Genome-wide association analysis for fumonisin content in maize kernels

Background: Plant breeding has been proposed as one of the most effective and environmentally safe methods to control fungal infection and to reduce fumonisin accumulation. However, conventional breeding can be hampered by the complex genetic architecture of resistance to fumonisin accumulation and marker-assisted selection is proposed as an efficient alternative. In the current study, GWAS has been performed for the first time for detecting high-resolution QTL for resistance to fumonisin accumulation in maize kernels complementing published GWAS results for Fusarium ear rot. Results: Thirty-nine SNPs significantly associated with resistance to fumonisin accumulation in maize kernels were found and clustered into 17 QTL. Novel QTLs for fumonisin content would be at bins 3.02, 5.02, 7.05 and 8.07. Genes with annotated functions probably implicated in resistance to pathogens based on previous studies have been highlighted. Conclusions: Breeding approaches to fix favorable functional variants for genes implicated in maize immune response signaling may be especially useful to reduce kernel contamination with fumonisins without significantly interfering in mycelia development and growth and, consequently, in the beneficial endophytic behavior of Fusarium verticillioides.Xunta de Galicia | Ref. IN607A/013Ministerio de Economía | Ref. AGL2015–67313-C2–1-RMinisterio de Economía | Ref. AGL2015–67313-C2–2-

Environmental factors related to fungal infection and fumonisin accumulation during the development and drying of white maize kernels

In Southern Europe where whole maize kernels are ground and used for making bread and other food products, infection of the kernels by Fusarium verticillioides and subsequent fumonisin contamination pose a serious safety issue. The influence of environmental factors on this fungal infection and mycotoxin accumulation as the kernel develops has not been fully determined, especially in such food grade maize. The objectives of the present study were to determine which environmental factors may contribute to kernel invasion by F. verticillioides and fumonisin accumulation as kernels develop and dry in naturally infected white maize. Three maize hybrids were planted at two different sowing dates and kernel samples were collected 20, 40, 60, 80 and 100. days after silking. The percentage of kernels infected, and ergosterol and fumonisin contents were recorded for each sampling. F. verticillioides was the most prevalent species identified as the kernels developed. Temperature and moisture conditions during the first 80. days after silking favored natural kernel infection by F. verticillioides rather than by Aspergillus or Penicillium species. Fumonisin was found in kernels as early as 20. days after silking however significant fumonisin accumulation above levels acceptable in the EU did not occur until after physiological maturity of the kernel indicating that kernel drying in the field poses a high risk. Our results suggest that this could be due to increasing kernel damage by insects that favor fungal development, such as the damage by the moth Sitotroga cerealella, and to the occurrence of stress conditions for F. verticillioides growth that could trigger fumonisin biosynthesis, such as exposure to suboptimal temperatures for growth simultaneously with low water activity. © 2013 Elsevier B.V.MICINNSpanish Council of ResearchAutonomous Government of GaliciaEuropean Social FundThis research was supported by the National Plan for Research and Development of Spain (AGL2009-12770). A. Cao acknowledges funding from the JAE Program of the Spanish Council of Research. R. Santiago acknowledges postdoctoral contract “Isidro Parga Pondal” supported by the Autonomous Government of Galicia and the European Social Fund.Peer Reviewe

Defensive changes in maize leaves induced by feeding of Mediterranean corn borer larvae

