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A subsample of each horizon was extracted immediately with 40 mL of 2M KCl for 1 h on a reciprocal shaker.
A subsample of each treatment (0.5 g) was mixed with 4.5 mL of autoclaved deionized water and vortexed for 2 min. One hundred microliters of diluted suspension for each treatment triplicate was plated for either bacteria or fungi counts under a laminar flow hood and incubated at 25°C in the dark.
Shoots were combined into a bulk sample for each density × date combination, and analyses were performed on a subsample of each bulk sample.
A subsample of each search was independently checked by BC to ensure consistency when applying inclusion criteria.
A subsample of each axillary process from adults or whole caudal fin from diploid offspring was added to the lysis buffer directly.
A subsample of each soil was also oven dried at 105 °C for 48 h to determine soil water content; all final data are reported on an oven-dried mass basis.
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Proximate analyses were also used to determine proportions of moisture, protein, lipid, and energy within a subsample of fish from each treatment.
Shortly following hatching, a subsample of offspring from each spawn was placed in 1.5-mL microcentrifuge tubes with 99% ethanol and stored in a −20°C freezer.
Using the Velvet genome assembler (v. 1.0.19) [ 24], we assembled the genomes on a subsample of reads from each isolate (69x-176x coverage when mapped using MAQ to the N16961 reference genome).
The average standard deviation of split frequencies was checked for a subsample of runs for each analysis to ensure it was < 0.01; this is the suggested diagnostic for determining when the different runs will produce a good sample from the posterior probability distribution.
# all values non-significant The Bayesian phylogenetic analysis shows B. balearicus to be reciprocally monophyletic on the mitochondrial level, albeit using a subsample of individuals within each group, with respect to all other green toads; thus our data corroborate the proposal that B. balearicus be given the status of a separate species (see Additional file 2 for details on nomenclature).
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