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Phototactic behaviourThe optimization results of the proposed behavioural setup allowed the phototactic behaviour of the studied D. magna clone and the effects of FK treatment on this behaviour to be monitored and quantified. Furthermore, the effects of the FKs were evident only upon light exposure, were more apparent after a short (5 min) acclimation to light and were tightly regulated by the light intensity. The above-mentioned factors agree with previous studies, which found a marked positive phototactism of clone P132,8528 and that the effects of the FKs become consistent after 5 min of light exposure28. Moreover, it has also been reported that light intensity controls anti-predatory defences in Daphnia29.The effects of the pharmacological treatments were consistent for GABAergic and muscarinic cholinergic compounds across two or three identical non-consecutive experiments performed over more than one year. Consistency of the toxicological results and, in particular, of the behavioural responses should be compulsory in toxicological studies to increase the credibility and robustness of the findings30,31. Agonists of these two neurotransmitter receptors (DZP, PILO) and the antagonist of the GABA receptor (PICRO) affected the induction of the phototactic behavioural changes (i.e., interfered with fish recognition). The receptor agonists DZP and PILO counteracted the negative phototactism evoked by the FKs, whereas PICRO enhanced the effect of the FKs, increasing the negative phototactism. None of the three applied substances when applied alone induced anti-predatory fish phototactic behaviour, indicating that these compounds interfered with the FK sensorial pathway. Alternatively, the muscarinic cholinergic antagonist SCOP interfered with phototaxis itself, almost completely abolishing the positive phototactic behaviour of the studied clone under both control and FK conditions. This indicates that the muscarinic cholinergic signalling pathway could potentially be a major regulator of anti-predatory fish phototactic behaviour. In D. pulex and D. galeata, the formation of neck teeth or helmets in response to predatory kairomones released by invertebrate predators has been related to a series of biological reactions that involve kairomone perception and neuronal signals, which are converted into endocrine signals and subsequently induce changes in the expression of morphogenetic factors32,33. We previously showed that DZP, PILO, PICRO and SCOP were neuroactive in D. magna, affecting sensitization and/or habituation motile responses to repetitive light stimuli34; thus, it is likely that these compounds disrupted neurological signalling pathways related to the phototactism shifts caused by FK perception or to the phototaxis itself.Little is known about how phototaxis is neuronally coded. In D. pulex, both in silico and experimental works have shown that histaminergic neurons may mediate phototactic responses to UV irradiation12. By using histamine immunohistochemistry, the previous authors labelled putative photoreceptors in the compound eye and neuronal projections from these cells to the brain. The D. pulex genome also has a putative Drosophila orthologue of histidine decarboxylase (the rate-limiting biosynthetic enzyme for histamine), as well as two putative histamine-gated chloride channels (hclA and hclB orthologues). Exposure of D. magna to cimetidine, an H2 receptor antagonist known to block both hclA and hclB in D. melanogaster, inhibited the negative phototactic responses of these orthologues to UV irradiation. In another study, it was found that short-day photoperiods induced a significant increase in light-avoidance behaviours relative to controls and increased glutamate signalling, which is a critical pathway in arthropod light-avoidance behaviour35. It has also been reported that a group of serotonergic cells located in the protocerebrum probably control phototactic behaviour16. Notably, the perception of predatory kairomones and neuronal and cellular wiring is largely unknown in Daphnia2. For example, the receptors that detect invertebrate cues from Notonecta in D. longicephala were shown to be located on the first antennae, from which neurites extend into the deutocerebrum of the brain. However, key olfactory neuronal structures, such as olfactory glomeruli in the deutocerebrum, were not found2.Our results obtained for DZP, an agonist of the GABAA receptor, agree with those of Weiss et al.11, who found that co-exposure to FKs and exogenous GABA ameliorated life history changes to FKs in a D. pulex