DNA sequencing
DNA was extracted individually from 1–12 specimens (in most cases 4)
per population following the Chelex® 100 resin
(Bio-Rad) extraction method by Casquet, Thebaud, & Gillespie (2012),
with modifications by Stec, Smolak, Kaczmarek, & Michalczyk (2015).
After extraction, exoskeletons, if found in the vial, were mounted on
permanent slide as voucher specimens (hologenophores; Pleijel et al.,
2008; 77 populations in total). Otherwise, vouchers were
isogenophores/progenophores (offspring of sequenced mothers; 10
populations) or paragenophores (individuals from the same population; 40
populations). Four molecular markers, three nuclear and one
mitochondrial, were Sanger-sequenced: the small ribosomal subunit
(18S rRNA), the large ribosomal subunit (28S rRNA), Internal Transcribed
Spacer 2 (ITS-2), and cytochrome oxidase c subunit 1 (COI). The PCR
protocol followed Stec, Kristensen, & Michalczyk (2020); primers and
PCR protocols with relevant references are listed in Table 1. All
chromatograms were manually checked in BioEdit ver. 7.2.5 (Hall, 1999).
COI sequences were translated into amino acids to test for potential
pseudogenes using MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018).
All sequences are deposited in GenBank (accession numbers are listed in
Table S2).