Loreta B. Freitas

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 Passifloras 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.

Diversity and natural hybridization in a highly endemic species of Petunia (Solanaceae): a molecular and ecological analysis

Intrinsic reproductive barriers among the species of Petunia are weak and genetic isolation is obtained mainly by geographical separation and ecological diversification. The Serra do Sudeste region in the extreme south of Brazil is one of the centres of diversity of this genus and is characterized by the presence of species with different pollination syndromes. Petunia exserta is known only from four sandstone towers in a restricted area of this region (about 500 km2) and is characterized by its differentiated habitat (shelters in the sandstone towers) and by its floral characteristics adapted to ornithophily. In towers where this species is sympatric with the sphingophilous Petunia axillaris, phylogenetically close to P. exserta, we found plants with intermediate floral morphology, suggesting hybridization between them. To test this hypothesis and to better understand its consequences we analysed the sequences of the plastid trnH‐psbA, trnS‐trnG and psbB‐psbH intergenic spacers in 121 individuals sampled all over the P. exserta distribution. The joint analysis of the three markers revealed 13 haplotypes and the network showed two main genetic clades, which probably represent the original gene pool of the two species in the region. In general, individuals of a given population presented the same haplotype, independently of phenotype, corroborating the hybridization hypothesis. Field observations suggest that hummingbirds are responsible for the interspecific gene flow. Analysis of molecular variance revealed high interpopulational diversity among the towers. The low gene flow between populations is possibly related to the autochoric seed dispersion system.

The Genus Petunia

The common garden petunia, Petunia hybrida, is derived from P. integrifolia and P. axillaris, two of many Petunia species endemic to South America. The geographic distribution includes temperate and subtropical regions of Argentina, Uruguay, Paraguay, Bolivia, and Brazil, with a center of diversity in southern Brazil. The presence of seven chromosomes and a number of morphological, anatomical, and biochemical characteristics differentiate the genus from its sister taxon, Calibrachoa. Included in this chapter is a taxononomic guide for the 14 currently recognized species, some of them restricted to very small geographic areas. Species diversity is in danger of diminishing significantly due to human intervention, particularly in the form of grassland destruction.

Caracterização do germoplasma de tangerineiras do Sul do Brasil mediante análises morfológicas e moleculares

Abstract – The objective of this work was to characterize mandarin (Citrus spp.) germplasm fromSouthern Brazil by morphological and molecular analyses. Thirty seven cultivars from 34 distinctmandarin varieties were evaluated by morphological and agronomic traits of leaves, flowers and fruits,and by microsatellite markers. The morphological and agronomic characteristics suggested that almostall varieties can be produced for commercial use, and some, as the Satsuma variety, are recommended forbreeding programs. Pooled DNA samples from 1-5 plants belonging to each cultivar were tested. Eightof the nine primers detected polymorphisms. Specific markers were found for some accessions. Thedendrogram constructed with the morphological results divided the 37 cultivars into four groups, whilethat obtained with the microsatellites clustered 35 of the 37 cultivars into three groups only. Generally,intervarietal differences are not high, and this lack of agreement in the two multifactorial analysesindicates that diverse evolutionary factors are acting at these two levels of investigation.Index terms: citrus, genetic variation, microsatellites, breeding methods.Caracterizacao do germoplasma de tangerineiras do Sul do Brasilmediante analises morfologicas e molecularesResumo – O objetivo deste trabalho foi caracterizar o germoplasma de tangerineiras ( Citrus spp.) pormeio de analises morfologicas e moleculares. Trinta e sete cultivares, pertencentes a 34 variedadesdistintas de tangerineiras, foram avaliadas pelas caracteristicas morfologicas e agronomicas de folhas,flores e frutos, bem como por analises de microssatelites. As caracteristicas morfologicas e agronomicassugerem que praticamente todas as variedades podem ser usadas comercialmente e algumas delas, comoa variedade Satsuma, sao recomendadas para uso em cruzamentos dirigidos. O DNA foi obtido deamostras extraidas de uma a cinco plantas de cada cultivar. Oito de nove primers detectaram polimorfismos.Foram encontrados marcadores especificos para alguns acessos. O dendrograma construido com osresultados morfologicos dividiu as 37 cultivares em quatro grupos, enquanto o obtido com osmicrossatelites agrupou 35 das 37 cultivares em tres grupos. As diferencas intervarietais nao sao gran-des, e esta falta de concordância nas duas analises multivariadas indica que fatores evolutivos diversosestao agindo nesses dois niveis de investigacao.Termos para indexacao: citros, variacao genetica, microsatelites, metodo de melhoramento.