clone, whereas co-exposure with the GABAA antagonist PICRO did not have any effect. The ineffectiveness of PICRO on the modulation of FK effects in D. pulex found by Weiss et al.11 might indicate species differences resulting from different receptor amino acid sequences. For example, GABAA receptor subtypes with a single amino acid replacement make the Drosophila GABAA receptor PICRO-insensitive36. Indeed, in crustaceans, lobster GABAA receptors were also found to be insensitive to PICRO37. There is also the possibility that FK-mediated changes in phototactic behaviour and life history traits may be controlled by different mechanisms6.Reported information on the modulatory effects of cholinergic compounds on anti-predatory defences in Daphnia is limited to invertebrate predatory cues, which, according to previous studies, should be regulated by neurological mechanisms distinct from those of fish2,11. Our results showed that the neurological cholinergic mechanisms that modulate induced defence responses against invertebrate predators or that mimic these responses are also able to do the same for fish predation but in the opposite way. Physostigmine and carbaryl, which are acetylcholinesterase inhibitors that increase acetylcholine receptor activity, enhanced and mimicked, respectively, the morphogenetic effects of invertebrate kairomones in several Daphnia species11,21,23. Conversely, atropine, which is a muscarinic acetylcholine receptor (AChR) inhibitor like SCOP, diminished neck tooth formation in D. pulex11,21. In our study, SCOP alone abolished the positive phototactism of the studied clone, which mimicked the effects of the FKs. Conversely, PILO, which is a muscarine AChR agonist, ameliorates the phototactic responses to FKs.The nicotinic AChR agonists (NICO, IMI) and antagonist (MEC) only marginally affected the phototactic responses to the FKs. This indicates that muscarinic cholinergic signalling but not nicotinic signalling is involved in phototaxis/phototactic behaviour. It is therefore possible that both FK and SCOP treatment, through inhibition of muscarinic cholinesterase receptor activity, diminished the positive phototaxis of the studied clone, and PILO activation of these receptors ameliorated the effects of the FKs. In insects, neurons that connect olfactory inputs to higher-order brain areas that coordinate behavioural responses are thought to be under cholinergic control38.In general, GABA is known to have inhibitory functions. It has been proposed that the continuous activation of the GABAergic neuronal pathway by endogenous GABA without predatory cues prevents life history shifts11, which in our case would be the transition from positive to negative phototaxis. FKs and PICRO relieve inhibition, which can be re-established by the experimental application of GABAA receptor agonists such as DZP or GABA itself. Our results and those of Weiss et al.11 agree with the previous argument.Equi-effective mixtures of the tested agonists and antagonists had similar effects on D. magna responses to FKs as the single mixture compound treatments did, indicating that the joint effects of agonists and antagonists of the GABAergic and cholinergic signalling pathways can act cooperatively and probably independently, modulating the effects of FKs. This is in line with other findings that showed that key ecophysiological responses in Daphnia are regulated by several signalling receptor pathways, which likely ensures more robust control. This is the case for the storage lipid dynamics associated with moulting and reproduction39.The involvement of additional neurotransmitter signalling pathways, such as the serotonergic pathway, can also be taken into consideration despite being less consistent. Agonists of the serotonin receptor (such as serotonin) or treatments that increase serotonin levels (such as fluoxetine) ameliorated the effects of the FKs in only one experiment, but treatments that decreased serotonin, such as PCPA, increased the effects of the FKs in two out of the three experiments. Previously, we reported that serotonin activity in the brains of D. magna increased with algae food levels, and thus, the effects of fluoxetine on the enhancement of brain serotonin levels could only be observed under limited food conditions24. This indicates that the high levels of food used in our experiments probably prevented fluoxetine from increasing the already high serotonin levels in the central nervous system. Interestingly, inducible fish kairomone changes in phototactic behaviour in Daphnia increased with food level40, which is probably related to high levels of