José A. Godoy

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.

Seed dispersal by animals: exact identification of source trees with endocarp DNA microsatellites

A long‐standing challenge in studies of seed dispersal by animal frugivores has been the characterization of the spatial relationships between dispersed seeds and the maternal plants, i.e. the seed shadow. The difficulties to track unambiguously the origin of frugivore‐dispersed seeds in natural communities has been considered an unavoidable limitation of the research field and precluded a robust analysis of the direct consequences of zoochory. Here we report that the multilocus genotype at simple sequence repeat (SSR; microsatellite) loci of the woody endocarp, a tissue of maternal origin, provides an unequivocal genetic fingerprint of the source tree. By comparing the endocarp genotype against the complete set of genotypes of reproductive trees in the population, we could unambiguously identify the source tree for 82.1% of the seeds collected in seed traps and hypothesize that the remaining 17.9% of sampled seeds come from other populations. Identification of the source tree for Prunus mahaleb seeds dispersed by frugivores revealed a marked heterogeneity in the genetic composition of the seed rain in different microhabitats, with a range of 1–5 distinct maternal trees contributing seeds to a particular landscape patch. Within‐population dispersal distances ranged between 0 and 316 m, with up to 62% of the seeds delivered within 15 m of the source trees. Long distance dispersal events, detected by the exclusion of all reproductive trees in the population, accounted for up to 17.9% of the seeds sampled. Our results indicate strong distance limitation of seed delivery combined with infrequent long‐distance dispersal events, extreme heterogeneity in the landscape pattern of genetic makeup, and a marked mosaic of multiple parentage for the seeds delivered to a particular patch.

A probable lipid transfer protein gene is induced by NaCl in stems of tomato plants.

A full-length tomato cDNA clone, TSW12, which is developmentally and environmentally regulated, has been isolated and characterized. TSW12 mRNA is accumulated during tomato seed germination and its level increases after NaCl treatment or heat shock. In mature plants, TSW12 mRNA is only detected upon treatment with NaCl, mannitol or ABA and its expression mainly occurs in stems. The nucleotide sequence of TSW12 includes an open reading frame coding for a basic protein of 114 amino acids; the first 23 amino acids exhibit the sequence characteristic of a signal peptide. The high similarity between the TSW12-deduced amino acid sequence and reported lipid transfer proteins suggests that TSW12 encodes a lipid transfer protein.

Faecal genetic analysis to determine the presence and distribution of elusive carnivores: design and feasibility for the Iberian lynx

Noninvasive methods using genetic markers have been suggested as ways to overcome difficulties associated with documenting the presence of elusive species. We present and assess a novel, reliable and effective molecular genetic technique for the unequivocal genetic identification of faeces from the endangered Iberian lynx (Lynx pardinus). From mitochondrial DNA (mtDNA) cytochrome b and D‐loop region sequences, we designed four species‐specific primers (for products 130–161 bp long) that were considered to be likely to amplify degraded DNA. We compared two DNA extraction methods, various DNA amplification conditions and the robustness and specificity of the primer pairs with 87 lynx samples from 5 potentially different lynx populations and with 328 samples of other carnivore species. The utility of the identification technique was tested with faeces of different ages, with faeces from controlled field experiments, and with faeces collected from locales with possible lynx populations from throughout the state of Andalusia, Spain (8052 km2). Faecal mtDNA extraction was more efficient using PBS wash of the faeces instead of a faeces homogenate. Our assay increased from 92.6 to 99% efficiency with a second amplification and a reduction in template concentration to overcome polymerase chain reaction (PCR) inhibition. Our assay never produced false positives, and correctly identified all lynx faeces. Of 252 faeces samples of unknown species collected throughout Andalusia, 26.6% (from three different areas) were classified as Iberian lynx, 1.4% showed evidence of PCR inhibition and 1.2% were of uncertain origin. This method has proven to be a reliable technique that can be incorporated into large‐scale surveys of Iberian lynx populations and exemplifies an approach that can easily be extended to other species.

