What species make up the Nike fish assemblages at the macrotidal estuary in Gorontalo Bay , Indonesia ?

No study has documented the species composition of Nike Background: fish (fam: Gobiidae) schools. The aim of this study is to document the species composition of the Nike-fish schooling. All samples were collected randomly from fisher’s catch during Methods: the fishing season on 5th–11th October 2018 at macrotidal area in Leato. Then, all specimens were identified morphologically by melanophore pattern differences. Subsequently, all identified-samples by melanophores pattern differences were sent to the genetic laboratory for identification. The morphological results show there are five individuals with a Results: different melanophores pattern. On the contrary, the genetic results only show four species from those five individuals. They are Sicyopterus and . pugnans, S. cynocephalus, Belobranchus segura, Bunaka gyrinoides Our findings show that there are only four species that Conclusions: compose the Nike fish schooling in Gorontao Bay. They are Sicyopterus and pugnans, Sicyopterus cynocephalus, Belobranchus segura, Bunaka . gyrinoides


Introduction
Estuaries are a crucial habitat for biota and small fish, in particular juveniles of commercially relevant species. They are considered as the most productive and dynamic ecosystem in the world (Cantera & Blanco, 2001;Lahjie et al., 2019;McHugh, 1967;Sreekanth et al., 2017). They also perform the most crucial role in the population dynamic for a lot of invertebrate and fish species. These ecosystems also significantly contribute to provide some ecological services such as nursery ground, feeding ground and breeding habitats for both freshwater and marine species (Beck et al., 2001;McLusky & Elliott, 2004;Sun et al., 2019). The most well-known species that occupy the seas and estuary area in Gorontalo Bay is Nike fish.
Nike (pronounced nee-K) is a local name for transparent juvenile of unknown fish. These fish are approximately 2-4 cm in length; they appear seasonally and fished at estuary waters around the Gorontalo Bay. These juvenile fish has been fished and marketed traditionally for a long time. They are preferable for consumption by the local people than other fisheries products. As a consequence, fishing activity has increased over time to supply local demand for Nike (Wolok et al., 2019).
However, the impact of fishing activities is unknown. A recent paper concerning Nike only reports the seasonal appearance during the fishing season (Pasisingi & Abdullah, 2018), total length and morphometric measurements (Zakaria, 2018), nutrition content (Liputo et al., 2013), and mercury contamination of these fish (Salam et al., 2016). To our knowledge, no studies have documented the species diversity that composed the schooling of Nike. Although, Yamasaki et al. (2011) have reported that species in juvenile form can be determined by its melanophores pattern and genetic determination.
The objective of the present study is to address this lack of knowledge by identifying the fish species that composed a Nike fish schooling. This information is very urgent and required for fisheries management. Therefore, we aimed to identify the species that composed the schooling of Nike fish in Gorontalo Bay by melanophores pattern and genetic identification.

Methods
This study was conducted in October 2018 at Leato (0°30'0.58"N, 123°3'55.42"E), Gorontalo Bay, Indonesia ( Figure 2). Approximately 100 g of the Nike-Fish Assemblages ( Figure 1) were collected randomly from the fishermen's catch at fishing grounds during the catch-season (on October 5 th -11 th ). All samples were transported using a cool-box to the lab for measurement. Immediately after collection, all samples were identified visually according to Yamasaki et al. (2011). and the specimens with different melanophore patterns were separated according to their melanophore display. We assummed that those separated individuals were diferent on species.
Then, we selected one individual from each group and labeled these as N1, N2, N3, N4, N5, for genetic identification. Images of the selected samples were captured using Canon EOS 100d with 58 mm pro Digital Wide Converter 0.45X Lens and subsequently converted to black and white using CorelDraw Graphic Suite 2019.
After selection, all of the individuals with different melanophores were preserved with ethanol 70% in a separate bottle and sent to the Genetics Laboratory at Manokwari for genetic identification by Sanger sequencing. The DNA cytochrome oxidase subunit I (CO1) of the sample was isolated with a Geneaid™ DNA Isolation Kit. Editing, and proofreadingof seqeunces,, and construction of the the phylogenetic tree was generated with MEGA 5.0 software.

Results
Five unspecified individuals of Nike-fish were identified morphologically by melanophore differences, as shown in Figure 3. N1 was revealed as Sicyopterus pugnans; N2 as Sicyopterus cynocephalus; N3 and N5 as Belobranchus segura; and N4 as Bunaka gyrinoides. The specimens with melanophores differences of each group is shown in Figure 4.

Melanophores pattern
Nike-fish schools consist of various species with the same body-shape, but different melanophore displays. Moreover, from 100 g (~145 individuals) of the total specimens that we identified, only five individuals with different melanophore patterns were identified (Figure 3). Figure 3 shows the genetic identification among the individuals (species). The outcomes of genetic identification for N3 and N5 shows that both samples are the same species: Belobranchus segura.

Discussion
Although the melanophore patterns in N3 and N5 are different, their genetics are identical, meaning they are the same species (Belobranchus segura). This dissimilarity might be affected by the changes of melanophore during the development of the larvae. Valade et al. (2009) report that such melanophores chang on Sicyopterus langocephalus during the larvae stage. These changes could represent a problem for morphological identification. We can not count the species by morphological differences. Therefore, for the next examination we strongly recommended determining the species composition of the Nike fish schools by genetic rather than morphological identification because for that reason.

Conclusion
Our findings show that there are four species that compose Nike fish schooling. They are Sicyopterus pugnans, Sicyopterus cynocephalus, Belobranchus segura, and Bunaka gyrinoides.
Group N5, Belobranchus segura isolate N5_LEATO_1 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial. GenBank accession number MN069308. The first sentence should be "Nike (pronounced nee-k) is a local name for transparent postflexion larvae of fish, but it has not been identified to the species as well as the genus or family level." ○ If you or the local people actually used to know what it is, for example, they are young gobies, please write it. ○ Does the "length" mean standard length or total length? Please specify, they are significantly different.

Results
First sentence: Please replace "five unspecified individuals" with "five unspecified types".

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Sicyopterus pugnans is a species in Polynesia. So probably it is a misidentification. Please remind that the information in the database is not always correct. Indeed the Sicyopterus pugnans in the figure 4 is divided in to two clades. If they are different species, at least one of them is not the S. pugnans. Please consider the meaning of the results before trusting the information of the database blindly. Please suggest the possibility of misidentification in the Discussion.

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Melanophore patterns: As I wrote above, if the nike material was composed of Sicyopterus, Bunaka, and Belobranchus species, the larvae can be identified at least at the genus level by their morphologies. Please observe the specimens carefully before saying "same body shape".

Discussion
I don't agree with the last two sentences.

Figure 1
Please write status of the larvae. Are they living, on ice, or fixed in 70% ethanol?

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The scale bar must be an error. The larvae are too big, if the bar indicates 3 cm. Please confirm.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility? Partly Are the conclusions drawn adequately supported by the results? Yes