First Record of Colonial Ascidian, Botrylloides diegensis Ritter and Forsyth, 1917 (Ascidiacea, Stolidobranchia, Styelidae), in South Korea

: Botrylloides species are important members of the fouling community colonizing artiﬁcial substrates in harbors and marinas. During monitoring in 2017–2020 of non-indigenous species in Korea, one colonial ascidian species was distinctly different from other native colonial ascidians, such as B. violaceus and Botryllus schlosseri , in South Korea. This species was identiﬁed as B. diegensis . DNA barcodes with mitochondrial COI were used to identify one-toned and two-toned colonies of B. diegensis . Intraspeciﬁc variations between Korean and other regions of B. diegensis from the NCBI ranged from 0.0% to 1.3%. The Korean B. diegensis was clearly distinct from other species of Botrylloides at 15.8–24.2%. In phylogenetic analysis results, Korean B. diegensis was established as a single clade with other regions of B. diegensis and was clearly distinct from Korean B. violaceus . After reviewing previous monitoring data, it was found that two-toned B. diegensis was already found in six harbors by July 2017. It has now spread into 14 harbors along the coastal line of South Korea. This means that B. diegensis might have been introduced to South Korea between 1999 and 2016.


Introduction
Introductions of non-indigenous species (NIS) have occurred at an increasing rate since the 20th century, showing increasing ranges and intensity of vectors [1]. However, identifying new or recently introduced NIS can be challenging if only traditional methods are used [2]. Many marine animal NIS in introduction hotspots (e.g., marinas and harbors) belong to taxonomic groups (especially colonial ascidians) that require substantial taxonomic expertise [3]. In this sense, the usefulness of a molecular barcoding approach has been well documented. Such an approach can be used to ascertain the presence of new NIS [4] to reveal false morphology-based NIS identification [5] and to determine the taxonomic status of previously unrecognized NIS [6]. An increasing number of studies have recommended the use of molecular tools to complement traditional methods (e.g., morphological taxonomic approach) for achieving reliable taxonomic identification of marine NIS [2,7,8], including those considered to be cryptic species, which have been widely reported for colonial ascidians [3,[9][10][11][12].
Botrylloides and Botryllus (class Ascidiacea, order Stolidobranchia, family Styelidae) are ascidians belonging to a group of colonial species, of which 53 species have been described [13]. Among them, Botrylloides species are important members of the fouling community colonizing artificial substrates on the Pacific coast of the United States (for instance, in harbors and marinas) [14,15]. In Europe, one putatively native species, B. leachii (Savigny, 1816), has also been recognized, often showing coloration somewhat similar to the two-toned color pattern seen in B. diegensis [3]. One-toned B. diegensis has also been found to be misidentified as B. violaceus in the NCBI database. Recently, rearrangement of mitochondrial COI data of each species has been accomplished [3]. In Korea, the marine NIS research program was initiated by the Ministry of Oceans and Fisheries in 2008. Many mitochondrial COI data of each species has been accomplished [3]. In Korea, the marine NIS research program was initiated by the Ministry of Oceans and Fisheries in 2008. Many ascidians inhabit many harbors in South Korea. Among them, a number of non-indigenous ascidians have been newly reported via this research program [16,17]. However, these new reports were focused on solitary ascidians. The identification of colonial ascidians, such as species identification in the field, remains a challenging task.
Thus, the objectives of this study were the following: (i) to identify botryllids ascidians in South Korea based on DNA barcoding, (ii) to provide mitochondrial COI data for B. diegensis from South Korea.

Sample Collection and Identification
Samples were collected from 11 May 2020 to 15 May 2020 in 14 harbors along the coastal line of South Korea (Figure 1, Table 1). All samples were taken from acrylic plates designed for monitoring non-indigenous and harmful organisms. The dimensions of the acrylic plates were 30 × 30 cm 2 with a thickness of 5 mm. Each plate was connected with polypropylene rope and the distance between each plate was 20 cm. A monitoring set was composed of 10 acrylic plates, and the first acrylic plate was situated 1 m below the surface of the water. The plates were installed from July 2017 to October 2020. Colonies were photographed with a digital camera (TG-5, Olympus, Tokyo, Japan) and labeled before the sample collection. We collected the sample from a colony of botryllids (0.5 × 0.5 cm 2 ) on a settlement plate. The samples were preserved immediately with an ethyl alcohol solution (>95%). They were then assigned voucher numbers (SYA200501-SYA200556), and stored in the Marine Biological Resource Institute, Sahmyook University, Korea. The collected samples used for DNA barcoding were identified based on their zooid morphological features from Tokioka [18] and Rho [19] under microscopes.

