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miR156/157 plays multiple pivotal roles during plant growth and development. In this study, we identified 11 miR156- and 5 miR157-encoding loci from the genome of Petunia axillaris and Petunia inflata, designated as PaMIR0156/157s and PiMIR0156/157s, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated that PhmiR156/157 was expressed predominantly in cotyledons, germinating seeds, flower buds, young fruits and seedlings. PhmiR156/157 levels declined in shoot apical buds and leaves of petunia before flowering as the plant ages; moreover, the temporal expression patterns of most miR156/157-targeted PhSPLs were complementary to that of PhmiR156/157. Ectopic expression of PhMIR0157a in Arabidopsis and petunia resulted in delayed flowering, dwarf plant stature, increased branches and reduced organ size. However, PhMIR0156f-overexpressing Arabidopsis and petunia plants showed only delayed flowering. In addition, downregulation of PhmiR156/157 level by overexpressing STTM156/157 led to taller plants with less branches, longer internodes and precocious flowering. qRT-PCR analysis indicated that PhmiR156/157 modulates these traits mainly by downregulating their PhSPL targets and subsequently decreasing the expression of flowering regulatory genes. Our results demonstrate that the PhmiR156/157-PhSPL module has conserved but also divergent functions in growth and development, which will help us decipher the genetic basis for the improvement of flower transition, plant architecture and organ development in petunia.

Cytosolic acetyl-CoA is an intermediate of the synthesis of most secondary metabolites and the source of acetyl for protein acetylation. The formation of cytosolic acetyl-CoA from citrate is catalysed by ATP-citrate lyase (ACL). However, the function of ACL in global metabolite synthesis and global protein acetylation is not well known. Here, four genes, PaACLA1, PaACLA2, PaACLB1, and PaACLB2, which encode the ACLA and ACLB subunits of ACL in Petunia axillaris, were identified as the same sequences in Petunia hybrida ‘Ultra’. Silencing of PaACLA1-A2 and PaACLB1-B2 led to abnormal leaf and flower development, reduced total anthocyanin content, and accelerated flower senescence in petunia ‘Ultra’. Metabolome and acetylome analysis revealed that PaACLB1-B2 silencing increased the content of many downstream metabolites of acetyl-CoA metabolism and the levels of acetylation of many proteins in petunia corollas. Mechanistically, the metabolic stress induced by reduction of acetyl-CoA in PaACL-silenced petunia corollas caused global and specific changes in the transcriptome, the proteome, and the acetylome, with the effect of maintaining metabolic homeostasis. In addition, the global proteome and acetylome were negatively correlated under acetyl-CoA deficiency. Together, our results suggest that ACL acts as an important metabolic regulator that maintains metabolic homeostasis by promoting changes in the transcriptome, proteome. and acetylome., PaACL silencing reduced anthocyanin contents, accelerated flower senescence, and changed the proteome and acetylome to maintain metabolic homeostasis in petunia

Floral morphological traits are frequently used to identify species, including those that are closely related and show low genetic diversity, and floral shape and colour are known to play an important role in diversification and species isolation. Floral morphology in Petunia (Solanaceae) is considered a driver of diversification because of its association with pollinators. Here, flower morphology was characterized through morphometric analyses and floral pigments. Our main aim was to determine corolla shape in populations of Petunia axillaris and P. exserta and their natural hybrids and how floral display, size and colour are involved in pollinator attraction. In addition, we investigated floral pigments in P. exserta and different hybrid classes. The results from morphometric analyses revealed that each species has a specific floral shape, independent of the collection site. By contrast, in two contact zones, a mosaic of floral phenotypes was observed with some hybrid classes based on corolla colour being placed close to P. exserta. The results suggest that several generations of hybrids or backcrossing could have given rise to this floral diversity in contact zones.

