The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Expanding the Distribution of Prosthechea jauana (Orchidaceae) in the Pantepui and Highlighting the Urgent Need for Conservation Strategies in the Region in Face of Climate Change.

Prosthechea jauana has been recognized as an orchid species endemic to the Venezuelan tepui. The first record of P. jauana in Brazil is presented here, also from a tepui in the Southern phytogeographical district of Pantepui in the Serra do Aracá, at the northern border of the Amazonas state. A detailed morphological description and images of the specimen are presented, as well as an updated distribution map, preliminary conservation status assessment, and taxonomic notes about the species. In addition, we provide species' distribution models for P. jauana based on current and future bioclimatic data. Future projections suggest that the geographic distribution of P. jauana will likely be severely affected, with ~79% of its suitable habitat being reduced by 2041-2060 and ~92% by 2061-2080. Prosthechea jauana could represent a flag species and an example of how climate change may affect the endemic Pantepui flora.

Complete chloroplast genomes of six neotropical palm species, structural comparison, and evolutionary dynamic patterns.

The Arecaceae family has a worldwide distribution, especially in tropical and subtropical regions. We sequenced the chloroplast genomes of Acrocomia intumescens and A. totai, widely used in the food and energy industries; Bactris gasipaes, important for palm heart; Copernicia alba and C. prunifera, worldwide known for wax utilization; and Syagrus romanzoffiana, of great ornamental potential. Copernicia spp. showed the largest chloroplast genomes (C. prunifera: 157,323 bp and C. alba: 157,192 bp), while S. romanzoffiana and B. gasipaes var. gasipaes presented the smallest (155,078 bp and 155,604 bp). Structurally, great synteny was detected among palms. Conservation was also observed in the distribution of single sequence repeats (SSR). Copernicia spp. presented less dispersed repeats, without occurrence in the small single copy (SSC). All RNA editing sites were C (cytidine) to U (uridine) conversions. Overall, closely phylogenetically related species shared more sites. Almost all nodes of the phylogenetic analysis showed a posterior probability (PP) of 1.0, reaffirming the close relationship between Acrocomia species. These results elucidate the conservation among palm chloroplast genomes, but point to subtle structural changes, providing support for the evolutionary dynamics of the Arecaceae family.

Complete chloroplast genomes and phylogeny in three Euterpe palms (E. edulis, E. oleracea and E. precatoria) from different Brazilian biomes.

The Brazilian palm fruits and hearts-of-palm of Euterpe edulis, E. oleracea and E. precatoria are an important source for agro-industrial production, due to overexploitation, conservation strategies are required to maintain genetic diversity. Chloroplast genomes have conserved sequences, which are useful to explore evolutionary questions. Besides the plastid DNA, genome skimming allows the identification of other genomic resources, such as single nucleotide polymorphisms (SNPs), providing information about the genetic diversity of species. We sequenced the chloroplast genome and identified gene content in the three Euterpe species. We performed comparative analyses, described the polymorphisms among the chloroplast genome sequences (repeats, indels and SNPs) and performed a phylogenomic inference based on 55 palm species chloroplast genomes. Finally, using the remaining data from genome skimming, the nuclear and mitochondrial reads, we identified SNPs and estimated the genetic diversity among these Euterpe species. The Euterpe chloroplast genomes varied from 159,232 to 159,275 bp and presented a conserved quadripartite structure with high synteny with other palms. In a pairwise comparison, we found a greater number of insertions/deletions (indels = 93 and 103) and SNPs (284 and 254) between E. edulis/E. oleracea and E. edulis/E. precatoria when compared to E. oleracea/E. precatoria (58 indels and 114 SNPs). Also, the phylogeny indicated a closer relationship between E. oleracea/E. precatoria. The nuclear and mitochondrial genome analyses identified 1,077 SNPs and high divergence among species (FST = 0.77), especially between E. edulis and E. precatoria (FST = 0.86). These results showed that, despite the few structural differences among the chloroplast genomes of these Euterpe palms, a differentiation between E. edulis and the other Euterpe species can be identified by point mutations. This study not only brings new knowledge about the evolution of Euterpe chloroplast genomes, but also these new resources open the way for future phylogenomic inferences and comparative analyses within Arecaceae.

Localized Phylogenetic Discordance Among Nuclear Loci Due to Incomplete Lineage Sorting and Introgression in the Family of Cotton and Cacao (Malvaceae).

