Genetic diversity and population structure of Capitulum mitella (Linnaeus, 1767) in Fujian (China) revealed by mtDNA COI sequences

Background: Capitulum mitella is a widely distributed and ecologically important stalked barnacle that settles extensively on rocky shores. This species contributes to the structural complexity of intertidal habitats and plays a critical role in the marine ecosystem. This study aimed to reveal the genetic diversity and population structure of C. mitella by analyzing the mitochondrial cytochrome oxidase I (COI) gene. Methods: A 683bp fragment of the COI gene was sequenced from 390 individuals sampled from six localities in Fujian, China. Results: A total of 84 distinct haplotypes were identified through the analysis of 82 polymorphic sites, resulting in an average haplotype diversity (h) of 0.660 and nucleotide diversity (π) of 0.00182. Analysis of molecular variance (AMOVA) and pairwise F ST statistics showed no significant population structure. Neutrality tests and mismatch distributions provided evidence of recent population expansion for the species. Conclusions: We suggest that the species' high dispersal ability, and ocean currents coupled with limited physical barriers in the region, contribute to its current phylogeographic structure. These findings enhance our comprehension of the genetic diversity and population structure of C. mitella, providing valuable insights for future conservation efforts.


Introduction
Barnacles are a key group of crustaceans that occupy the intertidal zone and have a vital effect in shaping the ecology of intertidal communities (Lim and Hwang, 2006). Capitulum mitella (Linnaeus, 1767), the single species within the genus Capitulum Gray (Crustacea, Maxillopoda, Cirripedia, Thoracica), is an ecolgically significant stalked barnacle that aggregates and settles extensively on rocky shores (Jones, 1994;Lee et al., 2000). C. mitella is a dominant organism in intertidal coastal ecosystems with a widespread distribution throughout warmer regions of the Indo-Pacific, from Korea through India to the West Pacific Ocean. C. mitella it like other barnacles, has a biphasic life history: sessile adults and planktonic larvae. They have six naupliar stages and one cyprid stage, when it fixes itself in place, undergoes metamorphosis, and becomes a sessile juvenile (Lee et al., 2000). It is commonly found attached to rocks in the lower part of the intertidal zone, particularly in areas with strong currents. It tends to occur in dense populations, often crowded together in cracks and grooves on otherwise smooth rocky surfaces. Its attachment to rocks provides shelter and refuge for various organisms, influencing their distribution and interactions. Additionally, the population density of C. mitella in cracks and grooves can shape the physical structure of the intertidal zone. C. mitella also plays a vital role in intertidal ecosystems by filtering food particles from the water. This feeding behavior contributes significantly to nutrient cycling and energy flow within the ecosystem. By consuming organic detritus, algae, and small invertebrates, C. mitella helps maintain the overall productivity and balance of the intertidal community. In addition, it is also considered a commercially valuable species due to its high protein content, low-fat levels, and rich mineral content. It has strong market demand, particularly in the Fujian province, where it is widely consumed as a seafood product. However, C. mitella populations have declined in recent years due to overfishing, habitat destruction, and slow growth. To ensure the effective management and protection of this economically valuable species, a comprehensive understanding of its population genetic structure and genetic diversity is crucial (Ortega-Villaizán Romo et al., 2006). Unfortunately, the population genetic structure of C. mitella in the Fujian province coast has yet to be extensively studied. This knowledge gap underscores the urgency for further investigation to grasp the genetic diversity and population structure of this key species and secure its sustainable utilization and conservation.
Mitochondrial DNA (mtDNA) has emerged as a valuable tool for studying genetic diversity and population structure in various organisms (Ren et al., 2017;Xu et al., 2019). The Cytochrome c oxidase subunit I (COI) gene is particularly widely utilized as a molecular marker for investigating phylogeographic structures in marine invertebrates, due to its rapid evolutionary rate, non-recombining nature, and simple amplification procedure (Ajao et al., 2021;Xu et al., 2019;Yuan et al., 2016). By employing COI as a molecular marker, we can gain insights into the historical processes, genetic connectivity, and population dynamics of marine invertebrates. This study aimed to explore the genetic diversity and population structure of C. mitella populations along the coast of Fujian province utilizing the COI gene. The results would be important for the conservation and sustainable management of this species.

Ethical considerations
Ethical review and approval were not required for this study because this research is about Capitulum mitella, a common invertebrate and a seafood species that are not protected. After collection, we immediately placed them in 95% ethanol for preservation and all efforts were made to ameliorate any suffering of the animals.