For ornamental annual bedding plants, flowering performance is critical. Flowering performance includes the length of the flowering period, the longevity of individual flowers, and the number of flowers produced during the flowering period, or flowering capacity. Flowering capacity is a function of several component traits, including the number of branches producing flowers, the number of inflorescences per flowering branch, and the number of flower buds per inflorescence. We employed an F7Petunia axillaris × P. exserta recombinant inbred line population to identify QTL for flowering capacity component traits. The population was phenotyped at 14, 17, and 20° over two years. Fifteen robust QTL (rQTL; QTL detected in two or more temperatures/years) were identified across six of the seven Petunia chromosomes (Chr) for total flower bud number (FlBud), branch number (Branch), flowering branch number (FlBranch), and primary shoot flower bud number (FlBudPS). The largest effect QTL explained up to 28.8, 34.9, 36, and 23.1% of the phenotypic variation for FlBub, FlBudPS, Branch, and FlBranch, respectively. rQTL for FlBud and FlBranch co-localized on Chr 1, and rQTL for FlBud, FlBudPS, and FlBranch co-localized on Chr 4. These regions in particular should be useful for identifying genes controlling flowering capacity of this important ornamental plant.

Petunia axillaris subsp. axillaris and P. axillaris subsp. parodii (Solanaceae) are closely related taxa primarily distinguished by their geographical distribution and floral morphology. Both are found in the southern South American Pampas ecoregion, with the latter also occurring in the Chaco ecoregion; in the Pampas, taxa occupying adjacent areas and intermediary morphologies have been observed. Here, we explored morphological traits and genetic polymorphisms to test the occurrence of hybrid zones between these taxa along the Negro River (Uruguay) and southern Brazil. Morphological data showed variation among the groups, although they could have some overlap. Genetic data also support the differences between them and hybrid zones. These findings suggest that the divergence between P. axillaris subspecies occurred with occasional hybridization in their contact zones.

A lot of researches have been focused on the evolution and function of MYB transcription factors (TFs). For revealing the formation of petunia flower color diversity, MYB gene family in petunia was identified and analyzed. In this study, a total of 155 MYB genes, including 40 1R-MYBs, 106 R2R3-MYBs, 7 R1R2R3-MYBs and 2 4R-MYBs, have been identified in the Petunia axillaris genome. Most R2R3 genes contain three exons and two introns, whereas the number of PaMYB introns varies from 0 to 12. The R2R3-MYB members could be divided into 28 subgroups. Analysis of gene structure and protein motifs revealed that members within the same subgroup presented similar exon/intron and motif organization, further supporting the results of phylogenetic analysis. Genes in subgroup 10, 11 and 21 were mainly expressed in petal, not in vegetative tissues. Genes in subgroup 9, 19, 25 and 27 expressed in all tissues, but the expression patterns of each gene were different. According to the promoter analysis, five R2R3-MYB and two MYB-related genes contained MBSI cis-element, which was involved in flavonoid biosynthetic regulation. PaMYB100/DPL has been reported to positively regulate to pigmentation. However, although PaMYB82, PaMYB68 and Pa1RMYB36 contained MBSI cis-element, their function in flavonoid biosynthesis has not been revealed. Consistent with existing knowledge, PaMYBs in subgroup 11 had similar function to AtMYBs in subgroup 6, genes in which played an important role in anthocyanin biosynthesis. In addition, PaMYB1 and PaMYB40 belonged to P9 (S7) and were potentially involved in regulation of flavonoid synthesis in petunia vegetative organs. This work provides a comprehensive understanding of the MYB gene family in petunia and lays a significant foundation for future studies on the function and evolution of MYB genes in petunia.