[Background] Plants can respond to insect attack via defense mechanisms that reduce insect performance. In this study, we examined the effects of several treatments applied to two maize genotypes (one resistant, one susceptible) on the subsequent growth and survival of Sesamia nonagrioides Lef. (Mediterranean corn borer, MCB) larvae. The treatments were infestation with MCB larvae, application of MCB regurgitant upon wounding, wounding alone, or exposure to methyl jasmonate, and they were applied at the V6–V8 stage of maize development. We also monitored changes in the concentrations of compounds known to be involved in constitutive resistance, such as cell wall-bound hydroxycinnamates and benzoxazinoids.[Results] In both maize genotypes, the leaves of plants pre-infested with MCB larvae were less suitable for larval development than those from untreated plants. Application of MCB regurgitant upon wounding, and wounding itself, resulted in leaf tissues becoming less suitable for larval growth than those of pre-infested plants, suggesting that there could be herbivore-associated effector molecules that suppress some wounding responses. A single application of MCB regurgitant did not seem to mimic feeding by MCB larvae, although the results suggested that regurgitant deposited during feeding may have enhanced ferulates and diferulates synthesis in infested vs. control plants. Jasmonic acid may play a role in mediating the maize response to MCB attack, but it did not trigger hydroxycinnamate accumulation in the leaves to a level comparable to that induced by larval leaf feeding. The EP39 maize genotype showed an increase in leaf cell wall strength by increasing hemicellulose cross-linking in response to MCB attack, while induced defenses in the EP42 plants appeared to reflect a broader array of resistance mechanisms.[Conclusions] The results indicated that leaf feeding by MCB larvae can increase leaf antibiosis against MCB in two maize genotypes with contrasting levels of resistance against this borer. Also, the larval regurgitant played a positive role in eliciting a defense response. We determined the effects of the plant response on larval growth, and detected defense compounds related to borer resistance.This research was supported by the National Plan for Research and Development of Spain (AGL2012-33415, AGL2015-67313-C2-1-R, and AGL2015-67313-C2-2-R) co-financed by the European Social Fund (FEDER). R. Santiago acknowledges postdoctoral contract “Ramón y Cajal” financed by the Ministry of Economy and Competitiveness (Spain), Vigo University, and the European Social Fund.Peer reviewe

Germline variation at 8q24 and prostate cancer risk in men of European ancestry

Chromosome 8q24 is a susceptibility locus for multiple cancers, including prostate cancer. Here we combine genetic data across the 8q24 susceptibility region from 71,535 prostate cancer cases and 52,935 controls of European ancestry to define the overall contribution of germline variation at 8q24 to prostate cancer risk. We identify 12 independent risk signals for prostate cancer (p < 4.28 × 10−15), including three risk variants that have yet to be reported. From a polygenic risk score (PRS) model, derived to assess the cumulative effect of risk variants at 8q24, men in the top 1% of the PRS have a 4-fold (95%CI = 3.62–4.40) greater risk compared to the population average. These 12 variants account for ~25% of what can be currently explained of the familial risk of prostate cancer by known genetic risk factors. These findings highlight the overwhelming contribution of germline variation at 8q24 on prostate cancer risk which has implications for population risk stratification

Fine-mapping of prostate cancer susceptibility loci in a large meta-analysis identifies candidate causal variants

Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Peer reviewe

Prevención da contaminación con fumonisinas no millo

Trabajo de investigación para optar el grado de Doctor por la Universidad de Vigo.- 198 páginas.[EN] Fusarium species are the most common moulds found in cereal crops in temperate regions. Many of these species produce mycotoxins, a diverse group of compounds that are toxic to humans and animals. Fumonisins are a family of mycotoxins found mainly in maize. They are produced mostly by F. verticillioides and F. proliferatum. Fumonisins were classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC), and maximum levels for fumonisin content in food and feed have been setting in the European Union and other countries. In Galicia, even though weather is favorable for fungal growth, there has been scarce research on the occurrence of mycotoxigenic fungi or contamination with mycotoxin in maize or other cereals. Currently, there is no specific information on the environmental and/or genotypic factors that determine fungal infection and fumonisin content in kernels at harvest. This knowledge is important and necessary when assessing the risk and make decisions to prevent fumonisin contamination. For this reason, in this study we investigated what these factors are, the weight of each of them and at what times during the growing season are critical for infection by F. verticillioides and fumonisin contamination of maize kernels. It has also been determined the occurrence of Fusarium species on maize kernels to assess the risk of contamination with other mycotoxins. Moreover, the influence of environmental factors in the process of fungal infection and fumonisin accumulation throughout the physiological development and drying of maize kernels has been studied. Finally, we evaluated the resistance to fumonisins accumulation four white corn hybrids after artificial inoculation with a local isolate of F. verticillioides.[EN] As a result of this study, we can confirm that F. verticillioides is the most abundant Fusarium species and the main producer of fumonisins in maize kernels grown in Pontevedra. Other Fusarium species were found in smaller percentage, showing there is a potential risk of contamination with other mycotoxins. Late sowings and early harvests are recommended to reduce contamination with fumonisins because they were less favorable for infection by F. verticillioides, fungal development and fumonisins accumulation in maize kernels. Fumonisin contamination was mainly influenced by environmental factors during flowering and kernel drying. Special attention should be given when weather during flowering are drier and warmer. Heavy rainfall before harvest and greatest kernel damage produced by insects should be avoided by performing earlier harvests. The use of maize varieties resistant to the Angoumois grain moth, to corn borers and with good husk coverage can be a useful tool to reduce the risk of contamination with fumonisins. Throughout kernel development, the significant increase of fumonisin content took place from physiological maturity and kernel drying in the field. This increase was favored by lower temperatures and higher fungal growth, favored, in turn, by the Angoumois grain moth damage. These results showed that besides the stage of kernel development, local environmental conditions during field drying can be decisive in the accumulation of fumonisins. Finally, it has been verified the standard behavior of the local isolate of F. verticillioides and its ability to produce fumonisins, and the partial resistance of the white maize hybrid EP10 x EC22 to the accumulation of fumonisin in kernel was confirmed by artificial inoculation.[ES] Las especies del género Fusarium son los mohos encontrados más frecuentemente en cultivos de cereales en las regiones templadas. Muchas de estas especies poseen la capacidad de producir micotoxinas, un grupo diverso de sustancias tóxicas para seres humanos y animales. Las fumonisinas son una familia de micotoxinas que se encuentran predominantemente en maíz y sus productores principales son F. verticillioides y F. proliferatum. Estas micotoxinas han sido clasificadas como “agente posiblemente carcinogénico en humanos” por la Agencia Internacional para la Investigación del Cáncer y se han delimitado sus contenidos máximos en los alimentos destinados al consumo humano y animal en la Unión Europea y en otros países. En Galicia, a pesar de existir un clima favorable para el desarrollo de mohos, apenas se ha investigado la presencia de especies micotoxigénicas o la contaminación con micotoxinas en maíz u otros cereales. Actualmente, no existe información concreta sobre qué factores ambientales y/o genotípicos determinan el nivel de infección fúngica y el contenido de fumonisinas en los granos en el momento de cosecha. Este conocimiento es importante y necesario a la hora de valorar el riesgo y tomar decisiones de prevención de la contaminación con fumonisinas. Por esta razón, en el presente trabajo se ha estudiado cuáles son estos factores, el peso que tiene cada uno de ellos y en qué momentos a lo largo del periodo de cultivo son críticos para la infección por F. verticillioides y la contaminación con fumonisinas de los granos de maíz. También se ha determinado el grado de presencia de otras especies de Fusarium en los granos para valorar el riesgo de contaminación con otras micotoxinas. Al mismo tiempo, se ha querido averiguar cómo influyen los factores ambientales, bióticos y abióticos, en el proceso de infección fúngica y de acumulación de fumonisinas a lo largo del desarrollo fisiológico y secado del grano en campo. Por último, y de forma paralela, se ha evaluado la resistencia a la acumulación de fumonisinas de cuatro híbridos de maíz blanco tras inoculación artificial con un aislado local de F. verticillioides.