Sample collection
A total of 390 individual C. mitella were collected from six locations in Fujian Province, China, during a survey conducted between July 2020 and September 2021. The collection site information is depicted in Figure 1 [Ningde (ND), Fuzhou (FZ), Putian (PT), Quanzhou (QZ), Xiamen (XM), Zhangzhou (ZZ)]. The specimens were stored in 95% ethanol at À20°C and muscle tissue was then extracted for DNA isolation.

DNA extraction and PCR amplification
The DNeasy Tissue Kit (QIAGEN) was employed to extract DNA from tissue samples preserved in ethanol, following the standard protocol for animal tissues. The quality and quantity of the extracted DNA are measured using BioDrop (BioDrop, UK). The amplification of the mitochondrial COI gene was carried out using the Lco1490/Hco2198 primers (Folmer et al., 1994) in a 25 μL reaction volume. The reaction consisted of 12 μL Taq plus Master Mix II (Dye Plus), 1 μL

REVISED Amendments from Version 1
In this new version (version 2), some major modifications have been done based on the suggestions and comments of the reviewers. We have revised the background and details of the article and made additions to the methodology, as requested.
Any further responses from the reviewers can be found at the end of the article each of the 10 μM primer concentration, 1 μL of DNA extract, and 11 μL nuclease-free water. The PCR thermal cycling profile was as follows: 94°C for 1 min, 15 cycles of denaturation at 94°C for 45 sec, annealing at 43°C (+0.5°C per cycle) temperature for 35 sec, extension at 72°C for 45 sec, followed by 20 cycles annealing at 50°C, with a final extension at 72°C for 10 mins. The PCR products were screened on a 1.0% agarose gel for quality control purposes. The sequencing in both directions was carried out by Sangon Biotech (Shanghai).
MEGA 11.0 was used to edit and align the sequences and calculate their base content. The identification of haplotypes was performed using the software DnaSP version 5.0 (Rozas et al., 2003), and the results were submitted to the GenBank database (accession numbers: ON495446 -ON495585). To investigate the relationships among haplotypes, we utilized NETWORK software version 4.613 (Bandelt et al., 1999) for visualization, and constructed a phylogenetic tree using the neighbor-joining (NJ) method with 1000 bootstrap replicates to assess branch reliability. We then calculated molecular diversity parameters using DNASP version 5.10.01 (Librado and Rozas, 2009) and Arlequin version 3.5 (Excoffier and Lischer, 2010), including haplotype diversity (h), nucleotide diversity (π) for each population, and analysis of molecular variance (AMOVA). Population pairwise F ST values were also analyzed by Arlequin. The significance of the F ST values comparison was tested by permutation tests (10,000 replicates). We evaluated the mismatch distribution and neutrality statistics to study demographic history, such as Tajima's D (Tajima, 1989) and Fu's F S test (Fu, 1997). In the event of a population expansion, we estimated the time of expansion (t) using τ=2μt (Rogers and Harpending, 1992), where we assumed a mutation rate of 3.1% per million years and a generation time of 1 year (Campo et al., 2010).

Genetic diversity
In this study, a 683 base pair (bp) segment of the COI gene was obtained from 390 individuals sampled from six populations. The average composition of the four nucleotides (A, T, C, and G) was found to be 18.19%, 42.93%, 14.65%, and 24.22%, respectively. It was determined that none of the sequences contained premature stop codons, insertions, or deletions. A nucleotide pair frequency analysis of the entire dataset revealed the presence of 82 variable sites (12.00%) among 683 sites, including 38 parsimony informative sites and 44 singleton sites.
A total of 84 haplotypes were identified among 390 individuals, with 59 of them being private and 25 being shared (Table 1, Figure 2). The most dominant haplotype H2 was identified in all six populations, accounting for 57.44%

Population genetic structure
In order to analyze the genetic structure of C. mitella populations, molecular variation analysis (AMOVA) and pairwise F ST values were employed. Results from the AMOVA analysis indicated that 99.77% of the genetic variation was found within populations, however, 0.23% have corresponded to among-population variation ( Table 3). The Φ ST values were not significantly different from zero in the six populations (Φ ST =0.00225), indicating a lack of significant genetic variation among these populations. The pairwise population F ST estimates obtained through an exact test were generally low, ranging from 0.00574 to 0.01144 among the six populations (Table 4). A neighbor-joining (NJ) tree constructed using 84 haplotypes demonstrated a shallow genetic structure (illustrated in Figure 3).