The economically important cotton and cacao family (Malvaceae sensu lato) have long been recognized as a monophyletic group. However, the relationships among some subfamilies are still unclear as discordant phylogenetic hypotheses keep arising when different sources of molecular data are analyzed. Phylogenetic discordance has previously been hypothesized to be the result of both introgression and incomplete lineage sorting (ILS), but the extent and source of discordance have not yet been evaluated in the context of loci derived from massive sequencing strategies and for a wide representation of the family. Furthermore, no formal methods have been applied to evaluate if the detected phylogenetic discordance among phylogenomic datasets influences phylogenetic dating estimates of the concordant relationships. The objective of this research was to generate a phylogenetic hypothesis of Malvaceae from nuclear genes, specifically we aimed to (1) investigate the presence of major discordance among hundreds of nuclear gene histories of Malvaceae; (2) evaluate the potential source of discordance; and (3) examine whether discordance and loci heterogeneity influence on time estimates of the origin and diversification of subfamilies. Our study is based on a comprehensive dataset representing 96 genera of the nine subfamilies and 268 nuclear loci. Both concatenated and coalescence-based approaches were followed for phylogenetic inference. Using branch lengths and topology, we located the placement of introgression events to directly evaluate whether discordance is due to introgression rather than ILS. To estimate divergence times, concordance and molecular rate were considered. We filtered loci based on congruence with the species tree and then obtained the molecular rate of each locus to distribute them into three different sets corresponding to shared molecular rate ranges. Bayesian dating was performed for each of the different sets of loci with the same parameters and calibrations. Phylogenomic discordance was detected between methods, as well as gene histories. At deep coalescent times, we found discordance in the position of five subclades probably due to ILS and a relatively small proportion of introgression. Divergence time estimation with each set of loci generated overlapping clade ages, indicating that, even with different molecular rate and gene histories, calibrations generally provide a strong prior.

Coalescent-Based Species Delimitation in Herbaceous Bamboos (Bambusoideae, Olyreae) from Eastern Brazil: Implications for Taxonomy and Conservation in a Group with Weak Morphological Divergence Coupled with Low Genetic Diversity.

Species delimitation in herbaceous bamboos has been complex and, in some genera, a great part of its diversity has been confirmed only based on genetic information, as is the case of the genus Raddia. It includes nine species, all occurring in Brazil, but only R. portoi predominates in dry forests of the Northeast associated with the Caatinga phytogeographic domain. This species is morphologically close to R. angustifolia, which is known for a single location in the Atlantic Forest in Southern Bahia, and is considered to be threatened by extinction. Besides problems with taxonomic focus, actions for its conservation are complicated because it is not certain if it must be considered an independent species or included in the more widespread R. portoi. In this study, we used coalescent multispecies (MSC) theory approaches combined with genetic structure analyses in an attempt to delimit these two species. Different analyses were congruent and the species delimitation using MSC inferred distinct lineages supporting their recognition as two species. These results solved the taxonomic doubts and also showed the power of these approaches to delimit species as lineages, even in groups with weak morphological divergence and low genetic variability, and also impacting our knowledge for conservation purposes.

Biogeography of Stigmaphyllon (Malpighiaceae) and a Meta-Analysis of Vascular Plant Lineages Diversified in the Brazilian Atlantic Rainforests Point to the Late Eocene Origins of This Megadiverse Biome.

We investigated the biogeography of Stigmaphyllon, the second-largest lianescent genus of Malpighiaceae, as a model genus to reconstruct the age and biogeographic history of the Brazilian Atlantic Rainforest (BAF). Few studies to date have focused on the tertiary diversification of plant lineages in the BAFs, especially on Stigmaphyllon. Phylogenetic relationships for 24 species of Stigmaphyllon (18 ssp. From the Atlantic forest (out of 31 spp.), three spp. from the Amazon Rainforest, two spp. from the Caatinga biome, and a single species from the Cerrado biome) were inferred based on one nuclear DNA (PHYC) and two ribosomal DNA (ETS, ITS) regions using parsimony and Bayesian methods. A time-calibrated phylogenetic tree for ancestral area reconstructions was additionally generated, coupled with a meta-analysis of vascular plant lineages diversified in the BAFs. Our results show that: (1) Stigmaphyllon is monophyletic, but its subgenera are paraphyletic; (2) the most recent common ancestor of Stigmaphyllon originated in the Brazilian Atlantic Rainforest/Caatinga region in Northeastern Brazil ca. 26.0 Mya; (3) the genus colonized the Amazon Rainforest at two different times (ca. 22.0 and 6.0 Mya), the Caatinga biome at least four other times (ca. 14.0, 9.0, 7.0, and 1.0 Mya), the Cerrado biome a single time (ca. 15.0 Mya), and the Southern Atlantic Rainforests five times (from 26.0 to 9.0 Mya); (4) a history of at least seven expansion events connecting the Brazilian Atlantic Rainforest to other biomes from 26.0 to 9.0 Mya, and (5) a single dispersion event from South America to Southeastern Asia and Oceania at 22.0 Mya via Antarctica was proposed. Compared to a meta-analysis of time-calibrated phylogenies for 64 lineages of vascular plants diversified in the Brazilian Atlantic Rainforests, our results point to a late Eocene origin for this megadiverse biome.

