Unlocking Antarctic molecular time-capsules - Recovering historical environmental DNA from museum-preserved sponges.

Marine sponges have recently emerged as efficient natural environmental DNA (eDNA) samplers. The ability of sponges to accumulate eDNA provides an exciting opportunity to reconstruct contemporary communities and ecosystems with high temporal and spatial precision. However, the use of historical eDNA, trapped within the vast number of specimens stored in scientific collections, opens up the opportunity to begin to reconstruct the communities and ecosystems of the past. Here, we define the term 'heDNA' to denote the historical environmental DNA that can be obtained from the recent past with high spatial and temporal accuracy. Using a variety of Antarctic sponge specimens stored in an extensive marine invertebrate collection, we were able to recover information on Antarctic fish biodiversity from specimens up to 20 years old. We successfully recovered 64 fish heDNA signals from 27 sponge specimens. Alpha diversity measures did not differ among preservation methods, but sponges stored frozen had a significantly different fish community composition compared to those stored dry or in ethanol. Our results show that we were consistently and reliably able to extract the heDNA trapped within marine sponge specimens, thereby enabling the reconstruction and investigation of communities and ecosystems of the recent past with a spatial and temporal resolution previously unattainable. Future research into heDNA extraction from other preservation methods, as well as the impact of specimen age and collection method, will strengthen and expand the opportunities for this novel resource to access new knowledge on ecological change during the last century.

The effects of transcription and recombination on mutational dynamics of short tandem repeats.

Short tandem repeats (STR) are ubiquitous components of the genomic architecture of most living organisms. Recent work has highlighted the widespread functional significance of such repeats, particularly around gene regulation, but the mutational processes underlying the evolution of these highly abundant and highly variable sequences are not fully understood. Traditional models assume that strand misalignment during replication is the predominant mechanism, but empirical data suggest the involvement of other processes including recombination and transcription. Despite this evidence, the relative influences of these processes have not previously been tested experimentally on a genome-wide scale. Using deep sequencing, we identify mutations at >200 microsatellites, across 700 generations in replicated populations of two otherwise identical sexual and asexual Saccharomyces cerevisiae strains. Using generalized linear models, we investigate correlates of STR mutability including the nature of the mutation, STR composition and contextual factors including recombination, transcription and replication origins. Sexual capability was not a significant predictor of microsatellite mutability, but, intriguingly, we identify transcription as a significant positive predictor. We also find that STR density is substantially increased in regions neighboring, but not within, recombination hotspots.

The accuracy, feasibility and challenges of sequencing short tandem repeats using next-generation sequencing platforms.

To date we have little knowledge of how accurate next-generation sequencing (NGS) technologies are in sequencing repetitive sequences beyond known limitations to accurately sequence homopolymers. Only a handful of previous reports have evaluated the potential of NGS for sequencing short tandem repeats (microsatellites) and no empirical study has compared and evaluated the performance of more than one NGS platform with the same dataset. Here we examined yeast microsatellite variants from both long-read (454-sequencing) and short-read (Illumina) NGS platforms and compared these to data derived through Sanger sequencing. In addition, we investigated any locus-specific biases and differences that might have resulted from variability in microsatellite repeat number, repeat motif or type of mutation. Out of 112 insertion/deletion variants identified among 45 microsatellite amplicons in our study, we found 87.5% agreement between the 454-platform and Sanger sequencing in frequency of variant detection after Benjamini-Hochberg correction for multiple tests. For a subset of 21 microsatellite amplicons derived from Illumina sequencing, the results of short-read platform were highly consistent with the other two platforms, with 100% agreement with 454-sequencing and 93.6% agreement with the Sanger method after Benjamini-Hochberg correction. We found that the microsatellite attributes copy number, repeat motif and type of mutation did not have a significant effect on differences seen between the sequencing platforms. We show that both long-read and short-read NGS platforms can be used to sequence short tandem repeats accurately, which makes it feasible to consider the use of these platforms in high-throughput genotyping. It appears the major requirement for achieving both high accuracy and rare variant detection in microsatellite genotyping is sufficient read depth coverage. This might be a challenge because each platform generates a consistent pattern of non-uniform sequence coverage, which, as our study suggests, may affect some types of tandem repeats more than others.

