The complete mitochondrial genome of the photosymbiotic sea slug Berghia stephanieae (Valdés, 2005) (Gastropoda, Nudibranchia).

Berghia stephanieae (Nudibranchia, Cladobranchia) is a photosymbiotic sea slug that feeds exclusively on sea anemones from the genus Exaiptasia. It then specifically incorporates dinoflagellates belonging to the Symbiodiniaceae obtained from their prey. Here, we present the complete mitochondrial genome sequence of B. stephanieae combining Oxford Nanopore long read and Illumina short-read sequencing data. The mitochondrial genome has a total length of 14,786 bp, it contains the 13 protein-encoding genes, 23 tRNAs, and two rRNAs and is similar to other nudibranchs except for the presence of a duplicated tRNA-Ser 1.

The aeolid nudibranch Berghia stephanieae (Valdés 2005) (Nudibranchia, Cladobranchia) is a stenophagous species that preferentially preys on the photosymbiotic sea anemone Exaiptasia diaphana (Rapp 1829). The slug digests all the anemones’ tissue and incorporates the dinoflagellate symbionts Symbiodiniaceae Fensome et al. 1993, in epithelial cells of the digestive gland system (Valdés 2005; Carmona et al. 2014). Once ingested, the symbionts are retained photosynthetically active for about 10 days (Mies et al. 2017), but the slugs are even able to overcome prolonged starvation periods up to 48 days apo-symbiotically (symbiont-free) (Bleidissel 2010). Further, because apo-symbiotic adults lose their biomass in the same manner as photosymbiotic ones, the photosymbiotic relationship of B. stephanieae and Symbiodiniaceae is rather considered as non-mutalistic than a stable one (Mies et al. 2017; Monteiro et al. 2019). Nevertheless, B. stephanieae is an important species to understand the evolution of photosymbiosis in Cladobranchia, because the species seems to be in a transitional state between non-photosymbiotic and photosymbiotic. To better understand the genomic adaptations needed to evolve a stable photosymbiosis (Melo Clavijo et al. 2018), the metabolism of the mitochondrial genome can give valuable insights into a potential connectivity of the host and the symbiont (Rauch et al. 2017). As a first step toward more comprehensive studies, we sequenced the mitochondrial genome of B. stephanieae using a combination of Oxford Nanopore long-read and Illumina short-read sequencing.

Specimens of B. stephanieae were purchased from a local provider (Seepferdchen24 Meeresaquaristik GmbH, Posthausen) in February 2019 and cultivated in our lab at 25 °C, at a day/night cycle of 12 h/12h. Voucher material was preserved in 96% ethanol and stored in the Biobank at the Zoological Research Museum Alexander Koenig (Bonn, Germany, voucher no. ZFMK-TIS-53240, biobank@leibniz-zfmk.de). Seven specimens of B. stephanieae were frozen in liquid nitrogen and total DNA was extracted using a modified protocol based on the E.Z.N.A®Mollusc DNA Kit, Omega (Georgia, USA) and after Schalamun et al. (2019) (Supplementary material S1). The genomic library preparation was performed using the 1 D Ligation Sequencing Kit SQK-LSK109, Oxford Nanopore Technologies (Oxford, UK) for long-read sequencing on a MinION device, using a modified manufacturer’s protocol (Supplementary material S1) generating about 13 GB of long reads. An additional library (insert size 100 bp, single end) was prepared using the Nextera DNA Library Prep Kit (California, USA) for Illumina sequencing on a HiSeq2500 platform resulting in approximately 5.5 GB of data. A hybrid assembly was done using SPAdes V3.14.1 (Nurk et al. 2013; Antipov et al. 2016), the assembled genome was annotated using the MITOS2 webserver (Bernt et al. 2013; Donath et al. 2019), and annotations were manually edited using Geneious 9.1.5. (https://www.geneious.com). Duplicated tRNAs were further confirmed with ARWEN v.1.2 (Laslett and Canback 2008).

The mitochondrial genome of B. stephanieae (GenBank accession number: MW027646) has a total length of 14,786 bp and consists of 13 protein-coding genes, two ribosomal RNA (rRNA) genes, and 23 tRNA genes. The base composition of the mitogenome is 26% A, 15% C, 21% G, and 38% T. The gene order is as follows: tRNA-Lys (aaa), cox1, tRNA-Val (gta), the large-subunit rRNA (rrnL), tRNA-Leu (cta) 1, tRNA-Ala (gca), tRNA-Pro (cca), nad6, nad5, nad1, tRNA-Tyr (tac), tRNA-Trp (tga), nad4L, cob, tRNA-Asp (gac), tRNA-Phe (ttc), cox2, tRNA-Gly (gga), tRNA-His (cac), tRNA-Cys (tgc), -tRNA-Gln (caa), -tRNA-Leu (tta) 2, -atp8, -tRNA-Asn (aac), -atp6, -tRNA-Arg (cga), -tRNA-Glu (gaa), -the small-subunit rRNA (rrnS), -tRNA-Met (atg), -nad3, -tRNA-Ser (tca) 2, tRNA-Ser (agc) 1, tRNA-Ser (aga) 1, nad4, -tRNA-Thr (aca), -cox3, tRNA-Ile (atc), nad2. The mitogenome of B. stephanieae is similar in size, base composition, has the same coding regions and gene arrangement compared to all publicly available nudibranch mitochondrial genomes (Sevigny et al. 2015; Karagozlu, Sung, Lee, Kim, et al. 2016; Karagozlu, Sung, Lee, Kwak, et al. 2016; Xiang, Lin, Wang, et al. 2016; Xiang, Lin, Zhao, et al. 2016; Lin et al. 2017; Yu et al. 2018; Dinh Do, Choi, et al. 2019; Dinh Do, Kim, et al. 2019) and only differs in the presence of a duplicated tRNA-Ser 1.

Full-length mitochondrial genome sequences of 20 Nudipleura species were downloaded from NCBI and aligned using MAFFT (Auto mode) V7.222 (Katoh and Stanley 2013). A phylogenetic tree was built based on the maximum likelihood criterion using IQ-TREE version 2.0.5 (Minh et al. 2020) with the Model Finder Plus option (-m TEST), 1000 bootstrap replicates, and Aplysia californica J. G. Cooper 1863 set as outgroup. Berghia stephanieae clustered with the other Cladobranchia species, and forms a monophyletic clade with Sakuraeolis japonica (Baba 1937) and Hermissenda emurai (Baba 1937), that corresponds to the superfamily Aeolidioidea (Figure 1).

Figure 1.

The molecular phylogeny of Berghia stephanieae and other nudibranchs based on the whole mitochondrial genome. The phylogenetic tree was calculated under the maximum-likelihood optimality criterion and 1,000 bootstrap replicates using Aplysia californica as outgroup. The accepted names (WoRMS Editorial Board, 2020) for Tyrannodoris europaea (synonym Roboastra europaea) and Tritonia tetraquetra (synonym Tritonia diomedea) were used (*).

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