Expression, tissue distribution and subcellular localization of dehydrin TAS14 in salt-stressed tomato plants

We previously isolated and characterized TAS14, an mRNA that is induced in tomato upon osmotic stress or abscisic acid (ABA) treatment and that shares expression and sequence characteristics with other dehydrin genes in different species. Affinity-purified antibodies against TAS14 protein were used to study the expression of TAS14 protein, both in seedlings and mature plants, its tissue distribution and its subcellular localization. TAS14 protein was not detected in 4-day-old seedlings but accumulated after ABA, NaCl or mannitol treatments. In NaCl-treated seedlings, some protein was detectable after 6 h of treatment and reached maximal levels between 24 and 48 h. Concentrations ranging from 5 to 12.5 g/l NaCl induced the protein to similar levels. In salt-stressed mature plants, TAS14 was expressed abundantly and continuously in aerial parts, but only slightly and transiently in roots. Immunocytochemical analysis of salt-treated plants showed TAS14 accumulated in adventitious root primordia and associated to the provascular and vascular tissues in stems and leaves. Immunogold electron microscopy localized TAS14 protein both in the cytosol and in the nucleus, associated to the nucleolus and euchromatin. Since TAS14 is a phosphoprotein in vivo, the classes of protein kinases potentially responsible for its in vivo phosphorylation were tested in in vitro phosphorylation assays. TAS14 protein was phosphorylated in vitro by both casein kinase II and cAMP-dependent protein kinase.

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.

Contemporary pollen and seed dispersal in a Prunus mahaleb population: patterns in distance and direction

Pollination and seed dispersal determine the spatial pattern of gene flow in plant populations and, for those species relying on pollinators and frugivores as dispersal vectors, animal activity plays a key role in determining this spatial pattern. For these plant species, reported dispersal patterns are dominated by short‐distance movements with a significant amount of immigration. However, the contribution of seed and pollen to the overall contemporary gene immigration is still poorly documented for most plant populations. In this study we investigated pollination and seed dispersal at two spatial scales in a local population of Prunus mahaleb (L.), a species pollinated by insects and dispersed by frugivorous vertebrates. First, we dissected the relative contribution of pollen and seed dispersal to gene immigration from other parts of the metapopulation. We found high levels of gene immigration (18.50%), due to frequent long distance seed dispersal events. Second, we assessed the distance and directionality for pollen and seed dispersal events within the local population. Pollen and seed movement patterns were non‐random, with skewed distance distributions: pollen tended moved up to 548 m along an axis approaching the N–S direction, and seeds were dispersed up to 990 m, frequently along the SW and SE axes. Animal‐mediated dispersal contributed significantly towards gene immigration into the local population and had a markedly nonrandom pattern within the local population. Our data suggest that animals can impose distinct spatial signatures in contemporary gene flow, with the potential to induce significant genetic structure at a local level.

Phylogeography, genetic structure and diversity in the endangered bearded vulture (Gypaetus barbatus, L.) as revealed by mitochondrial DNA

Bearded vulture populations in the Western Palearctic have experienced a severe decline during the last two centuries that has led to the near extinction of the species in Europe. In this study we analyse the sequence variation at the mitochondrial control region throughout the species range to infer its recent evolutionary history and to evaluate the current genetic status of the species. This study became possible through the extensive use of museum specimens to study populations now extinct. Phylogenetic analysis revealed the existence of two divergent mitochondrial lineages, lineage A occurring mainly in Western European populations and lineage B in African, Eastern European and Central Asian populations. The relative frequencies of haplotypes belonging to each lineage in the different populations show a steep East–West clinal distribution with maximal mixture of the two lineages in the Alps and Greece populations. A genealogical signature for population growth was found for lineage B, but not for lineage A; futhermore the Clade B haplotypes in western populations and clade A haplo‐types in eastern populations are recently derived, as revealed by their peripheral location in median‐joining haplotype networks. This phylogeographical pattern suggests allopatric differentiation of the two lineages in separate Mediterranean and African or Asian glacial refugia, followed by range expansion from the latter leading to two secondary contact suture zones in Central Europe and North Africa. High levels of among‐population differentiation were observed, although these were not correlated with geographical distance. Due to the marked genetic structure, extinction of Central European populations in the last century re‐sulted in the loss of a major portion of the genetic diversity of the species. We also found direct evidence for the effect of drift altering the genetic composition of the remnant Pyrenean population after the demographic bottleneck of the last century. Our results argue for the management of the species as a single population, given the apparent ecological exchangeability of extant stocks, and support the ongoing reintroduction of mixed ancestry birds in the Alps and planned reintroductions in Southern Spain.

