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.