Phylogenomic and morphological data reveal hidden patterns of diversity in the national tree of Brazil, Paubrasilia echinata.

PREMISE: Paubrasilia echinata (common names, pau brasil, brazilwood) is the national tree of Brazil and an endangered species endemic to the Brazilian Atlantic Forest. Over its wide distribution of 2000 km, its leaflets morphology exhibits extensive plasticity. Three morphotypes are commonly identified based on leaf size, but it is unclear if they represent distinct taxa or a single polymorphic species. This study aims to clarify the taxonomic position of the three morphotypes to inform conservation decisions. METHODS: A morphometric study of leaf characters of herbarium specimens was coupled with genetic analyses using genotype-by-sequencing data. We used maximum-likelihood and coalescent methods to evaluate the phylogenetic and population structure of the species. We compared these with a morphological dendrogram built from hierarchical clustering. RESULTS: Two of the three morphotypes formed separately evolving lineages, the third morphotype formed two geographically separate lineages, and northern trees with intermediate leaf morphology formed a separate fifth lineage. Leaflet size varied by over 35-fold, and although morphological clustering generally matched the genetic patterns, there were some overlaps, highlighting the cryptic diversity within this group. CONCLUSIONS: Our genetic and morphological results provide some evidence that cultivated trees from different states in Brazil seem to have a limited genetic origin and do not reflect the broader genetic and geographical diversity of the species. As a result, more care is likely needed to preserve the overall genomic diversity of this endangered and iconic species.

Plastome evolution in the Caesalpinia group (Leguminosae) and its application in phylogenomics and populations genetics.

MAIN CONCLUSION: The chloroplast genomes of Caesalpinia group species are structurally conserved, but sequence level variation is useful for both phylogenomic and population genetic analyses. Variation in chloroplast genomes (plastomes) has been an important source of information in plant biology. The Caesalpinia group has been used as a model in studies correlating ecological and genomic variables, yet its intergeneric and infrageneric relationships are not fully solved, despite densely sampled phylogenies including nuclear and plastid loci by Sanger sequencing. Here, we present the de novo assembly and characterization of plastomes from 13 species from the Caesalpinia group belonging to eight genera. A comparative analysis was carried out with 13 other plastomes previously available, totalizing 26 plastomes and representing 15 of the 26 known Caesalpinia group genera. All plastomes showed a conserved quadripartite structure and gene repertoire, except for the loss of four ndh genes in Erythrostemon gilliesii. Thirty polymorphic regions were identified for inter- or intrageneric analyses. The 26 aligned plastomes were used for phylogenetic reconstruction, revealing a well-resolved topology, and dividing the Caesalpinia group into two fully supported clades. Sixteen microsatellite (cpSSR) loci were selected from Cenostigma microphyllum for primer development and at least two were cross-amplified in different Leguminosae subfamilies by in vitro or in silico approaches. Four loci were used to assess the genetic diversity of C. microphyllum in the Brazilian Caatinga. Our results demonstrate the structural conservation of plastomes in the Caesalpinia group, offering insights into its systematics and evolution, and provides new genomic tools for future phylogenetic, population genetics, and phylogeographic studies.

How diverse is heterochromatin in the Caesalpinia group? Cytogenomic characterization of Erythrostemon hughesii Gagnon & G.P. Lewis (Leguminosae: Caesalpinioideae).

MAIN CONCLUSION: Cytogenomic characterization of Erythrostemon hughesii reveals a heterogeneity of repeats in its subtelomeric heterochromatin. Comparative analyses with other Caesalpinia group species reveal a significant reduction in the abundance of Ty3-gypsy/Chromovirus Tekay retrotransposons during its evolution. In numerically stable karyotypes, repetitive DNA variability is one of the main causes of genome and chromosome variation and evolution. Species from the Caesalpinia group (Leguminosae) are karyotypically characterized by 2n = 24, with small chromosomes and highly variable CMA+ heterochromatin banding patterns that correlate with environmental variables. Erythrostemon hughesii differs from other species of the group examined to date for having subtelomeric CMA+ bands; this contrasts with most species in the group which have proximal bands. Here we analyse the repeatome of E. hughesii using genome skimming and chromosomal mapping approaches to characterize the identity of the most abundant repetitive elements and their physical location. The repetitive fraction of E. hughesii comprises 28.73% of the genome. The most abundant elements were retrotransposons (RT) with long terminal repeats (LTR-RT; 9.76%) and satellite DNAs (7.83%). Within the LTR-RTs, the most abundant lineages were: Ty1/copia-Ale (1%), Ty3/gypsy CRM (0.88%) and Ty3/gypsy Athila (0.75%). Using fluorescent in situ hybridization four satellite DNAs and several LTR-RT elements were shown to be present in most subtelomeric CMA+ bands. These results highlight how the repeatome in E. hughesii, a species from Oaxaca state in Mexico, is clearly distinct from Northeast Brazilian species of the Caesalpinia group, mainly due to its high diversity of repeats in its subtelomeric heterochromatic bands and low amount of LTR-RT Ty3/gypsy-Tekay elements. Comparative sequence analysis of Tekay elements from different species is congruent with a clade-specific origin of this LTR-RT after the divergence of the Caesalpinia group. We hypothesize that repeat-rich heterochromatin may play a role in leading to faster genomic divergence between individuals, increasing speciation and diversification.

