## 4 Discussion  
### Congruence of Taxonomic Information between eDNA Metabarcoding and CMI  
Many of recent studies have strived to profile and quantify taxonomic composition of plankton communities using either eDNA metabarcoding or CMI [14; 24; 56]. Among them, a few studies have reported a degree of disagreement between the two pronged identification methods [32; 57]. In this respect, our study also presented some disagreement, between eDNA and CMI samples, in community  
Figure 4: Clustering result (**a**) of the data of water eDNA and CMI based on the self-organizing map. The right panels (**b**) present the corresponding physical, chemical, and biological conditions in Gwangyang Bay. The horizontal lines of zero indicate corresponding grand average values (water temperature: 25.6 \({}^{\circ}\)C, salinity: 29.3 psu, TP: 0.049 mg L\({}^{-1}\), TN: 0.45 mg L\({}^{-1}\), TC: 22.4 mg L\({}^{-1}\), Chl-_a_: 4.36 mg L\({}^{-1}\)). composition (Figure 3a,b as well as in relationships of biotic information (Figure 3e,f). Some pieces of literature on eDNA monitoring have enumerated possible reasons to explain the discrepancy between the two identification methods. It is reported that the capacity of identification between molecular and morphological datasets could have mainly caused the disagreement [24; 58]. That is, specimen identification can vary along accuracy of molecular reference databases [59]. Therefore, the establishment of well-curated databases of reference DNA sequences for identified specimens is essential in the field of eDNA metabarcoding to make the taxonomic information congruent with CMI. Additionally, there is another concern with the drawback of eDNA metabarcoding associated with technical biases/difficulties, such as copy number variation in the process of polymerase chain reaction (PCR) [60]. Related to a primer, its amplification and binding affinity are critical factors to bring about taxonomic biases in eDNA detection [61; 62; 63; 64; 65]. In terms of sensitivity of species detection, CMI-based assessment is also subject to an unpredictable, but probably significant, bias due to the presence of cryptic species [66]. Particularly in our study, marine calanoid copepods, _Candacia_, were only detected by eDNA metabarcoding in a very low proportion of \(<\)5%. However, we also admit that taxonomic misclassification due to lack of expertise and difficult to impossible taxonomic determination rather than just cryptic species also causes bias. With these concerns in mind, our results on the community composition might be influenced by the primer amplification effects (Figure 3a,b). The previous related research reported some technical biases against low-abundant taxa in delineating microbial diversity [63]. In fact, while Cnidaria comprised 3% in CMI, they were 14% in eDNA samples. Likewise, Mollunks occupied 5% in CMI, but did 10% in eDNA samples (Figure 3a,b). In contrast to these differences, the compositional changes between the two samples were not significant for the rest low-abundant taxa containing Annelida, Chaetognatha, Echinoderm, and fish (Figure 3a,b). Namely, our results showed that low-abundant taxa could always be overestimated in eDNA metabarcoding. These results of difference and variation might be associated with several reasons. Firstly, eDNA metabarcoding is highly sensitive to detecting species. This high sensitivity is advantageous in identifying low-abundant/cryptic species. However, it can also lead to variations originating not only from organisms that are a few miles away from the sampling site but also from food items hidden in organisms. In addition, abundance estimates are possibly erroneous because many small organisms could generate the same number of sequence reads as a few large organisms. Secondly, although it is relatively unexplored, the copy number variation derived from the technical bias during the PCR process is another factor leading to inaccurate estimation [60; 63]. Lastly, CMI is also error-prone depending on expertise/experience and specimen size.