Demographic history
The neutrality tests, including Tajima's D and Fu's F S showed significantly negative results for all populations of C. mitella, indicating a recent population expansion or evidence of purifying selection (Table 5). The unimodal pattern observed in the mismatch distribution analysis of COI haplotypes (Figure 4) supports the hypothesis of a sudden population expansion. Furthermore, the populations displayed no significant values for the Sum of Squared Deviation (SSD) and raggedness index analysis (Rg), ranging from 0.00075 to 0.09248 and 0.045 to 0.135, respectively (Table 5). These findings provide evidence of a good fit between the observed and expected distributions. Using the molecular clock estimates of other barnacle species, the population expansion of C. mitella is estimated to have taken place approximately 15,000 years ago.

Discussion
The investigation of genetic diversity is the foundation for understanding the evolution of life and species diversity. By examining genetic diversity, we gain insights into the genetic composition of a population, its evolutionary history,  and the mechanisms behind variation and evolution Zheng et al., 2019). A major method for studying genetic diversity is molecular genetics techniques, such as sequencing the DNA of individuals or populations. In this research, the mitochondrial COI gene was used to examine the genetic diversity and population structure of C. mitella in the Fujian province. Results showed an average haplotype diversity (h) of 0.660 and a nucleotide diversity (π) of 0.00182, with 84 haplotypes identified and a star-like haplotype network (Figure 2). Out of the haplotypes, 59 were detected only at single localities, while the other 25 were present in two or more locations ( Table 2). The results indicate that the C. mitella in Fujian province has a medium to high level of genetic diversity, with a low nucleotide diversity. This is comparable to the findings in other invertebrates, such as Portunus trituberculatus (h=0.582, π=0.00158) (Liu et al., 2009), but higher than those observed in China (h=0.490, π=0.00158), and lower than the Korean population (h=0.909, π=0.00550) (Yoon et al., 2013). The results of this study suggest that the C. mitella in Fujian province experienced a rapid population expansion from an ancestral population with a small effective size. This is indicated by the presence of rare haplotypes and low nucleotide diversity. This phenomenon could be attributed to a sudden increase in population size, which resulted in the preservation of rare haplotypes that would otherwise have been lost due to genetic drift (Zane et al., 2006). The small effective population size also suggests that this process of expansion occurred relatively recently, as a larger population size would have resulted in the elimination of these rare haplotypes over time (Nehemia et al., 2019).
This study of the genetic diversity of C. mitella populations in Fujian province found no evidence of a phylogeographic structure, as supported by the pairwise F ST statistics and AMOVA analyses.
The results of the neighbor-joining tree analysis indicate that the haplotype relationships of C. mitella in Fujian province are shallow and there is no clear geographic association. This may be due to high gene flow among populations. The findings of this study suggest that the high dispersal capability of C. mitella's planktonic larvae is a key factor in promoting gene flow across vast geographic areas among invertebrate populations, thus maintaining or increasing genetic diversity. The duration of the larval stage, which can last up to 14 days (Yuan et al., 2016), enables C. mitella to disperse over long distances. The distribution of C. mitella populations is also influenced by a range of physical oceanographic factors, such as the presence of physical barriers, ocean currents, and wind patterns (Schilling et al., 2020).
The results of Tajima's D and Fu's Fs neutrality tests in all localities of C. mitella showed negative and significant values (Table 5), indicating a recent population expansion. This conclusion is further supported by the unimodal mismatch distribution, high haplotype diversity, and low nucleotide diversity. The estimated date of the population expansion is estimated to be around 15,000 years ago, during the Pleistocene. The Pleistocene glaciations have been shown to significantly impact the population structure of marine species, with a reduction in population size during glacial periods and rapid expansion during interglacial periods (Wilson and Eigenmann Veraguth, 2010). This pattern of demographic fluctuations has directly influenced the distribution and population size of the C. mitella species.
In summary, the present study aimed to investigate the genetic diversity of C. mitella populations along the Fujian coast using mitochondrial COI gene analysis. Results revealed medium to high levels of haplotype diversity and low nucleotide diversity, with 84 haplotypes identified and no significant genetic structure among populations. These findings suggest a high degree of gene flow and a lack of geographic associations. The demographic history of the species, including the influence of Pleistocene glaciations, may have played a role in shaping its current distribution and population size. The findings of this study emphasize the significance of genetic studies to a comprehensive understanding of the population genetics of C. mitella, particularly to inform its conservation and management. Further research using more populations and more sensitive molecular markers is needed to gain a more complete picture.

