Alison G. Nazareno

Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome

Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq.) L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp) with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp) and a small single copy region (SSC, 17,586 bp) separated by inverted repeat regions (IRs, 25,789 bp). In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs) with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering plants is complex. The discovery of tandem repeats within T. tetragonolobum and the presence of divergent regions between chloroplast genomes of Lamiales provides the basis for the development of markers at various taxonomic levels. The newly developed markers have the potential to greatly improve the resolution of molecular phylogenies.

Transferability and characterization of microsatellite markers in two Neotropical Ficus species

Microsatellite markers were transferred and characterized for two Neotropical fig tree species, Ficus citrifolia and Ficus eximia. Our study demonstrated that microsatellite markers developed from different subgenera of Ficus can be transferred to related species. In the present case, 12 of the 15 primer pairs tested (80%) were successfully transferred to both of the above species. Eleven loci were polymorphic when tested across 60 F. citrifolia and 60 F. eximia individuals. For F. citrifolia, there were 4 to 15 alleles per locus, whereas expected heterozygosities ranged from 0.31 to 0.91. In the case of F. eximia, this was 2 to 12 alleles per locus and expected heterozygosities from 0.42 to 0.87.

Minimum sample sizes for population genomics: an empirical study from an Amazonian plant species

High‐throughput DNA sequencing facilitates the analysis of large portions of the genome in nonmodel organisms, ensuring high accuracy of population genetic parameters. However, empirical studies evaluating the appropriate sample size for these kinds of studies are still scarce. In this study, we use double‐digest restriction‐associated DNA sequencing (ddRADseq) to recover thousands of single nucleotide polymorphisms (SNPs) for two physically isolated populations of Amphirrhox longifolia (Violaceae), a nonmodel plant species for which no reference genome is available. We used resampling techniques to construct simulated populations with a random subset of individuals and SNPs to determine how many individuals and biallelic markers should be sampled for accurate estimates of intra‐ and interpopulation genetic diversity. We identified 3646 and 4900 polymorphic SNPs for the two populations of A. longifolia, respectively. Our simulations show that, overall, a sample size greater than eight individuals has little impact on estimates of genetic diversity within A. longifolia populations, when 1000 SNPs or higher are used. Our results also show that even at a very small sample size (i.e. two individuals), accurate estimates of FST can be obtained with a large number of SNPs (≥1500). These results highlight the potential of high‐throughput genomic sequencing approaches to address questions related to evolutionary biology in nonmodel organisms. Furthermore, our findings also provide insights into the optimization of sampling strategies in the era of population genomics.

Linking Phenology to Mating System: Exploring the Reproductive Biology of the Threatened Palm Species Butia eriospatha

The reproductive biology of the vulnerable palm species Butia eriospatha was studied to provide important information that contributes to our understanding and conservation of the species. In order to determine when and how B. erisopatha reproduces, we combined data from 7 nuclear microsatellite loci with ecological data on flowering and fruiting phenology collected between 2009 and 2011 from a population (N = 515) in the Atlantic Rainforest, Southern Brazil. Periods of flowering and fruit production were seasonal and variable across reproductive events. Mating system analyses indicate that B. eriospatha is a predominantly outcrossing species, ((m) = 0.961), since a certain degree of biparental inbreeding does occur. The species is self-compatible and reproduction may also occur by geitonogamy, indicating the ability of isolated populations to survive and persist. Open-pollinated seeds varied in relatedness, including mainly half-sibs and full-sibs. The effective population size was lower than that expected for panmictic populations. Hence, seeds for conservation programs must be collected from a large number of seed-trees to ensure an adequate effective population in the sample. The collection of germplasm is a high-priority strategy that should be employed to maintain the genetic variability that remains.

At Risk of Population Decline? An Ecological and Genetic Approach to the Threatened Palm Species Butia eriospatha (Arecaceae) of Southern Brazil

To estimate the risk of population decline for the threatened palm species Butia eriospatha, we investigated the patterns of demography, natural regeneration, herbivory, and the levels of genetic diversity using 9 microsatellite loci from both adults and seedlings sampled from 4 populations in Southern Brazil (n = 330). Our results indicate that cattle grazing in B. eriospatha population areas severely affect their demographic structure. Three B. eriospatha populations showed a bimodal age structure made up of adult plants and seedlings and high rates (>77%) of livestock herbivory. For 1 population, we describe and quantify for the first time the occurrence of 6 ontogenetic stages for this threatened palm species. Populations of B. eriospatha showed high levels of genetic differentiation (F ST adult plants = 0.287, F ST seedlings = 0.175). The amount of observed heterozygosity differed significantly between small (H O = 0.329) and large populations (H O = 0.461), indicating that small populations can be more susceptible to genetic drift. With no recruitment and a mortality rate of 2.0%, we show that the populations investigated in this study would be at an extremely high risk of local extinction, with a greater than 50% reduction in the effective population size, in the next 40 years. Although this study highlights the importance of analyzing both population ecology parameters and genetic data to better understand the level of risk facing threatened species, we emphasize that policy actions are urgently needed for effective conservation of this vulnerable biological resource.

