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\begin{document}

\title{Runcinidae and Facelinidae: Two complete mitogenomes of understudied and
misleading heterobranch families (Gastropoda, Mollusca)~~~~~~~~~~~~}



\author[1]{Carles Galià-Camps}%
\author[1]{Ana Karla Araujo}%
\author[1]{Leila Carmona}%
\author[1]{Ferran Palero}%
\author[1]{María del Rosario Martín-Hervás}%
\author[1]{Marta Pola}%
\author[1]{Juan Lucas Cervera}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Here, we present the mitochondrial sequences of two `opistobranch'
heterobranchs: \emph{Runcina aurata} Garc\selectlanguage{ngerman}ía-Gómez, López, Luque \&
Cervera, 1986 and \emph{Facelina auriculata} (O. F. Müller, 1776), the
latter type taxon of the genus. The mitochondrial genomes were 14,282
and 14,171bp in length respectively, the two of them with a complete set
of 13 CDS, 2 rRNAs, and 22 tRNAs. None of the mitogenomes showed gene
reorganization, keeping the standard heterobranch mitogenomic structure.
The base composition was completely distant between them, with a 25.7\%
GC for \emph{R. aurata}, becoming the mitogenome with more AT content to
the date, and 35\% for \emph{F. auriculata,} supposing one case of
extremely rich GC content in ``opistobranch'' mitochondrial genomes.%
\end{abstract}\selectlanguage{ngerman}%



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\textbf{Hosted file}

\verb`GaliaCamps_MainBody_preprint.docx`  available at \url{https://authorea.com/users/492660/articles/617135-runcinidae-and-facelinidae-two-complete-mitogenomes-of-understudied-and-misleading-heterobranch-families-gastropoda-mollusca}

\textbf{INTRODUCTION}

Although traditional systematics used to rely on morphological features,
molecular methods have demonstrated to provide a subjective tool to
compare individuals, where only the stochasticity of mutation is taken
in consideration, and the overweight of some morphological characters is
somehow avoided. In this scenario, these disciplines have merged to give
birth to an integrative taxonomy, which has become a key element in
modern systematics (Dayrat, 2005). In this regard, integrative taxonomy
allows the categorization of species according to both molecular and
morphological differences and resemblances. Thus, it is of primal
importance to know the taxonomical, but also the molecular nature of
species, since the second provides a reliable standard template to
compare with and to be compared to other organisms. Here, we aim to fill
a knowledge gap in mitogenomes of poorly studied and misleading taxa. On
one hand, we have \emph{Runcina aurata}Garc\selectlanguage{ngerman}ía-Gómez, López, Luque \&
Cervera, 1986 (Fig. 1), genus belonging to the order Runcinidae, which
englobe close to 100 small sized species (1-4mm) described so far,
although it is thought to include much more biodiversity (Araujo et al.,
2022). The members of such order feed on macroalgae, contributing to the
decomposition of dead matter, being a key element in the balance of the
ecosystem (Araujo et al., 2019). However, due to the small size and
difficulty to find them in their natural environment, few studies have
focused on them so far. On the other hand, \emph{Facelina auriculata}
(O. F. Müller, 1776) (Fig. 1) is the type taxon of the family
Facelinidae, which is considered one of the most diverse heterobranch
families with more than 200 species (Karmeinski et al., 2021). Its
distribution, ranging from the English coasts to the Spanish Atlantic
with some introgression in the Mediterranean, overlaps with the\emph{R.
aurata} 's one. Nevertheless, they do not compete with each other since
\emph{F. auriculata} feeds on cnidarians, from which they ``steal'' and
store the cnidoblasts in specialized organs named ceratas (Goodheart et
al., 2018). In view of the needs and scientific circumstances of both
taxa, we present here the reference mitogenomes for the two species,
which will allow direct comparisons of future genetic information, as
well as provide a template for the synthesis of new specific primers.

