Microevolución de esclerófilas mediterráneas : diferenciación fenotípica y genética en Quercus coccifera L. y Olea europaea L

La esclerofilia es un síndrome característico de muchas especies mediterráneas. Aparece en grupos taxonómicos diversos, con aparente independencia de su origen filogenético o del grupo funcional al que pertenezcan. Históricamente, se ha considerado como el resultado de la adaptación a las condiciones propias del ambiente mediterráneo. Actualmente sin embargo está bien establecido su origen Terciario, anterior al advenimiento del clima mediterráneo. Cabe entonces plantarse cuál es la habilidad de las plantas esclerófilas para expresar fenotipos alternativos en respuesta a cambios ambientales, espaciales y/o temporales y en qué medida estos fenotipos alternativos con síntoma de microevolución. En la presente Memoria Doctoral se ha abordado el estudio de procesos microeevolutivos en especies esclerófilas a través del análisis de la estructura genética y fenotípica de dos especies emblemáticas del mediterráneo, cómo son la coscoja (Quercus Coccifera L.) y el acebuche (Olea europaea var. Sylvestris L.). Los posibles mecanismos causales del cambio evolutivo se han tomado en consideración de forma independiente y sintética intentando dar respuesta a una serie de interrogantes: ¿En qué medida la estabilidad fenotípica implica una falta de diversificación genética? O de otra manera ¿es posible detectar procesos de divergencia genética en esclerófilas mediterráneas? Si la respuesta es afirmativa ¿Cuáles son?. La aparente estabilidad evolutivo del síndrome esclerófilo ¿supone la no existencia de procesos de divergencia fenotípica entre poblaciones?. Si estos procesos existen ¿ha jugado en ellos algún papel la plasticidad fenotípica? ¿Es la plasticidad fenotípica un factor importante en la respuesta evolutiva de las esclerófilas mediterráneas?. Los resultados obtenidos demuestran que las plantas esclerófilas han sufrido procesos de divergencia microevolutiva recientes. Estos procesos han desembocado en la formación de ecotipos diferenciados genéticamente y que presentan rasgos funcionales distintos. La diferenciación tiene lugar en un contexto genético en el que la introgresión está generalizada y el flujo génico es relativamente frecuente. Uno de los elementos vinculados con la aparición de nuevos ecotipos es la variación en la plasticidad fenotípica. Diferenciación genética de esclerólias mediterráneas: En las dos especies estudiadas se han producido procesos de diferenciación fenotípica recientes. La diferenciación fenotípica observada correlaciona con los patrones genéticos. La congruencia entre fenotipo y genotipo, aunque esperable, no deja de ser interesante, ya que de muestra que los fenotipos observados son ecotipos fijados genéticamente. Así, diferentes situaciones ambientales tienen su reflejo en la estructura fenotípica de las poblaciones estudiadas, que a su vez está condicionada genéticamente. La modificación del fenotipo de respuesta a la luz a de O.europaea y Q.coccifera supone la aparición de dos tipos de hojas en la copa de cada individuo. El primero de ellos es el característico de las hojas de las partes más expuestas de la copa (fenotipo Sol), mientras que el otro se genera en la zona más interior de la misma (fenotipo Sombra). El papel de la plasticidad fenotípica: La correlación observada en contextos genéticos y ambientales distintos entre patrones moleculares, fenotípicos y ecológicos pone de relieve la capacidad de las esclerófilas mediterráneas para generar nuevos ecotipos. La plasticidad aparece como el agente causal de la aparición de estos nuevos ecotipos y cómo responsable del mantenimiento del síndrome esclerófilo

A set of primers for length and nucleotide-substitution polymorphism in chloroplastic DNA of Olea europaea L. (Oleaceae)

Chloroplastic DNA (cpDNA) variation at five microsatellite motifs, two insertion-deletion sites, and eight nucleotide substitution sites was investigated in the Olea europaea complex. Primers were designed for flanking regions of these sites to amplify short cpDNA regions. They provided polymorphism when polymerase chain reaction (PCR) products from a representative sample of 128 O. europaea individuals were either resolved by size into polyacrylamide gels (length polymorphism) or digested with restriction enzymes (nucleotide-substitution polymorphism). These polymorphisms serve to distinguish most of the cytoplasmic haplotypes previously recognized. Potential application of these markers in O. europaea includes phylogeography, conservation and germplasm identification, even when using poorly preserved material from herbarium specimens or forensic and archaeological materials.This work was supported by the project BIOD-IBERIA (A82).Peer Reviewe