[ES] Como resultado de este trabajo, podemos confirmar que F. verticillioides es la especie más abundante y la principal productora de fumonisinas en los granos de maíz cultivado en Pontevedra. Con menor frecuencia, se detectaron otras especies micotoxigénicas del género Fusarium por lo que existe un riesgo potencial de contaminación con otras micotoxinas. Las siembras tardías y las cosechas tempranas fueron menos favorables para la infección por F. verticillioides, su desarrollo y la acumulación de fumonisinas en los granos, por lo que su aplicación es recomendable para reducir la contaminación con estas micotoxinas. Se observó que la contaminación con fumonisinas estuvo especialmente influenciada por las condiciones ambientales durante la floración y durante el secado del grano. Es necesario ser cauteloso cuando las condiciones climáticas durante la floración son más secas y calurosas, y tratar de evitar, mediante recolecciones tempranas, las precipitaciones intensas antes de la cosecha y daños mayores en los granos, producidos principalmente por insectos. El uso de variedades con cierta resistencia a la polilla, al taladro y con una buena cobertura de brácteas puede ser una herramienta útil para reducir el riesgo de contaminación con fumonisinas. A lo largo del desarrollo de los granos, el aumento significativo de la concentración de fumonisinas se produjo a partir de la madurez fisiológica y durante el secado en campo. Este aumento fue favorecido por la disminución de las temperaturas y por el crecimiento fúngico, favorecido, a su vez, por el daño de polilla, e indicando que, además del estado de desarrollo del grano, las condiciones ambientales locales durante el secado en campo pueden ser decisivas en la acumulación de fumonisinas. Finalmente, se ha verificado el comportamiento estándar del aislado local de F. verticillioides y su capacidad de producir fumonisinas, y se ha confirmado, mediante inoculación artificial, la resistencia parcial del híbrido de maíz blanco EP10 x EC22 a la acumulación de fumonisinas en los granos.[GL] As especies do xénero Fusarium son os mofos atopados máis frecuentemente nos cultivos de cereais nas rexións temperadas. Moitas destas especies posúen a capacidade de producir micotoxinas, un grupo diverso de substancias tóxicas para seres humanos e animais. As fumonisinas son unha familia de micotoxinas que se atopa predominantemente no millo e os seus produtores principais son F. verticillioides e F. proliferatum. Estas micotoxinas foron clasificadas como "axente posiblemente carcinoxénico en humanos" pola Axencia Internacional para a Investigación do Cancro e delimitáronse os seus contidos máximos nos alimentos destinados ó consumo humano e animal na Unión Europea e noutros países. En Galicia, a pesar de existir un clima favorable para o desenvolvemento dos mofos, non se investigou apenas a presenza de especies micotoxixénicas ou a contaminación con micotoxinas no millo ou noutros cereais. Actualemente, non existe información concreta sobre qué factores ambientais e/ou xenotípicos determinan o nivel de infección fúnxica e o contido de fumonisinas nos grans no momento da colleita. Este coñecemento é preciso e importante á hora de valorar o risco e tomar decisións de prevención da contaminación con fumonisinas. Por esta razón, no presente traballo estudouse cales son estes factores, o peso que ten cada un deles e en que momentos ó longo do período de cultivo son críticos para a infección por F. verticillioides e a contaminación con fumonisinas dos grans de millo. Tamén se determinou a presenza doutras especies de Fusarium nos grans para valorar o risco de contaminación con outras micotoxinas. Ó mesmo tempo, quíxose saber como inflúen os factores ambientais, bióticos e abióticos, no proceso de infección fúnxica e de acumulación de fumonisinas ó longo do desenvolvemento fisiolóxico e secado do gran no campo. Por último, e de forma paralela, avaliouse a resistencia á acumulación de fumonisinas de catro híbridos de millo branco tras inoculación artificial cun illado local de F. verticillioides.[GL] Como resultado deste traballo, podemos confirmar que F. verticillioides é a especie máis abundante e a principal produtora de fumonisinas nos grans do millo cultivado en Pontevedra. Con menor frecuencia, detectáronse outras especies micotoxixénicas do xénero Fusarium polo que existe un risco potencial de contaminación con outras micotoxinas. As sementeiras tardías e as colleitas temperás foron menos favorables para a infección por F. verticillioides, o seu desenvolvemento e a acumulación de fumonisinas nos grans, polo que a súa aplicación é recomendable para reduci-la contaminación con estas micotoxinas. Observouse que a contaminación con fumonisinas estivo especialmente influenciada polas condicións ambientais durante a floración e durante o secado do gran. É preciso ser cauteloso cando as condicións climáticas durante a floración son máis secas e calidas, e tratar de evitar, mediante recoleccións temperás, as precipitacións intensas antes da colleita e danos maiores nos grans, producidos principalmente por insectos. O uso de variedades con certa resistencia ós barrenos, á traza do millo e cunha boa cobertura de brácteas pode ser unha ferramenta útil para reducir o risco de contaminación con fumonisinas. Ó longo do desenvolvemento dos grans, o aumento significativo da concentración de fumonisinas produciuse a partir da madureza fisiolóxica e durante o secado en campo. Este aumento foi favorecido pola diminución das temperaturas e polo crecemento fúnxico, favorecido, á súa vez, polo dano da traza do millo, e indicando que, ademais do estado de desenvolvemento do gran, as condicións ambientais locais durante o secado en campo poden ser decisivas na acumulación de fumonisinas. Finalmente, verificouse o comportamento estándar do illado local de F. verticillioides e a súa capacidade de producir fumonisinas, e confirmouse, mediante inoculación artificial, a resistencia parcial do híbrido de millo branco EP10 x EC22 á acumulación de fumonisinas nos grans.Me gustaría recordar que, tanto la beca con la que he podido trabajar en ciencia, como todo el trabajo de investigación realizado en la presente Tesis, han sido posibles gracias a la financiación pública. Agradezco haber tenido esta oportunidad.Peer reviewe