\textbf{MATERIAL AND METHODS}

The \emph{Runcina aurata} specimen was collected in Swanage (S England,
50°36'28''N 1°56'45''W) at intertidal depth, whereas the \emph{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 \emph{F. auriculata} and the whole individual
of\emph{R. 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, \emph{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:\emph{javiermol@mncn.csic.es}),
and the \emph{R. aurata} DNA extraction was deposited in the same museum
with the code MNCN:ADN118948 (Curator: Isabel Rey Fraile,
mail:\emph{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 \emph{Runcina aurata} and \emph{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 (\emph{F. auriculata} :
OP661154,\emph{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.

\textbf{RESULTS AND DISCUSSION}

The mitogenome of \emph{Runcina aurata} spanned a total length of
14,282\selectlanguage{english} bp. It was found to contain the typical 13 coding genes (CDS),
the large and small ribosomal RNA genes, and 22 tRNA genes, all of them
with the standard gene order of heterobranchs (Fig. 2). The overall GC\%
content was 25.7\%, one of the lowest values in heterobranchs. When
focusing on CDS, rRNA and tRNA this value shifted to 26.3\%, 23.6\% and
25.5\% respectively. Parallelly, \emph{Facelina auriculata}mitochondrial
genome length was 14.171, comprising the ordinary 13 coding genes, 2
ribosomal RNA and 22 tRNA, with the standard gene order of heterobranchs
(Fig. 2). The overall GC\% content of the mitogenome was 35\%, in
contraposition to the \emph{R. aurata} 's one. The CDS of\emph{F.
auriculata} mitogenome were composed of 35.5\% GC, the rRNA were 32\% GC
and the tRNA were 35.8\% GC.

The phylogenetic analysis indicated, as previously reported (Varney et
al., 2021), that\emph{Runcina aurata} and therefore Runcinida is sibling
taxon to a clade composed by Aplysiida and Cephalaspidea with a BS
support value of 100 (Fig. 3). Following, \emph{Facelina auriculata} is
sibling to the other facelinid species \emph{Sakuraeolis japonica}
(Baba, 1937), with a bootstrap support value of 100, being facelinids
sibling taxon to the other aeolidiaceans \emph{Protaeollidiella atra}
Baba, 1955 and\emph{Hermissenda emurai} (Baba, 1937) (BS = 100) (Fig. 3)
(Carmona et al., 2013). With the \emph{R. aurata} and \emph{F.
auriculata} mitochondrial reference genomes, the inner systematic
relationships can be properly assessed and explored, while providing a
valuable tool to design specific primers for mitochondrial targeted
markers of these groups, which have been hampered when using the
standard Folmer set for cytochrome oxidase I, and Palumbi set for 16S.

\textbf{AUTHOR CONTRIBUTIONS}

FP, MP, and JLC conceived the study. CG, FP, LC, AKA and MRM performed
the data analysis. CG drafted the manuscript. All authors critically
reviewed the article. All authors approved the final version of the
manuscript.

\textbf{REFERENCES}

Andrews S. (2010). FastQC: a quality control tool for high throughput
sequence data. \emph{Babraham Bioinformatics} .

Araujo, A. K., Pola, M., Malaquias, M. A. E., Ballesteros, M., Vitale,
F., \& Cervera, J. L. (2022). Molecular phylogeny of European Runcinida
(Gastropoda, Heterobranchia): the discover of an unexpected pool of
complex species, with special reference to the case of \emph{Runcina
coronata}.\emph{Zoological Journal of the Linnean Society},
\emph{194}(3), 761--788.

Araujo, A. K., Pola, M., Malaquias, M. A. E., \& Cervera, J. L. (2019).
To be or not to be? What molecules say about Runcina brenkoae Thompson,
1980 (Gastropoda: Heterobranchia: Runcinida). \emph{Scientia Marina},
\emph{83}(3), 223.

Carmona, L., Pola, M., Gosliner, T. M., \& Cervera, J. L. (2013). A tale
that morphology fails to tell: a molecular phylogeny of Aeolidiidae
(Aeolidida, Nudibranchia, Gastropoda). \emph{PloS One}, \emph{8}(5),
e63000.

