Approaches and methods to study wildlife cancer.

The last few years have seen a surge of interest from field ecologists and evolutionary biologists to study neoplasia and cancer in wildlife. This contributes to the One Health Approach, which investigates health issues at the intersection of people, wild and domestic animals, together with their changing environments. Nonetheless, the emerging field of wildlife cancer is currently constrained by methodological limitations in detecting cancer using non-invasive sampling. In addition, the suspected differential susceptibility and resistance of species to cancer often make the choice of a unique model species difficult for field biologists. Here, we provide an overview of the importance of pursuing the study of cancer in non-model organisms and we review the currently available methods to detect, measure and quantify cancer in the wild, as well as the methodological limitations to be overcome to develop novel approaches inspired by diagnostic techniques used in human medicine. The methodology we propose here will help understand and hopefully fight this major disease by generating general knowledge about cancer, variation in its rates, tumour-suppressor mechanisms across species as well as its link to life history and physiological characters. Moreover, this is expected to provide key information about cancer in wildlife, which is a top priority due to the accelerated anthropogenic change in the past decades that might favour cancer progression in wild populations.

LAMP-based molecular sexing in a gonochoric marine bivalve (Macoma balthica rubra) with divergent sex-specific mitochondrial genomes.

Taking advantage of the unique system of doubly uniparental inheritance (DUI) of mitochondria, we developed a reliable molecular method to sex individuals of the marine bivalve Macoma balthica rubra. In species with DUI (~100 known bivalves), both sexes transmit their mitochondria: males bear both a male- and female-type mitogenome, while females bear only the female type. Male and female mitotypes are sufficiently divergent to reliably PCR-amplify them specifically. Loop-mediated isothermal amplification (LAMP) is a precise, economical and portable alternative to PCR for molecular sexing and we demonstrate its application in this context. We used 154 individuals sampled along the Atlantic coast of France and sexed microscopically by gonad examination to test for the congruence among gamete type, PCR sexing and LAMP sexing. We show an exact match among the sexing results from these three methods using the male and female mt-cox1 genes. DUI can be disrupted in inter-specific hybrids, causing unexpected distribution of mitogenomes, such as homoplasmic males or heteroplasmic females. To our knowledge, DUI disruption at the intra-specific scale has never been tested. We applied our sexing protocol to control for unexpected heteroplasmy caused by hybridization between divergent genetic lineages and found no evidence of disruption in the mode of mitochondrial inheritance in M. balthica rubra. We propose LAMP as a useful tool to accelerate eco-evolutionary studies of DUI. It offers the opportunity to investigate the potential role of, previously unaccounted-for, sex-specific patterns such as sexual selection or sex-specific dispersal bias in the evolution of free-spawning benthic species.

Complex mitogenomic rearrangements within the Pectinidae (Mollusca: Bivalvia).

BACKGROUND: Scallops (Bivalvia: Pectinidae) present extraordinary variance in both mitochondrial genome size, structure and content, even when compared to the extreme diversity documented within Mollusca and Bivalvia. In pectinids, mitogenome rearrangements involve protein coding and rRNA genes along with tRNAs, and different genome organization patterns can be observed even at the level of Tribes. Existing pectinid phylogenies fail to resolve some relationships in the family, Chlamydinae being an especially problematic group. RESULTS: In our study, we sequenced, annotated and characterized the mitochondrial genome of a member of Chlamydinae, Mimachlamys varia-a species of commercial interest and an effective bioindicator-revealing yet another novel gene arrangement in the Pectinidae. The phylogeny based on all mitochondrial protein coding and rRNA genes suggests the paraphyly of the Mimachlamys genus, further commending the taxonomic revision of the classification within the Chlamydinae subfamily. At the scale of the Pectinidae, we found that 15 sequence blocks are involved in mitogenome rearrangements, which behave as separate units. CONCLUSIONS: Our study reveals incongruities between phylogenies based on mitochondrial protein-coding versus rRNA genes within the Pectinidae, suggesting that locus sampling affects phylogenetic inference at the scale of the family. We also conclude that the available taxon sampling does not allow for understanding of the mechanisms responsible for the high variability of mitogenome architecture observed in the Pectinidae, and that unraveling these processes will require denser taxon sampling.

Development and characterization of novel SSR markers in the endangered endemic species Ferula sadleriana.

PREMISE: Ferula sadleriana (Apiaceae) is a polycarpic, perennial herb with a very limited range and small populations. It is listed as "endangered" on the IUCN Red List of Threatened Species. Microsatellite markers can contribute to conservation efforts by allowing the study of the genetic structure of its shrinking populations. METHODS AND RESULTS: We used a microsatellite-enriched library combined with an Illumina sequencing approach to develop simple sequence repeat markers in our target species. Out of 44 tested primer pairs, 22 provided specific products, and 13 showed heterologous amplification in the target species. Cross-species amplification was achieved at 20 and 19 loci in two congeneric species, F. soongarica and F. tatarica, respectively. CONCLUSIONS: The primers described here are the first tools that enable the population genetic characterization of F. sadleriana. Our results suggest a wider applicability in the genus Ferula.

Roadsides provide refuge for orchids: characteristic of the surrounding landscape.

Seminatural habitats are declining throughout the world; thus, the role of small anthropogenic habitats in the preservation of plants is becoming increasingly appreciated. Here, we surveyed the orchid flora of roadside verges in five Central European countries (Austria, Hungary, Romania, Slovakia, and Slovenia) and tested how the surrounding landscape matrix affects the overall number of species and individuals, and also different functional groups of orchids. We found more than 2,000 individuals of 27 orchid species during our surveys. According to our results, the increasing coverage of agricultural and urban areas negatively affects both the number of orchid species and individuals on roadsides. Our study further suggests that differences in the surrounding habitats affect which species are found on roadsides, since the increasing coverage of grasslands or forested areas around orchid occurrences had a significant positive effect on the number of grassland or forest-dwelling species and individuals, respectively. Most variance in orchid numerosity and diversity was explained by the cover of the suitable habitat types of the respective taxa in the surrounding landscape of the sampling points. This highlights the importance of roadsides acting as refugia for numerous species and valuable plant communities as well as in supporting biodiversity in general.

Isolation and characterization of 15 SSR loci for the endangered European tetraploid species Gladiolus palustris (Iridaceae).

PREMISE: Gladiolus palustris (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species. METHODS AND RESULTS: We utilized a 454 pyrosequencing approach to identify simple sequence repeat (SSR) regions in a microsatellite-enriched library. Of all SSR regions, 46 were screened for specific PCR amplification, and 15 were found to be applicable in the target species. We found 1.62-3.08 alleles per population (effective alleles: 1.58-2.08) that indicated moderate to high genetic diversity values (0.28-0.44) in three pilot populations. Cross-species amplification was less effective in G. imbricatus and G. tenuis. CONCLUSIONS: The primers reported here can be used for the population genetic characterization of G. palustris. They will help us to better understand the conservation genetics of this highly endangered species.

Bioaccumulation of persistent organic pollutants in the deepest ocean fauna.

The legacy and reach of anthropogenic influence is most clearly evidenced by its impact on the most remote and inaccessible habitats on Earth. Here we identify extraordinary levels of persistent organic pollutants in the endemic amphipod fauna from two of the deepest ocean trenches (>10,000 metres). Contaminant levels were considerably higher than documented for nearby regions of heavy industrialization, indicating bioaccumulation of anthropogenic contamination and inferring that these pollutants are pervasive across the world's oceans and to full ocean depth.