A Repertory of Rearrangements and the Loss of an Inverted Repeat Region in Passiflora Chloroplast Genomes.

Chloroplast genomes (cpDNA) in angiosperms are usually highly conserved. Although rearrangements have been observed in some lineages, such as Passiflora, the mechanisms that lead to rearrangements are still poorly elucidated. In the present study, we obtained 20 new chloroplast genomes (18 species from the genus Passiflora, and Dilkea retusa and Mitostemma brevifilis from the family Passifloraceae) in order to investigate cpDNA evolutionary history in this group. Passiflora cpDNAs vary in size considerably, with ∼50 kb between shortest and longest. Large inverted repeat (IR) expansions were identified, and at the extreme opposite, the loss of an IR was detected for the first time in Passiflora, a rare event in angiosperms. The loss of an IR region was detected in Passiflora capsularis and Passiflora costaricensis, a species in which occasional biparental chloroplast inheritance has previously been reported. A repertory of rearrangements such as inversions and gene losses were detected, making Passiflora one of the few groups with complex chloroplast genome evolution. We also performed a phylogenomic study based on all the available cp genomes and our analysis implies that there is a need to reconsider the taxonomic classifications of some species in the group.

Four raised to one equals one: A genetic approach to the Pseudolaelia vellozicola complex does not follow a math rule.

Pseudolaelia is a genus endemic to the eastern Brazilian Atlantic Forest, consisting of 12 accepted species. Some Pseudolaelia species, such as P. vellozicola, P. aguadocensis, P. oliveirana, and P. regentii, referred to here as the PV complex, present extensive intra- and interpopulation morphological polymorphism, raising uncertainty regarding their circumscriptions. Although previous morphological analyses were used to solve the generic boundaries in the PV complex, persuasive genetic evidence is lacking. In order to test the hypothesis that the group under investigation contains only one taxon, amplification profiles of five intersimple sequence repeat (ISSR) markers were used to evaluate genetic diversity, genetic structure, and the relationships among the PV complex species. A total of 134 reproductive individuals were sampled in eight insular populations. Intrapopulation genetic analysis indicated low levels of genetic diversity. Analysis of genetic structure revealed that each of the eight sample locations can be considered unique biological populations as they are highly differentiated from each other. The Mantel test showed a high and positive correlation between genetic and geographic distance (r = .841, p < .002), indicating isolation by distance. The results are consistent with that expected for plants with insular geographical distribution. When testing for the null hypothesis, the low levels of genetic variation among species (F CT = 0.155) suggest that the populations constitute only one highly polymorphic species with a wide distribution.

Phylogenetic relationships within Parianinae (Poaceae: Bambusoideae: Olyreae) with emphasis on Eremitis: Evidence from nuclear and plastid DNA sequences, macromorphology, and pollen ectexine patterns.