Dayrat, B. (2005). Towards integrative taxonomy. \emph{Biological
Journal of the Linnean Society. Linnean Society of London},
\emph{85}(3), 407--415.

Donath, A., Juhling, F., Al-Arab, M., Bernhart, S. H., Reinhardt, F.,
Stadler, P. F., Middendorf, M., \& Bernt, M. (2019). Improved annotation
of protein-coding genes boundaries in metazoan mitochondrial genomes.
\emph{Nucleic Acids Research}, \emph{47}(20), 10543--10552.

Goodheart, J. A., Bleidissel, S., Schillo, D., Strong, E. E., Ayres, D.
L., Preisfeld, A., Collins, A. G., Cummings, M. P., \& Wagele, H.
(2018). Comparative morphology and evolution of the cnidosac in
Cladobranchia (Gastropoda: Heterobranchia: Nudibranchia).
\emph{Frontiers in Zoology}, \emph{15}, 43.

Karmeinski, D., Meusemann, K., Goodheart, J. A., Schroedl, M., Martynov,
A., Korshunova, T., Wagele, H., \& Donath, A. (2021). Transcriptomics
provides a robust framework for the relationships of the major clades of
cladobranch sea slugs (Mollusca, Gastropoda, Heterobranchia), but fails
to resolve the position of the enigmatic genus Embletonia. \emph{BMC
Ecology and Evolution}, \emph{21}(1), 226.

Minh, B. Q., Schmidt, H. A., Chernomor, O., Schrempf, D., Woodhams, M.
D., von Haeseler, A., \& Lanfear, R. (2020). IQ-TREE 2: New Models and
Efficient Methods for Phylogenetic Inference in the Genomic Era.
\emph{Molecular Biology and Evolution}, \emph{37}(5), 1530--1534.

Varney, R. M., Brenzinger, B., Malaquias, M. A. E., Meyer, C. P.,
Schrodl, M., \& Kocot, K. M. (2021). Assessment of mitochondrial genomes
for heterobranch gastropod phylogenetics. \emph{BMC Ecology and
Evolution}, \emph{21}(1), 6.

Zerbino, D. R., \& Birney, E. (2008). Velvet: algorithms for de novo
short read assembly using de Bruijn graphs. \emph{Genome Research},
\emph{18}(5), 821--829.

\textbf{DISCLOSURE STATEMENT}

No potential conflict of interest was reported by the authors.

\subsection*{ETHICS STATEMENTS}

{\label{ethics-statements}}

None of the species use in the present study is included in any
protected or threatened species on the IUCN Red List or the Spanish and
Portuguese governments. Therefore, no specific permissions or licenses
were needed for the sampling. We followed ethic procedures to ensure no
substantial harm to the collecting individual.

{\label{none-of-the-species-use-in-the-present-study-is-included-in-any-protected-or-threatened-species-on-the-iucn-red-list-or-the-spanish-and-portuguese-governments.-therefore-no-specific-permissions-or-licenses-were-needed-for-the-sampling.-we-followed-ethic-procedures-to-ensure-no-substantial-harm-to-the-collecting-individual.}}

\subsection*{DATA AVAILABILITY
STATEMENT}

{\label{data-availability-statement}}

The data that support the findings of this study are openly available in
GenBank of NCBI at https://www.ncbi.nlm.nih.gov/genbank/, under the
accession numbers OP661154 for \emph{Facelina auriculata} and OP661155
for \emph{Runcina aurata} . The associated **BioProject**, **SRA**, and
**Bio-Sample** numbers are PRJNXXXXX, SXXXXXX, and SXXXXX
respectively.''