serotonin. On the other hand, the effects of PCPA, which decreases serotonin concentrations26, are unlikely to be modulated by food since this drug inhibits tryptophan hydrolase, the serotonin synthesis rate-limiting enzyme in D. magna41. This is apparently the case in our study.Neurophysiological stimulation experiments with dopaminergic/adrenergic agonists and antagonists were inconclusive since in only one out of two experiments the dopaminergic agonist APO diminish negative phototaxis after FK exposure. We also did not find any effects from the glutamatergic agonists and antagonists on phototactism. This could be related to the low stability of dopaminergic compounds in water and the reported small effects of glutaminergic compounds on the Daphnia motile response to light34.Consistent failure of the tested antihistaminergic drugs to modulate phototactism to visible light disagrees with previous findings that discovered that these drugs affected phototactism but at much higher doses12.Metabolomic changesThe study of metabolomic changes across the treatments that modulated FK-mediated phototactic changes or altered phototaxis provided further experimental evidence of the involvement of key neurological signalling metabolic pathways. Caution must be exercised, however, since the studied receptor agonist and antagonist drugs do not change the neurotransmitters or their related metabolites. Nevertheless, little is known about how these drugs may affect the Daphnia neuronal metabolome. The cholinergic neurotransmitter system is one of the most important systems that plays a pivotal role in learning and memory in animal species, including D. magna34,42. Whole-body concentrations of acetylcholine decreased in females exposed to FKs and those exposed to SCOP and increased in those exposed to the agonists PILO and DZP. Thus, it is possible to establish a direct link between the decreased levels of acetylcholine and decreased positive phototactism in the studied clone. The results obtained for the GABAergic and serotonergic signalling pathways were less convincing, as FKs alone did not consistently affect the levels of GABA and serotonin. However, co-exposure to FK and the GABAA receptor agonist DZP increased endogenous GABA levels, which is in line with the results reported by Weiss et al.11, who also found that the addition of exogenous GABA ameliorated FK effects. Interestingly, the summarized results depicted in Fig. 4 showed that serotonin levels dereased upon exposure to SCOP, PICRO and PILO but PILO also increase the levels of the serotonin degradation metabolite 5-HIAA. This may indicate that PILO may affect the turnover rather than the levls of serotionin.Previous findings have reported altered responses to light in D. magna individuals lacking serotonin16. Therefore, it is possible to establish a link between the observed marked negative phototactism of females exposed to SCOP and low levels of serotonin.Dopaminergic- and adrenergic-related metabolites deserve special attention, although there is only evidence that dopamine is involved in the proliferation and structural formation of morphological defences in Daphnia for invertebrate kairomones22. In some invertebrates, adrenergic signalling is considered to be absent, and the analogous functions are performed by octopamine43. In our study, fish kairomones and SCOP decreased the levels of dopamine and octopamine, whereas females co-treated with the agonists DZP and PILO and FKs showed relatively high levels of dopamine. In the insect Drosophila melanogaster, which shares many gene signalling pathways with Daphnia44, individuals deficient in dopamine show reduced positive phototactism45. Unfortunately, it is not possible to know whether the observed changes in DA in the whole bodies of D. magna indicate that DA is less used or used in excess. Figure 4 indicates that FK and SCOP reduced both DA and its intermediary metabolite L-DOPA. SCOP also increased the DA degradation metabolite 3-MT and two norepinephrine metabolites/neurotransmitters (NOEM, EPPY) that ultimately depend on DA. This means that FK decreased DA probably decreasing its intermediary metabolite L-DOPA, whereas SCOP decreased DA to a greater extent decreasing its intermediary L-DOPA but also increasing its turnover rate. Our neurophysiological stimulation experiments with dopaminergic active compounds are also not conclusive. This suggests that further research is needed to study the involvement of dopaminergic signalling in the response to fish. Existing studies on adrenergic signalling in daphnids indicated that β-blockers such as propranolol diminish