José Climent

Chloroplast microsatellites reveal colonization and metapopulation dynamics in the Canary Island pine

Chloroplast microsatellites are becoming increasingly popular markers for population genetic studies in plants, but there has been little focus on their potential for demographic inference. In this work the utility of chloroplast microsatellites for the study of population expansions was explored. First, we investigated the power of mismatch distribution analysis and the FS test with coalescent simulations of different demographic scenarios. We then applied these methods to empirical data obtained for the Canary Island pine (Pinus canariensis). The results of the simulations showed that chloroplast microsatellites are sensitive to sudden population growth. The power of the FS test and accuracy of demographic parameter estimates, such as the time of expansion, were reduced proportionally to the level of homoplasy within the data. The analysis of Canary Island pine chloroplast microsatellite data indicated population expansions for almost all sample localities. Demographic expansions at the island level can be explained by the colonization of the archipelago by the pine, while population expansions of different ages in different localities within an island could be the result of local extinctions and recolonization dynamics. Comparable mitochondrial DNA sequence data from a parasite of P. canariensis, the weevil Brachyderes rugatus, supports this scenario, suggesting a key role for volcanism in the evolution of pine forest communities in the Canary Islands.

Nursery fertilization enhances survival and physiological status in Canary Island pine (Pinus canariensis) seedlings planted in a semiarid environment

We tested the hypothesis that fertilized containerized Pinus canariensis seedlings increases survival when planted in semiarid sites through the improvement of their physiological status during the establishment phase by an increment in root growth. Seedlings were cultured under two different regimes: traditional (in non-fertilized natural soil) and alternative (in fertilized peat). Morphological attributes and nitrogen content were measured before planting. Measurements of survival and growth in the plantation were made periodically for 2xa0years and physiological plant responses (leaf water potential, gas exchange and chlorophyll fluorescence) during the third summer after planting were tested and finally a set of plants were excavated to measure the same parameters as before planting. Seedlings cultivated using fertilized peat achieved the highest values for all of evaluated parameters. During the third dry season, big seedlings exhibited better physiological status. Therefore, enhanced root growth can result in better water uptake during the dry period thereby increasing survival and growth in the next few years after planting. A feed-back physiological model is proposed to explain P. canariensis establishment in a semiarid environment.

Freezing injury in primary and secondary needles of Mediterranean pine species of contrasting ecological niches.

Abstract• Pine seedlings show a marked ontogenetic difference between primary and secondary needles, the latter prevailing with a different timing among species.• Using the electrolyte leakage method following an artificial freezing test, we aimed at (1) determining the differences in freezing tolerance between primary and secondary needles in eight pines of contrasting thermal habitats: P. canariensis, P. pinea, P. halepensis, P. brutia, P. pinaster, P. nigra, P. sylvestris and P. radiata, (2) evaluating the relation between freezing tolerance and sclerophylly and (3) relating freezing tolerance with the climate of origin.• Primary needles were significantly more sensitive to freezing than secondary needles in Pinus halepensis, P. brutia, P. pinaster and P. nigra, whereas no differences were found in P. canariensis, P. pinea and P. radiata. LT50 was uncorrelated with needle sclerophylly but very highly correlated with the mean temperature of the coldest month at the seed source.• Results support an adaptive role of secondary needles in the mountain Mediterranean pines P. nigra, P. pinaster and P. brutia, while the more complex responses in coastal Mediterranean pines can be interpreted in the light of seedling ontogeny and species’ ecological niches.Résumé• Les jeunes plants de pin présentent une nette différence ontogénétique entre aiguilles primaires et secondaires, les secondes devenant dominantes après des délais variables selon les espèces.• À l’aide d’une expérience de gel artificiel et à l’aide de la méthode de perte d’électrolyte, nous avons visé à : (1) déterminer les différences de tolérance au gel entre aiguilles primaires et secondaires de huit espèces de pins présentant des habitats thermiques contrastés : P. canariensis, P. pinea, P. halepensis, P. brutia, P. pinaster, P. nigra, P. sylvestris et P. radiata, (2) évaluer la relation entre tolérance au gel et degré de sclérophyllie, et (3) mettre en relation tolérance au gel et climat d’origine.• Les aiguilles primaires étaient beaucoup plus sensibles au gel que les aiguilles secondaires chez Pinus halepensis, P. brutia, P. pinaster et P. nigra, alors qu’aucune différence n’a été constatée chez P. canariensis, P. pinea et P. radiata. La température induisant 50 % de mortalité n’était pas corrélée avec la sclérophyllie des aiguilles mais très fortement avec la température moyenne du mois le plus froid de la provenance des semences.• Les résultats corroborent l’idée d’un rôle adaptatif des aiguilles secondaires des pins méditerranéens montagnards P. nigra, P. pinaster et P. brutia, tandis que les réponses plus complexes des pins méditerranéens côtiers peuvent être interprétées à la lumière de l’ontogénie des semis et des niches écologiques des espèces.

