Rumen microbial ecology in reindeer-adaptations to a unique diet

Svalbard reindeer are isolated on the high-arctic archipelago of Svalbard under austere nutritional conditions, while the semi-domesticated Norwegian reindeer migrate between lush coastal summer pastures and inland taiga and boreal forest habitats with ground lichens in winter on mainland Norway. We propose that interactions between reindeer and Arctic plants through 15 million years of evolution have resulted in a unique rumen microbial ecosystem, and present data on rumen bacterial diversity analysed by PCR amplifi cation and sequencing of 16S rDNA clone libraries from these sub-species of reindeer. Resulting sequences represent novel bacteria different from those reported in other ruminants.


INTRODUCTION
The Svalbard reindeer (Rangifer tarandus platyrhynchus) lives under extremely austere nutritional conditions at the arctic archipelago of Svalbard (74-81°N), where snow and ice cover the vegetation for more than eight months of the year. They have never been domesticated, are non-migratory and forage on tundra vegetation which is locally lush with herbs, dwarf shrubs, grasses and sedges in summer, while a polar desert vegetation of mosses and poor quality vascular plants dominate the winter diet (Sørmo, 1998). Norwegian reindeer (R. t. tarandus) on mainland Norway are herded by the Saami and migrate seasonally up to 350 km between lush coastal summer pastures and inland taiga and boreal forest habitats in winter, including a signifi cant proportion of ground lichens (Cladonia spp.) in their diet (Storeheier et al., 2002a,b). Seasonal variations in viable numbers of rumen bacteria as well as the composition of the viable population are found in both sub-species (Orpin et al., 1985;Olsen et al., 1997). The purpose of this project was to gain a better understanding of the natural rumen microbial ecology and hence the digestive adaptations in these Arctic ruminants, using new molecular techniques based on sequence heterogeneity of the 16S rDNA. Furthermore, the effect of rumen manipulations through artifi cial feeding of semi-domesticated Norwegian reindeer, on the rumen bacterial diversity was also studied.

Animals and diet
Rumen samples for molecular ecological analysis were obtained July 2001 from three male Norwegian reindeer calves on natural summer pastures on the coast of northern Norway and fi ve male Norwegian reindeer fed a commercially available pelleted concentrate diet for reindeer (RF-80 with a chemical composition of, %: crude protein 10.3, water soluble carbohydrates 8.2, cellulose 15.9, and hemicellulose 27.9) (Storeheier et al., 2002b(Storeheier et al., , 2003. Likewise, fi ve adult female Svalbard reindeer were slaughtered and sampled October 2001 in Colesdalen, Svalbard on natural late summer pasture.

Cloning and sequencing
16S rDNA amplicons from each group of animals were cloned using a pGEM-T vector system (Promega) and transformation was done by heat-and electro-shock resulting in 300-1000 colonies/plate (LB medium with X-gal, IPTG and Ampicillin). Transformants were picked at random and clones cultured in 3-6 ml of LB amp (50 mg/ml) over night. Recombinant plasmids were extracted using a QIA prep Spin Miniprep Kit, and tested for insert (~1.5Kb) on a 1% agarose gel. Enzymatic digest with SauA3 I was performed to check for diversity among the recombinants. DNA sequence analysis of the 16S rDNA insert was carried out using the primers M13 FW-21, M13 RW-48 and 338Bact (5' ACTCCTACGGGAGGCAGC 3') with a 337 DNA Sequencer (Applied Biosystems).

Sequence analysis
Assemblage of sequences was done using SeqMan TM II v.5.05 (DNASTAR Inc.). The sequences were aligned using CLUSTAL X v. 1.83 and phylogenetic analysis was performed using the neighbor-joining method. Statistical signifi cance of branching was verifi ed by bootstrapping involving construction and analysis of 1000 trees from the data set. Reference sequences (previously cultured and characterized rumen bacteria) were obtained from the GenBank Database.

RESULTS
A total of 120 16S rDNA sequences (1.5 kb) were analysed from the three libraries prepared from whole rumen contents from Norwegian reindeer (on natural summer pasture and fed RF-80) and Svalbard reindeer (on natural late summer pasture). In the fi rst library (Norwegian reindeer on natural summer pasture) of 34 clones, 67.6% of the sequences belonged to the low G+C Gram-positive bacteria phylum and 32.4% to the Cytophaga-Flavobacter-Bacteroides bacteria. In the second library (Norwegian reindeer fed concentrate diet) of 56 clones, as much as 91.1% of the clones belonged to the low G+C Gram-positive bacteria phylum and only 7.1% to the Cytophaga-Flavobacter-Bacteroides bacteria. One of the clones belonged to the Proteobacteria. And in the third library (Svalbard reindeer on natural pasture) of 30 clones, 56.7% of the sequences belonged to the low G+C Gram-positive bacteria phylum and 43.3% to the Cytophaga-Flavobacter-Bacteroides bacteria. Bacterial 16S rDNA sequences differed between the three groups of animals.

DISCUSSION AND CONCLUSIONS
Mechanistic knowledge of the rumen microbial ecosystem is crucial in order to manipulate the effi ciency of ruminant digestion, but may also be of importance to measure future impacts of human intervention and environmental changes. We propose that adapting to a heterogenous diet of arctic plants including lichens through 15 million years of evolution (Randi et al., 1998) has resulted in a unique rumen microbial ecosystem in reindeer. In support of our hypothesis, a recent study by Mackie et al. (2003) on the ecology of the uncultivated bacterium Oscillospira showed that the diversity of Oscillospira was greater in reindeer compared to other ruminants examined (sheep and cattle). Interestingly, some of the Oscillospira phylotypes were also common to all of these geographically SUNDSET M.A. ET AL. distant ruminants. Supplementary feeding of reindeer during winter has increased in the Saami reindeer husbandry in Norway during the past 10-15 years, a factor that may infl uence the natural gastrointestinal microbiota of these animals. This is illustrated by the increase in proportion of clones representing the low G+C gram positive bacteria in artifi cially fed (91.1%) compared with naturally fed (67.6%) Norwegian reindeer. The composition of the rumen bacterial population also varied between the two sub-species of reindeer studied, possibly due to dietary differences. The 16S rDNA sequences analysed in this study represented novel rumen bacteria not yet isolated or characterised. Their functional role, however, remains unknown because of our inability to cultivate them under laboratory conditions in order to establish their metabolic properties.