DNA Extraction and Amplification of DNA Barcoding Region
Total genomic DNA was extracted from a single zooid in a colony using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Partial sequences of COI were amplified using two primer pairs as follows: LCO1490-HCO2198 [20] and dinF-Nux1R [21]. All genomic DNA samples were stored at −72 • C until use. Polymerase chain reaction was performed with a total reaction volume of 20.0 µL, including AccuPower PCR PreMix and Master Mix (Bioneer, Seoul, Korea), 1.0 µL of each primer (10 mM), and 0.3 µL of DNA template (>50 ng/µL), with the following thermal cycling conditions: one cycle at 94 • C for 3 min, 35 cycles of 94 • C for 30 s, 50 • C for 45 s, 72 • C for 60 s, and a final extension step at 72 • C for 7 min. The PCR products were directly sequenced with the forward and reverse primers used for amplification (Cosmogenetech, Seoul, Korea). The assemblies and alignments of sequencing results were performed using Geneious v. 11.1.5 (Biomatters, Auckland, New Zealand).

DNA Barcoding Data Analysis
All COI sequences obtained in this study were deposited in GenBank. The accession numbers are shown in Table 2. Genetic distances and phylogenetic relationships of Korean B. diegensis with B. diegensis from other regions (12 localities of 6 countries; Supplementary Table S1) and 11 other species of Botrylloides and Botryllus schlosseri were investigated. All data, except for Korean botryllids, were obtained from the NCBI. The best-fit model of nucleotide substitution for the COI dataset was selected using Modeltest v. 2.1.1 [22] with the Akaike Information Criterion (AIC) for maximum likelihood (ML). The ML tree was constructed using PhyML 3.0 [23] under the TrN + I + G model and 1000 replicate bootstrapping for the COI dataset. Bayesian inference (BI) was performed using 1,000,000 generations of Markov Chain Monte Carlo chains. One in every 1000 generations was sampled. The initial 250 generations were discarded as burn-in. All processes were executed with MrBayes 3.2.6 [24] under the TrN + I + G model. Botryllus schlosseri was determined to belong to the Botrylloides group in the ML and BI analyses. Pairwise distances were calculated using the Kimura 2-parameter model (K2P) [25] in MEGA 7.0 [26].

DNA Barcoding Analysis for B. diegensis from South Korea and Other Colonial Ascidians
This study presents the first report of Botrylloides diegensis in South Korea. It was not clearly identifiable from B. violaceus or Botryllus schlosseri in the field survey (Figures 2 and 3, Supplementary Figure S1). Thus, we needed to compare it with more species of botryllids using DNA barcoding. We obtained 16 and 10 partial COI sequences of Korean B. diegensis and B. schlosseri at 672 bp and 858 bp, respectively (Table 2). We calculated the pairwise distances based on 396 bp sequences of COI genes of 11 species of Botrylloides and Botryllus schlosseri (Table 3, Supplementary Table S1).
The intraspecific variation range of the Korean B. diegensis group was 0.0-1.3%, with a mean of 0.4% (Supplementary Table S1). The intraspecific variation between Korean and other regions of B. diegensis from NCBI was 0.0% to 1.3%. Variations for other regions were 0.0-1.0% (Supplementary Table S1). Intraspecific variations in the Korean group seemed to be higher than those in other regions. The mean variation of the Korean group was 0.4%, which was slightly higher than that for other regions group at 0.2% (Table 3). The interspecific variation between Korean B. diegensis and other species of Botrylloides was 15.8-24.2% (Table 3). The intraspecific variation of other Botrylloides species, except for B. diegensis, was 0.0-1.3%, similar to the intraspecific variation of B. diegensis in this study (Supplementary Table S1). Additionally, the phylogenetic trees of ML and BI show the same results ( Figure 4). All species of Botrylloides were distinct from B. schlosseri, an outgroup ( Figure 4). Botrylloides diegensis formed a single clade with Korean B. diegensis and B. diegensis from GenBank ( Figure 4). This B. diegensis clade showed a clear, single clade, although several localities data were included: 20 localities in 9 countries (Figure 4, Supplementary Table S1). The posterior probability support values for several resolved   Table S1). The intraspecific variation between Korean and other regions of B. diegensis from NCBI was 0.0% to 1.3%. Variations for other regions were 0.0-1.0% (Supplementary Table S1). Intraspecific variations in the Korean group seemed to be higher than those in other regions. The mean variation of the Korean group was 0.4%, which was slightly higher than that for other regions group at 0.2% (Table 3). The interspecific variation between Korean B. diegensis and other species of Botrylloides was   Table S1). The intraspecific variation between Korean and other regions of B. diegensis from NCBI was 0.0% to 1.3%. Variations for other regions were 0.0-1.0% (Supplementary Table S1). Intraspecific variations in the Korean group seemed to be higher than those in other regions. The mean variation of the Korean group was 0.4%, which was slightly higher than that for other regions group at 0.2% (Table 3). The  Table S1). Additionally, the phylogenetic trees of ML and BI show the same results ( Figure 4). All species of Botrylloides were distinct from B. schlosseri, an outgroup (Figure 4). Botrylloides diegensis formed a single clade with Korean B. diegensis and B. diegensis from GenBank ( Figure 4). This B. diegensis clade showed a clear, single clade, although several localities data were included: 20 localities in 9 countries (Figure 4, Supplementary Table S1). The posterior probability support values for several resolved nodes were >0.8, although some bootstrapping support values in the ML tree were not well supported (<70) in the clade of Botrylloides (Figure 4).