Genetic factors involved in fumonisin accumulation in maize kernels and their implications in maize agronomic management and breeding

Contamination of maize with fumonisins depends on the environmental conditions; the maize resistance to contamination and the interaction between both factors. Although the effect of environmental factors is a determinant for establishing the risk of kernel contamination in a region, there is sufficient genetic variability among maize to develop resistance to fumonisin contamination and to breed varieties with contamination at safe levels. In addition, ascertaining which environmental factors are the most important in a region will allow the implementation of risk monitoring programs and suitable cultural practices to reduce the impact of such environmental variables. The current paper reviews all works done to address the influence of environmental variables on fumonisin accumulation, the genetics of maize resistance to fumonisin accumulation, and the search for the biochemical and/or structural mechanisms of the maize plant that could be involved in resistance to fumonisin contamination. We also explore the outcomes of breeding programs and risk monitoring of undertaken projects.This research was supported by the National Plan for Research and Development of Spain (AGL2009-12770). R. Santiago acknowledges postdoctoral contracts “Isidro Parga Pondal” supported by the Autonomous Government of Galicia and the European Social Fund and “Ramón y Cajal” supported by the Ministry of Economy and Competitiveness of Spain.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Is it possible to control fumonisin contamination in maize kernels by using genotypes resistant to the mediterranean corn borer?

This article is the copyright property of the Entomological Society of America and may not be used for any commercial or other private purpose without specific written permission of the Entomological Society of AmericaInsect activity has long been associated with Fusarium infection. The objectives of the current study were 1) to estimate the impact of Mediterranean corn borer, Sesamia nonagrioides Lefèbvre, damage on fumonisin contamination in the maize kernel by comparing fumonisin contamination under infestation and protected conditions, and 2) to measure the potential use of genotypes resistant to this borer as controlling factors of fumonisin contamination. Genotypes with increased kernel damage by borers tended to increase fumonisin accumulation under infestation conditions. In particular environments, other factors influenced fumonisin contamination more than damage by borers. When ear damage by borers is significant, maize resistance to ear damage could contribute to the reduction of fumonisin contamination in the kernels. Genotype such as EP42 × EP77 that combines low ear damage by borers and low fumonisin level across environments is a good choice to control fumonisin contamination. The use of an applicable methodology to identify Mediterranean corn borer-resistant genotypes to ear attack under artificial infestations might be a promising approach. © 2013 Entomological Society of America.National Plan for Research and Development of SpainAutonomous Government of GaliciaJAE Program of the Spanish Council of ResearchEuropean Social FundThis research was supported by the National Plan for Research and Development of Spain (AGL2006-13140, AGL2009-09611) and the Autonomous Government of Galicia(PGIDIT06TAL40301PR). A. Cao acknowledges funding from the JAE Program of the Spanish Council of Research. R. Santiago acknowledges postdoctoral contract “Isidro Parga Pondal” supported by the Autonomous Government of Galicia and the European Social Fund.Peer Reviewe

