A mitochondrial genome assembly of the opal chimaera, Chimaera opalescens Luchetti, Iglésias et Sellos 2011, using PacBio HiFi long reads

Abstract Chondrichthyans (sharks, rays and chimeras) are a fascinating and highly vulnerable group of early branching gnathostomes. However, they remain comparatively poorly sampled from the point of view of molecular resources, with deep water taxa being particularly data deficient. The development of long-read sequencing technologies enables the analysis of phylogenetic relationships through a precise and reliable assembly of complete mtDNA genomes. The sequencing and characterization of the complete mitogenome of the opal chimera Chimera opalescens Luchetti, Iglésias et Sellos 2011, using the long-read technique PacBio HiFi is presented. The entire mitogenome was 23,411 bp long and shows the same overall content, i.e. 13 protein-coding genes, 22 transfer RNA and 2 ribosomal RNA genes, as all other examined Chondrichthyan mitogenomes. Phylogenetic reconstructions using all available Chondrichthyan mitogenomes, including 11 Holocephali (chimeras and ratfishes), places C. opalescens within the Chimaeridae family. Furthermore, the results reinforce previous findings, showing the genus Chimera as paraphyletic and thus highlighting the need to expand molecular approaches in this group of cartilaginous fishes.

Chondrichthyans are a monophyletic clade with two sister taxa, the Elasmobranchii (sharks and rays) and Holocephali (chimeras). Their K-selective reproductive traits, such as large body size and slow growth rate (Calis et al. 2005;Dagit et al. 2007;Kraft et al. 2020;Kousteni 2021), make them vulnerable to human-mediated threats such as overfishing, particularly elasmobranchs (Cavanagh and Gibson 2007;Dulvy et al. 2014;Oliver et al. 2015;Dulvy and Trebilco 2018). Chimaerid are also a frequent by-catch of deep-water fisheries (Blasdale and Newton, 1998;Moura et al., 2004;Catarino et al. 2020). Holocephalans comprise a single surviving order, the Chimaeriformes (Wyffels et al. 2014). The described species are allocated into three different families: Callorhinchidae, Rhinochimaeridae and Chimaeridae (Weigmann 2016). Furthermore, the family Chimaeridae only includes two genera: Chimera Linnaeus 1758 and Hydrolagus Gill 1862 (Weigmann 2016 (Luchetti et al. 2011). This species is widely distributed in the eastern Atlantic, with records in the British Isles and France (Luchetti et al. 2011), on the banks of Greenland, Gorringe and Galicia (Bañon et al. 2016;Luchetti et al. 2011;Vieira and Cunha 2014), Madeira, northwestern slopes of Africa (Freitas et al. 2017) and Azores (Catarino et al. 2020). The species is listed has Least Concern (LC) according with the Red List of Threatened species of the IUCN (https://www.iucnredlist.org/ species/18901743/48862329). However, previous records of C. opalescens were erroneously classified as Chimera monstrosa (Luchetti et al. 2011;Catarino et al. 2020), due to the similar morphology (Luchetti et al. 2011;Didier et al. 2012;Freitas et al. 2017). This type of problems highlights the critical importance of molecular approaches to support species identification. In this context, the development of long-read sequencing technology has been instrumental, since it allows phylogenetic analysis utilizing complete mtDNA genomes (Satoh et al. 2016;Formenti et al. 2021).
A female of C. opalescens of 665 mm in total length was captured on 14 October 2020 in the Porcupine Bank (NE Atlantic; Lat:51.1731, Long:-13.5604) at 1037 m depth during the Bottom Trawl Survey PORCUPINE 2020 carried out by the Spanish Institute of Oceanography (IEO, CSIC). Morphological identification was performed onboard, the specimen was frozen, and a muscle tissue sample was stored in absolute ethanol. The specimen is stored at the Spanish Institute of Oceanography in Vigo, with the code voucher C.OPLSCENS_1_P20 (Nair Vilas-Arrondo, nair_vilasarrondo@ hotmail.com). The muscle sample is stored at the DNA bank of CIIMAR -Interdisciplinary Center of Marine and Environmental Research with the same voucher code. A small section of the muscle tissue was sent to the Brigham Young University DNA Sequencing Center (BYU), where genomic DNA extraction and whole genome PacBio HiFi library preparation and sequencing were performed, following the manufacturer's recommendations (Pacific Biosciences; https://www. pacb.com/wp-content/uploads/Procedure-Checklist-Preparing-Hi Fi-SMRTbell-Libraries-using-SMRTbell-Express-Template-Prep-Kit-2. 0.pdf). This work has been approved by the CIIMAR ethical committee and by CIIMAR Managing Animal Welfare Body (ORBEA) according to the European Union Directive 2010/ 63/EU.
The phylogeny (Figure 1) is divided into two main subclasses: the Holocephali and the Elasmobranchii, reciprocally monophyletic (Boisvert et al. 2019).
Within the Holocephali, there are three well-supported clades, Chimaeridae, Rhinochimaeridae and Callorhinchidae (Arnason et al. 2001;Inoue et al. 2010). As expected, C. opalescens is placed within the family Chimaeridae. However, as previously observed (Gomes-dos-Santos et al. 2020, 2021), neither Hydrolagus nor Chimera genera were recovered as monophyletic, which highlights the importance of revising the taxonomy. Indeed, previous authors had already suggested that the distinction between Chimera and Hydrolagus based on the presence or absence of a notch separating the anal from the caudal fin, respectively, needed revision (Didier et al. 2012 and references therein).

Disclosure statement
The authors declare no financial interest or benefit from the direct applications of our research.

Funding
The Spanish Bottom Trawl Survey on the Porcupine Bank (SP-PORC-Q3) was funded in part by the EU through the European Maritime and Fisheries Fund (EMFF) within the Spanish National Program of collection, management and use of data in the fisheries sector and support for scientific advice regarding the Common Fisheries Policy. FCT -Foundation for Science and Technology supported A.G.S. (SFRH/BD/137935/2018). This work was implemented in the framework of the project Ocean3R (reference NORTE-01-0145-FEDER-000064), supported by the North Portugal Regional Operational Programme (NORTE2020), through the European Regional Development Fund (ERDF). It was also supported by the strategic funding UIDB/04423/2020 and UIDP/04423/2020 through national funds provided by FCT.  Data availability statement