Eurycea_bislineata_AFLP_Data

AFLP data for Eurycea bislineata individuals at downstream and upstream sites in 6 streams in the Hubbard Brook Watershed, New Hampshire, USA

Gyrinophilus_porphyriticus_AFLP_Data

AFLP data for Gyrinophilus porphyriticus individuals at downstream and upstream sites in 6 streams in the Hubbard Brook Watershed, New Hampshire, USA

Appendix A. A table showing descriptive statistics for amplified fragment length polymorphism variation at 18 sites in the Hubbard Brook watershed, New Hampshire, USA, where Gyrinophilus porphyriticus tissue samples were collected.

A table showing descriptive statistics for amplified fragment length polymorphism variation at 18 sites in the Hubbard Brook watershed, New Hampshire, USA, where Gyrinophilus porphyriticus tissue samples were collected

Kovach_etal_WCTRBT_hybridization

The excel file contains the genotypic data for all individual trout (Oncorhynchus) that were sampled from 2003 - 2007. Fish were genotyped at 8 species diagnostic microsatellite loci. Columns D-S are the raw allele lengths, while columns T - AI describe whether each allele at each locus is of westslope cutthroat trout ancestry (denoted by 0) or rainbow trout ancestry (denoted by 1). Other descriptive columns include individual ID, maturity (spawning adult or outmigrating juvenile), date captured (i.e., migration date), length (in mm), and age (for Langford juveniles only). Data for Langford and Cyclone are on two separate sheets

Assay sequences and measures of specificity.

<p>Primer and probe sequences and location of mismatches (underlined) with non-target taxa for the three assays. Proportional S/N is the normalized fluorescence (Rn) at 40 cycles divided by the Rn at 1 cycle, standardized by the S/N of a positive control (n = 3; mean and std).</p

eDNA Data

Excel file containing raw eDNA data

Non-target template competition in the bull trout assay.

<p>Part A shows the amplification curves of <i>BUT1</i> when using DNA solutions from bull trout, lake trout, and bull trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Part B shows the amplification curves of <i>BUT1</i> when using DNA solutions from bull trout, brook trout, and bull trout mixed into brook trout at 1∶10 and 1∶100 dilutions. The assay primers have nine and five primer base pair mismatches with brook trout and lake trout respectively. Four of the mismatches with brook trout are within five base pairs of the 3′ primer ends, but none of the mismatches with lake trout are near the 3′ ends. The assay is not influenced by the presence of brook trout, but produces an ambiguous signal when bull trout represents a small portion of the sample.</p

Impact of base pair mismatches on fluorescence.

<p>The relationship between end-point fluorescence (measured as proportional S/N; the fluorescence at cycle 40 divided by the fluorescence at cycle 1 as a proportion of a positive control) and base pair mismatches in the primer regions (A), probe region (B), and primers and probe regions combined (C). End-point fluorescence decreases as the number of primer base pair mismatches increases.</p

Assay <i>BRK2</i> sensitivity results.

<p>The DNA source (brook trout tissue, Plant Creek and Miller Creek filter extractions, and a mixed brook/bull trout DNA solution diluted 1∶10), estimated concentration, number of replicates, and proportion of replicates with amplification for the assay sensitivity experiments. All DNA sources had 100% amplification success when concentrations were ≥2.5 copies/µl, and >70% amplification success when concentrations were 0.5 copies/µl.</p

Non-target template competition in the brook trout assays.

<p>Part A shows the amplification curves of <i>BRK1</i> when using DNA solutions from brook trout, bull trout, and brook trout mixed into bull trout at 1∶10 and 1∶100 dilutions. Part B shows the amplification curves of <i>BRK1</i> when using DNA solutions from brook trout, lake trout, and brook trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Part C shows the amplification curves of <i>BRK2</i> when using DNA solutions from brook trout, bull trout, and brook trout mixed into bull trout at 1∶10 and 1∶100 dilutions. Part D shows the amplification curves of <i>BRK2</i> when using DNA solutions from brook trout, lake trout, and brook trout mixed into lake trout at 1∶10 and 1∶100 dilutions. Assay <i>BRK1</i> has a single primer-base-pair mismatch and three probe-base-pair mismatches with bull trout, but produces an ambiguous signal when brook trout represent a small proportion of the sample. <i>BRK2</i> has nine primer base pair mismatches and a single probe base pair mismatch with bull trout, and is still sensitive even when brook trout represents a small proportion of the sample. The presence of lake trout DNA does not appear to influence the sensitivity of either assay.</p