real’ estimate (from the full model population). For both marker types, sampling ranges were evident, with lower limits below which estimation was poorly correlated and upper limits above which sampling became inefficient. Lower limits (correlation of 0.9) were 100 individuals, 10 loci for microsatellites and 150 individuals, 100 loci for AFLPs. Upper limits were 200 individuals, five loci for microsatellites and 200 individuals, 100 loci for AFLPs. The limits indicated by simulation were compared with data sets from real species. Instances where sampling effort had been either insufficient or inefficient were identified. The model results should form practical boundaries for studies aiming to detect SGS. However, greater sample sizes will be required in cases where SGS is weaker than for our simulated population, for example, in species with effective pollen/seed dispersal mechanisms.

Monitoring genetic diversity in tropical trees with multilocus dominant markers

Since no universal codominant markers are currently available, dominant genetic markers, such as amplified fragment length polymorphism (AFLP), are valuable tools for assessing genetic diversity in tropical trees. However, the measurement of genetic diversity (H) with dominant markers depends on the frequency of null homozygotes (Q) and the fixation index (F) of populations. While Q can be estimated for AFLP loci, F is less accessible. Through a modelling approach, we show that the monolocus estimation of genetic diversity is strongly dependent on the value of F, but that the multilocus diversity estimate is surprisingly robust to variations in F. The robustness of the estimate is due to a mechanistic effect of compensation between negative and positive biases of H by different AFLP loci exhibiting contrasting frequency profiles of Q. The robustness was tested across contrasting theoretical frequency profiles of Q and verified for 10 neotropical species. Practical recommendations for the implementation of this analytical method are given for genetic surveys in tropical trees, where such markers are widely applied.

Morphology of Gambierdiscus excentricus (Dinophyceae) with emphasis on sulcal plates

Abstract: Gambierdiscus excentricus is an epibenthic dinoflagellate able to produce ciguatoxin and maitotoxin-like compounds that are responsible for ciguatera fish poisoning. Morphological descriptions and molecular characterization of two G. excentricus strains isolated from Brazil and maintained in culture were provided. The most complete description of the morphology of the sulcal region of Gambierdiscus based on light and scanning electron microscopy was presented. The sulcal area morphology and nomenclature used by different authors to name the sulcal plates in Gambierdiscus were reviewed. Two small sulcal plates (S.m.a. and S.m.p.) were shown for the first time. Phylogenetic trees based on D1–D3 and D8–D10 large subunits of ribosomal RNA gene sequences showed that the strains of G. excentricus from Brazil clustered with strains of G. excentricus isolated from its type locality, the Canary Islands. Both phylogenetic trees reconstructed the same relationships among all the formally described Gambierdiscus species and Gambierdiscus sp. ribotype 2 and Gambierdiscus sp. type 2.

Prorocentrum lima from the South Atlantic: Morphological, molecular and toxicological characterization

Morphological descriptions using light and scanning electron microscopy and molecular characterization of two Prorocentrum lima strains (UNR-01 and UNR-09) isolated from Armação dos Búzios, Rio de Janeiro, Brazil are provided. Okadaic acid (OA), dinophysistoxin-1 (DTX1) and DTX2 production by strain UNR-01 was investigated using liquid chromatography with mass spectrometry. Toxins were extracted from heat-treated (boiled) and non-boiled cell pellets to obtain respective quantities of free and total OA and DTX1. Growth parameters (growth rate and mean generation time) were determined for strain UNR-01. Prorocentrum lima cells were oblong-to-ovate in shape, broad in the middle region, and narrow at the anterior end. The periflagellar area was triangular, set into a V-shaped depression and was composed of eight periflagellar platelets of different sizes. The morphology fits well the characterization of the species isolated from elsewhere. Phylogenetic analysis based on internal transcribed spacer - ITS - and D1-D3 large subunit - LSU - of ribosomal RNA gene sequences revealed that both strains were identical and closely related to P. lima isolates from the Caribbean Sea and USA. The growth rate of strain UNR-01 was 0.24divday-1. OA concentrations were on average 15.2 and 38.5pg[OA]cell-1 for heat-treated and non-treated cells respectively, while DTX1 mean concentration was 0.5pg[DTX1]cell-1 for both heat-treated and non-treated cells. DTX2 was not detected. To date, these are the first strains of P. lima from the south Atlantic that have been characterized.