Population genetics after fragmentation: the case of the endangered Spanish imperial eagle ( Aquila adalberti )

The highly endangered Spanish imperial eagle, Aquila adalberti, has suffered from both population decline and fragmentation during the last century. Here we describe the current genetic status of the population using an extensive sampling of its current distribution range and both mitochondrial control region sequences and nuclear microsatellite markers. Results were evaluated in comparison to those obtained for the Eastern imperial eagle, Aquila heliaca, its nearest extant relative. Mitochondrial haplotype diversity was lower in the Spanish than in the Eastern species whereas microsatellite allelic richness and expected heterozygosity did not differ. Both allelic richness and expected heterozygosity were lower in the small Parque Nacional de Doñana breeding nucleus compared to the remaining nuclei. A signal for a recent genetic bottleneck was not detected in the current Spanish imperial eagle population. We obtained low but significant pairwise FST values that were congruent with a model of isolation by distance. FST and exact tests showed differentiation among the peripheral and small Parque Nacional de Doñana population and the remaining breeding subgroups. The centrally located Montes de Toledo population did not differ from the surrounding Centro, Extremadura and Sierra Morena populations whereas the latter were significantly differentiated. On the other hand, a Bayesian approach identified two groups, Parque Nacional de Doñana and the rest of breeding nuclei. Recent migration rates into and from Parque Nacional de Doñana and the rest of breeding nuclei were detected by assignment methods and estimated as 2.4 and 5.7 individuals per generation, respectively, by a Bayesian approach. We discuss how management strategies should aim at the maintenance of current genetic variability levels and the avoidance of inbreeding depression through the connection of the different nuclei.

Frugivore-generated seed shadows: a landscape view of demographic and genetic effects.

A seed shadow is the spatial pattern of seed distribution relative to parent trees and other conspecifics; it results from the process of seed dispersal and represents the starting template for plant regeneration. Janzen (1970) and Connell (1971) consider it the population recruitment surface. For animal-dispersed, endozoochorous species the seed shadow results primarily from movement patterns of frugivores. Presumably, frugivores can dramatically affect both the demography and genetic make-up of animal-dispersed plant species. These effects, however, have rarely been documented in an integrated way. In this chapter we focus on how frugivores influence the number and spatial pattern of propagules that reach the soil, and their simultaneous influence on gene flow via seed dispersal. We advocate an integrated view of both demographic and genetic effects to understand the role of frugivores on plant recruitment (Alvarez-Buylla et al., 1996). Given that multiple influences sequentially alter after this initial effect of frugivores (i.e. post-dispersal seed predation, germination, seedling mortality), we need to quantitatively assess the relative importance of dispersal by frugivores for plant population biology. Seed dispersal by frugivores is the link in the demographic transition between the ripe fruit crop on the trees and, after delivery, the subsequent stages of establishment of germinated seeds, seedlings, saplings and established adults, i.e. the whole recruitment cycle. Thus, seed dispersal may play a pivotal role in the demography of plant populations (Harper, 1977) by simultaneously influencing not only the numerical dynamics of recruitment from dispersal to establishment, but also the genetic make-up of the seed shadow. The difficulty of tracking the origin of frugivore-dispersed seeds has precluded a robust analysis of vertebrate seed dispersal (Levey and Sargent, 2000). Indeed, the difficulties in measuring and analysing the dispersal of seeds in natural communities has been considered an unavoidable limitation of the field (Wheelwright and Orians, 1982). Recent developments in molecular biology (Carvalho, 1998), however, have resulted in a series of molecular tools based on DNA analysis that allow analysis of gene-flow patterns via seed dispersal (Ouborg et al., 1999) and the statistical analysis of the resulting patterns of genetic structure (Schnabel et al., 1998a; Luikart and England, 1999). More specifically, for animal-dispersed species, gene flow via seeds can be estimated