Plasticity in reproduction and growth among 52 range-wide populations of a Mediterranean conifer: adaptive responses to environmental stress

A plastic response towards enhanced reproduction is expected in stressful environments, but it is assumed to trade off against vegetative growth and efficiency in the use of available resources deployed in reproduction [reproductive efficiency (RE)]. Evidence supporting this expectation is scarce for plants, particularly for long‐lived species. Forest trees such as Mediterranean pines provide ideal models to study the adaptive value of allocation to reproduction vs. vegetative growth given their among‐population differentiation for adaptive traits and their remarkable capacity to cope with dry and low‐fertility environments. We studied 52 range‐wide Pinus halepensis populations planted into two environmentally contrasting sites during their initial reproductive stage. We investigated the effect of site, population and their interaction on vegetative growth, threshold size for female reproduction, reproductive–vegetative size relationships and RE. We quantified correlations among traits and environmental variables to identify allocation trade‐offs and ecotypic trends. Genetic variation for plasticity was high for vegetative growth, whereas it was nonsignificant for reproduction. Size‐corrected reproduction was enhanced in the more stressful site supporting the expectation for adverse conditions to elicit plastic responses in reproductive allometry. However, RE was unrelated with early reproductive investment. Our results followed theoretical predictions and support that phenotypic plasticity for reproduction is adaptive under stressful environments. Considering expectations of increased drought in the Mediterranean, we hypothesize that phenotypic plasticity together with natural selection on reproductive traits will play a relevant role in the future adaptation of forest tree species.

Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster

BACKGROUND AND AIMSnThe study of local adaptation in plant reproductive traits has received substantial attention in short-lived species, but studies conducted on forest trees are scarce. This lack of research on long-lived species represents an important gap in our knowledge, because inferences about selection on the reproduction and life history of short-lived species cannot necessarily be extrapolated to trees. This study considers whether the size for first reproduction is locally adapted across a broad geographical range of the Mediterranean conifer species Pinus pinaster. In particular, the study investigates whether this monoecious species varies genetically among populations in terms of whether individuals start to reproduce through their male function, their female function or both sexual functions simultaneously. Whether differences among populations could be attributed to local adaptation across a climatic gradient is then considered.nnnMETHODSnMale and female reproduction and growth were measured during early stages of sexual maturity of a P. pinaster common garden comprising 23 populations sampled across the species range. Generalized linear mixed models were used to assess genetic variability of early reproductive life-history traits. Environmental correlations with reproductive life-history traits were tested after controlling for neutral genetic structure provided by 12 nuclear simple sequence repeat markers.nnnKEY RESULTSnTrees tended to reproduce first through their male function, at a size (height) that varied little among source populations. The transition to female reproduction was slower, showed higher levels of variability and was negatively correlated with vegetative growth traits. Several female reproductive traits were correlated with a gradient of growth conditions, even after accounting for neutral genetic structure, with populations from more unfavourable sites tending to commence female reproduction at a lower individual size.nnnCONCLUSIONSnThe study represents the first report of genetic variability among populations for differences in the threshold size for first reproduction between male and female sexual functions in a tree species. The relatively uniform size at which individuals begin reproducing through their male function probably represents the fact that pollen dispersal is also relatively invariant among sites. However, the genetic variability in the timing of female reproduction probably reflects environment-dependent costs of cone production. The results also suggest that early sex allocation in this species might evolve under constraints that do not apply to other conifers.

Adaptive evolution of Mediterranean pines

Mediterranean pines represent an extremely heterogeneous assembly. Although they have evolved under similar environmental conditions, they diversified long ago, ca. 10 Mya, and present distinct biogeographic and demographic histories. Therefore, it is of special interest to understand whether and to what extent they have developed specific strategies of adaptive evolution through time and space. To explore evolutionary patterns, the Mediterranean pines phylogeny was first reconstructed analyzing a new set of 21 low-copy nuclear genes with multilocus Bayesian tree reconstruction methods. Secondly, a phylogenetic approach was used to search for footprints of natural selection and to examine the evolution of multiple phenotypic traits. We identified two genes (involved in pines defense and stress responses) that have likely played a role in the adaptation of Mediterranean pines to their environment. Moreover, few life-history traits showed historical or evolutionary adaptive convergence in Mediterranean lineages, while patterns of character evolution revealed various evolutionary trade-offs linking growth-development, reproduction and fire-related traits. Assessing the evolutionary path of important life-history traits, as well as the genomic basis of adaptive variation is central to understanding the past evolutionary success of Mediterranean pines and their future response to environmental changes.

