Abstract Delimiting species is challenging in recently diverged species, and adaptive radiation is fundamental to understanding the evolutionary processes because it requires multiple ecological opportunities associated with adaptation to biotic and abiotic environments. The young Petunia genus (Solanaceae) is an excellent opportunity to study speciation because of its association with pollinators and unique microenvironments. This study evaluated the phylogenetic relationships among a Petunia clade species with different floral syndromes that inhabit several environments. We based our work on multiple individuals per lineage and employed nuclear and plastid phylogenetic markers and nuclear microsatellites. The phylogenetic tree revealed two main groups regarding the elevation of the distribution range, whereas microsatellites showed high polymorphism-sharing splitting lineages into three clusters. Isolation by distance, migration followed by new environment colonization, and shifts in floral syndrome were the motors for lineage differentiation, including infraspecific structuring, which suggests the need for taxonomic revision in the genus.

Abstract The association between plants and their pollinators is essential for increasing the diversity in angiosperms. Morphological and physiological traits, mainly floral scent, can influence the pollination dynamics and select pollinators for each plant species. In this work, we studied two proteins involved in producing volatile organic compounds in plants, conyferyl alcohol acyltransferase (CFAT) and benzoyl-CoA:benzyl alcohol/phenyl ethanol benzoyl transferase (BPBT) genes. We aimed to understand these proteins with respect to evolutionary and structural aspects and functions in Solanaceae using phylogenetic methods and comparative molecular modeling. We used Bayesian inference to describe the proteins’ evolutionary history using Petunia x hybrida as a query to search for homologs in the Solanaceae family. Theoretical 3D models were obtained for both proteins using Panicum virgatum as a template. The phylogenetic tree included several different enzymes with diverse biological roles in Solanaceae, displaying the transferase domain. We identified only one sequence of CFAT in the databases, which belongs to Petunia x hybrida, and found several BPBT sequences from the genera Nicotiana, Solanum, and Capsicum. The 3D structures of CFAT and BPBT have two different domains, and we have identified the amino acid residues essential for the enzymatic activity and interaction with substrates.

Abstract Genetic polymorphism sharing between closely related and sympatric plant species could result from common ancestry, ancient or recent hybridization. Here we analyzed four Petunia species from the subtropical highland grasslands in southern South America based on nuclear diversity to disentangle the causes of high polymorphism sharing between them. We genotyped microsatellite loci, employed population genetic methods to estimate variability, species limits, and ancient and recent gene flow, and assigned individuals to genetic and taxonomic groups. Finally, we modeled evolutionary processes to determine the impact of Quaternary climate changes on species phylogenetic relationships. Our results indicated that genetic diversity was strongly influenced by expansion and habitat fragmentation during the Quaternary cycles. The extensive polymorphism sharing is mainly due to species’ common ancestry, and we did not discard ancient hybridization. Nowadays, niche differentiation is the primary driver for maintaining genetic and morphological limits between the four analysed Petunia species and there is no recent gene flow between them.

ABSTRACT The long-term success of populations is dependent on individual reproductive success, and in general, increased population size and genetic diversity contribute to population maintenance, reducing the risk of local extinction. Interspecific hybridization has consequences that can vary according to the hybrids' fate, which can be strongly influenced by the reproductive capacity of hybrids and canonical individuals from the contact zone. We examined the reproductive biology and morphology of two closely related Petunia (Solanaceae) species and their interspecific hybrids from Serra do Sudeste, Southern Brazil, and we measured their reproductive success under controlled conditions based on seed production and germination from five pollination treatments. We found differences in self-compatibility degree among individuals, lineages, and pollination treatments based on high total seed production (> 204,000 seeds) and germination (630 seedlings evaluated). No correlation was observed between corolla colour and reproductive success or between floral morphological traits and compatibility. High self-compatibility and inter-lineage compatibility can explain the hybrid populations' maintenance and origin, favouring the two analysed species' introgression.

