Putting small and big pieces together: a genome assembly approach reveals the largest Lamiid plastome in a woody vine.

The plastid genome of flowering plants generally shows conserved structural organization, gene arrangement, and gene content. While structural reorganizations are uncommon, examples have been documented in the literature during the past years. Here we assembled the entire plastome of Bignonia magnifica and compared its structure and gene content with nine other Lamiid plastomes. The plastome of B. magnifica is composed of 183,052 bp and follows the canonical quadripartite structure, synteny, and gene composition of other angiosperms. Exceptionally large inverted repeat (IR) regions are responsible for the uncommon length of the genome. At least four events of IR expansion were observed among the seven Bignoniaceae species compared, suggesting multiple expansions of the IRs over the SC regions in the family. A comparison with 6,231 other complete plastomes of flowering plants available on GenBank revealed that the plastome of B. magnifica is the longest Lamiid plastome described to date. The newly generated plastid genome was used as a source of selected genes. These genes were combined with orthologous regions sampled from other species of Bignoniaceae and all gene alignments concatenated to infer a phylogeny of the family. The tree recovered is consistent with known relationships within the Bignoniaceae.

There Is No 'Rule of Thumb': Genomic Filter Settings for a Small Plant Population to Obtain Unbiased Gene Flow Estimates.

The application of high-density polymorphic single-nucleotide polymorphisms (SNP) markers derived from high-throughput sequencing methods has heralded plenty of biological questions about the linkages of processes operating at micro- and macroevolutionary scales. However, the effects of SNP filtering practices on population genetic inference have received much less attention. By performing sensitivity analyses, we empirically investigated how decisions about the percentage of missing data (MD) and the minor allele frequency (MAF) set in bioinformatic processing of genomic data affect direct (i.e., parentage analysis) and indirect (i.e., fine-scale spatial genetic structure - SGS) gene flow estimates. We focus specifically on these manifestations in small plant populations, and particularly, in the rare tropical plant species Dinizia jueirana-facao, where assumptions implicit to analytical procedures for accurate estimates of gene flow may not hold. Avoiding biases in dispersal estimates are essential given this species is facing extinction risks due to habitat loss, and so we also investigate the effects of forest fragmentation on the accuracy of dispersal estimates under different filtering criteria by testing for recent decrease in the scale of gene flow. Our sensitivity analyses demonstrate that gene flow estimates are robust to different setting of MAF (0.05-0.35) and MD (0-20%). Comparing the direct and indirect estimates of dispersal, we find that contemporary estimates of gene dispersal distance (σ r t = 41.8 m) was ∼ fourfold smaller than the historical estimates, supporting the hypothesis of a temporal shift in the scale of gene flow in D. jueirana-facao, which is consistent with predictions based on recent, dramatic forest fragmentation process. While we identified settings for filtering genomic data to avoid biases in gene flow estimates, we stress that there is no 'rule of thumb' for bioinformatic filtering and that relying on default program settings is not advisable. Instead, we suggest that the approach implemented here be applied independently in each separate empirical study to confirm appropriate settings to obtain unbiased population genetics estimates.

One Step Away From Extinction: A Population Genomic Analysis of A Narrow Endemic, Tropical Plant Species.

