Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae).

Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

What actually is Vriesea? A total evidence approach in a polyphyletic genus of Tillandsioideae (Bromeliaceae, Poales).

The taxonomic delimitation of Vriesea is challenging because several diagnostic traits of the genus are shared with other genera in Tillandsioideae. The genus has no unique morphological features that distinguish it from other taxa. Earlier phylogenetic studies suggested a polyphyletic status of this genus, but taxon sampling was limited, and tests of congruence for morphological characters have not yet been performed. Here we used a total evidence approach and a more comprehensive Brazilian taxon sampling to help circumscribe Vriesea s.s. (the lineage that contains the type species). We concatenated morphological and molecular datasets for the first time to reconstruct phylogenetic hypotheses. We used 99 morphological characters and ~4 kb of plastid data (psbA-trnH, trnK5'-rps16, matK, trnL-trnF) for 172 terminals, including nine genera of Tillandsioideae. We analysed datasets by parsimony analyses, with several weighting schemes considered for character-state changes (equal weights and an implied weighting scheme that we used as a sensitivity analysis). The concatenated data resulted in a well-resolved phylogenetic inference that provided strong evidence that the genus is not monophyletic as traditionally circumscribed. Given the congruence between morphological, molecular and geographical sources of data, we separated Vriesea into distinct lineages, and proposed a new circumscription that includes only species of Vriesea s.s. found in the Chacoan and Parana subregions.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae)

Abstract Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

Multilocus phylogeny and phylogenomics of Eriochrysis P. Beauv. (Poaceae-Andropogoneae): Taxonomic implications and evidence of interspecific hybridization.

Species delimitation is a vital issue concerning evolutionary biology and conservation of biodiversity. However, it is a challenging task for several reasons, including the low interspecies variability of markers currently used in phylogenetic reconstructions and the occurrence of reticulate evolution and polyploidy in many lineages of flowering plants. The first phylogeny of the grass genus Eriochrysis is presented here, focusing on the New World species, in order to examine its relationships to other genera of the subtribe Saccharinae/tribe Andropogoneae and to define the circumscriptions of its taxonomically complicated species. Molecular cloning and sequencing of five regions of four low-copy nuclear genes (apo1, d8, ep2-ex7 and ep2-ex8, kn1) were performed, as well as complete plastome sequencing. Trees were reconstructed using maximum parsimony, maximum likelihood, and Bayesian inference analyses. The present phylogenetic analyses indicate that Eriochrysis is monophyletic and the Old World E. pallida is sister to the New World species. Subtribe Saccharinae is polyphyletic, as is the genus Eulalia. Based on nuclear and plastome sequences plus morphology, we define the circumscriptions of the New World species of Eriochrysis: E. laxa is distinct from E. warmingiana, and E. villosa is distinct from E. cayennensis. Natural hybrids occur between E. laxa and E. villosa. The hybrids are probably tetraploids, based on the number of paralogues in the nuclear gene trees. This is the first record of a polyploid taxon in the genus Eriochrysis. Some incongruities between nuclear genes and plastome analyses were detected and are potentially caused by incomplete lineage sorting and/or ancient hybridization. The set of low-copy nuclear genes used in this study seems to be sufficient to resolve phylogenetic relationships and define the circumscriptions of other species complexes in the grass family and relatives, even in the presence of polyploidy and reticulate evolution. Complete plastome sequencing is also a promising tool for phylogenetic inference.

Phylogenetic analysis of Saccharum s.l. (Poaceae; Andropogoneae), with emphasis on the circumscription of the South American species.