Eremitis, Pariana, and Parianella are herbaceous bamboos (tribe Olyreae) included in the subtribe Parianinae, which is characterized by the presence of fimbriae at the apex of the leaf sheaths and exclusively spiciform synflorescences. We analyzed 43 samples of herbaceous and woody bamboos in order to infer relationships within the Parianinae, based on combined data from the nuclear ribosomal internal transcribed spacer (ITS) and plastid DNA (rpl32-trnL and trnD-trnT spacers). Bayesian inference, maximum likelihood, and maximum parsimony methods were applied, and macro- and micromorphological aspects were also analyzed, including the ectexine patterns of pollen grains. Parianinae is represented by three well-supported lineages in our analyses: (1) Parianella, endemic to southern Bahia, Brazil; (2) Pariana sensu stricto with a broad distribution in southern Central America and northern South America, especially in the Amazon region; and (3) Eremitis, endemic to the Brazilian Atlantic Forest, from the states of Pernambuco to Rio de Janeiro, including one species previously described as a member of Pariana. Our molecular phylogeny showed that Pariana, as historically circumscribed, is not monophyletic, by recovering Pariana sensu stricto as strongly supported and sister to Eremitis + Pariana multiflora, with Parianella sister to the Pariana-Eremitis clade. Morphological features of their synflorescences and differences in ectexine patterns characterize each lineage. Based on all these characters and the phylogenetic results, Pariana multiflora, endemic to the state of Espírito Santo, Brazil, is transferred to Eremitis.

Plastomes resolve generic limits within tribe Clusieae (Clusiaceae) and reveal the new genus Arawakia.

Clusieae is an exclusively Neotropical tribe in the family Clusiaceae sensu stricto. Although tribes within Clusiaceae are morphologically and phylogenetically well-delimited, resolution among genera within these tribes remains elusive. The tribe Clusieae includes an estimated ∼500 species distributed among five genera: Chrysochlamys, Clusia, Dystovomita, Tovomita, and Tovomitopsis. In this study, we used nearly complete plastid genomes from 30 exemplar Clusieae species representing all genera recognized, plus two outgroups to infer the phylogeny of the tribe using Maximum Likelihood and Bayesian Inference. For comparison, we also inferred a phylogeny from the nuclear Internal Transcribed Spacer (ITS) region using the same methods. Our study corroborates earlier findings that Clusia is monophyletic while Tovomita is not. It also provides additional support to the hypothesis that Chrysochlamys and Tovomitopsis are not closely related despite gross morphological similarity. Tovomita is divided into three distantly related clades: (i) core Tovomita (including the type T. guianensis), (ii) T. croatii, and (iii) the T. weddelliana species complex. Members of the T. weddelliana complex are isolated from the core Tovomita, and placed in a well-supported clade that is sister to a clade composed of Chrysochlamys plus Clusia. Tovomita croatii is nested within Chrysochlamys. We propose taxonomic revisions to accommodate our phylogenetic findings, including the description of the new genus Arawakia, which includes the 18 species formerly recognized in the T. weddelliana species complex. Lectotypes are also designated for nine species (i.e., Arawakia angustata, A. lanceolata, A. lingulata, A. longicuneata, A. macrocarpa, A. oblanceolata, A. pithecobia, A. rhizophoroides, and A. weddelliana), and a taxonomic key for the identification of the six genera of Clusieae recognized is presented.

Target Nuclear and Off-Target Plastid Hybrid Enrichment Data Inform a Range of Evolutionary Depths in the Orchid Genus Epidendrum.

Universal angiosperm enrichment probe sets designed to enrich hundreds of putatively orthologous nuclear single-copy loci are increasingly being applied to infer phylogenetic relationships of different lineages of angiosperms at a range of evolutionary depths. Studies applying such probe sets have focused on testing the universality and performance of the target nuclear loci, but they have not taken advantage of off-target data from other genome compartments generated alongside the nuclear loci. Here we do so to infer phylogenetic relationships in the orchid genus Epidendrum and closely related genera of subtribe Laeliinae. Our aims are to: 1) test the technical viability of applying the plant anchored hybrid enrichment (AHE) method (Angiosperm v.1 probe kit) to our focal group, 2) mine plastid protein coding genes from off-target reads; and 3) evaluate the performance of the target nuclear and off-target plastid loci in resolving and supporting phylogenetic relationships along a range of taxonomical depths. Phylogenetic relationships were inferred from the nuclear data set through coalescent summary and site-based methods, whereas plastid loci were analyzed in a concatenated partitioned matrix under maximum likelihood. The usefulness of target and flanking non-target nuclear regions and plastid loci was assessed through the estimation of their phylogenetic informativeness. Our study successfully applied the plant AHE probe kit to Epidendrum, supporting the universality of this kit in angiosperms. Moreover, it demonstrated the feasibility of mining plastome loci from off-target reads generated with the Angiosperm v.1 probe kit to obtain additional, uniparentally inherited sequence data at no extra sequencing cost. Our analyses detected some strongly supported incongruences between nuclear and plastid data sets at shallow divergences, an indication of potential lineage sorting, hybridization, or introgression events in the group. Lastly, we found that the per site phylogenetic informativeness of the ycf1 plastid gene surpasses that of all other plastid genes and several nuclear loci, making it an excellent candidate for assessing phylogenetic relationships at medium to low taxonomic levels in orchids.