Parallel tagged next-generation sequencing on pooled samples - a new approach for population genetics in ecology and conservation.

Next-generation sequencing (NGS) on pooled samples has already been broadly applied in human medical diagnostics and plant and animal breeding. However, thus far it has been only sparingly employed in ecology and conservation, where it may serve as a useful diagnostic tool for rapid assessment of species genetic diversity and structure at the population level. Here we undertake a comprehensive evaluation of the accuracy, practicality and limitations of parallel tagged amplicon NGS on pooled population samples for estimating species population diversity and structure. We obtained 16S and Cyt b data from 20 populations of Leiopelma hochstetteri, a frog species of conservation concern in New Zealand, using two approaches - parallel tagged NGS on pooled population samples and individual Sanger sequenced samples. Data from each approach were then used to estimate two standard population genetic parameters, nucleotide diversity (π) and population differentiation (FST), that enable population genetic inference in a species conservation context. We found a positive correlation between our two approaches for population genetic estimates, showing that the pooled population NGS approach is a reliable, rapid and appropriate method for population genetic inference in an ecological and conservation context. Our experimental design also allowed us to identify both the strengths and weaknesses of the pooled population NGS approach and outline some guidelines and suggestions that might be considered when planning future projects.

Mixing plants from different origins to restore a declining population: ecological outcomes and local perceptions 10 years later.

Populations of the Large-flowered Sandwort (Arenaria grandiflora L.) in the Fontainebleau forest (France) have declined rapidly during the last century. Despite the initiation of a protection program in 1991, less than twenty individuals remained by the late 1990s. The low fitness of these last plants, which is likely associated with genetic disorders and inbreeding depression, highlighted the need for the introduction of non-local genetic material to increase genetic diversity and thus restore Fontainebleau populations. Consequently, A. grandiflora was introduced at three distant sites in the Fontainebleau forest in 1999. Each of these populations was composed of an identical mix of individuals of both local and non-local origin that were obtained through in vitro multiplication. After establishment, the population status (number of individuals, diameter of the plants, and number of flowers) of the introduced populations was monitored. At present, two populations (one of which is much larger than the other) persist, while the third one became extinct in 2004. Analyses of the ecological parameters of the introduction sites indicated that differences in soil pH and moisture might have contributed to the differences in population dynamics. This introduction plan and its outcome attracted interest of local community, with those who supported the plan and regarded its 10-year result as a biological success (i.e., persistent populations were created), but also those who expressed reservations or disapproval of the plan and its outcome. To understand this controversy, a sociological study involving 27 semi-structured interviews was carried out. From these interviews emerged three areas of controversy: alteration of the identity of the plant, alteration of the identity of its territory, and the biological and ethical consequences of the techniques used for the experimental conservation.

Changes in methylation patterns of kiss1 and kiss1r gene promoters across puberty.

The initiation of mammalian puberty is underpinned by an increase in Kisspeptin (Kiss1) signaling via its receptor (Kiss1r/GPR54) on gonadotropin-releasing hormone (GnRH) neurons. Animals and humans with loss-of-function mutations in Kiss1 or Kiss1r fail to go through puberty. The timing of puberty is dependent on environmental factors, and malleability in puberty timing suggests a mechanism that can translate environmental signals into patterns of Kiss1/Kiss1r gene expression. Epigenetics is a powerful mechanism that can control gene expression in an environment-dependent manner. We investigated whether epigenetic DNA methylation is associated with gene expression changes at puberty. We used bisulfite-PCR-pyrosequencing to define the methylation in the promoters of Kiss1 and Kiss1r before and after puberty in female rats. Both Kiss1 and Kiss1r showed highly significant puberty-specific differential promoter methylation patterns. By identifying key differentially methylated residues associated with puberty, these findings will be important for further studies investigating the control of gene expression across the pubertal transition.

Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae).