Flowering is the first committed step of plant sexual reproduction. While the developing flower is a strong sink requiring large quantity of sugars from photosynthetic source tissues, this process is under-temper-spatially controlled via hormone signaling pathway and nutrient availability. Sugar transporters SUT/SUC and SWEET mediate sugars movement across membranes and play a significant role in various physiological processes, including reproductive organ development. In Petunia axillaris, a model ornamental plant, 5 SUT/SUC and 36 SWEET genes are identified in the current version of the genome. Analysis of their gene structure and chromosomal locations reveal that SWEET family is moderately expanded. Most of the transporter genes are abundantly expressed in the flower than in other organs. During the five flower developmental stages, transcript levels of PaSUT1, PaSUT3, PaSWEET13c, PaSWEET9a, PaSWEET1d, PaSWEET5a and PaSWEET14a increase with the maturation of the flower and reach their maximum in the fully open flowers. PaSWEET9c, the nectar-specific PhNEC1 orthologous, is expressed in matured and fully opened flowers. Moreover, determination of sugar concentrations and phytohormone dynamics in flowers at the five developmental stages shows that glucose is the predominant form of sugar in young flowers at the early stage but depletes at the later stage, whereas sucrose accumulates only in maturated flowers prior to the corolla opening. On the other hand, GA3 content and to a less extent IAA and zeatin decreases with the flower development, however, JA, SA and ABA display a remarkable peak at mid- or later flower developmental stage.

Self-incompatibility in Petunia is controlled by the polymorphic S-locus, which contains S-RNase encoding the pistil determinant and 16-20 S-locus F-box (SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2 -haplotype of the S-locus in Petunia inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S-RNase. Two SLF pseudogenes and 28 potential protein-coding genes were identified, 20 of which were also found at the S-loci of both the S6a -haplotype of P. inflata and the SN -haplotype of self-compatible Petunia axillaris, but not in the S-locus remnants of self-compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses of S-locus sequences of these three S-haplotypes revealed potential genetic exchange in the flanking regions of SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of different S-haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at the S-loci of all three S-haplotypes and in the flanking regions of all S-locus genes examined. We also found evidence of the association of transposable elements with SLF pseudogenes. Based on the hypothesis that SLF genes were derived by retrotransposition, we identified 10 F-box genes as putative SLF parent genes. Our results shed light on the importance of non-coding sequences in the evolution of the S-locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLF genes.

A major bottleneck in plant breeding has been the much limited genetic base and much reduced genetic diversity in domesticated, cultivated germplasm. Identification and utilization of favorable gene loci or alleles from wild or progenitor species can serve as an effective approach to increasing genetic diversity and breaking this bottleneck in plant breeding. This study was conducted to identify quantitative trait loci (QTL) in wild or progenitor petunia species that can be used to improve important horticultural traits in garden petunia. An F7 recombinant inbred population derived between Petunia axillaris and P. exserta was phenotyped for plant height, plant spread, plant size, flower counts, flower diameter, flower length, and days to anthesis in Florida in two consecutive years. Transgressive segregation was observed for all seven traits in both years. The broad-sense heritability estimates for the traits ranged from 0.20 (days to anthesis) to 0.62 (flower length). A genome-wide genetic linkage map consisting of 368 single nucleotide polymorphism bins and extending over 277 cM was searched to identify QTL for these traits. Nineteen QTL were identified and localized to five linkage groups. Eleven of the loci were identified consistently in both years; several loci explained up to 34.0% and 24.1% of the phenotypic variance for flower length and flower diameter, respectively. Multiple loci controlling different traits are co-localized in four intervals in four linkage groups. These intervals contain desirable alleles that can be introgressed into commercial petunia germplasm to expand the genetic base and improve plant performance and flower characteristics in petunia.

The toxicity of nanoparticles in a biological system is an integration of effects arising from surface functionality, particle size, ionic dissolution, etc. This complexity suggests that generalization of a material’s toxicity may be inappropriate. Moreover, from a medicinal point of view, toxicity can be used for treatment of malignant cells, such as cancer. In this study, highly biocompatible carbon nanodots (gCDs) were synthesized by reacting citric acid and urea in glycerol, which resulted in abundant hydroxyl functional groups on the particle surface. gCDs show excitation-dependent photoluminescence but with bright green to yellow emission. Importantly, a series of toxicity assessments showed that as-synthesized gCDs possessed exceptional biocompatibilities to various biological entities including 18 bacteria species, Petunia axillaris seedlings, and Artemia franciscana nauplii. Furthermore, the particles were shown to have low to no toxic effects on human embryonic kidney (HEK-293), breast (MCF-7), and oral squamous (CAL-27) carcinoma cell lines. Of particular interest, the gCDs displayed antiproliferative activities against ovarian choriocarcinoma cells (JAr/Jeg-3 cell lines), which may be further explored for cancer drug discovery.