Evolutionary convergence or homology? Comparative cytogenomics of Caesalpinia group species (Leguminosae) reveals diversification in the pericentromeric heterochromatic composition.

MAIN CONCLUSION: We demonstrated by cytogenomic analysis that the proximal heterochromatin of the Northeast Brazilian species of Caesalpinia group is enriched with phylogenetically conserved Ty3/Gypsy-Tekay RT, but diverge in the presence of Ty3/Gypsy-Athila RT and satDNA. The Caesalpinia Group includes 225 species and 27 monophyletic genera of which four occur in Northeastern Brazil: Erythrostemon (1 sp.), Cenostigma (7 spp.), Libidibia (1 sp.), and Paubrasilia (1 sp.). The last three genera are placed in different clades in the Caesalpinia Group phylogeny, and yet they are characterized by having a numerically stable karyotype 2n = 24 (16 M+8A) and GC-rich heterochromatic bands (chromomycin A3 positive/CMA+ bands) in the proximal chromosome regions. To characterize the composition of their heterochromatin and test for the homology of these chromosomal regions, genomic DNA was extracted from Cenostigma microphyllum, Libidibia ferrea, and Paubrasilia echinata, and sequenced at low coverage using the Illumina platform. The genomic repetitive fractions were characterized using a Galaxy/RepeatExplorer-Elixir platform. The most abundant elements of each genome were chromosomally located by fluorescent in situ hybridization (FISH) and compared to the CMA+ heterochromatin distribution. The repetitive fraction of the genomes of C. microphyllum, L. ferrea, and P. echinata were estimated to be 41.70%, 38.44%, and 72.51%, respectively. Ty3/Gypsy retrotransposons (RT), specifically the Tekay lineage, were the most abundant repeats in each of the three genomes. FISH mapping revealed species-specific patterns for the Tekay elements in the proximal regions of the chromosomes, co-localized with CMA+ bands. Other species-specific patterns were observed, e.g., for the Ty3/Gypsy RT Athila elements which were found in all the proximal heterochromatin of L. ferrea or restricted to the acrocentric chromosomes of C. microphyllum. This Athila labeling co-localized with satellite DNAs (satDNAs). Although the Caesalpinia Group diverged around 55 Mya, our results suggest an ancestral colonization of Tekay RT in the proximal heterochromatin. Thus, the present-day composition of the pericentromeric heterochromatin in these Northeast Brazilian species is a combination of the maintenance of an ancestral Tekay distribution with a species-specific accumulation of other repeats.

Genetic and phytochemical investigations for understanding population variability of the medicinally important tree Saraca asoca to help develop conservation strategies.

Saraca asoca (Roxb.) De Wilde (Caesalpiniaceae) is a highly traded IUCN red listed tree species used in Ayurvedic medicines for the treatment of various disorders, especially gynaecological problems. However, information about the genetic variations between populations and corresponding variation in specialized metabolites of S. asoca remains unclear. To address this issue, we analysed 11 populations of S. asoca with 106 accessions collected from Western Ghats of India using ISSR markers along with selected phytocompounds using RP-HPLC. Twenty primers were screened, out of which seven were selected for further analysis based on generation of clear polymorphic banding patterns. These seven ISSR primers produced 74 polymorphic loci. AMOVA showed 43% genetic variation within populations and 57% among the populations of S. asoca. To estimate the genetic relationships among S. asoca populations, UPGMA and Bayesian Models were constructed, which revealed two clusters of similar grouping patterns. However, excluding minor deviations, UPGMA and dissimilarity analysis showed close association of genotypes according to their geographical locations. Catechin (CAT), epicatechin (EPI) and gallic acid (GA) were quantified from bark and leaf samples of corresponding genotypes collected from 106 accessions. ROC plots depicted the sensitivity and specificity of the concentrations of tested phytocompounds at various cut-off points. Although, multiple logistic regression analysis predicted some association between few loci with GA, EPI and CAT, but PCA for phytochemical data failed to distinguish the populations. Overall, there were no significant trends observed to distinguish the populations based on these phytocompounds. Furthermore, the study advocates the delineate provenance regions of S. asoca genotypes/chemotype snapshots for in-situ conservation and ex-situ cultivation.