PREMISE OF THE STUDY: Polyploidy and reticulate evolution are often a complication for discovering phylogenetic relationships between genera and species. Despite the huge economic importance of sugarcane (Saccharum officinarum-Poaceae, Andropogoneae), the limits of the genus Saccharum and its species are complex and largely unresolved, involving both polyploidy and reticulate evolution. This study aimed to assess the phylogenetic relationships of Saccharum s.l., including Erianthus and Tripidium, as well as investigate the taxonomic circumscription of the South American species of the genus. METHODS: Molecular cloning and sequencing of five regions of four low-copy nuclear loci were performed, including Aberrant panicle organization1 (apo1), Dwarf8 (d8), two exons of Erect panicle2 (ep2-ex7 and ep2-ex8), and Retarded palea1 (rep1). Concatenated trees were reconstructed using Maximum Parsimony, Maximum Likelihood, and Bayesian Inference analyses. KEY RESULTS: The allopolyploid origin of Saccharum was demonstrated using evidence from nuclear genes. The samples of Saccharum s.l. grouped in two distinct clades, with S. arundinaceum and S. ravennae (= Tripidium, or Erianthus sect. Ripidium) apart from all other species analyzed of the genus. Saccharum angustifolium, S. asperum, and S. villosum correspond to distinct clades (different species). The plants with intermediate morphology between S. angustifolium and S. villosum presented a pattern of paralogues consistent with a hybrid origin. CONCLUSIONS: Saccharum s.l. is polyphyletic and Tripidium should be recognized as a distinct genus. However, no strong evidence was found to support the segregation of Erianthus. The taxonomic circumscription of the South American species of the genus was resolved and the occurrence of natural hybrids was documented. Better understanding of the phylogenetic relationships of Saccharum and relatives may be useful for sugarcane breeders to identify potential taxa for interspecific and intergeneric crosses in the genetic improvement of sugarcane.

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus.

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH-psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity-based methods and tree-based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH-psbA. The ITS provided better results with 30.61-38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree-based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.

Genetic variation in polyploid forage grass: assessing the molecular genetic variability in the Paspalum genus.

BACKGROUND: Paspalum (Poaceae) is an important genus of the tribe Paniceae, which includes several species of economic importance for foraging, turf and ornamental purposes, and has a complex taxonomical classification. Because of the widespread interest in several species of this genus, many accessions have been conserved in germplasm banks and distributed throughout various countries around the world, mainly for the purposes of cultivar development and cytogenetic studies. Correct identification of germplasms and quantification of their variability are necessary for the proper development of conservation and breeding programs. Evaluation of microsatellite markers in different species of Paspalum conserved in a germplasm bank allowed assessment of the genetic differences among them and assisted in their proper botanical classification. RESULTS: Seventeen new polymorphic microsatellites were developed for Paspalum atratum Swallen and Paspalum notatum Flüggé, twelve of which were transferred to 35 Paspalum species and used to evaluate their variability. Variable degrees of polymorphism were observed within the species. Based on distance-based methods and a Bayesian clustering approach, the accessions were divided into three main species groups, two of which corresponded to the previously described Plicatula and Notata Paspalum groups. In more accurate analyses of P. notatum accessions, the genetic variation that was evaluated used thirty simple sequence repeat (SSR) loci and revealed seven distinct genetic groups and a correspondence of these groups to the three botanical varieties of the species (P. notatum var. notatum, P. notatum var. saurae and P. notatum var. latiflorum). CONCLUSIONS: The molecular genetic approach employed in this study was able to distinguish many of the different taxa examined, except for species that belong to the Plicatula group, which has historically been recognized as a highly complex group. Our molecular genetic approach represents a valuable tool for species identification in the initial assessment of germplasm as well as for characterization, conservation and successful species hybridization.

Development and characterization of nine microsatellite loci for Sisyrinchium micranthum (Iridaceae).

PREMISE OF THE STUDY: Sisyrinchium micranthum is an herb with wide distribution and morphological variation, presenting three known cytotypes (2x, 4x, and 6x), which is naturally occurring from Mexico to Argentina. The development of microsatellite markers for this species is important to provide molecular tools to investigate polyploidy origin, as well as its relationship with phenotypic and genetic variability. • METHODS AND RESULTS: Nine microsatellite markers were isolated using an enriched genomic library and characterized in a diploid population. Eight markers generated polymorphic loci in 27 plants of a population from southern Brazil. The number of alleles per locus ranged from two to nine, and the observed and expected heterozygosities varied from 0.192 to 0.741 and from 0.370 to 0.884, respectively. • CONCLUSIONS: These species-specific DNA markers may be useful for further investigations of the involved polyploidization process and the population genetics of S. micranthum.