Palynological studies on ten species of angiosperms family namely Hibiscus rosa- sinensis L . (Malvaceae), Lilium candidum L. (Liliaceae), Petunia axillaris (Lam.) B.S.P.(Solanaceae) and seven species from family asteraceae namely Artemisia indica Willd. , Aster ageratoides Kitam., Calendula offcinalis L., Cerpis japonica L. (Benth.), Sonchus asper (L.) Hill , Tagetus patula L., and Taraxicum officinale F.H.Wigg. was carried out. Result obtained from this investigation showed that pollen shape is spheroidal in Artemisia indica, Aster ageratoides, Calendula offcinalis, Hibiscus rosa- sinensis and P. axillaris; elliptical in L. candidum , hexagonal in S. asper and pentagonal in T. officinale ; tricolpater in C.japonica. Pollen aperture is porate in all except in L.candidum where it is elliptic. Pollen wall is echinate in H.rosa-sinensis, A.ageratoides, T. patula; spinulate in P. axillaries, T. officinale, S. asper, C. japonica, C. officinalis and T. officinale and smooth in L. candidum . The general appearance of the pollen grain is circular in P . axillaries but longer than wide in L.candidum . The circular, echinate, large and triporate pollens seem to be primitive ones and spharoidal, hexaporate features have been regarded as comparatively advanced ones. Polyporate pollens are considered to be secondarily derived. The nature of the pollen grain in these species could be evolutionary significance. The similarities in structure of the pollen grain showed the inter- species relationships among the species and that’s why put in same family while the differences in structures showed reasons for them to exist as distinct species. Only one species namely L.candidum from monocot family (Liliaceae) included in this study. Int. J. Appl. Sci. Biotechnol. Vol 5(3): 361-365

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato (Solanum lycopersicum), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3', and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petuniaaxillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris, tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris, PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3' position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3' position in tomato, is absent in P. axillaris Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis.

Galactose (Gal) is incorporated into cell wall polysaccharides as flowers open, but then is lost because of β-galactosidase activity as flowers mature and wilt. The significance of this for flower physiology resides in the role of galactan-containing polysaccharides in the cell wall, which is still largely unresolved. To investigate this, transcript accumulation of six cell wall-associated β-galactosidases was simultaneously knocked down in ‘Mitchell’ petunia (Petunia axillaris x (P. axillaris x P. hybrida)) flower petals. The multi-PhBGAL RNAi construct targeted three bud- and three senescence-associated β-galactosidase genes. The petals of the most down-regulated line (GA19) were significantly disrupted in galactose turnover during flower opening, and at the onset of senescence had retained 86% of their galactose compared with 20% in the controls. The Gal content of Na2CO3-soluble cell wall extracts and the highly insoluble polysaccharides associated with cellulose were particularly affected. Immunodetection with the antibody LM5 showed that much of the cell wall Gal in GA19 was retained as galactan, presumably the side-chains of rhamnogalacturonan-I. The flowers of GA19, despite having retained substantially more galactan, were no different from controls in their internal cell arrangement, dimensions, weight or timing of opening and senescence. However, the GA19 petals had less petal integrity (as judged by force required to cause petal fracture) after opening and showed a greater decline in this integrity with time than controls, raising the possibility that galactan loss is a mechanism for helping to maintain petal tissue cohesion after flower opening.

