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Measuring Levels of MHC diversity between free-ranging and potentially isolated populations of Black-backed jackals(Canis mesomelas)

Final Report Summary - JACKALS (Measuring Levels of MHC diversity between free-ranging and potentially isolated populations of Black-backed jackals(Canis mesomelas))

Project overview:
The aim of this project was to investigate levels of genetic variation associated with genes that are directly related to survival in a globally distributed species. To this end levels of allele diversity expressed by two immune system associated class II MHC genes were described for the most widely distributed terrestrial carnivore, the red fox (Vulpes vulpes). Genetic samples were sourced from locations throughout the red fox global distribution incorporating tundra, taiga forest, temperate forest, grassland and desert biomes. This was accomplished in order to develop a data set that would encompass as many external environmental parameters as possible for one single species. The study has identified a large number of previously unknown alleles some of which demonstrate trans-species polymorphism and possibly show evidence of adaptation to specific environmental conditions. Additionally data from North American populations provide further evidence for the taxonomic separation of native California red fox. As such the study has lead to the development of several additional collaborative investigations with universities both within Europe and the United States.
Rationale:
Immune function genes of the major histocompatibility complex (MHC) were selected as appropriate markers for this study as previous investigations have strongly linked MHC allelic diversity and heterozygosity to species survival. Overall it is hoped that understanding which genes and allelic profiles are expressed under a variety of external conditions (such as disease risk) will prove invaluable for the development of future endangered species captive breeding programmes. Maintenance of appropriate levels of genetic diversity should be a priority for such programmes as it can significantly enhance post release survival.
The red fox was chosen as a suitable study species due to its wide global distribution and therefore high potential for investigating differing responses to a variety of external environmental factors.
Target groups for the findings of this study include the general research community but specifically conservation geneticists and conservation institutions focusing on captive management of endangered species such as captive breeding and release facilities, zoological gardens, wildlife reserves and national parks.
Samples:
Genetic samples have been obtained from 11 countries and 4 continents (Ireland, USA, Sweden, Poland, Belarus, France, UK. Egypt, Libya, UAE and Australia). The overall sample size is approximately 600 individual red fox from all biomes mentioned above. In addition a further 150 samples of arctic fox (Vulpes lagopus) representing the entire circumpolar distribution of this species were also included to further investigate if the specific molecular techniques developed for this study could be applied to other species.
Polymerase Chain Reaction (PCR) was used to amplify the genomic regions of interest (exon 2 MHC class II genes DRA and DRB), with the aid of a new set of primers designed during this study for foxes. Sequencing was achieved using Sanger sequencing techniques. Sequence alignment and allele assignment was carried out using SBTengine®.
New primer design:
New robust red fox specific DRB primers where developed for the purposes of this project. These new primers were designed from the regions flanking the exon 2 to ensure the entire exon could be targeted and also make sure other closely related MHC genes are not cross amplified giving falsely inflated impressions of allelic diversity. The resultant primers have proven successful by ensuring that only DRB sequences are amplified for both red and arctic fox.
Results:
MHC class II gene DRA was discovered to show little variation throughout the entire range of the red fox thus far tested. One global allele (DRA1*00101) seems to exist with two variants of this allele (001v1 and 001v2) occurring in low numbers (5 and 1 copies respectively) in two regions of the United States. However, in contrast, DRB has proven to be a remarkably variable gene with 52 new alleles identified. This gene also shows striking global structure. Unique DRB alleles have been found in Ireland, Scandinavia and North America.
Unique regional patterns:
22 new DRB alleles have been found in foxes from the United States (including Alaska), 20 of which are unique to the region. Further structuring was observed with three unique Alaskan alleles. Additionally, allele differences observed between purported native, (Sacramento valley) and non-native Californian foxes have also been found. Native Californian foxes possess six alleles found nowhere else . Fst values show little evidence of introgression between these populations. These findings may prove significant with regards to the ongoing debate over the taxonomic identity of dwindling native California red fox populations. As such it is planned that the analysis will be extended to also include Sierra Nevada red fox, a critically endangered and likely separate red fox species.
A total of 19 alleles have been identified for the Fennoscandia region, 10 of which are unique to this locale with Norway and Finland both possessing 3 seemingly exclusive alleles. Phylogenetic analysis identified a number alleles from Fennoscandia and Alaska that group together. This group is of particular interest as it may suggest the possibility that these alleles confer immune system adaptations to cold climates.
14 alleles have been observed in red foxes from Ireland with eight of these proving thus far unique. This surprising level of allelic diversity from a relatively isolated geographic region may provide further evidence for Southwestern Ireland acting as refugia during periods of glaciation.
Wide-ranging alleles.
The distinct geographical structure reported here would seem to point to adaptation to local conditions. Such information can prove vital for conservation efforts. However alleles that seem to prove successful over a wide range of habitat types are equally valuable. Two alleles (DRB1*001 and 010) observed as common in the British Isles dominate the relatively meagre Australian fox profiles. However given the success of the introduction of red fox to Australia, these alleles should warrant further investigations into their functionality and range of antigenic reactivity.

Project evolution.
The collaborative nature of the project has lead to the development of a number of new projects between the fellow and researchers at universities in both Europe and the United States. In each case the new study is based on the findings of the original study and includes postgraduate research opportunities for students at either the host or the collaborative institution that is either supervised or co-supervised by the fellow. These student projects are designed to produce publishable results and therefore enhance the student experience and introduce a new generation of potential researchers to the field MHC analysis.

1. Successful amplification of arctic fox alleles has facilitated the development of a Masters level investigation into MHC variability in an inbred arctic fox population conducted by a student at Stockholm. This project has been completed.
2. MHC variation in the Sierra Nevada red fox. Researchers at the University of California at Davis will conduct laboratory work based on the protocols established during the course of “Jackals”. In addition the fellow will supervise a postgraduate researcher at the host institute who will conduct sequence alignment, allele assignment and phylogenetic and population analysis. Ongoing
3. South Western Ireland as a potential ice age refugia. A collaborative investigation between the fellow and researchers at Durham University (UK) to further investigate patterns of MHC diversity in UK and European red foxes (including urban vs rural). Ongoing

Conclusion.
This project has brought together researchers from all over the world with a view to understanding what makes the worlds widest ranging terrestrial carnivore, the red fox, so successful. We have uncovered a high degree of allelic diversity within a particular genomic region relating to pathogen recognition. This diversity points towards the ability to generate and maintain adaptations to specific regional conditions. However, our findings in Australian foxes add complexity to the picture by demonstrating that the red fox can persist and succeed when diversity in this genomic region is lacking. This work has spawned the development of several off shoot investigations that will hopefully add further insight into this field.