Diversification of plant species in a subtropical region of eastern South American highlands: a phylogeographic perspective on native Petunia (Solanaceae)

In the Southern and Southeastern Brazilian highlands, a clade of seven species of Petunia that are endemic to the region (P. altiplana, P. bonjardinensis, P. guarapuavensis, P. mantiqueirensis, P. reitzii, P. saxicola and P. scheideana) exists in association with grassland formations. These formations are isolated in high‐altitude regions, being surrounded by forested areas, and experienced contraction–expansion cycles associated with the glacial cycles of the Pleistocene. To understand the evolutionary history of this group, the divergence of which is probably linked to these past shifts in habitat, we analysed the sequences of the plastidial intergenic spacers trnH‐psbA and trnS‐trnG from populations throughout the known distributions of all seven species. The common ancestor of this highland clade started to differentiate ∼0.9 million years (Myr) ago, which corresponds to a high diversification rate of 2.06 species per Myr in the intervening period. The high level of haplotype sharing among several species in the clade and the absence of reciprocal monophyly suggest the persistence of ancestral polymorphisms during speciation events and/or past hybridization, because no hybrid was found. Four of the five species displayed very low genetic diversity and possessed either one or two haplotypes, which is consistent with long‐term isolation in restricted areas. The three more diverse species displayed significant population structure, and P. altiplana showed a clear signs of population growth during the last glacial period. These results suggest that diversification occurred as a result of expansion of the ancestral species of the clade during glacial periods followed by fragmentation and isolation during retraction in interglacial periods.

Alu insertions versus blood group plus protein genetic variability in four Amerindian populations.

Background : Do the population relationships obtained using DNA or blood group plus protein markers remain the same or do they reveal different patterns, indicating that the factors which influence genetic variation at these two levels of analysis are diverse? Can these markers shed light on the biological classification of the Aché, a Paraguayan tribe which only recently established more permanent contacts with non-Indians? Subjects and methods : To consider these questions we typed 193 individuals from four Amerindian tribes in relation to 12 Alu polymorphisms (five of them never studied in these populations), while 22 blood group plus protein systems were studied among the Aché. These data were then integrated with those previously available (blood groups plus proteins) for the three other populations. DNA extraction and amplification, as well as the other laboratory procedures, were performed using standard methods currently in use in our laboratory. The genetic relationships were obtained using the D A distance, and the trees were constructed by the neighbour-joining method, both developed by M. Nei and collaborators. Reliability of the trees was tested by bootstrap replications. Other population variability values were also determined using Neis methods. Results : Alu polymorphism was observed in all populations and for most of the loci; in the seven systems from which we could compare our results with those of other Amerindian groups agreement was satisfactory. Unusual findings on the blood group plus protein systems of the Aché were a very low (5%) HP*1 frequency and the presence of the C W phenotype in the Rh blood group. The intertribal patterns of relationship and other aspects of their variation were remarkably congruent in the two sets ( Alu; blood group plus protein) of systems. Conclusions : The answer to the first question posed above is affirmative. However, the problem of whether the Aché derived from a Gê group that preceded the Guarani colonization of Paraguay, or are just a differentiated Guarani group, could not be answered with the genetic information available; the second hypothesis seems more likely at present, but the point to be emphasized is the striking genetic distinctiveness of the Aché as compared to other Amerindians.

Extremely limited mitochondrial DNA variability among the Aché Natives of Paraguay.