Karyotype diversity and 2C DNA content in species of the Caesalpinia group.

BACKGROUND: The Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species' phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry. RESULTS: The fluorochrome banding indicated that the chromomycin A3+/4',6-diamidino-2-phenylindole- blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering. CONCLUSIONS: The chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be done through integrative evaluation.

Brazilwood, sappanwood, brazilin and the red dye brazilein: from textile dyeing and folk medicine to biological staining and musical instruments.

Brazilin is a nearly colorless dye precursor obtained from the heartwood of several species of trees including brazilwood from Brazil, sappanwood from Asia and the Pacific islands, and to a minor extent from two other species in Central America, northern South America and the Caribbean islands. Its use as a dyeing agent and medicinal in Asia was recorded in the 2(nd) century BC, but was little known in Europe until the 12(th) century AD. Asian supplies were replaced in the 16(th) century AD after the Portuguese discovered vast quantities of trees in what is now Brazil. Overexploitation decimated the brazilwood population to the extent that it never fully recovered. Extensive environmental efforts currently are underway to re-create a viable, sustainable population. Brazilin is structurally similar to the better known hematoxylin, thus is readily oxidized to a colored dye, brazilein, which behaves like hematein. Attachment of the dye to fabric is by hydrogen bonding or in conjunction with certain metallic mordants by coordinative bonding. For histology, most staining procedures involve aluminum (brazalum) for staining nuclei. In addition to textile dyeing and histological staining, brazilin and brazilein have been and still are used extensively in Asian folk medicine to treat a wide variety of disorders. Recent pharmacological studies for the most part have established a scientific basis for these uses and in many cases have elucidated the biochemical pathways involved. The principal use of brazilwood today is for the manufacture of bows for violins and other stringed musical instruments. The dye and other physical properties of the wood combine to produce bows of unsurpassed tonal quality.

Comparison of methods to isolate DNA from Caesalpinia ferrea.

Molecular markers are important for characterizing the genetic diversity of plants and can provide the basis for strategies to protect and conserve endangered populations. However, numerous molecular techniques are used, requiring an evaluation of fast and efficient methods to extract DNA. Since molecular studies of Caesalpinia ferrea are rare, it is important to develop and/or adapt a DNA extraction protocol that produces quality DNA samples to enable the design of strategies for the conservation of this threatened species. This study aimed to compare five methods for DNA extraction and to determine the most efficient protocol for C. ferrea. Sufficient genomic DNA was obtained from the leaves of C. ferrea using all the tested protocols to perform techniques involving molecular markers. Two protocols based on the detergent cetyl trimethyl ammonium bromide, as well as a commercial kit, yielded high concentrations of pure DNA. However, when polymerase chain reaction amplifications were performed, DNA was only successfully amplified from extractions performed with the commercial kit, which produced sufficient genomic DNA of good quality from the leaves of C. ferrea to perform techniques involving molecular markers.

Phylogenetic relationships among morphotypes of Caesalpinia echinata Lam. (Caesalpinioideae: Leguminosae) evidenced by trnL intron sequences.

Caesalpinia echinata (brazilwood or Pernambuco wood) comprises a complex of three morphological leaf variants, characterized by differences in the number and size of the pinnae and leaflets, and occurring in allopatric and sympatric populations. The present study evaluates the utility of the chloroplast DNA trnL intron in a phylogenetic analysis of the three leaf variants along with other species of Caesalpinia and generic relatives. Our study supports the hypothesis that the name C. echinata designates a species complex and provides evidence that one of the forms, the highly divergent C. echinata large-leafleted variant, represents a distinct taxon.

Cryptic speciation in the Caesalpinia hintonii complex (Leguminosae: Caesalpinioideae) in a seasonally dry Mexican forest.