Floral rewards in the tribe Sisyrinchieae (Iridaceae): oil as an alternative to pollen and nectar?

Iridaceae is one of the few families in which floral oils are produced and collected by pollinators as a resource. Perigonal nectaries and trichomal elaiophores are highly unusual within the tribe Sisyrinchieae. Both structures occur mainly on the staminal column, while they are usually distributed on the tepals in the other tribes of the subfamily Iridoideae. Sisyrinchieae is the largest tribe of Iridaceae present on the American continent, and the diversity observed may be related to the exceptional development of trichomal elaiophores within the genus Sisyrinchium, but knowledge concerning the other types of nuptial glandular structures within the tribe is still limited, preventing us from estimating their implication for species diversity. Structural observations and histochemical tests were performed to identify and characterize glandular structures and pollen rewards within the flowers of the genera Orthrosanthus, Sisyrinchium and Solenomelus. Perigonal nectaries were detected only in Solenomelus segethi, and trichomal elaiophores were characterized only within Sisyrinchium. All species showed large amounts of additional resources available for pollinators in the form of pollenkitt and polysaccharides present in the cytoplasm of the pollen grains. The results are discussed in a phylogenetic context, with regard to pollinators and floral rewards reported for the tribe Sisyrinchieae.

Oil-producing flowers within the Iridoideae (Iridaceae): evolutionary trends in the flowers of the New World genera.

BACKGROUND AND AIMS: Oil-producing flowers related to oil-bee pollination are a major innovation in Neotropical and Mexican Iridaceae. In this study, phylogenetic relationships were investigated among a wide array of New World genera of the tribes Sisyrinchieae, Trimezieae and Tigridieae (Iridaceae: Iridoideae) and the evolution of floral glandular structures, which are predominantly trichomal elaiophores, was examined in relation to the diversification of New World Iridaceae. METHODS: Phylogenetic analyses based on seven molecular markers obtained from 97 species were conducted to produce the first extensive phylogeny of the New World tribes of subfamily Iridoideae. The resulting phylogenetic hypothesis was used to trace the evolutionary history of glandular structures present in the flowers of numerous species in each tribe. Hypotheses of differential diversification rates among lineages were also investigated using both topological and Binary-State Speciation and Extinction methods. KEY RESULTS AND CONCLUSIONS: Floral glandular structures and especially trichomal elaiophores evolved multiple times independently in the American tribes of Iridoideae. The distribution pattern of species displaying glandular trichomes across the phylogeny reveals lability in the pollination system and suggests that these structures may have played a significant role in the diversification of the Iridoideae on the American continent.

Population genetic structure of Sisyrinchium micranthum Cav. (Iridaceae) in Itapuã State Park, Southern Brazil.

Sisyrinchium micranthum Cav. is a member of the family Iridaceae, which is distributed over the American continent. In Brazil, this species is found, not only in disturbed areas and coastal regions, but is also very common in urban centers, such as public parks, during the spring. Chromosome counts for North American specimens are 2n = 32 and 2n = 48, whereas in southern Brazil, there is a polyploidy series with three chromosome numbers, 2n = 16, 2n = 32, and 2n = 48. Population analyses using DNA molecular markers are inexistent for this species, in spite of its wide distribution and morphological variation. To study the genetic population structure of S. micranthum, five natural populations were accessed in a conservation park within the Atlantic Rain Forest Biome in southern Brazil. Here, the chromosome numbers 2n = 16 and 2n = 48 had already been described. Molecular analysis showed that the populations are highly structured with low gene flow among them. The population with 2n = 48 was genetically less variable than and distinct from the other populations. Population genetics in relation to cytogenetic data provided new insights regarding the genetic diversification and mating system of S. micranthum.

Studies on diversity and evolution of Iridaceae species in southern Brazil.