Open Peer Review I confirm that I have read this submission and believe that I have an appropriate level of
effective population size of Capitulum mitella, the authors can estimate this population size using software like n-MIGRATES.
Additionally, authors must confirm whether they used pairwise population F ST (use conversional F-statistics) or Φ ST (calculate distance matrix) for subpopulation comparisons.

6.
Is the work clearly and accurately presented and does it cite the current literature? Yes

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes sessile adults and planktonic larvae. They have six naupliar stages and one cyprid stage, when it fixes itself in place, undergoes metamorphosis, and becomes a sessile juvenile (Lee et al., 2000). It is commonly found attached to rocks in the lower part of the intertidal zone, particularly in areas with strong currents. It tends to occur in dense populations, often crowded together in cracks and grooves on otherwise smooth rocky surfaces. Its attachment to rocks provides shelter and refuge for various organisms, influencing their distribution and interactions. Additionally, the population density of C. mitella in cracks and grooves can shape the physical structure of the intertidal zone. C. mitella also plays a vital role in intertidal ecosystems by filtering food particles from the water. This feeding behavior contributes significantly to nutrient cycling and energy flow within the ecosystem. By consuming organic detritus, algae, and small invertebrates, C. mitella helps maintain the overall productivity and balance of the intertidal community.
2. I advise utilizing MEGA software that is more recent, such as MEGA 11, rather than the old version MEGA 7.
Thank you very much for your suggestion， we have made the changes as per your request.
3. Although the authors performed a pairwise FST test, I advise doing additional statistical tests to support the findings of population genetic structure. As a result, I advise using hierarchical AMOVA. To investigate every clustering possibility, SAMOVA would be the best choice. In order to determine whether isolation by distance (IBD) may be influencing the genetic structure of the population in this species, the author may also need to undertake testing for IBD.
Thank you very much for your feedback. This suggestion is excellent and addresses the question of whether isolation by distance (IBD) is influencing the genetic structure of the population. In our study, we focused only on resources within a single province, where the geographic distances were relatively small, and no genetic differentiation was observed among populations. Therefore, we did not employ this method. However, moving forward, we will adopt your recommended approach to investigate the distribution, dispersal, genetic structure, and variations of this species along the Chinese coast. By doing so, we hope to obtain more comprehensive and in-depth results.
4. I recommend performing post hoc analysis, such as sequential Bonferroni correction for pairwise analysis, because it is advised to perform a multiple-comparison correction when several dependent or independent statistical tests are being performed simultaneously. The authors indicated how molecular variation analysis (AMOVA) was performed in the methodology part, but did not describe how pairwise population FST estimates were carried out; instead, the description is shown in the section of results.
Thank you for your suggestion. We have already supplemented that section.
5. In order to compare the results of the genetic diversity and demographic history with the effective population size of Capitulum mitella, the authors can estimate this population size using software like n-MIGRATES.
Thank you for your suggestion. We highly appreciate your proposed approach, and similar to the third point, we will adopt this method to study the distribution, dispersal, genetic structure, and variations of this species along the Chinese coast, to achieve more comprehensive and in-depth results through this approach.
6. Additionally, authors must confirm whether they used pairwise population FST (use conversational F-statistics) or ΦST (calculate distance matrix) for subpopulation comparisons.
Thank you for your suggestion. We have already confirmed and supplemented this section.
Thank you once again for your valuable suggestions.
2."Mitochondrial DNA (mtDNA) is widely used as an ideal marker for investigating genetic diversity and population structure due to its rapid evolutionary rate, non-recombining nature, and simple amplification procedure (Ren et al., 2017;Xu et al., 2019)." The authors should explain the importance of using different genetic markers, including COI gene, to better understand the current population structure.
Thank you for your suggestion. We appreciate the importance of incorporating different genetic markers, including the COI gene, to enhance our understanding of the current population structure. We have already rewritten that section and made some additions.
3. Please could you include the populations to be studied in the main text? They are only detailed in the caption of figure. Please include the full name in addition to the abbreviation. Some information describing the study area would be great.
Thank you for your suggestion. We have made the necessary revisions.
4. In the section "DNA extraction and PCR amplification", how the DNA was extracted is missing. Please indicate the methodology used. Also indicate the commercial name of the reagents used.
Thank you. We have already added that section as per your request.
5. In the sentence, 'MEGA 7.0 was used to edit and align the sequences,' the word 'aligned' should be changed to 'align'.
Thank you. The word 'aligned' has been changed to 'align'.
6. Please specify what you mean by SSD before the acronym (sum of squared deviation) and include the acronym after raggedness index analysis (Rg).
Thank you. We have made the necessary revisions as this suggestion.
Competing Interests: No competing interests were disclosed.