Juan L. García-Castaño

Differential contribution of frugivores to complex seed dispersal patterns

Frugivores are highly variable in their contribution to fruit removal in plant populations. However, data are lacking on species-specific variation in two central aspects of seed dispersal, distance of dispersal and probability of dispersal among populations through long-distance transport. We used DNA-based genotyping techniques on Prunus mahaleb seeds dispersed by birds (small- and medium-sized passerines) and carnivorous mammals to infer each seeds source tree, dispersal distance, and the probability of having originated from outside the study population. Small passerines dispersed most seeds short distances (50% dispersed <51 m from source trees) and into covered microhabitats. Mammals and medium-sized birds dispersed seeds long distances (50% of mammals dispersed seeds >495 m, and 50% of medium-sized birds dispersed seeds to >110 m) and mostly into open microhabitats. Thus, dispersal distance and microhabitat of seed deposition were linked through the contrasting behaviors of different frugivores. When the quantitative contribution to fruit removal was accounted for, mammals were responsible for introducing two-thirds of the immigrant seeds into the population, whereas birds accounted for one-third. Our results demonstrate that frugivores differ widely in their effects on seed-mediated gene flow. Despite highly diverse coteries of mutualistic frugivores dispersing seeds, critical long-distance dispersal events might rely on a small subset of large species. Population declines of these key frugivore species may seriously impair seed-mediated gene flow in fragmented landscapes by truncating the long-distance events and collapsing seed arrival to a restricted subset of available microsites.

Population structure of Hypochaeris salzmanniana DC. (Asteraceae), an endemic species to the Atlantic coast on both sides of the Strait of Gibraltar, in relation to Quaternary sea level changes

To detect potential Pleistocene refugia and colonization routes along the Atlantic coast, we analysed amplified fragment length polymorphisms (AFLPs) in 140 individuals from 14 populations of Hypochaeris salzmanniana (Asteraceae), an annual species endemic to the southwestern European and northwestern African coastal areas. Samples covered the total distributional range of the species, with eight populations in southwestern Spain and six populations in northwestern Morocco. Using nine primer combinations, we obtained 546 fragments in H. salzmanniana and its sister species H. arachnoidea of which 487 (89.2%) were polymorphic. The neighbour‐joining tree shows that the populations south of the Loukos river in Morocco are clearly differentiated, having more polymorphic, private, and rare fragments, and higher genetic diversity, than all the other populations. The southernmost populations in Morocco, south of the river Sebou, form a large panmictic population. They are probably the oldest populations that have been relatively unaffected by stochastic processes resulting from Pleistocene glaciations. Northward migration of populations during this period may have resulted in loss of genetic diversity in specific regions, perhaps due to bottlenecks caused by rise in sea level during interglacial periods, and, in some cases, by changes in the breeding system.

Self-incompatibility and floral parameters in Hypochaeris sect. Hypochaeris (Asteraceae)

We studied the relationships between self-incompatibility mechanisms and floral parameters in the genus Hypochaeris L. sect. Hypochaeris (consisting of H. glabra, H. radicata, H. arachnoidea, and H. salzmanniana). We assessed at intra- and interspecific levels (1) the self-incompatibility (SI) mechanism and its distribution among populations, (2) the relationship between SI and floral parameters, and (3) the relationship of SI to reproductive success. Hypochaeris salzmanniana is semi-incompatible, H. glabra is self-compatible, and H. arachnoidea and H. radicata are self-incompatible. Floral parameters differed among populations of H. salzmanniana: plants in self-compatible populations had fewer flowers per head, a smaller head diameter on the flower, and a shorter period of anthesis than self-incompatible populations. We also detected this pattern within a semi-compatible population of H. salzmanniana, and these differences were also found between species with different breeding mechanisms. Fruit to flower ratio in natural populations was generally high (>60%) in all species, regardless of breeding system. It is hypothesized that self-compatibility may have arisen through loss of allelic diversity at the S locus due to bottleneck events and genetic drift.