Phylogenetics of Olea (Oleaceae) based on plastid and nuclear ribosomal DNA sequences: Tertiary climatic shifts and lineage differentiation times

Background and Aims The genus Olea (Oleaceae) includes approx. 40 taxa of evergreen shrubs and trees classified in three subgenera, Olea, Paniculatae and Tetrapilus, the first of which has two sections (Olea and Ligustroides). Olive trees (the O. europaea complex) have been the subject of intensive research, whereas little is known about the phylogenetic relationships among the other species. To clarify the biogeographical history of this group, a molecular analysis of Olea and related genera of Oleaceae is thus necessary. Methods A phylogeny was built of Olea and related genera based on sequences of the nuclear ribosomal internal transcribed spacer-1 and four plastid regions. Lineage divergence and the evolution of abaxial peltate scales, the latter character linked to drought adaptation, were dated using a Bayesian method. Key Results Olea is polyphyletic, with O. ambrensis and subgenus Tetrapilus not sharing a most recent common ancestor with the main Olea clade. Partial incongruence between nuclear and plastid phylogenetic reconstructions suggests a reticulation process in the evolution of subgenus Olea. Estimates of divergence times for major groups of Olea during the Tertiary were obtained. Conclusions This study indicates the necessity of revising current taxonomic boundaries in Olea. The results also suggest that main lines of evolution were promoted by major Tertiary climatic shifts: (1) the split between subgenera Olea and Paniculatae appears to have taken place at the Miocene-Oligocene boundary; (2) the separation of sections Ligustroides and Olea may have occurred during the Early Miocene following the Mi-1 glaciation; and (3) the diversification within these sections (and the origin of dense abaxial indumentum in section Olea) was concomitant with the aridification of Africa in the Late Miocen

Lack of ITS sequence homogenization in Erysimum species (Brassicaceae) with different ploidy levels

Funding This research is supported by grants from FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento A-RNM-505-UGR18 and P18-FR-3641. This research was also funded by the Spanish Ministry of Science and Innovation (CGL2013-47558-P and PID2021-126456NB-C22), including EU FEDER funds. COM was supported by the Ministry of Economy and Competitiveness (BES-2014-069022). This is a contribution to the Research Unit Modeling Nature, funded by the Consejería de Economía, Conocimiento, Empresas y Universidad, and European Regional Development Fund (ERDF), reference QUALIFICA 00011.Acknowledgements The authors thank the Tatiana López Pérez and the Evoflor group for helping us during several phases of the study. We also thank the Sierra Nevada National Park headquarters for providing access to sampling in the National Park.The internal transcribed spacers (ITS) exhibit concerted evolution by the fast homogenization of these sequences at the intragenomic level. However, the rate and extension of this process are unclear and might be conditioned by the number and divergence of the different ITS copies. In some cases, such as hybrid species and polyploids, ITS sequence homogenization appears incomplete, resulting in multiple haplotypes within the same organism. Here, we studied the dynamics of concerted evolution in 85 individuals of seven plant species of the genus Erysimum (Brassicaceae) with multiple ploidy levels. We estimated the rate of concerted evolution and the degree of sequence homogenization separately for ITS1 and ITS2 and whether these varied with ploidy. Our results showed incomplete sequence homogenization, especially for polyploid samples, indicating a lack of concerted evolution in these taxa. Homogenization was usually higher in ITS2 than in ITS1, suggesting that concerted evolution operates more efficiently on the former. Furthermore, the hybrid origin of several species appears to contribute to the maintenance of high haplotype diversity, regardless of the level of ploidy. These findings indicate that sequence homogenization of ITS is a dynamic and complex process that might result in varying intra- and inter-genomic diversity levels.FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento A-RNM-505-UGR18 and P18-FR-3641Spanish Ministry of Science and Innovation (CGL2013-47558-P and PID2021-126456NB-C22)Ministry of Economy and Competitiveness (BES-2014-069022)Research Unit Modeling Nature, funded by the Consejería de Economía, Conocimiento, Empresas y Universidad, and European Regional Development Fund (ERDF), reference QUALIFICA 0001