Amazon plant diversity revealed by a taxonomically verified species list.

Recent debates on the number of plant species in the vast lowland rain forests of the Amazon have been based largely on model estimates, neglecting published checklists based on verified voucher data. Here we collate taxonomically verified checklists to present a list of seed plant species from lowland Amazon rain forests. Our list comprises 14,003 species, of which 6,727 are trees. These figures are similar to estimates derived from nonparametric ecological models, but they contrast strongly with predictions of much higher tree diversity derived from parametric models. Based on the known proportion of tree species in neotropical lowland rain forest communities as measured in complete plot censuses, and on overall estimates of seed plant diversity in Brazil and in the neotropics in general, it is more likely that tree diversity in the Amazon is closer to the lower estimates derived from nonparametric models. Much remains unknown about Amazonian plant diversity, but this taxonomically verified dataset provides a valid starting point for macroecological and evolutionary studies aimed at understanding the origin, evolution, and ecology of the exceptional biodiversity of Amazonian forests.

Microsatellite markers for Senna spectabilis var. excelsa (Caesalpinioideae, Fabaceae).

PREMISE OF THE STUDY: Senna spectabilis var. excelsa (Fabaceae) is a South and Central American tree of great ecological importance and one of the most common species in several sites of seasonally dry forests. Our goal was to develop microsatellite markers to assess the genetic diversity and structure of this species. METHODS AND RESULTS: We designed and assessed 53 loci obtained from a microsatellite-enriched library and an intersimple sequence repeat library. Fourteen loci were polymorphic, and they presented a total of 39 alleles in a sample of 61 individuals from six populations. The mean values of observed and expected heterozygosities were 0.355 and 0.479, respectively. Polymorphism information content was 0.390 and the Shannon index was 0.778. CONCLUSIONS: Polymorphism information content and Shannon index indicate that at least nine of the 14 microsatellite loci developed are moderate to highly informative, and potentially useful for population genetic studies in this species.

Phylogenetic relationships of Echinolaena and Ichnanthus within Panicoideae (Poaceae) reveal two new genera of tropical grasses.

Echinolaena and Ichnanthus are two tropical grass genera distributed mostly in the Americas, characterized by the presence of rachilla appendages in the shape of convex swellings, scars or wings at the base of the upper anthecium. However, recent studies have shown that rachilla appendages arose several times independently in several groups within Paniceae and Paspaleae (Panicoideae). Thus, this study aimed to assess the monophyly of Echinolaena and Ichnanthus and their relationship to other genera of Paniceae and Paspaleae, especially those including species with rachilla appendages. Parsimony and Bayesian analyses of the cpDNA regions ndhF, rpl16, trnH-(rps19)-psbA, trnL-trnF, trnS-(psbZ)-trnG, and the rDNA ITS region included 29 of the 39 known species of Echinolaena and Ichnanthus, 23 of which were sampled for the first time. The multiple loci analyses indicated that Echinolaena and Ichnanthus are polyphyletic in their current circumscriptions, with species in four distinct lineages within subtribe Paspalinae, each one characterized by a single type of rachilla appendage. Thus, Echinolaena and Ichnanthus are each circumscribed in a narrow sense, and the other two lineages excluded from them are proposed as the new genera Hildaea and Oedochloa, resulting in 15 new combinations and the restablishment of I. oplismenoides Munro ex Döll.

DNA barcoding in Atlantic Forest plants: What is the best marker for Sapotaceae species identification?

The Atlantic Forest is a phytogeographic domain with a high rate of endemism and large species diversity. The Sapotaceae is a botanical family for which species identification in the Atlantic Forest is difficult. An approach that facilitates species identification in the Sapotaceae is urgently needed because this family includes threatened species and valuable timber species. In this context, DNA barcoding could provide an important tool for identifying species in the Atlantic Forest. In this work, we evaluated four plant barcode markers (matK, rbcL, trnH-psbA and the nuclear ribosomal internal transcribed spacer region - ITS) in 80 samples from 26 species of Sapotaceae that occur in the Atlantic Forest. ITS yielded the highest average interspecific distance (0.122), followed by trnH-psbA (0.019), matK (0.008) and rbcL (0.002). For species discrimination, ITS provided the best results, followed by matK, trnH-psbA and rbcL. Furthermore, the combined analysis of two, three or four markers did not result in higher rates of discrimination than obtained with ITS alone. These results indicate that the ITS region is the best option for molecular identification of Sapotaceae species from the Atlantic Forest.