Molecular evidence of the presence of Dictyota dichotoma (Dictyotales: Phaeophyceae) in Argentina based on sequences from mtDNA and cpDNA and a discussion of its possible origin

ABSTRACT Dictyota is a brown algae genus inhabiting tropical to warm temperate environments where it is an important food source, shelter and substrate to several species of invertebrates and other algae. The taxonomy of this genus is troublesome, with poor species delimitation and doubtful records in the literature. Dictyota dichotoma, the type species of the genus, was commonly accepted as a cosmopolitan species because of the inaccurate geographical distributional range as a consequence of misidentification in several parts of the world. Recent studies with molecular data revealed new species and a new understanding of the evolution and biogeography of the genus Dictyota. These studies confined natural populations of D. dichotoma to Europe (Atlantic and Mediterranean coasts) and Macaronesian islands (Azores, Madeira and Canary archipelagos). Also, they confirmed its presence in South Africa, but whether the species was native or introduced in South Africa could not be verified. In the present study two regions of cpDNA (psbA, rbcL) and one region of mtDNA (nad1) from Argentinian samples, identified morphologically as D. dichotoma, were analysed and compared to other Dictyota species. The identity of these samples as D. dichotoma was confirmed. A haplotype network analysis using all available psbA sequences distinguished seven haplotypes divided into two geographic groups: Atlantic–Mediterranean and Canarian. In Argentina and South Africa only the most common haplotype of the Atlantic–Mediterranean group was observed. According to the paleoceanographic currents and the presence of a European haplotype, the introduced nature of D. dichotoma is indicated.

Phylogeography of the disjunct Schizolobium parahyba (Fabaceae-Caesalpinioideae)

This work aims to analyze the phylogeography of Schizolobium parahyba(Fabaceae), which includes two varieties with a disjunct distribution, from Southern Brazil to Central America. Neotropical rain forests, focus on four largest wet forests: Atlantic forest, Amazonian forests, Andean forest and Central America forest. The genetic diversity and differentiation of populations among S. parahyba populations using sequences of three cpDNA regions (psbA-trnH, trnL-trnFandmatK) and one nrDNA region (ITS) were analyzed. The presence of the significant phylogeographic structure was inferred by testing if GST and NST were significantly different and a spatial analysis of molecular variance was made with both markers.Using cpDNA (matK) sequences of the S. parahyba and other Fabaceae species and fossils, we estimated de divergent time for Schizolobium clade and using the average ITS substitution rate reported for a range of woody plants, w estimated the divergence time between the two varieties. The high levels of genetic diversity in some populations of S. parahyba and two centres of genetic diversity that correlate with the two known varieties: one in the southeast Atlantic forest, and the other in the Amazonian basin. In contrast, the populations from Northeast Atlantic forest and Andean-Central America forests showed low level of genetic diversity and divergent haplotypes, probably because the founder effect after dispersion.The results suggest dispersion from southeast Atlantic forest to Amazonian, Andean and Central America forests. We verifya high level of genetic structure, with 68% (nrDNA) and 82% (cpDNA) of the total genetic diversity due to differences among populations. Twenty-one haplotypes were found with cpDNA and four with nrDNA and no haplotypes were shared between varieties.The age for Schizolobiumclade using matK sequences was estimated ranging from 8.4-23.0 My and the divergence between two varieties using ITS sequence variation was of the 6.5 My. In conclusion, the variation pattern of cpDNA (maternally inherited) and nrDNA (biparentally inherited) markers provides different insights into the phylogeographic structure and gene flow in S. parahyba. This comparative analysis of cpDNA and nrDNA markerscan help a deeper understanding of the dynamics responsible for both ancient and more recent events that have shaped the current distribution of genetic variability in Neotropical plants. Theresults are relevant to conservation efforts and ongoing work on the genetics of population divergence and speciation in these Neotropical rainforests. Also, for the long-term conservation of the genetic diversity of S. parahyba, including the divergent lineages of the two varieties, it would be important to design strategies that aim to preserve most of its lineages.

Phylogeographic patterns of Calophyllum Braziliense Camb. (Calophyllaceae)based on the psbA-trnH cpDNA locus

