Isoetes are ancient quillworts members of the only genus of the order Isoetales. The genus is slow evolving but is resilient, and widespread worldwide. Two recently described species occur in the Eastern Brazilian Amazon, Isoetes serracarajensis and Isoetes cangae. They are found in the ironstone grasslands known as Canga. While I. serracarajensis is present mostly in seasonal water bodies, I. cangae is known to occur in a single permanent lake at the South mountain range. In this work, we undertake an extensive morphological, physiological and genetic characterization of both species to establish species boundaries and better understand the morphological and genetic features of these two species. Our results indicate that the morphological differentiation of the species is subtle and requires a quantitative assessment of morphological elements of the megaspore for diagnosis. We did not detect differences in microspore output, but morphological peculiarities may establish a reproductive barrier. Additionally, genetic analysis using DNA barcodes and whole chloroplast genomes indicate that although the plants are genetically very similar both approaches provide diagnostic characters. There was no indication of population structuring I. serracarajensis. These results set the basis for a deeper understanding of the evolution of the Isoetes genus.

Cangas (ironstone outcrops) host a specialized flora, characterized by high degree of edaphic endemism and an apparent lack of natural history knowledge of its flora. Due to intense pressure from iron ore mining this ecosystem is under threat and in need of restoration. We studied seed functional traits that are relevant for restoration, translocation and ex situ conservation in 48 species from cangas in eastern Amazon. Were determined the thermal niche breadth, classified seed dormancy and determined methods to overcome it, determined the effect of seed storage on germination, tested the association between germination traits and functional groups, and tested whether seed traits are phylogenetically conserved. We found a broad interspecific variation in most seed traits, except for seed water content. Large interspecific variation in the temperature niche breadth was found among the studied species, but only four species, showed optimum germination at high temperatures of 35–40°C, despite high temperatures under natural conditions. Only 35% of the studied species produced dormant seeds. Mechanical scarification was effective in overcoming physical dormancy and application of gibberellic acid was effective in overcoming physiological dormancy in five species. For the 29 species that seeds were stored for 24 months, 76% showed decreases in the germination percentage. The weak association between germination traits and life-history traits indicate that no particular plant functional type requires specific methods for seed-based translocations. Exceptions were the lianas which showed relatively larger seeds compared to the other growth-forms. Dormancy was the only trait strongly related to phylogeny, suggesting that phylogenetic relatedness may not be a good predictor of regeneration from seeds in cangas. Our study provides support to better manage seed sourcing, use, storage and enhancement techniques with expected reduced costs and increased seedling establishment success.

Background Two endemic lycophyte species Isoetes cangae and Isoetes serracarajensis have been recently described in the State of Pará in the Amazon forest located in northern Brazil. Isoetes L. has survived through three mass extinctions. Plants are considered small-sized, heterosporous, and can display a great diversity of physiological adaptations to different environments. Thus, the current study aimed to estimate the genetic variation of the populations of I. cangae and I. serracarajensis to generate information about their different mechanisms for survival at the same geographical location that could point to different reproductive, adaptative and dispersal strategies and should be considered for effective conservation strategies. Methods The genetic diversity and population structure of I. cangae and I. serracarajensis were investigated using Inter Simple Sequence Repeat (ISSR) molecular markers. Total genomic DNA was isolated, and the genetic diversity parameters were calculated. Results The sixteen primers produced 115 reproducible bands, 87% of which were polymorphic. A high level of polymorphic loci (81.74% and 68.48%) and a high Shannon index (Sh = 0.376 and 0.289) were observed for I. cangae and I. serracarajensis, respectively. The coefficient of genetic differentiation between population areas (GST) showed a higher value in I. serracarajensis (0.5440). Gene flow was higher in I. cangae (1.715) and lower in I. serracarajensis populations (0.419). Overall, the results further show that I. serracarajensis and I. cangae are two species with considerable genetic variation and that these differences may reflect their habitats and modes of reproduction. These results should be considered in the development of effective conservation strategies for both species.

Advancing functional ecology depends fundamentally on the availability of data on reproductive traits, including those from tropical plants, which have been historically underrepresented in global trait databases. Although some valuable databases have been created recently, they are mainly restricted to temperate areas and vegetative traits such as leaf and wood traits. Here, we present Rock n' Seeds, a database of seed functional traits and germination experiments from Brazilian rock outcrop vegetation, recognized as outstanding For affiliation refer to page 2

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The application of the mitigation hierarchy (MH) to mining projects is challenging in situations of locational overlap between endemic flora and mineral deposits. We review flora surveys conducted in connection with the environmental impact assessment of several iron ore mining projects in an area of high degree of endemism in Eastern Amazon to discuss the practical implications of anticipating conservation strategies. Desktop studies and secondary data review were conducted to guide field searches to determine the distribution of endemic flora, resulting in 45 out of 46 endemic plant species having their known distribution extended to new areas. A framework for positioning flora conservation strategies in the MH is presented. Specific habitat requirements and scarce knowledge about endangered and endemic flora species are a conservation obstacle, since essential information to define species conservation strategies may be lacking. We show that anticipating conservation strategies can minimize time-lag uncertainties related to restoration success and biodiversity offsets. The more effort is placed in the preventative steps of the MH, the smaller the time-lag between impact (biodiversity losses) and conservation outcomes (biodiversity gains), decreasing uncertainties and reducing risks to biodiversity.

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We examined the reproductive strategy of two Amazonian quillworts (Isoëtes cangae and Isoëtes serracarajensis), endemic and threatened species of canga ecosystems. Sexual propagation was examined by in vitro fertilization assays, while asexual propagation was examined by tiller emission. Isoëtes cangae is an outcrossing species that reproduces exclusively by spore germination and is able to propagate by self- and cross-fertilization. Isoëtes serracarajensis reproduces asexually by emitting tillers from the plant corm, despite producing male and female sporangia. These distinct reproductive strategies in the different species may be linked to their contrasting habitats. Isoëtes cangae inhabit a permanent oligotrophic lake with mild environmental changes, while I. serracarajensis are found in temporary ponds facing severe seasonal drought, where asexual propagation may represent an adaptive advantage to the short growth period during access to water. We also observed different relationships between plant growth and reproductive traits between the species, despite their common production of sporophytes with high survival rates. Together, these results are of paramount importance for establishing conservation plans for both species considering the advantages of sexual propagation to maintain the genetic diversity of I. cangae and the diligent management required to do the same with asexually propagated I. serracarajensis.