Abstract Interspecific hybridization has been fundamental in plant evolution. Nevertheless, the fate of hybrid zones throughout the generations remains poorly addressed. We analyzed a pair of recently diverged, interfertile, and sympatric Petunia species to ask what fate the interspecific hybrid population has met over time. We analyzed the genetic diversity in two generations from two contact sites and evaluated the effect of introgression. To do this, we collected all adult plants from the contact zones, including canonicals and intermediary colored individuals, and compared them with purebred representatives of both species based on seven highly informative microsatellite loci. We compared the genetic diversity observed in the contact zones with what is seen in isolated populations of each species, considering two generations of these annual species. Our results have confirmed the genetic differentiation between the species and the hybrid origin of the majority of the intermediary colored individuals. We also observed a differentiation related to genetic variability and inbreeding levels among the populations. Over time, there were no significant differences per site related to genetic diversity or phenotype composition. We found two stable populations kept by high inbreeding and backcross rates that influence the genetic diversity of their parental species through introgression.

Abstract Phylogenetic niche conservatism can be investigated at multiple scales on an explicit geographical context. Haplotype-based comparative analyses of lineages occupying the same region, and thus subjected to similar environmental factors, allow decoupling shared evolutionary and ecological patterns, as well as multiple dimensions of adaptive diversification. Here we aimed to assess the role of environmental drivers on diversification of subtropical grassland, based on haplotypic diversity of two plant genera. We sampled two closely related and co-distributed grassland plant genera, Petunia and Calibrachoa, across their entire distribution area. Eigenvectors extracted from pairwise distances based on chloroplast DNA haplotypes were used to fit Phylogenetic Signal-Representation (PSR) curves to estimate evolutionary patterns in 19 bioclimatic variables and altitude. The PSR curves showed that altitude, precipitation, and temperature variables changed at different rates with haplotype differentiation. Altitude and temperature traits evolved under conditions closer to a neutral dynamics, whereas precipitation traits differentiated following more complex models. Our results indicated that the diversification in the two genera was more limited by precipitation conditions. Based on these novel findings, we suggest that future studies should test the possible impact of precipitation variables on the process of ecological differentiation in these genera.

ABSTRACT Studying the role of hybridization in the speciation of plants is one of the most thrilling areas of evolutionary biology. Hybridization in natural populations can act in opposition to divergence, contribute to adaptation through introgression or foster the emergence of new lineages via hybrid speciation. Species of the plant genus Petunia grow in open areas in southern South America. Some natural interspecific hybrid events have been described for the genus, such as between the endemic P. exserta and the widespread P. axillaris. Both species occur in sympatry in Serra do Sudeste (Brazil), where they occur in diverse habitats and exhibit floral divergence, which has been related to the attraction of different primary pollinators. The present study evaluates the maintenance of the species boundaries front of hybridization and introgression. Direct and indirect methods of estimating gene exchange employed genotyping 720 reproductive plants and 611 progenies of both species with eight microsatellite loci. Gene exchange was found to be frequent and bidirectional between the species, indicating that introgression changes their genetic constitution in areas of sympatry. Limits of the studied species are being maintained because of the high level of inbreeding and backcrosses that are habitat-dependent.

ABSTRACT Calibrachoa pygmaea is a unique species of Calibrachoa, especially concerning its flower morphology and the environment where it occurs. The species is self-incompatible and is narrowly distributed in wet and flooded fields of the Pampas region. We characterize the genetic diversity of the species based on traditional plastid markers and newly developed nuclear microsatellites to identify drivers that guide its evolution. Our results identified markers that are informative and useful for studying the population structure of C. pygmaea, as well as that of other species of Calibrachoa. Both marker sets were congruent in developing conclusions regarding the evolutionary scenario of C. pygmaea, and revealed that the genetic variability and population structure of the species could be explained by common allele fixation or shared ancestral polymorphism, while its diversification can be attributed mainly to the species’ dispersal ability and certain ecological features.