Petunia, a commercially important ornamental plant worldwide, has been subjected to breeding programs that have yielded a high number of varieties. One of the key factors in the commercial value of these varieties is plant compactness. Currently, compact petunias are obtained through the application of expensive, harmful and short-lasting chemicals. To avoid the use of these chemicals, transgenic plants that over-express dwarf-inducing genes have been recently proposed as an alternative, but the current legislation regarding transgenic plants restricts their commercialization. In this work, we studied the effect of polyploidization in the plant architecture of Petunia axillaris, an Argentine native petunia. We developed a new polyploidization protocol that consisted in culturing petunia leaves in RL medium (MS medium supplemented with 30 g l-1 sucrose, 1mg l-1 BA and 0.2 mg l-1 IAA) supplemented with 0.2 g l-1 colchicine for 15 days. This protocol allowed the regeneration of stable autotetraploid petunias (polyploidization rate: 29.0 ± 8,2%), which were 54% more compact than the diploid ones. Furthermore, they exhibited no variations in agronomical traits compared to the initial genotypes, except for a short delay in blooming. These autotetraploid plants can be used in different breeding programs and the polyploidization method developed can be tested in others cultivars of the genus Petunia for the same purpose. Fil: Regalado González, Jose Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Carmona Martín, E.. Consejo Superior de Investigaciones Científicas; España Fil: Querol, V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Vélez, Carlos Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina Fil: Encina, C. L.. Consejo Superior de Investigaciones Científicas; España Fil: Pitta-Alvarez, Sandra Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Micología y Botánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Micología y Botánica; Argentina

Plant hybridization is frequently observed in nature and considered an important driver of angiosperm diversity. Species are thought to arise through the accumulation of morphological and genetic differences that promote their evolutionary independence, even in the presence of hybridization. Natural hybrid zones yield an excellent system to study the outcomes of hybridization in terms of species diversity. Two recently diverged species, Petunia axillaris and P. exserta, show floral differentiation attributed to attraction of varying pollinators. Previous studies suggested natural hybridization between these species to explain morphological floral polymorphisms found among individuals of P. exserta. Here, we analyse genetic and morphological diversity in plants from the contact zone between these species and from isolated populations of each species to evaluate natural hybridization and its consequences. We found that the species' integrity is maintained despite interspecific hybridization and introgression that drove the origin of a new lineage in P. exserta in the contact zones.

Angiosperm petal fusion (sympetaly) has evolved multiple times independently and is associated with increased specificity between plants and their pollinators. To undercover developmental genetic changes that might have led to the evolution of sympetaly in the asterid core eudicot genus Petunia (Solanaceae), we carried out global and fine-scale gene expression analyses in different regions of the corolla. We found that, despite several similarities with the choripetalous model species Arabidopsis thaliana in the proximal-distal transcriptome, the Petunia axillaris fused and proximal corolla tube expresses several genes that in A. thaliana are associated with the distal petal region. This difference aligns with variation in petal shape and fusion across ontogeny of the two species. Moreover, differential gene expression between the unfused lobes and fused tube of P. axillaris petals revealed three strong candidate genes for sympetaly based on functional annotation in organ boundary specification. Partial silencing of one of these, the HANABA TARANU (HAN)-like gene PhGATA19, resulted in reduced fusion of Petunia hybrida petals, with silencing of both PhGATA19 and its close paralog causing premature plant senescence. Finally, detailed expression analyses for the previously characterized organ boundary gene candidate NO APICAL MERISTEM (NAM) supports the hypothesis that it establishes boundaries between most P. axillaris floral organs, with the exception of boundaries between petals.

Jasmonate ZIM-domain (JAZ) family proteins are the key repressors in the jasmonate signaling pathway and play crucial roles in plant development, defenses, and responses to stresses. However, our knowledge about the JAZ protein family in petunia is limited. This research respectively identified 12 and 16 JAZ proteins in two Petunia progenitors, Petunia axillaris and Petunia inflata. Phylogenetic analysis showed that the 28 proteins could be divided into four groups (Groups A&ndash, D) and further classified into six subgroups (A1, A2, B1, B3, C, and D1), members in the same subgroup shared some similarities in motif composition and sequence structure. The Ka/Ks ratios of seven paralogous pairs were less than one, suggesting the petunia JAZ family might have principally undergone purifying selection. Quantitative real-time PCR (qRT-PCR) analysis revealed that PaJAZ genes presented differential expression patterns during the development of flower bud and anther in petunia, and the expression of PaJAZ5, 9, 12 genes was generally up-regulated after MeJA treatment. Subcellular localization assays demonstrated that proteins PaJAZ5, 9, 12 were localized in nucleus. Yeast two hybrid (Y2H) elucidated most PaJAZ proteins (PaJAZ1-7, 9, 12) might interact with transcription factor MYC2. This study provides insights for further investigation of functional analysis in petunia JAZ family proteins.