Phylogeography of the invasive weed Hypochaeris radicata (Asteraceae): from Moroccan origin to worldwide introduced populations

In an attempt to delineate the area of origin and migratory expansion of the highly successful invasive weedy species Hypochaeris radicata, we analysed amplified fragment length polymorphisms from samples taken from 44 populations. Population sampling focused on the central and western Mediterranean area, but also included sites from Northern Spain, Western and Central Europe, Southeast Asia and South America. The six primer combinations applied to 213 individuals generated a total of 517 fragments of which 513 (99.2%) were polymorphic. The neighbour‐joining tree presented five clusters and these divisions were supported by the results of Bayesian analyses: plants in the Moroccan, Betic Sierras (Southern Spain), and central Mediterranean clusters are all heterocarpic. The north and central Spanish, southwestern Sierra Morena, and Central European, Asian and South American cluster contain both heterocarpic (southwestern Sierra Morena) and homocarpic populations (all other populations). The Doñana cluster includes two homocarpic populations. Analyses of fragment parameters indicate that the oldest populations of H. radicata are located in Morocco and that the species expanded from this area in the Late Quaternary via at least three migratory routes, the earliest of which seems to have been to the southwestern Iberian Peninsula, with subsequent colonizations to the central Mediterranean area and the Betic Sierras. Homocarpic populations originated in the southwestern Iberian Peninsula and subsequently spread across north and central Spain, Central Europe and worldwide, where they became a highly successful weed.

Spatial variation of post-dispersal seed removal by rodents in highland microhabitats of Spain and Switzerland

Few data are available and little is known about spatial variation in post-dispersal seed removal at different levels throughout the geographic range of a plant species. Here, we compare post-dispersal seed removal by rodents within and among sites in two distinct regions, the south-eastern Spanish Mediterranean highlands and the Swiss Jura. Seed removal was assessed experimentally for four fleshyfruited species (Berberis vulgaris, Crataegus monogyna, Rosa spp. and Taxus baccata )i n heterogeneous sites with distinct microhabitats. A factorial general linear model (GLM) analysis was used to evaluate the relative influence of speciesspecific seed traits on removal at three spatial levels (microhabitat, site and region). In both highland ecosystems, live trapping revealed that rodents were the main seed removers: Apodemus sylvaticus was observed in both regions, whereas A. flavicollis and Clethrionomys glareolus occurred only at the Swiss sites. There was a significant difference in seed removal between regions, and the preferences of the rodents varied among plant species and microhabitats. Variation in rodent presence explained some differences in seed removal between regions and among microhabitats. Finally, the effect of rodent presence on seed removal differs due to both regional and microhabitat effects.

Big thistle eats the little thistle: does unidirectional introgressive hybridization endanger the conservation of Onopordum hinojense?

Hybridization is known to have a creative role in plant evolution. However, it can also have negative effects on parental species. Onopordum is a large genus whose species frequently hybridize. In the Southwest Iberian Peninsula, the rare O. hinojense co-occurs with the widely distributed O. nervosum, and hybrids between these two taxa have been described as O. × onubense. In this study we determine the extinction risk in a hybrid zone, both for hybrids and parentals, using analyses of morphological and cytogenetic traits as well as genetic markers and demographic models. To investigate the introgression process we used amplified fragment length polymorphism (AFLP) markers, Bayesian analyses and genome scan methods. Morphology, genome size and molecular markers confirmed homoploid hybridization and also indicated unidirectional backcrossing of F₁ hybrids with O. nervosum, which is likely to swamp O. hinojense, the parental with lower pollen size and a very low fruit set (8%). Genome scan methods revealed several loci significantly deviating from neutrality. Finally, our demographic modeling indicated that the higher fitness of O. nervosum threats the survival of O. hinojense by demographic swamping. Our study provides strong new evidence for a scenario of rapid extinction by unidirectional introgression and demographic swamping. The multifaceted approach used here sheds new light on the role of introgression in plant extinctions.

Nuclear Microsatellite Primers for the Endangered Relict Fir, Abies pinsapo (Pinaceae) and Cross-Amplification in Related Mediterranean Species

Twelve nuclear microsatellite primers (nSSR) were developed for the endangered species Abies pinsapo Boiss. to enable the study of gene flow and genetic structure in the remaining distribution areas. Microsatellite primers were developed using next-generation sequencing (454) data from a single Abies pinsapo individual. Primers were applied to thirty individuals from the three extant localities. The number of alleles per locus ranged from one to four. Cross-amplification was tested for other Abies species from the Mediterranean Basin, and most of the loci showed higher polymorphisms in the Mediterranean species than in A. pinsapo. These microsatellite markers provide tools for conservation genetic studies in Abies pinsapo as well other Abies species from the Mediterranean Basin.