Plants of the family Iridaceae are well represented in the grassland vegetation of southern Brazil, occurring in the Pampa and Atlantic Forest biomes. Nevertheless, little is known about the taxonomy and evolution of Iridaceae species in southern Brazil. The main goal of this review is to compile published information about South American Iridaceae, and to discuss the evolution and genetic diversity of the family presenting our own research data in the light of the published literature. The main focus is on the genera Calydorea, Cypella, Herbertia, and Sisyrinchium. Aspects of reproductive system and of pollinator attraction are also discussed.

Population genetic structure of Sisyrinchium micranthum Cav. (Iridaceae) in Itapuã State Park, Southern Brazil

Sisyrinchium micranthum Cav. is a member of the family Iridaceae, which is distributed over the American continent. In Brazil, this species is found, not only in disturbed areas and coastal regions, but is also very common in urban centers, such as public parks, during the spring. Chromosome counts for North American specimens are 2n = 32 and 2n = 48, whereas in southern Brazil, there is a polyploidy series with three chromosome numbers, 2n = 16, 2n = 32, and 2n = 48. Population analyses using DNA molecular markers are inexistent for this species, in spite of its wide distribution and morphological variation. To study the genetic population structure of S. micranthum, five natural populations were accessed in a conservation park within the Atlantic Rain Forest Biome in southern Brazil. Here, the chromosome numbers 2n = 16 and 2n = 48 had already been described. Molecular analysis showed that the populations are highly structured with low gene flow among them. The population with 2n = 48 was genetically less variable than and distinct from the other populations. Population genetics in relation to cytogenetic data provided new insights regarding the genetic diversification and mating system of S. micranthum.

Evolution of oil-producing trichomes in Sisyrinchium (Iridaceae): insights from the first comprehensive phylogenetic analysis of the genus.

BACKGROUND AND AIMS: Sisyrinchium (Iridaceae: Iridoideae: Sisyrinchieae) is one of the largest, most widespread and most taxonomically complex genera in Iridaceae, with all species except one native to the American continent. Phylogenetic relationships within the genus were investigated and the evolution of oil-producing structures related to specialized oil-bee pollination examined. METHODS: Phylogenetic analyses based on eight molecular markers obtained from 101 Sisyrinchium accessions representing 85 species were conducted in the first extensive phylogenetic analysis of the genus. Total evidence analyses confirmed the monophyly of the genus and retrieved nine major clades weakly connected to the subdivisions previously recognized. The resulting phylogenetic hypothesis was used to reconstruct biogeographical patterns, and to trace the evolutionary origin of glandular trichomes present in the flowers of several species. KEY RESULTS AND CONCLUSIONS: Glandular trichomes evolved three times independently in the genus. In two cases, these glandular trichomes are oil-secreting, suggesting that the corresponding flowers might be pollinated by oil-bees. Biogeographical patterns indicate expansions from Central America and the northern Andes to the subandean ranges between Chile and Argentina and to the extended area of the Paraná river basin. The distribution of oil-flower species across the phylogenetic trees suggests that oil-producing trichomes may have played a key role in the diversification of the genus, a hypothesis that requires future testing.

Overview of the phylogenetic relationships of some southern Brazilian species from section Torva and related sections of "spiny Solanum" (Solanum subgenus Leptostemonum, Solanaceae).

One of the major concerns of conservation biology is the knowledge on biodiversity. Many taxa have not yet been described and plant taxonomists are working hard to change this situation. This work presents a taxonomic overview of Solanum section Torva, from South Brazil, which still poorly understood. The section includes species known as "jurubeba" which are of considerable interest because of their pharmacological properties. Despite their higher economic value, it is difficult to identify the "jurubebas" correctly due to their taxonomic problems. A few questions on evolution concerning phylogenetic relationships in this section and other related sections of subgenus Leptostemonum were investigated to clarify the species circumscription and taxonomic issues. Eighty samples were analysed based on sequence variability of the internal transcribed spacers (ITS) of the nuclear ribosomal DNA, as well as the chloroplast intron trnL, trnL-trnF and trnS-trnG spacers. Five different matrixes were analyzed as the basis for a phylogenetic approach. The combined data from the chloroplast analyses formed well-supported trees. Solanum section Torva was proposed as a monophyletic group and it is close to S. melongena, S. jamaicense and S. sisymbriifolium.