the heart rate46 and motile responses to light27, which are related to the known role of adrenergic signalling that regulates blood pressure47 and other fight-or-flight responses to stress48. Future research is needed to elucidate the involvement of OCT, EPPY and NORM in the phototactic response of D. magna to FKs.In summary, this study provides consistent results that muscarinic cholinergic and GABAergic receptor agonists and antagonists are able to ameliorate or enhance, respectively, the phototactic response of adult females from the studied D. magna clone to FKs. Furthermore, inhibition of the muscarinic acetylcholine receptor by SCOP induced the phototactic response to fish kairomones. This may indicate that muscarinic cholinergic antagonists changed phototaxis, whereas muscarinic cholinergic agonists and GABAergic agonists and antagonists changed the perception of FKs. Serotonergic agonists and antagonists were also able to diminish and increase FK effects, respectively, but only in half of the trials performed. The fact that we could not observe effects from the remaining neuroactive agents (i.e., dopaminergic, histaminergic, glutamatergic) could simply be because they are not relevant for predator-induced anti-phototaxis. The study of neurotransmitters and their related metabolite changes allowed us to identify acetylcholine and GABA as putative key metabolites associated with the observed phototactic modulatory effects of FK and cholinergic and GABAergic compounds. Increased and decreased levels of dopamine in the whole bodies of D. magna were related to positive and negative phototactic behaviours, respectively, but could not be related to neurophysiological studies with the tested dopaminergic drugs. More

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Our measure of interest was whether females ate more of the flake items (i.e., cheated less quickly) when the male had perceptual access than when he did not. Consistent with our predictions, females ate fewer flake items in the male not visible condition (Mean = 3.6 items, SD = 3.2) than in the male visible condition (Mean = 4.5, SD = 3.1; Fig. 2A; for flake items eaten by pairs see Supplementary Fig. S1). Additionally, females tended to cheat less over rounds (Supplementary Fig. S2). Indeed, the number of flake items eaten was predicted by both conditions (LRT, X21 = 8.13, p = 0.004; Fig. 2A; Supplementary Table S1) and round (LRT, X21 = 12.46, p  More

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In banana, bract opening behavior depends on the time of the day, the position of the bract, and sex of the flowers enclosed by the bract. Bract opening is a continuous process especially in the first bracts subtending female flowers of some genotypes; it starts in the evening and continues through the night (Table 1). In cases where bracts did not fully open, the process was halted early morning and resumed in the evening. It is therefore not obvious to judge whether such bracts have opened or not. However, opening is permanent as opposed to some plant species which open and close their flowers at specific times. Ssebuliba et al.16 considered East African Highland bananas ready for pollination when bracts were half way open with stigmas having a creamy white appearance. According to observations made in the current study, it can be said that bract lifting is indicative of flower opening thus pollination can start.Bract lift and bract roll seemed to be a response of a certain light quality6, the response time and speed are genotype dependent. Finger curling also seems to be triggered by the same factors that lead to bract opening. Bract opening and finger curling are likely to be a response of changes in turgor pressures in cells that lead to tissues being pushed in a given direction17. This was evident with upward movement of the inflorescence from the horizontal-pendent toward the horizontal position in the evening and downward movement towards the pendent position by mid-morning. These movements were genotype dependent and small, maximum oscillation was about 10˚. A similar pattern was observed for leaf folding to influence relative canopy cover18.Generally, bracts subtending female flower lifted and started rolling earlier than those subtending male flowers. However, male flowers ended opening before female flowers, resulting in faster bract opening for male flowers (Table 1 and t-test). This might be due to the smaller bract size of male flowers (Fig. 1) or an adaption for female flowers to find male flowers open with ready pollen. Consequently, the strategy ensures maximum pollination success