ABSTRACT Interspecific hybridization has several consequences for parental species, from blurring species limits to the emergence of new taxa. Petunia axillaris and P. exserta occur in sympatry in southernmost Brazil and naturally hybridize despite their different pollination syndromes and habitat requirements. We employed genetic and morphological analyses to characterize two contact zones between the species with the aim of determining the effect of interspecific hybridization. Microsatellite loci and a morphometric evaluation of the corolla shape were used to classify individuals based on their origin as pure parental or hybrids. Corolla color was used to classify individuals a priori (white, red or intermediate, for P. axillaris, P. exserta or hybrid, respectively). Corolla color was found to be a good indicator of the genetic component of each species and their hybrid, while the shape of the corolla did not always correspond to genetic origin. Hybridization increased the variability, and introgression occurred in both directions in this system.

Abstract Calibrachoa is a South-American genus comprising 27 species, several considered endemic or rare; few were subjects in genetic studies. We attempted to generate new data about the phylogenetically related and rare species C. eglandulata, C. sendtneriana, C. serrulata, and C. spathulata concerning their genetic diversity and population structure, which, coupled with their known restricted distribution, could help access their conservation status and contribute to the study of the Brazilian biodiversity. We sequenced 88 individuals for plastid intergenic spacers and genotyped 186 individuals for five microsatellite loci. Compared among each other, C. sendtneriana and C. serrulata presented the highest values of genetic diversity [π% (sd) = 0.23 (0.14) and 0.43 (0.25), respectively], followed by C. spathulata [π% (sd) = 0.19 (0.12)] and C. eglandulata [π% (sd) = 0.02 (0.03)]. Population differentiation was evident for these latter species, whereas it was not significant for C. sendtneriana and C. serrulata. Factors such as habitat specificity and fragmentation, pollination syndrome, and life history could explain the observed patterns. Based on the new genetic data and the species’ biology, a conservation status was assigned for C. sendtneriana and the status of the other three species was reviewed.

ABSTRACT The reproductive system of flowering plants can be highly variable, affecting their biology, gene flow and genetic variability among populations. Petunia secreta is a rare annual endemic species of Pedra do Segredo, located in the municipality of Caçapava do Sul, state of Rio Grande do Sul, Brazil. Although rare, the species possesses a high level of genetic variability. We investigated the reproductive system of P. secreta, including fruit production and seed germinability, in order to determine if its reproductive system can explain its genetic diversity. We sampled five populations and conducted five greenhouse hand-pollination treatments: 1) autonomous apomixis; 2) self-pollination; 3) hand self-pollination; 4) geitonogamy; and 5) cross-pollination. We analysed a total of 40 plants, 468 flowers, and 6,500 seeds. Only autonomous apomixis and self-pollination did not produce fruit. No differences in fruit weight were observed among pollination treatments (P > 0.05). Seeds of two colours were produced, with no differences in germinability. Considering all plants, populations, and treatments, the average germinability was 73 % (range 9 % to 100 %). These results, along with other previous studies, indicate that the reproductive systems of P. secreta, and its large effective population size, can explain its high genetic diversity.

Abstract Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.

Abstract The Tropical Niche Conservatism hypothesis is one of the most relevant theories to explain why tropical diversity is high, although the mechanisms underlying this hypothesis require further clarification. A possible research avenue to address the underlying mechanisms includes determining population-level processes associated with such a hypothesis, in particular by trying to identify how adaptation may occur in extreme niche conditions at the edges of species ranges. However, the determinants of molecular diversity at the edges of geographical distributions of tropical taxa are still poorly known. Here we assessed which environmental variables determine diversity in nuclear and plastid genetic markers for populations of four Passiflora species in the southern limit of their geographical distributions. Climatic factors can drive genetic diversity, and their importance varies according to the marker. The primary predictors are variables representing higher temperatures during cold periods of the year and higher precipitation during dry periods. We concluded that, although these species are present in colder areas at the edge of their range, Tropical Niche Conservatism acts as a restraining force on genetic diversity in southern populations of Passiflora.

Abstract Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.

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.