Development and characterization of microsatellite loci in Ulex parviflorus Pourr. And its cross-transferability to other Genisteae.

BACKGROUND: The genus Ulex is composed by 15 species distributed in Europe and Africa, but the majority of them are restricted to the Iberian Peninsula and Northwest Africa. Some of these species are common elements at the landscape level, and others contribute to global biodiversity as narrow endemics. Assayed nuclear and plastid Sanger-sequenced regions do not provide enough resolution to perform evolutionary studies on the genus, neither at the intraspecific population level nor at the interspecific phylogenetic level. Thus, we have developed and characterized a set of nuclear microsatellite loci in U. parviflorus to provide new highly polymorphic molecular markers for the genus Ulex. METHODS AND RESULTS: Genomic DNA enriched in microsatellite motifs using streptavidin-coated M-280 magnetic beads attached to 5'-biotinylated oligonucleotides was sequenced in a 454GS Junior System. After primer design, fluorescent-dyed amplicons were analyzed through capillary sequencing (ABI3730XL). Here we present twelve new high polimorphic SSRs markers developed in U. parviflorus specimens and tested in 120 individuals. The 12 SSR loci amplified a total of 152 alleles, and detected expected heterozygosities that ranged from 0.674 to 0.725 in the genotyped populations. Successful cross-species transferability of the 12 SSR loci to the rest of species included in the genus Ulex and three other representative Genisteae was achieved. CONCLUSIONS: The 12 novel proposed SSRs loci will contribute to perform evolutionary studies and genetic research on the genus Ulex and in other Genisteae.

Latitudinal environmental niches and riverine barriers shaped the phylogeography of the Central Chilean endemic Dioscorea humilis (Dioscoreaceae).

The effects of Pleistocene glaciations and geographical barriers on the phylogeographic patterns of lowland plant species in Mediterranean-climate areas of Central Chile are poorly understood. We used Dioscorea humilis (Dioscoreaceae), a dioecious geophyte extending 530 km from the Valparaíso to the Bío-Bío Regions, as a case study to disentangle the spatio-temporal evolution of populations in conjunction with latitudinal environmental changes since the Last Inter-Glacial (LIG) to the present. We used nuclear microsatellite loci, chloroplast (cpDNA) sequences and environmental niche modelling (ENM) to construct current and past scenarios from bioclimatic and geographical variables and to infer the evolutionary history of the taxa. We found strong genetic differentiation at nuclear microsatellite loci between the two subspecies of D. humilis, probably predating the LIG. Bayesian analyses of population structure revealed strong genetic differentiation of the widespread D. humilis subsp. humilis into northern and southern population groups, separated by the Maipo river. ENM revealed that the ecological niche differentiation of both groups have been maintained up to present times although their respective geographical distributions apparently fluctuated in concert with the climatic oscillations of the Last Glacial Maximum (LGM) and the Holocene. Genetic data revealed signatures of eastern and western postglacial expansion of the northern populations from the central Chilean depression, whereas the southern ones experienced a rapid southward expansion after the LGM. This study describes the complex evolutionary histories of lowland Mediterranean Chilean plants mediated by the summed effects of spatial isolation caused by riverine geographical barriers and the climatic changes of the Quaternary.

Population genetic structure of a sandstone specialist and a generalist heath species at two levels of sandstone patchiness across the Strait of Gibraltar.

Many habitat specialist species are originally composed of small, discontinuous populations because their habitats are naturally fragmented or patchy. They may have suffered the long-term effects of natural patchiness. Mediterranean heathlands, a representative habitat in the Strait of Gibraltar region, are associated with nutrient-poor, acidic sandstone soils. Sandstone soil patches in the African side of the Strait (Tangier) are, in general, smaller and more scattered than in the European side (Algeciras). In this study, we analyze the effect of this sandstone patchiness on the population genetic diversity and structure of two Erica species from these Mediterranean heathlands that differ in their edaphic specificity, E. australis, sandstone specialist, and E. arborea, generalist. Average levels of within-population genetic diversity and gene flow between populations were significantly lower in Tangier (high sandstone patchiness) than in Algeciras (low patchiness) for the sandstone specialist, whereas no differences between both sides of the Strait were detected in the edaphic generalist. Since most endemic species in Mediterranean heathlands of the Strait of Gibraltar are sandstone specialists, these results highlight an increased vulnerability to loss of genetic diversity and local extinction of the heathland endemic flora in the Tangier side of the Strait of Gibraltar.

Novel microsatellite loci for Sebaea aurea (Gentianaceae) and cross-amplification in related species.

PREMISE OF THE STUDY: Microsatellite loci were developed in Sebaea aurea (Gentianaceae) to investigate the functional role of diplostigmaty (i.e., the presence of additional stigmas along the style). • METHODS AND RESULTS: One hundred seventy-four and 180 microsatellite loci were isolated through 454 shotgun sequencing of genomic and microsatellite-enriched DNA libraries, respectively. Sixteen polymorphic microsatellite loci were characterized, and 12 of them were selected to genotype individuals from two populations. Microsatellite amplification was conducted in two multiplex groups, each containing six microsatellite loci. Cross-species amplification was tested in seven other species of Sebaea. The 12 novel microsatellite loci amplified only in the two most closely related species to S. aurea (i.e., S. ambigua and S. minutiflora) and were also polymorphic in these two species. • CONCLUSIONS: These results demonstrate the usefulness of this set of newly developed microsatellite loci to investigate the mating system and population genetic structure in S. aurea and related species.

Microsatellite loci in the Gypsophyte Lepidium subulatum (Brassicaceae), and transferability to other Lepidieae.

Polymorphic microsatellite markers were developed for the Ibero-North African, strict gypsophyte Lepidium subulatum to unravel the effects of habitat fragmentation in levels of genetic diversity, genetic structure and gene flow among its populations. Using 454 pyrosequencing 12 microsatellite loci including di- and tri-nucleotide repeats were characterized in L. subulatum. They amplified a total of 80 alleles (2-12 alleles per locus) in a sample of 35 individuals of L. subulatum, showing relatively high levels of genetic diversity, H(O) = 0.645, H(E) = 0.627. Cross-species transferability of all 12 loci was successful for the Iberian endemics Lepidium cardamines, Lepidium stylatum, and the widespread, Lepidium graminifolium and one species each of two related genera, Cardaria draba and Coronopus didymus. These microsatellite primers will be useful to investigate genetic diversity, population structure and to address conservation genetics in species of Lepidium.

Unravelling genetics at the top: mountain islands or isolated belts?

BACKGROUND AND AIMS: In mountain plant populations, local adaptation has been described as one of the main responses to climate warming, allowing plants to persist under stressful conditions. This is especially the case for marginal populations at their lowest elevation, as they are highly vulnerable. Adequate levels of genetic diversity are required for selection to take place, while high levels of altitudinal gene flow are seen as a major limiting factor potentially precluding local adaptation processes. Thus, a compromise between genetic diversity and gene flow seems necessary to guarantee persistence under oncoming conditions. It is therefore critical to determine if gene flow occurs preferentially between mountains at similar altitudinal belts, promoting local adaptation at the lowest populations, or conversely along altitude within each mountain. METHODS: Microsatellite markers were used to unravel genetic diversity and population structure, inbreeding and gene flow of populations at two nearby altitudinal gradients of Silene ciliata, a Mediterranean high-mountain cushion plant. KEY RESULTS: Genetic diversity and inbreeding coefficients were similar in all populations. Substantial gene flow was found both along altitudinal gradients and horizontally within each elevation belt, although greater values were obtained along altitudinal gradients. Gene flow may be responsible for the homogeneous levels of genetic diversity found among populations. Bayesian cluster analyses also suggested that shifts along altitudinal gradients are the most plausible scenario. CONCLUSIONS: Past population shifts associated with glaciations and interglacial periods in temperate mountains may partially explain current distributions of genetic diversity and population structure. In spite of the predominance of gene flow along the altitudinal gradients, local genetic differentiation of one of the lower populations together with the detection of one outlier locus might support the existence of different selection forces at low altitudes.

In vitro micropropagation of rare and endangered moss Entosthodon hungaricus (Funariaceae) / Micropropagação in vitro do musgo raro Entosthodon hungaricus, ameaçado de extinção (Funariaceae)

The moss Entosthodon hungaricus (Boros) Loeske is an European endemic species typical of dry and saline soils extending from the Iberian Peninsula to Aral-Caspian steppes, similarly to some other xerothermic bryophytes. However, the distribution range is fragmented and localities are quite scattered and the species is considered as rare and vulnerable because of its ephemeral characteristics and specialized ecology. With the aim to develop an active protection plan for this species, the ex situ conservation requirements of E. hungaricus were developed. The axenic culture in in vitro conditions were established, and the optimal growth parameters were adjusted to achieve fully developed gametophytes ready to be reintroduced to its native range and other potentially native areas, where this species was once reported but has not been collected in recent times, suggesting its local extinction (i.e. some areas in Vojvodina, N. Serbia). Starting materials were derived from recent herbarium specimens and from fresh materials collected from Hungarian populations. Several means for sterilization of stating material and growing nutritive media were assayed in different regimes of light and temperature. Here we describe the conditions to achieve full plant development and for its micropropagation. Such materials are adequate for ex situ conservation purposes and for experimental introductions in native and potentially native areas. The first axenical culture of E. hungaricus was successfully established, and the first in vitro micropropagation of this rare and endangered species was achieved. Our study contributes to the conservation biology as well as for the potential use of this moss species in biotechnological research.

O musgo Entosthodon hungaricus (Boros) Loeske é uma espécie endêmica Européia típica de solos secos e salinos que se estendem da Península Ibérica até as estepes Aral-Cáspias, similar a outras briófitas de clima seco. Entretanto, a distribuição é bastante dispersa e fragmentada e a espécie é considerada muito rara e vulnerável devido às suas características efêmeras e ecologia especializada. Com o intuito de desenvolver um plano de proteção a essa espécie, foram elaborados os requisitos de preservação ex situ das E. hungaricus. As condições para a cultura axênica in vitro foram estabelecidas e os parâmetros ideais de crescimento foram atingidos para conseguir gametófitos completamente desenvolvidos, prontos para serem reintroduzidos em suas áreas nativas e em outras áreas potencialmente nativas, onde essa espécie já foi relatada. Porém, não houve coleta da mesma nos últimos anos, o que sugere uma extinção local (por exemplo, algumas áreas em Voivodina, Norte da Sérvia). Os materiais iniciais foram derivados de espécies recentes de herbários e de materiais frescos coletados de populações Húngaras. Várias formas de assepsia do material inicial e dos meios de crescimento nutritivo foram ensaiadas em diferentes regimes de luz e temperatura. No trabalho descrevemos as condições para obter desenvolvimento completo da planta e sua micropropagação. Os materiais são adequados para os fins de conservação ex situ e para as introduções experimentais em áreas nativas e/ou potencialmente nativas. O estudo contribui para a conservação biológica bem como para o potencial uso dessa espécie de musgo em pesquisas biotecnológicas.

Characterization of microsatellites in the mountain plant Armeria caespitosa (Plumbaginaceae) and transferability to congeners.

PREMISE OF THE STUDY: The focus of this study is to develop microsatellite markers in Armeria caespitosa, a narrow endemic of central Spain. Microsatellite loci are sought to clarify population structure and estimate gene flux among populations. METHODS AND RESULTS: Enriched microsatellite genomic libraries were used for microsatellite isolation. Sixteen microsatellite loci were characterized, eight of which can be used for successful genotyping. Allele number ranged from two to seven per locus. Observed and expected heterozygosity ranged from 0.300 to 0.800 and from 0.296 to 0.733, respectively. Cross-amplification of seven and six loci was successful for A. maritima and A. cantabrica, respectively. CONCLUSIONS: These microsatellites are suitable in the study of population genetics and gene flow among A. caespitosa populations. The information provided by these markers may be useful in the study of this plant's response to global warming.

Characterization of microsatellite loci in Festuca gautieri (Poaceae) and transferability to F. eskia and F. xpicoeuropeana.

PREMISE OF THE STUDY: Enriched genomic libraries were used to isolate and characterize microsatellite loci in Festuca gautieri, an important plant component of subalpine calcareous grasslands of the eastern Iberian Peninsula, the Pyrenees, and the Cantabrian Mountains. Microsatellites were required to investigate landscape genetics across its distribution range and at a narrower geographical scale within the Ordesa y Monte Perdido, Aigüestortes, and Picos de Europa Spanish national parks. METHODS AND RESULTS: Ten polymorphic microsatellite loci were characterized. They amplified a total of 116 alleles in a sample of 30 individuals of F. gautieri, showing high levels of genetic diversity (expected heterozygosity = 0.821). Cross-species transferability to two other close congeners, F. eskia and F ×picoeuropeana, increased the total number of alleles to 137. These taxa showed lower numbers of alleles but similar levels of genetic diversity to F. gautieri. CONCLUSIONS: These microsatellite primers will be useful in population and landscape genetics and in establishing conservation strategies for these characteristic elements of subalpine pastures.

Postfire response and genetic diversity in Erica coccinea: connecting population dynamics and diversification in a biodiversity hotspot.

Understanding the processes of biological diversification is a central topic in evolutionary biology. The South African Cape fynbos, one of the major plant biodiversity hotspots out of the tropics, has prompted several hypotheses about the causes of generation and maintenance of biodiversity. Fire has been traditionally invoked as a key element to explain high levels of biodiversity in highly speciose fynbos taxa, such as the genus Erica. In this study, we have implemented a microevolutionary approach to elucidate how plant-response to fire may contribute to explain high levels of diversification in Erica. By using microsatellite markers, we investigated the genetic background of seeder (fire-sensitive) and resprouter (fire-resistant) populations of the fynbos species Erica coccinea. We found higher within-population genetic diversity and higher among-population differentiation in seeder populations and interpreted these higher levels of genetic diversification as a consequence of the comparatively shorter generation times and faster population turnover in the seeder form of this species. Considering that genetic divergence among populations may be seen as the initial step to speciation, the parallelism between these results and the pattern of biodiversity at the genus level offers stimulating insights into understanding causes of speciation of the genus Erica in the Cape fynbos.

New microsatellite loci in the dwarf yams Dioscorea group Epipetrum (Dioscoreaceae).

PREMISE OF THE STUDY: Microsatellite loci were isolated and characterized from enriched genomic libraries of two taxa of the Chilean Epipetrum group of Dioscorea to assess their levels of genetic diversity and population differentiation. • METHODS AND RESULTS: Eleven microsatellite loci were identified. Six out of nine microsatellites from D. biloba amplified in D. humilis, and the two microsatellites from D. humilis amplified in both taxa. Two different sets of eight loci amplified in each of the two tested taxa, D. biloba and D. humilis. The average number of alleles was 5.75 and 5 for D. biloba and D. humilis, respectively. Higher levels of mean genetic diversity were found in D. biloba (H(E) = 0.639) than in D. humilis (H(E) = 0.414). • CONCLUSIONS: These microsatellite primers will be useful in population genetic studies and to establish conservation strategies in the endangered taxa of the Epipetrum group of Dioscorea.

Siring success and paternal effects in heterodichogamous Acer opalus.

BACKGROUND AND AIMS: Heterodichogamy (a dimorphic breeding system comprising protandrous and protogynous individuals) is a potential starting point in the evolution of dioecy from hermaphroditism. In the genus Acer, previous work suggests that dioecy evolved from heterodichogamy through an initial spread of unisexual males. Here, the question is asked as to whether the different morphs in Acer opalus, a species in which males co-exist with heterodichogamous hermaphrodites, differ in various components of male in fitness. METHODS: Several components of male fertility were analysed. Pollination rates in the male phase were recorded across one flowering period. Pollen viability was compared among morphs through hand pollinations both with pollen from a single sexual morph and also simulating a situation of pollen competition; in the latter experiment, paternity was assessed with microsatellite markers. It was also determined whether effects of genetic relatedness between pollen donors and recipients could influence the siring success. Finally, paternal effects occurring beyond the fertilization process were tested for by measuring the height reached by seedlings with different sires over three consecutive growing seasons. KEY RESULTS: The males and protandrous morphs had higher pollination rates than the protogynous morph, and the seedlings they sired grew taller. No differences in male fertility were found between males and protandrous individuals. Departures from random mating due to effects of genetic relatedness among sires and pollen recipients were also ruled out. CONCLUSIONS: Males and protandrous individuals are probably better sires than protogynous individuals, as shown by the higher pollination rates and the differential growth of the seedlings sired by these morphs. In contrast, the fertility of males was not higher than the male fertility of the protandrous morph. While the appearance of males in sexually specialized heterodichogamous populations is possible, even in the absence of a fitness advantage, it is not clear that males can be maintained at an evolutionary equilibrium with two classes of heterodichogamous hermaphrodites.

Dated historical biogeography of the temperate Loliinae (Poaceae, Pooideae) grasses in the northern and southern hemispheres.

Divergence times and biogeographical analyses have been conducted within the Loliinae, one of the largest subtribes of temperate grasses. New sequence data from representatives of the almost unexplored New World, New Zealand, and Eastern Asian centres were added to those of the panMediterranean region and used to reconstruct the phylogeny of the group and to calculate the times of lineage-splitting using Bayesian approaches. The traditional separation between broad-leaved and fine-leaved Festuca species was still maintained, though several new broad-leaved lineages fell within the fine-leaved clade or were placed in an unsupported intermediate position. A strong biogeographical signal was detected for several Asian-American, American, Neozeylandic, and Macaronesian clades with different affinities to both the broad and the fine-leaved Festuca. Bayesian estimates of divergence and dispersal-vicariance analyses indicate that the broad-leaved and fine-leaved Loliinae likely originated in the Miocene (13My) in the panMediterranean-SW Asian region and then expanded towards C and E Asia from where they colonized the New World. Further expansions in America (10-3.8My) showed a predominant migratory route from North to South (N America<-->the Andes<-->Patagonia). This late Tertiary scenario of successive colonizations and secondary polyploid radiations in the southern hemisphere from the northern hemisphere was accompanied by occasional transcontinental long-distance dispersal events between South America and New Zealand. Multiple Pliocene dispersal events (3.6-2.5My) from the near SW European and NW African continents gave rise to the Macaronesian Loliinae flora, while a more recent Pleistocene origin (2-1My) is hypothesized for the high polyploid lineages that successfully colonized newly deglaciated areas in both hemispheres.

Burning phylogenies: fire, molecular evolutionary rates, and diversification.

Mediterranean-type ecosystems are among the most remarkable plant biodiversity "hot spots" on the earth, and fire has traditionally been invoked as one of the evolutionary forces explaining this exceptional diversity. In these ecosystems, adult plants of some species are able to survive after fire (resprouters), whereas in other species fire kills the adults and populations are only maintained by an effective post-fire recruitment (seeders). Seeders tend to have shorter generation times than resprouters, particularly under short fire return intervals, thus potentially increasing their molecular evolutionary rates and, ultimately, their diversification. We explored whether seeder lineages actually have higher rates of molecular evolution and diversification than resprouters. Molecular evolutionary rates in different DNA regions were compared in 45 phylogenetically paired congeneric taxa from fire-prone Mediterranean-type ecosystems with contrasting seeder and resprouter life histories. Differential diversification was analyzed with both topological and chronological approaches in five genera (Banksia, Daviesia, Lachnaea, Leucadendron, and Thamnochortus) from two fire-prone regions (Australia and South Africa). We found that seeders had neither higher molecular rates nor higher diversification than resprouters. Such lack of differences in molecular rates between seeders and resprouters-which did not agree with theoretical predictions-may occur if (1) the timing of the switch from seeding to resprouting (or vice versa) occurs near the branch tip, so that most of the branch length evolves under the opposite life-history form; (2) resprouters suffer more somatic mutations and therefore counterbalancing the replication-induced mutations of seeders; and (3) the rate of mutations is not related to shorter generation times because plants do not undergo determinate germ-line replication. The absence of differential diversification is to be expected if seeders and resprouters do not differ from each other in their molecular evolutionary rate, which is the fuel for speciation. Although other factors such as the formation of isolated populations may trigger diversification, we can conclude that fire acting as a throttle for diversification is by no means the rule in fire-prone ecosystems.

A Bayesian approach for discriminating among alternative inheritance hypotheses in plant polyploids: the allotetraploid origin of genus Borderea (Dioscoreaceae).

Polyploidy is a common phenomenon occurring in a vast number of land plants. Investigations of patterns of inheritance and the origins of plants (i.e., autopolyploidy vs. allopolyploidy) usually involve cytogenetic and molecular studies of chromosome pairing, chromosome mapping, and marker segregation analysis through experimental crosses and progeny tests. Such studies are missing for most wild species, for which artificial crosses are difficult, not feasible, or unaffordable. We report here a Bayesian method to discriminate between alternative inheritance patterns in the two extant, tetraploid species of the monocot genus Borderea (Dioscoreaceae), which does not involve progeny array tests. Our approach is based on the screening of a large number of SSR genotypes, which were obtained from successful amplifications of 17 microsatellite regions in individuals of both B. chouardii and B. pyrenaica. We tested for tetrasomic vs. disomic modes of inheritance, using the Bayes factor test. Assignment of genotypes under both alternatives could be unequivocally done for 14 and 11 of the 17 studied microsatellite regions in B. chouardii and B. pyrenaica, respectively, totaling 9502 analyzed genotypes. The comparison of posterior probabilities for the two competing hypotheses across the surveyed loci clearly favored a disomic inheritance pattern. Linkage tests indicated that none of the studied SSR loci were in linkage disequilibrium, thus representing independent samples of the Borderea genome. These results, along with previous allozyme data, support the allotetraploid origin of this paleoendemic genus and reveal the lowest reported chromosome base number for the family of the yams.

Genetic fingerprinting of germplasm accessions as an aid for species conservation: a case study with Borderea chouardii (Dioscoreaceae), one of the most critically endangered Iberian plants.

BACKGROUND AND AIMS: Molecular markers have changed previous expectations about germplasm collections of endangered plants, as new perspectives aim at holding a significant representation of all the genetic diversity in the studied species to accomplish further conservation initiatives successfully. Borderea chouardii is a critically endangered allotetraploid dioecious member of Dioscoreaceae, known from a single population in the Iberian pre-Pyrenees. This population was reported to be highly structured into two genetically distinct groups of individuals corresponding to their spatial separation along the vertical cliff where it grows. In 1999, the Spanish Government of Aragón launched the first conservation programme for the ex situ preservation of this species, and since then a seed collection has been conserved at the Germplasm Bank of the Universidad Politécnica de Madrid. However, as some seed samples had not been labelled clearly at the time of collection, their origin was uncertain. METHODS: Genetic variation in germplasm accessions of B. chouardii was investigated using microsatellite (simple sequence repeat; SSR) markers. KEY RESULTS: The 17 primer pairs used detected 62 SSR alleles in the 46 samples analysed from five different germplasm stocks. Eight alleles scored from the wild population were not detected in the germplasm samples analysed. The relatedness of the germplasm samples to the wild subpopulations through neighbour-joining clustering, principal coordinates analysis (PCO) and assignment tests revealed a biased higher representation of the genetic diversity of the lower cliff (43 samples) subpopulation than that of the upper cliff (three samples). CONCLUSIONS: The collection of additional samples from the upper cliff is recommended to achieve a better representation of the genetic diversity of this subpopulation. It is also recommended that these stocks should be managed separately according to their distinct microspatial origin in order to preserve the genetic substructuring of the wild population.