4.1. Efficacy of DNA extraction protocols and primers
We first tested two different DNA extraction methods using a series of novel and published nuclear and mitochondrial primer pairs. Although primer amplification success varied between 0% and ~98% (Table S2), the average amplification success of our “legacy” primers (Fig 3; Tables 3, S1) was ~80%. The lowest amplification rates overall were seen for legacy primers 16S (average ~62%) and H3 (average ~60%), which is likely caused by divergent priming sites or too low copy numbers for individual y-larvae, respectively. Importantly, maximum amplification rates were not restricted to nuclear loci as amplification with the COX1 “Folmer” and “Leray” primers yielded ~93% success. In combination with nuclear ribosomal markers that demonstrated high amplification rates, this will likely positively influence phylogeny estimation and robust species delimitation in future systematic efforts.
DNA extraction relied on either the GeneReleaser kit, which had previously been used to retrieve high-quality DNA templates from minute zooplankton specimen (Schizas et al., 1997; Böttger-Schnack and Machida, 2011; Watanabe et al., 2016), including y-larvae (Olesen et al., 2022), or a simplified yet highly effective modification of the QIAGEN DNeasy Blood and Tissue kit. There are important advantages and disadvantages related to both protocols. On the one hand, GeneReleaser yields higher amplification rates for certain loci. On the other hand, the GeneReleaser kit only yields ~15-20 µL DNA extract and its thermal incubation program passes through high temperatures (>90°C). This may have a severe impact on successful retrieval voucher “exuviae” because (1) in initial trials with various crustacean larvae (data not shown), incubation at such temperatures almost always resulted in the disintegration of the “exuviae”, and (2) the “exuviae” may easily become trapped in the GeneReleaser powder grains when the tubes are spun down after incubation. Less than 5% of the vials contained “exuviae” after transferring the GeneReleaser supernatant to new vials. Moreover, the GeneRelaser-kit is more expensive (US$125 for a kit on 20 October 2022), considering that it does not come with buffers and protease K included, which thus have to be acquired elsewhere. By contrast, the simplified DNeasy kit contains all relevant products for fast and efficient DNA extraction and produces bright gel-bands in the expected size ranges, clean chromatograms, and high rates of both overall and locus-specific amplification (Figs. 2, 3; Table S2). For these reasons, we recommend using our extraction method with the simplified DNeasy kit and with the legacy primers. We are currently designing a series of primers for nuclear protein-coding genes in order to expand this approach to higher-level phylogenetics.