The complete mitochondrial genome of Membranipora villosa Hincks, 1880 (Bryozoa: Gymnolaemata: Cheilostomatida): phylogenetic relationship of two kelp-encrusting bryozoans within the suborder Membraniporina

Abstract The two commonest kelp-encrusting bryozoans, Membranipora villosa and M. membranacea, are difficult to distinguish morphologically. Molecular studies of M. villosa should thus be helpful for the identification of both species because the mitogenome of M. membranacea was already sequenced. The complete mitogenome of M. villosa collected from Sinjido was determined in this study through Illumina NovaSeq sequencing. Maximum-likelihood (ML) analysis was based on concatenated 13 protein-coding genes dataset from nine bryozoan species. The mitogenome length was 15,407 bp, and its gene arrangement was similar to those of the mitogenome of other membraniporids, having 13 PCGs, two ribosomal RNAs, and 22 tRNAs. It had an overall A + T content of 63.7% (29.7% A, 16.7% C, 19.6% G, and 34.0% T). M. villosa and M. membranacea showed sequence differences of 20% for the total length of mitogenome and 16.1.% for 13 PCGs. Molecular data definitely consider them to be separate species. Phylogenetic analyses based on the amino acids of 13 PCGs indicated that M. villosa has the closest relationship with another kelp-encrusting bryozoan, M. membranacea of membraniporids. The phylogenetic position of genera and families within the suborder Membraniporina coincides with the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions.


Introduction
Two nominal common kelp-encrusting species of membraniporids belonging to the phylum Bryozoa, Membranipora villosa Hincks, 1880 (Bryozoa: Cheilostomatida: Membraniporina: Membraniporidae) from the North Pacific including Korean waters and Membranipora membranacea (Linnaeus, 1767) from mainly the Atlantic, are known.Of the two, M. villosa is the most predominant epialgal organism in Korean kelp farms (Kim et al. 2017) (Figure 1(A)); M. membranacea sensu stricto has yet to be found in Korean waters.As currently understood, M. villosa typically has a moderately to coarsely serrated cryptocyst and cuticular spines on the frontal membrane, whereas M. membranacea is normally characterized by a thinner lateral cryptocyst that is nearly smooth or only weakly denticulate and there are no frontal cuticular spines (Dick et al. 2005;Seo 2010) (Figure 1(B)).Both species have a short, blunt, hollow, conical, spine-like knob at each proximal corner.The two species, if such they are, are so morphologically similar that several studies have sought to distinguish them (Yoshioka 1982;Soule et al. 1995;Schwaninger 1999;Dick et al. 2005).In addition, Korean Membranipora used in a biochemical study was taken to be M. membranacea and unaccompanied by any morphological description (Getachew et al. 2014), highlighting the confusion between these two nominal species.
The complete mitochondrial genome of M. membranacea from the Atlantic Ocean has been sequenced (GenBank Accession No.: OU612079), while no sequencing was conducted with M. villosa.Previous molecular studies have focused on the overall phylogenetic analysis of bryozoans (Orr et al. 2020(Orr et al. , 2021;;Grant et al. 2023) or the phylogenetic relationships between bryozoans and other animals (Jang and Hwang 2009;Shen et al. 2012;Gim et al. 2018).In this study, to distinguish the morphologically similar species, we describe the complete mitochondrial genome of M. villosa through Illumina NovaSeq sequencing for the first time.The phylogenetic relationships of the families and genera within the suborder Membraniporina are also analyzed herein.
The mt DNA enrichment procedure is divided into three steps (mitochondria isolation, mt DNA extraction, and mt DNA amplification).First, the mitochondria were isolated using the Qproteome Mitochondrial Isolation Kit (Qiagen Co., Hilden, Germany), and mt DNA was extracted using an E.Z.N.A Mollusc DNA Kit (Omega Co., Norcross, GA).The REPLI-g Mitochondrial DNA Kit (Qiagen Co., Hilden, Germany) was used for amplification.Amplified mt DNA of M. villosa was sequenced using the Illumina NovaSeq platform at NICEM (Seoul, South Korea).Short-read DNA sequences were assembled and analyzed by NOVOplasty v.4.3.3 (Dierckxsens et al. 2017) and Geneious 9.1.8(Biomatters Ltd, Auckland, New Zealand) using M. membranacea mitogenome data as a reference sequence.Of nine species used in this study, comprising six Membraniporina species and two bugulids (outgroup), the annotated genes of B. grandicella (Canu & Bassler, 1929), B. neritina (Linnaeus, 1758), and M. membranacea were available from the GenBank.The authors annotated the remaining six species, including M. villosa, using MITOS Web Server (Bernt et al. 2013).
The mitogenome sequences (13 protein-coding genes as amino acids, excluding stop codon) of nine species, were aligned using the multiple sequence alignment program, MAFFT v.7 (Katoh and Standley 2013) and were concatenated for phylogenetic analysis.PartitionFinder v. 2.1.0(Lanfear et al. 2012) was used to find the best partition scheme and the best-fit model for amino acids sequence alignments.A phylogenetic tree was reconstructed based on the bryozoan concatenated dataset using the maximum-likelihood (ML) method with the GTR þ G þ I model in raxmlGUI 2.0 (Edler et al. 2021), and the bootstrap values were calculated from 10,000 replicates.
The ML tree (Figure 3) in the current study showed that M. villosa nests within the genus Membranipora and has a close relationship with M. membranacea with very high nodal support (100% BP in ML).The Membraniporidae is fully supported as a monophyletic family in our phylogenetic analysis (100% BP in ML) and the suborder Membraniporina forms two clades: Electridae is not clustered with the other two families, Membraniporidae and Sinoflustridae.