Background: The Aché Natives are an especially interesting group of people, due to their distinctive morphological aspect and the fact that only in the last three decades have they established more permanent contact with outside populations. The objectives of the present study were: (a) to verify their distinctiveness in relation to mitochondrial DNA (mtDNA) variability; (b) to ascertain whether the pattern observed was congruent with other genetic studies performed among them; and (c) to establish historical inferences that would explain the eventual similarities or differences. Subjects and methods: Sample collection was made at two localities in eastern Paraguay. DNA from 64 maternally unrelated subjects were tested in relation to the mtDNA hypervariable segment 1 (HVS-1) by automatic sequencing. Results: Fifty-six individuals presented exactly the same haplogroup B founder haplotype; another differed from it by a single transition polymorphism at site 16362, while six other subjects showed an identical haplogroup A founding haplotype. An A/G heteroplasmy at the 16269 site was seen in one haplogroup B individual, probably due to a somatic mutation. Conclusions: The Aché present distinctive differences and reduced mtDNA HVS-1 variability compared to other South American Natives. Similar differences were observed for other genetic systems. At present it is not clear whether their peculiarities already existed in their founding populations or whether they were secondarily acquired due to a long period of isolation in the humid, subtropical forest.

MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation

Adaptations to new pollinators involve multiple floral traits, each requiring coordinated changes in multiple genes. Despite this genetic complexity, shifts in pollination syndromes have happened frequently during angiosperm evolution. Here we study the genetic basis of floral UV absorbance, a key trait for attracting nocturnal pollinators. In Petunia, mutations in a single gene, MYB-FL, explain two transitions in UV absorbance. A gain of UV absorbance in the transition from bee to moth pollination was determined by a cis-regulatory mutation, whereas a frameshift mutation caused subsequent loss of UV absorbance during the transition from moth to hummingbird pollination. The functional differences in MYB-FL provide insight into the process of speciation and clarify phylogenetic relationships between nascent species.

Phylogeny, biogeography and divergence times in Passiflora (Passifloraceae).

As part of a long-term investigation on the evolution of Passiflora L., we investigated the divergence ages of the genus and diversification of its subgenera, relating them with biogeographical and/or historical events, and other characteristics of this taxon. The main aim of the present work was to evaluate the biogeographic distribution of this genus to better understand its evolutionary history. This is the first time that representatives from South American and Old World Passifloraceae genera have been studied as a group comprising a total of 106 widely distributed species, with representative samples of the four suggested subgenera. Seven DNA regions were studied, comprising 7,431 nucleotides from plastidial, mitochondrial and nuclear genomes. Divergence time estimates were obtained by using a Bayesian Markov Chain Monte Carlo method and a random local clock model for each partition. Three major subgenera have been shown to be monophyletic and here we are proposing to include another subgenus in the Passiflora infrageneric classification. In general, divergence among the four subgenera in Passiflora is very ancient, ranging from ∼32 to ∼38 Mya, and Passifloraceae seems to follow a biogeographic scenario proposed for several plant groups, originating in Africa, crossing to Europe/Asia and arriving in the New World by way of land bridges. Our results indicated that Passiflora ancestors arrived in Central America and diversified quickly from there, with many long distance dispersion events.

Does Variation in Genome Sizes Reflect Adaptive or Neutral Processes? New Clues from Passiflora

One of the long-standing paradoxes in genomic evolution is the observation that much of the genome is composed of repetitive DNA which has been typically regarded as superfluous to the function of the genome in generating phenotypes. In this work, we used comparative phylogenetic approaches to investigate if the variations in genome sizes (GS) should be considered as adaptive or neutral processes by the comparison between GS and flower diameters (FD) of 50 Passiflora species, more specifically, within its two most species-rich subgenera, Passiflora and Decaloba. For this, we have constructed a phylogenetic tree of these species, estimated GS and FD of them, inferred the tempo and mode of evolution of these traits and their correlations, using both current and phylogenetically independent contrasted values. We found significant correlations among the traits, when considering the complete set of data or only the subgenus Passiflora, whereas no correlations were observed within Decaloba. Herein, we present convincing evidence of adaptive evolution of GS, as well as clues that this pattern is limited by a minimum genome size, which could reduce both the possibilities of changes in GS and the possibility of phenotypic responses to environment changes.