and survival of the Musa spp. Studies have revealed that pollen viability reduces with time after flower opening1. This is in agreement that controlled pollination should be done between 07:00 and 10:00 h7. In comparison to lilies, some flowers were observed also to open starting at 17:00 h while others open during day. Both nocturnal and diurnal pollinators were found to be active flower visitors19. This implies that pollination in banana can start in the evening as long as bracts for parents in the cross of interest lift in time.In Musa itinerans, two nectar production peaks were found, that is between 08:00 to 12:00 h and 20:00 to 24:00 h20. This maybe a close depiction of what happens in edible bananas thus emphasizing the potential importance of diurnal and nocturnal pollinators. Bats, bees, and birds were found to be among the most important pollinators of bananas at Onne, Nigeria10. However, natural pollinators were not the main focus of the study though they are good indicators of when stigmas might be highly receptive. Since nectar quality and quantity varies between different agro-ecologies and seasons21, flower visitations and seed set are also expected to vary accordingly. Different agro-ecologies are also expected to experience variable BOTs due to variable solar radiation. Likewise the different growing seasons (rainy and dry) might also affect BOTs and therefore seed set22. However, a comparison of time from sunrise to beginning of bract lift of Musa AAA Cavendish cultivars in a glasshouse and M. basjoo in the garden in Belgium revealed no significant difference6. But comparison of bract curling time in Mchare in Arusha with short days and Cavendish cultivars in a glasshouse in Belgium with long days in summer, there was early curling in the glasshouse. However, bract lift time may be a better event to use for comparison than bract curling or rolling time.Bracts of both female and male flowers of different genotypes completed opening at different times and this may be partly the reason for variable pollen viability and stigma receptivity (Table 1). Female flowers that finish opening much earlier may set less seed compared to those that finish opening closer to the routine time of hand pollination between 07:00 and 10:00 h. Conversely, male flowers that are ready shortly before the time of hand pollination are expected to have higher pollen viability. This probably explains the high fertility of ‘Calcutta 4’ as it finished opening at 06:30 h. Some male flowers like those of Matooke finished opening as early as 21:54 h (Table 1) and are expected to have pollen with low viability at the time it is measured the next day.All observed inflorescences opened one female bract on the first day, increasing to multiple bracts opening on subsequent days (Fig. 2). One to three bracts subtending female flowers were observed to open per day from the second bract position of the inflorescence. The pattern of opening took on a hyperbolic shape with up to four bracts opening on the fourth day in the midsection of the inflorescence. For hand pollination, more clusters are therefore expected to be pollinated per day during bract opening in the mid-section of the inflorescence. The different clusters of female flowers that open on the same day are likely to have stigmas with varying receptivity. The darker appearance of stigmas of former clusters compared to creamy stigmas in latter clusters reflects higher receptivity in the latter2. This may explain why some clusters set more seed especially in the mid-section of a seemingly fertile inflorescence.Upon complete opening of female and transitional bracts, inflorescences went into a pause period before male flowers opened (Table 2). In additional to spatial separation of flowers, this is temporal separation to promote cross pollination in banana. However, temporal separation of male and female flowers is not very effective for genotypes that had less than 24 h of separation. With aid of crawling insects, self-pollination may happen between the last female cluster and the first male cluster as stigmas are likely to be receptive for more than one day. Once male flowers started opening, one bract opened per day and occasionally two bracts were observed to open on the same day. For highly fertile genotypes like ‘Calcutta 4’, ample pollen is produced to pollinate many female flowers. Male flowers are also produced throughout the inflorescence growth period which ensures constant supply of pollen especially for controlled hand pollination. Averages of bracts subtending male flowers opening per day could not be calculated as there