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Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

The influences of wind and snow distribution at the tree line near Churchill, Manitoba, were studied. Wind profiles above the snow surface, snow crystal size and quantity were examined during abrasion experiments with white spruce branchlets. For white spruce, the abrasion zone was evidently within 80 cm above the snow surface, and needle removal by abrasion was strongly influenced by branch age. Removal by abrasion of similar-aged needles was highest in new branches and declined with branch age up to 7 or 8 years, and then In forest-tundra environments the matted forms of trees were snow covered early in winter and received little abrasion. Sparsely needled islands of trees were largely covered with snow at the base. More densely needled trees and some trees within woodlands were less exposed to abrasion. The blockage of air flow resulted in high-speed jets, which cleared out snow within a "horseshoe-shaped vortex" around the base of the tree. Both in much of the woodlands and in the open forest, snow abrasion was minimal or non-existent and snow load on the branches (qali) becomes the prevalent stress. During winter, a large proportion of the snowfall was initially blown off the exposed surfaces of Hudson Bay and the coastal tundra regions, into the woodlands, and eventually across the tree line and into the open forest. As the woodlands filled up with snow in midwinter , the rate of snow accumulation in the forest increased from double to triple the snowfall. Variations in the rate of accumulation occurred with wind speed and direction. During May, snowmelt began on exposed tundra first and usually ran off the frozen surface. Snowmelt occurred about three weeks later in the open forest and was characterized there by less runoff, as the water more readily permeated the thawing peat. The late snow beds are characteristic of the forest-tundra woodlands and were usually gone by mid-July. The woodlands were snow free for 1.5-2 months during the year, while the open forest was snow free for about 3 months and the tundra was largely snow free for 6 months or more.

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