Background Past climate changes have severely influenced the current distribution of species and their genetic diversity. Phylogeography is the study of the principles and processes governing the geographic distributions of genealogical lineages [1]. South America has the world’s largest area of swamps, floodplains and wetlands in general [2]. Brazil’s major wetlands cover 2% of the country’s huge territory [3]. However, from a plant ecology standpoint, Brazilian freshwater wetlands are largely unknown and the scarce data available refer mostly to flooded forests of the Amazon [4]. Calophyllum Braziliense Camb. (Calophyllaceae), also know as guanandi, jacareuba or landim, is a canopy tree species typical of waterlogged areas from South and Central America. This species occurs in the humid tropical forests of Central America, Amazon Forests, Atlantic Forest (including restingas); and in the riverines forests of the Cerrado biome (brazilian savannah) [5,6]. However, unlike other species typical of flooded areas, C. Braziliense shows none of the morphological features common to flood-adapted plants. Calophyllum Braziliense is a hermaphroditic tree pollinated by bees. Its seeds are animal (mainly by bats) or water dispersed. The timber has excellent characteristics and is widely used. Calophyllum. Braziliense is also used in vegetation restoration programs and its leaves extract presents antiinflammatory activity [7]. This study examines the phylogeographic patterns of C. Braziliense based on the cpDNA intergenic region psbA-trnH. Methods Twenty four populations of C. Braziliense were sampled from Costa Rica (10° 12’N, 83° 47’W) to the Parana State in Brazil (25° 34’S, 48° 27’W). Samples were collected from about 5-10 adult trees in each population, totaling 192 individuals. Total DNA was extracted from leaves or cambium using the CTAB procedure described by Doyle & Doyle [8]. After a screening for cpDNA amplification and polymorphism in C. Braziliense, the psbA-trnH intergenic regions was selected [9]. Sequences were aligned using CLUSTAL-W implemented in the MEGA 4 software. The cpDNA haplotypes were defined by analyzing the sequences with DNASP 4.01. The genetic diversity indexes were estimated in ARLEQUIN 3.01. The phylogenetic relationships among the haplotypes were estimated using the median-joining algorithm implemented in NETWORK 4.1. An analysis of molecular variance (AMOVA) was performed in ARLEQUIN. A spatial analysis of molecular variance (SAMOVA) was conduced using the SAMOVA 1.0 software. To evaluate the hypothesis of population expansion, neutrality tests were computed in DNASP and ARLEQUIN.

mtDNA analysis of Mytilopsis (Bivalvia, Dreissenidae) invasion in Brazil reveals the existence of two species

The genus Mytilopsis includes some of the most invasive estuarine bivalves in the world, and M. leucophaeata (native to the Gulf of Mexico) and M. sallei (indigenous in the Caribbean Sea) are the most often reported species. Molecular investigations of the COI gene in invasive populations of M. leucophaeata in Europe found only one haplotype, whereas the invasive M. sallei in Asia had high haplotype diversity. The present study investigated COI variability of invasive populations previously identified as M. leucophaeata in Brazil, located at the cities of Recife and Rio de Janeiro. Our genetic analysis revealed that the record of M. leucophaeata from Recife appears erroneous; this population forms a clade adjacent to but genetically separated from the Asian M. cf. sallei. Similarly, samples from Rio de Janeiro clustered close to but not within European/North American sequences of M. leucophaeata. The Automatic Barcode Gap Discovery test suggested the existence of three species: M. leucophaeata, M. cf. sallei (Asia), and M. cf. sallei (Recife), which merit confirmation with nuclear DNA sequences. The discovery of two lineages of Mytilopsis in Brazil augments concerns about their expansion.

Chloroplast DNA variation and phylogeography of Eugenia uniflora L. (Myrtaceae) in the Brazilian Atlantic forest

The Atlantic Forest (AF) is considered the second largest tropical forest in South America with high species richness and endemisms, harboring a large diversity of animals, plants and habitats types [1]. This biome covers an area of more than one million square kilometres along the Brazilian coast and extending to eastern Paraguay and northeastern Argentina [2]. AF is considered one of the most threatened ecosystems on Earth due to intense disturbance, having been reduced to only 7.5% of its original area [1]. Despite an increase in research efforts in the past few years, studies of AF species diversification and knowledge about its evolutionary history is still scarce. Vegetation changes in the Atlantic Forest related to climatic changes during the Pleistocene have been registered in paleopalinological studies, with the replacement of large areas of forests by subtropical grasslands and savannas during cooler and drier conditions [3,4]. Also, studies of paleoclimatic models, predicted the presence of historically stable areas (refugia) in the Atlantic Forest during the Late Quaternary [5]. Eugenia uniflora L. (Myrtaceae), a shrubby tree with edible cherry-like fruits which is locally known as pitanga or Brazilian cherry. This species is one of the key species in the Atlantic rain forest geomorphological domain, which includes the Atlantic forest and the adjacent Restinga ecosystem [6]. E. unifloraoccurs in areas of medium and large levels of rainfall and can also be found in different vegetation types and ecosystems. This species present economic and folk medicinal applications and is an important pioneer species in the Restinga ecosystem and has been used to recover and manage disturbed and fragmented areas. Our aim in this study was to investigate the phylogeography and genetic diversity of Eugenia uniflora to help elucidate the evolutionary history of this species as a model for gain insights into past vegetation patterns in the Brazilian Atlantic Forest.