Genomic libraries of Arenaria grandiflora enriched for di- and trinucleotide repeats were used for the development of novel microsatellite markers. The subset of 13 polymorphic markers was characterized on 40 individuals of A. grandiflora originating from lowland locations in France. The loci amplified 3 to 10 alleles per locus and expected heterozygosities ranged from 0.46 to 0.83. The newly developed markers will be used for population genetic studies and for assessing genetic composition of a restoration experiment of lowland A. grandiflora populations that are protected in France.

Effects of intermediate host genetic background on parasite transmission dynamics: a case study using Schistosoma mansoni.

For parasites that require multiple hosts to complete their development, genetic interplay with one host may impact parasite transmission and establishment in subsequent hosts. In this study, we used microsatellite loci to address whether the genetic background of snail intermediate hosts influences life-history traits and transmission patterns of dioecious trematode parasites in their definitive hosts. We performed experimental Schistosoma mansoni infections utilizing two allopatric populations of Biomphalaria glabrata snails and assessed intensities and sex ratios of adult parasites in mouse definitive hosts. Our results suggest that the genetic background of hosts at one point in a parasite's life cycle can influence the intensities and sex ratios of worms in subsequent hosts.

Interplay between host genetic variation and parasite transmission in the Biomphalaria glabrata-Schistosoma mansoni system.

Genetic variability is often predicted to enhance host fitness in the face of parasitism, yet this idea is rarely tested in an experimental setting, particularly with animal hosts. To assess this question, we used a relatively resistant line of snail hosts (Biomphalaria glabrata) to generate inbred and outcrossed progeny that were then either exposed or sham-exposed to the trematode parasite, Schistosoma mansoni. Results showed no difference in prevalence between the groups; however, large differences appeared in other host life history traits, particularly reproduction. Outcrossed progeny produced large numbers of eggs relative to inbred progeny especially in the face of infection. Furthermore, eggs produced by outcrossed snails took less time to hatch and exhibited greater hatching success compared to their inbred counterparts. Parasite reproduction demonstrated the opposite trend, with fewer parasites emerging from outcrossed snails compared to inbred individuals. This work shows that the introduction of genetic variation into inbred snail populations can have important implications for the viability of host populations and disease transmission.

Pollinating fig wasps: genetic consequences of island recolonization.

The levels of genetic diversity and gene flow may influence the long-term persistence of populations. Using microsatellite markers, we investigated genetic diversity and genetic differentiation in island (Krakatau archipelago, Indonesia) and mainland (Java and Sumatra, Indonesia) populations of Liporrhopalum tentacularis and Ceratosolen bisulcatus, the fig wasp pollinators of two dioecious Ficus (fig tree) species. Genetic diversity in Krakatau archipelago populations was similar to that found on the mainland. Population differentiation between mainland coastal sites and the Krakatau islands was weak in both wasp species, indicating that the intervening 40 km across open sea may not be a barrier for wasp gene flow (dispersal) and colonization of the islands. Surprisingly, mainland populations of the fig waSPS may be more genetically isolated than the islands, as gene flow between populations on the Javan mainland differed between the two wasp species. Contrasting growth forms and relative 'immunity' to the effects of deforestation in their host fig trees may account for these differences.

Sex ratio strategies and the evolution of cue use.

Quantitative tests of sex allocation theory have often indicated that organism strategies deviate from model predictions. In pollinating fig wasps, Lipporrhopalum tentacularis, whole fig (brood) sex ratios are generally more female-biased than predicted by local mate competition (LMC) theory where females (foundresses) use density as a cue to assess potential LMC. We use microsatellite markers to investigate foundress sex ratios in L. tentacularis and show that they actually use their clutch size as a cue, with strategies closely approximating the predictions of a new model we develop of these conditions. We then provide evidence that the use of clutch size as a cue is common among species experiencing LMC, and given the other predictions of our model argue that this is because their ecologies mean it provides sufficiently accurate information about potential LMC that the use of other more costly cues has not evolved. We further argue that the use of these more costly cues by other species is due to the effect that ecological differences have on cue accuracy. This implies that deviations from earlier theoretical predictions often indicate that the cues used to assess environmental conditions differ from those assumed by models, rather than limits on the ability of natural selection to produce "perfect" organisms.