Difference in cuticular transpiration and sclerophylly in juvenile and adult pine needles relates to the species-specific rates of development

Pinus species show remarkable ontogenetic differences in needle morphology (heterophylly) between juvenile and adult vegetative phases. This developmental shift may play an adaptative role in their success under diverse habitats. As a first step to know the functional differences between each vegetative phase, we compared water loss through the cuticles of juvenile and adult needles of 21-month-old nursery-grown seedlings of nine hard pine species. Cuticular transpiration (CT), calculated after complete stomatal closure, was obtained by leaf-drying curves, and was related to leaf, ontogenetic and climatic parameters. The rate of cuticular transpiration (RCT) between juvenile and adult needles differed across pine species, and in particular segregated the Mediterranean species Pinus canariensis and P. halepensis, from the Eurasian P. uncinata and introduced species P. radiata. For these species, RCT was always higher in juvenile needles. The different leaf and ontogenic parameters studied were correlated with the variation in RCT among the nine pine species. We discuss this relationship in the light of the species ecology. Besides their possible adaptive interpretation, these results suggest an underlying need to consider the ontogenetic heterophylly when assessing functional traits in hard pine seedlings, in particular those traits that govern water relations.

Contrasting ecotypic differentiation for growth and survival in Pinus canariensis

We tested genetic and environmental effects and their interaction on the behaviour of 21 provenances of Pinus canariensis Chr. Sm. Ex DC from the Canary Islands and three seed sources from Israel at seven study sites covering a wide range of ecological conditions. Survival and growth traits (height, diameter and polycyclism) and their relationship with environmental parameters were assessed to evaluate their adaptive value and establish patterns of variation of the species inside and outside its natural distribution area. The results showed a high level of ecotypic differentiation for survival. As a general pattern, seed sources from favourable environments exhibited lower survival rates at dry sites than those from harsh environments (r =− 0.76, P < 0.05, between survival and site index at the seed source). By contrast, growth traits presented a high phenotypic plasticity, scarce differentiation among seed sources and a negligible genotype × environment interaction. With few exceptions, local provenances from the Canary Islands did not stand out for survival or growth when compared with the rest at each trial site, whereas local seed sources from Israel were among the best growing and survived better at the experimental sites in this country. Therefore, the possibility of a locally adapted land race in Israel is discussed. We also discuss the low geographic differentiation for growth in the Canary Islands pine in the light of the current knowledge of the species life history and the potential use of this species in reforestation programs in semi-arid environments.

Heritability and quantitative genetic divergence of serotiny, a fire-persistence plant trait

BACKGROUND AND AIMSnAlthough it is well known that fire acts as a selective pressure shaping plant phenotypes, there are no quantitative estimates of the heritability of any trait related to plant persistence under recurrent fires, such as serotiny. In this study, the heritability of serotiny in Pinus halepensis is calculated, and an evaluation is made as to whether fire has left a selection signature on the level of serotiny among populations by comparing the genetic divergence of serotiny with the expected divergence of neutral molecular markers (QST-FST comparison).nnnMETHODSnA common garden of P. halepensis was used, located in inland Spain and composed of 145 open-pollinated families from 29 provenances covering the entire natural range of P. halepensis in the Iberian Peninsula and Balearic Islands. Narrow-sense heritability (h(2)) and quantitative genetic differentiation among populations for serotiny (QST) were estimated by means of an animal model fitted by Bayesian inference. In order to determine whether genetic differentiation for serotiny is the result of differential natural selection, QST estimates for serotiny were compared with FST estimates obtained from allozyme data. Finally, a test was made of whether levels of serotiny in the different provenances were related to different fire regimes, using summer rainfall as a proxy for fire regime in each provenance.nnnKEY RESULTSnSerotiny showed a significant narrow-sense heritability (h(2)) of 0·20 (credible interval 0·09-0·40). Quantitative genetic differentiation among provenances for serotiny (QST = 0·44) was significantly higher than expected under a neutral process (FST = 0·12), suggesting adaptive differentiation. A significant negative relationship was found between the serotiny level of trees in the common garden and summer rainfall of their provenance sites.nnnCONCLUSIONSnSerotiny is a heritable trait in P. halepensis, and selection acts on it, giving rise to contrasting serotiny levels among populations depending on the fire regime, and supporting the role of fire in generating genetic divergence for adaptive traits.

The role of developmental stage in frost tolerance of Pinus pinea L. seedlings and saplings

ContextAlthough drought is generally considered the main environmental constraint in Mediterranean environments, the ability to acclimate to and tolerate frost in early developmental stages can be a determinant for seedling survival of many Mediterranean tree species like stone pine (Pinus pinea L.).AimsThe aim of this study was to assess the impact of the developmental stage of naturally regenerated stone pine individuals on tolerance to low temperature (LT) from summer to late autumn and in spring, at a highly continental site in central Spain. Specifically, we tested to what extent the differences in tolerance are related to shoot heteroblasty.MethodsWe assessed LT tolerance of needles from individuals at three age classes (class C1: seedlings, class C2: 4- to 8-year-old saplings and class C3: >9-year-old saplings) over nine dates from summer to spring.ResultsLT tolerance displayed severe seasonal trends and differed between age classes. It usually increased with sapling age. Such differences were tightly related to heteroblasty of the shoots. Our results point to a higher LT tolerance associated with larger leaf dry mass per unit area (LMA) values. No impact of late frosts on shoot growth rates was detected during this study.ConclusionsDevelopmental changes during early plant growth seem to play a role in frost tolerance of stone pine seedlings, a finding which furthers our understanding of regeneration dynamics in this species in areas with continental influence.