were two to three observed bracts subtending male flowers for most genotypes.It appears that proximal bracts subtending female flowers are less stimulated to lift and roll compared to distal bracts subtending female flowers and all bracts subtending male flowers. This was revealed by low vigour of bract lift and the small angle of lift at 08:00 h especially in the first female flower cluster (Figs. 2, 3). The bract angle of lift increases from proximal to distal end and this has been linked to reduced fertility in proximal clusters2. But it may not be the case since highly female (in all clusters) and male fertile ‘Calcutta 4’ showed the same pattern as edible bananas. The high R2 for female bract roll scores compared to bracts subtending male flowers was a result of more bracts used to calculate averages for bracts subtending female flowers compared to bracts subtending male flowers (Fig. 3). For bracts subtending male flowers, two to three bracts were observed for most genotypes thus the first three data points were close to the trend line. Since the number of female clusters varies, reducing number of data points were used to calculate average bract lift angles in the distal end or larger inflorescences. Besides, bract lift angles of some clusters could not be measured because of obscurity or being in awkward positions. This led to the last two points being far off the trend line for angle of lift and hence a low R2.Flower opening time is said to be genetically and environmentally controlled, results from this study are in agreement since light had considerable influence on bract opening events (Tables 1, 3). Significant effects of temperature, solar radiation, and vapor pressure deficit on flower opening time have been observed in rice11. For Musa spp., only light has a significant relationship with BOT. However, there was early curling under long summer days in the glasshouse in Belgium compared to short days in Arusha field conditions6. This suggested a particular light signal for BOT in Musa spp. It is unclear why high light intensity led to early lift of bracts subtending male flowers and this calls for farther investigation. Since bracts subtending male flowers instinctively open later than bracts subtending female flowers, light intensity had less effect on the former bracts. The small sample size could have also had an impact on the results in the study, the light flush from the camera could have also affected the results. The extent of weather effects on BOT in banana need to be studied in field conditions of locations with significantly different day length for a more reliable conclusion. More

Study site, field survey, and in situ filtration
The field survey was performed in Tateyama Bay (34° 60′ N, 139° 48′ E), central Japan, in the proximity of the Kuroshio warm current facing the Pacific Ocean (Fig. 1). This area has many artificial reefs (ARs) created to improve fishing efficiency for fishers. Among the ARs, we focused on one high-rise steel AR (AR1), with a height of 30 m, where fish tended to aggregate (Fig. 1 and S1). Sampling stations were set up at the AR1 and at six linear distant points extending northeast and southwest. These stations were named E150, E500, E750, W150, W500, and W750, where “W” or “E” and the number of each station name represented northeast or southwest and distance in meters from the AR1, respectively (Table S1 and Fig. 1). Another station was set up at a second AR (AR2: 25 m height) 220 m from AR1 because we found AR2 by chance during the survey (Table S1 and Fig. 1), and it might affect the eDNA concentration at other stations.Figure 1(a) Location of sampling stations, cruise track, and a set net in Tateyama Bay. Gray areas indicate landmasses, a gray bold line indicates cruise track, and gray thin lines indicate depth contours with an interval of 20 m. The maps were created using ArcGIS Software 10.6.0.8321 by ESRI (https://www.esri.com/) based on the municipal boundary data of Japan (Esri Japan) and Global Map Japan (Geospatial information Authority of Japan) as well as the M7000-series isobath data set (Japan Hydrographic Association). A picture of the artificial reef (AR1) (b) taken one year after this survey (June 2019) and pictures of the dominant species, (c) splendid alfonsino (Beryx splendens), (d) chicken grunt (Parapristipoma trilineatum), (e) chub mackerel (Scomber japonicus), (f) red seabream (Pagrus major), and (g) jack mackerel (Trachurus japonicus). Photograph credits: (b) Nariaki Inoue, (c) Fumie Yamaguchi, (d, e, g) Yutaro Kawano, and (f) Masaaki Sato.Full size imageWe conducted water sampling at