Assessment of corn resistance to fumonisin accumulation in a broad collection of inbred lines

Genetic improvement is an effective and environmentally safe method to reduce the levels of fumonisin mycotoxins in corn kernels infected with Fusarium verticillioides. In order to find new sources of resistance, a wide collection of corn inbred lines were evaluated for Fusarium ear rot and fumonisin accumulation after inoculation of the kernels with Fusarium verticillioides. Augmented designs were used for testing 240 un-replicated inbreds and 6 inbred checks in 2010 and 2011. Sixty-one inbreds were found to have the highest levels of resistance to Fusarium ear rot and fumonisin accumulation across years. Inbreds differing in kernel color, use, kernel type and heterotic group were all represented in this group of 61 inbreds. White corn inbreds had higher levels of fumonisin than yellow corn inbreds, but it was still possible to find white inbreds with comparable resistance to fumonisin accumulation to that of the most resistant yellow inbreds. Similarly, although the sweet corn inbreds evaluated in this study were less resistant to infection by F. verticillioides than the field corn inbreds, there were some which were grouped in the most resistant 61 inbreds. Many of these inbreds can be used to improve resistance to F. verticillioides infection and fumonisin accumulation by crossing the most resistant inbreds of each subgroup.MICINNSpanish Council of Research.Autonomous Government of GaliciaEuropean Social FundThis research was supported by the National Plan for Research and Development of Spain (AGL2009-12770). A. Cao acknowledges funding from the JAE Program of the Spanish Council of Research. R. Santiago acknowledges postdoctoral contract “Isidro Parga Pondal” supported by the Autonomous Government of Galicia and the European Social Fund.Peer reviewe

Assessing white maize resistance to fumonisin contamination

Genetic improvement is an emerging method to reduce the levels of fumonisin (FB) contamination in maize, but breeding advances depend on the development of suitable methods to accurately assess the performance of different cultivars. Our study focused on characterizing a local isolate of Fusarium verticillioides; comparing artificial inoculation techniques with this isolate (injection into kernels and down the silk channel); and assessing white maize resistance under artificial vs. natural inoculation. The fungal growth rate significantly increased with temperature and water activity a w. The optimum growth rate, corresponding with the shortest phase of initial growth, occurred at 25-30 °C and 0.99 a w. Under silk inoculation with this isolate, the hybrid EP10 × EC22 accumulated significantly less FBs than the other hybrids, whereas, under kernel inoculation, differences among hybrids were not significant (P ≤ 0.05). The local isolate of F. verticillioides produced FBs and responded to the usual environmental conditions during maize kernel ripening in northwestern Spain. Inoculation with this isolate is recommended because it is aggressive, toxigenic, and adapted to the local environment. Silk inoculation was the only method that allowed a clear distinction among genotypes based on differences in resistance to FB accumulation. Resistance to natural and artificial inoculations was confirmed for the hybrid EP10 × EC22. © 2013 KNPV.MINECOAutonomous Government of GaliciaExcma. Diputación Provincial de PontevedraSpanish Council of ResearchEuropean Social FundThis research was supported by the National Plan for Research and Development of Spain (AGL2009-12770), the Autonomous Government of Galicia (PGIDIT06TAL40301PR) and the Excma. Diputación Provincial de Pontevedra. A. Cao acknowledges funding from the JAE Program of the Spanish Council of Research. R.Santiago acknowledges postdoctoral contract “Isidro Parga Pondal” supported by the Autonomous Government of Galicia and the European Social Fund.Peer Reviewe