Dioecy, more than monoecy, affects plant spatial genetic structure: the case study of Ficus

In this analysis, we attempt to understand how monoecy and dioecy drive spatial genetic structure (SGS) in plant populations. For this purpose, plants of the genus Ficus were used as a comparative model due to their particular characteristics, including high species diversity, variation in life histories, and sexual systems. One of the main issues we assessed is whether dioecious fig tree populations are more spatially genetically structured than monoecious populations. Using the Sp statistic, which allows for quantitative comparisons among different studies, we compared the extent of SGS between monoecious and dioecious Ficus species. To broaden our conclusions we used published data on an additional 27 monoecious and dioecious plant species. Furthermore, genetic diversity analyses were performed for two monoecious Ficus species using 12 microsatellite markers in order to strengthen our conclusions about SGS. Our results show that dioecy, more than monoecy, significantly contributes to SGS in plant populations. On average, the estimate of Sp was six times higher for dioecious Ficus species than monoecious Ficus species and it was two times higher in dioecious than monoecious plant species. Considering these results, we emphasize that the long-distance pollen dispersal mechanism in monoecious Ficus species seems to be the dominant factor in determining weak spatial genetic structure, high levels of genetic diversity, and lack of inbreeding. Although Ficus constitute a model species to study SGS, a more general comparison encompassing a wider range of plants is required in order to better understand how sexual systems affect genetic structure.

Microsatellite markers for Butia eriospatha (Arecaceae), a vulnerable palm species from the Atlantic Rainforest of Brazil

PREMISE OF THE STUDY Microsatellite markers were developed for the vulnerable palm species Butia eriospatha (Mart. ex Drude) Becc. to investigate genetic diversity, spatial genetic structure, mating system, and population dynamics. METHODS AND RESULTS From a genomic library enriched for GA/CA repeats, 14 sets of primers were isolated and characterized for 50 B. eriospatha samples from two populations. The number of alleles per locus ranged from 2 to 6 (with amplified dinucleotide repeat-based primers); the observed and expected heterozygosities ranged from 0.000 to 1.000 and from 0.120 to 0.690, respectively. At least 86% of primers were also amplified for Butia catarinensis Noblick & Lorenzi, another threatened palm species from the Atlantic Rainforest in Brazil. CONCLUSIONS The new marker set described here will be useful for studies of population genetics of B. eriospatha, and they have been shown to be applicable for other species from the Butia genus.

Species–genetic diversity correlations in habitat fragmentation can be biased by small sample sizes

Predicted parallel impacts of habitat fragmentation on genes and species lie at the core of conservation biology, yet tests of this rule are rare. In a recent article in Ecology Letters, Struebig et al. (2011) report that declining genetic diversity accompanies declining species diversity in tropical forest fragments. However, this study estimates diversity in many populations through extrapolation from very small sample sizes. Using the data of this recent work, we show that results estimated from the smallest sample sizes drive the species–genetic diversity correlation (SGDC), owing to a false‐positive association between habitat fragmentation and loss of genetic diversity. Small sample sizes are a persistent problem in habitat fragmentation studies, the results of which often do not fit simple theoretical models. It is essential, therefore, that data assessing the proposed SGDC are sufficient in order that conclusions be robust.

Wide but not impermeable: Testing the riverine barrier hypothesis for an Amazonian plant species

Wallaces riverine barrier hypothesis postulates that large rivers, such as the Amazon and its tributaries, reduce or prevent gene flow between populations on opposite banks, leading to allopatry and areas of species endemism occupying interfluvial regions. Several studies have shown that two major tributaries, Rio Branco and Rio Negro, are important barriers to gene flow for birds, amphibians and primates. No botanical studies have considered the potential role of the Rio Branco as a barrier, while a single botanical study has evaluated the Rio Negro as a barrier. We studied an Amazon shrub, Amphirrhox longifolia (A. St.‐Hil.) Spreng (Violaceae), as a model to test the riverine barrier hypothesis. Twenty‐six populations of A. longifolia were sampled on both banks of the Rio Branco and Rio Negro in the core Amazon Basin. Double‐digest RADseq was used to identify 8,010 unlinked SNP markers from the nuclear genome of 156 individuals. Data relating to population structure support the hypothesis that the Rio Negro acted as a significant genetic barrier for A. longifolia. On the other hand, no genetic differentiation was detected among populations spanning the narrower Rio Branco, which is a tributary of the Rio Negro. This study shows that the strength of riverine barriers for Amazon plants is dependent on the width of the river separating populations and species‐specific dispersal traits. Future studies of plants with contrasting life history traits will further improve our understanding of the landscape genetics and allopatric speciation history of Amazon plant diversity.

The same but different: Monomorphic microsatellite markers as a new tool for genetic analysis

PREMISE OF THE STUDY The nucleotide variation at a microsatellite locus lacking length polymorphisms among its alleles was assessed to generate an informative tool for genetic analysis. METHODS AND RESULTS From a set of microsatellite markers, a monomorphic microsatellite locus developed for the palm species Butia eriospatha was used to elucidate whether there are polymorphic sites in its flanking regions. DNA sequences ≈133 bp long were obtained. Aligned sequences show variation at 17 polymorphic sites with both insertions and nucleotide substitutions. Fourteen distinct sequences (alleles) among 22 individuals were identified. The percent sequence difference varied from 0.0 to 5%, indicating that there is significant variation among sequences. CONCLUSIONS Due to significant levels of information and sequence diversity on a simple sequence repeat (SSR) locus of identical size, our study highlights that this molecular marker class can be a useful tool for population genetics and evolutionary studies for many plant species.