A preliminary approach to the phylogeny of the genus Paspalum (Poaceae).

The present work intends to clarify the phylogenetic relationships among the species of Paspalum L. belonging to the informal groups Notata/Linearia and Dilatata, and to raise some preliminary hypotheses on the phylogeny of the genus as a whole. A combined dataset including morphological and molecular characters was used to analyze 28 species of Paspalum plus some representatives of related genera of the tribe Paniceae. Analyses were performed using both parsimony and maximum likelihood. The monophyly of Paspalum is not supported nor contradicted. The circumscription of informal groups of Paspalum is discussed, as well as the cladistic treatment of allopolyploid taxa, especially those comprising the Dilatata group. The relationships of members of the Dilatata with their putative progenitors is confirmed, but the monophyly of the group as a whole is not. A close relationship between P. dilatatum Poir. and P. lividum Trin. ex Schltdl. is shown. Our analysis is consistent with the monophyly of a group comprising Notata+Linearia, with a monophyletic Notata group nested within it. The delimitation of the core Notata is proposed by including P. conduplicatum Canto-Dorow, Valls and Longhi-Wagner, P. notatum Flüggé, P. minus E. Fourn., P. pumilum Nees and P. subciliatum Chase.

Genetic diversity among Paspalum L. species (Poaceae) belonging to the Notata and Linearia groups based on restriction fragment length polymorphism analyses.

A taxonomic study of Paspalum L. was carried out using a genetic diversity approach. Thirty accessions representing twenty one different species from the Notata and Linearia groups of Paspalum were studied using restriction fragment length polymorphism analysis of the amplified ITS ribosomal DNA (rDNA) and from the psbA-trnH of the chloroplast genome (cpDNA). The combined analysis of the internal transcribed spacer (ITS) and the chloroplast spacer region between the psbA and trnH genes identified genetic polymorphisms. A distance analysis of the molecular data generated a dendrogram which showed the relationships of the two informal groups of Paspalum studied here. Although the distribution of species in the dendrogram was found to be roughly in agreement with previous works based on morphological and cytological data, the results obtained reveal the current artificiality in Paspalum taxonomy. Based on molecular data, a new circumscription of the Notata and Linearia groups is proposed here in order to provide a more accurate delimitation of these groups and contribute to the taxonomy of Paspalum. This study, although preliminary, reveals the potential utility of such a molecular approach for clarifying the taxonomy of closely related taxa.

A first molecular phylogenetic analysis of Passiflora (Passifloraceae).

Passiflora, a genus with more than 400 species, exhibits a high diversity of floral and vegetative structures and a complex taxonomy, which includes 23 subgenera and many sections and series. To better understand Passiflora's variability and interspecific relationships, the phylogeny of 61 species, classified in 11 of 23 suggested subgenera, was investigated. Three molecular markers were used, the nuclear ribosomal internal transcribed spacers (nrITS), the plastid trnL-trnF spacer regions (∼1000 bp), and the rps4 plastid gene (∼570 bp). Three major clades were highly supported, independent of the marker and phylogenetic method used; one included the subgenera Distephana, Dysosmia, Dysosmioides, Passiflora, and Tacsonioides, a second, the subgenera Adopogyne, Decaloba, Murucuja, and Pseudomurucuja, and a third, the subgenus Astrophea. We call these the Passiflora, Decaloba, and Astrophea clades, respectively. The position of subgenus Deidamioides is undefined. The monophyly of Passiflora could not be statistically corroborated, and the relationships among the major clades and of these clades with the related genera remain unresolved. Our results indicate that a reevaluation of the monophyly of Passiflora and its infrageneric classification is necessary.