{\label{the-data-that-support-the-findings-of-this-study-are-openly-available-in-genbank-of-ncbi-at-httpswww.ncbi.nlm.nih.govgenbank-under-the-accession-numbers-op661154-for-facelina-auriculata-and-op661155-for-runcina-aurata.-the-associated-bioproject-sra-and-bio-sample-numbers-are-prjnxxxxx-sxxxxxx-and-sxxxxx-respectively.}}

\textbf{FUNDING AND AKNOWLEDGEMENTS}

This study was supported by the Fundacion Biodiversidad of the Spanish
Ministry for the Ecological Transition, {[}Project BIOCALETA{]}, Spanish
Ministry of Science, Innovation and Universities, and by ERDF ``A way of
making Europe'' {[}PRE-2018-085227 - MCIN/AEI/10.13039501100011033{]},
the University of Cadiz {[}PR2018-039{]}, the Andalusian Plan of
Research, Development, and Innovation (PAIDI), from the Andalusian
Autonomic Government {[}RNM-213{]}, the Generalitat de Catalunya\selectlanguage{english}-AGAUR
{[}SGR2017-1120{]}, and the Conselleria d'Innovacio, Universitats,
Ciencia i Societat Digital. {[}CIDEGENT/2019/028{]}

Figure 1: Species reference images of the present study. A)\emph{Runcina
aurata} under the binocular lens. B) \emph{Facelina auriculata} on its
natural environment. C) \emph{Facelina auriculata}under the binocular
lens. Note that due to the small size of\emph{Runcina auriculata} , it
was not possible to take a picture on nature.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 2. Gene map of the two newly generated mitogenomes. Above, the
gene map of \emph{Runcina aurata} . Below, the gene map of
\emph{Facelina auriculata} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 3. Best Maximum Likelihood phylogeny recovered from 46
heterobranch CDS+rRNA sequences. In bold font are indicated the species
newly included in this study. Accession numbers: \emph{A.
californica:}NC\_005827.1, \emph{A. dactylomela} : NC\_015088.1,
\emph{A. kurodai:}NC\_024260.1, \emph{A. vulgaris:} NC\_046044.1,
\emph{Berthellina} sp.: NC\_015091.1, \emph{B. exarata} : NC\_042169.1,
\emph{C. uva:}NC\_034226.1, \emph{C. annae} : NC\_036426.1, \emph{C.
magnifica} : NC\_015096.1, \emph{C. orientalis} : NC\_041196.1, \emph{C.
quadricolor:}NC\_030004.1, \emph{C. obtusus:} NC\_017872.1, \emph{D.
otome} : NC\_062675.1, \emph{D. reticulatum:} NC\_035495.1, \emph{D.
atromarginata} : NC\_057611.1, \emph{E. timida} : NC\_035490.1, \emph{E.
ornata:} NC\_030537.1, \emph{H. flavescens} :
NC\_059879.1,\emph{Haminoea japonica} : NC\_053883.1, \emph{H. pomatia:}
NC\_041247.1,\emph{H. emurai:} NC\_048494.1, \emph{Homoidoris
japonica:}NC\_034006.1, \emph{H. apolegma} : NC\_036425.1, \emph{H.
festiva} : NC\_030774.1, \emph{M. leonina:} NC\_026987.1, \emph{M.
undata:}NC\_015106.1, \emph{M. myosotis:} NC\_012434.1, \emph{N.
kubaryana} : NC\_034920.1, \emph{N. gardineri:} NC\_015111.1, \emph{O.
hiroi:}NC\_056185.1, \emph{O. celtica:} NC\_012376.1, \emph{P.
redfieldi:}NC\_040969.1, \emph{P. ocellata:} NC\_030039.1, \emph{P.
pustulosa} : NC\_048495.1, \emph{P. krempfi:} NC\_052698.1,
\emph{Placida} sp.: NC\_020343.1, \emph{P. atra} : NC\_053882.1,
\emph{P. strigosa:}NC\_002176.1, \emph{R. europaea:} NC\_004321.1,
\emph{R. otaheitana:}NC\_044784.1, \emph{S. japonica:} NC\_033968.1,
\emph{S. gigas} : NC\_016188.1, \emph{T. diomedea:} NC\_026988.1,
\emph{V. pusilla:}NC\_026045.1.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Supplementary Figure 1: Base by base coverage along the mitogenome. On
the left panel, it is displayed the \emph{Runcina aurata} mitogenome
coverage. On the right panel, it is displayed the \emph{Facelina
auriculata} mitogenome coverage.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\selectlanguage{english}
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\end{document}

