Dinámica de la variación de las especies en declive: Variación neutral y funcional en el lince ibérico

Programa de Doctorado en Medio Ambiente y SociedadLínea de Investigación: Biodiversidad y Biología de la ConservaciónClave Programa: DAMCódigo Línea: 83El declive y fragmentación de las poblaciones implica un aumento de la deriva genética que puede resultar en pérdidas de diversidad genética y acumulación de consanguinidad. Las disminuciones del potencial adaptativo y de eficacia biológica que éstos procesos pueden llevar asociados se reconocen como riesgos para la persistencia de las especies. Las pérdidas de diversidad se han documentado en la gran mayoría de los casos a través de marcadores moleculares supuestamente neutrales, quedando la duda de hasta que punto éstos representan la variación adaptativa, por los posibles efectos de la selección sobre ésta. Una parte muy importante de la variación claramente adaptativa, que puede ser especialmente relevante para contrarrestar los riesgos de extinción derivados de enfermedades, es la relacionada con la respuesta inmune. La capacidad de respuesta inmune está condicionada en buena medida a la variación presente en genes claves para el reconocimiento y presentación de antígenos. Entre éstos, los genes clase I y clase II del Complejo Mayor de Histocompatibilidad (MHC) son los más estudiados, pero no los únicos elementos importantes. El lince ibérico (Lynx pardinus) ha sufrido un declive y una fragmentación muy acusadas en las últimas décadas que le ha colocado al borde de la extinción y le ha llevado a ser una de las especies con diversidad genética más baja. En cambio, hemos podido constatar que la selección balanceadora ha sido capaz de contrarrestar, al menos para estos genes de inmunidad, el devastador efecto que la deriva genetica que ha tenido para la especie a nivel neutral.Universidad Pablo de Olavide de Sevilla. Departamento de Biología Molecular e Ingeniería BioquímicaPostprin

Biología molecular en la conservación del lince ibérico

La conservación de la biodiversidad en un escenario de cambio global acelerado constituye uno de los grandes retos de la humanidad para las próximas décadas. La genética ha pasado a ser un elemento fundamental del enfoque multidisciplinar que esta tarea requiere, dando origen al área de conocimiento de la genética de la conservación.N

PCR Strategies for Complete Allele Calling in Multigene Families Using High-Throughput Sequencing Approaches

The characterization of multigene families with high copy number variation is often approached through PCR amplification with highly degenerate primers to account for all expected variants flanking the region of interest. Such an approach often introduces PCR biases that result in an unbalanced representation of targets in high-throughput sequencing libraries that eventually results in incomplete detection of the targeted alleles. Here we confirm this result and propose two different amplification strategies to alleviate this problem. The first strategy (called pooled-PCRs) targets different subsets of alleles in multiple independent PCRs using different moderately degenerate primer pairs, whereas the second approach (called pooled-primers) uses a custom-made pool of non-degenerate primers in a single PCR. We compare their performance to the common use of a single PCR with highly degenerate primers using the MHC class I of the Iberian lynx as a model. We found both novel approaches to work similarly well and better than the conventional approach. They significantly scored more alleles per individual (11.33 ± 1.38 and 11.72 ± 0.89 vs 7.94 ± 1.95), yielded more complete allelic profiles (96.28 ± 8.46 and 99.50 ± 2.12 vs 63.76 ± 15.43), and revealed more alleles at a population level (13 vs 12). Finally, we could link each allele’s amplification efficiency with the primer-mismatches in its flanking sequences and show that ultra-deep coverage offered by high-throughput technologies does not fully compensate for such biases, especially as real alleles may reach lower coverage than artefacts. Adopting either of the proposed amplification methods provides the opportunity to attain more complete allelic profiles at lower coverages, improving confidence over the downstream analyses and subsequent applications

Bottleneck-associated changes in the genomic landscape of genetic diversity in wild lynx populations

Demographic bottlenecks generally reduce genetic diversity through more intense genetic drift, but their net effect may vary along the genome due to the random nature of genetic drift and to local effects of recombination, mutation, and selection. Here, we analyzed the changes in genetic diversity following a bottleneck by comparing whole-genome diversity patterns in populations with and without severe recent documented declines of Iberian (Lynx pardinus, n = 31) and Eurasian lynx (Lynx lynx, n = 29). As expected, overall genomic diversity correlated negatively with bottleneck intensity and/or duration. Correlations of genetic diversity with divergence, chromosome size, gene or functional site content, GC content, or recombination were observed in nonbottlenecked populations, but were weaker in bottlenecked populations. Also, functional features under intense purifying selection and the X chromosome showed an increase in the observed density of variants, even resulting in higher θW diversity than in nonbottlenecked populations. Increased diversity seems to be related to both a higher mutational input in those regions creating a large collection of low-frequency variants, a few of which increase in frequency during the bottleneck to the point they become detectable with our limited sample, and the reduced efficacy of purifying selection, which affects not only protein structure and function but also the regulation of gene expression. The results of this study alert to the possible reduction of fitness and adaptive potential associated with the genomic erosion in regulatory elements. Further, the detection of a gain of diversity in ultra-conserved elements can be used as a sensitive and easy-to-apply signature of genetic erosion in wild populations.Peer reviewe

