MATERIAL AND METHODS
The Runcina aurata specimen was collected in Swanage (S England,
50°36’28”N 1°56’45”W) at intertidal depth, whereas the Facelina
auriculata individual was collected in Lagos (S Portugal, 37°05’00”N
8°39’57”W) intertidally. Both specimens were preserved in 96º ethanol. A
piece of foot of F. auriculata and the whole individual ofR. aurata were retrieved for DNA extraction, which was carried
out with the kit QIAamp DNA mini Kit (QIAGEN Inc) following
manufacturers’ indications. Due to methodolocial procedures, F.
auriculata tissue was deposited in the Museo Nacional de Ciencias
Naturales de Madrid (MNCN) with the code MNCN15.05/94859 (Curator:
Francisco Javier de Andrés Cobeta, mail:javiermol@mncn.csic.es),
and the R. aurata DNA extraction was deposited in the same museum
with the code MNCN:ADN118948 (Curator: Isabel Rey Fraile, mail:isabel.rey@csic.es ). The genomic DNA was quantified using the
NanoDrop One (ThermoScientific) and QubIt HS DNA kit (Invitrogen)
systems. Total DNA was then sent out for library construction and
sequencing to the Get-PlaGe core facilities of GenoToul (Toulouse,
France). Library preparation was carried out for each species DNA sample
using the Illumina TruSeq Nano DNA Library Prep Kit (Illumina) and
sequenced in 2x150pb paired-ends on the high-speed sequencer Illumina
HiSeq 3,000. Paired-end reads of both genomic libraries were then
subjected to quality inspection using the FastQC software (Andrews,
2010), and the Velvet-1.2.10 de novo assembler
(Zerbino & Birney, 2008)
was used on each dataset independently in order to obtain contigs (long
sequences). A local database including nudibranch mitochondrial genomes
available in GenBank was used to screen the contigs collection after
sequence assembly by running a BLAST search. The complete mitochondrial
genome of Runcina aurata and Facelina auriculata was thus
identified as single contigs of approximately 14 kb, with a mean depth
coverage above 1500x in both cases (Supp Fig. 1). Contigs presumed to be
the mitogenomes of the two species, were selected and annotated using
MITOS2 (Donath et al.,
2019). The annotation files were manually checked and curated in
Geneious Prime. The mitogenomes sequence and their respective
annotations were uploaded to Genbank (F. auriculata : OP661154,R. aurata : OP661155). We downloaded 44 heterobranch additional
mitogenomes from Genbank, and generated a supermatrix of the nucleotide
sequence of 13 CDS + 2 rRNA for the 46 gathered species. We used the
maximum likelihood software IQtree-2
(Minh et al., 2020) to
generate a phylogeny of the superorder, partitioning the genes according
to their codon position, and rooting the topology with the superclass
Acteonimorpha to highlight the clades Tectipleura and Ringipleura.