Type it microsatellite kit

To assign individuals to species, we amplified a partial sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene, which is species-specific in Daphnia²⁸ , from a subset of current-day populations (16 and 10 individuals from the heated and control lakes, respectively) and the resting eggs from sediment cores (6 eggs). The following primers were used: bcdF01: 5′-CATTTTCHACTAAYCATAARGATATTGG-3′ and bcdR04: 5′-TATAAACYTCDGGATGNCCAAAAAA-3′^{67 (link)}. COI gene sequences were amplified from 1 μL DNA template in 5 μL of PCR reaction mix, containing 2.5 μL Type-it Microsatellite Kit (Qiagen GmbH, Hilden, Germany), 0.25 μmol/L of each primer, and 2 μL of water. Thermocycling parameters were: one cycle of 5 min at 95 °C; 35 steps of 30 s at 95 °C, 1 min at 50 °C, and 1 min at 72 °C; and a final step of 5 min at 72 °C. The PCR product was visualised by electrophoresis on 1% agarose gel, purified using Exonuclease I and Alkaline Phosphatase (Thermo Scientific, San Jose, USA) for 15 min at 37 °C and 15 min at 80 °C, and Sanger sequenced using the BigDye Terminator v3.1 kit and an ABI Prism 3130XL Analyzer (Applied Biosystems, Foster City, CA, USA).

Allelic variation was determined at 16 microsatellite loci (Supporting information Table S1) for all 120 individuals (Table 1). For each sample, two multiplex PCR reactions were performed using the Qiagen Type‐it Microsatellite kit in a 10‐μl reaction volume with 3 μl of extracted DNA, 5 μl of kit master mix and primers with concentrations and dyes as presented in Supporting information. PCR reactions were carried out in PTC200 Thermal Cyclers (MJ Research), and the temperature profile of the PCR program was suggested in the Type‐it Microsatellite kit manual. The annealing temperature was 56°C. The amplification products were separated by capillary electrophoresis on AB3130 Genetic Analyzers (Applied Biosystems, Foster City, CA). The sizes of the microsatellite alleles were determined using Genemapper v. 4.0 software (Applied Biosystems, Foster City, CA), and manually checked. Deviations from Hardy‐Weinberg equilibrium in each population were tested using function hw.test from pegas package (Paradis, 2010) in R environment. A few loci showed a significant deviation from equilibrium within populations (2 in Tuhkajoki, 1 in Pohjajoki, 4 in Vaarainjoki and 2 in hatchery stock). However, as all microsatellite loci were in equilibrium in at least two populations, none were excluded from analysis.

We sequenced DNA from a single A. hymenopus individual using Illumina 250 bp paired-end v2 chemistry. We characterised eleven novel tri- and tetra-nucleotide microsatellite markers according to Griffiths et al. [35 (link)] (Supplementary Information, Table S1). We conducted PCRs in five multiplex reactions with the Type-it® Microsatellite Kit (Qiagen) with the following conditions: 95 °C for 5 min; 30–35 × 95 °C for 30 s, 60–66 °C for 90 s, 72 °C for 30 s; 60 °C for 30 min (Table S1). We sized PCR amplicons using the 3730 DNA Analyzer and the GeneScan™ LIZ1200 size standard (Thermo-Fisher Scientific), scored alleles in Genemapper v.3.7 (Thermo-Fisher Scientific), and binned alleles in MsatAllele v1.02 [36 (link)] in R v3.3.3 [31 ].

We extracted DNA from 116 workers per colony, as well as the mother queen, by placing a single antenna or leg in 100 μl of 10% Chelex solution (Chelex 100, 100–200 mesh, Bio-Rad), then incubating for 20 minutes at 95°C. We refrigerated or froze the supernatant before PCR.
We genotyped each worker at five variable loci (LIST2004, RUFA05, RUFA13, RUFA19, VMA3) using two multiplex PCR reactions [24 (link)–26 ]. Products at each locus were distinguished by employing simple dye-labeled primers (Applied Biosystems) and a 3-primer method [27 (link)], as well as fragment size for those labeled with the same dye. Each 10 μl PCR reaction included 1 μl of extracted DNA, 5 μl Qiagen master mix (Qiagen Type-It Microsatellite Kit, Qiagen Inc.), 0.2 μl of each reverse primer, 0.2 μl (dye-labeled) or 0.1 μl (3-primer labeled) of each forward primer, 0.15 μl FAM-labeled 3-primer tag for each 3-primer-labeled primer pair, and water. PCR reaction conditions were 95°C for 15 minutes, 35 cycles of 95°C for 30 seconds, 50°C for 90 seconds, 72°C for 60 seconds, followed by 60°C for 30 minutes. The fragment analysis was performed on an ABI-3730xl sequencer using 0.5 μl PCR product combined with 15 μl HiDi Formamide and 0.15 μl LIZ 500 internal size standard (Applied Biosystems). Allele sizes were called using GeneMarker (SoftGenetics LLC) and checked twice by eye.

We extracted DNA from approximately twenty workers or gynes per colony, as well as the mother queen when present, by placing a single antenna or leg in 100 μL of 10% Chelex solution (Chelex 100, 100–200 mesh, Bio-Rad), then incubating for 20 minutes at 95°C. We then refrigerated or froze the supernatant before PCR. Variable loci were selected based on preliminary screening of published loci [31 -33 (link)]. We used dye-labeled primers (Applied Biosystems) in combination with a 3-primer labeling method [34 (link)] to perform multiplex PCR with 4–6 primers, depending on the species (Additional file 1: Table S1). Each 10 μL PCR reaction included 1ul extracted DNA, 5 μL Qiagen master mix (Qiagen Type-It Microsatellite Kit, Qiagen Inc.), 0.2 μL of each reverse primer, 0.2 μL (dye-labeled) or 0.1 μL (3-primer labeled) of each forward primer, 0.15 μL FAM-labeled 3-primer tag for each 3-primer-labeled primer pair, and water to total 10 μL. PCR reaction conditions were 95°C for 15 minutes, 35 cycles of 95°C for 30 seconds, 50°C for 90 seconds, 72°C for 60 seconds, followed by 60°C for 30 minutes. Fragment analysis was performed on an ABI-3730 × l sequencer using 0.5 μL PCR product combined with 15 μL HiDi Formamide and 0.15 μL LIZ 500 internal size standard (Applied Biosystems). Allele sizes were called using GeneMarker (SoftGenetics LLC) and checked twice by eye.