Distributions of B. diegensis and Other Similar Native Colonial Ascidians in South Korea
One-toned Botrylloides diegensis was quite similar to B. violaceus (Figures 2 and 3, Supplementary Figure S1). Thus, the existence of B. diegensis was not clearly recognized before this study. We carefully reexamined all settlement plate photographs and checked the distribution of two-toned B. diegensis (Table 4). From July 2017, two-toned B. diegensis appeared at six harbors (Table 4). It was newly observed in Incheon in January 2018 and appeared in Gwangyang and Dangjin in August 2018 and May 2020, respectively (Table  4). Two-toned B. diegensis and other botryllid species (B. violaceus and B. schlosseri) were observed in 12 of the 14 harbors, not including Busan and Yangpo ( Figure 5). Among them, four harbors (Gunsan, Wando, Yeosu, and Ulsan) showed the existence of three botryllid species, including two-toned B. diegensis ( Figure 5).

Distributions of B. diegensis and Other Similar Native Colonial Ascidians in South Korea
One-toned Botrylloides diegensis was quite similar to B. violaceus (Figures 2 and 3, Supplementary Figure S1). Thus, the existence of B. diegensis was not clearly recognized before this study. We carefully reexamined all settlement plate photographs and checked the distribution of two-toned B. diegensis (Table 4). From July 2017, two-toned B. diegensis appeared at six harbors (Table 4). It was newly observed in Incheon in January 2018 and appeared in Gwangyang and Dangjin in August 2018 and May 2020, respectively (Table 4). Two-toned B. diegensis and other botryllid species (B. violaceus and B. schlosseri) were observed in 12 of the 14 harbors, not including Busan and Yangpo ( Figure 5). Among them, four harbors (Gunsan, Wando, Yeosu, and Ulsan) showed the existence of three botryllid species, including two-toned B. diegensis ( Figure 5).

Discussion
Several widely distributed botryllids, including B. diegensis, have been misidentified, and the correct identification of these species is critical for understanding their biology and spread, as well as for detecting the spread of additional species [27]. Preliminary molecular analyses revealed that these one-toned color colonies included specimens attribut-

Discussion
Several widely distributed botryllids, including B. diegensis, have been misidentified, and the correct identification of these species is critical for understanding their biology and spread, as well as for detecting the spread of additional species [27]. Preliminary molecular analyses revealed that these one-toned color colonies included specimens attributable to B. diegensis. Thus, B. diegensis might be misidentified in the field as B. violaceus based on the criterion of possessing one-toned color rather than two-toned color [3]. In addition, one-toned color B. diegensis is morphologically very similar to B. violaceus in Korea. In this study, we selected the mitochondrial cytochrome c oxidase subunit 1 (COI) for the detection of one-and two-toned color B. diegensis in South Korea. The COI was identified as the marker of choice for species discrimination [28] and has been effectively used for detecting NIS [8,29] and botryllids [27,30]. As a result, we recognized the presence of B. diegensis in South Korea based on DNA barcoding analysis. Thus, we needed to know when and where this species first appeared. We reviewed the monitoring data from 2017-2019, focusing on two-toned colonies of B. diegensis. As a result, B. diegensis was found to be present in six harbors in July 2017. It has now spread to 14 harbors along the coastal line of South Korea. Botrylloides diegensis was not present in the Northwest Pacific region, including Korea and Japan [18,31,32], according to previous ascidian studies (~2020). Professor Rho, a great ascidian taxonomist in Korea, did not report this species either. Only two Botrylloides, B. magnicoecum and B. violaceus, have been reported by Rho [19,[33][34][35][36][37][38][39][40]. However, in 2021, Nydam et al. [27] first reported B. diegensis in Japan and these specimens were collected in 2005-2009 in three localities of Japan. Thus, we supposed that B. diegensis was introduced to the Northwest Pacific region before 2006. Botrylloiodes violaceus and B. diegensis are both native to the North Pacific [3]. While the former is native to the Northwest Pacific, there is more uncertainty regarding the native range of the latter [41]. Although B. diegensis was originally described from the Northeast Pacific (southern California), it might have been introduced from the Indo-Pacific [31,42]. This remains unclear. The presence of B. diegensis was confirmed through this study, and therefore, the investigation of the introductory route of B. diegensis is urgently needed, and also investigate the ecological and economic impact from B. diegensis in South Korea.

Conclusions
Based on our DNA barcoding results, one-and two-toned color B. diegensis has spread along all coastal lines of South Korea. It was possibly introduced to South Korea between 1999 and 2016 based on field monitoring data and previous studies. Further studies are needed to analyze the specific route of its introduction to South Korea based on the population genetic studies and previous monitoring data.