Population genetics of the endemic and endangered Vriesea minarum (Bromeliaceae) in the Iron Quadrangle, Espinhaço Range, Brazil.

• Premise of the study: Knowledge about genetic variability in plant populations is one of the main branches of conservation genetics, linking genetic data to conservation strategies. Vriesea minarum is a bromeliad endemic to the Iron Quadrangle region (southeastern Brazil), occurring on mountaintop rock outcrops. It is listed as endangered due to habitat loss, particularly from iron ore mining. Thus, determining the structure and genetic diversity of V. minarum populations could help develop strategies to conserve the species.• Methods: We studied the genetic structure of 12 populations of V. minarum using 10 microsatellite loci transferred from other species of Bromeliaceae. Statistical analyses to compare and describe the genetic diversity of each population were performed, and genetic structure within and among populations, isolation by distance, and Bayesian structure were also analyzed.• Key results: Our results show high inbreeding (GIS = 0.376) and low population structure (FST = 0.088), possibly related to high gene flow due to great pollinator efficiency and/or efficient seed dispersal, thus leading to high connectivity among populations of these fragmented rock outcrops. Two clusters were observed, corresponding to the basins of rivers São Francisco and Doce.• Conclusions: Gene flow among populations is high but, given the rate of habitat loss to mining, most populations are vulnerable and will become increasingly isolated if no action is taken to preserve them. Thus, conservation of this species depends on in situ and ex situ actions, such as controlling overexploitation and creating a germoplasm bank.

A molecular phylogeny of Raddia and its allies within the tribe Olyreae (Poaceae, Bambusoideae) based on noncoding plastid and nuclear spacers.

The plastid spacer trnD-trnT and the nuclear ribosomal internal transcribed spacer (ITS) were sequenced for 37 samples of herbaceous bamboos (Poaceae: Olyreae), including all Raddia species and allied genera, as well as two members of the woody bamboos (tribes Bambuseae and Arundinarieae), in order to examine their relationships. The sequences were analyzed using maximum parsimony and Bayesian inference. Both the individual and combined analyses of ITS and trnD-trnT supported Olyreae as a monophyletic group. All species of Raddia also formed a well-supported monophyletic group, and combined datasets allowed us to outline some relationships within this group. Individual analyses indicated incongruence regarding the sister group of Raddia, with ITS data weakly indicating Raddiella malmeana whereas trnD-trnT data supported Sucrea maculata in this position. However, the combined analysis supported Sucrea as sister to Raddia, although the monophyly of Sucrea is not well supported. Parodiolyra is paraphyletic to Raddiella in all analyses; Olyra is also paraphyletic, with species of Lithachne, Arberella and Cryptochloa nested within it. Eremitis and Pariana appeared as an isolated clade within Olyreae, and the position of the New Guinean Buergersiochloa remains uncertain within this tribe.

Microsatellite markers for an endemic Atlantic Forest tree, Manilkara multifida (Sapotaceae).

Manilkara multifida is a tropical tree that is endemic to the Atlantic forests of southern Bahia, Brazil. Currently, populations of this species are restricted to fragmented landscapes that are susceptible to anthropogenic disturbances. Considering this issue, and that there is no genetic information available for this endangered species, we developed microsatellite markers for M. multifida to provide resources for future conservation genetics studies. Using an enriched genomic library, we isolated eight polymorphic microsatellite loci and optimized the amplification conditions for M. multifida. For each locus, we estimated the number of alleles, H E and H O, paternity exclusion Q, individual identity I and fixation index F, and examined the presence of null alleles. The mean number of alleles was 11.9, and the heterozygosity was high at all loci (average H E = 0.809 and H O = 0.777). The combined values for both paternity exclusion and individual identity were Q = 0.9959 and I = 5.45 × 10(-11), respectively. No evidence of null alleles was detected. The results of our analysis indicated that all eight microsatellites are promising for assessing questions involving inbreeding, gene flow, co-ancestry and mating patterns in M. multifida.