BACKGROUND AND AIMS: [corrected] The Caesalpinia hintonii group comprises six species of endemic shrubs or trees, C. epifanioi, C. hintonii, C. laxa, C. macvaughii, C. melanadenia and C. oyamae, found in scattered patches of seasonally dry forest in the Río Balsas depression and the neighbouring Tehuacán-Cuicatlán valley, which are part of the Mexican morphotectonic province of Sierra Madre del Sur. An evaluation is made of phylogeographic patterns and genetic diversity with a phylogenetic analysis of the C. hintonii complex in order to study the dynamics of speciation in this endemic group of legumes. METHODS: A phylogeographic study based on four highly variable non-coding plastid regions (trnL intron, trnL-F intergenic spacer, trnH-psbA intergenic spacer, and accD-psaI intergenic spacer) was carried out for the Caesalpinia hintonii complex. Five of the six taxa of the C. hintonii complex were included. KEY RESULTS AND CONCLUSIONS: The plastid analyses involving multiple accessions of each taxon from throughout their ranges resolved C. epifanioi and C. hintonii as well-supported clusters, but C. oyamae has two unexpectedly divergent lineages. Two well-supported geographic clades: eastern (C. epifanioi, C. melanadenia and C. oyamae) and western (C. hintonii and C. macvaughii) were established. The analyses performed provide evidence of recent morphostatic radiation in C. oyamae resulting from isolation and local adaptation. This pattern of genetic differentiation without morphological divergence may be a model that fits many groups of tropical woody taxa inhabiting similarly dry forests subjected to shifting selection.

Identification of RAPD markers, in situ DNA content and structural chromosomal diversity in some legumes of the mangrove flora of Orissa.

Randomly amplified polymorphic DNA (RAPD) markers, karyotypes and 4C DNA content were analyzed in five legume mangroves belonging to the sub-family Papilinoideae (Dalbergia spinosa, Derris heterophylla and D. indica) and Caesalpinioideae (Caesalpinia crista, Cynometra ramiflora) of the family Fabaceae to establish the genetic variability and phylogenetic affinities. Somatic chromosome numbers were reported for the first time in D. spinosa (2n = 20), C. ramiflora (2n = 26) and D. heterophylla (2n = 24) with reconfirmation of the somatic chromosome number in D. indica (2n = 22) and C. crista (2n = 24). Significant intergeneric and interspecific variation of 4C DNA content was observed and that varied from 8.970 pg in C. ramiflora to 28.730 pg in D. indica. From the RAPD analysis, the dendogram showed clustering of Caesalpinia crista and Cynomitra ramiflora into one group (81.80). In the second groups Derris indica and Derris heterophylla were more similar (83.10) than Dalbergia spinosa (85.80). Species-specific DNA markers (900 bp) obtained in D. spinosa from OPN15; 700 and 2000 bp in C. ramiflora from OPN4 and 400 and 800 bp in D. heterophylla and 500 bp DNA fragment in C. crista obtained from OPN-11 were found characteristic RAPD markers of these species. C. crista found more closer affinity to C. ramiflora of the sub-family Caesalpinioideae [genetic distance (1-F) = 0.847]. Derris indica showed closer genetic relation with D. heterophylla [genetic distance (1-F) = 0.856] than D. spinosa [genetic distance (1-F) = 0.876] where Derris and Dalbergia belongs to the sub-family Papilionoideae. By employing these markers the present study has helped to resolve the relationship between the taxonomically diverse leguminous mangroves and study their ability to coexist with mangroves that would shed light on the evolution of mangroves from terrestrial species.

Long-term population isolation in the endangered tropical tree species Caesalpinia echinata Lam. revealed by chloroplast microsatellites.

Habitat fragmentation represents the single most serious threat to the survival of tropical ecosystems. In formulating strategies to counteract the detrimental effects of fragmentation, knowledge of the levels and patterns of genetic diversity within and between natural populations is vital to the establishment of any conservation programme. We utilized polymorphic chloroplast microsatellite markers to analyse genetic diversity in populations of the endangered tropical tree Caesalpinia echinata Lam. representing the entire extant range of the species. Levels of within-population diversity were low, with only two of seven populations studied displaying any variation. The vast majority of the genetic variation was partitioned between geographical regions (36%) and between populations within regions (55%). These levels of genetic structuring, coupled with a calculated pollen-to-seed flow ratio of approximately 6.7:1, suggest that there has been little gene flow between the three major geographical regions over an extended period. Thus, the current tripartite distribution of the species is more consistent with the existence of separate glacial refugia, rather than reflecting any anthropogenic effects.