Discussion and conclusions
Alpha-level taxonomy in the bryozoan order Cheilostomatida relies almost exclusively on hard-part morphology (Dick and Mawatari 2005).Two morphologically similar species of the genus Membranipora show a preference for kelp.M. villosa is a major kelp-encrusting bryozoan in Japan, Hong Kong, and the northeastern Pacific Ocean, including Korean waters, settling on such species as Saccharina japonica (Osburn 1950;Huang et al. 1986;Dick et al. 2005;Chae and Seo 2019), whereas M. membranacea mainly occurs in the Atlantic Ocean (Hayward and Ryland 1998;Saunders and Metaxas 2008).M. villosa is nominally distinct but, owing to close similarity with M. membranacea, its status has been questioned.In this study, M. villosa and M. membranacea showed sequence differences of 20% for the total length of mitogenome and 16.1% for 13 PCGs.Molecular data definitely consider them to be separate species.
A ML tree based on a concatenated 13 PCGs sequence dataset from nine bryozoan species in the current study shows that M. villosa and M. membranacea have the closest relationship with very high nodal support (100% BP in ML).Interestingly, the two species having close molecular relationships show morphological and ecological similarities.
A phylogenetic analysis of the mitochondrial genome sequence of M. villosa was also conducted to determine the phylogenetic relationships within the suborder Membraniporina.Three genera belonging to Membraniporidae are in the same position in the phylogenetic tree as the previous studies (Hao et al. 2005;Orr et al. 2020;Grant et al. 2023).Electridae, one clade within the suborder Membraniporina, has a sister group relationship to the other clade, comprising Membraniporidae and Sinoflustridae, showing similar results to previous studies (Hao et al. 2005;Orr et al. 2020;Grant et al. 2023).The phylogenetic tree also shows that Sinoflustridae is not nested in Membraniporidae or Electridae, and this result coincides with the fact that Gordon (2009) proposed a new family with morphological data and Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions (Grant et al. 2023).

Figure 2 .
Figure 2. Circular map of the mitogenome of M. villosa.