Additional file 2: Figure S1. of Retention of functional variation despite extreme genomic erosion: MHC allelic repertoires in the Lynx genus

Phylogenetic network of Lynx MHCI alleles. We constructed a network with the Neighbor-Net method and Jukes-Cantor distances in SplitsTree4. Alleles are colored by species of origin. Figure S2. Phylogenetic network of Lynx MHCII-DRB alleles. We constructed a network with the Neighbor-Net method and Jukes-Cantor distances in SplitsTree4. Alleles are colored by species of origin. Figure S3. Phylogenetic tree of Felidae MHCI alleles. The tree was constructed with RaxML. Tips are colored by genus of origin. Figure S4. Phylogenetic tree of Felidae MHCII_DRB alleles. The tree was constructed with RaxML. Tips are colored by genus of origin. Figure S5. Felidae MHCI supertype definition. We used average hierarchical clustering and a cut-off threshold of Euclidean distance ≥15 to classify alleles into distinctive functional groups (supertypes, red boxes) based on functional similarity at their ABS. Alleles with identical amino acids at ABS are represented as tip polytomies at 0 Euclidean distance. Figure S6. Felidae MHCII-DRB supertype definition. We used average hierarchical clustering and a cut-off threshold of Euclidean distance ≥15 to classify alleles into distinctive functional groups (supertypes, red boxes) based on functional similarity at their ABS. Alleles with identical amino acids at ABS are represented as polytomies at 0 euclidean distance. (PDF 408 kb

Allelic profile completeness obtained with increasing coverage.

<p>The same set of individuals was assayed with the three strategies and the reads obtained for each amplicon were bootstrapped to simulate lower coverages (increasing steps of 10 reads, 100 iterations). Profile completeness is defined as the proportion of the alleles in the individual’s inferred profile (from the pooling of all available data) that were scored in each iteration. Both the average value and its confidence interval (95%) are represented. Note that increasing the coverage does not compensate for highly biased amplification efficiencies (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157402#pone.0157402.s002" target="_blank">S2 Fig</a> for larger sampling sizes).</p

Comparison of the general performance of the three approaches and expected values.

<p>Comparison of the general performance of the three approaches and expected values.</p

Diagram depicting the three alternative multigene family amplification strategies compared in this study.

<p>When amplifying multigene families, or other complex targets, some alleles may contain variants in the priming region, here reflected by colored bars. (a) Primer design: The conventional strategy tries to match all known variation by designing highly degenerate primers, but some variants might be missed because of unknown variation or in an attempt to avoid highly-degenerate primers. Degenerate nucleotides in the primers are represented by bars with more than one color. In the pooled-PCRs strategy, allele groups with similar priming regions are targeted separately with low-degeneracy primers and by taking into account the observed phase. In the pooled-primer approach, non-degenerate primers targeting each known flanking region are pooled according to the expected number of targeted alleles. (b) PCR amplification: The conventional and pooled-primers strategies amplify all alleles in a single PCR while in the pooled-PCRs strategy an independent PCR is performed for each primer pair. Both the conventional and pooled-PCRs approaches yield unbalanced libraries, but in the case of pooled-PCRs each library is biased toward a different set of alleles. Biases in the pooled-primers approach are minimized because all alleles are primed by perfectly matching primers at the right concentration. (c) PCR yield pooling: This step is exclusive to the pooled-PCRs approach and attempts to produce a final balanced sequencing library by pooling independent PCRs taking into account the number of perfectly targeted alleles. Note that this diagram is a sketch for illustrative purposes only and does not reflect the alleles, primers or libraries used in this study.</p

Reproductive biology and genealogy in the endangered Iberian lynx: Implications for conservation

For endangered species, the availability of genealogies and a good knowledge of mating patterns are valuable resources for conservation purposes. Here we studied mating patterns in the endangered Iberian lynx, Lynx pardinus, and reconstructed a partial genealogy of the intensively monitored population in Doñana from 1990 to 2013. Using microsatellites in combination with field information we assigned 146 out of the 175 analysed individuals to at least one parent with confidence above 0.9. We detected breeding events for 30% of individuals in the population (n = 181), which follows a positively skewed distribution (maximum: 7 breeding events for females, 9 for males). On average, individuals reproduce with approximately 1.6 mates in their life, from 2 years-old to a maximum of 10 years for males and 11 years for females. This broadening of the previously reported breeding age is likely due to stochastic changes in the demography that resulted in lack of competition and a high turnover of the territories. We identified several crosses between close relatives (e.g. full-sibs) which resulted in highly inbred offspring. To our knowledge, this is one of the most comprehensive studies on reproductive patterns of an endangered felid in the wild. This novel information highlights the importance of both field and genetic data to broaden the knowledge of the species and to improve conservation programs.Peer Reviewe

Standardized amplification efficiencies for each allele obtained with the three amplification strategies.

<p>For easier comparison alleles are ranked from one (the lowest) to 13 (the highest) within each strategy, and thus numbers do not identify specific alleles. The range of standardized amplification efficiencies is approximately two times and four times larger using the conventional strategy than the pooled-PCRs and pooled-primers approaches, respectively.</p