Transmitting tissue-specific proteins (TTS proteins) are abundant in the extracellular matrix of Nicotiana pistils, and vital for optimal pollen tube growth and seed set. We have identified orthologs from several species in the Solanaceae, including Petunia axillaris axillaris and Petunia integrifolia. We refer to TTS proteins and their orthologs as histidine domain-arabinogalactan proteins (HD-AGPs). HD-AGPs have distinctive domains, including a small histidine-rich region and a C-terminal PAC domain. Pairwise comparisons between HD-AGPs of 15 species belonging to Petunia, Nicotiana, and Solanum show that the his-domain and PAC domain are under purifying selection. In contrast, a proline-rich domain (HV2) is conserved among cross-hybridizing species, but variant in species-pairs that are reproductively isolated by post-pollination pre-fertilization reproductive barriers. In particular, variation in a tetrapeptide motif (XKPP) is systematically correlated with the presence of an interspecific reproductive barrier. Ka/Ks ratios are not informative at the infrageneric level, but the ratios reveal a clear signature of positive selection on two hypervariable domains (HV1 and HV2) when HD-AGPs from five solanaceous genera are compared. We propose that sequence divergence in the hypervariable domains of HD-AGPs reinforces sympatric speciation in incipient species that may have first diverged as a consequence of pollinator preferences or other ecological factors.

Most animal-pollinated plants produce nectar as a pollinator reward. Despite the main role that nectar plays in plant-pollinator interactions, the impact of natural variation in nectar traits on realized male fitness is poorly known. Here, we assessed this relation for a wild Petunia axillaris population using paternity-based direct selection gradient analysis, which allowed us also to infer pollen dispersal patterns. Because male fecundity may depend on other traits which could be associated with nectar characteristics (i.e. volume and concentration), we also considered selection on other key reproductive traits. The analysis revealed that P. axillaris was a strict outcrosser, but that successful pollination occurred mainly among neighbours. Individual plants varied greatly in their male fecundity. Nectar concentration, a key feature of nectar that determines its profitability, was subjected to stabilizing selection. Selection through male function also affected corolla area (positive directional selection), corolla tube length (negative directional selection), and floral display size (stabilizing selection), but none of these traits were phenotypically correlated with nectar characteristics. Because nectar concentration affects the ability and foraging efficiency of different flower visitors to feed on nectar, stabilizing selection may reflect either the preference of the most effective pollinators, or antagonistic selection driven by pollinators and non-pollinating nectar consumers. Fil: Gleiser, Gabriela Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; Argentina. University of Neuchâtel; Suiza Fil: Internicola, Antonina I.. University of Neuchâtel; Suiza Fil: Austerlitz, Frédéric. Centre National de la Recherche Scientifique; Francia Fil: Bernasconi, Giorgina. University of Neuchâtel; Suiza