Genomic Resources for Erysimum spp. (Brassicaceae): Transcriptome and Chloroplast Genomes

Funding was provided by the Spanish Ministry of Science and Competitiveness (CGL2016-79950-R; CGL2017-86626-C22-P), including FEDER funds. This research was also funded by the Consejeria de Economia, Conocimiento, Empresas y Universidad, and European Regional Development Fund (ERDF), ref. SOMM17/6109/UGR and A-RNM-505-UGR18. COM was supported by the Ministry of Economy and Competitiveness (BES-2014-069022).We are grateful to Modesto Berbel Cascales and José M. Gómez for their help in sampling and DNA/RNA extractions.Erysimum (Brassicaceae) is a genus of more than 200 species (Al-Shehbaz, 2012). It is widely distributed in the Northern Hemisphere and has been the focus of active research in ecology, evolution, and genetics (Gómez and Perfectti, 2010; Gómez, 2012; Valverde et al., 2016). Despite long-standing interest in Erysimum, its taxonomy has yet to be properly established, partly due to a complex and reticulated evolutionary history that renders phylogenetic reconstructions highly challenging (Ancev, 2006; Marhold and Lihová, 2006; Abdelaziz et al., 2014; Gomez et al., 2014; Moazzeni et al., 2014; Züst et al., 2020). The Baetic Mountains (South-Eastern Iberia) are among the most critical glacial refugia in Europe. The waxing and waning of plant populations following climatic fluctuations have likely complicated the distribution and genetic variation of extant diversity in this region. Isolation and posterior secondary contact between taxa may have favored hybridization and introgression (Médail and Diadema, 2009). The Erysimum species that inhabit these mountains have been a particularly fruitful system for plant evolutionary ecology [e.g., Gómez et al., 2006, 2008; Gómez and Perfectti, 2010; Gómez, 2012; Valverde et al., 2016]. However, the relationships among these species remain unresolved, hampering comparative and evolutionary studies. Genome duplications, incomplete lineage sorting, and hybridization have compromised the phylogenetic reconstructions within Erysimum (Marhold and Lihová, 2006; Osuna-Mascaró, 2020). Additionally, clarifying this group's complex evolution requires extensive genomic resources, which are currently being produced but are mostly lacking. The fast development of high-throughput sequencing technologies has led to a rapid increase in genomic and transcriptomic for many plant species (Dong et al., 2004; Duvick et al., 2007; Sundell et al., 2015; Boyles et al., 2019). However, obtaining complete genome sequencing remains a challenge with large, repetitive-DNA enriched genomes. Transcriptome sequencing is comparatively more accessible, providing a relatively cheap and fast method to obtain large amounts of functional genomic data (Timme et al., 2012; Yang and Smith, 2013; Wickett et al., 2014; Léveillé-Bourret et al., 2017). Accordingly, global initiatives such as the 1,000 plants (1KP) project have generated transcriptomic resources for over 1,000 plant species (Matasci et al., 2014; Leebens-Mack et al., 2019). In addition, the use of RNA-Seq could be useful in obtaining complete chloroplast genomes in a reliable and accessible way, making possible the use of complete molecules in phylogenomic analyses (Smith, 2013; Osuna-Mascaró et al., 2018; Morales-Briones et al., 2021). Here, we report the annotation of 18 floral transcriptomes assembled de novo from total RNA-Seq libraries and nine chloroplast genomes from seven Erysimum species inhabiting the Baetic Mountains. The chloroplast genomes were assembled from total RNA-Seq data following a previously-validated reference assemble approach (Osuna-Mascaró et al., 2018). The data presented here represent reliable genomic resources for transcriptomic, proteomic, and phylotranscriptomic studies. These data contribute to the ecological and genetic resources available for Brassicaceae in general and the genus Erysimum in particular, being the only genomic resources for these species coming from flower buds.Spanish Ministry of Science and Competitiveness CGL2016-79950-R CGL2017-86626-C22-PEuropean CommissionConsejeria de Economia, Conocimiento, Empresas y UniversidadEuropean Commission SOMM17/6109/UGR A-RNM-505-UGR18Ministry of Economy and Competitiveness BES-2014-06902

Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses

Background Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. Methods Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. Key Results Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. Conclusions This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europae