Phylogeography of SW Mediterranean firs: Different European origins for the North African Abies species

The current distribution of Western Mediterranean Abies species is a result of complex geodynamic processes and climatic oscillations that occurred in the past. Abies sect. Piceaster offers a good study model to explore how geo-climatic oscillations might have influenced its expansion and diversification on both sides of the W Mediterranean basin. We investigated the genetic variation within and among nine populations from five Abies species by molecular markers with high and low mutation rates and contrasting inheritance (AFLP and cpSSR). Analyses revealed the opening of the Strait of Gibraltar as an effective barrier against gene flow between the Southern Iberian (A. pinsapo) and North African (A. marocana and A. tazaotana) firs. The A. pinsapo populations in Spain and likewise those of the A. marocana - A. tazaotana population complex were not differentiated, and no evidence was found to distinguish A. tazaotana at the species level. Diversification of Abies across North Africa could occur by way of at least two vicariant events from Europe, in the west, giving rise to the A. marocana - A. tazaotana complex, and in the east, giving A. numidica. Secondary contacts among species from Abies sect. Piceaster (A. pinsapo and A. numidica), and with A. alba (Abies sect. Abies) are also indicated. However, there is a closer relationship between the Algerian fir (A. numidica) and the North Mediterranean widespread A. alba, than with the Moroccan firs (A. marocana and A. tazaotana) or the Southern Iberian (A. pinsapo). We also discuss the distribution range of these taxa in its paleogeological and paleoclimatic context, and propose that part of the modern geography of the South-Western Mediterranean firs might be traced back to the Tertiary.

AFLP and breeding system studies indicate vicariance origin for scattered populations and enigmatic low fecundity in the Moroccan endemic Hypochaeris angustifolia (Asteraceae), sister taxon to all of the South American Hypochaeris species.

We used Amplified Fragment Length Polymorphism markers (AFLP) and breeding system studies to investigate the population structure and reproductive biology of Hypochaeris angustifolia (Asteraceae: Cichorieae). This species is endemic to altiplanos of the Atlas Mountains (Morocco) where it occurs in scattered populations, and it is the sister species to c. 40 species of this genus in South America. PCoA, NJ, and Bayesian clustering, revealed that the populations are very isolated whilst AFLP parameters show that almost all populations have marked genetic divergence. We contend that these features are more in accord with a vicariance origin for the scattered populations of H. angustifolia, rather than establishment by long-distance dispersal. The breeding system studies revealed that H. angustifolia is a self-incompatible species, with low fecundity in natural and in experimental crosses, probably due to a low frequency of compatible phenotypes within and between the populations.

Effects of tree architecture on pollen dispersal and mating patterns in Abies pinsapo Boiss. (Pinaceae)

Plant architecture is crucial to pollination and mating in wind‐pollinated species. We investigated the effect of crown architecture on pollen dispersal, mating system and offspring quality, combining phenotypic and genotypic analyses in a low‐density population of the endangered species Abies pinsapo. A total of 598 embryos from three relative crown height levels (bottom, middle and top) in five mother plants were genotyped using eleven nuclear microsatellite markers (nSSRs). Paternity analysis and mating system models were used to infer mating and pollen dispersal parameters. In addition, seeds were weighed (N = 16 110) and germinated (N = 736), and seedling vigour was measured to assess inbreeding depression. Overall, A. pinsapo shows a fat‐tailed dispersal kernel, with an average pollen dispersal distance of 113–227 m, an immigration rate of 0.84–26.92%, and a number of effective pollen donors (Nep) ranging between 3.5 and 11.9. We found an effect of tree height and relative crown height levels on mating parameters. A higher proportion of seeds with embryo (about 50%) and a higher rate of self‐fertilization (about 60%) were found at the bottom level in comparison with the top level. Seed weight and seedling vigour are positively related. Nevertheless, no differences were found in seed weight or in seedling‐related variables such as weight and length of aerial and subterranean parts among the different relative crown height levels, suggesting that seeds from the more strongly inbred bottom level are not affected by inbreeding depression. Our results point to vertical isotropy for outcross‐pollen and they suggest that self‐pollen may ensure fertilization when outcross‐pollen is not available in low‐density population.