eight stations for eDNA analysis and performed an acoustic survey for estimating relative fish density using research vessel Takamaru (Japan Fisheries Research and Education Agency: FRA) on May 23, 2018. We started the echo sounder survey at the eastern part of the bay and continued it during the water sampling (Fig. 1). Although the echo sounder survey could not differentiate between fish species, we collected this data to assess the association between the estimated concentration of fish eDNA and the echo intensity measured by the echo sounder. Water sampling began at E750, then continued along the transect line to E150, AR1, W150, W500, W750, before going back to AR2. At each sampling station, we collected 10 L of seawater from both the middle and bottom layers by one cast of two Niskin water samplers (5L × 2 samples) and measured vertical profiles of water temperature and salinity with a conductivity-temperature-depth sensor (RINKO profiler, JFE Advantech Co., Ltd.). We subsampled 2L seawater from the 5 L seawater of Niskin sampler using measuring bottle and remaining 3 L seawater was used for pre-wash of measuring bottle and filtration devices. Two 2L samples were collected from two Niskin water samples, and then immediately filtered using a combination of Sterivex filter cartridges (nominal pore size = 0.45 μm; Merck Millipore) through an aspirator (i.e., the two filters were subsets of a single water collection) in a laboratory on the research vessel. After filtration (average time of 15 min), an outlet port of the filter cartridge was sealed with an outlet luer cap, 1.5 ml RNAlater (Thermo Fisher Scientific Inc., Waltham, MA) was injected into the cartridge using a filtered pipette tip to prevent eDNA degradation, and an inlet port was also sealed with an inlet luer cap14. The Niskin water samplers were bleached before each water collection using a commercial bleach solution while filtering devices (i.e., filter funnels and measuring cups used for filtration) were bleached after every filtration. We filtered 2L MilliQ water with a filter funnel and measuring cup as a field negative control to test for possible contamination. The filter cartridges were placed in a freezer immediately after filtration until eDNA extraction. In total we collected and filtered 32 eDNA samples (eight stations × two depth layers × two replicates). Disposable latex or nitrile gloves were worn during sampling and replaced between each sampling station.DNA extraction and purificationWorkspaces were sterilized prior to DNA extraction using 10% commercial bleach, and filter tip pipettes were used to safeguard against cross-contamination. Following the method developed by Miya et al.15, the eDNA was extracted and purified. Briefly, after removing RNAlater inside the cartridge using a centrifuge, proteinase-K solution was injected into the cartridge from the inlet port, and the port was re-capped with the inlet lure cap. The eDNA captured on the filter membrane was extracted by constant stirring of the cartridge at a speed of 20 rpm using a roller shaker placed in an incubator heated at 56 °C for 20 min. The eDNA extracts were transferred to a 2-ml tube from the inlet of the filter cartridges by centrifugation. The collected DNA was purified using a DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol. After the purification, DNA was eluted using 100 μl of the elution buffer (buffer AE). All DNA extracts were frozen at − 20 °C until paired-end library preparation.Preparation of internal standard DNAsFive artificially designed and synthetic internal standard DNAs, which were similar but not identical to the region of any existing fish mitochondrial 12S rRNA, were included in the library preparation process to estimate the number of fish DNA copies [i.e., quantitative MiSeq sequencing (qMiseq)]7,16. They were designed to have the MiFish primer‐binding regions as those of known existing fishes and to have the conserved regions in the insert region. Variable regions in the insert region were replaced with random bases so that no known existing fish sequences had the same sequences as the standard sequences. The standard DNA size distribution of the library was estimated using an Agilent 2100 BioAnalyzer (Agilent, Santa Clara, CA, USA), and the concentration of double-stranded DNA of the library was quantified using a Qubit dsDNA HS assay kit and a Qubit fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Based on the quantification values obtained using the Qubit fluorometer, the copy number of the standard DNAs was adjusted as follows: Std. A (100 copies/µl), Std. B (50 copies/µl), Std. C (25 copies/µl), Std. D (12.5 copies/µl) and Std. E (2.5 copies/µl). Then, these standard DNAs were mixed.Paired-end library preparationTwo PCR‐level negative controls (i.e., each with and without internal standard DNAs) were employed for MiSeq run to monitor contamination during the experiments. The first-round PCR (1st PCR) was carried out with a 12-µl reaction volume containing 6.0 µl of 2 × KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), 0.7 µl of each primer (5 µM), 2.6 µl of sterilized distilled H2O, 1.0 µl of standard DNA mix and 1.0 µl of template. Note that the standard DNA mix was included for each sample. The final concentration of each primer was 0.3 µM. We used a mixture of the following four PCR primers modified from original MiFish primers16: MiFish-U-forward (5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NNG TCG GTA AAA CTC GTG CCA GC-3′) and MiFish-U-reverse (5′-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TNN NNN CAT AGT GGG GTA TCT AAT CCC AGT TTG-3′), MiFish-E-forward (5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT NNN NNG TTG GTA AAT CTC GTG CCA GC-3′), and MiFish-E-reverse (5′-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TNN NNN CAT AGT GGG GTA TCT AAT CCT AGT TTG-3′). These primer pairs co-amplify a hypervariable region of the fish mitochondrial 12S rRNA gene (around 172 bp) and append primer-binding sites (5′ ends of the sequences before five Ns) for sequencing at both ends of the amplicon. The five random bases were used to enhance cluster separation on the flow cells during initial base call calibrations on the MiSeq platform. The thermal cycle profile after an initial 3 min denaturation at 95 (^circ)C was as follows (35 cycles): denaturation at 98 (^circ)C for 20 s; annealing at 65 (^circ)C for 15 s; and extension at 72 (^circ)C for 15 s, with a final extension at the same temperature for 5 min. Eight replications were performed for the 1st PCR, and the replicates were pooled to minimize the PCR dropouts. The 1st PCR products from the eight tubes were pooled in a single 1.5-ml tube. Then, we sent the 1st PCR products to IDEA consultants, Inc. to outsource the following MiSeq sequencing processes. The pooled products were purified and size-selected for 200–400 bp using a SPRIselect (Beckman Coulter, Inc.) to remove dimers and monomers following the manufacturer’s protocol.The second-round PCR (2nd PCR) was carried out with a 24 µl reaction volume containing 12 µl of 2 × KAPA HiFi HotStart ReadyMix, 2.8 µl of each primer (5 µM), 4.4 µl of sterilized distilled H2O, and 2.0 µl of template. We used the following two primers to append the dual-index sequences (8 nucleotides indicated by Xs) and flowcell-binding sites for the MiSeq platform (5′ ends of the sequences before eight Xs): 2nd-PCR-forward (5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACX XXX XXX XAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T-3′); and 2nd- PCR-reverse (5′-CAA GCA GAA GAC GGC ATA CGA GAT XXX XXX XXG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT-3′). The thermal cycle profile after an initial 3 min denaturation at 95 (^circ)C was as follows (12 cycles): denaturation at 98 (^circ)C for 20 s; combined annealing and extension at 72 (^circ)C for 15 s, with a final extension at 72 (^circ)C for 5 min. The concentration of each second PCR product was measured by quantitative PCR using TB Green Fast qPCR Mix (Takara inc.). Each sample was diluted to a fixed concentration and combined (i.e., one pooled 2nd PCR product that included all samples). The pooled 2nd PCR product was size-selected to approximately 370 bp using BluePippin (Sage Science). The size-selected library was purified using the Agencourt AMPure XP beads, adjusted to 4 nM by quantitative PCR using TB Green Fast qPCR Mix (Takara Bio Inc.), and sequenced on the MiSeq platform using a MiSeq v2 Reagent Kit (2 × 150 bp) (Illumina, Inc.).Data preprocessing and taxonomic assignmentThe raw MiSeq data were converted into FASTQ files using the bcl2fastq program provided by Illumina (bcl2fastq v2.18). The FASTQ files were then demultiplexed using the command implemented in Claident17. We adopted this process rather than using FASTQ files demultiplexed by the Illumina MiSeq default program in order to remove sequences with low-quality scores and PCR artifacts (chimeras).The processed reads were subjected to a BLASTN search against the full NCBI database. We excluded unique sequences of the following settings: the sequence belonged to organisms other than bony fishes, sharks, and rays; the sequence similarity between queries and the top BLASTN hit was  More