Os padrões de distribuição da diversidade genética são moldados por um conjunto de diferentes fatores que influenciam a dinâmica evolutiva dos taxa. Entender quais processos evolutivos levaram à diferenciação entre populações e à manutenção de linhagens genéticas isoladas permite a compreensão do papel de diferentes forças atuando na manutenção da história evolutiva das espécies e um melhor entendimento dos processos de diversificação. O objetivo geral desta tese foi identificar e compreender os processos de diversificação que moldaram a diversidade nos campos do Sul da América do Sul através da variabilidade genética de Petunia axillaris subsp. parodii, táxon amplamente distribuído na região estudada. Os objetivos específicos deste trabalho foram (a) inferir como as oscilações climáticas do Pleistoceno influenciaram a dinâmica populacional e a história evolutiva do complexo P. axillaris, (b) investigar as potenciais zonas híbridas entre P. axillaris subsp. parodii e P. P. axillaris subsp. axillaris, buscando evidências genéticas e morfológicas de migração e/ou hibridação entre estes taxa, e (c) identificar linhagens evolutivas de P. axillaris subsp. parodii e compreender quais processos influenciaram a dinâmica evolutiva da diferenciação destas linhagens. Para tanto, realizamos experimentos e análises envolvendo a detecção de polimorfismos genéticos e dados ecológicos através das seguintes técnicas: modelagem de nicho ecológico e identificação de biorregiões, amplificação e análise de loci de microssatélites e sequenciamento de ampla cobertura do genoma. Os resultados obtidos foram divididos em três artigos científicos, contemplando cada um dos objetivos específicos da tese, e permitiram identificar como os eventos climáticos do Pleistoceno moldaram os padrões de diversificação dos campos do Sul da América do Sul através de processos ecológicos, eventos de hibridação e quebras filogeográficas. Essa tese trouxe evidências para o entendimento dos mecanismos de diversificação e especiação em plantas do Cone Sul, além de abrir perspectivas de trabalhos futuros. A set of different factors influences the evolutionary dynamics of taxa and shapes the genetic diversity distribution. Understanding which evolutionary processes led to differentiation between populations and the maintenance of isolated genetic lineages allows the comprehension of the evolutionary forces acting on the diversification processes. The main aim of this thesis was to identify and understand witch diversification processes shaped the diversity in the grasslands of the southern South America through the genetic variability of Petunia axillaris subsp. parodii, a widely distributed taxon from this region. The specifics goals of this study were (a) to infer how Pleistocene climate fluctuations influenced on the population dynamics and evolutionary history of the P. axillaris complex, (b) to investigate the potential hybrid zones between P. axillaris subsp. parodii and P. axillaris subsp. axillaris, searching for genetic and morphological evidences of migration and/or hybridization between these taxa, and (c) to identify P. axillaris subsp. parodii evolutionary lineages and understand which processes influenced on the evolutionary dynamics of the differentiations among those lineages. To do that, we performed experiments and analyzes involving the detection of genetic polymorphisms and ecological data through the following techniques: ecological niche modeling and bioregion identification; amplification and analysis of microsatellite loci; and broad genome sequencing. The results obtained were divided into three scientific articles, covering each of the specific goals and allowed to identify how the Pleistocene climatic events shaped the diversification patterns of the southern South American fields through ecological processes, climate events, hybridization, and phylogeographic breaks. This thesis has provided some further evidence on the mechanisms of diversification and speciation in plants from the Southern Cone and also opened some perspectives for future works.

Pollination success of deceptive orchids is affected by the density and distribution of nectar providing plant species and overall plant density. Here we extended the framework of how plant density can affect pollination to examine how it may promote the success of plant intraspecific cheaters. We compared hawkmoth behaviour in two native populations of Petunia axillaris, where we simultaneously offered rewarding and manually depleted P. axillaris. We asked whether pollinator foraging strategies change as a function of plant density and whether such changes may differentially affect nectarless plants. We observed the first choice and number of flowers visited by pollinators and found that in the dense population, pollinators visited more flowers on rewarding plants than on nectar-depleted plants. In the sparse population, such discrimination was absent. As we found no differences in nectar volume between plants of the two populations, the observed differences in plant density may be temporal. We reason that if differences were more permanent, an equivalent of the remote habitat hypothesis prevails: in a sparse population, cheating plants benefit from the absence of inter- and intraspecific competitors because pollinators tend to visit all potential resources. In a denser population, a pollinator’s optimal foraging strategy involves more selectivity. This would cause between-plant competition for pollinators in a pollinator-limited context, which applies to most hawkmoth-pollinated systems. We propose that nectar-provisioning of plants can be density-dependant, with cheaters able to persist in low density areas.

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants.