Evaluating the impact of scoring parameters on the structure of intra-specific genetic variation using RawGeno, an R package for automating AFLP scoring

<p>Abstract</p> <p>Background</p> <p>Since the transfer and application of modern sequencing technologies to the analysis of amplified fragment-length polymorphisms (AFLP), evolutionary biologists have included an increasing number of samples and markers in their studies. Although justified in this context, the use of automated scoring procedures may result in technical biases that weaken the power and reliability of further analyses.</p> <p>Results</p> <p>Using a new scoring algorithm, RawGeno, we show that scoring errors – in particular "bin oversplitting" (i.e. when variant sizes of the same AFLP marker are not considered as homologous) and "technical homoplasy" (i.e. when two AFLP markers that differ slightly in size are mistakenly considered as being homologous) – induce a loss of discriminatory power, decrease the robustness of results and, in extreme cases, introduce erroneous information in genetic structure analyses. In the present study, we evaluate several descriptive statistics that can be used to optimize the scoring of the AFLP analysis, and we describe a new statistic, the information content per bin (I_bin) that represents a valuable estimator during the optimization process. This statistic can be computed at any stage of the AFLP analysis without requiring the inclusion of replicated samples. Finally, we show that downstream analyses are not equally sensitive to scoring errors. Indeed, although a reasonable amount of flexibility is allowed during the optimization of the scoring procedure without causing considerable changes in the detection of genetic structure patterns, notable discrepancies are observed when estimating genetic diversities from differently scored datasets.</p> <p>Conclusion</p> <p>Our algorithm appears to perform as well as a commercial program in automating AFLP scoring, at least in the context of population genetics or phylogeographic studies. To our knowledge, RawGeno is the only freely available public-domain software for fully automated AFLP scoring, from electropherogram files to user-defined working binary matrices. RawGeno was implemented in an R CRAN package (with an user-friendly GUI) and can be found at <url>http://sourceforge.net/projects/rawgeno</url>.</p

Genetic Diversity in an Invasive Clonal Plant? A Historical and Contemporary Perspective

Introduced populations of Eichhornia crassipes (Pontederiaceae) possess extremely low levels of genetic diversity due to severe bottleneck events and clonal reproduction. While populations elsewhere have been well studied, North American populations of E. crassipes remain understudied. We used Amplified Fragment Length Polymorphism markers to assess genetic diversity and population structure in North American E. crassipes populations. Patterns of diversity over the past fifty years were analyzed using herbarium specimens. Furthermore, we sampled populations across the Gulf Coast of the United States throughout a year to determine contemporary genetic diversity and assess potential seasonal effects. Genetic diversity was found to be scant in the United States without population structure, agreeing with previous studies from other regions. Genetic diversity has remained consistently low over the past fifty years despite significant changes in selection pressure. However, evidence for and against population structure between seasons was found and the consequences of this are discussed

Morphological, ecological and genetic aspects associated with endemism in the Fly Orchid group

The European genus Ophrys (Orchidaceae) is famous for its insect-like floral morphology, an adaptation for a pseudocopulatory pollination strategy involving Hymenoptera males. A large number of endemic Ophrys species have recently been described, especially within the Mediterranean Basin, which is one of the major species diversity hotspots. Subtle morphological variation and specific pollinator dependence are the two main perceptible criteria for describing numerous endemic taxa. However, the degree to which endemics differ genetically remains a challenging question. Additionally, knowledge regarding the factors underlying the emergence of such endemic entities is limited. To achieve new insights regarding speciation processes in Ophrys, we have investigated species boundaries in the Fly Orchid group (Ophrys insectifera sensu lato) by examining morphological, ecological and genetic evidence. Classically, authors have recognized one widespread taxon (O. insectifera) and two endemics (O. aymoninii from France and O. subinsectifera from Spain). Our research has identified clear morphological and ecological factors segregating among these taxa; however, genetic differences were more ambiguous. Insights from cpDNA sequencing and amplified fragment length polymorphisms genotyping indicated a recent diversification in the three extant Fly Orchid species, which may have been further obscured by active migration and admixture across the European continent. Our genetic results still indicate weak but noticeable phylogeographic clustering that partially correlates with the described species. Particularly, we report several isolated haplotypes and genetic clusters in central and southeastern Europe. With regard to the morphological, ecological and genetic aspects, we discuss the endemism status within the Fly Orchid group from evolutionary, taxonomical and conservation perspectives

Preliminary analysis of genetic variation of Escallonia alpina and E. rubra

Escallonia alpina y E. rubra (Escalloniaceae) comparten su área de distribución en el sur de los Andes y se diferencian por pocos caracteres morfológicos diagnósticos, tales como la disposición de las flores y la presencia y tipo de glándulas; sin embargo, en algunas poblaciones no es posible asignar los individuos a una u otra especie. Además, se han reconocido y descrito como híbridos varias formas de transición entre ambas. El objetivo de este trabajo es examinar la variabilidad genética poblacional de Escallonia alpina y E. rubra, y analizar si se corresponde con la variabilidad morfológica observada. Se estudió la morfología en 39 individuos de cinco poblaciones sobre los que también se realizó un análisis de AFLP. Se investigó la relación entre los individuos mediante análisis de ordenación, agrupamiento y de asignación bayesiana. Además se calcularon índices de diversidad y se realizó una prueba de Mantel. A partir del análisis morfológico, se asignaron dos poblaciones a cada especie, mientras que una población con individuos con morfología intermedia no se pudo asignar unívocamente a ninguna de las dos especies. Se observó una fuerte correspondencia entre las especies delimitadas mediante caracteres morfológicos diagnósticos tradicionales y el patrón de AFLP. La evidencia morfológica y molecular preliminar nos permitió reconocer a Escallonia alpina y E. rubra como dos especies distintas, y a una población con caracteres morfológicos y genéticos intermedios que podrían ser el resultado de flujo génico interespecífico o de una separación incompleta de linajes.Escallonia alpina and E. rubra (Escalloniaceae) are distributed in the same geographical region in the southern Andes; they are differentiated by few diagnostic morphological characters such as flower arrangement, and type and presence of glands. A morphological gap between both species is absent in some populations; in addition, intermediate forms have been recognized and described as hybrids. The aim of this work is to examine the genetic variation in populations of Escallonia alpina and E. rubra, and to analyze if there is a correspondence between genetic and morphological variability. We studied the morphology of 39 individuals belonging to five populations, which were also used to perform an AFLP analysis. We conducted ordination, clustering and Bayesian assignment analyses to investigate the relationship among individuals, and we calculated genetic diversity indices and a Mantel test. As a result of the morphological analysis, two populations were assigned to each species, while the remaining population could not be assigned unequivocally to any of the two species, as the individuals exhibited an intermediate morphology. A strong correspondence between traditional morphological characters and the AFLP pattern was observed. Our preliminary morphological and molecular evidence support Escallonia alpina and E. rubra as two distinct species. The variability found in the intermediate population might be the result of inter-specific hybridization or incomplete lineage sorting.Fil: Morello, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion (i); Argentina;Fil: Giussani, Liliana Monica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion (i); Argentina;Fil: Sede, Silvana Mabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica Darwinion (i); Argentina

High levels of effective long-distance dispersal may blur ecotypic divergence in a rare terrestrial orchid

Background: Gene flow and adaptive divergence are key aspects of metapopulation dynamics and ecological speciation. Long-distance dispersal is hard to detect and few studies estimate dispersal in combination with adaptive divergence. The aim of this study was to investigate effective long-distance dispersal and adaptive divergence in the fen orchid (Liparis loeselii (L.) Rich.). We used amplified fragment length polymorphism (AFLP)-based assignment tests to quantify effective long-distance dispersal at two different regions in Northwest Europe. In addition, genomic divergence between fen orchid populations occupying two distinguishable habitats, wet dune slacks and alkaline fens, was investigated by a genome scan approach at different spatial scales (continental, landscape and regional) and based on 451 AFLP loci. Results: We expected that different habitats would contribute to strong divergence and restricted gene flow resulting in isolation-by-adaptation. Instead, we found remarkably high levels of effective long-distance seed dispersal and low levels of adaptive divergence. At least 15% of the assigned individuals likely originated from among-population dispersal events with dispersal distances up to 220 km. Six (1.3%) ‘outlier’ loci, potentially reflecting local adaptation to habitat-type, were identified with high statistical support. Of these, only one (0.22%) was a replicated outlier in multiple independent dune-fen population comparisons and thus possibly reflecting truly parallel divergence. Signals of adaptation in response to habitat type were most evident at the scale of individual populations. Conclusions: The findings of this study suggest that the homogenizing effect of effective long-distance seed dispersal may overwhelm divergent selection associated to habitat type in fen orchids in Northwest Europe

Transgenerational effects in asexually reproduced offspring of Populus

The response of trees to a changing climate can be affected by transgenerational phenotypic plasticity, i.e. phenotypic variation that is conserved and transferred to the offspring. Transgenerational plasticity that is influenced by epigenetics (heritable changes in gene function that do not result from changes in DNA sequence) during both sexual and asexual reproduction are of major relevance for adaptation of plants to climate change. To understand the transgenerational effects on the responses of vegetatively propagated poplar (Populus deltoides and P. trichocarpa) ramets (cuttings) to a changing environment, we tested whether the temperature and photoperiod experienced by the mother trees (genets) persistently affects the phenology of the cuttings grown in a common environment. We weekly monitored the bud phenology of the cuttings collected from the parent trees that have been growing across Europe along a >2100 km latitudinal gradient for at least 18 years. In addition, we asked whether there was variation in DNA methylation as measured by Methylation Sensitive Amplified Fragment Length Polymorphism (MSAPs) in the clones due to the different environmental conditions experienced by the parent trees. Our results indicate a transgenerational effect on bud phenology in the asexually reproduced offspring (vegetative cuttings). The temperatures experienced by the parent tree clones (from different geographic regions) altered the bud flush of the cuttings in the common garden. However, no significant epigenetic variation was detected in the cuttings of the parent trees within single genotypes growing under different climates. In sum, our results show that trees have the potential to respond to rapid climate change but the mechanism behind these changes needs to be further investigated by more powerful molecular methods like whole-genome bisulphite sequencing techniques

Phylogeography of western Mediterranean Cymbalaria (Plantaginaceae) reveals two independent long-distance dispersals and entails new taxonomic circumscriptions

The Balearic Islands, Corsica and Sardinia (BCS) constitute biodiversity hotspots in the western Mediterranean Basin. Oligocene connections and long distance dispersal events have been suggested to cause presence of BCS shared endemic species. One of them is Cymbalaria aequitriloba, which, together with three additional species, constitute a polyploid clade endemic to BCS. Combining amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequences and morphometrics, we inferred the phylogeography of the group and evaluated the species' current taxonomic circumscriptions. Based on morphometric and AFLP data we propose a new circumscription for C. fragilis to additionally comprise a group of populations with intermediate morphological characters previously included in C. aequitriloba. Consequently, we suggest to change the IUCN category of C. fragilis from critically endangered (CR) to near threatened (NT). Both morphology and AFLP data support the current taxonomy of the single island endemics C. hepaticifolia and C. muelleri. The four species had a common origin in Corsica-Sardinia, and two long-distance dispersal events to the Balearic Islands were inferred. Finally, plastid DNA data suggest that interspecific gene flow took place where two species co-occur

Amplified fragment length homoplasy: in silico analysis for model and non-model species

<p>Abstract</p> <p>Background</p> <p>AFLP markers are widely used in evolutionary genetics and ecology. However the frequent occurrence of non-homologous co-migrating fragments (homoplasy) both at the intra- and inter-individual levels in AFLP data sets is known to skew key parameters in population genetics. Geneticists can take advantage of the growing number of full genome sequences available for model species to study AFLP homoplasy and to predict it in non-model species.</p> <p>Results</p> <p>In this study we performed <it>in silico </it>AFLPs on the complete genome of three model species to predict intra-individual homoplasy in a prokaryote (<it>Bacillus thuringiensis </it>ser. <it>konkukian</it>), a plant (<it>Arabidopsis thaliana</it>) and an animal (<it>Aedes aegypti</it>). In addition, we compared <it>in silico </it>AFLPs to empirical data obtained from three related non-model species (<it>Bacillus thuringiensis </it>ser. <it>israelensis, Arabis alpina </it>and <it>Aedes rusticus</it>). Our results show that homoplasy rate sharply increases with the number of peaks per profile. However, for a given number of peaks per profile, genome size or taxonomical range had no effect on homoplasy. Furthermore, the number of co-migrating fragments in a single peak was dependent on the genome richness in repetitive sequences: we found up to 582 co-migrating fragments in <it>Ae. aegypti</it>. Finally, we show that <it>in silico </it>AFLPs can help to accurately predict AFLP profiles in related non-model species.</p> <p>Conclusions</p> <p>These predictions can be used to tackle current issues in the planning of AFLP studies by limiting homoplasy rate and population genetic estimation bias. ISIF (In SIlico Fingerprinting) program is freely available at <url>http://www-leca.ujf-grenoble.fr/logiciels.htm</url>.</p

Effects of cold stress on reproductive biology and epigenetic profiles of the alpine plant Ranunculus kuepferi (Ranunculaceae)

Alpine habitats are shaped by harsh abiotic conditions and cold climates. Plant life in such habitats is challenging, as environmental influence can alter the conditions for development and reproduction. More specifically, phenotypic plasticity of morphological traits can be influenced by temperature stress. Temperature stress can also affect epigenetic and gene expression profiles, which may have an impact on acclimation and adaptation of the species. Polyploidy seems to affect the DNA methylation profiles, while distribution patterns suggest that it could be advantageous under cold conditions. Nevertheless, little is known about non -model plants, whether temperature stress can induce methylation changes depending on the cytotypes of the individuals, to what extent a treatment shift can induce epigenetic responses and how they are depicted in phenotypic plasticity and reproduction of the species. Furthermore, it remains vague how cold stress is translated in gene expression changes under different cytotypes and how such a putative response is framed through gene set pathways and epigenetic control. The perennial alpine plant Ranunculus kuepferi was utilized to investigate the correlations of cold stress with polyploidy, mode of reproduction, phenotypic plasticity, epigenetics, gene expression and geographical parthenogenesis . The species is mainly found in the wild with diploid and autotetraploid cytotypes, which are mostly sexual and facultative apomicts, respectively. Diploid and autotetraploid individuals were placed in two climate chambers and exposed to cold (+7°C day/+2°C night, -1°C cold shocks for three nights per week) and warm (control) (+15°C day/+10°C night) temperature treatments in climate growth chambers for four consecutive flowering periods and shifted from one condition to the other after the first flowering period. Methylation-sensitive amplified fragment- length polymorphism markers were applied for the first two years, to screen possible genome-wide methylation alterations triggered by temprerature treatments and treatment shifts. For the second year of t emperature treatments, morphological traits (height, leaves and flowers) and the proportion of well- developed seeds were measured as fitness indicators, while flow cytometric seed screening (FCSS) was utilized to determine the reproduction mode. Subsequently, comparisons with patterns of methylation-sensitive amplified fragment- length polymorphisms (MS-AFLPs/MSAPs) regarding the same year of treatment were conducted. Finally, for the last year of treatment, both cytotypes were investigated for their gene expression profiles via transcriptome sequencing and qRT-PCR. The datasets were analyzed for four predefined groups with respect to treatment (Cold/Warm) and ploidy level (Diploid/Tetraploid). DNA methylation profiles showed temperature sensitivity and propose a ploidy effect for both years of analysis. Likewise, the treatment shift had an impact on both cytotypes, resulting in significantly less epiloci, regardless of the shift’s direction. Such correlations of ploidy level and epigenetic profiles may reflec t DNA methylation dynamics during cold acclimation. The AMOVA results are in line with the hypothesis of cold stress influencing the epigenetic patterns, while they also depict the DNA methylation dynamics of tetraploids, as a response to temperature treatment shift. Concerning the phenotypic plasticity of the species under temperature treatments, the potential of acclimation under environmental conditions is underlined, as diploids grow better under warm conditions and tetraploids perform better in cold treatments, while the expressed morphological traits are linked with epigenetic patterns. Moreover, cold stress reduced the reproduction fitness but did not induce apomixis in diploid individuals. These results confirm the different niche preferences of cytotypes in natural populations and empower the geographical parthenogenesis scenario, which is previously proposed for the species. Cold acclimation of the cytotypes is further indicated by gene expression profiles. Overall, diploid individuals altered more gene set pathways than tetraploid ones, and suppressed pathways involved in ion/cation homeostasis. Gene Set Pathways mostly activated under tetraploids are related to cell wall and plasma membrane. Thus, tetraploids seem to be better acclimated to cold conditions, enabling them to colonize colder climatic areas in the Alps. Finally, an epigenetic background for gene regulation in response to temperature conditions is indicated.2022-01-2