Intraspecific genetic variation plays a fundamental role in maintaining the evolutionary potential of wild populations. Hence, the assessment of genetic diversity patterns becomes essential to guide biodiversity conservation policies, particularly for threatened species. To inform management strategies for conservation of Mimosa catharinensis - a narrow endemic, critically endangered plant species - we identified 1,497 unlinked SNP markers derived from a reduced representation sequencing method (i.e., double digest restriction site associated DNA sequencing, or ddRADseq). This set of molecular markers was employed to assess intrapopulation genetic parameters and the demographic history of one extremely small population of M. catharinensis (N=33) located in the Brazilian Atlantic Forest. Contrary to what is expected for narrow endemic and threatened species with small population sizes, we observed a moderate level of genetic diversity for M. catharinensis [uH E(0%missing data)=0.205, 95% CI (0.160, 0.250); uH E(30%missing data)=0.233, 95% CI (0.174, 0.292)]. Interestingly, M. catharinensis, which is a lianescent shrub with no indication of seed production for at least two decades, presented high levels of outcrossing [t (0%missing data)=0.883, SE±0.0483; t (30%missing data)=0.909, SE±0.011] and an apparent absence of inbreeding [F (0%missing data)=-0.145, 95% CI (-0.189, -0.101); F (30%missing data)=-0.105, 95% CI (-0.199, -0.011)]. However, the reconstruction of demographic history of M. catharinensis indicated that the population should be suffered a recent bottleneck. Our population genomic study tackles a central issue in evolution and conservation biology and we expect that it will be useful to help safeguard the remaining genetic diversity reported for this unique genetic resource.

By Animal, Water, or Wind: Can Dispersal Mode Predict Genetic Connectivity in Riverine Plant Species?

Seed dispersal is crucial to gene flow among plant populations. Although the effects of geographic distance and barriers to gene flow are well studied in many systems, it is unclear how seed dispersal mediates gene flow in conjunction with interacting effects of geographic distance and barriers. To test whether distinct seed dispersal modes (i.e., hydrochory, anemochory, and zoochory) have a consistent effect on the level of genetic connectivity (i.e., gene flow) among populations of riverine plant species, we used unlinked single-nucleotide polymorphisms (SNPs) for eight co-distributed plant species sampled across the Rio Branco, a putative biogeographic barrier in the Amazon basin. We found that animal-dispersed plant species exhibited higher levels of genetic diversity and lack of inbreeding as a result of the stronger genetic connectivity than plant species whose seeds are dispersed by water or wind. Interestingly, our results also indicated that the Rio Branco facilitates gene dispersal for all plant species analyzed, irrespective of their mode of dispersal. Even at a small spatial scale, our findings suggest that ecology rather than geography play a key role in shaping the evolutionary history of plants in the Amazon basin. These results may help improve conservation and management policies in Amazonian riparian forests, where degradation and deforestation rates are high.

A Biogeographic Barrier Test Reveals a Strong Genetic Structure for a Canopy-Emergent Amazon Tree Species.

Wallace's (1854) Riverine Barrier hypothesis is one of the earliest explanations for Amazon biotic diversification. Despite the importance of this hypothesis for explaining speciation in some animal groups, it has not been studied extensively for plant species. In this study we use a prominent Amazon tree, Buchenavia oxycarpa (Mart.) Eichler (Combretaceae), to evaluate Wallace's hypothesis along the Rio Negro, a major Amazon tributary that has driven allopatric speciation for several animal taxa. We sampled six individuals from sixteen localities along both river banks, and used a modified ddRADseq protocol to identify SNP markers. Our population genomic data revealed strong genetic structure for B. oxycarpa sampled across banks of the Rio Negro (ϕCT = 0.576, P < 0.001), supporting the hypothesis that the Rio Negro acted as a significant genetic barrier for B. oxycarpa. Our study shows that gene flow for this large and well-dispersed Amazon tree is impeded by riverine barriers, though this has not yet resulted in speciation. Future studies focused on species with different life histories, including species restricted to non-flooded forests, are needed to further advance our understanding of Amazon rivers as drivers of biotic diversification.

Tangled banks: A landscape genomic evaluation of Wallace's Riverine barrier hypothesis for three Amazon plant species.

Wallace's Riverine Barrier hypothesis is one of the earliest biogeographic explanations for Amazon speciation, but it has rarely been tested in plants. In this study, we used three woody Amazonian plant species to evaluate Wallace's Hypothesis using tools of landscape genomics. We generated unlinked single-nucleotide polymorphism (SNP) data from the nuclear genomes of 234 individuals (78 for each plant species) across 13 sampling sites along the Rio Branco, Brazil, for Amphirrhox longifolia (8,075 SNPs), Psychotria lupulina (9,501 SNPs) and Passiflora spinosa (14,536 SNPs). Although significantly different migration rates were estimated between species, the population structure data do not support the hypothesis that the Rio Branco-an allopatric barrier for primates and birds-is a significant genetic barrier for Amphirrhox longifolia, Passiflora spinosa or Psychotria lupulina. Overall, we demonstrated that medium-sized rivers in the Amazon Basin, such as the Rio Branco, are permeable barriers to gene flow for animal-dispersed and animal-pollinated plant species.

Chloroplast genomes of Byrsonima species (Malpighiaceae): comparative analysis and screening of high divergence sequences.

Byrsonima is the third largest genus (about 200 species) in the Malpighiaceae family, and one of the most common in Brazilian savannas. However, there is no molecular phylogeny available for the genus and taxonomic uncertainties at the generic and family level still remain. Herein, we sequenced the complete chloroplast genome of B. coccolobifolia and B. crassifolia, the first ones described for Malpighiaceae, and performed comparative analyses with sequences previously published for other families in the order Malpighiales. The chloroplast genomes assembled had a similar structure, gene content and organization, even when compared with species from other families. Chloroplast genomes ranged between 160,212 bp in B. crassifolia and 160,329 bp in B. coccolobifolia, both containing 115 genes (four ribosomal RNA genes, 28 tRNA genes and 83 protein-coding genes). We also identified sequences with high divergence that might be informative for phylogenetic inferences in the Malpighiales order, Malpighiaceae family and within the genus Byrsonima. The phylogenetic reconstruction of Malpighiales with these regions highlighted their utility for phylogenetic studies. The comparative analyses among species in Malpighiales provided insights into the chloroplast genome evolution in this order, including the presence/absence of three genes (infA, rpl32 and rps16) and two pseudogenes (ycf1 and rps19).

Using genomic data to develop chloroplast DNA SSRs for the Neotropical liana Stizophyllum riparium (Bignonieae, Bignoniaceae).

PREMISE OF THE STUDY: We developed chloroplast microsatellite markers (cpSSRs) to be used to study the patterns of genetic structure and genetic diversity of populations of Stizophyllum riparium (Bignonieae, Bignoniaceae). METHODS AND RESULTS: We used genomic data obtained through an Illumina HiSeq sequencing platform to develop a set of cpSSRs for S. riparium. A total of 36 primer pairs were developed, of which 28 displayed polymorphisms across 59 individuals from three populations. Two to 12 alleles were recorded, and the unbiased haploid diversity per locus ranged from 0.037 to 0.905. All 28 cpSSRs presented transferability to two closely related species, S. inaequilaterum and S. perforatum. CONCLUSIONS: We report a set of 28 cpSSRs for S. riparium. All markers were shown to be variable in S. riparium, indicating that these markers will be valuable for population genetic studies across S. riparium and congeneric species.

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

Wallace's 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.

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.

A genomic approach for isolating chloroplast microsatellite markers for Pachyptera kerere (Bignoniaceae).

PREMISE OF THE STUDY: In this study, we developed chloroplast microsatellite markers (cpSSRs) for Pachyptera kerere (Bignoniaceae) to investigate the population structure and genetic diversity of this species. METHODS AND RESULTS: We used Illumina HiSeq data to reconstruct the chloroplast genome of P. kerere by a combination of de novo and reference-guided assembly. We then used the chloroplast genome to develop a set of cpSSRs from intergenic regions. Overall, 24 primer pairs were designed, 21 of which amplified successfully and were polymorphic, presenting three to nine alleles per locus. The unbiased haploid diversity per locus varied from 0.207 (Pac28) to 0.817 (Pac04). All but one locus amplified for all other taxa of Pachyptera. CONCLUSIONS: The markers reported here will serve as a basis for studies to assess the genetic structure and phylogeographic history of Pachyptera.

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.

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.